U.S. patent application number 15/111384 was filed with the patent office on 2016-11-17 for chimeric antigen receptors (cars) having mutations in the fc spacer region and methods for their use.
The applicant listed for this patent is Christine E. Brown, Stephen J. Forman, Umamaheswararao Jonnalagadda, Armen Mardiros. Invention is credited to Christine E. Brown, Stephen J. Forman, Umamaheswararao Jonnalagadda, Armen Mardiros.
Application Number | 20160333108 15/111384 |
Document ID | / |
Family ID | 53524229 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160333108 |
Kind Code |
A1 |
Forman; Stephen J. ; et
al. |
November 17, 2016 |
CHIMERIC ANTIGEN RECEPTORS (CARs) HAVING MUTATIONS IN THE FC SPACER
REGION AND METHODS FOR THEIR USE
Abstract
Adoptive immunotherapy using T cells genetically redirected via
expression of chimeric antigen receptors (CARs) is a promising
approach for cancer treatment. However, this immunotherapy is
dependent in part on the optimal molecular design of the CAR, which
involves an extracellular ligand-binding domain connected to an
intracellular signaling domain by spacer and/or transmembrane
sequences.
Inventors: |
Forman; Stephen J.; (Duarte,
CA) ; Brown; Christine E.; (Duarte, CA) ;
Jonnalagadda; Umamaheswararao; (Troy, MI) ; Mardiros;
Armen; (Glendale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Forman; Stephen J.
Brown; Christine E.
Jonnalagadda; Umamaheswararao
Mardiros; Armen |
Duarte
Duarte
Troy
Glendale |
CA
CA
MI
CA |
US
US
US
US |
|
|
Family ID: |
53524229 |
Appl. No.: |
15/111384 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US2014/028961 |
371 Date: |
July 13, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61926881 |
Jan 13, 2014 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/526 20130101;
C07K 16/30 20130101; C07K 2319/00 20130101; C07K 2319/33 20130101;
A61K 2039/5156 20130101; C07K 16/00 20130101; C07K 14/7051
20130101; C07K 16/2803 20130101; C07K 2319/30 20130101; C07K
2317/92 20130101; C07K 14/70521 20130101; C07K 2317/524 20130101;
C07K 2319/03 20130101; A61P 35/00 20180101; C07K 2317/56 20130101;
C07K 2317/622 20130101; C07K 14/70517 20130101; C07K 16/2866
20130101; A61K 45/06 20130101; C07K 14/71 20130101; C07K 2319/02
20130101; A61K 39/39558 20130101; C07K 2317/53 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 14/705 20060101
C07K014/705; C07K 14/725 20060101 C07K014/725 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] The present invention was made with government support under
Grant Nos P50 CA107399 and P01 CA030206 awarded by the National
Institutes of Health (NIH). The Government has certain rights in
the invention.
Claims
1. A recombinant chimeric antigen receptor (CAR) having impaired
binding to an Fc receptor (FcR) comprising: an antigen recognition
domain; a spacer domain derived from a modified immunoglobulin Fc
region having one or more mutations in its CH2 region resulting in
impaired binding to an FcR; and an intracellular signaling
domain.
2. The method of claim 1, wherein the antigen recognition domain is
an scFv.
3. The method of claim 1, wherein the antigen recognition domain
targets a cancer associated antigen selected from the group
consisting of 5T4, 8H9, .alpha.v.beta.6 integrin, alphafetoprotein
(AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22,
CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171,
carcionoembryonic antigen (CEA), EGFrvIII, epithelial
glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40),
ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor
antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate
receptor-.alpha., G250/CAIX, ganglioside 2 (GD2), ganglioside 3
(GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated
antigen (HMW-MAA), IL-13 receptor .alpha.2, KDR, k-light chain,
Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated
antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1
(MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D)
ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal
antigen (h5T4), prostate stem cell antigen (PSCA),
prostate-specific membrane antigen (PSMA), receptor-tyrosine
kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE,
tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular
endothelial growth factor (VEGF) receptors.
4. The method of claim 1, wherein the modified immunoglobulin Fc
region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.
5. The method of claim 1, wherein the one or more mutations of the
modified immunoglobulin Fc region comprise one or more amino acid
substitutions selected from an S228P amino acid substitution, an
L235E amino acid substitution, an N297Q amino acid substitution, or
a combination thereof.
6. The method of claim 1, wherein the one or more mutations of the
modified immunoglobulin Fc region comprise one or more
deletions.
7. The method of claim 1, further comprising a transmembrane
domain.
8. The method of claim 1, wherein the intracellular signaling
domain is a T cell receptor (TCR) zeta chain signaling domain.
9. The method of claim 8, further comprising one or more
costimulatory intracellular signaling domain derived from CD28,
inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck,
or 2B4.
10. The method of claim 1, wherein the CAR is encoded by a nucleic
acid sequence which that is inserted within a viral vector.
11. A population of human immune cells transduced by a viral vector
comprising an expression cassette that includes a CAR gene, the
gene comprising a nucleotide sequence that encodes: an antigen
recognition domain; a spacer domain derived from a modified
immunoglobulin Fc region having one or more mutations in its CH2
region resulting in impaired binding to an FcR; and an
intracellular signaling domain; wherein the population of human
immune cells expresses the CAR gene.
12. The method of claim 11, wherein the antigen recognition domain
targets a cancer associated antigen selected from the group
consisting of 5T4, 8H9, .alpha.v.beta.6 integrin, alphafetoprotein
(AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22,
CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171,
carcionoembryonic antigen (CEA), EGFrvIII, epithelial
glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40),
ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor
antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate
receptor-.alpha., G250/CAIX, ganglioside 2 (GD2), ganglioside 3
(GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated
antigen (HMW-MAA), IL-13 receptor .alpha.2, KDR, k-light chain,
Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated
antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1
(MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D)
ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal
antigen (h5T4), prostate stem cell antigen (PSCA),
prostate-specific membrane antigen (PSMA), receptor-tyrosine
kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE,
tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular
endothelial growth factor (VEGF) receptors.
13. The method of claim 11, wherein the modified immunoglobulin Fc
region is a modified IgG1, IgG2, IgG3, or IgG4 Fc region.
14. The method of claim 11, wherein the one or more mutations of
the modified immunoglobulin Fc region comprise one or more amino
acid substitutions selected from an S228P amino acid substitution,
an L235E amino acid substitution, an N297Q amino acid substitution,
or a combination thereof.
15. The method of claim 11, wherein the one or more mutations of
the modified immunoglobulin Fc region comprise one or more
deletions.
16. The method of claim 11, wherein the intracellular signaling
domain is a T cell receptor (TCR) zeta chain signaling domain.
17. The method of claim 16, further comprising one or more
costimulatory intracellular signaling domain derived from CD28,
inducible costimulatory (ICOS), OX40, CD27, DAP10, 4-1BB, p56lck,
or 2B4.
18. A method of treating a cancer in a subject comprising
administering a population of human immune cells transduced with a
CAR gene to the subject, wherein the CAR gene comprises a
nucleotide sequence that encodes: an antigen recognition domain
that targets a cancer associated antigen specific to the cancer; a
spacer domain derived from a modified immunoglobulin Fc region
having one or more mutations in its CH2 region resulting in
impaired binding to an FcR; and an intracellular signaling
domain.
19. The method of claim 18, wherein the impaired binding to the FcR
results in improved persistence of the human immune cells as
compared to human immune cells transduced with a CAR gene
comprising a nucleotide sequence that encodes a spacer domain
derived from an unmodified immunoglobulin Fc region.
20. The method of claim 18, further comprising administering the
population of human immune cells transduced with the CAR gene in
combination with one or more anti-cancer therapy selected from stem
cell transplantation, radiation therapy, surgical resection,
chemotherapeutics, immunotherapeutics, targeted therapeutics or a
combination thereof.
21. A recombinant chimeric antigen receptor (CAR) having impaired
binding to an Fc receptor (FcR) comprising: an antigen recognition
domain comprising an scFv; a spacer domain derived from a modified
immunoglobulin Fc region having one or more mutations in its CH2
region resulting in impaired binding to an FcR, wherein the one or
more mutations are selected from an S228P amino acid substitution,
an L235E amino acid substitution, an N297Q amino acid substitution,
or a combination thereof; and an intracellular signaling domain.
Description
PRIORITY CLAIM
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/926,881, filed Jan. 13, 2014, which is
incorporated herein in its entirety, including the drawings.
BACKGROUND
[0003] Adoptive immunotherapy using chimeric antigen receptor (CAR)
expressing T cells is a promising cancer treatment, because these
cells can directly recognize and kill antigen-expressing tumor
cells in a human leukocyte antigen (HLA)-independent manner.
However, besides a careful choice of the target tumor associated
antigen, this therapeutic approach is highly dependent on the
optimal molecular design of the CAR.
[0004] Although CARs that contain a TAA-specific scFv that produces
an intracellular signal via a cytoplasmic costimulatory (e.g., CD28
or 4-1BB) domain fused to CD3-zeta have been shown in various
systems to exhibit significant anti-tumor potency (Brentjens et al.
2013; Brentjens et al. 2011; Grupp et al. 2013; Kalos et al. 2011;
Kochenderfer et al. 2012), immunological rejection and clearance by
the host remains a challenge to effective cancer treatment.
[0005] Certain modifications in CAR design have been used to
prevent the FcR-mediated clearance of therapeutic cells. For
example, hinge/spacer sequences that do not originate from Ig Fc
domains may be used, such as those from CD8.alpha. or CD28
(Brentjens et al. 2007; Kalos et al. 2011; Imai et al. 2004;
Kochenderfer et al. 2009). Although these spacer sequences may
alleviate FcR binding, their length may not endow CAR T cells with
optimal potency when targeting certain antigens. For instance, when
targeting 5T4, NCAM and MUC1 using CAR T cells, longer linker
regions (i.e., longer than those derived from CD8.alpha. or CD28)
were required for optimal potency (Wilkie et al. 2008; Guest et al.
2005). Thus, it would be desirable to design a CAR that addresses
these challenges, while maintaining its efficacy in killing cancer
cells.
SUMMARY
[0006] According to some embodiments, recombinant chimeric antigen
receptors (CAR) having impaired binding to an Fc receptor (FcR) are
provided. Such CARs may include, but are not limited to, an antigen
recognition domain, a spacer domain derived from a modified
immunoglobulin Fc region having one or more mutations in its CH2
region resulting in impaired binding to an FcR, and an
intracellular signaling domain.
[0007] In another embodiment, a population of human immune cells
transduced by a viral vector comprising an expression cassette that
includes a CAR gene is provided. In some aspects, the CAR gene
comprises a nucleotide sequence that encodes an antigen recognition
domain, a spacer domain derived from a modified immunoglobulin Fc
region having one or more mutations in its CH2 region resulting in
impaired binding to an FcR, and an intracellular signaling domain,
wherein the population of human immune cells expresses the CAR
gene.
