Note: Descriptions are shown in the official language in which they were submitted.
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TRANSGENIC MACROPHAGES, CHIMERIC ANTIGEN RECEPTORS,
AND ASSOCIATED METHODS
TECHNICAL FIELD
The present disclosure relates generally to biotechnology. More specifically,
the
present disclosure relates to chimeric antigen receptors, nucleic acids
encoding chimeric
antigen receptors, macrophages harboring chimeric antigen receptors and/or
nucleic acids
encoding, and associated methods.
BACKGROUND
Cancer consists of a group of diseases which involve unregulated cell growth
and
death, genome instability and mutations, tumor-promoting inflammation,
induction of
angiogenesis, immune system evasion, deregulation of metabolic pathways,
immortal cell
replication, and metastatic tissue invasion [1]. Cancer is the second leading
cause of death
in the United States after heart disease [2]. More than 1.6 million new cases
of cancer are
projected to be diagnosed each year, with more than 580,000 Americans expected
to die
(about 1600 cancer deaths per day), accounting for nearly 1 in 4 of all
American deaths
[2,3].
The immune system plays an important role in the development and progression
of
cancer. Immune cell infiltration to the tumor site can adversely affect
malignancy
progression and metastasis [4, 51. Infiltration of macrophages into the tumor
site has been
shown to account for more than 50% of the tumor mass in certain breast cancer
cases
suggesting macrophages have a significant role in tumor progression [6-8].
Macrophages are cells derived from the myeloid lineage and belong to the
innate
immune system. They are derived from blood monocytes that migrate into tissue.
One of
their main functions is to phagocytose microbes and clear cellular debris.
They also play
an important role in both the initiation and resolution of inflammation [9,
101. Moreover,
macrophages can display different responses, ranging from pro-inflammatory to
anti-
inflammatory, depending on the type of stimuli they receive from the
surrounding
microenvironment [111 Two major macrophage phenotypes have been proposed which
correlate with extreme macrophage responses: M1 and M2.
M1 pro-inflammatory macrophages are activated upon contact with certain
molecules such as lipopolysaccharide (LPS), IFN-y, IL-1(3, TNF-a, and Toll-
like receptor
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engagement. M1 macrophages constitute a potent arm of the immune system
deployed to
fight infections. They are capable of either direct (pathogen pattern
recognition receptors)
or indirect (Fe receptors, complement receptors) recognition of the pathogen.
They are
also armed in their ability to produce reactive oxygen species (ROS) as means
to help
killing pathogens. In addition, M1 macrophages secrete pro-inflammatory
cytokines and
chemokines attracting other types of immune cells and
integrating/orchestrating the
immune response. Mi activation is induced by IFN-g, TNFa, GM-CSF, LPS and
other
toll-like receptors (TLR) ligands.
In contrast, M2 anti-inflammatory macrophages, also known as alternatively
activated macrophages, are activated by anti-inflammatory molecules such as IL-
4, IL-13,
and IL-10 [12, 131. M2 macrophages exhibit immunomodulatory, tissue repair,
and
angiogenesis properties which allow them to recruit regulatory T cells to
sites of
inflammation. M2 macrophages do not constitute a uniform population and often
are
further subdivided into M2a, M2b and M2c categories. The common denominator of
all
three subpopulations is high IL-10 production accompanied by low production of
IL-12.
One of their signatures is production of enzyme Arginase-1 that depletes L-
arginine
thereby suppressing T cell responses and depriving iNOS of its substrate.
The in vivo molecular mechanisms of macrophage polarization are poorly
characterized because of the variety of signals macrophages experience in the
cellular
microenvironment [10, 141. In recent years, progress has been made in
identifying in vivo
macrophage polarization under physiological conditions such as ontogenesis,
pregnancy,
and pathological conditions such as allergies, chronic inflammation, and
cancer. We do
know, however, that in vitro macrophage polarization is plastic and
macrophages, with the
help of cytokines, can be polarized back and forth to either phenotype [15,
161. Interferon
gamma (IFN-y) and IL-4 are two cytokines that can polarize macrophages to M1
and M2
phenotypes, respectively [15].
The presence of macrophages is crucial for tumor progression and growth, and
has
implications in determining prognosis [17, 181. Because macrophages can
exhibit both
pro-inflammatory and anti-inflammatory properties, it is important to
understand their
polarization and function in tumor progression and metastasis.
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Macrophage polarization
The tumor microenvironment can affect macrophage polarization. The process of
polarization can be diverse and complex because of the hostile environment of
IL-10,
glucocorticoid hormones, apoptotic cells, and immune complexes that can
interfere with
innate immune cells function [11, 191. The mechanisms of polarization are
still unclear but
we know they involve transcriptional regulation. For example, macrophages
exposed to
LPS or IFN-y will polarize towards an M1 phenotype, whereas macrophages
exposed to
IL-4 or IL-13 will polarize towards an M2 phenotype. LPS or IFN-y can interact
with Toll-
like receptor 4 (TLR4) on the surface of macrophages inducing the Trif and
MyD88
pathways, inducing the activation of transcription factors IRF3, AP-1, and
NEKB and thus
activating TNFs genes, interferon genes, CXCL10, NOS2, IL-12, etc., which are
necessary
in a pro-inflammatory M1 macrophage response [20]. Similarly, IL-4 and IL-13
bind to
IL-4R, activation the Jak/5tat6 pathway, which regulates the expression of
CCL17, ARG1,
IRF4, IL-10, 50053, etc., which are genes associated with an anti-inflammatory
response
(M2 response).
Additional mechanisms of macrophage polarization include microRNA (miRNA)
micromanagement. miRNAs are small non-coding RNA of 22 nucleotides in length
that
regulate gene expression post-transcriptionally, as they affect the rate of
mRNA
degradation. Several miRNAs have been shown to be highly expressed in
polarized
macrophages, especially miRNA-155, miRNA-125, miRNA-378 (M1 polarization), and
miRNA let-7c, miRNA-9, miRNA-21, miRNA-146, miRNA147, miRNA-187 (M2
polarization) [21].
Macrophage polarization is a complex process, were macrophages behave and
elicit
different responses depending on microenvironment stimuli. Therefore,
macrophage
polarization is better represented by a continuum of activation states where
M1 and M2
phenotypes are the extremes of the spectrum. In recent years, there has been
much
controversy on the definition/description of macrophage activation and
macrophage
polarization. A recent paper published by Murray et al., in which they
describe a set of
standards to be considered for the consensus definition/description of
macrophage
activation, polarization, activators, and markers. This publication was much
needed for the
definition and characterization of activated/polarized macrophages [22].
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MI phenotype
M1 pro-inflammatory macrophages or classically activated macrophages are
aggressive, highly phagocytic, and produce large amounts of reactive oxygen
and nitrogen
species, thereby promoting a Thl response [11]. M1 macrophages secrete high
levels of
.. two important inflammatory cytokines, IL-12 and IL-23. IL-12 induces the
activation and
clonal expansion of Th17 cells, which secrete high amounts of IL-17, which
contributes to
inflammation [23]. These characteristics allow M1 macrophages to control
metastasis,
suppress tumor growth, and control microbial infections [24]. Moreover, the
infiltration
and recruitment of M1 macrophages to tumor sites correlates with a better
prognosis and
higher overall survival rates in patients with solid tumors [17, 18, 25-281.
Polarization of macrophages to the M1 phenotype is regulated in vitro by
inflammatory signals such as IFN-y, TNF-a, IL-1B and LPS as well as
transcription factors
and miRNAs [29, 301. Classically activated macrophages initiate the induction
of the
STAT1 transcription factor which targets CXCL9, CXCL10 (also known as IP-10),
IFN
regulatory factor-1, and suppressor of cytokine signaling-1 [31].Cytokine
signaling-1
protein functions downstream of cytokine receptors, and takes part in a
negative feedback
loop to attenuate cytokine signaling In the tumor microenvironment, Notch
signaling
plays an important role in the polarization of M1 macrophages, as it allows
transcription
factor RBP-J to regulate classical activation. Macrophages that are deficient
in Notch
signaling express an M2 phenotype regardless of other extrinsic inducers [32].
One crucial
miRNA, miRNA-155, is upregulated when macrophages are transitioning from M2 to
Ml;
M1 macrophages overexpressing miRNA-155 are generally more aggressive and are
associated with tumor reduction [33]. Moreover, miRNA-342-5p has been found to
foster
a greater inflammatory response in macrophages by targeting Aktl in mice. This
miRNA
also promotes the upregulation of Nos2 and IL-6, both of which act as
inflammatory
signals for macrophages [34]. Other miRNAs such as miRNA-125 and miRNA-378
have
also been shown to be involved in the classical activation pathway of
macrophages (M1)
[35].
Classically activated macrophages are thought to play an important role in the
recognition and destruction of cancer cells as their presence usually
indicates good
prognosis. After recognition, malignant cells can be destroyed by M1
macrophages
through several mechanisms, which include contact-dependent phagocytosis and
cytotoxicity (i.e., cytokine release such as TNF-a) [24]. Environmental
signals such as the
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tumor microenvironment or tissue-resident cells, however, can polarize M1
macrophages to
M2 macrophages. In vivo studies of murine macrophages have shown that
macrophages
are plastic in their cytokine and surface marker expression and that re-
polarizing
macrophages to an M1 phenotype in the presence of cancer can help the immune
system
reject tumors [191.
M2 phenotype
M2 macrophages are anti-inflammatory and aid in the process of angiogenesis
and
tissue repair. They express scavenger receptors and produce large quantities
of IL-10 and
.. other anti-inflammatory cytokines [33, 361. Expression of IL-10 by M2
macrophages
promotes a Th2 response. Th2 cells consequently upregulate the production of
IL-3 and
IL-4. IL-3 stimulates proliferation of all cells in the myeloid lineage
(granulocytes,
monocytes, and dendritic cells), in conjunction with other cytokines, e.g.,
Erythropoietin
(EPO), Granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-6. IL-
4 is
.. an important cytokine in the healing process because it contributes to the
production of the
extracellular matrix [23]. M2 macrophages exhibit functions that may help
tumor
progression by allowing blood vessels to feed the malignant cells and thus
promoting their
growth. The presence of macrophages (thought to be M2) in the majority of
solid tumors
negatively correlates with treatment success and longer survival rates [37].
Additionally,
the presence of M2 macrophages has been linked to the metastatic potential in
breast
cancer. Lin and colleagues found that early recruitment of macrophages to the
breast
tumor sites in mice increase angiogenesis and incidence of malignancy [38]. It
is thought
that the tumor microenvironment helps macrophages maintain an M2 phenotype
[23, 391.
Anti-inflammatory signals present in the tumor microenvironment such as
adiponectin and
IL-10 can enhance an M2 response [41].
Tumor-associated macrophages (TAMs)
Cells exposed to a tumor microenvironment behave differently. For example,
tumor-associated macrophages found in the periphery of solid tumors are
thought to help
promote tumor growth and metastasis, and have an M2-like phenotype [42]. Tumor-
associated macrophages can be either tissue resident macrophages or recruited
macrophages derived from the bone marrow (macrophages that differentiate from
monocytes to macrophages and migrate into tissue). A study by Cortez-Retamozo
found
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that high numbers of TAM precursors in the spleen migrate to the tumor stroma,
suggesting
this organ as a TAM reservoir also [43]. TAM precursors found in the spleen
were found
to initiate migration through their CCR2 chemokine receptor [43]. Recent
studies have
found CSF-1 as the primary factor that attracts macrophages to the tumor
periphery, and
that CSF-1 production by cancer cells predicts lower survival rates and it
indicates an
overall poor prognosis [44-46]. Other cytokines such as TNF-a and IL-6 have
been also
linked to the accumulation/recruitment of macrophages to the tumor periphery
[45].
