NZ745372B2 - Method and compositions for cellular immunotherapy - Google Patents
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- NZ745372B2 NZ745372B2 NZ745372A NZ74537213A NZ745372B2 NZ 745372 B2 NZ745372 B2 NZ 745372B2 NZ 745372 A NZ745372 A NZ 745372A NZ 74537213 A NZ74537213 A NZ 74537213A NZ 745372 B2 NZ745372 B2 NZ 745372B2
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Abstract
The present invention provides chimeric antigen receptors comprising: (a) an extracellular domain consisting of: an extracellular ligand binding domain consisting of a single chain variable fragment (scFv) that binds to a CD19, wherein the scFv comprises a variable light chain (VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence encoded by SEQ ID NO:3; and an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); (b) a transmembrane domain; and (c) an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided is a chimeric receptor, the chimeric receptor comprising: (a) a single chain variable fragment (scFv) that binds to CD19, wherein the scFv comprises: a variable light chain (VL) domain comprising a VL domain present in the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a VH domain present in the amino acid sequence encoded by SEQ ID NO:3; or the amino acid sequence encoded by SEQ ID NO:3; (b) a transmembrane domain; (c) a polypeptide spacer located between the scFv and the transmembrane domain, wherein the polypeptide spacer is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided are cells comprising the chimeric receptor, and related compositions and uses. ng a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence encoded by SEQ ID NO:3; and an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); (b) a transmembrane domain; and (c) an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided is a chimeric receptor, the chimeric receptor comprising: (a) a single chain variable fragment (scFv) that binds to CD19, wherein the scFv comprises: a variable light chain (VL) domain comprising a VL domain present in the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a VH domain present in the amino acid sequence encoded by SEQ ID NO:3; or the amino acid sequence encoded by SEQ ID NO:3; (b) a transmembrane domain; (c) a polypeptide spacer located between the scFv and the transmembrane domain, wherein the polypeptide spacer is about 15 amino acids or less in length and comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) a polynucleotide encoding an intracellular signaling domain that comprises a CD3ζ signaling domain and a costimulatory domain. Also provided are cells comprising the chimeric receptor, and related compositions and uses.
Description
METHOD AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY
This application is a divisional of New Zealand patent ation 738636,
which is a divisional of New Zealand application 705475, which is the national phase
entry in New Zealand of PCT international application
(published as WO 2014//031687), and claims the benefit of priority to U.S. patent
application serial no. 61/691,117 filed on 20 August 2012, the disclosure of which is
incorporated herein by nce in its entirety.
Field of the Invention
The present ion relates to the field of biomedicine and specifically
methods useful for cancer therapy. In particular, embodiments of the invention relate
to methods and compositions for carrying out cellular immunotherapy comprising T
cells modified with tumor ing receptors.
Statement ing Federally red Research
This invention was made with government support in the form of grants from
the United States Department of Health and Human Services and from the ia
and Lymphoma Society. The United States government has certain rights in the
invention.
Background of the Invention
The adoptive transfer of human T lymphocytes that are engineered by gene
transfer to express chimeric antigen receptors (chimeric receptors) ic for
surface molecules expressed on tumor cells has the potential to effectively treat
advanced malignancies. Chimeric receptors are synthetic ors that include an
extracellular ligand binding domain, most commonly a single chain variable
fragment of a monoclonal antibody (scFv) linked to intracellular ing
components, most commonly CD3? alone or combined with one or more
costimulatory domains. Much of the research in the design of chimeric receptors has
focused on defining scFvs and other ligand binding elements that target malignant
cells without causing s toxicity to essential normal tissues, and on defining the
optimal composition of intracellular ing modules to activate T cell effector
functions. r, it is uncertain whether the variations in chimeric or
design that mediate superior in vitro function will translate reproducibly into
improved in vivo therapeutic ty in clinical applications of chimeric receptormodified
T cells.
There is a need to identify methods for determining elements of chimeric
receptor design that are important for therapeutic activity and cell populations to
genetically modify and adoptively transfer that e enhanced survival and
efficacy in vivo. It is an object of the present invention to go some way towards
meeting this need and/or to provide the public with a useful choice.