[0008] In another embodiment, a method of treating a cancer in a
subject is provided. Such a method includes administering a
population of human immune cells transduced with a CAR gene to the
subject. In some aspects, the CAR gene comprises a nucleotide
sequence that encodes an antigen recognition domain that targets a
cancer associated antigen specific to the cancer, a spacer domain
derived from a modified immunoglobulin Fc region having one or more
mutations in its CH2 region resulting in impaired binding to an
FcR, and an intracellular signaling domain.
[0009] Designing a CAR having a spacer domain that has decreased or
impaired binding to FcRs (such as those described herein) helps
prevent the FcR-expressing cells from recognizing and destroying,
or unintentionally activating, the CAR-expressing immunotherapeutic
cells in vivo. Therefore, such CARs help prevent immunological
rejection and clearance of the cells meant to provide therapeutic
benefit to patients
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows that CD19-specific CAR-expressing T cells do
not efficiently engraft in NSG mice according to one embodiment.
FIG. 1a shows the schematics of the CD19R/EGFRt (top) and EGFRt
(bottom) expression constructs that were used to gene modify T
cells for engraftment studies. Sequence portions of the
CD19-specific, CD28-costimulatory CAR (CD19R), the self-cleavable
T2A, the huEGFRt, and the drug resistance DHFR.sup.FS and
IMPDH2.sup.IY genes are indicated, along with the Elongation Factor
1 promoter sequences (EF-1p), the GM-CSF receptor alpha chain
signal sequences (GMCSFRss), and the 3 nucleotide stop codons. FIG.
1b is a flow cytometric analysis of T cells administered to NSG
mice for engraftment studies. T.sub.CM-derived cells were left
non-transduced (Non-Txd), or were transduced with lentiviral
vectors containing the CD19R/EGFRt (CD19R) or EGFRt/DHFRFS/IMPDH2IY
(EGFRt) constructs described in (A) and immunomagnetically selected
for EGFRt-expression. The cells were then expanded in vitro for 19
days and analyzed for surface phenotype. Percentages of cells
staining with antibodies specific for CD4 (top) or CD8 (bottom) vs.
EGFRt are indicated in each histogram, using quadrants that were
created based on negative control staining. In FIG. 1c, 10.sup.7
T.sub.CM-derived cells as described in (B) were administered i.v.
to NSG mice with irradiated NS0-IL15 support. Day 7 and 14
peripheral blood leukocytes that were harvested from each group
(n=3-5 mice) were stained using FITC-conjugated anti-human CD45,
and biotinylated-cetuximab followed by PE-conjugated streptavidin.
Percentages of lymphocyte-gated, huCD45+ and huCD45+EGFRt+ cells
are indicated in each histogram, using quadrants that were created
based on negative control staining. Data are representative of 4
different experiments performed with T.sub.CM-derived cells from
multiple donors.
[0011] FIG. 2 illustrates that CD19-specific CAR-expressing T cells
bind soluble Fc.gamma.R1 according to one embodiment. The same T
cells described in FIG. 1 were stained with the indicated volume
titration of biotinylated soluble human Fc gamma receptor 1
followed by PE-conjugated streptavidin (SA-PE, grey histogram). For
CD19R-expressing cells, percentages of immune reactive cells are
indicated in each histogram, and based on an M1 gate set to detect
.ltoreq.1% of that stained with SA-PE alone (black line).
[0012] FIG. 3 shows that a mutated IgG4 spacer does not affect
CD19-specific effector function of CAR-expressing T cells according
to one embodiment. FIG. 3a shows the schematics of the parental
CD19-specific CAR (CD19R), the CD19-specific CAR that contains the
2 point mutations, L235E and N297Q, in the CH2 portion of the IgG4
spacer (CD19R(EQ)), and the CD19-specific CAR that contains a
truncated IgG4 spacer, where the whole CH2 domain is removed
(CD19Rch2.DELTA.). The ligand-binding scFv domain derived from the
FMC63 mAb, the transmembrane and cytoplasmic signaling domains
derived from huCD28, and the cytoplasmic signaling domain of
huCD3.zeta. are also depicted. In FIG. 3b, T.sub.CM-derived,
EGFRt-enriched and expanded cells expressing either the parental
CD19R, the EGFRt marker alone, the CD19R that has a single IgG4
point mutation at either amino acid 235 (CD19R(L235E)) or amino
acid 297 (CD19R(N297Q)), the double-mutated CD19R(EQ) or the
CH2-deleted CD19Rch2.DELTA., were analyzed for transgene
expression. Percentages of cells staining with antibodies specific
for the Fc-containing CAR (top) or EGFRt (bottom) are indicated in
each histogram, and based on an M1 gate set to detect .ltoreq.1% of
that stained with SA-PE alone (black line). In FIG. 3c, the same
cells used in FIG. 3b were used as effectors in a 4-hour chromium
release assay against .sup.51Cr-labeled CD19.sup.+ LCL or SupB15
targets. LCL expressing the CD3 agonist OKT3 (LCL-OKT3) and
CD19-negative K562 cells were used as positive- and
negative-control targets, respectively. Mean percent chromium
release .+-.S.D. of triplicate wells at the indicated E:T ratios
are depicted.
[0013] FIG. 4 shows that CARs with a mutated IgG4 spacer exhibit
inhibited Fc.gamma.R binding according to one embodiment.
TCM-derived, EGFRt-enriched, expanded cell lines expressing either
the EGFRt marker alone, the parental CD19R, the single
point-mutated CD19R(L235E) or CD19R(N297Q), the double
point-mutated CD19R(EQ), or the CH2-deleted CD19Rch2.DELTA. were
stained with the following biotinylated reagents: anti-Fc antibody
(to detect the CAR), cetuximab (to detect EGFRt), or the indicated
human (Hu) or murine (Mu) soluble Fc receptors (Fc.gamma.R1, R2a,
or R2b); followed by PE-conjugated streptavidin (SA-PE, grey
histogram). Percentages of immune reactive cells are indicated in
each histogram, and based on an M1 gate set to detect .ltoreq.1% of
that stained with SA-PE alone (black line).
[0014] FIG. 5 shows that T cells expressing CARs with mutated IgG4
spacer exhibit enhanced in vivo engraftment according to one
embodiment. 10.sup.7 T.sub.CM-derived, EGFRt-enriched cells
expressing either the parental CD19R, the EGFRt marker alone, the
single point-mutated CD19R(L235E) or CD19R(N297Q), the double
point-mutated CD19R(EQ), or the CH2-deleted CD19Rch2.DELTA. (see
phenotype FIG. 3b) were infused i.v. into NSG mice on day 0 with
irradiated NS0-IL15 support. Day 7 and 14 peripheral blood
leukocytes harvested from each group (n=5 mice) were stained using
PerCP-conjugated anti-human CD45, and biotinylated-cetuximab
followed by PE-conjugated streptavidin. In FIG. 5a, mean
percentages of CD45+ EGFRt+ cells in the viable lymphocyte-gated
population .+-.S.E.M. are indicated. *, p<0.034 when compared to
mice given CD19R-expressing cells using an unpaired Student's
t-test. FIG. 5b shows representative histograms (i.e., median 3 of
each group of 5 mice) that are depicted with quadrants created
based on control staining. Percentages of huCD45+ EGFRt+ cells are
indicated in each histogram.
[0015] FIG. 6 shows that T.sub.CM-derived cells expressing CARs
with mutated IgG4 spacer exhibit enhanced therapeutic efficacy
according to some embodiments. 1.5.times.10.sup.6 ffLuc.sup.+ LCL
cells were administered i.v. into NSG mice on day 0, and then
5.times.10.sup.6 CAR+ T.sub.CM-derived cells (10.sup.7 cells total)
expressing either the EGFRt marker alone, the parental CD19R, the
double point-mutated CD19R(EQ), or the CH2-deleted CD19Rch2.DELTA.
were infused i.v. into NSG mice on day 3. LCL tumor growth was then
monitored by Xenogen imaging. FIG. 6a shows a flow cytometric
analysis depicting the CAR profiles of the input T.sub.CM-derived
cells (used at day 23 after bead stimulation and
lentitransduction). Percentages of immunoreactive cells are
indicated in each histogram, and based on an M1 gate set to detect
.ltoreq.1% of that stained with SA-PE alone (black line). FIG. 6b
shows mean flux levels (.+-.S.E.M.) of luciferase activity are
depicted for each group (n=6). FIG. 6c shows representative
bioluminescence images of NSG mice at day 21 are depicted for each
group. FIG. 6d shows mean percentages (+S.E.M.) of CD45.sup.+
EGFRt.sup.+ cells in the viable lymphocyte-gated population of
peripheral blood at day 21 are indicated. *, p<0.035 when
compared to mice given CD19R-expressing cells using an unpaired
Student's t-test. FIG. 6e shows a Kaplan Meier analysis of survival
for each group. Log-rank (Mantel-COX) tests were used to perform
statistical analyses of survival between groups; *, p=0.0009 when
compared to mice that received T cells expressing the parental
CD19R.
[0016] FIG. 7 shows that bulk T cells expressing CD19R(EQ) exhibit
enhanced therapeutic efficacy according to one embodiment.
1.5.times.10.sup.6 ffLuc.sup.+ LCL cells were administered i.v.
into NSG mice on day 0, and then 5.times.10.sup.6 CAR.sup.+ T cells
expressing either the parental CD19R or the double point-mutated
CD19R(EQ) were infused i.v. into NSG mice on day 2. LCL tumor
growth was then monitored by Xenogen imaging. FIG. 7a shows a flow
cytometric analysis of the CAR (top), EGFRt vs. CD3 (middle) and
CD4 vs CD8 (bottom) profiles of the input T cells (used at day 21
after bead stimulation and lentitransduction). Percentages of
immunoreactive cells as determined by histogram subtraction (top),
or based on quadrants that were drawn according to the staining of
mock-transduced cells and isotype control staining (middle, bottom)
are depicted in each histogram. FIG. 7b shows representative
bioluminescence images of NSG mice at day 2, 11 and 23 are depicted
for each group. FIG. 7c shows mean flux levels (.+-.S.E.) of
luciferase activity are depicted for each group (n=3). FIG. 7d
shows a Kaplan-Meier analysis of survival for each group. Log-rank
(Mantel-COX) tests were used to perform statistical analyses of
survival between groups; *, p=0.0295 when compared to mice that
received T cells expressing the parental CD19R.