It is thought that macrophages that are recruited around the tumor borders are
regulated by an "angiogenic switch" that is activated in the tumor. The
angiogenic switch
is defined as the process by which the tumor develops a high density network
of blood
vessels that potentially allows the tumor to become metastatic, and is
necessary for
malignant transition. In a breast cancer mouse model, it was observed that the
presence of
macrophages was required for a full angiogenic switch. When macrophage
maturation,
migration, and accumulation around the tumor was delayed, the angiogenic
switch was also
delayed suggesting that the angiogenic switch does not occur in the absence of
macrophages and that macrophage presence is necessary for malignancy
progression [47].
Moreover, the tumor stromal cells produce chemokines such as CSF1, CCL2, CCL3,
CCL5, and placental growth factor that will recruit macrophages to the tumor
surroundings.
These chemokines provide an environment for macrophages to activate the
angiogenic
switch, in which macrophages will produce high levels of IL-10, TGF-I3, ARG-1
and low
levels of IL-12, TNF-a, and IL-6. The level of expression of these cytokines
suggests
macrophages modulate immune evasion. It is important to note that macrophages
are
attracted to hypoxic tumor environments and will respond by producing hypoxia-
inducible
factor-la (HIF-1a) and HIF-2a, which regulate the transcription of genes
associated with
angiogenesis. During the angiogenic switch, macrophages can also secrete VEGF
(stimulated by the NF-KB pathway), which will promote blood vessel maturation
and
vascular permeability [48].
Tumor-associated macrophages are thought to be able to maintain their M2-like
phenotype by receiving polarization signals from malignant cells such as IL-1R
and
MyD88, which are mediated through IkB kinase 13 and NF-kB signaling cascade.
Inhibition of NF-kB in TAMs promotes classical activation [40]. Moreover,
another study
suggested that p50 NF-kB subunit was involved in suppression of M1
macrophages, and
reduction of inflammation promoted tumor growth. A p50 NF-KB knock-out mouse
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generated by Saccani et al. suggested that M1 aggressiveness was restored upon
p50 NF-
kB knockout, reducing tumor survival [49].
Because the tumor mass contains a great number of M2-like macrophages, TAMs
can be used as a target for cancer treatment. Reducing the number of TAMs or
polarizing
them towards an M1 phenotype can help destroy cancer cells and impair tumor
growth [50-
52]. Luo and colleagues used a vaccine against legumain, a cysteine protease
and stress
protein upregulated in TAMs thought to be a potential tumor target [52]. When
the vaccine
against legumain was administered to mice, genes controlling angiogenesis were
downregulated and tumor growth was halted [52].
Metabolism and activation pathways
Metabolic alterations present in tumor cells are controlled by the same
genetic
mutations that produce cancer [53]. As a result of these metabolic
alterations, cancer cells
are able to produce signals that can modify the polarization of macrophages
and promote
tumor growth [54, 551.
M1 and M2 macrophages demonstrate distinct metabolic patterns that reflect
their
dissimilar behaviors [56]. The M1 phenotype increases glycolysis and skews
glucose
metabolism towards the oxidative pentose phosphate pathway, thereby decreasing
oxygen
consumption and consequently producing large amounts of radical oxygen and
nitrogen
species as well as inflammatory cytokines such as TNF-a, IL-12, and IL-6 [56,
571. The
M2 phenotype increases fatty acid intake and oxidation, which decreases flux
towards the
pentose phosphate pathway while increasing the overall cell redox potential,
consequently
upregulating scavenger receptors and immunomodulatory cytokines such as IL-10
and
TGF-r3 [56].
Multiple metabolic pathways play important roles in macrophage polarization.
Protein kinases, such as Aktl and Akt2, alter macrophage polarization by
allowing cancer
cells to survive, proliferate, and use an intermediary metabolism [58]. Other
protein
kinases can direct macrophage polarization through glucose metabolism by
increasing
glycolysis and decreasing oxygen consumption [57, 591. Shu and colleagues were
the first
to visualize macrophage metabolism and immune response in vivo using a PET
scan and a
glucose analog [60].
L-arginine metabolism also exhibits discrete shifts important to cytokine
expression
in macrophages and exemplifies distinct metabolic pathways which alter TAM-
tumor cell
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interactions [61]. Classically activated (M1) macrophages favor inducible
nitric oxide
synthase (iNOS). The iNOS pathway produces cytotoxic nitric oxide (NO), and
consequently exhibits anti-tumor behavior. Alternatively activated (M2)
macrophages
have been shown to favor the arginase pathway and produce ureum and 1-
ornithine, which
contribute to progressive tumor cell growth [61, 621.
Direct manipulation of metabolic pathways can alter macrophage polarization.
The
carbohydrate kinase-like protein (CARKL) protein, which plays a role in
glucose
metabolism, has been used to alter macrophage cytokine signatures [56, 571.
When
CARKL is knocked down by RNAi, macrophages tend to adopt an Ml-like metabolic
pathway (metabolism skewed towards glycolysis and decreased oxygen
consumption).
When CARKL is overexpressed, macrophages adopt an M2-like metabolism
(decreased
glycolytic flux and more oxygen consumption) [56]. When macrophages adopt an
Ml-like
metabolic state through LPS/TLR4 engagement, CARKL levels decrease, genes
controlled
by the NFic13 pathway are activated (TNF-a, IL-12, and IL-6), and cell redox
potential
increases due to growing concentrations of NADH:NAD+ and GSH:GSSSG complexes.
During an M2-like metabolic state, macrophages upregulate CARKL and genes
regulated
by STAT6/IL-4 (IL-10 and TGF-13).
Obesity can also affect macrophage polarization. Obesity is associated with a
state
of chronic inflammation, an environment that drives the IL4/STAT6 pathway to
activate
NKT cells, which drive macrophages towards an M2 response. During late-stage
diet-
induced obesity, macrophages migrate to adipose tissue, where immune cells
alter levels of
TH1 or TH2 cytokine expression in the adipose tissue, causing an M2 phenotype
bias and
possibly increased insulin sensitivity [63].
M1 phenotype bias by targeting metabolic pathways in TAMS may offer an
.. alternative means of reducing tumor growth and metastasis.
Macrophage immunotherapy approaches against cancer
The role of cancer immunotherapy is to stimulate the immune system to
recognize,
reject, and destroy cancer cells. Cancer immunotherapy with
monocytes/macrophages has
the goal to polarize macrophages towards a pro-inflammatory response (M1),
thus allowing
the macrophages and other immune cells to destroy the tumor. Many cytokines
and
bacterial compounds can achieve this in vitro, although the side effects are
typically too
severe in vivo. The key is to find a compound with minimal or easily managed
patient side
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effects. Immunotherapy using monocytes/macrophages has been used in past
decades and
new approaches are being developed every year [64, 651. Early immunotherapy
has
established a good foundation for better cancer therapies and increased
survival rate in
patients treated with immunotherapies [66].
Some approaches to cancer immunotherapy include the use of cytokines or
chemokines to recruit activated macrophages and other immune cells to the
tumor site
which allow for recognition and targeted destruction of the tumor site [67,
681. IFN- a and
IFN-r3 have been shown to inhibit tumor progression by inducing cell
differentiation and
apoptosis [69]. Also, IFN treatments are anti-proliferative and can increase S
phase time in
the cell cycle [70, 711. Zhang and colleagues performed a study in nude mice
using IFN-r3
gene therapy to target human prostate cancer cells. Their results indicate
that adenoviral-
delivered IFN-r3 gene therapy involves macrophages and helps suppress growth
and
metastasis [72].
The macrophage inhibitory factor (MIF) is another cytokine that can be used in
cancer immunotherapy. MIF is usually found in solid tumors and indicates poor
prognosis.
MIF inhibits aggressive macrophage function and drives macrophages toward an
M2
phenotype, which can aid tumor growth and progression. Simpson, Templeton &
Cross
(2012) found that MIF induces differentiation of myeloid cells, macrophage
precursors,
into a suppressive population of myeloid cells that express an M2 phenotype
[73]. By
targeting MIF, they were able to deplete this suppressive population of
macrophages,
inhibiting their growth and thus control tumor growth and metastasis [73].
The chemokine receptor type 2, CCR2, is crucial to the recruitment of
monocytes to
inflammatory sites and it has been shown as a target to prevent the
recruitment of
macrophages to the tumor site, angiogenesis, and metastasis. Sanford and
colleagues
(2013) studied a novel CCR2 inhibitor (PF-04136309) in a pancreatic mouse
model,
demonstrating that the CCR2 inhibitor depleted monocyte/macrophage recruitment
to the
tumor site, decreased tumor growth and metastasis, and increased antitumor
immunity [74].
Another recent study by Schmall et al. showed that macrophages co-cultured
with 10
different human lung cancers upregulated CCR2 expression. Moreover, they
showed that
tumor growth and metastasis were reduced in a lung mouse model treated with a
CCR2
antagonist [75].
Other studies have used liposomes to deliver drugs to deplete M2 macrophages
from tumors and to stop angiogenesis. Cancer cells that express high levels of
IL-113 grow
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faster and induce more angiogenesis in vivo. Kimura and colleagues found that
macrophages exposed to tumor cells expressing IL-1(3 produced higher levels of
angiogenic
factors and chemokines such as vascular endothelial growth factor A (VEG-A),
IL-8,
monocyte chemoattractant protein 1, etc., facilitating tumor growth and
angiogenesis [76].
When they used clodronate liposomes to deplete macrophages, they found fewer
IL-113-
producing tumor cells. They also found that by inhibiting NF-KB and AP-1
transcription
factors in the cancer cells, tumor growth and angiogenesis were reduced. These
findings
may suggest that macrophages that surround the tumor site may be involved in
promoting
tumor growth and angiogenesis [76].
Compounds such as methionine enkephalin (MENK) have anti-tumor properties in
vivo and in vitro. MENK has the ability to polarize M2 macrophages to M1
macrophages
by downregulating CD206 and arginase-1 (M2 markers) while upregulating CD64,
MHC-
II, and the production of nitric oxide (M1 markers). MENK can also upregulate
TNF-a and
downregulate IL-10 [77].
Recent studies have focused on bisphosphonates as a potential inhibitor of M2
macrophages. Bisphosphonates are commonly used to treat metastatic breast
cancer
patients to prevent skeletal complications such as bone resorption [78]. While
bisphosphonates stay in the body for short periods of time, bisphosphonates
can target
osteoclasts, cells in the same family as macrophages, due to their high
affinity for
hydroxyapatite. Once bisphosphonates bind to the bones, the bone matrix
internalizes the
bisphosphonates by endocytosis. Once in the cytoplasm, bisphosphonates can
inhibit
protein prenylation, an event that prevents integrin signaling and endosomal
trafficking,
thereby forcing the cell to go apoptotic [69]. Until recently, it was unknown
whether
bisphosphonates could target tumor-associated macrophages but a recent study
by Junankar
et al. has shown that macrophages uptake nitrogen-containing bisphosphonate
compounds
by pinocytosis and phagocytosis, an event that does not occur in epithelial
cells
surrounding the tumor [79]. Forcing TAMs to go apoptotic using bisphosphonates
could
reduce angiogenesis and metastasis.