Summary of the Invention
In a first aspect the present invention provides a chimeric receptor, the chimeric
or comprising:
(a) an extracellular domain consisting of:
an extracellular ligand binding domain consisting of a single chain variable
nt (scFv) that binds to a CD19, wherein the scFv comprises a variable light chain
(VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence
encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1,
a CDRH2, and a CDRH3 of the amino acid ce encoded by SEQ ID NO:3; and
an extracellular polypeptide spacer that is about 15 amino acids or less in length and
comprises an amino acid sequence of X1PPX2P, wherein X1 is a cysteine, glycine, or
arginine and X2 is a cysteine or a threonine (SEQ ID NO:1);
(b) a transmembrane domain; and
(c) an intracellular ing domain that comprises a CD3? signaling domain
and a ulatory domain.
In a second aspect the present invention provides a chimeric receptor, the ic
receptor comprising:
(a) a single chain variable fragment (scFv) that binds to CD19, wherein the
scFv comprises:
the amino acid sequence encoded by SEQ ID NO:3;
(b) a transmembrane domain;
(c) a polypeptide spacer located between the scFv and the transmembrane
domain, wherein the polypeptide spacer is about 15 amino acids or less in length and
comprises an amino acid ce of X1PPX2P, wherein X1 is a cysteine, glycine, or
arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and
(d) an intracellular signaling domain that comprises a CD3? signaling domain
and a costimulatory domain.
In a third aspect the present invention provides an isolated host cell, comprising the
chimeric receptor of the first or second aspects.
In a fourth aspect the present invention provides a composition, comprising the host
cell of the third , in a pharmaceutically acceptable excipient.
In a fifth aspect the present invention provides a use of a pharmaceutical
composition, sing the chimeric receptor of the first or second aspects, the host cell of
the third aspect, or the composition of the fourth aspect in the manufacture of a medicament
for the treatment of a tumor sing CD19.
In a sixth aspect the invention provides a use of a population of immune cells
comprising the chimeric or of the first or second aspects in the manufacture of a
medicament for the treatment of a tumor expressing CD19.
In a h aspect the present invention provides a use of a population of CD8+ T
cells comprising the ic or of the first or second aspects in the manufacture of a
medicament for the treatment of a tumor expressing CD19.
In an eighth aspect the present invention provides a use of a population of CD4+ T
cells comprising the ic receptor of the first or second aspect in the manufacture of a
medicament for the treatment of a tumor expressing CD19.
In an ninth aspect the present invention provides a use of a combination comprising
a population of CD8+ T cells sing the chimeric receptor of the first or second aspects
and a population of CD4+ T cells comprising the chimeric receptor of the first or second
aspects in the manufacture of a medicament for the treatment of a tumor expressing CD19.
Also described are methods and compositions to confer and/or augment
immune responses mediated by cellular immunotherapy, such as by adoptively
transferring specific, genetically ed subsets of CD8+ or CD4+ T cells
alone, or in combination. The disclosure describes chimeric receptor c acids,
and vectors and host cells including such nucleic acids. The nucleic acid sequence
that encodes the chimeric receptor links er a number of modular components
that can be excised and replaced with other components in order to customize the
chimeric receptor for efficient T cell activation and recognition of a specific target
le or an epitope on the target le.
In embodiments described herein, a chimeric receptor nucleic acid
comprises a polynucleotide coding for a ligand binding domain, wherein the ligand
is a molecule expressed on malignant or infected cells, a polynucleotide coding for a
polypeptide spacer n the polypeptide spacer is about 200 amino acids or less,
a polynucleotide coding for a transmembrane domain; and a polynucleotide coding
for ellular signaling domains. In embodiments, the polypeptide spacer
comprises a modified IgG4 hinge region containing an amino acid sequence
X1PPX2P that may be linked to other amino acid sequences including but not limited
to the CH2 and CH3 or CH3 only sequences of the Ig Fc. It has been surprisingly
found that the length of the spacer region that is presumed not to have signaling
lity affects the in vivo efficacy of the T cells modified to express the chimeric
receptor and needs to be ized for individual target molecules for optimal
tumor or target cell recognition.