[0017] FIG. 8 shows that non-enriched T.sub.CM-derived cells
expressing CARs with mutated IgG4 spacer exhibit enhanced in vivo
engraftment according to some embodiments. 10.sup.7
T.sub.CM-derived cells expressing either the EGFRt marker alone,
the parental CD19R, or the double point-mutated CD19R(EQ) were
infused i.v. into NSG mice on day 0 with irradiated NS0-IL15
support. Day 7 and 14 peripheral blood leukocytes harvested from
each group (n=4-6 mice) were stained using PerCP-conjugated
anti-human CD45, and biotinylated-cetuximab followed by
PE-conjugated streptavidin. FIG. 8A shows a flow cytometric
analysis depicting the CAR profiles of the input T.sub.CM-derived
cells (used at day 26 after bead stimulation and
lentitransduction). Percentages of cells staining with antibodies
specific for the Fc-containing CAR (top) or EGFRt (bottom) are
indicated in each histogram, and based on an M1 gate set to detect
1% of that stained with SA-PE alone (black line). FIG. 8B shows
mean percentages of CD45+EGFRt+ cells in the viable
lymphocyte-gated population .+-.S.E.M. are indicated. *, p=0.004
and **, p=0.057 when using an unpaired Student's t-test to compare
mice infused with T.sub.CM-derived cells expressing the parental
CD19R vs. CD19R(EQ). FIG. 8C shows representative histograms (i.e.,
median 2 of each group of 4-6 mice) are depicted with quadrants
created based on control staining. Percentages of huCD45+ EGFRt+
cells are indicated in each histogram.
DETAILED DESCRIPTION
[0018] The following examples are intended to illustrate various
embodiments of the invention. As such, the specific embodiments
discussed are not to be construed as limitations on the scope of
the invention. It will be apparent to one skilled in the art that
various equivalents, changes, and modifications may be made without
departing from the scope of invention, and it is understood that
such equivalent embodiments are to be included herein. Further, all
references cited in the disclosure are hereby incorporated by
reference in their entirety, as if fully set forth herein.
Chimeric Antigen Receptors
[0019] According to the embodiments described herein, recombinant
chimeric antigen receptors (CARs) to target cancer-related antigens
and methods for their use are provided. As described by the
embodiments below, a CAR may include a series of protein or peptide
domains including, but not limited to, one or more of an antigen
binding domain, a spacer domain, a transmembrane domain, an
intracellular signaling domain and an intracellular costimulatory
domain.
[0020] In some embodiments, a gene encoding the CAR is provided,
wherein the gene includes a nucleotide or nucleic acid sequence
which includes a series of regions which encode an amino acid
sequence corresponding to the protein or peptide domains of the CAR
described herein. Because the degeneracy of the genetic code is
known, any amino acid sequences disclosed herein are also
indicative of all degenerate nucleic acid codons corresponding to
each amino acid in said sequences. As such, it is understood that
the embodiments describing CARs and their domains may be provided
as a gene comprising a nucleic acid sequence as well as the amino
acid sequences encoded by said genes.
[0021] In one embodiment, a CAR may include, but is not limited to,
an antigen binding domain, a spacer domain, optionally at least one
intracellular signaling domain and optionally at least one
intracellular costimulatory domain.
[0022] In other embodiments, a CAR may include, but is not limited
to, an antigen binding domain, a spacer domain, and at least one
intracellular signaling domain.
[0023] In other embodiments, a CAR may include, but is not limited
to, an antigen binding domain, a spacer domain, at least one
intracellular signaling domain and at least one intracellular
costimulatory domain.
[0024] Antigen Binding Domain
[0025] A CAR antigen binding domain may include a nucleotide
sequence that, when expressed as a peptide or polypeptide, binds an
epitope of a cancer-related antigen. In some embodiments, a
cancer-related antigen may be any antigen expressed by or
overexpressed by a cancer cell (e.g., a tumor cell, a neoplastic
cell, a malignant cell, or any other cancerous cell), and may be a
protein, peptide, carbohydrate, glycoprotein, ganglioside,
proteoglycan, or any combination or complex thereof. In some
aspects, the cancer-related antigen is a tumor specific antigen
(TSA) that may be expressed only on cancer or tumor cells, while in
other aspects, the cancer-related antigen is a tumor-associated
antigen (TAA) that may be expressed on both tumor cells and normal
cells. In other aspects, the cancer-related antigen may be a
product of a mutated oncogene or tumor suppressor gene, or a
product of another mutated gene (e.g., overexpressed or aberrantly
expressed cellular proteins, tumor antigens produced by oncogenic
viruses, oncofetal antigens, altered cell surface glycolipids or
glycoproteins, or cell type-specific differentiation antigens).
[0026] According to the embodiments described herein,
cancer-related antigens that may be targeted by a CAR antigen
binding domain described herein include, but are not limited to,
5T4, 8H9, .alpha..sub.v.beta..sub.6 integrin, alphafetoprotein
(AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22,
CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171,
carcionoembryonic antigen (CEA), EGFrvIII, epithelial
glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40),
ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor
antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate
receptor-.alpha., G250/CAIX, ganglioside 2 (GD2), ganglioside 3
(GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated
antigen (HMW-MAA), IL-13 receptor .alpha.2, KDR, k-light chain,
Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated
antigen (MAGE-A1), mesothelin, Murine CMV infected cells, mucin-1
(MUC1). mucin-16 (MUC16), natural killer group 2 member D (NKG2D)
ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal
antigen (h5T4), prostate stem cell antigen (PSCA),
prostate-specific membrane antigen (PSMA), receptor-tyrosine
kinase-like orphan receptor 1 (ROR1), TAA targeted by mAb IgE,
tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular
endothelial growth factor (VEGF) receptors. In some embodiments,
the antigen binding domain that is part of a CAR described herein
targets CD19 or CD123.
[0027] An antigen binding domain may be any targeting moiety which
targets an antigen associated with cancer. In some embodiments, the
antigen binding domain is an antibody or functional fragment of an
antibody. An antibody refers to an immunoglobulin molecule that
specifically binds to, or is immunologically reactive with an
antigen or epitope, and includes both polyclonal and monoclonal
antibodies, as well as functional antibody fragments, including but
not limited to fragment antigen binding (Fab) fragments, F(ab')2
fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG)
fragments, single chain variable fragments (scFv) and single domain
antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term
"antibody or functional fragment thereof" also includes genetically
engineered or otherwise modified forms of immunoglobulins, such as
intrabodies, peptibodies, chimeric antibodies, fully human
antibodies, humanized antibodies, and heteroconjugate antibodies
(e.g., bispecific antibodies, diabodies, triabodies, tetrabodies,
tandem di-scFv, tandem tri-scFv). Unless otherwise stated, the term
"antibody" should be understood to encompass functional antibody
fragments thereof. In one embodiment, the antigen binding domain is
an scFv having a heavy (V.sub.H) and light chain (V.sub.L). In
other embodiments, the antigen binding domain is an scFv that
targets CD19 or CD123. In such embodiments, the scFv that targets
CD19 may have the following amino acid sequence:
TABLE-US-00001 CD19R V.sub.L (SEQ ID NO: 1) DIQMTQTTSS LSASLGDRVT
ISCRASQDIS KYLNWYQQKP DGTVKLLIYH TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ
EDIATYFCQQ GNTLPYTFGG GTKLEIT CD19R V.sub.H (SEQ ID NO: 2)
EVKLQESGPG LVAPSQSLSV TCTVSGVSLP DYGVSWIRQP PRKGLEWLGV IWGSETTYYN
SALKSRLTII KDNSKSQVFL KMNSLQTDDT AIYYCAKHYY YGGSYAMDYW
GQGTSVTVSS
[0028] And the scFv that targets CD123 may have one of the
following amino acid sequences:
TABLE-US-00002 CD123 V.sub.H1 (SEQ ID NO: 3) QIQLVQSGPE LKKPGETVKI
SCKASGYIFT NYGMNWVKQA PGKSFKWMGW INTYTGESTY SADFKGRFAF SLETSASTAY
LHINDLKNED TATYFCARSG GYDPMDYWGQ GTSVTVSS CD123 V.sub.H2 (SEQ ID
NO: 4) QVQLQQPGAE LVRPGASVKL SCKASGYTFT SYWMNWVKQR PDQGLEWIGR
IDPYDSETHY NQKFKDKAIL TVDKSSSTAY MQLSSLTSED SAVYYCARGN WDDYWGQGTT
LTVSS CD123 V.sub.L1 (SEQ ID NO: 5) DIVLTQSPAS LAVSLGQRAT
ISCRASESVD NYGNTFMHWY QQKPGQPPKL LIYRASNLES GIPARFSGSG SRTDFTLTIN
PVEADDVATY YCQQSNEDPP TFGAGTKLEL K CD123 V.sub.L2 (SEQ ID NO: 6)
DVQITQSPSY LAASPGETIT INCRASKSIS KDLAWYQEKP GKTNKLLIYS GSTLQSGIPS
RFSGSGSGTD FTLTISSLEP EDFAMYYCQQ HNKYPYTFGG GTKLEIK
[0029] Spacer Domain
[0030] The spacer domain (also referred to as a "hinge region" or
"spacer/hinge region") may be derived from or include at least a
portion of an immunoglobulin Fc region, for example, an IgG1 Fc
region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an
IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain
embodiments, the spacer domain includes at least a portion of an
IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA
immunoglobulin Fc region that falls within its CH2 and CH3 domains.
In some embodiments, the spacer domain may also include at least a
portion of a corresponding immunoglobulin hinge region. In some
embodiments, the spacer domain is derived from or includes at least
a portion of a modified immunoglobulin Fc region, for example, a
modified IgG1 Fc region, a modified IgG2 Fc region, a modified IgG3
Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a
modified IgM Fc region, or a modified IgA Fc region. The modified
immunoglobulin Fc region may have one or more mutations (e.g.,
point mutations, insertions, deletions, duplications) resulting in
one or more amino acid substitutions, modifications, or deletions
that cause impaired binding of the spacer domain to an Fc receptor
(FcR). In some aspects, the modified immunoglobulin Fc region may
be designed with one or more mutations which result in one or more
amino acid substitutions, modifications, or deletions that cause
impaired binding of the spacer domain to one or more FcR including,
but not limited to, Fc.gamma.RI, Fc.gamma.R2A, Fc.gamma.R2B1,
Fc.gamma.R2B2, Fc.gamma.R3A, Fc.gamma.R3B, Fc.epsilon.RI,
Fc.epsilon.R2, Fc.alpha.RI, Fc.alpha./.mu.R, or FcRn.
[0031] Some amino acid sequences within the Fc CH2 domain have been
identified as having involvement in antibody-FcR interaction
(Strohl, 2009). FcRs, such as Fc.gamma.RI, are integral membrane
proteins located on immune cells including natural killer (NK)
cells and macrophages, which then use this Fc-targeting ability to
carry out various immune functions such as antibody-dependent
cell-mediated cytotoxicity (ADCC) and phagocytosis.