Additional approaches to cancer immunotherapy include the use of biomaterials
that may elicit an immune response. Cationic polymers are used in
immunotherapy
because of their reactivity once dissolved in water. Chen et al. used cationic
polymers
including PEI, polylysine, cationic dextran and cationic gelatin to produce a
strong Thl
immune response [77]. They were also able to induce proliferation of CD4+
cells and
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secretion of IL-12 typical of M1 macrophages [77]. Huang and colleagues also
used
biomaterials to trigger TAMs to produce an anti-tumor response by targeting
TLR4 [80].
This study found that TAMs were able to polarize to an M1 phenotype and
express IL-12.
They found that these cationic molecules have direct tumoricidal activity and
demonstrate
tumor reduction in mice [80].
TLR4
Toll-like receptor 4 is a protein in humans that is encoded by the TLR4 gene.
TLR
4 detects lipopolysaccharide (LPS) on gram negative bacteria and thus plays a
fundamental
role in the recognition of danger and the activation of the innate immune
system (FIG. 7).
It cooperates with LY96 (MD-2) and CD14 to mediate signal transduction when
macrophages are induced by LPS. The cytoplasmic domain of TLR4 is responsible
for the
activation of M1 macrophages when they detect the presence of LPS. This is the
functional
portion of the receptor that would be coupled to the MOTO-CAR (i.e., chimeric
receptor)
to induce activation of the monocyte/macrophage when the CAR binds its target
protein.
The adaptor proteins MyD88 and TIRAP contribute to the activation of several
and
possibly all pathways via direct interactions with TLR4's Toll/interleukin-1
receptor (IL-
1R) (TIR) domain. However, additional adaptors that are required for the
activation of
specific subsets of pathways may exist, which could contribute to the
differential regulation
of target genes.
Thymidine Kinase
Human Thymidine Kinase 1 (TK1) is a well-known nucleotide salvage pathway
enzyme that has largely been studied in the context of its overexpression in
tumors. Since
TK1 was initially popularized by its expression in the serum of cancer
patients (sTK), its
diagnostic and prognostic potential has been studied extensively. For example,
several
studies have demonstrated that sTK1 in many different cancer patients is
elevated in a
stage-like manner with a higher level of TK1 indicating a more advanced tumor
[81].
Other studies have investigated the prognostic potential of TK1. One such
study
demonstrates that the TK1 levels in primary breast tumors can be used to
predict
recurrence. Other exciting TK1 prognostic studies show significant reductions
in sTK1
levels when patients respond to treatment while sTK1 levels continue to rise
in patients
who do not appear to respond to their treatment. It is also known that sTK1
levels begin to
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rise prior to recurrence and noted in some cases sTK1 levels could predict
recurrence "1-6
months before the onset of clinical symptoms." Several other studies confirm
the rich
potential of TK1 as a diagnostic and prognostic indicator of cancer [82].
Although the diagnostic and prognostic potential of TK1 has been well
established,
the therapeutic potential of TK1 remains veiled in comparison. While it is
true that HSV-
TK has been used in gene therapy and PET imaging utilizes TK1 to identify
proliferating
cancer cells, few, if any studies address the possibility of a TK1
immunotherapy. Perhaps
this is primarily because TK1 is a known cytosolic protein. It has been
recently discovered
that TK1 is expressed not only in cancer cells but also on the surface
membrane of multiple
tumor types and is therefore a very viable target for tumor immunotherapy.
DISCLOSURE
Described herein are chimeric receptors. Chimeric receptors comprise a
cytoplasmic
domain; a transmembrane domain; and an extracellular domain. In embodiments,
the
cytoplasmic domain comprises a cytoplasmic portion of a receptor that when
activated
polarizes a macrophage. In further embodiments, a wild-type protein comprising
the
cytoplasmic portion does not comprise the extracellular domain of the chimeric
receptor (see,
.e.g., FIG. 21). In embodiments, the binding of a ligand to the extracellular
domain of the
chimeric receptor activates the intracellular portion of the chimeric receptor
(see, . e.g., FIG.
22). Activation of the intracellular portion of the chimeric receptor may
polarize the
macrophage into an M1 or M2 macrophage (see, . e.g., FIGs. 23 and 24(A) and
25).
In certain embodiments, the extracellular domain may comprise an antibody or a
fragment there of that specifically binds to a ligand. In embodiments, the
chimeric receptor
may contain a linker. In embodiments, the chimeric receptor may contain a
hinge region.
Further embodiments include cells comprising a chimeric receptor or nucleic
acids
encoding a chimeric receptor.
Embodiments include methods of polarizing a macrophage by contacting a
macrophage comprising a chimeric receptor with a ligand for the extracellular
domain of the
chimeric receptor; binding the ligand to the extracellular domain of the
chimeric receptor.
The binding of the ligand to the extracellular domain of the chimeric receptor
activates the
cytoplasmic portion and the activation of the cytoplasmic portion polarizes
the macrophage.
These and other aspects of the disclosure will become apparent to the skilled
artisan in
view of the teachings contained herein.
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BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT01. FIG. 1B depicts the sequence of TK1-MOTO1 (SEQ ID NO:35). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-313 are a TLR4 transmembrane domain,
and amino
acids 314-496 are a TLR4 cytosolic domain.
FIG. 2A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT02. FIG. 2B depicts the sequence of TK1-MOTO2 (SEQ ID NO:36). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-295 are a LRR short hinge, amino
acids 296-318
are a TLR4 transmembrane domain, and amino acids 319-500 are a TLR4 cytosolic
domain.
FIG. 3A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT03. FIG. 3B depicts the sequence of TK1-MOTO3 (SEQ ID NO:37). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-345 are a LRR long hinge, amino acids
346-368 are
a TLR4 transmembrane domain, and amino acids 269-501 are a TLR4 cytosolic
domain.
FIG. 4A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT04. FIG. 4B depicts the sequence of TK1-MOTO4 (SEQ ID NO:38). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-302 are an IgG4 short hinge, amino
acids 303-325
are a TLR4 transmembrane domain, and amino acids 326-508 are a TLR4 cytosolic
domain.
FIG. 5A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT05. FIG. 5B depicts the sequence of TK1-MOTO5 (SEQ ID NO:39). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-409 are an IgG 119 amino acid medium
hinge,
amino acids 410-432 are a TLR4 transmembrane domain, and amino acids 433-615
are a
TLR4 cytosolic domain.
FIG. 6A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT06. FIG. 6B depicts the sequence of TK1-MOTO6 (SEQ ID NO:40). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-518 are an IgG4 long hinge, amino
acids 519-541
are a TLR4 transmembrane domain, and amino acids 542-724 are a TLR4 cytosolic
domain.
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FIG. 7A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT07. FIG. 7B depicts the sequence of TK1-MOTO7 (SEQ ID NO:41). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
276-290 are a GS linker, amino acids 291-358 are mutated CD8 hinge with C3395
and
C3565, amino acids 359-381 are a TLR4 transmembrane domain, and amino acids
382-564
are a TLR4 cytosolic domain.
FIG. 8A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MOT08. FIG. 8B depicts the sequence of TK1-MOTO8 (SEQ ID NO:42). Amino
acids 1-18 are a signal peptide (SP), amino acids 19-275 are an anti-TK1 ScFv,
amino acids
.. 276-290 are a GS linker, amino acids 291-358 are a portion of a CD8 hinge,
amino acids 359-
381 are a TLR4 transmembrane domain, and amino acids 382-564 are a TLR4
cytosolic
domain.
FIG. 9A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-1. FIG. 9B depicts the sequence of TK1-MO-FCGRA-CAR-1 (SEQ
ID NO:43). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275 are
an anti-TK1
ScFv, amino acids 276-290 are a GS linker, amino acids 291-311 are a FCGR3A
transmembrane domain, amino acids 312-336 are a FCGR3A cytosolic domain, and
amino
acids 337-378 are a FCER1G cytosolic domain.
FIG. 10A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-2. FIG. 10B depicts the sequence of TK1-MO-FCGRA-CAR-2
(SEQ ID NO:44). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-358 are
mutated CD8
hinge with C3395 and C3565, amino acids 359-379 are a FCGR3A transmembrane
domain,
amino acids 380-404 are a FCGR3A cytosolic domain, and amino acids 405-446 are
a
FCER1G cytosolic domain.
FIG. 11A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-3. FIG. 11B depicts the sequence of TK1-MO-FCGRA-CAR-3
(SEQ ID NO:45). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-358 are a
portion of a
CD8 hinge, amino acids 359-379 are a FCGR3A transmembrane domain, amino acids
380-
404 are a FCGR3A cytosolic domain, and amino acids 405-446 are a FCER1G
cytosolic
domain.
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FIG. 12A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-4. FIG. 12B depicts the sequence of TK1-MO-FCGRA-CAR-4
(SEQ ID NO:46). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-303 are a
IgG4 short
hinge, amino acids 304-324 are a FCGR3A transmembrane domain, amino acids 325-
349 are
a FCGR3A cytosolic domain, and amino acids 350-391 are a FCER1G cytosolic
domain.
FIG. 13A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-5. FIG. 13B depicts the sequence of TK1-MO-FCGRA-CAR-5
(SEQ ID NO:47). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-409 are a
IgG4 119
amino acid hinge, amino acids 410-430 are a FCGR3A transmembrane domain, amino
acids
431-455 are a FCGR3A cytosolic domain, and amino acids 456-497 are a FCER1G
cytosolic
domain.
FIG. 14A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCGRA-CAR-6. FIG. 14B depicts the sequence of TK1-MO-FCGRA-CAR-6
(SEQ ID NO:48). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-519 are a
IgG4 long
hinge, amino acids 520-540 are a FCGR3A transmembrane domain, amino acids 541-
565 are
a FCGR3A cytosolic domain, and amino acids 566-607 are a FCER1G cytosolic
domain.
FIG. 15A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-1. FIG. 15B depicts the sequence of TK1-MO-FCG2A-CAR-1
(SEQ ID NO:49). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-312 are a
FCGR2A
transmembrane domain, amino acids 313-390 are a FCGR2A cytosolic domain.
FIG. 16A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-2. FIG. 16B depicts the sequence of TK1-MO-FCG2A-CAR-2
(SEQ ID NO:50). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-358 are
mutated CD8
hinge with C3395 and C3565, amino acids 359-380 are a FCGR2A transmembrane
domain,
amino acids 381-458 are a FCGR2A cytosolic domain.
FIG. 17A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-3. FIG. 17B depicts the sequence of TK1-MO-FCG2A-CAR-3
(SEQ ID NO:51). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
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anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-358 are a
portion of a
CD8 hinge, amino acids 359-380 are a FCGR2A transmembrane domain, amino acids
381-
458 are a FCGR2A cytosolic domain.
FIG. 18A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-4. FIG. 18B depicts the sequence of TK1-MO-FCG2A-CAR-4
(SEQ ID NO:52). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-303 are a
IgG4 short
hinge, amino acids 304-325 are a FCGR2A transmembrane domain, amino acids 326-
403 are
a FCGR2A cytosolic domain.
FIG. 19A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-5. FIG. 19B depicts the sequence of TK1-MO-FCG2A-CAR-5
(SEQ ID NO:53). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-409 are a
IgG4 119
amino acid hinge, amino acids 410-431 are a FCGR2A transmembrane domain, amino
acids
432-509 are a FCGR2A cytosolic domain.
FIG. 20A depicts a block diagram of the order of elements in the chimeric
receptor
TK1-MO-FCG2A-CAR-6. FIG. 20B depicts the sequence of TK1-MO-FCG2A-CAR-6
(SEQ ID NO:54). Amino acids 1-18 are a signal peptide (SP), amino acids 19-275
are an
anti-TK1 ScFv, amino acids 276-290 are a GS linker, amino acids 291-519 are a
IgG4 long
hinge, amino acids 520-541 are a FCGR2A transmembrane domain, amino acids 542-
619 are
a FCGR2A cytosolic domain.