r aspect of the disclosure describes an isolated chimeric receptor
nucleic acid comprising: a polynucleotide coding for a ligand binding domain,
wherein the ligand is a tumor specific n, viral antigen, or any other molecule
expressed on a target cell population that is suitable to mediate recognition and
elimination by a lymphocyte; a polynucleotide coding for a polypeptide spacer
wherein the polypeptide spacer is of a customized length that is specific for each
targeted ligand, wherein the spacer provides for enhanced T cell proliferation and/
or cytokine production as compared to a nce chimeric receptor; a
polynucleotide coding for a transmembrane domain; and a polynucleotide coding for
one or more intracellular signaling domains. In embodiments, a long spacer is
employed if the epitope on the target ligand is in a membrane proximal position and
a short spacer is employed if the epitope on the target ligand is in a membrane distal
on. The disclosure includes expression vectors and host cells comprising the
isolated ic receptor as described herein.
Another aspect of the disclosure bes a chimeric receptor polypeptide
comprising a ligand binding domain, wherein the ligand is a tumor specific antigen,
viral n or any other molecule that is expressed on a target cell population and
can be targeted to mediate recognition and elimination by lymphocytes; a
polypeptide spacer wherein the polypeptide spacer is about 10-229 amino acids; a
transmembrane domain; and one or more intracellular signaling domains. In
embodiments, the polypeptide spacer comprises a modified IgG hinge region
containing the amino acid sequence X1PPX2P.
In another aspect, the present disclosure describes compositions to confer
and/or augment immune responses mediated by cellular immunotherapy, such as by
adoptively transferring tumor-specific, subset specific genetically ed CD4+ T
cells, wherein the CD4+ T cells confer and/or t the ability of CD8+ T cells to
sustain anti-tumor reactivity and increase and/or ze tumor-specific
proliferation. In embodiments, the CD4+ cells are genetically modified to express a
chimeric or nucleic acid and/or chimeric receptor ptide as described
herein.
In another aspect, described are compositions to confer and/or augment
immune responses mediated by cellular immunotherapy, such as by adoptively
transferring tumor-specific, subset specific cally modified CD8+ T cells. In
ments, the CD8+ cells s a chimeric receptor nucleic acid and/or
chimeric receptor polypeptide as described herein.
Also bed is an adoptive cellular therapy composition having a
genetically modified CD8+ cytotoxic T lymphocyte cell preparation to confer and/or
augment immune responses, wherein the cytotoxic T lymphocyte cell preparation
comprises CD8+ T cells that express a ic receptor comprising a ligand
binding domain for a ligand associated with the disease or disorder, a customized
spacer region, a transmembrane ; and an intracellular signaling domain of a T
cell or other receptors, such as a costimulatory domain, and/or a genetically
modified helper T cyte cell preparation, n the helper T lymphocyte
cell preparation has CD4+ T cells that express a chimeric receptor comprising an
antibody variable domain specific for the ligand associated with the disease or
disorder, a customized spacer region, a transmembrane domain; and one or more
intracellular signaling domains.
Also described is a method of performing ar immunotherapy in a
subject having a disease or disorder by administering to the subject a genetically
modified cytotoxic T lymphocyte cell preparation that provides a cellular immune
response, wherein the cytotoxic T lymphocyte cell ation ses CD8+ T
cells that have a chimeric receptor comprising a polynucleotide coding for a ligand
binding domain, wherein the ligand is a tumor specific antigen, viral antigen, or any
other molecule expressed on a target cell population that is suitable to mediate
recognition and elimination by a lymphocyte; a polynucleotide coding for a
polypeptide spacer wherein the polypeptide spacer is of a customized length that is
specific for each targeted ligand, n the spacer provides for enhanced T cell
proliferation and/or cytokine production as compared to a reference chimeric
receptor; a polynucleotide coding for a transmembrane domain; and a
polynucleotide coding for one or more intracellular signaling domains. In
embodiment, the ligand binding domain is an extracellular antibody variable domain
ic for a ligand associated with the disease or disorder. An embodiment
es a genetically modified helper T lymphocyte cell ation that wherein
the helper T lymphocyte cell preparation comprises CD4+ T cells that have a
chimeric receptor comprising an a polynucleotide coding for a ligand binding
domain, wherein the ligand is a tumor specific antigen, viral antigen, or any other
molecule expressed on a target cell population that is suitable to mediate recognition
and elimination by a lymphocyte; a polynucleotide coding for a polypeptide spacer
wherein the polypeptide spacer is of a customized length that is ic for each
targeted ligand, wherein the spacer provides for ed T cell proliferation and/or
cytokine production as compared to a reference chimeric receptor; a polynucleotide
coding for a embrane domain; and a polynucleotide coding for one or more
intracellular signaling domains. In embodiments, the genetically ed CD8+
and genetically modified CD4+ cell population are coadministered. In embodiments,
the T cells are autologous or allogeneic T cells.