[0032] Impairment of binding to FcRs by the spacer domain prevents
the FcR-expressing cells from recognizing and destroying, or
unintentionally activating, the CAR-expressing immunotherapeutic
cells in vivo, thereby helping to prevent immunological rejection
and clearance of the cells meant to provide therapeutic benefit to
patients. The mutations described herein also contribute to
reducing the CAR's off-target effects and, thereby increasing its
specificity and efficacy.
[0033] An "amino acid modification" or an "amino acid substitution"
or a "substitution," as used herein, mean an amino acid
substitution, insertion, and/or deletion in a protein or peptide
sequence. An "amino acid substitution" or "substitution" as used
herein, means a replacement of an amino acid at a particular
position in a parent peptide or protein sequence with another amino
acid. For example, the substitution S228P refers to a variant
protein or peptide, in which the serine at position 228 is replaced
with proline.
[0034] Amino acid substitutions can be made by a mutation such that
a particular codon in the nucleic acid sequence encoding the
protein or peptide is changed to a codon which codes for a
different amino acid. Such a mutation is generally made by making
the fewest nucleotide changes possible. A substitution mutation of
this sort can be made to change an amino acid in the resulting
protein in a non-conservative manner (i.e., by changing the codon
from an amino acid belonging to a grouping of amino acids having a
particular size or characteristic to an amino acid belonging to
another grouping) or in a conservative manner (i.e., by changing
the codon from an amino acid belonging to a grouping of amino acids
having a particular size or characteristic to an amino acid
belonging to the same grouping). Such a conservative change
generally leads to less change in the structure and function of the
resulting protein.
[0035] The following are examples of various groupings of amino
acids:
[0036] Amino acids with nonpolar R groups: Alanine, Valine,
Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan,
Methionine.
[0037] Amino acids with uncharged polar R groups: Glycine, Serine,
Threonine, Cysteine, Tyrosine, Asparagine, Glutamine.
[0038] Amino acids with charged polar R groups (negatively charged
at Ph 6.0): Aspartic acid, Glutamic acid.
[0039] Basic amino acids (positively charged at pH 6.0): Lysine,
Arginine, Histidine (at pH 6.0).
[0040] Another grouping may be those amino acids with phenyl
groups: Phenylalanine, Tryptophan, Tyrosine.
[0041] Another grouping may be according to molecular weight (i.e.,
size of R groups) as shown below:
TABLE-US-00003 Glycine 75 Alanine 89 Serine 105 Proline 115 Valine
117 Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131
Asparagine 132 Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic
acid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165
Arginine 174 Tyrosine 181 Tryptophan 204
[0042] In certain embodiments, the spacer domain is derived from a
modified IgG1, IgG2, IgG3, or IgG4 Fc region that includes one or
more amino acid residues substituted with an amino acid residue
different from that present in an unmodified hinge. The one or more
substituted amino acid residues are selected from, but not limited
to one or more amino acid residues at positions 220, 226, 228, 229,
230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280,
290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332,
333, 334, 336, 339, or a combination thereof.
[0043] In some embodiments, the spacer domain is derived from a
modified IgG1, IgG2, IgG3, or IgG4 Fc region that includes, but is
not limited to, one or more of the following amino acid residue
substitutions: C220S, C226S, S228P, C229S, P230S, E233P, V234A,
L234V, L234F, L234A, L235A, L235E, G236A, G237A, P238S, S239D,
F243L, P2471, S267E, H268Q, S280H, K290S, K290E, K290N, R292P,
N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I ,
V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S,
P331S, 1332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or a
combination thereof.
[0044] In some embodiments, the spacer domain is derived from an
IgG Fc region having one or more modifications made to its CH2-CH3
region, wherein the unmodified IgG CH2-CH3 region corresponds to
one of the following amino acid sequences:
TABLE-US-00004 IgG1
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK IgG2
APP-VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK IgG3
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTK IgG4
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK IgG1
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT IgG2
PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYT IgG3
PREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYT IgG4
PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT IgG1
LPPSRISKAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL IgG2
LPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDKDGSFFLYSKL IgG3
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKL IgG4
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL IgG1
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7) IgG2
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) IgG3
TVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 9) IgG4
TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10)
[0045] In some embodiments, the spacer domain is derived from an
IgG Fc region having one or more modifications made to its hinge
region, wherein the unmodified IgG hinge region corresponds to one
of the following amino acid sequences:
TABLE-US-00005 IgG1 (SEQ ID NO: 11) EPKSCDKTHTCPPCP IgG2 (SEQ ID
NO: 12) ERKCCVECPPCP IgG3 (SEQ ID NO: 13)
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PEPKSCDTPPPCPRCP
IgG4 (SEQ ID NO: 14) ESKYGPPCPSCP
[0046] In some embodiments, the spacer domain is derived from an
IgG4 Fc region having the following amino acid sequence:
TABLE-US-00006 Pos. 219 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE
VTCVVVDVSQ EDPEVQFNWY Pos. 279 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV
LHQDWLNGKE YKCKVSNKGL PSSIEKTISK Pos. 339 AKGQPREPQV YTLPPSQEEM
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL Pos. 399 DSDGSFFLYS
RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 15)
[0047] In certain embodiments, the spacer domain is derived from a
modified IgG4 Fc that includes one or more amino acid residues
substituted with an amino acid residue different from that present
in an unmodified IgG4 Fc region. The one or more substituted amino
acid residues are selected from, but not limited to one or more
amino acid residues at positions 220, 226, 228, 229, 230, 233, 234,
235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297,
298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336,
339, 396, or a combination thereof.
[0048] In some embodiments, the spacer domain is derived from a
modified IgG4 Fc region that includes, but is not limited to, one
or more of the following amino acid residue substitutions: 220S,
226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E,
236A, 237A, 238S, 239D, 243L, 2471, 267E, 268Q, 280H, 290S, 290E,
290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I,
309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E,
333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combination thereof,
wherein the amino acid in the unmodified IgG4 Fc region is
substituted with the above identified amino acids at the indicated
position.
[0049] In some embodiments, the spacer domain is derived from a
modified IgG4 Fc region that includes, but is not limited to, two
or more (i.e., "double mutated"), three or more (i.e., "triple
mutated"), four or more, five or more, or more than five of the
following amino acid residue substitutions: 220S, 226S, 228P, 229S,
230S, 233P, 234A, 234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S,
239D, 243L, 2471, 267E, 268Q, 280H, 290S, 290E, 290N, 292P, 297A,
297Q, 298A, 298G, 298D, 298V, 299A, 300L, 305I, 309L, 318A, 326A,
326W, 326E, 328F, 330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S,
334A, 339D, 339Q, 396L, or a combination thereof, wherein the amino
acid in the unmodified IgG4 Fc region is substituted with the above
identified amino acids at the indicated position.
[0050] In some embodiments, the spacer domain is derived from a
modified IgG4 Fc region that includes, but is not limited to, a
substitution of proline (P) in place of serine (S) at position 228
(S228P), a substitution of leucine (L) in place of glutamic acid
(E) at position 235 (L235E), a substitution of asparagine (N) in
place of glutamine (Q) at position 297 (N297Q), or a combination
thereof. In certain embodiments, a modified IgG4 Fc region has a
single mutation, as indicated in the following amino acid sequences
(mutations are in bold and underlined):
TABLE-US-00007 (L235E mutation; SEQ ID NO: 16) ESKYGPPCPS
CPAPEFEGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK
TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV
YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS
RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (N297Q mutation; SEQ ID
NO: 17) ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ
EDPEVQFNWY VDGVEVHNAK TKPREEQFQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL
PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE
NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ
KSLSLSLGK
[0051] In other embodiments, the spacer domain is derived from a
modified IgG4 Fc region that is double mutated to include an L235E
substitution and an N297Q substitution ("EQ"). In another
embodiment, the modified IgG4 Fc region is triple mutated to
include an S228P substitution, an L235E substitution, and an N297Q
substitution ("S228P+L235E+N297Q"). In certain embodiments, a
modified IgG4 Fc and/or hinge region may include a nucleotide
sequence which encodes an amino acid sequence selected from the
following (mutations are in bold and underlined):
TABLE-US-00008 (EQ mutation; SEQ ID NO: 18) ESKYGPPCPS CPAPEFEGGP
SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFQS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ
EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (S228P + L235E + N297Q mutation;
SEQ ID NO: 19) ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMISRTPE
VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFQS TYRVVSVLTV LHQDWLNGKE
YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA
VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ
KSLSLSLGK
[0052] In certain embodiments, the spacer domain is derived from a
modified immunoglobulin Fc region that includes one or more
deletions of all of a part of its CH2 domain. In one embodiment,
the spacer domain is derived from a modified IgG4 Fc region that
includes one or more deletions of all of a part of its CH2 domain
("ch2.DELTA."). In one aspect of such an embodiment, the spacer
domain may include a nucleotide sequence which encodes the
following amino acid sequence:
TABLE-US-00009 (ch2.DELTA. mutation/deletion; SEQ ID NO: 20)
ESKYGPPCPP CPGGGSSGGG SGGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA
VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ
KSLSLSLGK
[0053] In some embodiments, the spacer domain may be modified to
substitute the immunoglobulin Fc region for a spacer that does not
have the ability to bind FcR, such as the hinge region of CD8a.
Alternatively, the Fc spacer region of the hinge may be deleted.
Such substitutions would reduce or eliminate Fc binding.
[0054] The term "position," as used herein, is a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example a Kabat position or
an EU position or EU index as in Kabat. For all positions discussed
herein, numbering is according to the EU index or EU numbering
scheme (Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Service, National
Institutes of Health, Bethesda, hereby entirely incorporated by
reference). The EU index or EU index as in Kabat or EU numbering
scheme refers to the numbering of the EU antibody (Edelman et al.,
1969, Proc Natl Acad Sci USA 63:78-85, which is hereby entirely
incorporated by reference). Kabat positions, while also well known
in the art, may vary from the EU position for a given position. For
example, the S228P and L235E substitutions described above refer to
the EU position. However, these substitutions may also correspond
to Kabat positions 241 (5241 P) and 248 (L248E).
Transmembrane and Signaling Domains
[0055] The intracellular signaling domain may include any suitable
T cell receptor (TCR) complex signaling domain, or portion thereof.