FIG. 21 is a schematic illustrating a chimeric receptor.
FIG. 22 is a schematic showing a macrophage expressing a chimeric receptor. As
depicted, the chimeric receptor comprises the cytosolic domain of a toll like
receptors, a
transmembrane domain, and a ScFv specific for a ligand. The arrows depict
signaling to
polarize the macrophage upon the ScFv binding the ligand.
FIG. 23 is a schematic showing different macrophage receptors that could be
utilized to build a chimeric receptor.
FIGs. 24A through 24C. FIG. 24A is a schematic showing the Fc Gamma Receptor
III signaling cascade leading to cell activation. FIG. 24B is a schematic
showing the Fc
Gamma Receptor III signaling cascade leading to inhibition of calcium flux and
proliferation. FIG. 24C is a schematic showing the Fc Gamma Receptor III
signaling
cascade leading to apoptosis.
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FIG. 25 is a schematic illustrating the Toll Like Receptor Signaling cascade.
FIG. 26 presents graphs illustrating flow cytometry confirming that an
expressed
antibody fragment binds the ligand of interest.
FIG. 27 presents two images showing a phenotype change in macrophages after
transduction with a chimeric receptor.
FIG. 28 presents two images confirming the expression of a chimeric receptor
in
mono cytes.
FIG. 29 presents three scatter plots of fluorescence activated cell sorting
demonstrating the expression of dTomato. The left most plot shows a control
wherein only
0.58% of cells show fluorescence which would indicate expression of dTomato.
The right
two plots show a transduction efficiency of 27.1 percent after transduction.
FIG. 30 presents six scatter plots of fluorescence activated cell sorting
demonstrating the retention of dye (Alexa 647), and the expression of CD80,
CD163,
CD206, and CD14 in macrophages transduced with a chimeric receptor.
FIG. 31 presents a histogram demonstrating the relative expression levels of
CD80,
CD163, CD206, and CD14 in macrophages transduced with a chimeric receptor.
FIG. 32 presents six images of transduced macrophages expressing a chimeric
receptor detecting, attacking, and inducing cell death in a lung cancer cell
line (NCI-H460).
MODE(S) FOR CARRYING OUT THE INVENTION
Described herein are chimeric receptors. Chimeric receptors comprise a
cytoplasmic
domain; a transmembrane domain; and an extracellular domain. In embodiments,
the
cytoplasmic domain comprises a cytoplasmic portion of a receptor that when
activated
polarizes a macrophage. In further embodiments, a wild-type protein comprising
the
cytoplasmic portion does not comprise the extracellular domain of the chimeric
receptor. In
embodiments, the binding of a ligand to the extracellular domain of the
chimeric receptor
activates the intracellular portion of the chimeric receptor. Activation of
the intracellular
portion of the chimeric receptor may polarize the macrophage into an M1 or M2
macrophage.
In certain embodiments, the cytoplasmic portion of the chimeric receptor may
comprise a cytoplasmic domain from a toll-like receptor, myeloid
differentiation primary
response protein (MYD88) (SEQ ID NO:19), toll-like receptor 3 (TLR3) (SEQ ID
NO:1),
toll-like receptor 4 (TLR4) (SEQ ID NO:3), toll-like receptor 7 (TLR7) (SEQ ID
NO:7), toll-
like receptor 8 (TLR8) (SEQ ID NO:9), toll-like receptor 9 (TLR9) (SEQ ID
NO:11), myelin
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and lymphocyte protein (MAL) (SEQ ID NO:21), interleukin-1 receptor-associated
kinase 1
(IRAK1) (SEQ ID NO:23), low affinity immunoglobulin gamma Fc region receptor
III-A
(FCGR3A) (SEQ ID NO:15), low affinity immunoglobulin gamma Fc region receptor
II-a
(FCGR2A) (SEQ ID NO:13), high affinity immunoglobulin epsilon receptor subunit
gamma
(FCER1G) (SEQ ID NO:19), or sequences having at least 90% sequence identity to
a
cytoplasmic domain of any one of the foregoing. In certain embodiments, the
cytoplasmic
portion is not a cytoplasmic domain from a toll-like receptor, FCGR3A, IL-1
receptor, or IFN-
gamma receptor. In embodiments, the cytosolic portion can be any polypeptide
that, when
activated, will result in the polarization of a macrophage.
In further embodiments, examples of ligands which bind to the extracellular
domain
may be, but are not limited to, Thymidine Kinase (TK1), Hypoxanthine-Guanine
Phosphoribosyltransferase (HPRT), Receptor Tyrosine Kinase-Like Orphan
Receptor 1
(ROR1), Mucin-16 (MUC-16), Epidermal Growth Factor Receptor vIII (EGFRvIII),
Mesothelin, Human Epidermal Growth Factor Receptor 2 (HER2), Carcinoembryonic
Antigen (CEA), B-Cell Maturation Antigen (BCMA), Glypican 3 (GPC3), Fibroblast
Activation Protein (FAP), Erythropoietin-Producing Hepatocellular Carcinoma A2
(EphA2), Natural Killer Group 2D (NKG2D) ligands, Disialoganglioside 2 (GD2),
CD19,
CD20, CD30, CD33, CD123, CD133, CD138, and CD171. In certain embodiments, the
ligand is not TK1 or HPRT.
Antibodies which may be adapted to generate extracellular domains of a
chimeric
receptor are well known in the art and are commercially available. Examples of
commercially
available antibodies include, but are not limited to: anti-HGPRT, clone
13H11.1 (EMD
Millipore), anti-ROR1 (ab135669) (Abcam), anti-MUC1 [EP1024Y1 (ab45167)
(Abcam),
anti-MUC16 [X75] (ab1107) (Abcam), anti-EGFRvIII [L8A41 (Absolute antibody),
anti-
Mesothelin [EPR2685(2)] (ab134109) (Abcam), HER2 [3B51 (ab16901) (Abcam), anti-
CEA
(LS-C84299-1000) (LifeSpan BioSciences), anti-BCMA (ab5972) (Abcam), anti-
Glypican 3
[9C2] (ab129381) (Abcam), anti-FAP (ab53066) (Abcam), anti-EphA2 [RM-0051-
8F211
(ab73254) (Abcam), anti- GD2 (LS-0546315 ) (LifeSpan BioSciences), anti-CD19
[2E2B6B101 (ab31947) (Abcam), anti-CD20 [EP459Y1 (ab78237) (Abcam), anti-CD30
[EPR41021 (ab134080) (Abcam), anti-CD33 [5P266] (ab199432) (Abcam), anti-CD123
(ab53698) (Abcam), anti-CD133 (BioLegend), anti-CD123 (1A3H4) ab181789
(Abcam), and
anti-CD171 (L1.1) (Invitrogen antibodies). Techniques for creating antibody
fragments, such
as ScFvs, from known antibodies are routine in the art. Further, generating
sequences
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encoding such fragments and recombinantly including them in as part of a
polynucleotide
encoding a chimeric protein is also routine in the art.
In certain embodiments, the extracellular domain may comprise an antibody or a
fragment there of that specifically binds to a ligand. Examples of antibodies
and fragments
thereof include, but are not limited to IgAs, IgDs, IgEs, IgGs, IgMs, Fab
fragments, F(ab1)2
fragments, monovalent antibodies, ScFv fragments, scRv-Fc fragments, IgNARs,
hcIgGs,
VhH antibodies, nanobodies, and alphabodies. In additional embodiments, the
extracellular
domain may comprise any amino acid sequence that allows for the specific
binding of a
ligand, including, but not limited to, dimerization domains, receptors,
binding pockets, etc.
In embodiments, the chimeric receptor may contain a linker. Without
limitation, the
linker may be located between the extracellular domain and the transmembrane
domain of the
chimeric receptor. Without limitation, the linker may be a G linker, a GS
linker, a G4S linker,
an EAAAK linker, a PAPAP linker, or an (Ala-Pro)õ linker. Other examples of
linkers are
well known in the art.
In embodiments, the chimeric receptor may contain a hinge region. Without
limitation, the hinge region may be located between the extracellular domain
and the
transmembrane domain of the chimeric receptor. In further embodiments, the
hinge region
may be located between a linker and the transmembrane domain. Without
limitation, the
linker may be a leucine rich repeat (LRR), or a hinge region from a toll-like
receptor, an IgG,
IgG4, CD8m or FcyllIa-hing. In embodiments, cysteines in the hinge region may
be replaced
with serines. Other examples of hinge regions are well known in the art.
Chimeric receptors as described herein may comprise one or more of SEQ ID
NOS:1,
3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-34, fragments of any of
thereof, and/or
polypeptides having at least 90% sequence identity to at least one of SEQ ID
NOS:1, 3, 4, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-34 or fragments thereof Examples of
chimeric
receptors include, but are not limited to, SEQ ID NOS:35-54, or a homologue or
fragment
thereof In another embodiment, the polypeptide comprises an amino acid
sequence selected
from the group consisting of a polypeptide having at least 90% sequence
identity to at least
one of SEQ ID NOS:35-54.
Embodiments include nucleic acid sequences comprising a nucleic acid sequence
encoding a chimeric receptor as described above. Examples of such nucleic
acids may
comprise one or more of SEQ ID NOS:2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and
24, fragments of
any of thereof, and/or nucleic acids having at least 90% sequence identity to
at least one of
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SEQ ID NOS:2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 or fragments thereof
Further
examples include nucleic acids encoding one or more of SEQ ID NOS:24-54 and
fragments
of any of thereof
In embodiments, the chimeric receptors may be glycosylated, pegylated, and/or
otherwise post-translationally modified. Further, the nucleic acid sequence
may be part of a
vector. By way of example, the vector may be a plasmid, phage, cosmid,
artificial
chromosome, viral vector, AAV vector, adenoviral vector, or lentiviral vector.
In certain
embodiments, a nucleic acid encoding a chimeric receptor may be operably
linked to a
promoter and/or other regulatory sequences (e.g., enhancers, silencers,
insulators, locus
control regions, cis-acting elements, etc.).
Further embodiments include cells comprising a chimeric receptor or nucleic
acids
encoding a chimeric receptor. Non-limiting examples of such cells include
myeloid cells,
myeloid progenitor cells, monocytes, neutrophils, basophils, eosinophils,
megakaryocytes, T
cells, B cells, natural killer cells, leukocytes, lymphocytes, dendritic
cells, and macrophages.
Embodiments include methods of polarizing a macrophage by contacting a
macrophage comprising a chimeric receptor with a ligand for the extracellular
domain of the
chimeric receptor; binding the ligand to the extracellular domain of the
chimeric receptor.
The binding of the ligand to the extracellular domain of the chimeric receptor
activates the
cytoplasmic portion and the activation of the cytoplasmic portion polarizes
the macrophage.
Nucleotide, polynucleotide, or nucleic acid sequence will be understood
according to
the present disclosure as meaning both a double-stranded or single-stranded
DNA or RNA in
the monomeric and dimeric (so-called in tandem) forms and the transcription
products of the
DNAs or RNAs.
Aspects of the disclosure relate nucleotide sequences which it has been
possible to
isolate, purify or partially purify, starting from separation methods such as,
for example, ion-
exchange chromatography, by exclusion based on molecular size, or by affinity,
or
alternatively fractionation techniques based on solubility in different
solvents, or starting from
methods of genetic engineering such as amplification, cloning, and subcloning,
it being
possible for the sequences to be carried by vectors.