Various modifications of the above method are possible. For e, the
chimeric receptor that is expressed by the CD4+ T cell and the CD8+ T cell can be
the same or different.
Also described is a method of cturing an adoptive immunotherapy
composition by obtaining a chimeric receptor modified tumor-specific CD8+
cytotoxic T cyte cell preparation that elicits a cellular immune response and
expresses an antigen-reactive chimeric receptor, wherein the ed cytotoxic T
lymphocyte cell preparation comprises CD8+ T cells that have a chimeric or
comprising a ligand binding domain, wherein the ligand is a tumor specific antigen,
viral antigen, or any other molecule expressed on a target cell population that is
suitable to mediate ition and elimination by a lymphocyte; a polypeptide
spacer wherein the polypeptide spacer is of a ized length that is specific for
each targeted ligand, wherein the spacer provides for enhanced T cell proliferation
and/or cytokine production as compared to a reference chimeric receptor; a
transmembrane domain; and one or more intracellular signaling domains.; and/or
obtaining a ed naïve or memory CD4+ T helper cell wherein the modified
helper T lymphocyte cell preparation comprises CD4+ cells that have a chimeric
or comprising a ligand binding domain, n the ligand is a tumor specific
antigen, viral antigen, or any other molecule expressed on a target cell population
that is suitable to mediate recognition and ation by a lymphocyte; a
polypeptide spacer wherein the polypeptide spacer is of a customized length that is
specific for each targeted , wherein the spacer provides for enhanced T cell
proliferation and/or cytokine production as compared to a reference chimeric
receptor; a transmembrane domain; and one or more intracellular signaling
domains.
These and other embodiments of the invention are described further in the
accompanying specification, drawings and claims.
Brief Description of the Drawings
Figure 1 Library of spacer sequences. We constructed a plasmid y
that contain codon optimized DNA sequences that encode ellular components
including of the IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 s, or
IgG4 hinge linked to the CH3 domain. Any scFV sequence (VH and VL) can be
cloned 5’ to the sequences encoding this library of variable spacer domains. The
spacer s are in turn linked to CD28 embrane and intracellular
signaling domains and to CD3 zeta. A T2A sequence in the vector separates the
chimeric receptor from a selectable marker encoding a truncated human epidermal
growth factor receptor (tEGFR).
Figure 2: In vitro cytotoxicity, cytokine production, and proliferation of
T-cells modified to s 2A2 ROR1 chimeric receptors with modified spacer
length. (A) Phenotype of purified CD8+ TCM-derived cell lines modified with each
of the 2A2 ROR1 chimeric receptors with long, intermediate and short spacer
domain. Staining with anti-F(ab) antibody that binds to an e in the 2A2 scFV
shows surface expression of ROR1 chimeric receptors with full length or truncated
spacer. (B) Cytolytic activity of T-cells sing the s 2A2 ROR1 chimeric
receptors with long (?), intermediate (?) and short spacer (?), or a tEGFR control
lentiviral vector against ROR1+ (x) and control target cells. The bar diagram
summarizes cytotoxicity data from 3 independent experiments (E:T = 30:1)
normalized to cytolytic activity by 2A2 ROR1 chimeric receptor ‘long’ = 1, and
analyzed by Student’s t-test. (C) CFSE dye dilution was used to measure
proliferation of 2A2 ROR1 ic receptor and tEGFR control T-cells, 72 hours
after stimulation with Raji/ROR1 (left panel) and primary CLL cells (right panel)
without addition of exogenous cytokines. For analysis, triplicate wells were pooled
and the proliferation of live (PI-), CD8+ T-cells analyzed. Numbers above each
histogram indicate the number of cell divisions the proliferating subset ent,
and the fraction of T-cells in each gate that underwent =4/3/2/1 cell divisions is
provided next to each plot. (D) Multiplex cytokine assay of supernatants obtained
after 24 hours from triplicate co-cultures of 5x104 T-cells expressing the various
2A2 ROR1 chimeric receptors with Raji/ROR1 and primary CLL cells. lex
cytokine data from 3 independent experiments were ized (cytokine release by
2A2 ROR1 chimeric receptor ‘long’ = 1) and analyzed by t’s t-test (right bar
diagram).