In some embodiments, the intracellular signaling domain is derived
from a CD3 complex. In some embodiments, the intracellular
signaling domain is a TCR zeta-chain (.zeta.-chain) signaling
domain. In certain embodiments, a .zeta.-chain signaling domain may
include a nucleotide sequence which encodes an amino acid sequence
as follows:
TABLE-US-00010 (SEQ ID NO: 21) RVKFSRSADA PAYQQGQNQL YNELNLGRRE
EYDVLDKRRG RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
QGLSTATKDT YDALHMQALP PR
[0056] The intracellular signaling domain may be associated with
any suitable costimulatory domain including, but not limited to, a
4-1BB costimulatory domain, an OX-40 costimulatory domain, a CD27
costimulatory domain, a CD28 costimulatory domain, a DAP10
costimulatory domain, an inducible costiumulatory (ICOS) domain, or
a 2B4 costimulatory domain. According to the embodiments described
herein, a CAR may include at least one costimulatory signaling
domain. In one aspect the CAR has a single costimulatory signaling
domain, or it may include two or more costimulatory signaling
domains such as those described above. In another aspect, the
costimulatory domain may be made up of a single costimulatory
domain such as those described above, or alternatively, may be made
up of two or more portions of two or more costimulatory domains.
Alternatively, in some embodiments, the CAR does not include a
costimulatory signaling domain. In one embodiment, the CAR includes
a costimulatory signaling domain which is a CD28 costimulatory
domain. In this embodiment, such a modified CD28 costimulatory
domain may have one or more amino acid substitutions or
modifications including, but not limited to a substitution of
leucine-leucine (LL) to glycine-glycine (GG). In certain
embodiments, a modified costimulatory signaling domain region may
include a nucleotide sequence which encodes an amino acid sequence
selected from the following:
TABLE-US-00011 (SEQ ID NO: 22) RSKRSRGGHS DYMNMTPRRP GPTRKHYQPY
APPRDFAAYR S
[0057] The signaling domain or domains may include a transmembrane
domain selected from a CD28 transmembrane domain, a CD3
transmembrane domain, or any other suitable transmembrane domain
known in the art. In some embodiments, the transmembrane domain is
a CD28 transmembrane domain. In certain embodiments, a modified
costimulatory signaling domain region may include a nucleotide
sequence which encodes an amino acid sequence selected from the
following:
TABLE-US-00012 (SEQ ID NO: 23) MFWVLVVVGG VLACYSLLVT VAFIIFWV
Expression of CAR Genes and Transduction of T Cells
[0058] In some embodiments, the CAR gene is part of an expression
cassette. In some embodiments, the expression cassette may--in
addition to the CAR gene--also include an accessory gene. When
expressed by a T cell, the accessory gene may serve as a transduced
T cell selection marker, an in vivo tracking marker, or a suicide
gene for transduced T cells.
[0059] In some embodiments, the accessory gene is a truncated EGFR
gene (EGFRt). An EGFRt may be used as a non-immunogenic selection
tool (e.g., immunomagnetic selection using biotinylated cetuximab
in combination with anti-biotin microbeads for enrichment of T
cells that have been lentivirally transduced with EGFRt-containing
constructs), tracking marker (e.g., flow cytometric analysis for
tracking T cell engraftment), and suicide gene (e.g., via
Cetuximab/Erbitux.RTM. mediated antibody dependent cellular
cytotoxicity (ADCC) pathways). An example of a truncated EGFR
(EGFRt) gene that may be used in accordance with the embodiments
described herein is described in International Application No.
PCT/US2010/055329, the subject matter of which is hereby
incorporated by reference as if fully set forth herein. In other
embodiments, the accessory gene is a truncated CD19 gene
(CD19t).
[0060] In another embodiment, the accessory gene is an inducible
suicide gene. A suicide gene is a recombinant gene that will cause
the cell that the gene is expressed in to undergo programmed cell
death or antibody mediated clearance at a desired time. In one
embodiment, an inducible suicide gene that may be used as an
accessory gene is an inducible caspase 9 gene (see Straathof et al.
(2005). An inducible caspase 9 safety switch for T-cell therapy.
Blood. June 1; 105(11): 4247-4254, the subject matter of which is
hereby incorporated by reference as if fully set forth herein).
[0061] In some embodiments, the expression cassette that include a
CAR gene described above may be inserted into a vector for
delivery--via transduction or transfection--of a target cell. Any
suitable vector may be used, for example, a bacterial vector, a
viral vector, or a plasmid. In some embodiments, the vector is a
viral vector selected from a retroviral vector, a lentiviral
vector, a poxvirus vector, an adenoviral vector, or an
adeno-associated viral vector. In some embodiments, the vector may
transduce a population of healthy immune cells, e.g., T cells.
Successfully transduced or transfected target cells express the one
or more genes that are part of the expression cassette.
[0062] As such, one or more populations of immune cells, such as T
cells, may be transduced with a CAR gene such as those described
above. The transduced T cells may be from a donor, or may be from a
subject having a cancer and who is in need of a treatment for the
cancer. In some embodiments, the transduced T cells are used in an
adoptive immunotherapy treatment for the treatment of the cancer
(residues in bold/underline indicate substitutions). In some
embodiments, the transduced T cells express a CAR gene that encodes
an amino acid sequence selected from SEQ ID NOS:24-27:
TABLE-US-00013 CD19R(L235E)28Z (SEQ ID NO: 24): MLLLVTSLLL
CELPHPAFLL IPDIQMTQTT SSLSASLGDR VTISCRASQD ISKYLNWYQQ KPDGTVKLLI
YHTSRLHSGV PSRFSGSGSG TDYSLTISNL EQEDIATYFC QQGNTLPYTF GGGTKLEITG
STSGSGKPGS GEGSTKGEVK LQESGPGLVA PSQSLSVTCT VSGVSLPDYG VSWIRQPPRK
GLEWLGVIWG SETTYYNSAL KSRLTIIKDN SKSQVFLKMN SLQTDDTAIY YCAKHYYYGG
SYAMDYWGQG TSVTVSSESK YGPPCPPCPA PEFEGGPSVF LFPPKPKDTL MISRTPEVTC
VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC
KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW
ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL
SLSLGKMFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR GGHSDYMNMT PRRPGPTRKH
YQPYAPPRDF AAYRSGGGRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD
PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD
ALHMQALPPR CD19R(N297Q)28Z (SEQ ID NO: 25): MLLLVTSLLL CELPHPAFLL
IPDIQMTQTT SSLSASLGDR VTISCRASQD ISKYLNWYQQ KPDGTVKLLI YHTSRLHSGV
PSRFSGSGSG TDYSLTISNL EQEDIATYFC QQGNTLPYTF GGGTKLEITG STSGSGKPGS
GEGSTKGEVK LQESGPGLVA PSQSLSVTCT VSGVSLPDYG VSWIRQPPRK GLEWLGVIWG
SETTYYNSAL KSRLTIIKDN SKSQVFLKMN SLQTDDTAIY YCAKHYYYGG SYAMDYWGQG
TSVTVSSESK YGPPCPPCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP
EVQFNWYVDG VEVHNAKTKP REEQFQSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS
IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY
KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGKMFWV
LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR GGHSDYMNMT PRRPGPTRKH YQPYAPPRDF
AAYRSGGGRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK
NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR
CD19R(EQ)28Z (SEQ ID NO: 26): MLLLVTSLLL CELPHPAFLL IPDIQMTQTT
SSLSASLGDR VTISCRASQD ISKYLNWYQQ KPDGTVKLLI YHTSRLHSGV PSRFSGSGSG
TDYSLTISNL EQEDIATYFC QQGNTLPYTF GGGTKLEITG STSGSGKPGS GEGSTKGEVK
LQESGPGLVA PSQSLSVTCT VSGVSLPDYG VSWIRQPPRK GLEWLGVIWG SETTYYNSAL
KSRLTIIKDN SKSQVFLKMN SLQTDDTAIY YCAKHYYYGG SYAMDYWGQG TSVTVSSESK
YGPPCPPCPA PEFEGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG
VEVHNAKTKP REEQFQSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG
QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD
GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGKMFWV LVVVGGVLAC
YSLLVTVAFI IFWVRSKRSR GGHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSGGGRV
KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL
QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR
CD19RCH2.DELTA.CD28Z (SEQ ID NO: 27): MLLLVTSLLL CELPHPAFLL
IPDIQMTQTT SSLSASLGDR VTISCRASQD ISKYLNWYQQ KPDGTVKLLI YHTSRLHSGV
PSRFSGSGSG TDYSLTISNL EQEDIATYFC QQGNTLPYTF GGGTKLEITG STSGSGKPGS
GEGSTKGEVK LQESGPGLVA PSQSLSVTCT VSGVSLPDYG VSWIRQPPRK GLEWLGVIWG
SETTYYNSAL KSRLTIIKDN SKSQVFLKMN SLQTDDTAIY YCAKHYYYGG SYAMDYWGQG
TSVTVSSESK YGPPCPPCPG GGSSGGGSGG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG
FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA
LHNHYTQKSL SLSLGKMFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR GGHSDYMNMT
PRRPGPTRKH YQPYAPPRDF AAYRSGGGRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY
DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG
LSTATKDTYD ALHMQALPPR
[0063] Further, the one or more populations of T cells may be part
of a pharmaceutically acceptable composition for delivery for
administration to a subject. In addition to the CAR-transduced T
cells, the pharmaceutically effective composition may include one
or more pharmaceutically effective carriers. A "pharmaceutically
acceptable carrier" as used herein refers to a pharmaceutically
acceptable material, composition, or vehicle that is involved in
carrying or transporting a treatment of interest from one tissue,
organ, or portion of the body to another tissue, organ, or portion
of the body. Such a carrier may comprise, for example, a liquid,
solid, or semi-solid filler, solvent, surfactant, diluent,
excipient, adjuvant, binder, buffer, dissolution aid, solvent,
encapsulating material, sequestering agent, dispersing agent,
preservative, lubricant, disintegrant, thickener, emulsifier,
antimicrobial agent, antioxidant, stabilizing agent, coloring
agent, or some combination thereof.
[0064] Each component of the carrier is "pharmaceutically
acceptable" in that it must be compatible with the other
ingredients of the composition and must be suitable for contact
with any tissue, organ, or portion of the body that it may
encounter, meaning that it must not carry a risk of toxicity,
irritation, allergic response, immunogenicity, or any other
complication that excessively outweighs its therapeutic
benefits.
[0065] Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) natural polymers such as
gelatin, collagen, fibrin, fibrinogen, laminin, decorin,
hyaluronan, alginate and chitosan; (7) talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil
and soybean oil; (10) glycols, such as propylene glycol; (11)
polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12) esters, such as trimethylene carbonate, ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid (or
alginate); (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) alcohol, such as ethyl alcohol and propane
alcohol; (20) phosphate buffer solutions; (21) thermoplastics, such
as polylactic acid, polyglycolic acid, (22) polyesters, such as
polycaprolactone; (23) self-assembling peptides; and (24) other
non-toxic compatible substances employed in pharmaceutical
formulations such as acetone.
[0066] The pharmaceutical compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents,
toxicity adjusting agents and the like, for example, sodium
acetate, sodium chloride, potassium chloride, calcium chloride,
sodium lactate and the like.