A nucleotide sequence fragment will be understood as designating any
nucleotide
fragment, and may include, by way of non-limiting examples, length of at least
8, 12, 20, 25,
50, 75, 100, 200, 300, 400, 500, 1000, or more, consecutive nucleotides of the
sequence from
which it originates.
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A specific fragment of a nucleotide sequence will be understood as designating
any
nucleotide fragment of, having, after alignment and comparison with the
corresponding wild-
type sequence, at least one less nucleotide or base.
Homologous nucleotide sequence as used herein is understood as meaning a
nucleotide sequence having at least a percentage identity with the bases of a
nucleotide
sequence of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%, this
percentage
being purely statistical and it being possible to distribute the differences
between the two
nucleotide sequences at random and over the whole of their length.
Specific homologous nucleotide sequences in the sense of the present
disclosure is
understood as meaning a homologous sequence having at least one sequence of a
specific
fragment, such as defined above. The "specific" homologous sequences can
comprise, for
example, the sequences corresponding to a genomic sequence or to the sequences
of its
fragments representative of variants of the genomic sequence. These specific
homologous
sequences can thus correspond to variations linked to mutations within the
sequence and
especially correspond to truncations, substitutions, deletions and/or
additions of at least one
nucleotide. The homologous sequences can likewise correspond to variations
linked to the
degeneracy of the genetic code.
The term "degree or percentage of sequence homology" refers to "degree or
percentage of sequence identity between two sequences after optimal alignment"
as defined in
the present application.
Two nucleotide sequences are said to be "identical" if the sequence of amino-
acids or
nucleotidic residues, in the two sequences is the same when aligned for
maximum
correspondence as described below. Sequence comparisons between two (or more)
peptides
or polynucleotides are typically performed by comparing sequences of two
optimally aligned
sequences over a segment or "comparison window" to identify and compare local
regions of
sequence similarity. Optimal alignment of sequences for comparison may be
conducted by
the local homology algorithm of Smith and Waterman, Ad. App. Math 2: 482
(1981), by the
homology alignment algorithm of Neddleman and Wunsch, I Mol. Biol. 48: 443
(1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
(U.S.A.) 85:
2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT,
FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group
(GCG), 575 Science Dr., Madison, Wis.), or by visual inspection.
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"Percentage of sequence identity" (or degree of identity) is determined by
comparing
two optimally aligned sequences over a comparison window, where the portion of
the peptide
or polynucleotide sequence in the comparison window may comprise additions or
deletions
(i.e., gaps) as compared to the reference sequence (which does not comprise
additions or
deletions) for optimal alignment of the two sequences. The percentage is
calculated by
determining the number of positions at which the identical amino-acid residue
or nucleic acid
base occurs in both sequences to yield the number of matched positions,
dividing the number
of matched positions by the total number of positions in the window of
comparison and
multiplying the result by 100 to yield the percentage of sequence identity.
The definition of sequence identity given above is the definition that would
be used by
one of skill in the art. The definition by itself does not need the help of
any algorithm, the
algorithms being helpful only to achieve the optimal alignments of sequences,
rather than the
calculation of sequence identity.
From the definition given above, it follows that there is a well defined and
only one
value for the sequence identity between two compared sequences which value
corresponds to
the value obtained for the best or optimal alignment.
In the BLAST N or BLAST P "BLAST 2 sequence," software which is available in
the web site worldwideweb.ncbi.nlm.nih.gov/gorf/b12.html, and habitually used
by the
inventors and in general by the skilled person for comparing and determining
the identity
between two sequences, gap cost which depends on the sequence length to be
compared is
directly selected by the software (i.e., 11.2 for substitution matrix BLOSUM-
62 for
length>85).
Complementary nucleotide sequence of a sequence as used herein is understood
as
meaning any DNA whose nucleotides are complementary to those of the sequences
and
.. whose orientation is reversed (antisense sequence).
Hybridization under conditions of stringency with a nucleotide sequence as
used
herein is understood as meaning hybridization under conditions of temperature
and ionic
strength chosen in such a way that they allow the maintenance of the
hybridization between
two fragments of complementary DNA.
By way of illustration, conditions of great stringency of the hybridization
step with the
aim of defining the nucleotide fragments described above are advantageously
the following.
The hybridization is carried out at a preferential temperature of 65 C in the
presence
of SSC buffer, 1 x SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate. The
washing
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steps, for example, can be the following: 2 x SSC, at ambient temperature
followed by two
washes with 2 x SSC, 0.5% SDS at 65 C.; 2 x 0.5 x SSC, 0.5% SDS; at 65 C for
10 minutes
each.
The conditions of intermediate stringency, using, for example, a temperature
of 42 C
in the presence of a 2 x SSC buffer, or of less stringency, for example a
temperature of 37 C
in the presence of a 2 x SSC buffer, respectively require a globally less
significant
complementarity for the hybridization between the two sequences.
The stringent hybridization conditions described above for a polynucleotide
with a
size of approximately 350 bases will be adapted by the person skilled in the
art for
oligonucleotides of greater or smaller size, according to the teaching of
Sambrook et al., 1989.
Among the nucleotide sequences described herein, are those which can be used
as a
primer or probe in methods allowing the homologous sequences to be obtained,
these
methods, such as the polymerase chain reaction (PCR), nucleic acid cloning,
and sequencing,
being well known to the person skilled in the art.
Among the nucleotide sequences are those which can be used as a primer or
probe in
methods allowing the presence of specific nucleic acids, one of their
fragments, or one of their
variants such as defined below to be determined. In embodiments, the
nucleotide sequences
may comprise fragments of SEQ ID NOS:2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and
24 which
encode a transmembrane domain, cytosolic domain, or a portion thereof Further
fragments
may include nucleotide sequences encoding linkers, hinges, or fragments
thereof such as
nucleotides encoding one or more of SEQ ID NOS :26-34. Further fragments may
include
fragments of nucleotide sequences encoding one or more of SEQ ID NOS:35-54.
The nucleotide sequence fragments can be obtained, for example, by specific
amplification, such as PCR, or after digestion with appropriate restriction
enzymes of
nucleotide sequences, these methods in particular being described in the work
of Sambrook et
al., 1989. Also, such fragments may be obtained with gene synthesis standard
technology
available from companies such as GENScRIPTO. Such representative fragments can
likewise
be obtained by chemical synthesis according to methods well known to persons
of ordinary
skill in the art.
Modified nucleotide sequence will be understood as meaning any nucleotide
sequence
obtained by mutagenesis according to techniques well known to the person
skilled in the art,
and containing modifications with respect to a wild-type sequence, for example
mutations in
the regulatory and/or promoter sequences of polypeptide expression, especially
leading to a
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modification of the rate of expression of the polypeptide or to a modulation
of the replicative
cycle.
Modified nucleotide sequence will likewise be understood as meaning any
nucleotide
sequence coding for a modified polypeptide such as defined below.
Disclosed are nucleotide sequences encoding a chimeric receptor, the
nucleotide
sequences comprising nucleotide sequences selected from SEQ ID NOS:2, 6, 8,
10, 12, 14,
16, 18, 20, 22, and 24 or one of their fragments. Such fragments may encode
particular
domains such as transmembrane domains or cytosolic domains or portions thereof
Further
nucleotide sequences encoding a chimeric receptor may include nucleotide
sequences
encoding linkers, hinges, or fragments thereof such as nucleotides encoding
one or more of
SEQ ID NOS:26-34. Nucleotide sequences encoding a chimeric receptor may
further
nucleotide sequences encoding one or more of SEQ ID NOS :35-54 or fragments
thereof
Embodiments likewise relate to nucleotide sequences characterized in that they
comprise a nucleotide sequence selected from: a) at least one of a nucleotide
sequence of SEQ
ID NOS:2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, a nucleotide sequence
encoding at least one
of SEQ ID NOS:25-54, or one of their fragments; b) a nucleotide sequence of a
specific
fragment of a sequence such as defined in a); c) a homologous nucleotide
sequence having at
least 80% identity with a sequence such as defined in a) or b); d) a
complementary nucleotide
sequence or sequence of RNA corresponding to a sequence such as defined in a),
b) or c); and
e) a nucleotide sequence modified by a sequence such as defined in a), b), c)
or d).
Among the nucleotide sequences are the nucleotide sequences of SEQ ID NOS:2,
6, 8,
10, 12, 14, 16, 18, 20, 22, and 24, a nucleotide sequence encoding at least
one of SEQ ID
NOS:25-54,or fragments thereof and any nucleotide sequences which have a
homology of at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%,
95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with the at least one
of the
sequences of SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 a
nucleotide sequence
encoding at least one of SEQ ID NOS:25-54, or fragments thereof The homologous
sequences can comprise, for example, the sequences corresponding to the wild-
type
sequences. In the same manner, these specific homologous sequences can
correspond to
variations linked to mutations within the wild-type sequence and especially
correspond to
truncations, substitutions, deletions and/or additions of at least one
nucleotide. As will be
apparent to one of ordinary skill in the art, such homologues are easily
created and identified
using standard techniques and publicly available computer programs such as
BLAST. As
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such, each homologue referenced above should be considered as set forth herein
and fully
described.
Embodiments comprise the chimeric receptors coded for by a nucleotide sequence
described herein, or fragments thereof, whose sequence is represented by a
fragment. Amino
acid sequences corresponding to the polypeptides which can be coded for
according to one of
the three possible reading frames of at least one of the sequences of SEQ ID
NOS:35-54.
Embodiments likewise relate to chimeric receptors, characterized in that they
comprise
a polypeptide selected from at least one of the amino acid sequences of SEQ ID
NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-54, or one of their fragments.
Among the polypeptides, according to embodiments, are the polypeptides of
amino
acid sequence SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-54, or
fragments
thereof or any other polypeptides which have a homology of at least 80%, 81%,
82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%,
99.5%, 99.6%, or 99.7% identity with at least one of the sequences of SEQ ID
NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, and 25-54 or fragments thereof As will be
apparent to one of
ordinary skill in the art, such homologues are easily created and identified
using standard
techniques and publicly available computer programs such as BLAST. As such,
each
homologue referenced above should be considered as set forth herein and fully
described.
Embodiments also relate to the polypeptides, characterized in that they
comprise a
polypeptide selected from: a) a specific fragment of at least 5 amino acids of
a polypeptide of
an amino acid sequence; b) a polypeptide homologous to a polypeptide such as
defined in a);
c) a specific biologically active fragment of a polypeptide such as defined in
a) or b); and d) a
polypeptide modified by a polypeptide such as defined in a), b) or c).
In the present description, the terms polypeptide, peptide and protein are
interchangeable.
In embodiments, the chimeric receptors may be glycosylated, pegylated, and/or
otherwise post-translationally modified. In further embodiments,
glycosylation, pegylation,
and/or other posttranslational modifications may occur in vivo or in vitro
and/or may be
performed using chemical techniques. In additional embodiments, any
glycosylation,
pegylation and/or other posttranslational modifications may be N-linked or 0-
linked.
In embodiments any one of the chimeric receptors may be enzymatically or
functionally active such that, when the extracellular domain is bound by a
ligand, a signal is
transduced to polarize a macrophage.
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As used herein, a "polarized macrophage" is a macrophage that correlates with
an M1
or M2 macrophage phenotype. M1 polarized macrophages secrete IL-12 and IL-23.
The
determination of a macrophage as polarized to M1 may be performed by measuring
the
expression of IL-12 and/or IL-23 using a standard cytokine assay and comparing
that
expression to the expression by newly differentiated unpolarized macrophages.
