U.S. patent application number 16/757410 was filed with the patent office on 2020-09-10 for hook fusion protein for regulating the cellular trafficking of a target protein.
The applicant listed for this patent is CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), INSTITUT CURIE. Invention is credited to Gaelle Boncompain, Zelia Gouveia, Franck Perez.
Application Number | 20200283485 16/757410 |
Document ID | / |
Family ID | 1000004913852 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283485 |
Kind Code |
A1 |
Perez; Franck ; et
al. |
September 10, 2020 |
HOOK FUSION PROTEIN FOR REGULATING THE CELLULAR TRAFFICKING OF A
TARGET PROTEIN
Abstract
The present invention relates to a hook fusion protein
comprising a hook domain and at least one cytoplasmic carboxy
terminal endoplasmic reticulum (ER) retention signal and/or at
least one cytoplasmic amino terminal endoplasmic reticulum (ER)
retention signal; wherein the hook fusion protein is a soluble
protein that localizes in the cytoplasm. The present invention also
relates to a nucleic acid system for intracellular targeting
control comprising a nucleic acid encoding a hook fusion protein as
herein disclosed, and a nucleic acid encoding a target fusion
protein comprising a hook-binding domain; wherein said target
fusion protein in a membrane protein; and wherein the hook fusion
protein localizes in the ER when bound to the target fusion
protein. The invention also encompasses a vector system, viral
particle system, host cell and kit comprising said nucleic acids.
The invention also includes the vector system, viral particle
system, host cell or kit for use as a medicament, in particular for
immunotherapy.
Inventors: |
Perez; Franck; (Paris,
FR) ; Gouveia; Zelia; (L'hay les Roses, FR) ;
Boncompain; Gaelle; (Chatillon, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT CURIE
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) |
Paris
Paris |
|
FR
FR |
|
|
Family ID: |
1000004913852 |
Appl. No.: |
16/757410 |
Filed: |
October 22, 2018 |
PCT Filed: |
October 22, 2018 |
PCT NO: |
PCT/EP2018/078930 |
371 Date: |
April 19, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/63 20130101;
C07K 2319/04 20130101; C07K 14/7056 20130101; C07K 2319/10
20130101; C07K 14/7051 20130101; C07K 14/36 20130101; C07K 14/70539
20130101 |
International
Class: |
C07K 14/36 20060101
C07K014/36; C07K 14/725 20060101 C07K014/725; C07K 14/74 20060101
C07K014/74; C07K 14/705 20060101 C07K014/705; C12N 15/63 20060101
C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 20, 2017 |
EP |
17306453.6 |
Claims
1. A hook fusion protein comprising a hook core at least one
cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention
signal and/or at least one cytoplasmic amino terminal endoplasmic
reticulum (ER) retention signal; wherein the hook fusion protein is
a soluble protein that localizes in the cytoplasm.
2. A hook fusion protein according to claim 1, wherein the hook
core is a streptavidin sequence.
3. A hook fusion protein according to claim 1 wherein the carboxy
terminal endoplasmic reticulum (ER) retention signal is K(X)KXX,
with X being any amino acids and/or the amino terminal endoplasmic
reticulum (ER) retention signal is a fragment of the isoform of the
human invariant chain of the major histocompatibility complex
protein Ii, optionally wherein the fragment of the isoform of the
human invariant chain of the major histocompatibility complex
protein Ii has an amino acid sequence selected from SEQ ID NO: 5 or
14.
4. A hook fusion protein according to claim 1 further comprising an
endocytosis signal, preferably consisting of YXXI with X being any
amino acids.
5. A nucleic acid comprising a nucleic acid sequence encoding the
hook fusion protein according to claim 1.
6. The nucleic acid according to claim 5 further comprising a
nucleic acid sequence encoding a target fusion protein comprising a
hook-binding domain, wherein said target fusion protein is a
chimeric antigen receptor comprising: a binding domain; a
hook-binding domain, and at least one activation domain; or
alternatively comprising: the full NKG2D or a functional variant
thereof, at least one activation domain, and a hook-binding
domain.
7. A nucleic acid system for intracellular targeting control
comprising (a) a nucleic acid encoding a hook fusion protein
according to claim 1, and (b) a nucleic acid encoding a target
fusion protein comprising a hook-binding domain; wherein said
target fusion protein is a membrane protein; and wherein the hook
fusion protein localizes in the ER when bound to the target fusion
protein; optionally wherein the hook fusion protein comprises a
streptavidin domain and the target fusion protein comprises a
streptavidin-binding domain, optionally wherein the target fusion
protein is a chimeric antigen receptor as defined in claim 6
comprising: a binding domain; a hook-binding domain, and at least
one activation domain; or alternatively comprising: the full NKG2D
or a functional variant thereof, at least one activation domain,
and a hook-binding domain.
8. A vector system comprising one or more vectors comprising (a)
the nucleic acid sequence of claim 6, and optionally (b) a nucleic
acid encoding a target fusion protein comprising a hook-binding
domain; wherein the nucleic acids (a) and (b) are located on the
same or on different vectors; optionally wherein the hook fusion
protein comprises a streptavidin domain and the target fusion
protein comprises a streptavidin-binding domain.
9. A vector system according to claim 8, wherein the nucleic acids
(a) and (b) are located on the same vector and wherein the nucleic
acid (a) is inserted upstream of an IRES sequence and the nucleic
acid (b) is inserted downstream of said IRES sequence.
10. A vector system according to claim 8, wherein the nucleic acids
(a) and (b) are located on the same vector and wherein: i) the
nucleic acid (a) comprises an Ii retention signal in its N terminal
sequence and is inserted upstream of a 2A peptide sequence, or ii)
the nucleic acid (a) comprises a K(X)KXX retention signal in its C
terminal sequence and is inserted downstream of a 2A peptide
sequence.
11. A vector system according to claim 10 further comprising the
nucleic acid sequence (b) wherein said nucleic acid sequence (b)
comprises a streptavidin-binding domain, and wherein said nucleic
acid sequence (b) is inserted downstream of the 2A peptide in the
i) configuration or upstream of the 2A peptide in the ii)
configuration.
12. A vector system according to claim 8, wherein the target fusion
protein encoded by the nucleic acid (b) is a chimeric antigen
receptor comprising: a binding domain; a hook-binding domain, and
at least one activation domain; or alternatively comprising: the
full NKG2D or a functional variant thereof, at least one activation
domain, and a hook-binding domain.
13. A viral particle system comprising a vector system according to
claim 8; optionally wherein the viral particle system is a
lentiviral particle.
14. An isolated cell comprising a vector system as defined in claim
8.
15. An in vitro method for regulating the intracellular trafficking
in a host cell of a target protein; wherein said target protein is
a fusion protein comprising a hook binding domain; and wherein the
method comprises expressing in said host cell a vector system
according to claim 8; wherein the hook fusion protein and the
target fusion protein are capable of conditional interaction in the
absence of a ligand for the hook core domain, optionally wherein
the hook core domain is streptavidin, the hook-binding domain is a
streptavidin-binding domain and the ligand is biotin.
16. (canceled)
17. (canceled)
18. (canceled)
Description
INTRODUCTION
[0001] The inventors of the present application have previously
described a molecular system named RUSH (Retention Using Selective
Hooks) capable of controlling the intracellular trafficking of a
target fusion protein and in particular that can be used to control
the targeting of a target protein in a given cellular
compartment.
[0002] RUSH was described as a two state system based on the
reversible interaction of a hook protein fused to streptavidin and
stably anchored in the donor compartment with a target fusion
protein fused to a streptavidin-binding peptide (SBP), which is
therefore immobilized in said donor compartment. Addition of biotin
caused a synchronous release of the target protein from the hook
which is therefore free to resume its journey to its final
compartment.
[0003] The RUSH has been successfully applied to Chimeric Antigen
Receptors (CARs) as a tool to selectively control the expression of
the CARs at the cell surface, to prevent adverse effects (see
WO2016/012623).
[0004] The various hooks that have been described contained a
mutant of a stromal interaction molecule 1 (STIM1-NN, a type I
protein) that localizes in the endoplasmic reticulum (ER), 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 being an ER retention signal; or a
C-terminal ER retention signal (KDEL). However these hooks had some
limitations.
[0005] In particular, it remains of high relevance to develop
hooks, which have the smallest size as possible. Indeed, it is
known that vector production, notably lentivirus production, is
inversely proportional to the size of the protein of interest.
Consequently higher size inserts, such as the ones containing the
hook and the reporter (above 4 kb) impaired the production of
lentivirus required for the transduction and/or generation of
stable cell lines, namely modified immune cells containing CAR.
[0006] Also, vector constructs are based on cellular mRNAs
translation through initiation at internal ribosome entry sites
(IRESs) to generate a bicistronic or multicistronic vector
containing the hook and the reporter. It has been reported that
transduction of the proteins through IRES can impair their
expression (Jones, Peng et al. 2009). Moreover, the large size of
the IRES sequence and differences in gene expression levels before
and after IRES are other limiting factors. New strategies have been
envisioned to overcome such limitation, such as the use of
self-cleaving 2A peptide (2A) or 2A peptides (Jones, Peng et al.
2009). 2A peptides allow simultaneous translation of the protein
upstream and downstream of the 2A peptide (Szymczak, Workman et al.
2004, Tan, Liang et al. 2010, Yen and Scheerlinck 2013, Wang, Wang
et al. 2015). However, while extremely powerful, 2A peptides impose
that a few amino acids be added at the extremity of the 5' and 3'
ORF which prevent the use of carboxy-terminal signals ER retention
signals, like -KDEL or -K(X)KXX, that cannot be extended to stay
active.
[0007] Furthermore, the previously described hooks are expressed in
the endoplasmic reticulum (ER) (i.e.: anchored in the ER membrane
or expressed in the ER lumen). Therefore such hooks do not support
a fully reversible system, wherein the target membrane protein
could be retrieved from the membrane. Similarly such hooks do not
allow preventing the target membrane protein to leak out from the
ER, and in particular the leaking of the target membrane protein to
the cell membrane.
[0008] Lastly, the hooks that were stably anchored in the membrane
from the ER can only retain in the ER proteins that are in their
close vicinity.
[0009] The present invention provides an innovative technical
solution which overcomes the limitations as previously
mentioned.
SUMMARY OF THE INVENTION
[0010] The inventors have surprisingly discovered that the
carboxy-terminal KKXX (or K(X)KXX) sequence ER retention signal
that is described in the art as working in "Cis", when present on
membrane proteins, is able to work in "trans" when present on a
soluble cytosolic protein, if said protein is recruited on a
membrane protein. Indeed, their results now show that a soluble
cytosolic protein bearing an ER retention signal is stably
expressed in the cytosol but is targeted in the endoplasmic
reticulum when bound to a membrane protein. They also showed that
this interaction can be rendered reversible, such that retention in
the ER can also be reversible.
[0011] Therefore the present invention relates to a hook fusion
protein comprising [0012] a hook domain [0013] at least one
cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention
signal and/or at least one cytoplasmic amino terminal endoplasmic
reticulum (ER) retention signal;
[0014] wherein the hook fusion protein is a soluble protein that
localizes in the cytoplasm.
[0015] Such soluble hooks support a fully reversible control of the
membrane expression of a membrane target protein. The hooks of the
present invention should also allow retrieving proteins that leaked
out from the ER as these new "trans" hooks can sample the full
cytosol and retrieve protein from the Golgi back to the ER and
potentially from later compartments.
[0016] The inventors have further shown that a targeting sequence
based on the invariable chain Ii of human major histocompatibility
complex (MHC), corresponding to the first 46 amino acids of the N
terminal portion, can also be used as an ER retention signal when
fused in the N-terminal part of a soluble hook of the invention.
The results of the present application also show for the first time
that such a short fragment of the invariable chain Ii of the MHC
can be used as a retention signal.
[0017] "Trans" hooks of the invention having such an N terminal ER
retention signal are also highly advantageous as they can be
included in a vector upstream of a 2A peptide, as their C terminal
is now free to accommodate the 2A peptide without compromising the
function of the hook.
[0018] Advantageously also such "trans" hooks according to the
invention are of much smaller size than the hooks that have been
previously described.
[0019] Lastly, as also further shown in the results, an endocytosis
signal such as copied from the LAMP1 protein can be further fused
to the hook protein to create a hook that can therefore induce
trans-signaling for endocytosis and ER retrograde transport and
retention in a reversible way. Such a hook should therefore solve
the problem of target proteins leaking from the ER down to the
plasma membrane.
[0020] Thus, the hook fusion protein of the inventions comprises a
hook domain (or a hook core) that is typically a streptavidin
sequence.