Figure 3. R11 ic receptor requires a long spacer for recognition of
ROR1+ tumor cells. The sequences encoding the scFV from the R11 monoclonal
antibody that is specific for an epitope in the membrane al Kringle domain of
the orphan tyrosine kinase receptor ROR1 were cloned upstream of IgG4 hinge only
(short), IgG4 hinge/CH3 (intermediate), and IgG4 hinge/CH2/CH3 sequences in our
chimeric receptor library containing the 4-1BB costimulatory domains and prepared
as lentiviral vectors. A). Human CD8+ T cells were transduced and the transduction
efficiency with each of the short, intermediate and long chimeric receptors was
determined by staining for the tEGFR marker. B). Transduced T cells expressing the
short (top), intermediate (middle), and long (bottom) were assayed for lysis of K562
leukemia cells alone or transfected to s ROR1. Only the T cells expressing
the long spacer chimeric receptor efficiently killed ROR1+ K562 cells. C).
Transduced T cells expressing the short (top), intermediate (middle), and long
(bottom) were labeled with CFSE, stimulated with K562 cells expressing ROR1 or
CD19 ol) and assayed for cell proliferation over 72 hours. The T cells
expressing the long spacer chimeric receptor proliferated specifically to the ROR1+
K562 cells. D). Transduced T cells expressing the short (top), intermediate (middle),
and long (bottom) were ated with Raji lymphoma cells and K562 cells that
expressed ROR1 or CD19 (control) and d for the secretion of interferon
gamma into the supernatant over 24 hours. The T cells expressing the long spacer
chimeric receptor proliferated and produced the highest levels of interferon gamma
in response to ROR1 positive target cells.
Figure 4: Design of ROR1 ic receptors with modified spacer
length and derived from the 2A2 and R12 scFV with different affinity. (A)
Design of lentiviral transgene inserts encoding a panel of ROR1 chimeric receptors
ning the 2A2 scFV, an c d spacer of -CH2-CH3’ (long
spacer, 229 AA), ‘Hinge-CH3’ (intermediate, 119 AA), or ‘Hinge’ only (short, 12
AA), and a signaling module with CD3? and CD28. Each chimeric or cassette
contains a truncated EGFR marker encoded downstream of a T2A element. (B)
Lentiviral transgene inserts encoding ROR1-specific chimeric receptors derived
from the R12 and 2A2 scFV with short IgG4-Fc ‘Hinge’ spacer (12 AA), and a
signaling module containing CD28 or 4-1BB and CD3? tively (total: 4
constructs).
Figure 5: Anti-tumor reactivity of T-cells modified with ROR1 chimeric
receptors derived from mAb R12 with higher affinity than 2A2. (A) tEGFR
expression on purified polyclonal CD8+ TCM-derived T-cell lines modified with each
of the R12 and 2A2 ROR1 chimeric receptors with short IgG4-Fc ‘Hinge’ spacer,
and CD28 or 4-1BB costimulatory domain. (B) Cytotoxicity against ROR1+ and
control target cells by T-cells sing R12(28-?; 4-1BB-?) and 2A2 ROR1
chimeric receptors (28-?; 4-1BB?) or a tEGFR control vector (x). (C) Multiplex
ne assay of atants obtained after 24 hours from co-cultures of 5x104 T-
cells sing the various ROR1 chimeric receptors with Raji/ROR1 tumor cells.