[0067] In one embodiment, the pharmaceutically acceptable carrier
is an aqueous carrier, e.g. buffered saline and the like. In
certain embodiments, the pharmaceutically acceptable carrier is a
polar solvent, e.g. acetone and alcohol.
[0068] The concentration of CAR-transduced T cells in these
formulations can vary widely, and will be selected primarily based
on fluid volumes, viscosities, organ size, body weight and the like
in accordance with the particular mode of administration selected
and the biological system's needs.
[0069] In certain embodiments, populations of T cells transduced
with a CAR gene (i.e., CAR-transduced T cells) such as those
described herein cells used in the methods for targeting and
killing cancer or tumor cells may be grown in a cell culture. In
certain aspects of this embodiment, the method may be used in an in
vitro or research setting to investigate the role of a particular
cancer-related antigen in the etiology of a cancer, or to evaluate
the targeting abilities of new CAR constructs.
Treatment of Cancer with CAR-Transduced T Cells
[0070] According to some embodiments, CAR genes and populations of
T cells that are transduced with CAR genes such as those described
above may be used in methods for treating cancer in a subject. Such
methods may include a step of administering a therapeutically
effective amount of at least one population of T cells transduced
with at least one CAR gene to the subject. In these embodiments,
the population of CAR-transduced T-cells expresses one or more CAR
genes, such as those described above. In certain embodiments, the T
cells are transduced with and express a single mutant gene
construct such as a CD19R(L235E) or CD19R(N297Q) construct as
described herein, a double mutant gene construct which has both a
L235E and N297Q mutation (e.g., CD19R(EQ)), as described herein, or
a deletion gene construct (e.g., CD19Rch2.DELTA.), as described
herein. When such cells are administered via an adoptive
immunotherapy treatment, the transduced T cells specifically target
and lyse the cancer-related antigen expressing cells (i.e., cancer
cells) in vivo, thereby delivering their therapeutic effect of
eliminating cancer cells.
[0071] Cancers that may be treated using the population of
transduced T cells may include, but are not limited to, Acute
Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),
Adrenocortical, Carcinoma, AIDS-Related Cancers, Anal Cancer,
Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor,
Central Nervous System, Basal Cell Carcinoma, Bile Duct Cancer,
Bladder Cancer, Bone Cancer, Osteosarcoma and Malignant Fibrous
Histiocytoma, Brain Stem Glioma, Brain Tumors, Breast Cancer,
Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumors, Central
Nervous System Cancers, Cervical Cancer, Chordoma, Chronic
Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML),
Chronic Myeloproliferative Disorders, Colon Cancer, Colorectal
Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Embryonal
Tumors, Central Nervous System, Endometrial Cancer,
Ependymoblastoma, Ependymoma, Esophageal Cancer,
Esthesioneuroblastoma, Ewing Sarcoma Family of Tumors Extracranial
Germ Cell Tumor, Extragonadal Germ Cell Tumor Extrahepatic Bile
Duct Cancer, Eye Cancer Fibrous Histiocytoma of Bone, Malignant,
and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors
(GIST)--see Soft Tissue Sarcoma, Germ Cell Tumor, Gestational
Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and Neck
Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis,
Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma,
Islet Cell Tumors (Endocrine Pancreas), Kaposi Sarcoma, Kidney
cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia,
Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular
Carcinoma In Situ (LCIS), Lung Cancer, Lymphoma, Macroglobulinemia,
Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and
Osteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel
Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with
Occult Primary Midline Tract Carcinoma Involving NUT Gene, Mouth
Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple
Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic
Syndromes, Myelodysplastic/Myeloproliferative Neoplasms,
Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML),
Myeloma, Multiple, Myeloproliferative Disorders, Nasal Cavity and
Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,
Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral
Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma and Malignant
Fibrous Histiocytoma of Bone, Ovarian Cancer, Pancreatic Cancer,
Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity
Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer,
Pheochromocytoma, Pineal Parenchymal Tumors of Intermediate
Differentiation, Pineoblastoma and Supratentorial Primitive
Neuroectodermal Tumors, Pituitary Tumor, Plasma Cell
Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and
Breast Cancer, Primary Central Nervous System (CNS) Lymphoma,
Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal
Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma,
Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Sezary Syndrome,
Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue
Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer, Stomach
(Gastric) Cancer, Supratentorial Primitive Neuroectodermal Tumors,
T-Cell Lymphoma, Cutaneous, Testicular Cancer, Throat Cancer,
Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell
Cancer of the Renal Pelvis and Ureter, Trophoblastic Tumor, Ureter
and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer, Uterine
Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom
Macroglobulinemia, and Wilms Tumor.
[0072] The population or populations of T cells transduced with the
CAR gene or genes that may be used in accordance with the methods
described herein may be administered, by any suitable route of
administration, alone or as part of a pharmaceutical composition. A
route of administration may refer to any administration pathway
known in the art, including but not limited to intracranial,
parenteral, or transdermal. "Parenteral" refers to a route of
administration that is generally associated with injection,
including infraorbital, infusion, intraarterial, intracapsular,
intracardiac, intradermal, intramuscular, intraperitoneal,
intrapulmonary, intraspinal, intrasternal, intrathecal,
intratumoral, intrauterine, intravenous, subarachnoid, subcapsular,
subcutaneous, transmucosal, or transtracheal. In certain
embodiments, transduced T cells are administered intravenously or
intrathecally.
[0073] The term "effective amount" as used herein refers to an
amount of an agent, compound, treatment or therapy that produces a
desired effect. For example, a population of cells may be contacted
with an effective amount of an agent, compound, treatment or
therapy to study its effect in vitro (e.g., cell culture) or to
produce a desired therapeutic effect ex vivo or in vitro. An
effective amount of an agent, compound, treatment or therapy may be
used to produce a therapeutic effect in a subject, such as
preventing or treating a target condition, alleviating symptoms
associated with the condition, or producing a desired physiological
effect. In such a case, the effective amount of a compound is a
"therapeutically effective amount," "therapeutically effective
concentration" or "therapeutically effective dose." The precise
effective amount or therapeutically effective amount is an amount
of the composition that will yield the most effective results in
terms of efficacy of treatment in a given subject or population of
cells. This amount will vary depending upon a variety of factors,
including but not limited to the characteristics of the compound
(including activity, pharmacokinetics, pharmacodynamics, and
bioavailability), the physiological condition of the subject
(including age, sex, disease type and stage, general physical
condition, responsiveness to a given dosage, and type of
medication) or cells, the nature of the pharmaceutically acceptable
carrier or carriers in the formulation, and the route of
administration. Further an effective or therapeutically effective
amount may vary depending on whether the compound is administered
alone or in combination with another compound, drug, therapy or
other therapeutic method or modality. One skilled in the clinical
and pharmacological arts will be able to determine an effective
amount or therapeutically effective amount through routine
experimentation, namely by monitoring a cell's or subject's
response to administration of a compound and adjusting the dosage
accordingly. For additional guidance, see Remington: The Science
and Practice of Pharmacy, 21.sup.st Edition, Univ. of Sciences in
Philadelphia (USIP), Lippincott Williams & Wilkins,
Philadelphia, Pa., 2005, which is hereby incorporated by reference
as if fully set forth herein. Agents, compounds treatments or
therapies that may be used in an effective amount or
therapeutically effective amount to produce a desired effect in
accordance with the embodiments described herein may include, but
are not limited to, a CAR gene, an expression cassette that
includes a CAR gene, a vector that delivers an expression cassette
that includes a CAR gene to a target cell such as a T cell, and a
population of T cells that are transduced with a CAR gene.
[0074] The terms "treating" or "treatment" of a condition may refer
to preventing the condition, slowing the onset or rate of
development of the condition, reducing the risk of developing the
condition, preventing or delaying the development of symptoms
associated with the condition, reducing or ending symptoms
associated with the condition, generating a complete or partial
regression of the condition, or some combination thereof. Treatment
may also mean a prophylactic or preventative treatment of a
condition.
[0075] The term "subject" as used herein refers to a human or
animal, including all mammals such as primates (particularly higher
primates), sheep, dog, rodents (e.g., mouse or rat), guinea pig,
goat, pig, cat, rabbit, and cow. In some embodiments, the subject
is a human.
[0076] In certain embodiments, the methods for treating cancer may
include a step of administering a therapeutically effective amount
of a first population of T cells transduced with a first CAR gene
in combination with a therapeutically effective amount of a second
population of T cells transduced with a second CAR gene.
[0077] In other embodiments, CAR-transduced T cells may be
administered in combination with one or more additional anti-cancer
therapies. "In combination" or "in combination with," as used
herein, means in the course of treating the same cancer in the same
subject using two or more agents, drugs, therapeutics, procedures,
treatment regimens, treatment modalities or a combination thereof,
in any order. This includes simultaneous administration, as well as
in a temporally spaced order of up to several days apart. Such
combination treatment may also include more than a single
administration of any one or more of the agents, drugs,
therapeutics, procedures, treatment regimens, and treatment
modalities. Further, the administration of the two or more agents,
drugs, therapeutics, procedures, treatment regimens, treatment
modalities or a combination thereof may be by the same or different
routes of administration.
[0078] Additional anti-cancer therapies that may be used in
accordance with the methods described herein may include one or
more anti-cancer procedures, treatment modalities, anti-cancer
therapeutics or a combination thereof. In some embodiments, the
CAR-transduced T cells may be administered in combination with one
or more anti-cancer procedures or treatment modalities including,
but not limited to, stem cell transplantation (e.g., bone marrow
transplant or peripheral blood stem cell transplant using allogenic
stem cells, autologous stem cells; or a non-myeloablative
transplant), radiation therapy, or surgical resection. In other
embodiments, the CAR-transduced T cells may be administered in
combination with one or more anti-cancer therapeutics or drugs that
may be used to treat cancer including, but not limited to,
chemotherapeutics and other anti-cancer drugs, immunotherapeutics,
targeted therapeutics, or a combination thereof.
[0079] Chemotherapeutics and other anti-cancer drugs that may be
administered in combination with the CAR-transduced T cells in
accordance with the embodiments described herein include, but are
not limited to, all-trans-retinoic acid (ATRA), arsenic trioxide,
anthracycline antibiotics and pharmaceutically acceptable salts
thereof (e.g., doxorubicin hydrochloride, daunorubicin
hydrochloride, idarubicin, mitoxantrone), alkylating agents (e.g.,
cyclophosphamide, laromustine), antimetabolite analogs (cytarabine,
6-thioguanine, 6-mercaptopurine, methotrexate), demethylating
agents (e.g., decitabine, 5-azacytidine), nucleic acid synthesis
inhibitors (e.g., hydroxyurea), topoisomerase inhibitors (e.g.,
etoposide), vinca alkaloids (e.g., vincristine sulfate), or a
combination thereof (e.g., "ADE," which is a combination treatment
that includes a combination of Cytarabine (Ara-C), Daunorubicin
Hydrochloride and Etoposide).