[0021] The carboxy terminal endoplasmic reticulum (ER) retention
signal of the hook fusion protein of the invention can be K(X)KXX,
and/or the amino terminal endoplasmic reticulum (ER) retention
signal can be a fragment of the isoform of the human invariant
chain of the major histocompatibility complex protein Ii as herein
defined.
[0022] The hook fusion protein of the invention preferably
comprises an endocytosis signal, preferably consisting of YXXI.
[0023] The invention also includes a nucleic acid comprising a
nucleic acid sequence encoding the hook fusion protein as herein
defined.
The nucleic acid of the invention can further comprise a nucleic
acid sequence encoding a target fusion protein comprising a
hook-binding domain, wherein said target fusion protein is a
chimeric antigen receptor comprising:
[0024] a binding domain;
[0025] a hook-binding domain, and
[0026] at least one activation domain;
or alternatively comprising:
[0027] the full NKG2D or a functional variant thereof,
[0028] at least one activation domain, and
[0029] a hook-binding domain.
[0030] The invention also includes a nucleic acid system for
intracellular targeting control comprising
(a) a nucleic acid encoding a hook fusion protein as herein
defined, and (b) a nucleic acid encoding a target fusion protein
comprising a hook-binding domain; wherein said target fusion
protein in a membrane protein; and wherein the hook fusion protein
localizes in the ER when bound to the target fusion protein;
optionally wherein the hook fusion protein comprises a streptavidin
domain and the target fusion protein comprises a
streptavidin-binding domain, optionally wherein the target fusion
protein is a chimeric antigen receptor as herein defined.
[0031] The present invention also includes a vector system
comprising one or more vectors comprising
(a) the nucleic acid sequence as herein defined, and optionally (b)
a nucleic acid encoding a target fusion protein comprising a
hook-binding domain; wherein the nucleic acids (a) and (b) are
located on the same or on different vectors; optionally wherein the
hook fusion protein comprises a streptavidin domain and the target
fusion protein comprises a streptavidin-binding domain.
[0032] In a vector system of the invention, the nucleic acids (a)
and (b) can be located on the same vector, wherein the nucleic acid
(a) is inserted upstream of an IRES sequence and the nucleic acid
(b) is inserted downstream of said IRES sequence.
[0033] In a vector system of the invention, the nucleic acids (a)
and (b) can be located on the same vector wherein:
i) the nucleic acid (a) comprises an Ii retention signal in its N
terminal sequence and is inserted upstream of a 2A peptide
sequence, or ii) the nucleic acid (a) comprises a K(X)KXX retention
signal in its C terminal sequence and is inserted downstream of a
2A peptide sequence. In one embodiment the vector system of the
invention comprises the nucleic acid sequence (b) wherein said
nucleic acid sequence (b) comprises a streptavidin-binding domain,
and wherein said nucleic acid sequence (b) is inserted downstream
of the 2A peptide in the i) configuration or upstream of the 2A
peptide in the ii) configuration.
[0034] Typically in a vector system according to the invention, the
target fusion protein encoded by the nucleic acid (b) is a chimeric
antigen receptor as herein defined
[0035] The present invention also includes a viral particle system
comprising a vector system as herein defined; optionally wherein
the viral particle is a lentiviral particle.
The present invention also includes an isolated cell comprising a
vector system or a viral particle system as herein defined. The
present invention also relates to an in vitro method for regulating
the intracellular trafficking in a host cell of a target protein;
wherein said target protein is a fusion protein comprising a hook
binding domain; and wherein the method comprises expressing in said
host cell a vector system or a viral particle system as herein
defined; wherein the hook fusion protein and the target fusion
protein are capable of conditional interaction in the absence of a
ligand for the hook core domain, optionally wherein the hook core
domain is streptavidin, the hook-binding domain is a
streptavidin-binding domain and the ligand is biotin.
[0036] The present invention also relates to a kit comprising a
nucleic acid encoding the hook fusion protein, a vector system, a
viral particle system, or a host cell as herein defined.
[0037] The present invention also relates to a hook fusion protein,
a nucleic acid system, a vector system, a viral particle system, a
host cell or a kit, for use as a medicament.
[0038] The present invention also relates to the use of a hook
fusion protein, a nucleic acid or a system, a vector, a viral
particle system, a host cell or a kit for controlling the
trafficking of a target fusion protein, wherein said target fusion
protein is a membrane protein comprising a hook-binding domain
DETAILED DESCRIPTION
Definitions
[0039] 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.
[0040] 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.
[0041] 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
(Gin, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie,
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).
[0042] 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). The term
"in vivo" refers to events that occur within an organism (e.g.,
animal, plant, or microbe).
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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. 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.
[0051] As used herein, "recombinant" may refer to a biomolecule,
e.g., a gene or protein, or to a cell or 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
and/or if it contains any modifications that do not naturally occur
to the corresponding nucleic acid in a genome. 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. 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.
[0052] 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.
[0053] A "synthetic" RNA, DNA or a mixed polymer is one created
outside of a cell, for example one synthesized chemically.
[0054] 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. In some embodiments
such a fragment encodes a polypeptide fragment (as defined herein)
of the protein encoded by the open reading frame nucleotide
sequence.
[0055] 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.
[0056] 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)).
[0057] As used herein a "functional variant" of a given protein
includes the wild-type version of said protein, a variant protein
belonging to the same family, a homolog protein, or a truncated
version, which preserves the functionality of the given protein.
Typically the functional variant exhibit at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98%, or 99% amino acid identity with the given
protein.
[0058] As used herein, a "regulatory sequence" also named an
"expression control sequence" refers to polynucleotide sequences
which affect the expression of coding sequences to which they are
operatively linked. Expression control sequences or regulatory
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" (also interchangeably named regulatory
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.
[0059] As used herein, "operatively linked" or "operably linked" to
a linkage in which the expression control sequence (e.g.:
regulatory sequences) is contiguous with the gene of interest to
control its expression of the gene of interest. This term also
include expression control sequences that act in trans or at a
distance to control the expression of the gene of interest.
[0060] As used herein, the term "vector", "transfer vector"
"recombinant transfer vector", or "gene transfer vector" is
intended to mean a nucleic acid molecule capable of transporting a
foreign nucleic acid (such as the polynucleotide or the nucleic
acid encoding a hook fusion protein or the target fusion protein)
to which it is linked.
[0061] One type of vector which can be used in the present
invention includes, in a non-limiting manner, a linear or circular
DNA or RNA molecule consisting of chromosomal, non-chromosomal,
synthetic or semi-synthetic nucleic acids, such as in particular a
cosmid, artificial chromosomes such as a bacterial artificial
chromosome (BAC) or a yeast artificial chromosome (YAC), a viral
vector, a plasmid or an RNA vector. One of skill in the art would
be well equipped to construct a vector through standard recombinant
techniques, which are described in Sambrook et al. (1989) and
Ausubel et al. (1994), both incorporated herein by reference.
[0062] Numerous vectors into which a nucleic acid molecule can be
inserted, in order to introduce it into and maintain it in a
eukaryotic host cell including hematopoietic cell, are known per
se; the choice of an appropriate vector depends on the use
envisioned for this vector (for example, replication of the
sequence of interest, expression of this sequence, maintaining of
this sequence in extrachromosomal form, or else integration into
the chromosomal material of the host), and also on the nature of
the host cell.
[0063] A "plasmid," 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.
Naked nucleic acid vectors such as plasmids are usually combined
with a substance which allows them to cross the host cell membrane,
such as a transporter, for instance a nanotransporter or a
preparation of liposomes, or of cationic polymers. Alternatively, a
naked nucleic acid may be introduced into said host cell using
physical methods such as electroporation or microinjection. In
addition, these methods can advantageously be combined, for example
using electroporation combined with liposomes.
[0064] Another type of vector is a viral vector, wherein additional
DNA segments may be ligated into the viral genome. Viral vectors
are by nature capable of penetrating into cells and delivering
polynucleotide(s) of interest into cells, according to a process
named as viral transduction. Therefore, the polynucleotide
sequences of interest are introduced into cells by contacting the
recombinant viral vector with said cells. Viral vectors include
retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus,
poxvirus, and other virus vectors. Retrovirus includes in
particular type c retrovirus, human T cell leukemia virus (HTLV-1,
HTLV-2) and lentivirus. Lentivirus includes in particular human
immunodeficiency virus, including HIV type 1 (HIV1) and HIV type 2
(HIV2), feline immunodeficiency virus (FIV), bovine
immunodeficiency virus (BIV), equine immunodeficiency virus (FIV),
simian immunodeficiency virus (SIV), visna-maedi and caprine
arthritis-encephalitis virus (CAEV).
[0065] 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.
[0066] Moreover, certain vectors are capable of directing the
expression of genes or nucleic acid sequences (i.e. encoding the
hook fusion protein and/or the target fusion protein) to which they
are operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" (or simply "expression vectors").
Expression vectors can contain a variety of "control sequences,"
which refer to nucleic acid sequences necessary for the
transcription and possibly translation of an operably linked coding
sequence in a particular host organism.
[0067] 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.
[0068] As used herein, "a hook protein" is usable in a system
referred to as RUSH (retention using selective hooks) (see
Boncompain et al., Nat. Methods 9:493-498, 2012, as well as
WO2010142785 and WO201612623, which also describe the RUSH system).
Typically the hook protein is a fusion protein, which allows
retaining a target protein containing a corresponding hook-binding
domain in a donor compartment (i.e. the compartment from which the
target protein originates) by a specific interaction with said
target protein. When released from the interaction with the hook
protein, the target protein is free to traffic toward its target
compartment (i.e. the compartment to which the target protein is
targeted). To control these two states, the specific interaction
between the target protein and the hook is mediated by a reversible
interaction between two interaction domains. In one embodiment, the
interaction only occurs in the presence of a given ligand
("molecule-dependant" set-up, "MD"). In another embodiment, the
interaction occurs by default and can be disrupted by a given
ligand ("interaction-by-default" setup, "ID"). The removal or
addition of the ligand acts like a switch to allow the synchronous
release of the target protein from the donor compartment. When
referring to a hook protein and a target fusion protein in a
nucleic acid system, a vector system, an isolated cell, a kit or in
a method or use of the invention, it is intended that the target
fusion protein comprises a hook-binding domain which corresponds to
the hook domain of said hook fusion protein. Suitable hook
domain/hook-binding domain couples are described below.
[0069] Hook Fusion Protein:
[0070] The present invention provides a new hook protein, which is
a fusion protein comprising: [0071] a hook domain [0072] at least
one cytoplasmic carboxy terminal endoplasmic reticulum (ER)
retention signal and/or at least one cytoplasmic amino terminal
endoplasmic reticulum (ER) retention signal; wherein the hook
fusion protein of the invention is a soluble protein that localizes
in the cytoplasm.
[0073] By soluble protein it is intended herein that the hook
protein does not comprise a transmembrane domain. Said protein is
further a cytoplasmic protein.
[0074] This soluble hook protein allows controlling the
localization of a given target membrane protein comprising a
hook-binding domain, according to the presence or absence of a
specific ligand. The previously described hooks were stably
anchored by default in the donor compartment (typically the ER or
the Golgi apparatus). To the contrary, the new hook of the
invention remains soluble in the cytosol in the absence of binding
to the target protein containing the hook-binding domain. It can
therefore retrieve any target membrane protein that leaked out from
the ER to the cytosol. When bound to the target protein, the hook
protein retains the target protein in the endoplasmic reticulum
(ER) (i.e. the ER lumen or the ER membrane). Upon addition or
removal of the ligand, the target protein is released. The release
of said target protein is fast and synchronous for all the
molecules of the target protein.
[0075] In one embodiment, the interaction between the hook protein
and the target protein occurs in a molecule-dependent way in the
presence of a specific ligand ("molecule-dependent" or "MD" setup),
and can be reversed by wash-out of the ligand. According to this
embodiment, the interaction between the hook domain of the hook
protein and the hook-binding domain of the target protein occurs
only in the presence of a given ligand. This embodiment is called
the "MD" mode. Regulation of the interaction, which results in the
release of the target protein from the hook fusion protein, can be
carried out by wash-out of the specific ligand with or without
competition by a competitor, which competes with the specific
ligand for binding to either the hook domain of the hook protein or
the hook-binding domain of the target protein.