Alternatively,
the determination can be made by determining if the cells are CD14+, CD80+,
CD206+, and
CDCD163-. M2 polarized macrophages secrete IL-10. The determination of a
macrophage
as polarized to M2 may be performed by measuring the expression of IL-10 using
a standard
cytokine assay and comparing that expression to the expression by newly
differentiated
unpolarized macrophages. Alternatively, the determination can be made by
determining if the
cells are CD14+, CD80-, CD206+, and CDCD163+
Aspects of the disclosure relate to chimeric receptors obtained by genetic
recombination, or alternatively by chemical synthesis and that they may thus
contain
unnatural amino acids, as will be described below.
A "polypeptide fragment" according to the embodiments is understood as
designating
a polypeptide containing at least 5 consecutive amino acids, preferably 10
consecutive amino
acids or 15 consecutive amino acids.
Herein, a specific polypeptide fragment is understood as designating the
consecutive
polypeptide fragment coded for by a specific fragment a nucleotide sequence.
"Homologous polypeptide" will be understood as designating the polypeptides
having,
with respect to the natural polypeptide, certain modifications such as, in
particular, a deletion,
addition, or substitution of at least one amino acid, a truncation, a
prolongation, a chimeric
fusion, and/or a mutation. Among the homologous polypeptides, those are
preferred whose
amino acid sequence has at least 80% or 90%, homology with the sequences of
amino acids of
polypeptides described herein.
"Specific homologous polypeptide" will be understood as designating the
homologous
polypeptides such as defined above and having a specific fragment of
polypeptide
polypeptides described herein.
In the case of a substitution, one or more consecutive or nonconsecutive amino
acids
are replaced by "equivalent" amino acids. The expression "equivalent" amino
acid is directed
here at designating any amino acid capable of being substituted by one of the
amino acids of
the base structure without, however, essentially modifying the biological
activities of the
corresponding peptides and such that they will be defined by the following. As
will be
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apparent to one of ordinary skill in the art, such substitutions are easily
created and identified
using standard molecular biology techniques and publicly available computer
programs such
as BLAST. As such, each substitution referenced above should be considered as
set forth
herein and fully described.
These equivalent amino acids can be determined either by depending on their
structural homology with the amino acids which they substitute, or on results
of comparative
tests of biological activity between the different polypeptides, which are
capable of being
carried out.
By way of nonlimiting example, the possibilities of substitutions capable of
being
carried out without resulting in an extensive modification of the biological
activity of the
corresponding modified polypeptides will be mentioned, the replacement, for
example, of
leucine by valine or isoleucine, of aspartic acid by glutamic acid, of
glutamine by asparagine,
of arginine by lysine etc., the reverse substitutions naturally being
envisageable under the
same conditions.
In a further embodiment, substitutions are limited to substitutions in amino
acids not
conserved among other proteins which have similar identified enzymatic
activity. For
example, one of ordinary skill in the art may align proteins of the same
function in similar
organisms and determine which amino acids are generally conserved among
proteins of that
function. One example of a program that may be used to generate such
alignments is
wordlwideweb.charite.de/bioinf/strap/ in conjunction with the databases
provided by the
NCBI.
Thus, according to one embodiment, substitutions or mutation may be made at
positions that are generally conserved among proteins of that function. In a
further
embodiment, nucleic acid sequences may be mutated or substituted such that the
amino acid
they code for is unchanged (degenerate substitutions and/mutations) and/or
mutated or
substituted such that any resulting amino acid substitutions or mutation are
made at positions
that are generally conserved among proteins of that function.
The specific homologous polypeptides likewise correspond to polypeptides coded
for
by the specific homologous nucleotide sequences such as defined above and thus
comprise in
the present definition the polypeptides which are mutated or correspond to
variants which can
exist in wild-type sequences, and which especially correspond to truncations,
substitutions,
deletions, and/or additions of at least one amino acid residue.
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"Specific biologically active fragment of a polypeptide" as used herein will
be
understood in particular as designating a specific polypeptide fragment, such
as defined
above, having at least one of the characteristics of polypeptides described
herein. In certain
embodiments the peptide is capable of behaving as chimeric antigen receptor
that when
activated polarizes a macrophage.
"Modified polypeptide" of a polypeptide as used herein is understood as
designating a
polypeptide obtained by genetic recombination or by chemical synthesis as will
be described
below, having at least one modification with respect to a wild-type sequence.
These
modifications may or may not be able to bear on amino acids at the origin of
specificity,
and/or of activity, or at the origin of the structural conformation,
localization, and of the
capacity of membrane insertion of the polypeptide as described herein. It will
thus be possible
to create polypeptides of equivalent, increased, or decreased activity, and of
equivalent,
narrower, or wider specificity. Among the modified polypeptides, it is
necessary to mention
the polypeptides in which up to 5 or more amino acids can be modified,
truncated at the N- or
C-terminal end, or even deleted or added.
The methods allowing the modulations on eukaryotic or prokaryotic cells to be
demonstrated are well known to the person of ordinary skill in the art. It is
likewise well
understood that it will be possible to use the nucleotide sequences coding for
the modified
polypeptides for the modulations, for example through vectors and described
below.
The preceding modified polypeptides can be obtained by using combinatorial
chemistry, in which it is possible to systematically vary parts of the
polypeptide before testing
them on models, cell cultures or microorganisms for example, to select the
compounds which
are most active or have the properties sought.
Chemical synthesis likewise has the advantage of being able to use unnatural
amino
.. acids, or nonpeptide bonds.
Thus, in order to improve the duration of life of the polypeptides, it may be
of interest
to use unnatural amino acids, for example in D form, or else amino acid
analogs, especially
sulfur-containing forms, for example.
Finally, it will be possible to integrate the structure of the polypeptides,
its specific or
modified homologous forms, into chemical structures of polypeptide type or
others. Thus, it
may be of interest to provide at the N- and C-terminal ends compounds not
recognized by
proteas es .
The nucleotide sequences coding for a polypeptide are likewise disclosed
herein.
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Embodiments likewise relates to nucleotide sequences utilizable as a primer or
probe,
characterized in that the sequences are selected from the nucleotide sequences
described
herein.
It is well understood that various embodiments likewise relate to specific
polypeptides
including chimeric receptors, coded for by nucleotide sequences, capable of
being obtained by
purification from natural polypeptides, by genetic recombination or by
chemical synthesis by
procedures well known to the person skilled in the art and such as described
in particular
below. In the same manner, the labeled or unlabeled mono- or polyclonal
antibodies directed
against the specific polypeptides coded for by the nucleotide sequences are
also encompassed
by this disclosure.
Embodiments additionally relate to the use of a nucleotide sequence as a
primer or
probe for the detection and/or the amplification of nucleic acid sequences.
The nucleotide sequences according to embodiments can thus be used to amplify
nucleotide sequences, especially by the PCR technique (polymerase chain
reaction) (Erlich,
.. 1989; Innis et al., 1990; Rolfs et al., 1991; and White et al., 1997).
These oligodeoxyribonucleotide or oligoribonucleotide primers advantageously
have a
length of at least 8 nucleotides, preferably of at least 12 nucleotides, and
even more
preferentially at least 20 nucleotides.
Other amplification techniques of the target nucleic acid can be
advantageously
employed as alternatives to PCR.
The nucleotide sequences described herein, in particular the primers, can
likewise be
employed in other procedures of amplification of a target nucleic acid, such
as: the TAS
technique (Transcription-based Amplification System), described by Kwoh et al.
in 1989; the
35R technique (Self-Sustained Sequence Replication), described by Guatelli et
al. in 1990; the
NASBA technique (Nucleic Acid Sequence Based Amplification), described by
Kievitis et al.
in 1991; the SDA technique (Strand Displacement Amplification) (Walker et al.,
1992); the
TMA technique (Transcription Mediated Amplification).
The polynucleotides, including chimeric receptors, can also be employed in
techniques
of amplification or of modification of the nucleic acid serving as a probe,
such as: the LCR
technique (Ligase Chain Reaction), described by Landegren et al. in 1988 and
improved by
Barany et al. in 1991, which employs a thermostable ligase; the RCR technique
(Repair Chain
Reaction), described by Segev in 1992; the CPR technique (Cycling Probe
Reaction),
described by Duck et al. in 1990; the amplification technique with Q-beta
replicase, described
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by Miele et al. in 1983 and especially improved by Chu et al. in 1986, Lizardi
et al. in 1988,
then by Burg et al. as well as by Stone et al. in 1996.
In the case where the target polynucleotide to be detected is possibly an RNA,
for
example an mRNA, it will be possible to use, prior to the employment of an
amplification
reaction with the aid of at least one primer or to the employment of a
detection procedure with
the aid of at least one probe, an enzyme of reverse transcriptase type in
order to obtain a
cDNA from the RNA contained in the biological sample. The cDNA obtained will
thus serve
as a target for the primer(s) or the probe(s) employed in the amplification or
detection
procedure.
The detection probe will be chosen in such a manner that it hybridizes with
the target
sequence or the amplicon generated from the target sequence. By way of
sequence, such a
probe will advantageously have a sequence of at least 12 nucleotides, in
particular of at least
nucleotides, and preferably of at least 100 nucleotides.
Embodiments also comprise the nucleotide sequences utilizable as a probe or
primer,
15
characterized in that they are labeled with a radioactive compound or with a
nonradioactive
compound.
The unlabeled nucleotide sequences can be used directly as probes or primers,
although the sequences are generally labeled with a radioactive isotope (32p,
35s, 3H, 1250 or
with a nonradioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5-
20
bromodeoxyuridine, fluorescein) to obtain probes which are utilizable for
numerous
applications.
Examples of nonradioactive labeling of nucleotide sequences are described, for
example, in French Patent No. 78.10975 or by Urdea et al. or by Sanchez-
Pescador et al. in
1988.
In the latter case, it will also be possible to use one of the labeling
methods described
in patents FR-2 422 956 and FR-2 518 755.
The hybridization technique can be carried out in various manners (Matthews et
al.,
1988). The most general method consists in immobilizing the nucleic acid
extract of cells on
a support (such as nitrocellulose, nylon, polystyrene) and in incubating,
under well-defined
conditions, the immobilized target nucleic acid with the probe. After
hybridization, the excess
of probe is eliminated and the hybrid molecules formed are detected by the
appropriate
method (measurement of the radioactivity, of the fluorescence or of the
enzymatic activity
linked to the probe).
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Various embodiments likewise comprise the nucleotide sequences or polypeptide
sequences described herein, characterized in that they are immobilized on a
support,
covalently or noncovalently.
According to another advantageous mode of employing nucleotide sequences, the
latter can be used immobilized on a support and can thus serve to capture, by
specific
hybridization, the target nucleic acid obtained from the biological sample to
be tested. If
necessary, the solid support is separated from the sample and the
hybridization complex
formed between the capture probe and the target nucleic acid is then detected
with the aid of a
second probe, a so-called detection probe, labeled with an easily detectable
element.
Another aspect is a vector for the cloning and/or expression of a sequence,
characterized in that it contains a nucleotide sequence described herein.
The vectors, characterized in that they contain the elements allowing the
integration,
expression and/or the secretion of the nucleotide sequences in a determined
host cell, are
likewise provided.
The vector may then contain a promoter, signals of initiation and termination
of
translation, as well as appropriate regions of regulation of transcription. It
may be able to be
maintained stably in the host cell and can optionally have particular signals
specifying the
secretion of the translated protein. These different elements may be chosen as
a function of
the host cell used. To this end, the nucleotide sequences described herein may
be inserted into
autonomous replication vectors within the chosen host, or integrated vectors
of the chosen
host.