[0080] Immunotherapeutics that may be administered in combination
with the CAR-transduced T cells in accordance with the embodiments
described herein include, but are not limited to, immune modulatory
reagents (e.g., STAT3 inhibitors, Lenalidomide) and therapeutic
monoclonal antibodies. The therapeutic monoclonal antibodies may be
designed to target one or more additional cancer-related
antigens
[0081] Targeted therapeutics that may be administered in
combination with the CAR-transduced T cells in accordance with the
embodiments described herein include, but are not limited to,
tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib,
sunitinib), farnesyl transferase inhibitors (e.g., tipifarnib), FLT
inhibitors, and c-Kit (or CD117) inhibitors (imatinib, dasatinib,
nilotinib).
[0082] The following examples are intended to illustrate various
embodiments of the invention. As such, the specific embodiments
discussed are not to be construed as limitations on the scope of
the invention. For example, although the example below relates to
an embodiment for a CAR that targets CD19, it is appreciated that a
CAR may be generated to target any antigen. It will be apparent to
one skilled in the art that various equivalents, changes, and
modifications may be made without departing from the scope of
invention, and it is understood that such equivalent embodiments
are to be included herein. Further, all references cited in the
disclosure are hereby incorporated by reference in their entirety,
as if fully set forth herein.
EXAMPLES
Example 1
Chimeric Antigen Receptors (CARs) Incorporating Mutations in the
IgG4 Fc Spacer Region Avoid Fc Receptor Mediated Recognition and
Clearance of CAR T Cells, Resulting in Improved T Cell Persistence
and Anti-Tumor Efficacy
[0083] To determine whether cellular FcR-mediated interactions play
a role in immunological rejection and clearance, or even
unintentional activation of adoptively transferred CAR-expressing T
cells, a CD19-specific CAR that has been mutated at one or two
sites within the CH2 region (L235E and/or N297Q) of its IgG4 Fc
spacer--referred to herein as CD19R(L235E), CD19R(N297Q) or
CD19R(EQ)--as well as a CD19-specific CAR that has a CH2 deletion
in its IgG4 Fc spacer--referred to herein as CD19Rch2.DELTA.. T
cells expressing these mutated CAR were then compared to T cells
expressing a non-mutated CAR (CD19R) or only a truncated EGFR
molecule (EGFRt) as a tracking marker (Wang et al. 2011), for in
vitro Fc.gamma.R binding and CAR-mediated cytolytic activity, as
well as in vivo engraftment and therapeutic efficacy. The results
provide evidence that elimination of cellular Fc.gamma.R
interactions improves the persistence and anti-tumor responses of
adoptively transferred CAR-expressing T cells.
[0084] Materials and Methods
[0085] DNA Constructs and Lentiviral Vectors. The
CD19R28Z-T2A-EGFRt_epHIV7 lentiviral construct contains a) the
chimeric antigen receptor (CAR) sequence consisting of the V.sub.H
and V.sub.L gene segments of the CD19-specific FMC63 mAb, an IgG4
hinge-C.sub.H2--C.sub.H3, the transmembrane and cytoplasmic
signaling domains of the costimulatory molecule CD28 that contains
gg mutations that enhance chimeric receptor expression and function
(Nguyen et al. 2003), and the cytoplasmic domain of the CD3.lamda.
chain (Kowolik et al. 2006); b) ribosomal skip T2A sequence
(Szymczak et al. 2004) and c) the truncated EGFR sequence (Wang et
al. 2011a). The EGFRt-T2A-DHFR.sup.FS-T2A-IMPDH2.sup.IY_epHIV7
lentiviral vector was generated as previously described
(Jonnalagadda et al. 2013). The CD19R(L235E)28Z-T2A-EGFRt_epHIV7,
CD19R(N297Q)28Z-T2A-EGFRt_epHIV7 and CD19R(EQ)28Z-T2A-EGFRt_epHIV7
vectors were generated by site directed mutagenesis using the
QuikChange II XL kit (Agilent Technologies, Santa Clara, Calif.) of
a codon optimized CD19R28Z_pGA plasmid that had been synthesized by
Geneart, digested with NheI/RsrII and ligated with a similarly
digested CD19R28Z-T2A-EGFRt_epHIV7. The
CD19Rch2.DELTA.28Z-T2A-EGFRt_epHIV7 vector was generated from a
codon optimized CD19R-HL-CH3(CO)_pMK-RQ plasmid that had been
synthesized by Geneart, digested with NheI/RsrII and ligated with a
similarly digested CD19R28Z-T2A-EGFRt_epHIV7.
[0086] Cell Lines and Maintenance.
[0087] Human peripheral blood mononuclear cells (PBMCs) were
isolated as described (Wang, 2011b) from heparinized peripheral
blood obtained from discard kits containing residual blood
components of healthy donors that had undergone apheresis at the
City of Hope National Medical Center (COHNMC). Because this was
de-identified discard blood material, informed consent was waived
with the approval of the COHNMC Internal Review Board (IRB protocol
#09025), and the COHNMC Office of Human Subjects Protection.
T.sub.CM isolation (using CD14- and CD45RA-depletion followed by
CD62L-selection), anti-CD3/CD28 bead stimulation and
lentiviral-mediated transduction was then done as previously
described (Wang et al. 2012). In some cases, transduced T cells
were immunomagnetically enriched for EGFRt expression as previously
described (Wang et al. 2011a).
[0088] EBV-transformed lymphoblastoid cell lines (LCL) and LCL that
expressed OKT3 (LCL-OKT3) (Wang et al. 2011 b) or ffLuc.sup.+ LCL
cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana,
Calif.) supplemented with 10% heat-inactivated fetal calf serum
(FCS, Hyclone, Logan, Utah) 2 mM L-glutamine (Irvine Scientific),
and 25 mM HEPES (Irvine Scientific). ffLuc+ LCL were generated by
transduction with lentiviral vector eGFP-ffluc_epHIV7 at an MOI of
20 in the presence of 5 .mu.g/mL polybrene in 500 uL medium, and
subsequent purification by sorting GFP+ cells.
[0089] Mouse myeloma cells secreting human homeostatic IL-15
cytokine (NSO-IL15) were generated as previously described (Wang et
al. 2011b).
[0090] SupB15 and K562 leukemia cell lines (ATCC) were grown in the
corresponding ATCC recommended media.
[0091] Antibodies and Flow Cytometry.
[0092] Fluorochrome-conjugated isotype controls, anti-CD3,
anti-CD4, anti-CD8, anti-CD45 and streptavidin were obtained from
BD Biosciences (San Jose, Calif.). Biotinylated anti-Fc was
purchased from Jackson ImmunoResearch Laboratories, Inc. (West
Grove, Pa.). Generation of biotinylated-cetuximab was previously
described (Wang et al. 2011a). Biotinylated huFc.gamma.R1,
muFc.gamma.R1, huFc.gamma.R2a, huFc.gamma.R2b, and muFc.gamma.R2b
were obtained from Sino Biological, Inc. (Beijing, P.R. China). The
percentage of immunofluorescent cells were analyzed by a
FACScalibur system (BD Biosciences), and the percentage of cells in
a region of analysis were calculated using FCS Express V3 (De Novo
Software, CA, USA).
[0093] In Vivo T Cell Engraftment and Therapy.
[0094] All mouse experiments were approved by the COHNMC Institute
Animal Care and Use Committee. For engraftment studies, 6-10 week
old NOD/Scid IL-2R.gamma.C.sup.null (NSG) mice were injected
intravenously (i.v.) on day 0 with 10.sup.7 of the indicated
T.sub.CM-derived cells, and intraperitoneal (i.p.) injections three
times a week of 2.times.10.sup.7 irradiated NS0-IL15 to provide a
systemic supply of human IL-15 in vivo. Peripheral blood was
harvested from retro-orbital bleeds, red blood cells were lysed and
cell suspensions were analyzed by flow cytometry. For the
therapeutic study, 1.5.times.10.sup.6 ffLuc.sup.+ LCL cells were
administered i.v. into 6-8 week old NSG mice on day 0, and then
5.times.10.sup.6 of the indicated CAR+ T.sub.CM-derived cells were
administered i.v. on day 3. Luciferase activity was measured by
Xenogen imaging as previously described (Kahlon et al. 2004).
[0095] Chromium-Release Assays.
[0096] 4-hour .sup.51Cr-release assays were performed as previously
described (Stastny et al. 2007) using the indicated effector/target
cell ratios.
[0097] Results
[0098] CD19R+ T Cells Fail to Engraft in NSG Mice.
[0099] Central memory T cells (T.sub.CM) as a T cell subpopulation,
have been characterized as having superior engraftment potential,
and thus therapeutic efficacy, after adoptive transfer (Wang et al.
2011 b). Further evidence has shown that CAR expression on the
T.sub.CM-derived cells seem to correlate with decreased in vivo
persistence in an in vivo xenograft model using NSG mice. As the
studies described herein indicate, this decrease in persistence was
shown in an experiment comparing the engraftment of non-transduced
T.sub.CM-derived cells to (i) T.sub.CM-derived cells that were
lentivirally transduced to express both a CD19-specific CAR (CD19R)
and a truncated EGFR (EGFRt) as a tracking marker, and (ii)
T.sub.CM-derived cells that were lentivirally transduced to express
just the EGFRt tracking marker on the cell surface (FIG. 1).
Looking at peripheral blood collected 7 and 14 days after the cells
were administered i.v. into mice, staining with anti-human CD45 mAb
allowed for detection of non-transduced T.sub.CM-derived cells
(FIG. 1c). However, upon co-staining for the EGFRt tracking marker
to detect gene-modified cells, it was apparent that, despite the
similar level of transduction and/or EGFRt-expression of the input
cells (FIG. 1b, 78-79% positive), there was significantly less
engraftment of cells in the peripheral blood of mice that received
CD19R/EGFRt+ TCM compared to those that received EGFRt+ TCM (FIG.
1c, p<0.0001 comparing percentages of CD45/EGFRt+ cells in each
group at either day 7 or day 14 using unpaired Student's t-tests).
Although low levels of T cells were detected for the CD19R/EGFRt+
TCM-treated mice, all of the persistent T cells at day 7 and 14
were CAR-negative. This impaired in vivo persistence is not
associated with lentiviral transduction of the T cells, as it is
specific to cells transduced to express the CAR transgene and not
the EGFRt transgene. Furthermore, the lack of CD19 antigen in these
NSG mice, and the fact that a similar phenomenon with T cells
expressing CARs of different antigen specificity has been seen
(data not shown), suggests that the lack of engraftment/persistence
in the peripheral blood is antigen independent.