[0076] In this embodiment, the MD interaction couple (hook
domain/hook binding domain or hook-binding domain/hook domain) can
be the FKBP-FK506 binding domain 12/FKBP-rapamycin associated
protein (FKBP 12/FRAP). FKBP12 (also known as FKBP1A) is a FK506
and rapamycin-binding protein of 12 kD (Standaert et al, 1990; Maki
et al, 1990). FRAP is a 245 kD which binds to the FKBP12-rapamycin
associated protein (Brown et al., 1994). In a preferred embodiment
of the RUSH system, only the rapamycin-binding domains are used. In
this embodiment, the interaction occurs only in the presence of
rapamycin or analogues thereof as a specific ligand L. Such ligand
can be any ligand able to mediate the interaction between FKBP 12
and FRAP and can be, in particular, selected from the group
consisting of FKl 012, FK-CsA and rapamycin. Analogs of Rapamycin
(Rapalog) may also be used in conjunction with mutants of FKBP 12
and FRAP domains (like AP21967, ARIAD Pharmaceutical Inc.) These
ligands have been extensively used in systems for controlling gene
expression at the transcriptional level (see Clackson 1997 for
review). Rapamycin (commercially available from Sigma-Aldrich for
example) can be used at concentrations ranging from 1.5 nM to 200
nM, preferably from 1.52 nM to 12.2 nM, even more preferably at
about 3.1 nM. FK506 can be used as a competitor and can therefore
be added when rapamycin is removed, in order to disrupt the
interaction between FKPB 12 and FRAP. FK506 (commercially available
from Cayman for example) can be used at concentrations ranging from
390 .mu.M to 1.25 .mu.M, preferably at about 3.3 .mu.M. Other
competitors can be used, such as Ascomycin (Sigma-Aldrich) at
concentrations ranging from 12.5 .mu.M to 1.6 .mu.M, preferably at
about 3.3 .mu.M or SLF (Cayman) at concentrations ranging from 28.6
.mu.M to 3.6 .mu.M and preferably at about 5 .mu.M.
[0077] Alternatively, the MD interaction couple (hook domain/hook
binding domain or hook-binding domain/hook domain) can be
FKBP-rapamycin binding domain 12/a protein that binds to FKBP12 in
a rapamycin-dependent manner. In this embodiment, the interaction
occurs only in the presence of rapamycin or analogues thereof as a
ligand L. Document U.S. Pat. No. 6,492,106 discloses methods for
identifying such proteins that bind to FKBP 12 in a
rapamycin-dependent manner
[0078] In a preferred embodiment, the interaction between the hook
protein and the target protein occurs by default in the absence of
a specific ligand ("interaction by default" or "ID" set-up) and is
inhibited in the presence of such specific ligand. In this
embodiment, the interaction between the hook domain of the hook
protein and the hook-binding domain of the target protein occurs by
default, in the absence of any ligand. The interaction is disrupted
by the presence of a specific ligand.
[0079] Suitable ID interaction domain couples (hook domain/hook
binding domain or hook-binding domain/hook domain) can be selected
for example from the group consisting of Streptavidin/SBP tag,
Ftsz/ZipA, HPV E1/E2, recombinant antibody/epitope, recombinant
epitope/hapten, proteinA/IgG domain, Fos/Jun. Interaction domain
couples for which a specific ligand inhibiting the interaction is
already known are preferred.
[0080] In one embodiment, the ID interaction domain couple (hook
domain/hook binding domain or hook-binding domain/hook domain) is
FtsZ/ZipA. FtsZ and ZipA are bacterial proteins which form part of
the septal ring which forms during the replication of certain
Gram-negative bacteria. Their interaction can be disrupted by
addition of a small molecule named "compound 1" as a ligand L (see
Wells et al. 2007 for review.). Compound 1 (Wyeth Research (NY,
USA)) can be used at concentrations ranging between 10 and 100
.mu.M.
[0081] In the preferred embodiment according to the invention, the
ID interaction domain couple (hook domain/hook binding domain or
hook-binding domain/hook domain) is streptavidin/streptavidin
binding peptides (SBPs) and free biotin can be used as a ligand.
Streptavidin is a bacterial protein that binds with very high
affinity to vitamin D-biotin. In vitro selection approaches have
led to the discovery of synthetic peptides (SBPs) that bind to
Streptavidin and that can be competed out by biotin or biotin
mimetic molecules from the ALiS (Artificial ligands of
streptavidin) series (these compound are described in Terai T,
Kohno M, Boncompain G, Sugiyama S, Saito N, Fujikake R, Ueno T,
Komatsu T, Hanaoka K, Okabe T, Urano Y, Perez F, Nagano T.
"Artificial Ligands of Streptavidin (ALiS): Discovery,
Characterization, and Application for Reversible Control of
Intracellular Protein Transport". J Am Chem Soc. 2015 Aug. 26;
137(33):10464-7 and in Tachibana R, Terai T, Boncompain G, Sugiyama
S, Saito N, Perez F, Urano Y. "Improving the Solubility of
Artificial Ligands of Streptavidin to Enable More Practical
Reversible Switching of Protein Localization in Cells".
Chembiochem: a European journal of chemical biology. 2017 Feb. 16;
18(4):358-62).
[0082] Accordingly, a hook protein of the invention preferably
comprises as a hook domain a streptavidin protein sequence. Such a
hook protein according to the invention is able to control the
trafficking of any membrane protein comprising a
streptavidin-binding domain (such as SBP).
[0083] Preferably, the hook comprises a streptavidin protein
sequence, most preferably core streptavidin, such as described in
U.S. Pat. No. 5,672,691, which is hereby incorporated by
reference.
[0084] Streptavidin protein sequences suitable to the present
invention typically encompass the Streptavidin protein sequences as
described below:
TABLE-US-00001 (SEQ ID NO: 1, wt streptavidin sequence)
MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGG
AEARINTQWLLTSGTTEAN AWKSTLVG H DTFTKVKPSAAS I DAAK KAGVN NG N
PLDAVQQ
[0085] Suitable hook domains can also be selected from low affinity
streptavidin mutant sequences. Such streptavidin mutant sequences
can bind reversibly to biotin while keeping a high affinity for the
streptavidin-binding protein (SBP). Accordingly, streptavidin
protein sequences suitable for use in the present invention also
encompass streptavidin sequences as described in Wu et al., PLoS
ONE 8(7): e69530 (2013) and WO2013/038272 U.S. Pat. No 9,353,161B2,
which are hereby incorporated by reference. In particular,
streptavidin sequences wherein the glycine at aa 49 (including the
first methionine amino acid, or amino acid 48 if excluding said
first methionine) of SEQ ID NO:1 or SEQ ID NO: 2 is replaced with a
bulkier residue (e.g., threonine) to reduce the biotin binding
affinity without affecting the SBP binding affinity are
encompassed. Another mutation can also be introduced to further
favor SBP binding over biotin (mutation S27A).
[0086] In particular, the skilled person in the art can create a
single mutant containing a single mutation of serine to alanine
substitution at residue 27, and a double mutant containing this
change as well as a glycine to threonine substitution at residue 48
corresponding to full-length wild-type streptavidin (SEQ ID NO: 1).
Although threonine is exemplified as a replacement residue for
glycine 48, other residues with bulky side chains and high
propensity for turns (Pt>0.83) are contemplated (e.g., Asp, Glu,
Asn, Gln).
[0087] A monomeric core Streptavidin has also been constructed by
Wu and Wong (2005) (see U.S. Pat. No. 7,265,205 B2 and SEQ ID NO: 2
below).
TABLE-US-00002 (SEQ ID NO: 2).
MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYTLTGRYDSAPATDGSGTALGWRVAWKNNYRNAHSATTWSGQYVGG
AEARINTQWTLTSGTTEANAWKSTLRGHDTFTKVKPSAASIDAAKKAGVN N GNPLDAVQQ.
[0088] As used herein, "Streptavidin" can refer to all forms of
streptavidin (tetramer, core or monomer). In a preferred
embodiment, a streptavidin sequence comprises the amino acid
sequence as set forth in SEQ ID NO: 1 or 2 as well as the low
affinity variants as described above, or a variant thereof having
at least 80% identity with SEQ ID NO: 1 or SEQ ID NO: 2, preferably
85%, 90, 95, 96, 97, 98, 99, 99.5% identity with such sequences.
"Streptavidin" can also encompass Streptavidin homologs from other
species, such as avidin or rhizavidin. Mutant of these natural
biotin-binding proteins may also be used.
[0089] The endoplasmic reticulum (ER) retention domain according to
the invention can be any protein or protein domain which is a
resident of the ER. The term "resident", when used herein applied
to a given protein or domain and to a given compartment, is
intended to mean that said protein or domain is in majority located
in a given compartment. Typically, at least 70%, preferably at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of said protein
or domain is located in said compartment at steady-state in a host
cell.
[0090] Suitable carboxy-terminal endoplasmic reticulum (ER)
retention sequence are typically C-terminal dilysine-based motifs,
such as the KXKXX sequence (SEQ ID NO: 3) and the KKXX sequence
(SEQ ID NO: 4), with X being any amino acid.
[0091] Suitable amino-terminal endoplasmic reticulum (ER) retention
sequences can be selected from fragments comprising the
localization domains of an isoform of the invariant chain which
resides in the ER (Ii, a type II protein), of Ribophorin I or II
(Strubin et al., 1986; Strubin et al., 1984; Schutze et al., 1994;
Fu et al. 2000), of SEC61, or of cytochrome b5 (Bulbarelli et al.,
2002). Preferred amino-terminal endoplasmic reticulum (ER)
retention sequences are fragments comprising the localization
domains of an isoform of the invariant chain which resides in the
ER (Ii). Typically a suitable fragments comprising the localization
domains of an isoform of the invariant chain which resides in the
ER (Ii) comprises, or consists in, the first 46 amino acids
starting from the N-terminal portion of the Ii protein. Said
fragment is represented by the amino acid of SEQ ID NO: 5:
TABLE-US-00003 MHRRRARACREDQKPVTDDQRDLISNNEQLPMLGRRPGAPESKCSR.
[0092] In one alternative sequence the "T" amino acid in position
17 (from the left of the sequence SEQ ID NO: 5) might be replaced
by an "I" (SEQ ID NO: 14).
[0093] Preferably, the hook protein according to the present
invention further comprises an endocytosis signal. Such a hook
protein is highly advantageous as it allows retrieving target
proteins from the plasma membrane. Such hook allows a fully
reversible control of the subcellular localization of the target
protein. Indeed, the target protein may be retained in the ER
compartment, let free to resume its journey to the plasma membrane
or retrieved from the plasma membrane.
[0094] Typically the endocytosis signal (also named internalization
signal) allows clathrin-dependent endocytosis of the target
membrane protein. Suitable internalization signals include
endocytotic signals as described in Traub L M, Bonifacino J S.
"Cargo recognition in clathrin-mediated endocytosis". Cold Spring
Harb Perspect Biol. 2013 Nov. 1; 5(11):a016790. Typically, linear
motifs consisting of short arrays of invariant and variant amino
acids, including "tyrosine-based" YXXO and [FY]XNPX[YF] motifs, and
"dileucine-based" [DE]XXXL[LI] motifs, acidic clusters and
[YF]XNPX[YF] motifs are well-suited to the present invention.
Typically the signal is a C terminal endocytosis signal. Suitable
endocytosis signals according to the invention include fragments of
the LAMP1 protein sequence containing the endocytosis signal, or
comprises the [DE]XXXLL consensus sequence. In this notation, amino
acids are represented in single-letter code, X indicates any amino
acid, O indicates an amino acid with a bulky hydrophobic side
chain, and the brackets mean that either amino acid is allowed at
that position. The endocytosis signal of the invention may be
selected from the group consisting of SEQ ID NO: 15 to 18.
Preferably the endocytosis signal is YXXI (X being any amino
acid).
[0095] The hook protein typically comprises at least one ER
retention signal and at least one retention signal. In one
embodiment the hook protein comprises at least a cytoplasmic
carboxy terminal ER retention signal and/or a cytoplasmic amino
terminal ER retention signal and an endoplasmic retention signal.
The hook protein can comprise both a cytoplasmic carboxy terminal
ER retention signal and a cytoplasmic amino terminal ER retention
signal in addition to the endocytosis signal.
[0096] Nucleic Acids and Vectors of the Invention
[0097] The present invention also encompasses a nucleic acid system
comprising one or more nucleic acids and comprising (a) at least
one nucleic acid sequence encoding a hook fusion protein as
previously, defined and wherein the hook fusion protein localizes
in the ER when bound to a target fusion membrane protein.
[0098] 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.