Such vectors will be prepared according to the methods currently used by the
person
skilled in the art, and it will be possible to introduce the clones resulting
therefrom into an
appropriate host by standard methods, such as, for example, calcium phosphate
precipitation,
lipofection, electroporation, and thermal shock.
The vectors according are, for example, vectors of plasmid or viral origin.
Examples
of vectors for the expression of polypeptides described herein are plasmids,
phages, cosmids,
artificial chromosomes, viral vectors, AAV vectors, baculovirus vectors,
adenoviral vectors,
lentiviral vectors, retroviral vectors, chimeric viral vectors, and chimeric
adenoviridae such as
ADS/F35.
These vectors are useful for transforming host cells in order to clone or to
express the
nucleotide sequences described herein.
Embodiments likewise comprise the host cells transformed by a vector.
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These cells can be obtained by the introduction into host cells of a
nucleotide sequence
inserted into a vector such as defined above, then the culturing of the cells
under conditions
allowing the replication and/or expression of the transfected nucleotide
sequence.
The host cell can be selected from prokaryotic or eukaryotic systems, such as,
for
example, bacterial cells (Olins and Lee, 1993), but likewise yeast cells
(Buckholz, 1993), as
well as plants cells, such as Arabidopsis sp., and animal cells, in particular
the cultures of
mammalian cells (Edwards and Aruffo, 1993), for example, HEK 293, cells, HEK
293T cells,
Chinese hamster ovary (CHO) cells, myeloid cells, myeloid progenitor cells,
monocytes,
neutrophils, basophils, eosinophils, megakaryocytes, T cells, B cells, natural
killer cells,
leukocytes, lymphocytes, dendritic cells, and macrophages, but likewise the
cells of insects in
which it is possible to use procedures employing baculoviruses, for example
sf9 insect cells
(Luckow, 1993).
Embodiments likewise relate to organisms comprising one of the transformed
cells.
The obtainment of transgenic organisms expressing one or more of the nucleic
acids
or part of the nucleic acids may be carried out in, for example, rats, mice,
or rabbits according
to methods well known to the person skilled in the art, such as by viral or
nonviral
transfections. It will be possible to obtain the transgenic organisms
expressing one or more of
the genes by transfection of multiple copies of the genes under the control of
a strong
promoter of ubiquitous nature, or selective for one type of tissue. It will
likewise be possible
to obtain the transgenic organisms by homologous recombination in embryonic
cell strains,
transfer of these cell strains to embryos, selection of the affected chimeras
at the level of the
reproductive lines, and growth of the chimeras.
The transformed cells as well as the transgenic organisms are utilizable in
procedures
for preparation of recombinant polypeptides.
It is today possible to produce recombinant polypeptides in relatively large
quantity by
genetic engineering using the cells transformed by expression vectors or using
transgenic
organisms.
The procedures for preparation of a polypeptide, such as a chimeric receptor,
in
recombinant form, characterized in that they employ a vector and/or a cell
transformed by a
vector and/or a transgenic organism comprising one of the transformed cells
are themselves
comprised in in the present disclosure.
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As used herein, "transformation" and "transformed" relate to the introduction
of
nucleic acids into a cell, whether prokaryotic or eukaryotic. Further,
"transformation" and
"transformed," as used herein, need not relate to growth control or growth
deregulation.
Among the procedures for preparation of a polypeptide, such as a chimeric
receptor, in
recombinant form, the preparation procedures employing a vector, and/or a cell
transformed
by the vector and/or a transgenic organism comprising one of the transformed
cells,
containing a nucleotide sequence, such as those encoding a chimeric receptor.
A variant according, as used herein, may consist of producing a recombinant
polypeptide fused to a "carrier" protein (chimeric protein). The advantage of
this system is
that it may allow stabilization of and/or a decrease in the proteolysis of the
recombinant
product, an increase in the solubility in the course of renaturation in vitro
and/or a
simplification of the purification when the fusion partner has an affinity for
a specific ligand.
More particularly, embodiments relate to a procedure for preparation of a
polypeptide
comprising the following steps: a) culture of transformed cells under
conditions allowing the
expression of a recombinant polypeptide of nucleotide sequence; b) if need be,
recovery of the
recombinant polypeptide.
When the procedure for preparation of a polypeptide, such as a chimeric
receptor,
employs a transgenic organism, the recombinant polypeptide may then extracted
from the
organism or left in place.
Embodiments also relate to a polypeptide which is capable of being obtained by
a
procedure such as described previously.
Embodiments also comprise a procedure for preparation of a synthetic
polypeptide,
characterized in that it uses a sequence of amino acids of polypeptides.
This disclosure likewise relates to a synthetic polypeptide, such as a
chimeric receptor,
obtained by a procedure.
The polypeptides, such as chimeric receptors, can likewise be prepared by
techniques
which are conventional in the field of the synthesis of peptides. This
synthesis can be carried
out in homogeneous solution or in solid phase.
For example, recourse can be made to the technique of synthesis in homogeneous
solution described by Houben-Weyl in 1974.
This method of synthesis consists in successively condensing, two by two, the
successive amino acids in the order required, or in condensing amino acids and
fragments
formed previously and already containing several amino acids in the
appropriate order, or
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alternatively several fragments previously prepared in this way, it being
understood that it will
be necessary to protect beforehand all the reactive functions carried by these
amino acids or
fragments, with the exception of amine functions of one and carboxyls of the
other or vice-
versa, which must normally be involved in the formation of peptide bonds,
especially after
activation of the carboxyl function, according to the methods well known in
the synthesis of
peptides.
Recourse may also be made to the technique described by Merrifield.
To make a peptide chain according to the Merrifield procedure, recourse is
made to a
very porous polymeric resin, on which is immobilized the first C-terminal
amino acid of the
chain. This amino acid is immobilized on a resin through its carboxyl group
and its amine
function is protected. The amino acids which are going to form the peptide
chain are thus
immobilized, one after the other, on the amino group, which is deprotected
beforehand each
time, of the portion of the peptide chain already formed, and which is
attached to the resin.
When the whole of the desired peptide chain has been formed, the protective
groups of the
different amino acids forming the peptide chain are eliminated and the peptide
is detached
from the resin with the aid of an acid.
These hybrid molecules can be formed, in part, of a polypeptide carrier
molecule or of
fragments thereof, associated with a possibly immunogenic part, in particular
an epitope of the
diphtheria toxin, the tetanus toxin, a surface antigen of the hepatitis B
virus (patent FR 79
21811), the VP1 antigen of the poliomyelitis virus or any other viral or
bacterial toxin or
antigen.
The polypeptides, including chimeric receptors, the antibodies described below
and
the nucleotide sequences encoding any of the foregoing can advantageously be
employed in
procedures for the polarization of a macrophage.
In embodiments, a nucleic acid sequence encoding a chimeric receptor is
provided to a
cell. The cell may then express the encoded chimeric receptor. The expressed
chimeric
receptor may be present on the surface of the cell or in the cytoplasm. In
particular
embodiments, the cell expressing the chimeric receptor is a macrophage. The
macrophage
expressed chimeric receptor may bind a ligand, and binding of the ligand may
activate the
chimeric receptor so as to induce polarization of the macrophage as previously
described.
In embodiments, the cell provided with the nucleic acid sequence encoding a
chimeric
receptor may be isolated from a subject. After the cell is provided with the
nucleic acid, the
cell may be returned to the subject from whom it was obtained, for example by
injection or
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transfusion. In other embodiments, the cell provided with the nucleic acid may
be provided
by a donor. After the donor cell is provided with the nucleic acid, the cell
may then be
provided to an individual other than the donor. Examples of donor cells
include, but are not
limited to primary cells from a subject and cells from a cell line.
In other embodiments, chimeric receptors may be introduced directly into
cells. Any
method of introducing a protein into cell may be used, including, but not
limited to,
microinjection, electroporation, membrane fusion, and the use of protein
transduction
domains. After the cell is provided with chimeric receptors, the cell may be
returned to the
subject from whom it was obtained, for example by injection or transfusion. In
other
embodiments, the cell provided with the chimeric receptors is provided by a
donor. After the
donor cell is provided with the nucleic acid, the cell may then be provided to
an individual
other than the donor. Examples of donor cells include, but are not limited to
primary cells
from a subject and cells from a cell line.
Embodiments likewise relates to polypeptides, such as chimeric receptors,
labeled
with the aid of an adequate label, such as, of the enzymatic, fluorescent or
radioactive type.
The polypeptides allow monoclonal or polyclonal antibodies to be prepared
which are
characterized in that they specifically recognize the polypeptide. It will
advantageously be
possible to prepare the monoclonal antibodies from hybridomas according to the
technique
described by Kohler and Milstein in 1975. It will be possible to prepare the
polyclonal
antibodies, for example, by immunization of an animal, in particular a mouse,
with a
polypeptide or a DNA, associated with an adjuvant of the immune response, and
then
purification of the specific antibodies contained in the serum of the
immunized animals on an
affinity column on which the polypeptide which has served as an antigen has
previously been
immobilized. The polyclonal antibodies can also be prepared by purification,
on an affinity
column on which a polypeptide has previously been immobilized, of the
antibodies contained
in the serum of an animal immunologically challenged by a chimeric receptor,
or a
polypeptide or fragment thereof
In addition, antibodies can be used to prepare other forms of binding
molecules,
including, but not limited to, IgAs, IgDs, IgEs, IgGs, IgMs, Fab fragments,
F(ab1)2 fragments,
monovalent antibodies, scFv fragments, scRv-Fc fragments, IgNARs, hcIgGs, VhH
antibodies, nanobodies, and alphabodies.
Embodiments likewise relates to mono- or polyclonal antibodies or their
fragments, or
chimeric antibodies, or fragments thereof, characterized in that they are
capable of specifically
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recognizing a polypeptide described herein or a ligand of a polypeptide and/or
chimeric
receptor.
It will likewise be possible for the antibodies to be labeled in the same
manner as
described previously for the nucleic probes, such as a labeling of enzymatic,
fluorescent or
radioactive type. It will be also be possible to include such antibodies
and/or fragments
thereof as part of a chimeric receptor. By way of non-limiting example, such
an antibody or
fragment thereof may make up a portion of the extracellular domain of a
chimeric receptor.
Embodiments are additionally directed at a procedure for the detection and/or
identification of chimeric receptor in a sample, characterized in that it
comprises the following
steps: a) contacting of the sample with a mono- or polyclonal (under
conditions allowing an
immunological reaction between the antibodies and the chimeric receptor
possibly present in
the biological sample); b) demonstration of the antigen-antibody complex
possibly formed.
The embodiments are described in additional detail in the following
illustrative
examples. Although the examples may represent only selected embodiments, it
should be
understood that the following examples are illustrative and not limiting.
EXAMPLES
Example 1: Isolation of ScFv fragments for specific ligands
cDNA was purified from a monoclonal antibody hybridoma cell (CB1) expressing
an
antibody specific to human TK1. The isolated cDNA was used to amplify the
heavy and light
chains of the CB1 variable region via polymerase chain reaction (PCR)
Sequences from the
heavy and light chain were confirmed using NCBI Blast. CB1 heavy and light
chains were
fused together via site overlap extension (SOE) PCR to form a single chain
fragment variable
(scFv) using a GLIS linker. The GLIS linker was codon optimized for yeast and
humans using
the Codon Optimization tool provided by IDT (htips://www.idtdna.com/CodonOpt)
in order
to maximize protein expression. The CB1 scFv was cut out using restriction
enzymes and
inserted into a pMP71 CAR vector.