The middle/right bar diagrams show normalized multiplex data from 3 independent
experiments ine release by ROR1 chimeric or 2A2 = 1) analyzed by
Student’s t-test. (D) Proliferation of ROR1 chimeric receptor T-cells and tEGFR
control T-cells 72 hours after stimulation with Raji/ROR1 cells and without addition
of exogenous cytokines was ed by CFSE dye dilution. Numbers above each
histogram indicate the number of cell divisions the proliferating subset underwent,
and the fraction of T-cells in each gate that underwent =4/3/2/1 cell divisions is
provided above each plot.
Figure 6: Analysis of cytokine production and proliferation of CD4+ T-
cells lines modified with a ROR1 chimeric receptor derived from mAb R12
with higher affinity than 2A2. (A-B) The 2A2 and R12 ROR1 chimeric ors
had the short spacer and a CD28 ulatory domain. (A) Multiplex cytokine
analysis from supernatants obtained 24 hours after stimulation of 5x104 CD4+ T-
cells expressing the 2A2 and R12 ROR1 chimeric receptor with OR1 tumor
cells. (B) Proliferation of CD4+ R12 and 2A2 ROR1 chimeric receptor T-cells and
tEGFR control T-cells 72 hours after stimulation with Raji/ROR1 cells and without
addition of ous cytokines was assessed by CFSE dye dilution. s
above each histogram indicate the number of cell divisions the proliferating subset
underwent, and the fraction of T-cells in each gate that ent =5/4/3/2/1 cell
ons is provided above the histograms.
Figure 7: Recognition of primary CLL by T-cells modified with 2A2 and
R12 ROR1 chimeric receptors with optimal short spacer and 4-1BB
costimulatory domain or with a CD19-specific chimeric receptor. (A)
Expression of ROR1/CD19 on primary CLL, and CD80/86 on primary CLL and
OR1 tumor cells (black dot plots) that can engage CD28 on ic or
T-cells (white histograms). Staining with matched isotype control mAbs is shown as
grey dot plots/histograms. (B) Cytolytic activity of s expressing the 2A2(?)
and R12 ROR1 chimeric receptor (¦), a CD19-specific chimeric receptor (?) and
T-cells modified with a tEGFR control vector (x) against primary CLL (left
diagram) and normal B cells (right diagram) analyzed by chromium release assay.
Cytotoxicity data against primary CLL from 4 independent experiments (E:T =
:1) were normalized (cytolytic ty by ROR1 ic receptor 2A2 = 1) and
analyzed by Student’s t-test (bar diagram). (C) Multiplex cytokine analysis after a
24-hour stimulation of 5x104 chimeric receptor T-cells with primary CLL cells.
ne release of ulated chimeric receptor T-cells was below 3.6 pg/ml
(detection limit) (left bar diagram). ELISA for IFN-? production by 5x104 2A2 and
R12 ROR1 chimeric receptor T-cells after a r co-culture with primary CLL.
O.D. of 1 corresponds to approximately 250 pg/ml (right bar diagram). (D)
Proliferation of CD8+ s modified with the 2A2 ROR1, R12 ROR1 and a CD19
chimeric receptor, 72 hours after stimulation with primary CLL cells. Numbers
above each histogram indicate the number of cell divisions, and the fraction of T-
cells in each gate that underwent =3/2/1 cell divisions is provided next to each plot.
Figure 8: The function of ROR1-chimeric receptor and CD19-chimeric
receptor modified CD8+ T-cells against primary CLL is ted by chimeric
receptor-modified CD4+ helper T-cells. (A) ELISA for IL-2 production from
triplicate co-cultures of 5x104 CD8+ and CD4+ s expressing the R12 ROR1
and CD19-chimeric receptor respectively, incubated with primary CLL for 24-hours.