[0099] As a matter of example, the nucleic acid comprises one or
more of the following nucleic acid sequences encoding a hook fusion
protein as previously described:
[0100] Soluble Cytoplasmic Hook Proteins Having N Terminal ER
Retention Signal
TABLE-US-00004 (SEQ ID NO: 6)
ATGCACAGGAGGAGAGCCAGGGCCTGTCGGGAAGATCAAAAGCCAGTCAC
tGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGG
GCCGGCGGCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGCTAGCGACCCT
AGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCATTACTGG
AACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTGGAGCCG
ATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCCGAATCA
CGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGACGGATC
TGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACCGGAATG
CTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCTGAGGCT
AGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGCAAATGC
TTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGAAACCCT
CCGCCGCTTCAATTGATGCCGCCAAAAAAGCCGGAGTCAACAACGGCAAT
CCTCTGGATGCCGTCCAGCAGTACCCCTACGACGTGCCCGACTACGCCGC
CGGCTACCAGACCATC.
The sequence SEQ ID NO: 6 having the following features: [0101]
HA-tag: nucleotides [622-648] [0102] LAMP1: nucleotides [649-666]
[0103] Streptavidin: nucleotides [146-621] [0104] mini-Ii-46aa:
nucleotides [1:138] [0105] Or
TABLE-US-00005 [0105] (SEQ ID NO: 7)
ATGCACAGGAGGAGAGCCAGGGCCTGTCGGGAAGATCAAAAGCCAGTCAT
CGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGG
GCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCCTCGAGGACCCT
AGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCATTACTGG
AACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTGGAGCCG
ATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCCGAATCA
CGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGACGGATC
TGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACCGGAATG
CTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCTGAGGCT
AGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGCAAATGC
TTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGAAACCCT
CCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAACAACGGCAAT
CCTCTGGATGCCGTCCAGCAG.
[0106] The sequence SEQ ID NO: 7 having the following features:
[0107] Core Streptavidin: nucleotides [145-621] [0108] mini Ii:
nucleotides [1-138]
[0109] Soluble Cytoplasmic Hook Proteins Having C Terminal ER
Retention Signal
TABLE-US-00006 (SEQ ID NO: 8)
ATGGACCCCAGCAAGGACAGCAAGGCCCAGGTGAGCGCCGCCGAGGCCGG
CATCACCGGCACCTGGTACAACCAGCTGGGCAGCACCTTCATCGTGACCG
CCGGCGCCGACGGCGCCCTGACCGGCACCTACGAGAGCGCCGTGGGCAAC
GCCGAGAGCAGATACGTGCTGACCGGCAGATACGACAGCGCCCCCGCCAC
CGACGGCAGCGGCACCGCCCTGGGCTGGACCGTGGCCTGGAAGAACAACT
ACAGAAACGCCCACAGCGCCACCACCTGGAGCGGCCAGTACGTGGGCGGC
GCCGAGGCCAGAATCAACACCCAGTGGCTGCTGACCAGCGGCACCACCGA
GGCCAACGCCTGGAAGAGCACCCTGGTGGGCCACGACACCTTCACCAAGG
TGAAGCCCAGCGCCGCCAGCATCGACGCCGCCAAGAAGGCCGGCGTGAAC
AACGGCAACCCCCTGGACGCCGTGCAGCAGGGCGGatcCTACCCCTACGA
CGTGCCCGACTACGCCGCCGGCTACCAGACCATCAAGAAGACCAAC
[0110] The sequence SEQ ID NO: 8 having the following features:
[0111] Streptavidin: [1:480] [0112] (GGS)-HA: [481:516] [0113]
LAMP1 tail: [517:534] [0114] ER Retention: [535:546] [0115] HA tag:
[490:516]
[0116] In a preferred embodiment the nucleic acid system further
comprises (b) at least one nucleic acid sequence encoding a target
fusion membrane protein comprising a hook-binding domain.
[0117] Said at least one nucleic acid sequences (a) and (b) can be
located on the same or different nucleic acids.
[0118] Preferably, the hook fusion protein comprises a streptavidin
domain and the target fusion protein comprises a
streptavidin-binding domain. In a preferred embodiment, the present
invention relates to a nucleic acid encoding at least one hook
fusion protein as previously described and a nucleic acid sequence
encoding a target fusion membrane protein comprising a hook-binding
domain.
[0119] Target fusion membrane proteins, which are well suited to
the present invention, are described in the following sections.
Thus the present invention also encompasses nucleic acids encoding
such target proteins.
[0120] A hook-binding domain is a domain that reversibly binds
directly or indirectly to a hook domain (as previously defined) of
a hook fusion protein inside a cell and which binding leads to the
retention of the target protein in the ER under appropriate
conditions. Suitable hook domains and corresponding hook-binding
domain have been described in the previous section (see hook fusion
protein). Therefore, an appropriate hook domain can be selected
from the above mentioned example depending on the selected hook
domain of the hook protein.
[0121] In a preferred embodiment, the hook domain of the hook
protein comprises a streptavidin sequence a previously mentioned.
Accordingly, the hook-binding domain comprises or consists in a
streptavidin binding peptide (SBP). Preferably, the hook-binding
domain comprises the following SBP amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 9)
MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP,
[0122] or is encoded by the nucleic acid sequence:
TABLE-US-00008 (SEQ ID NO: 10)
ATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGAAGGACT
GGCCGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACCCCCAGG
GCCAGAGAGAGCCC.
[0123] 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 crystallographies. Section D, Biological crystallography
69:879-887.
[0124] Well-suited short SBP (sSBP) versions can have the following
sequences:
TABLE-US-00009 (SEQ ID NO: 11) GHVVEGLAGELEQLRARLEHHPQGQREP, or
(SEQ ID NO: 12) GGHVVEGLAGELEQLRARLEHHPQGQREP
[0125] The target protein according to the invention can be any
protein for which is desirable to control the intracellular
trafficking from a given donor compartment to a final target
compartment, in particular for which it is desirable to reversibly
control cell membrane expression.
[0126] Examples of target proteins can be, but are not limited to:
plasma membrane markers and Major Histocompatibility (MHC)
molecules such as CD4, CD8; viral glycoproteins such as VSVG and
HA; signal transduction proteins; Transporter proteins like the
multidrug resistance protein ABCB 1. Typically, the target protein
can be any molecule of therapeutic interest, for which it is
desirable to tightly regulate the intracellular trafficking in
order to obtain a therapeutic effect. Conversely, the target
protein can be a pathological molecule, whose pathological effect
is linked to cell membrane expression. In a preferred embodiment,
the target protein is a chimeric antigen receptor (CAR).
[0127] In one embodiment, 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 as above defined; and
an intracellular signaling domain (activation domain). The CAR can
contain one, two, three, or more of each of these domains. The
invention encompasses individually all possible combinations of the
specific polypeptides and fragments thereof recited herein.
[0128] Typically, the binding domain of a CAR according to the
invention 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 antibody fragments (diabodies
and triabodies).
[0129] Preferably, the antibody is a single-chain Fv antibody or a
nanobody. In one embodiment, the antibody is monospecific. In one
embodiment, the antibody is multispecific for 2, 3, or 4
polypeptides, for example bispecific.
[0130] 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.
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.
[0131] 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.
[0132] 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.
[0133] 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., Cane. 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.
[0134] 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.
[0135] The activation domain of a CAR according to the invention
typically comprises CD3-.zeta. or Fc receptor .gamma. amino acid
sequences (see notably Sadelain et al., Cancer Discov. 2013 April;
3(4): 388-398, which is hereby incorporated by reference) or a
CD3-.zeta. chain and at least one cytoplasmic domain of a
costimulatory receptor. For example, costimulatory receptors
include CD28, 4-1 BB (CD137), DAP10, DAP12, OX40 (CD134), ICOS,
CD27, and CD40L.
[0136] 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 proteins having T-cell activating activity:
CD3-.zeta. chain and the costimulatory receptors CD28, 4-1 BB
(CD137), DAP10, DAP12, OX40 (CD134), ICOS, CD27, and CD40L.
[0137] In various embodiments, the 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 following
proteins having T-cell activating activity: CD3-.zeta. chain and
the costimulatory receptors CD28, 4-1 BB (CD137), DAP10, DAP12,
OX40 (CD134), ICOS, CD27, and CD40L.
[0138] 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 following proteins having T-cell
activating activity: CD3-.zeta. chain and the costimulatory
receptors CD28, 4-1 BB (CD137), DAP 10, DAP12, OX40 (CD134), ICOS,
CD27, and CD40L.
[0139] 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, FcRy, DAP10 and DAP12
transmembrane region.
[0140] Preferred CARs according to the above-mentioned description
and nucleic acids encoding such CARs are described in WO201612623.
In one embodiment, the nucleic acid encoding the hook fusion
protein as previously described further comprises a nucleic acid
sequence encoding a CAR as described in WO201612623.
[0141] In a distinct embodiment of the present invention, the
target fusion protein is an "NKG2D-based CAR" comprising: the full
NKG2D or a functional variant thereof, at least an activation
domain, and a hook-binding domain. The intracellular domain (i.e.
the cytoplasmic region) of the full NKG2D protein or functional
variant thereof can be fused to the activation domain or to the
hook-binding domain. Preferably, the intracellular domain (i.e. the
cytoplasmic region) of the full NKG2D protein or functional variant
thereof is fused to the hook-binding domain.
[0142] Natural killer (NK) cells attack tumor and virally infected
cells in the absence of major histocompatibility complex (MHC)
restriction, using a combination of signals from activating and
inhibitory receptors. One of these activating receptors is NKG2D,
which is expressed on all NK cells, NKT cells, .gamma..delta. T
cells, and some CD8+.alpha..beta. T cells. Ligands for human NKG2D
include MHC class I chain-related A (MICA), MICB, and several
UL-16-binding proteins (ULBPs). It has been found that NKG2D
ligands are primarily expressed on tumor cells but not on most
normal tissues. Thus, the NKG2D receptor-NKG2D ligand system
provides a relatively specific system for immune cells to recognize
tumor cells.
[0143] NKG2D according to the invention is preferably the human
NKG2D (UNIPROT ref P26718) encoded by the KLRK1 gene (killer cell
lectin like receptor K1) (Gene ID: 22914).
[0144] Suitable activation domains and hook-binding domains have
been described previously. Preferably the activation domain
comprises a CD3-.zeta. chain and/or at least one cytoplasmic domain
of a costimulatory receptor as defined above. For example,
costimulatory receptors include CD28, 4-1 BB (CD137), DAP10, DAP12,
OX40 (CD134), ICOS, CD27, and CD40L, as also defined above.
Preferably also, the hook-binding domain is a streptavidin binding
sequence as previously defined.
[0145] As a matter of example, a nucleic acid of the invention
encoding a hook fusion protein as previously described can further
encompasses a nucleic acid sequence encoding an NKG2D-based CAR
comprising the following construct: CD3z-SBP-NKG2D.
[0146] A nucleic acid sequence encoding a well-suited
CD3z-SBP-NKG2D construct is the following:
TABLE-US-00010 (SEQ ID NO: 13)
ATGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGG
CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG
ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG
CAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA
TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA
GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG
GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGAATTCCC
TGCAGGAGGCCGGCCAGACGAGAAGACCACCGGCTGGAGAGGCGGCCACG
TGGTGGAGGGCCTGGCCGGCGAGCTGGAGCAGCTGAGAGCCAGACTGGAG
CACCACCCCCAGGGCCAGAGAGAGCCCAGCGATCGCGGGTGGATTCGTGG
TCGGAGGTCTCGACACAGCTGGGAGATGAGTGAATTTCATAATTATAACT
TGGATCTGAAGAAGAGTGATTTTTCAACACGATGGCAAAAGCAAAGATGT
CCAGTAGTCAAAAGCAAATGTAGAGAAAATGCATCTCCATTTTTTTTCTG
CTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAGCAA
TATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATT
CCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTA
CAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGA
GCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGC
AAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGG
ACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCA
TTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGT
GCACTCTATGCATCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCC
AAATACATACATCTGCATGCAAAGGACTGTGTAATTA.
[0147] The present invention further encompasses a nucleic acid
system for the targeting control of a target protein comprising:
(a) a nucleic acid sequence encoding a hook fusion protein as
previously defined, and (b) a nucleic acid sequence encoding a
target fusion protein fused to a hook-binding domain as previously
defined. Typically as previously indicated the target fusion
protein in a membrane protein and the hook fusion protein localizes
in the ER when bound to the target fusion protein. Said nucleic
acid sequence (a) and (b) can be included in the same nucleic acid
or in separate nucleic acids. Preferably, the hook fusion protein
comprises a streptavidin domain and the target fusion protein
comprises a streptavidin binding domain. Preferably, also, the
target fusion protein is a CAR fusion protein as previously
described.
[0148] The present invention also encompasses an isolated nucleic
acid or nucleic acid system as described above inserted in one or
more vector(s).