[0100] HuFc.gamma.R Binds CD19R+ T Cells.
[0101] The CD19R construct includes a CD19-specific scFv derived
from mouse monoclonal antibody FMC63, a human IgG4 Fc linker, human
CD28 transmembrane and cytoplasmic domains, and a human CD3-zeta
cytoplasmic domain. Because the CAR construct includes a portion of
a human IgG4 Fc region, the propensity of FcR-mediated innate
immune responses to selectively clear the CD19R/EGFRt+ cells--but
not the EGFRt+ cells--was investigated. Indeed, a binding assay
using soluble human Fc.gamma.R1 revealed that, in contrast to
T.sub.CM-derived cells that were non-transduced or expressed only
the EGFRt, those that expressed CD19R exhibited binding of the
Fc.gamma.R1 molecules that could be titrated down with higher
dilutions (FIG. 2). Of note, NSG mice, while immunodeficient, are
known to still have FcR-expressing neutrophils and monocytes
(Ishikawa et al. 2005; Ito et al. 2002), thus providing a potential
rationale for the lack of CAR+ T cell persistence observed in prior
engraftment studies.
[0102] Generation of CD19R Mutants.
[0103] To further test the significance of potential FcR-mediated
effects on the CAR-expressing T.sub.CM population, the
CD19-specific CAR was mutated at amino acids within the IgG4 CH2
domain that may be involved with FcR binding--L235E and/or N297Q
(FIG. 3a). A CD19-specific CAR with a deletion of the IgG4 CH2
domain (i.e., a deletion of the domain that contains residues 235
and 297) was also generated (FIG. 3a). The resulting single
mutants, CD19R(L235E) and CD19R(N297Q), double mutant CD19R(EQ)
(having both L235E and N297Q mutations), and deletion
CD19Rch2.DELTA. sequences were incorporated into separate
lentiviral constructs, where they were each coordinately expressed
with EGFRt from a single transcript, using the T2A ribosome skip
sequence in a design similar to that described in FIG. 1a for the
non-mutated CD19R. After lentiviral transduction, immunomagnetic
enrichment of EGFRt-expressing cells, and a single round of rapid
expansion, each of the T.sub.CM-derived lines were 92-99% positive
for the expected transgenes (FIG. 3b), demonstrating that the
mutations do not adversely affect CAR expression. Furthermore, none
of these mutations altered the CD19 specific cytolytic potential of
these T.sub.CM-derived cells in 4 hour .sup.51Cr-release assays
(FIG. 3c).
[0104] huFc.gamma.R Binding to CARs with Mutated IgG4 Spacer is
Impaired.
[0105] To determine the efficacy of the different
mutations/deletion in the CAR to affect FcR binding, flow
cytometric analysis was performed using various human and murine
biotinylated soluble Fc.gamma.Rs, and PE-streptavidin (SA-PE) to
detect the binding of the Fc.gamma.Rs to the different cell
populations. T cells that expressed the non-mutated CD19R were
bound by human Fc.gamma.R1, Fc.gamma.R2a and Fc.gamma.R2b, as well
as murine Fc.gamma.R1 and Fc.gamma.R2b (FIG. 4). In contrast, T
cells that expressed only EGFRt were not bound by these
Fc.gamma.Rs, and T cells that expressed either the CD19R(N297Q),
CD19R(L235E) or CD19R(EQ) mutants, or the CD19Rch2.DELTA. deletion
all displayed significantly reduced binding to these
Fc.gamma.Rs.
[0106] T Cells with CD19R Mutants Exhibit Improved In Vivo
Engraftment and Persistence.
[0107] To determine whether the CD19R mutations or deletion which
helped prevent Fc.gamma.R binding would translate to an increased
in vivo persistence upon adoptive transfer, 10.sup.7 T cells
expressing either the parental CD19R, the EGFRt marker alone, the
CD19R(L235E), the CD19R(N297Q), the CD19R(EQ), or the
CD19Rch2.DELTA. were infused i.v. into NSG mice. One and two weeks
later, peripheral blood was assayed for CD45.sup.+ EGFRt.sup.+ cell
engraftment (FIG. 5). Engrafted EGFRt+ cells could be detected when
the T cells expressed the single mutated CD19R(L235E) or
CD19R(N297Q). Further, expression of the double point-mutated
CD19R(EQ) or CH2-deleted CD19Rch2.DELTA. rescued T cell
engraftment, as levels of CD45/EGFRt+ cells observed in these
groups of mice were similar to that seen when EGFRt alone was
expressed. This rescued engraftment and persistence of
gene-modified cells was also observed using TCM-derived cells that
were not EGFRt-enriched prior to adoptive transfer (FIG. 8).
[0108] T Cells with CD19R Mutants Exhibit Improved Therapeutic
Efficacy.
[0109] Based on the engraftment findings, the effects of the
CD19R(EQ) or CD19Rch2.DELTA. on the anti-tumor efficacy of the
T.sub.CM-derived cells were compared. LCL is a CD19-expressing
tumor cell line that was transduced to express firefly luciferase
(ffLuc) to allow for bioluminescent monitoring of in vivo tumor
growth. Three days after the ffLuc+ LCL were administered to NSG
mice i.v., the mice were treated i.v. with either PBS as a control
or 5.times.10.sup.6 T cells expressing either the non-mutated
CD19R, the EGFRt marker alone, the double point-mutated CD19R(EQ),
or the CH2-deleted CD19Rch2.DELTA.. Expression of either the
CD19R(EQ) or the CD19Rch2.DELTA. on the T.sub.CM-derived cells
resulted in significant control of tumor growth (FIG. 6). This
efficacy correlated with the presence/persistence of the
gene-modified cells in the peripheral blood at day 21 (FIG. 6d).
Indeed, while the PBS, CD19R and EGFRt control groups all had to be
euthanized at day 21, all of the mice in the CD19R(EQ) and
CD19Rch2.DELTA. groups survived past 100 days (FIG. 6e). While
these engraftment and efficacy studies focused on the TCM subset of
T cells, these findings suggest that the positive benefit of
IgG4-mutations for eliminating FcR interaction are independent of
the T cell population that is engineered. Indeed, expression of the
CD19R(EQ) in bulk PBMC-derived T cells, instead of TCM-derived
lines, also resulted in improved anti-tumor efficacy and long-term
survival (p=0.0295) (FIG. 7).
DISCUSSION
[0110] Clinically, the in vivo therapeutic efficacy of adoptive T
cell strategies directly correlates with engraftment and
persistence upon adoptive transfer (Heslop et al. 2003; Brenner
& Heslop 2010). Various approaches have been suggested to
improve transferred T cell persistence, including lymphodepletion
of the host prior to cell transfer (Gattinoni et al. 2005),
cytokine support after cell transfer (most recently reviewed in
(Overwijk & Schluns 2009), and use of the optimal T cell
population(s) for transfer (Berger et al. 2008; Hinrichs et al.
2011; Yang et al. 2013; Gattinoni et al. 2011; Cieri et al. 2013).
The studies described above provide further evidence that chimeric
antigen receptor (CAR) design plays a significant role in directing
the engraftment and persistence of therapeutic cells. Previously,
CAR design has been exploited to benefit engraftment and
persistence of therapeutic cells is by including costimulatory
signaling domains in second and third generation CARs (see
Cartellieri et al. 2010). However, as the data above also suggests,
sequences that are used to connect the ligand-binding domain to the
signaling domain(s) of the CAR (known as either the spacer, hinge
and/or linker) are of previously unappreciated importance for in
vivo therapeutic outcome in murine models of malignant disease.
Specifically, it was found that the use of an Ig Fc spacer can
potentially inhibit the engraftment and/or persistence of
CAR-expressing cells in NSG mouse models in a manner that
correlates with Fc.gamma.R binding. Prevention of Fc.gamma.R
binding by either point mutation or deletion of the relevant
sequences within the CAR Fc domain can then restore the in vivo
persistence of the adoptively transferred cells to that of cells
which do not express a CAR. The increased in vivo persistence that
is mediated by the spacer-optimized CAR then translates, into
significantly improved CAR-directed anti-tumor therapy in an in
vivo mouse model.
[0111] The immunological clearance of adoptively transferred T
cells is not a new issue. For example, cellular immune rejection
responses against the HyTK and NeoR selection genes have been shown
to be coordinately expressed with the CAR (Berger et al. 2006;
Jensen et al. 2010). However, the studies described above
highlights the importance of FcR-mediated responses against
CAR-expressing T cells for in vivo T cell persistence and
anti-tumor efficacy. Consequently, the studies also show that there
is a `fix` to avoid this form of immunogenicity--namely, the
incorporation of mutations in the CAR design to prevent
Fc.gamma.R-recognition.
[0112] Based on these results, the mutations described herein may
be extrapolated to humans and should therefore augment the
persistence and therapeutic efficacy of T cells expressing
IgG-spacer containing CAR in humans. Any discrepancy in CAR T cell
engraftment and in vivo anti-tumor efficacy is likely impacted by
the nature of the murine NSG model system. Human IgG4 has been
shown to efficiently bind murine FcRs to mediate potent antibody
dependent cell-mediated cytotoxicity (Isaacs et al. Steplewski et
al. 1988). In contrast, human FcRs have the strongest affinity
toward IgG1 and IgG3, and reduced affinity for IgG4 (Schroeder
& Cavacini 2010; Nirula et al. 2011). Additionally, given that
NSG mice lack serum antibodies, FcRs expressed by their innate
immune cells are unoccupied and thus have a greater potential to
bind the IgG-Fc spacer within the CAR. With the exception of
hypoglobulinemia cases, immunocompetent humans have high serum IgG
levels of approximately 10 mg/mL (Stoop et al., 1969), which could
potentially compete for recognition of IgG-containing CARs. Indeed,
several groups have administered IgG-Fc bearing CAR T cells to
humans, and in some cases low levels of CAR T cells were detectable
by quantitative PCR up to 6 weeks (Savoldo et al. 2011) and even
one year (Till et al. 2012) after administration. Incorporation of
the mutations described herein would likely further improve this
CAR T cell persistence in humans.
[0113] Overall, the studies reported here provide evidence that
CARs containing components of an Ig Fc spacer should incorporate
modifications that prevent the FcR-mediated recognition of the
cells in vivo. Such modifications can involve either point
mutations to change the amino acid sequence, or sequence deletions
such as that seen with the CD19R(EQ) and CD19Rch2.DELTA. constructs
described herein. Not only will such modifications prevent the
ability of FcR-expressing cells to recognize the CAR-expressing
immunotherapeutic cellular product in vivo, but they might also
prevent the unintentional activation of the transferred T cells
and/or the host immune responses (Hombach et al. 2010), which could
contribute to various unwanted side-effects of this
immunotherapeutic strategy.
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