TK-1 and HPRT-specific human scFv fragments were isolated from a yeast
antibody
library. TK-1 and HPRT proteins were isolated, His-tagged, and purified. TK-1
and HPRT
protein were labeled with an anti-His biotinylated antibody and added to the
library to select
for TK-1 and HPRT-specific antibody clones. TK-1 and HPRT antibody clones were
alternately stained with streptavidin or anti-biotin microbeads and enriched
using a magnetic
column. Two additional rounds of sorting and selection were performed to
isolate TK-1 and
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HPRT specific antibodies. For the final selection, possible TK-1 and HPRT
antibody clones
and their respective proteins were sorted by fluorescence-activated cell
sorting (FACS) by
alternately labeling with fluorescently-conjugated anti-HA or anti-c-myc
antibodies to isolate
TK-1 and HPRT specific antibodies. High affinity clones were selected for
chimeric receptor
construction. Other human antibodies or humanized antibodies from other
animals could be
selected or altered to be TK-1 or HPRT specific by using phage display or
other
recombination methods.
Selected scFv clones were then combined with human IgG1 constant domains to
create an antibody for use in applications such as Western blot or ELISA in
order to confirm
the binding specificity of the scFv. The antibody construct was inserted into
the pPNL9 yeast
secretion vector and YVH10 yeast were transformed with the construct and
induced to
produce the antibody. Other expression systems such as E. coli or mammalian
systems could
also be used to secrete antibodies.
Isolation and characterization of protein-specific antibody fragments.
Referring to FIG. 26, 105 yeast were incubated with 2.5ug of protein of
interest
labeled with the fluorescent tag APC. The higher left (red) peak indicates
yeast population
that was not binding to the protein of interest (negative control). The lower
left (blue) peak on
the left illustrates yeast not expressing their surface protein while the high
(blue) peak on the
right indicates binding of the expressed antibody fragment to the protein of
interest.
Structural Consensus among Antibodies Defines the Antigen 5 Binding Site, PLoS
Comput Biol. 8(2): e1002388. doi: 10.1371/j ournal.pcbi.1002388, Kunik V,
Ashkenazi S,
Ofran Y (2012). Paratome: An online tool for systematic identification of
antigen binding
regions in antibodies based on sequence or structure, Nucleic Acids Res. 2012
Jul; 40 (Web
Server issue):W521-4. doi: 10.1093/nar/gks480. Epub 2012 Jun 6.
Example 2: Creation of Chimeric Receptors
Construction of chimeric receptor vectors:
The first step in the process is the design of the nucleotide sequences for
synthetic
chimeric receptor genes and the selection of appropriate lentiviral vectors.
All the vector
design are carried out in genious software version 9.1.6. The sequences are
retrieved from the
Uniprot and the Human Protein Reference Data base and NCBI as well.
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Vectors are synthesized with a combination of recombinant DNA techniques and
gene
synthesis.
Sequences for the Single chain variable fragments are produced with a
humanized
antibody yeast display library or a phage display library. Nucleic acids
encoding ScFv
specific for each of TK1, HPRT, ROR1, MUC-16, EGFRvIII, Mesothelin, HER2, CEA,
BCMA, GPC3, FAP, EphA2, NKG2D ligands, GD2, CD19, CD20, CD30, CD33, CD123,
CD133, CD138, and CD171. All possible combinations of nucleic acids encoding
chimeric
receptors having at least one of each of a), b), c), d), and e), wherein a),
b), c), d), and e) are:
a) an ScFv specific for TK1, HPRT, ROR1, MUC-16, EGFRvIII,
Mesothelin, HER2, CEA, BCMA, GPC3, FAP, EphA2, NKG2D ligands, GD2,
CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171;
b) a GS linker or no GS linker;
c) A hinge region selected from an LRR 5 amino acid short hinge, a LRR
long hinge, an IgG4 short hinge, an IgG 119 amino acid medium hinge, and IgG4
long
hinge, a CD8 hinge, a CD8 hinge with cysteines converted to serines, and no
hinge;
d) a transmembrane domain selected from the transmembrane domains of
MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, FCGR2A, FCGR3A, and
FCER1G; and
e) a cytosolic domain selected from the cytosolic domains of MYD88,
TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, FCGR2A, FCGR3A, and
FCER1G.
The foregoing nucleic acids encoding chimeric receptors are synthesized with a
combination of recombinant DNA techniques and gene synthesis.
Macrophages are genetically modified with an integrated gene delivery method
via
lentiviral-mediated gene transfer to provide the nucleic acids encoding
chimeric receptors. A
third generation lentiviral system from addgene is used to package our
lentiviral vectors.
pHIV-dTomato (#21374) and pUltra-chilli (#48687) are the gene transfer
plasmids. pCMV-
VSV-G (#8454), pMDLg/pRRE (#12251), pRSV-Rev (#12253), pHCMV-AmphoEnv
(#15799) are the packaging plasmids. A lentiviral mediated gene transfer of
human
lymphocytes has been standardized previously getting efficiencies up to 50 %
transduction.
HEK293T cells are transfected with the calcium phosphate method (SIGMA
CAPHOS).
Around 10 pg of each packaging plasmid and 20 ug of vector encoding the
chimeric receptor
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are used per transfection. After 48-36 hours viral particles are harvested and
sterile filtered.
Viral titration are determined infecting HT1080 and U937 cells.
The analysis is performed by flow cytometry detecting a red fluorescent
protein. After
viral titration human monocytes are transduced using retronectin plates
(Clonetech, T1 00B)
and the spin infection method
Previous to lentiviral transduction, monocytes are isolated from whole PBMNCs
by
negative selection and magnetic sorting using the Monocyte Isolation Kit II,
human
(MACS130-091-153). After monocyte isolation cells are split in 2 nunclon 6-
well plates
(Thermo, 145380) seeding 1.5X106 cells in each well for each vector. One plate
is
immediately transduced while the second plate is used for ex-vivo
differentiation of
monocytes to M1 macrophages. The M1 macrophages are produced using the media
Ml-
Macrophage Generation Medium DFX (Promocell, C-28055). After 7 days,
macrophages are
transduced and activated at day 9 with LPS (500X) (Affimetryx, 00-4976-03) and
IFN-y
(Promokine, C-60724). The transduction efficiency is analyzed by flow
cytometry.
Transduced cells are separated by cell sorting using a FACS Aria cell sorter.
After cell sorting
transduced monocytes are ex vivo cultured for a couple of days before
differentiation while
differentiated macrophages can last a month.
Example 3: Polarization of macrophages through chimeric receptors
The transduced macrophages prepared in Example 2 are separately exposed to
TK1,
HPRT, ROR1, MUC-16, EGFRvIII, Mesothelin, HER2, CEA, BCMA, GPC3, FAP, EphA2,
NKG2D conjugated ligands, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138,
and
CD171 and tested for polarization to the M1 phenotype by monitoring the
secretion of IL-12
and IL-23 using a standard cytokine assay or by measuring RNA production.
Macrophages
bearing chimeric receptors are polarized to the M1 phenotype when exposed to
the ligand
specific for the particular chimeric receptor and determined by increased
secretion of IL-12
and/or IL-23. Ligands other than the specific ligand for the specific chimeric
receptor display
no increase in IL-12 and/or IL-21.
Example 4: Production of monocyte-derived macrophages and transduction
After 7 days of differentiation monocyte-derived macrophages had undergone
phenotype changes. These changes where compared between transduced and non-
transduced
cells. As can be observed in FIG. 27, transduced cells have a more aggressive
phenotype
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similar to M1 or classically activated macrophages. FIG. 27 shows images of
Non-transduced
and transduced monocyte-derived macrophages at day 8 of differentiation. No
Interferon
gamma and LPS was added at this point. It can be observed that the phenotype
of
macrophages transduced with a chimeric receptor is different from non-
transduced
macrophages. Transduced cells displayed a classically activated or Ml-like
phenotype
indicating macrophage activation. The altered phenotype may be a combined
effect of the
transduction process and the expression of the new synthetic receptor.
FIG 28 provides confirmation of the insertion and expression of constructs
encoding a
chimeric receptor as was confirmed by the expression of dTomato 48-72 hours
after
transduction. This demonstrates the successful transduction of human monocyte-
derived
macrophages.
Example 5: Transduction efficiency
After day 10 of differentiation the transduction efficiency was assessed and
macrophages expressing the chimeric receptor were cell sorted. Lentiviral
transduction is
challenging in macrophages. However, using HIV-1 based systems with EF1-a
promoters
almost 30% macrophage transduction was achieved. Transductions of the cells at
early stages
of macrophage differentiation displayed different transduction efficiencies.
Monocytes or
macrophages in earlier stages of differentiation are easier to transduce.
Adenoviral
transduction with the chimeric adenovirus ADS/F35 has emerged as another
alternative for
macrophage transduction. FIG. 29 shows the results of macrophages that were
transduced
being cell sorted using a FACSAria system. Around 30% of macrophage
transduction was
achieved using the lentiviral approach. The left most plot shows a control
wherein only
0.58% of cells show fluorescence which would indicate expression of dTomato.
The right
two plots show a transduction efficiency of 27.1 percent after transduction.
Example 6: Immunophenotyping of transduced macrophages
Immunophenotyping of macrophages transduced with vectors for the expression of
a
chimeric receptor was performed to identify the activation state of the
transduced cells. It has
been reported that modifications of the extracellular domain of TLR-4 may
induce constant
activation of its signaling domain (Gay et al., 2014). Constant activation of
the TLR-4
signaling could lead to macrophage activation or M1 phenotype. It is not know
if the
construct which was used, which is based on TLR-4, is able to trigger a
constant activation of
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the signaling through the TIR domain taken from TLR-4. However, after the
transduction
process, a change in the phenotype was observed and a change in the expression
of cell
surface markers in the macrophages. This is likely due to a combination of the
lentiviral
transduction and the expression of the chimeric receptor protein. Expression
of CD14, CD80,
D206 and low expression of CD163 were indicators of macrophage polarization
towards the
M1 phenotype. The expression of these cell surface markers in was observed in
the
transduced cells. FIG. 30 presents six scatter plots of fluorescence activated
cell sorting
demonstrating the retention of dye (Alexa 647), and the expression of CD80,
CD163,
CD206, and CD14 in macrophages transduced with a chimeric receptor.
FIG 31. Presents a histogram of the relative expression levels of M1 cells
surface
markers in macrophages transduced with a vector to express a chimeric
receptor.
Example 7: In-vitro toxicity of TK1 targeting chimeric receptor transduced
macrophages against NCI-H460 cells.
The tumoricidal activity of TK1 targeting chimeric receptor transduced
macrophages
was tested against NCI-H460-GFP cells. The E:T ratio used was 1:10. The
analysis was
performed with confocal microscopy. Detection of fluorescence was performed
every 5
minutes during a 12 hour period. It was observed during time lapse that TK1
targeting
chimeric receptor transduced macrophages migrate toward H460-GFP cells and
attack them.
After the synapsis, specific cell death is induced in the target cell. As
demonstrated by the
images in FIG. 32, TK1 targeting chimeric receptor transduced macrophages can
detect,
attack and induce cell death in lung cancer cell lines expressing TK1. NCI-
H460 cells were
modified to express GFP. The tumoricidal activity of TK1 targeting chimeric
receptor
transduced macrophages was detected with confocal microscopy as a loss of
fluorescence in
the target cell.
The disclosure can be further modified within the spirit and scope of this
disclosure.
This application is therefore intended to cover any variations, uses, or
adaptations of the
disclosure using its general principles. Further, this application is intended
to cover such
departures from the present disclosure as come within known or customary
practice in the art
to which this disclosure pertains and which fall within the limits of the
appended claims and
their legal equivalents.
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