[0149] Vectors of the Invention
[0150] The present invention encompasses a vector system comprising
one or more vectors comprising
[0151] (a) the nucleic acid sequence a hook fusion protein as
previously defined, and optionally
[0152] (b) a nucleic acid encoding a target fusion protein
comprising a hook-binding domain;
[0153] wherein the nucleic acids (a) and (b) are located on the
same or on separate vectors;
[0154] Preferably, as previously mentioned, the hook fusion protein
comprises a streptavidin sequence and the target fusion protein
comprises a streptavidin-binding domain.
[0155] A vector according to the present invention can be a
plasmid.
[0156] A vector according to the invention is preferably a vector
suitable for stable gene transfer and long-term gene expression
into mammalian cells, such as by replication of the sequence of
interest, expression of this sequence, maintaining of this sequence
in extrachromosomal form, or else integration into the chromosomal
material of the host. The recombinant vectors are constructed using
standard recombinant DNA technology techniques and produced using
conventional methods that are known in the art.
[0157] In some embodiments, a vector of the invention is an
integrating vector, such as an integrating viral vector, such as in
particular a retrovirus or AAV vector. Preferably, the viral vector
is a lentiviral vector.
[0158] Within the context of this invention, a "lentiviral vector"
means a non-replicating non-pathogenic virus engineered for the
delivery of genetical material into cells, and requiring lentiviral
proteins (e.g., Gag, Pol, and/or Env) that are provided in trans.
Indeed, the lentiviral vector lacks expression of functional Gag,
Pol, and Env proteins. The lentivirus vector is advantageously a
self-inactivating vector (SIN vector). The lentiviral vector
comprises advantageously a central polypurine tract/DNA FLAP
sequence (cPPT-FLAP), and/or insulator sequence (s) such as chicken
beta-globin insulator sequence(s) to improve expression of the
gene(s) of interest. The lentiviral vector is advantageously
pseudotyped with another envelope protein, preferably another viral
envelope protein, preferably the vesicular stomatis virus (VSV)
glycoprotein. In some preferred embodiments, said lentiviral vector
is a human immunodeficiency virus (HIV) vector.
[0159] 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. The lentiviral vector can
be in the form of a recombinant DNA molecule, such as a plasmid, or
in the form of a lentiviral vector particle (interchangeably named
lentiviral particle in the context of the present invention), such
as an RNA molecule(s) within a complex of lentiviral and other
proteins.
[0160] 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.
[0161] 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.
[0162] Efficient integration and replication in non-dividing cells
generally requires the presence of two c/s-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. 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.
[0163] 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 (AU3) (Miyoshi H et al, 1998, J Virol.
72(10):8150-7; Zufferey et al., 1998, J V/ro/72(12):9873-80).
[0164] 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 (AU3)
removing promoter and enhancer sequences in the 3' LTR.
[0165] The packaging sequence .psi. (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). In
one embodiment, the invention encompasses a lentiviral vector
comprising a lentiviral packaging sequence .psi. (psi).
[0166] 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. 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
[0167] Typically, lentiviral particles refer to the extracellular
infectious form of a virus composed of genetic material made from
either DNA or RNA (most preferably single stranded RNA) surrounded
by a protein coat, called the capsid, and in some cases an envelope
of lipids that surrounds the capsid. Thus a lentiviral vector
particle (or a lentiviral particle) comprises a lentiviral vector
as previously defined in association with viral proteins. The
vector is preferably an integrating vector.
[0168] The RNA sequences of the lentiviral particle 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. Appropriate methods for designing and
preparing lentiviral particles in particular for therapeutic
application are well-known in the art and are for example described
in Merten O W, Hebben M, Bovolenta C. Production of lentiviral
vectors. Mol Ther Methods Clin Dev. 2016 Apr. 13; 3:16017.
[0169] Preferably the lentiviral 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.
[0170] Preferably, a vector (i.e. a recombinant transfer vector) of
the invention is an expression vector comprising appropriate means
for expression of the hook fusion protein and/or the target fusion
protein in a host cell.
[0171] Various promoters may be used to drive high expression of
the nucleic acid sequence encoding the hook fusion protein and/or
the target fusion protein. The promoter may be a tissue-specific,
ubiquitous, constitutive or inducible promoter. Preferred promoters
are notably functional in T cells and/or NK cells, preferably human
T cells and human NK cells. In particular, preferred promoters are
able to drive high expression of the hook fusion protein and the
target fusion protein (notably a CAR as previously defined) from
lentivectors in T cells or NK cells, preferably human T cells or NK
T cells. For example, a promoter according to the invention can be
selected from phosphoglycerate kinase promoter (PGK), elongation
factor-1 alpha (EF-1 alpha) promoter including the short form of
said promoter (EFS), viral promoters such as cytomegalovirus (CMV)
immediate early enhancer and promoter, retroviral 5' and 3' LTR
promoters including hybrid LTR promoters, human ubiquitin promoter,
MHC class I promoter, MHC class II promoter, and 132 microglobulin
(.beta.2.eta..eta.) promoter. The promoters are advantageously
human promoters, i.e., promoters from human cells or human viruses.
Typically, the promoter can be a spleen focus-forming virus
promoter (SFPV). Human ubiquitin promoter, MHC class I promoter,
MHC class II promoter, and .beta.2 microglobulin
(.beta.2.eta..eta.) promoter are more particular preferred.
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 some embodiments the promoter is not a CMV
promoter/enhancer, or is not a dectin-2 or MHCII promoter. Such
promoters are well-known in the art and their sequences are
available in sequence data base.
[0172] In one embodiment of the present invention, the nucleic acid
encoding the hook fusion protein and the target fusion protein are
inserted into separate vectors.
[0173] In another embodiment, the nucleic acid encoding the hook
fusion protein and the target fusion protein are inserted into the
same vector.
[0174] When the vector system comprises more than one vector,
typically two or more vectors, said vectors are typically of the
same type (e.g.: a lentiviral vector). In the following sections
the vector can also be intended as "the one or more vector" or "the
vector system". Preferably the present invention encompasses a
lentiviral vector system and notably a lentiviral particle
system.
[0175] Each coding sequence (i.e. the nucleic acids encoding
respectively the hook fusion protein and the target fusion protein)
can be inserted in a separate expression cassette. Each expression
cassette therefore comprises the coding sequence (open reading
frame or ORF) functionally linked to the regulatory sequences which
allow the expression of the corresponding protein (hook fusion
protein and target fusion protein) in the host cell, such as in
particular promoter, promoter/enhancer, initiation codon (ATG),
codon stop, transcription termination signal.
[0176] Alternatively, the hook fusion protein and the target fusion
protein may also be expressed from a unique expression cassette
using an Internal Ribosome Entry Site (IRES), or a self-cleaving 2A
peptide inserted between the two coding sequences to allow
simultaneous expression.
[0177] Typically, nucleic acids encoding the hook fusion protein
and the target fusion protein can be inserted in a single
expression vector, said single vector comprising a bicistronic
expression cassette. Vectors containing biscitronic expression
cassette are well known in the art. Advantageously, bicistronic
expression cassettes contain an Internal Ribosome Entry Site (IRES)
that enables the expression of both fusion proteins from a single
promoter. Suitable commercially available bicistronic vectors can
include, but are not limited to plasmids of the pIRES (Clontech),
pBud (Invitrogen) and Vitality (Stratagene) series. Preferably, the
nucleic acid located upstream of the IRES sequence is
operably-linked to a promoter. Preferably the nucleic acid encoding
the hook fusion protein is inserted upstream of the IRES sequence
and the nucleic acid encoding the target fusion protein is inserted
downstream of said IRES sequence to ensure that enough the hook
fusion protein will be sufficiently expressed to retain every
target fusion protein. In some embodiments multicistronic
expression vectors may be used wherein more than one, typically at
least two, nucleic acids encoding each a distinct hook and at least
one nucleic acid encoding a target fusion protein are inserted. For
example such a vector may include a nucleic acid encoding a hook as
described in the present invention and a nucleic acid encoding a
hook as previously described in WO 201612623.
[0178] A self-cleaving 2A peptide can also be used in replacement
of IRES. Such strategy is highly advantageous because of its small
size and high cleavage and translation efficacy between nucleic
acid sequences upstream and downstream of the 2A peptide. Suitable
2A peptide according to the invention are notably described in Kim
J H, Lee S-R, Li L-H, et al. High Cleavage Efficiency of a 2A
Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines,
Zebrafish and Mice. PLoS ONE. 2011; 6(4):e18556. 2A peptides can be
selected from FMDV 2A (abbreviated herein as F2A); equine rhinitis
A virus (ERAV) 2A (E2A); porcine teschovirus-1 2A (P2A) and
Thoseaasigna virus 2A (T2A). P2A or T2A peptide is preferred.
Although the use of a self-cleaving 2A peptide is generally
recommended when a stoichiometric expression of the sequences
located upstream and downstream of the 2A peptide, the inventors
have found that it could still be used advantageously in the
present "RUSH" context.
[0179] Typically, the invention encompasses a vector notably and
expression vector comprising a nucleic acid encoding the hook
fusion protein as previously defined which is inserted upstream of
a 2A peptide sequence and a nucleic encoding a target fusion
protein which is inserted downstream of the 2A peptide. In this
configuration, the hook fusion protein comprises an amino-terminal
ER retention signal such as an Ii retention signal as previously
described. The invention also encompasses a vector notably and
expression vector comprising a nucleic acid encoding the hook
fusion protein as previously defined which is inserted downstream
of a 2A peptide sequence and a nucleic acid encoding a target
fusion protein which is inserted upstream of the 2A peptide
sequence. In this second configuration, the hook fusion protein
comprises a carboxy terminal ER retention signal such as a KXKXX
retention signal as previously described.
[0180] In the embodiment as above described the target fusion
protein is preferably a CAR as previously defined.
[0181] The present invention also encompasses a viral particle
system comprising a vector system as previously defined.
Preferably, the viral particle is a lentiviral particle. Preferably
the vector system is a lentiviral vector system. In one embodiment,
the vector system comprises one vector encoding both the hook
fusion protein and the target fusion protein. Thus a preferred
viral particle system according to the invention comprises a
lentiviral vector comprising a nucleic acid encoding the hook
fusion protein and a nucleic acid sequence encoding a target fusion
protein in association with viral proteins. The lentiviral vector
is preferably an integrating vector.
Isolated Cells of the Invention
[0182] The invention encompasses isolated cells, particularly cells
of the immune system, comprising vectors and notably a viral vector
particle system encoding at least a hook fusion protein as
previously described. Preferably the vector system and/or
lentiviral particle system further comprise a nucleic acid sequence
encoding a target fusion protein. Preferably, the cells are T cells
or NK cells.
[0183] In one embodiment, the cell contains the vector and/or viral
vector particle systems integrated into the cellular genome (stable
expression). In another embodiment, the cell contains the vector
transiently expressing the hook fusion protein and preferably also
the target fusion protein. In one embodiment, the cell produces
lentiviral vector particles encoding the hook fusion protein and
preferably also the target fusion protein. Preferably the target
fusion protein is a CAR.
[0184] In various embodiments, the invention encompasses a cell
line, a population of cells, or a cell culture comprising vectors,
notably viral vector particles, encoding the hook fusion protein
and preferably also the target fusion protein.
[0185] Kit According to the Invention:
[0186] The present invention also relates to a kit comprising a
nucleic acid comprising at least a nucleic acid system as above
defined and comprising at least a nucleic acid sequence encoding a
hook fusion protein of the invention. Preferably said nucleic acid
system further comprises a nucleic acid sequence encoding a target
fusion protein. Preferentially, said nucleic sequences are
comprises in the same nucleic acid.
[0187] The kit of the invention may alternatively comprise a vector
system, a viral particle system, or a host cell as previously
defined. Preferably the kit comprises a vector encoding a hook
fusion protein and its corresponding target fusion protein.
Preferably the vector is a viral vector notably a lentiviral
vector. In another advantageous embodiment, the kit comprises a
viral vector particle system comprising a viral vector system
according to the invention.
[0188] Preferably the hook fusion protein comprises a streptavidin
sequence. Most preferably the streptavidin sequence is the
streptavidin sequence as set forth in SEQ ID NO: 1 or 2. In another
embodiment, the streptavidin sequence is a low affinity
streptavidin sequence as previously described.
[0189] In a preferred embodiment wherein the hook fusion protein
comprises a streptavidin sequence, the kit further comprises a
specific ligand. When the streptavidin sequence is a low affinity
streptavidin sequence the ligand is preferably biotin. When the
streptavidin sequence is not a low affinity streptavidin sequence
the ligand is advantageously a biotin mimetic molecule selected
from ALiS.
[0190] Method and Use for Regulating the Intracellular Trafficking
of a Target Protein in a Host Cell:
[0191] The present invention also encompasses a method, typically
an in vitro method, for regulating the intracellular trafficking of
a target protein in a host cell. As mentioned previously, said
target protein is a fusion protein comprising a hook binding
domain.
[0192] This method comprises the expression in said host cell of a
vector system or a viral particle as previously described; wherein
the hook fusion protein and the target fusion protein are capable
of conditional interaction in the absence of a ligand for the hook
core domain.
[0193] Preferably the vector system comprises one vector comprising
at least a nucleic acid sequence encoding the hook fusion protein
and at least a nucleic acid encoding the target fusion protein.
Preferably also the vector is a viral vector, notably a lentiviral
vector.
[0194] Preferably, when a viral particle according to the invention
is expressed in a host cell, said viral particle comprises at least
a nucleic acid sequence encoding the hook fusion protein and at
least a nucleic acid encoding the target fusion protein. Typically
the viral particle is a lentiviral particle.
[0195] Preferably in the method of the invention, the hook domain
of the hook fusion protein comprises a streptavidin sequence as
previously mentioned the hook-binding domain comprises a
streptavidin-binding peptide. In such a configuration release of
the target fusion protein is achieved upon addition of biotin,
which reverses interaction of the streptavidin sequence with the
streptavidin-binding peptide. The use of such configuration is
advantageous as biotin is a vitamin known to be well tolerated by
the organism even at high doses.
[0196] Typically, to achieve full reversibility of the trafficking,
a low affinity streptavidin sequence, as previously described is
used. Alternatively, when wild-type streptavidin sequences or
variants thereof with high affinity for biotin are used, full
reversibility may be achieved by using biotin mimetic compounds
such artificial ligands of streptavidin (ALiS) (see Terai T et al.,
2015 and 2017 previously mentioned) exhibits fast dissociation
kinetics and excellent cell permeability allowing repeated
reversible cycling of the target protein localization between the
plasma membrane and the endoplasmic reticulum. Indeed in these both
configurations (using low affinity streptavidin or ALiS) the target
fusion protein can be retrieved from the cell membrane by simple
wash-out of the biotin or ALiS thanks to the use of the new
cytoplasmic "trans" hook as herein disclosed.
[0197] The present invention also relates to the use of a hook
fusion protein, or a nucleic acid or a nucleic acid system, or a
vector system or a viral particle or a host cell or a kit as herein
described for "trans control" of the trafficking of a target fusion
protein; wherein said target fusion protein is a membrane protein
comprising a hook-binding domain.
[0198] Medical Uses of the Invention:
[0199] The present invention further relates to a hook fusion
protein, or a nucleic acid or a nucleic acid system, or a vector
system or a viral particle or a host cell or a kit as herein
described as a medicament. In particular, the present invention
relates to the use of a vector system, notably a viral vector
system and in particular a lentiviral vector system as a
medicament. Said vector system comprises a nucleic acid sequence
encoding a hook fusion protein and a nucleic acid sequence encoding
a target fusion protein, which is preferably a CAR. Preferably also
the hook fusion protein has a hook domain comprising a streptavidin
sequence.
[0200] As previously mentioned the present invention based on an
innovative hook fusion protein design allows full control of the
expression of a corresponding target fusion protein to the cell
membrane. This innovation is of particular relevance when the
target fusion protein is a cytotoxic protein, such as a chimeric
antigen receptor, which cell exposure must therefore be timely
controlled.
[0201] 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.
[0202] Preferably the nucleic acid sequences as above mentioned are
included in the same vector, notably in the same integrating viral
vector, in particular in the same integrating lentiviral
vector.
[0203] Alternatively, the nucleic acid sequences as defined above
are present in two separate vectors, notably in two separate
integrating viral vectors, in particular two separate integrating
lentiviral vectors.
[0204] In a preferred embodiment, the invention relates to the
viral vector as above mentioned or to a viral vector particle
comprising said viral vector for use as a medicament. Said viral
vector or viral vector particle can be used for example in a
therapeutic composition or vaccines which are capable of inducing
or contributing to the occurrence or improvement of an
immunological reaction with the CAR encoded by the vector. The
invention therefore also encompasses an immunogenic composition
comprising a viral vector as previously defined.
[0205] The invention encompasses methods of administration of a
viral vector (notably a lentiviral vector) to a human. Preferred
modes of administration include reinfusion of the modified T cells,
preferably intravenously or intra-articular administration, most
preferably intra-tumoral administration.
[0206] In one embodiment, viral vector particles according to the
invention can be administered to T or NK cells. The obtained
modified T cells or NK cells can be further administered to a
human.
[0207] The viral vector and viral vector particles according to the
invention have the ability to redirect the specificity and function
of T lymphocytes and/or other immune cells such as NK cells. They
can rapidly generate T cells targeted to a specific tumor antigen
or an antigen relevant in other pathologies like auto-immune
diseases.
[0208] The viral vector and viral vector particles of the invention
can therefore be used in methods of treatment and methods of
inducing an immune response comprising administering the viral
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.
[0209] 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. As a result, the invention encompasses a
composition that could be used in therapeutic vaccination
protocols. In particular, it can be used in combination with
adjuvants, other immunogenic compositions, chemotherapy, or any
other therapeutic treatment.
[0210] The method can further comprise administering biotin or a
biotin mimetic as previously described to the human to release the
target fusion protein and in particular the CAR from the ER
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.
[0211] 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.
FIGURE LEGENDS
[0212] FIG. 1: Schematic representation of the bicistronic plasmid
coding for the known (Cs-Ii), newly Hooks and reporter gene. The
Hooks are (A) cytoplasmic hook (Str-Ii, streptavidin (str)) fused
to the isoform of the human invariant chain of the major
histocompatibility complex (Ii; a type II protein) containing an ER
retention arginine-based motif at the N-terminal); (B) soluble
Cytoplasmic Streptavidin with the endocytosis signal (YXXI, X any
aa) and ER retention signal (KKXX, X any aa); (C) cytoplasmic mini
Hook with a Ii retention signal in the N-terminal and the
endocytosis signal in C-terminal. These gene are expressed under
the control of the CMV promoter and separated by a synthetic
intron, i.e. intervening sequence (IVS) followed by an internal
ribosome entry site (IRES)(Boncompain, Divoux et al. 2012).
[0213] FIG. 2: Traffic of RUSH based constructs using the
streptavidin containing an endocytosis signal and the ER retention
signal KKXX hook. HeLa cells expressing A) scFv
(CD19)-myc-DAP10-sSBP reporter (anti-myc stained) or B)
CD3-SBP-NKG2D co-transfected with myc-DAP10 (DAP10 is required for
NKG2D traffic) (anti-NKG2D stained). The cells were non-treated
(NT) and treated with biotin and different time points were
recorded. Overnight treatment (ON) was performed by adding biotin
immediately after adding transduction solution and it is
representative of the protein steady state. Nucleus was stained
using DAPI.
[0214] FIG. 3: Traffic of RUSH based constructs using the soluble
mini hook containing an endocytosis signal. HeLa cells expressing
A) scFv (CD19)-GFP-DAP10-sSBP reporter or B) scFv
(CD19)-GFP-DAP10CD3-sSBP. Streptavidin in the mini hook was stained
with anti-Str. The cells were non-treated (NT) and treated with
biotin and different time points were recorded. Overnight treatment
(ON) was performed by adding biotin immediately after adding
transduction solution and it is representative of the protein
steady state. Nucleus was stained using DAPI.
[0215] FIG. 4: The soluble Str-endoKKXX Hook allows retention at
the level of the ER and retrieval from the cell surface. HeLa cells
were transfected by BACE1-GFP-SBP (BACE1-RUSH) together with an
Invariant chain-based Hook (Ii Hook, a-c) or a cytoplasmic Hook
bearing both an ER transport signal and an endocytosis signal
(Str-endoKKXX, d-f). Upon transfection in the absence of releasing
molecule (without biotin, a,d), BACE1-RUSH is retained in the ER.
Addition of the biotin-mimetic molecule ALiS-1 overnight (b, e)
allows efficient release of BACE1-RUSH and its transport to the
cell surface. Washing-out ALiS-1 for 1 hour (c,f) does not allow to
capture cell surface localized BALI-RUSH if transfected with the
Ii-based Hook (c) while it is efficiently transported back to the
ER when expressed with Str-endoKKXX highlighting the capacity of
the new Hook to mediate transport form the plasma membrane to the
ER in addition to its ability to retain proteins in the ER.
[0216] FIG. 5: The soluble miniIi Hook allows retention at the
level of the ER and retrieval from the cell surface. HeLa cells
were transfected by scFv (CD19)-myc-DAP10-sSBP together with a
cytoplasmic Hook bearing both an ER transport signal and an
endocytosis signal (Str-endoKKXX, a-c) or soluble mini hook
containing an endocytosis signal (mini hook, d-f). Upon
transfection in the absence of releasing molecule (without ALIS,
a,d), scFv (CD19)-myc-DAP10-sSBP is retained in the ER. Addition of
the biotin-mimetic molecule ALiS-1 45 min (b, e) allows efficient
release of scFv (CD19)-myc-DAP10-sSBP and its transport to the cell
surface. Washing-out ALiS-1 for 2 hours (c,f) efficiently
transported back scFv (CD19)-myc-DAP10-sSBP to the ER when
expressed with Str-endoKKXX or mini hook highlighting the capacity
of the new Hooks to mediate transport form the plasma membrane to
the ER in addition to its ability to retain proteins in the ER.
[0217] FIG. 6: Schematic representation of the NKG2D CAR. NKG2D
(type II protein) is fused to CD3 zeta domain and to SBP in two
distinct positions.
EXAMPLES
[0218] In the examples below, the term "Hook" refers to the hook
fusion protein comprising the hook domain, and the term "Reporter"
refers to the target membrane protein comprising the hook-binding
domain.
[0219] Methods and Material:
[0220] Constructs
[0221] FIG. 1 shows a schematic representation of the Hook
constructs by Boncompain et al (FIG. 1A), and of the new hooks
according to the present invention (FIG. 1B, 1C). Those are
inserted in the bicistronic vector using multicloning sites and the
reporter using the typical cloning cassettes of the previously
published RUSH vector. The soluble streptavidin containing an
endocytosis signal and the ER retention signal KKXX were built by
gene synthesis (gBlocks Gene Fragments--Integrated DNA Technologies
or GeneArt/Thermo-Fisher). The soluble mini hook containing a
endocytosis signal was synthetized by gene syntheses (gBlocks Gene
Fragments--Integrated DNA Technologies) was generated by PCR
amplification of the previously described luminal Ii-Str
(Boncompain, Divoux et al. 2012), using the primers
Fow-Nhe-IiMini-Str
(5'-CTAgctagccATGCACAGAAGAAGAAGCAGAAGCgaccctagcaaagactcaaaagc-3')(SEQ
ID NO: 19) and Rev-mini-Ii-2nd-Xho
(5'-CTCGAGgcggctgcacttgctctc-3')(SEQ ID NO: 20) for amplification
of the 46 aa of Ii and for streptavidin amplification the primer
Fow-Xho-Str (5'-CTCGAGGACCCTAGCAAAGACTCA-3')(SEQ ID NO: 21) and
REV-Ires (5'-GGATCAGTTATCTATGCG-3')(SEQ ID NO: 22). The fragments
generated were digested with the respective enzymes and clone into
the pCMV vector used previously in (Boncompain, Divoux et al.
2012). The sequences were evaluated and validate by sequencing. The
several reporters were used tagged with a fluorescent protein
(GFP). The sequence of some of the reporter were synthesized by
gene syntheses (gBlocks Gene Fragments--Integrated DNA
Technologies), other were previously generated in (Boncompain,
Divoux et al. 2012).
[0222] Cell Culture and Transfection:
[0223] HeLa cells were cultivated at 37.degree. C. and 5% of
CO.sub.2 in Dulbecco's modified Eagle medium (DMEM) supplemented
with 10% FBS (Biowest), 1 mM sodium Pyruvate and 100 .mu.M of
penicillin and streptomycin (Invitrogen). HeLa cells were
transfected with the plasmid of interest using Calcium phosphate
protocol in the presence of 25 mM of HEPES. Briefly, the plasmids
coding the sequence of CAR based RUSH such as CD3-SBP-NKG2D (SEQ ID
NO: 13), scFv(CD19)-GFP-DAP10CD3-sSBP (SEQ ID NO: 23),
scFv(CD19)-GFP-DAP10-SBPdel (SEQ ID NO: 24),
scFv(CD19)-mycDAP10-SBP (SEQ ID NO: 25) or BACE1-SBP-EGFP (SEQ ID
NO: 26) (2.5 ug per 1 mL of final volume) were add to 1 mM tris-HCl
pH 8.02 buffer followed by the addition of 10% of CaCl.sub.2 and
incubated for 5 min (RT). Then this mix was add drop by drop into
2.times. concentrate HEBS buffer (160 mM NaCl, 1.5 mM
Na.sub.2HPO.sub.4, 50 mM Hepes PH 7.04-7.05) while vortexing. The
cells were incubated with this solution overnight at 37.degree. C.
and 5% of CO.sub.2.
[0224] Time Course Release Using Biotin:
[0225] The cells were seeded into a glass coverslips for fixed cell
immunofluorescence and/or live imaging. In the next day the cells
were transfected with the plasmids coding the construct of interest
as previously described. For the steady state of the
protein/construct, 40 .mu.M final concentration of biotin was added
(4 mM stock solution) just after addition of the transfection
solution. The presence of biotin prevented the interaction of the
reporter (target membrane protein) with the hook, allowing the
normal traffic of the reporter. In the next day, the cells in the
coverslips were incubated at different time point with a final
concentration of 40 .mu.M of biotin, allowing the traffic of the
reporter and then prepared for immunofluorescence.
Biotin-mimetic molecule ALiS-1 was prepared in DMSO to 20 mM (stock
solution) and the cells were treated with 40 .mu.M final
concentration to prevent the interaction between the reporter
(target membrane protein) and the hook.
[0226] Immunofluorescence:
[0227] Cells coated in the coverslips were washed once in
1.times.PBS buffer, fixed in 3% of paraformaldehyde (PFA) (10-15
min, RT), washed (2.times.) and incubated with 50 mM of NH.sub.4Cl
(5 min, RT) to quench free aldehydes. The cells were then
permeabilized using a solution of PBS containing bovine serum
Albumin (BSA, 0.5%, Sigma-Aldrich) and saponin (Sapo, 0.05%
Sigma-Aldrich)(15 min, RT). When the protein was not fluorescent
labelled, we used antibodies for their detection. These include the
monoclonal anti human NKG2D (1/800, Biolegend), and anti-myc tag
from mouse (1/2000, clone 9E10) or anti-myc from rabbit (1/500,
Cell Signaling). The coverslip were mounted in Mowiol (Calbiochem)
supplemented with DAPI (4',6-Diamidino-2-phenylindole) for DNA
staining.
[0228] Results
[0229] Soluble Streptavidin Containing an Endocytosis Signal and
the ER Retention Signal KKXX:
[0230] The soluble streptavidin containing an endocytosis signal
and the ER retention signal KKXX was used to synchronized the
traffic of the CAR, scFvCD19-Myc-DAP10-sSBP (sSBP; small
streptavidin binding peptide, with 28 amino-acids (aa), instead of
the typical 36 aa) (FIG. 2, A) and the NKG2D based CAR, with SBP
into two different positions (FIG. 2, B). These include a SBP as
CD3-SBP-NKG2D (FIG. 2, B). The NKG2D based CARs were always
co-transfected with Myc-DAP10, since DAP10 is required for NKG2D
traffic. The scFvCD19-Myc-DAP10-sSBP was well retain in the ER by
this Hook and upon addition of biotin is released and 15 min later
reached the Golgi apparatus (FIG. 2, A). 30 min later, part of the
protein is localized in the cells surface although some remained in
the Golgi and at 60 min the majority is at the cell surface (FIG.
2, A). Overnight with biotin allow the traffic of the protein to
the cell surface, presumably as in its steady state (FIG. 2, A).
The CD3-SBP-NKG2D (FIG. 2, B) were co-transfected with DAP10 for
their traffic. Similar to the previous CAR, CD3-SBP-NKG2D (FIG. 2,
B) is retained in the ER by this Hook. The traffic behavior of the
CD3-SBP-NKG2D is very similar to the scFvCD19-Myc-DAP10-sSBP (FIG.
2, B). At 15 min the CD3-SBP-NKG2D is localized in the Golgi
apparatus, 30 min after it started to reach the cell surface and 60
min, the majority of the NKG2D is at the cell surface, although
some is still retain in the Golgi (FIG. 2, B).
[0231] Cytoplasmic Mini Hook:
[0232] To the cytoplasmic mini hook an endocytosis signal was added
or not in the C-terminal. The endocytosis signal is similar to the
one used for the soluble streptavidin containing an endocytosis
signal and the ER retention signal KKXX (FIG. 3). This newly
developed soluble mini hook is efficient to retain the CARs scFv
(CD19)-GFP-DAP10-sSBP reporter (FIG. 3, A) or scFv
(CD19)-GFP-DAP10CD3-sSBP (FIG. 3, B) in the endoplasmic reticulum.
To the construct scFv (CD19)-GFP-DAP10-sSBP was fused a CD3 zeta
domain (activation domain) after DAP10 that should increase the
activation capacity of the CAR (scFv (CD19)-GFP-DAP10CD3-sSBP). The
addition of the biotin leads to the release of the mentioned CARs
and at 15 min they reached the Golgi apparatus and at 30 min at
cell surface, although some still remain in the Golgi. At 60 min
with biotin, the majority of the CARs are at the cell surface
similar to the overnight treatment with biotin (FIG. 3). We also
observed the scFv (CD19)-GFP-DAP10CD3-sSBP CAR at 60 min and ON in
the presence of biotin, is still retained at the Golgi apparatus
even when the majority reached the cells surface.
[0233] Cytoplasmic Mini Hook and Soluble Str-endoKKXX Hook
Reversible Capacity:
[0234] We could observe that both cytoplasmic mini Hook and
str-endoKKXX allow the retention and release using a biotin-mimetic
molecule ALiS-1 (Terai et al, J. Am. Chem. Soc, 2015;
137(33):10464-7) (FIGS. 4 and 5). Washing-out ALiS-1 allows the
retrieval from surface localized BACE1-RUSH and scFv
(CD19)-myc-DAP10-sSBP back to the ER. These highlighting the
capacity of the new Hooks to mediate a reversible transport from
the plasma membrane to the ER while maintain their ability to
retain and thus release proteins in the ER.
Sequence CWU 1
1
261160PRTStreptomyces avidinii 1Met 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 1602160PRTArtificial Sequencemonomeric
streptavidin 2Met 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 16035PRTArtificial SequenceC ter retention
sequenceVARIANT(2)..(2)X means any amino acidVARIANT(4)..(5)X means
any amino acid 3Lys Xaa Lys Xaa Xaa1 544PRTArtificial SequenceC ter
retention sequenceVARIANT(3)..(4)X means any amino acid 4Lys Lys
Xaa Xaa1546PRTArtificial SequenceN ter retention sequence 5Met His
Arg Arg Arg Ala Arg Ala Cys Arg Glu Asp Gln Lys Pro Val1 5 10 15Thr
Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met 20 25
30Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg 35 40
456666DNAArtificial Sequencenucleotide sequence coding for soluble
cytoplasmic hook having N terminal ER retention signal 6atgcacagga
ggagagccag ggcctgtcgg gaagatcaaa agccagtcac tgatgatcag 60cgcgacctta
tctccaacaa tgagcaactg cccatgctgg gccggcggcc tggggccccg
120gagagcaagt gcagccgcgc tagcgaccct agcaaagact caaaagctca
ggtgtccgct 180gccgaggctg gcattactgg aacatggtac aatcagctcg
ggagcacctt tattgtgact 240gctggagccg atggagccct caccggaaca
tacgaatctg ctgtgggaaa cgccgaatca 300cggtacgtcc tcactggccg
atacgatagt gcccctgcca ccgacggatc tgggactgcc 360ctgggatgga
ctgtcgcttg gaaaaacaac taccggaatg ctcattctgc cacaacatgg
420agtggacagt acgtgggagg cgctgaggct agaatcaata cacagtggct
gctcacatct 480ggcacaaccg aggcaaatgc ttggaaatcc accctggtgg
gacatgacac attcaccaaa 540gtgaaaccct ccgccgcttc aattgatgcc
gccaaaaaag ccggagtcaa caacggcaat 600cctctggatg ccgtccagca
gtacccctac gacgtgcccg actacgccgc cggctaccag 660accatc
6667621DNAArtificial Sequencenucleotide sequence coding for soluble
cytoplasmic hook having N ter ER retention signal 7atgcacagga
ggagagccag ggcctgtcgg gaagatcaaa agccagtcat cgatgatcag 60cgcgacctta
tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg
120gagagcaagt gcagccgcct cgaggaccct agcaaagact caaaagctca
ggtgtccgct 180gccgaggctg gcattactgg aacatggtac aatcagctcg
ggagcacctt tattgtgact 240gctggagccg atggagccct caccggaaca
tacgaatctg ctgtgggaaa cgccgaatca 300cggtacgtcc tcactggccg
atacgatagt gcccctgcca ccgacggatc tgggactgcc 360ctgggatgga
ctgtcgcttg gaaaaacaac taccggaatg ctcattctgc cacaacatgg
420agtggacagt acgtgggagg cgctgaggct agaatcaata cacagtggct
gctcacatct 480ggcacaaccg aggcaaatgc ttggaaatcc accctggtgg
gacatgacac attcaccaaa 540gtgaaaccct ccgccgcttc aatcgatgcc
gccaaaaaag ccggagtcaa caacggcaat 600cctctggatg ccgtccagca g
6218546DNAArtificial Sequencenucleotide sequence coding for soluble
cytoplasmic hook having C ter ER retention sequence 8atggacccca
gcaaggacag caaggcccag gtgagcgccg ccgaggccgg catcaccggc 60acctggtaca
accagctggg cagcaccttc atcgtgaccg ccggcgccga cggcgccctg
120accggcacct acgagagcgc cgtgggcaac gccgagagca gatacgtgct
gaccggcaga 180tacgacagcg cccccgccac cgacggcagc ggcaccgccc
tgggctggac cgtggcctgg 240aagaacaact acagaaacgc ccacagcgcc
accacctgga gcggccagta cgtgggcggc 300gccgaggcca gaatcaacac
ccagtggctg ctgaccagcg gcaccaccga ggccaacgcc 360tggaagagca
ccctggtggg ccacgacacc ttcaccaagg tgaagcccag cgccgccagc
420atcgacgccg ccaagaaggc cggcgtgaac aacggcaacc ccctggacgc
cgtgcagcag 480ggcggatcct acccctacga cgtgcccgac tacgccgccg
gctaccagac catcaagaag 540accaac 546938PRTArtificial SequenceSBP
9Met 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 3510114DNAArtificial
SequenceSBP nucleotide sequence 10atggacgaga aaaccaccgg ctggcgggga
ggccacgtgg tggaaggact ggccggcgag 60ctggaacagc tgcgggccag actggaacac
cacccccagg gccagagaga gccc 1141128PRTArtificial Sequenceshort SBP
11Gly His Val Val Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala1
5 10 15Arg Leu Glu His His Pro Gln Gly Gln Arg Glu Pro 20
251229PRTArtificial Sequenceshort SBP variant 12Gly Gly His Val Val
Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg1 5 10 15Ala Arg Leu Glu
His His Pro Gln Gly Gln Arg Glu Pro 20 25131137DNAArtificial
SequenceCAR nucleotide sequence 13atgagagtga agttcagcag gagcgcagac
gcccccgcgt accagcaggg ccagaaccag 60ctctataacg agctcaatct aggacgaaga
gaggagtacg atgttttgga caagagacgt 120ggccgggacc ctgagatggg
gggaaagccg cagagaagga agaaccctca ggaaggcctg 180tacaatgaac
tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc
240gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac
agccaccaag 300gacacctacg acgcccttca catgcaggcc ctgccccctc
gcgaattccc tgcaggaggc 360cggccagacg agaagaccac cggctggaga
ggcggccacg tggtggaggg cctggccggc 420gagctggagc agctgagagc
cagactggag caccaccccc agggccagag agagcccagc 480gatcgcgggt
ggattcgtgg tcggaggtct cgacacagct gggagatgag tgaatttcat
540aattataact tggatctgaa gaagagtgat ttttcaacac gatggcaaaa
gcaaagatgt 600ccagtagtca aaagcaaatg tagagaaaat gcatctccat
tttttttctg ctgcttcatc 660gctgtagcca tgggaatccg tttcattatt
atggtagcaa tatggagtgc tgtattccta 720aactcattat tcaaccaaga
agttcaaatt cccttgaccg aaagttactg tggcccatgt 780cctaaaaact
ggatatgtta caaaaataac tgctaccaat tttttgatga gagtaaaaac
840tggtatgaga gccaggcttc ttgtatgtct caaaatgcca gccttctgaa
agtatacagc 900aaagaggacc aggatttact taaactggtg aagtcatatc
attggatggg actagtacac 960attccaacaa atggatcttg gcagtgggaa
gatggctcca ttctctcacc caacctacta 1020acaataattg aaatgcagaa
gggagactgt gcactctatg catcgagctt taaaggctat 1080atagaaaact
gttcaactcc aaatacatac atctgcatgc aaaggactgt gtaatta
11371446PRTArtificial SequenceN ter ER retention sequence 14Met His
Arg Arg Arg Ala Arg Ala Cys Arg Glu Asp Gln Lys Pro Val1 5 10 15Ile
Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met 20 25
30Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg 35 40
45154PRTArtificial Sequenceendocytosis signal
peptideVARIANT(2)..(3)X means any amino acidVARIANT(4)..(4)X
indicates an amino acid with a bulky hydrophobic side chain 15Tyr
Xaa Xaa Xaa1166PRTArtificial Sequenceendocytosis signal
peptideVARIANT(1)..(1)X indicates that either amino acid Y or F is
allowed at that positionVARIANT(2)..(2)X means any amino
acidVARIANT(5)..(5)X means any amino acidVARIANT(6)..(6)X indicates
that either amino acid Y or F is allowed at that position 16Xaa Xaa
Asn Pro Xaa Xaa1 5176PRTArtificial Sequenceendocytosis
signalVARIANT(1)..(1)X indicates that either amino acid D or E is
allowed at that positionVARIANT(2)..(4)X means any amino
acidVARIANT(6)..(6)X indicates that either amino acid L or I is
allowed at that position 17Xaa Xaa Xaa Xaa Leu Xaa1
5186PRTArtificial Sequenceendocytosis signal
peptideVARIANT(1)..(1)X indicates either amino acid Y or F is
allowed at that positionVARIANT(2)..(2)X means any amino
acidVARIANT(5)..(5)X means any amino acidVARIANT(6)..(6)X indicates
either amino acid Y or F is allowed at that position 18Xaa Xaa Asn
Pro Xaa Xaa1 51957DNAArtificial Sequenceprimer
Forward-Nhe-IiMini-Str 19ctagctagcc atgcacagaa gaagaagcag
aagcgaccct agcaaagact caaaagc 572024DNAArtificial SequencePrimer
Reverse-mini-Ii-2nd-Xho 20ctcgaggcgg ctgcacttgc tctc
242124DNAArtificial Sequenceprimer Fow-Xho-Str 21ctcgaggacc
ctagcaaaga ctca 242218DNAArtificial SequencePrimer REV-Ires
22ggatcagtta tctatgcg 18232187DNAartificial
sequencescFvCD19-GFP-DAP10CD3z-SBPd- 23atggccttac cagtgaccgc
cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggatatcc agatgacaca
gactacatcc tccctgtctg cctctctggg agacagagtc 120accatcagtt
gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa
180ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc
aggagtccca 240tcaaggttca gtggcagtgg gtctggaaca gattattctc
tcaccattag caacctggag 300caagaagata ttgccactta cttttgccaa
cagggtaata cgcttccgta cacgttcgga 360ggggggacca agctggagat
cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420ggcggatctg
aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc
480ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt
aagctggatt 540cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa
tatggggtag tgaaaccaca 600tactataatt cagctctcaa atccagactg
accatcatca aggacaactc caagagccaa 660gttttcttaa aaatgaacag
tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720cattattact
acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc
780gtctcctcac ctgcaggtat ggtgagcaag ggcgaggagc tgttcaccgg
ggtggtgccc 840atcctggtcg agctggacgg cgacgtaaac ggccacaagt
tcagcgtgtc cggcgagggc 900gagggcgatg ccacctacgg caagctgacc
ctgaagttca tctgcaccac cggcaagctg 960cccgtgccct ggcccaccct
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 1020taccccgacc
acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
1080caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
cgaggtgaag 1140ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
gcatcgactt caaggaggac 1200ggcaacatcc tggggcacaa gctggagtac
aactacaaca gccacaacgt ctatatcatg 1260gccgacaagc agaagaacgg
catcaaggtg aacttcaaga tccgccacaa catcgaggac 1320ggcagcgtgc
agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
1380ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
ccccaacgag 1440aagcgcgatc acatggtcct gctggagttc gtgaccgccg
ccgggatcac tctcggcatg 1500gacgagctgt acaagggccg gccacagacg
actccaggag agagatcatc actccctgcc 1560ttttaccctg gcacttcagg
ctcttgttcc ggatgtgggt ccctctctct gccgctcctg 1620gcaggcctcg
tggctgctga tgcggtggca tcgctgctca tcgtgggggc ggtgttcctg
1680tgcgcacgcc cacgccgcag ccccgcccaa gaagatggca aagtctacat
caacatgcca 1740ggcaggggcc ttaagagagt gaagttcagc aggagcgcag
acgcccccgc gtaccagcag 1800ggccagaacc agctctataa cgagctcaat
ctaggacgaa gagaggagta cgatgttttg 1860gacaagagac gtggccggga
ccctgagatg gggggaaagc cgcagagaag gaagaaccct 1920caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt
1980gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca
gggtctcagt 2040acagccacca aggacaccta cgacgccctt cacatgcagg
ccctgccccc tcgcaccggt 2100ggccacgttg ttgaaggact ggctggggaa
cttgaacaac ttcgtgcacg actggagcat 2160cacccacaag gtcaacgtga accatga
2187241833DNAartificial sequencescFvCD19-GFP-DAP10-SBPdel
24atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccggatatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc
120accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta
tcagcagaaa 180ccagatggaa ctgttaaact cctgatctac catacatcaa
gattacactc aggagtccca 240tcaaggttca gtggcagtgg gtctggaaca
gattattctc tcaccattag caacctggag 300caagaagata ttgccactta
cttttgccaa cagggtaata cgcttccgta cacgttcgga 360ggggggacca
agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc
420ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc
ctcacagagc 480ctgtccgtca catgcactgt ctcaggggtc tcattacccg
actatggtgt aagctggatt 540cgccagcctc cacgaaaggg tctggagtgg
ctgggagtaa tatggggtag tgaaaccaca 600tactataatt cagctctcaa
atccagactg accatcatca aggacaactc caagagccaa 660gttttcttaa
aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa
720cattattact acggtggtag ctatgctatg gactactggg gccaaggaac
ctcagtcacc 780gtctcctcac ctgcaggtat ggtgagcaag ggcgaggagc
tgttcaccgg ggtggtgccc 840atcctggtcg agctggacgg cgacgtaaac
ggccacaagt tcagcgtgtc cggcgagggc 900gagggcgatg ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg 960cccgtgccct
ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
1020taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
aggctacgtc 1080caggagcgca ccatcttctt caaggacgac ggcaactaca
agacccgcgc cgaggtgaag 1140ttcgagggcg acaccctggt gaaccgcatc
gagctgaagg gcatcgactt caaggaggac 1200ggcaacatcc tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg 1260gccgacaagc
agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
1320ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
cggccccgtg 1380ctgctgcccg acaaccacta cctgagcacc cagtccgccc
tgagcaaaga ccccaacgag 1440aagcgcgatc acatggtcct gctggagttc
gtgaccgccg ccgggatcac tctcggcatg 1500gacgagctgt acaagggccg
gccacagacg actccaggag agagatcatc actccctgcc 1560ttttaccctg
gcacttcagg ctcttgttcc ggatgtgggt ccctctctct gccgctcctg
1620gcaggcctcg tggctgctga tgcggtggca tcgctgctca tcgtgggggc
ggtgttcctg 1680tgcgcacgcc cacgccgcag ccccgcccaa gaagatggca
aagtctacat caacatgcca 1740ggcaggggcc acgttgttga aggactggct
ggggaacttg aacaacttcg tgcacgactg 1800gagcatcacc cacaaggtca
acgtgaacca tga 1833251120DNAartificial sequencescFvCD19-mycDAP10SBP
25atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccggatatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc
120accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta
tcagcagaaa 180ccagatggaa ctgttaaact cctgatctac catacatcaa
gattacactc aggagtccca 240tcaaggttca gtggcagtgg gtctggaaca
gattattctc tcaccattag caacctggag 300caagaagata ttgccactta
cttttgccaa cagggtaata cgcttccgta cacgttcgga 360ggggggacca
agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc
420ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc
ctcacagagc 480ctgtccgtca catgcactgt ctcaggggtc tcattacccg
actatggtgt aagctggatt 540cgccagcctc cacgaaaggg tctggagtgg
ctgggagtaa tatggggtag tgaaaccaca 600tactataatt cagctctcaa
atccagactg accatcatca aggacaactc caagagccaa 660gttttcttaa
aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa
720cattattact acggtggtag ctatgctatg gactactggg gccaaggaac
ctcagtcacc 780gtctcctcac ctgcaggaga gcagaagctg atctcagagg
aggacctggg ccggccacag 840acgactccag gagagagatc atcactccct
gccttttacc ctggcacttc aggctcttgt 900tccggatgtg ggtccctctc
tctgccgctc ctggcaggcc tcgtggctgc tgatgcggtg 960gcatcgctgc
tcatcgtggg ggcggtgttc ctgtgcgcac gcccacgccg cagccccgcc
1020caagaagatg gcaaagtcta catcaacatg ccaggcaggg gccacgttgt
tgaaggactg 1080gctggggaac ttgaacaact tcgtgcacga ctggagcatc
1120262295DNAartificial sequenceBACE1-SBP-EGFP 26atggcccaag
ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60ggcacccagc
acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg
120ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag
gggcagcttt 180gtggagatgg tggacaacct gaggggcaag tcggggcagg
gctactacgt ggagatgacc 240gtgggcagcc ccccgcagac gctcaacatc
ctggtggata caggcagcag taactttgca 300gtgggtgctg ccccccaccc
cttcctgcat cgctactacc agaggcagct gtccagcaca 360taccgggacc
tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag
420ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg
tgccaacatt 480gctgccatca ctgaatcaga caagttcttc atcaacggct
ccaactggga aggcatcctg 540gggctggcct atgctgagat tgccaggcct
gacgactccc tggagccttt ctttgactct 600ctggtaaagc agacccacgt
tcccaacctc ttctccctgc agctttgtgg tgctggcttc 660cccctcaacc
agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc
720gaccactcgc tgtacacagg cagtctctgg tatacaccca tccggcggga
gtggtattat 780gaggtgatca ttgtgcgggt ggagatcaat ggacaggatc
tgaaaatgga ctgcaaggag 840tacaactatg acaagagcat tgtggacagt
ggcaccacca
accttcgttt gcccaagaaa 900gtgtttgaag ctgcagtcaa atccatcaag
gcagcctcct ccacggagaa gttccctgat 960ggtttctggc taggagagca
gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 1020ttcccagtca
tctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc
1080atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca
agacgactgt 1140tacaagtttg ccatctcaca gtcatccacg ggcactgtta
tgggagctgt tatcatggag 1200ggcttctacg ttgtctttga tcgggcccga
aaacgaattg gctttgctgt cagcgcttgc 1260catgtgcacg atgagttcag
gacggcagcg gtggaaggcc cttttgtcac cttggacatg 1320gaagactgtg
gctacaacat tccacagaca gatgagtcaa ccctcatgac catagcctat
1380gtcatggctg ccatctgcgc cctcttcatg ctgccactct gcctcatggt
gtgtaggaat 1440tccatggacg agaagaccac tggttggcga ggtggacacg
ttgttgaagg actggctggg 1500gaacttgaac aacttcgtgc acgactggag
catcacccac aaggtcaacg tgaaccacct 1560gcaggtatgg tgagcaaggg
cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 1620ctggacggcg
acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
1680acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
cgtgccctgg 1740cccaccctcg tgaccaccct gacctacggc gtgcagtgct
tcagccgcta ccccgaccac 1800atgaagcagc acgacttctt caagtccgcc
atgcccgaag gctacgtcca ggagcgcacc 1860atcttcttca aggacgacgg
caactacaag acccgcgccg aggtgaagtt cgagggcgac 1920accctggtga
accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
1980gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
cgacaagcag 2040aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
tcgaggacgg cagcgtgcag 2100ctcgccgacc actaccagca gaacaccccc
atcggcgacg gccccgtgct gctgcccgac 2160aaccactacc tgagcaccca
gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 2220atggtcctgc
tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
2280aagggccggc cttaa 2295
* * * * *