O.D. of 1 corresponds to approx. 800 pg/ml. (B) Proliferation of chimeric receptor-
modified CD8+ T-cells in se to primary CLL is enhanced by addition of
chimeric receptor-modified CD4+ T-cells. CFSE-labeled CD8+ T-cells sing
the 2A2 ROR1, R12 ROR1 and CD19-chimeric receptor respectively, were cocultured
with tumor cells and with 2A2 ROR1, R12 ROR1 and CD19-chimeric
receptor transduced or control untranduced CD4+ T-cells (CD8+:CD4+ = 1:1).
Proliferation of the CD8+ subset was analyzed 72 hours after stimulation. Numbers
above each histogram indicate the number of cell divisions, and the fraction of T-
cells in each gate that underwent =3/2/1 cell divisions is provided above each plot.
Figure 9: In vivo anti-tumor efficacy of 2A2 ROR1, R12 ROR1 and
CD19 chimeric receptor T-cells. Cohorts of mice were inoculated with 0.5x106
JeKo-1/ffluc MCL via tail vein injection, and 5x106 2A2 ROR1, R12 ROR1 or
CD19 chimeric receptor T-cells, or T-cells expressing a tEGFR control vector were
administered 7 days after tumor inoculation. All chimeric receptor constructs had the
short IgG4 ‘Hinge-only’ spacer and a 4-1BB costimulatory domain. (A, B) Serial
bioluminescence imaging of tumor in cohorts of mice treated with T-cells expressing
the 2A2 ROR1 chimeric receptor (?), the high affinity R12 ROR1 chimeric
receptor (¦), a CD19-specific chimeric receptor (?), with T-cells uced with
tEGFR alone (?), and untreated mice. Bioluminescence imaging showed tumor
manifestations in the bone marrow and thorax and thus, signal intensity was
measured in regions of interest that encompassed the entire body and thorax of each
individual mouse. (C) Kaplan-Meier analysis of survival in individual treatment and
control groups. Statistical es were performed using the nk test. The data
shown in A-C are representative of results obtained in 2 independent experiments.
(D) Proliferation of 2A2 ROR1, R12 ROR1 and CD19 ic receptor T-cells in
vivo. Tumor bearing NSG/JeKo-1 mice received a single dose of 5x106 CFSE-
d 2A2 ROR1, R12 ROR1 or CD19 chimeric receptor T-cells on day 7 after
tumor inoculation, and 72 h later peripheral blood, bone marrow and spleen were
collected from each individual mouse. The frequency and proliferation of live (PI-),
CD45+ CD8+ tEGFR+ T-cells was analyzed. The frequency of 2A2 ROR1, R12
ROR1 and CD19 chimeric or T-cells tively is provided on the left of
each histogram as percentage of live cells, and the fraction of T-cells that underwent
=4/3/2/1 cell ons is ed above each plot.
Figure 10 Expression of ROR1 and NKG2D ligands on epithelial cancer
cell lines. (A) Expression of ROR1 on the triple negative breast cancer cell lines
-231 and 468, and the renal cell cancer lines FARP, TREP and RWL
(black histograms). Staining with d isotype control antibody is shown as grey
histograms. (B) Expression of CD80/86 and the NKG2D s MICA/B on MDA-
MB-231 and Raji/ROR1 tumor cells, and NKG2D (CD314) on 2A2 and R12 ROR1-
chimeric receptor T-cells. Staining with matched isotype control mAbs is shown as
grey dot plots/histograms.
Figure 11: ROR1-chimeric receptor ed s ize ROR1+
epithelial tumor cells in vitro. (A) Chromium release assay to evaluate the
cytolytic activity of R12 ROR1-chimeric receptor modified T-cells (short spacer/4-
1BB costimulatory domain, closed symbols) and tEGFR control T-cells (open
symbols) against ROR1+ breast cancer and renal cell cancer lines. (A-D) The 2A2
and R12 ROR1-chimeric receptors had the optimal short spacer and a 4-1BB
ulatory domain. (B) Multiplex cytokine is after stimulation of T-cells
expressing the 2A2 and R12 ROR1-chimeric receptor with MDA-MB-231 and
Raji/ROR1 tumor cells. (C) Proliferation of CD8+ T-cells modified with the 2A2 and
R12 ROR1-chimeric receptor 72 hours after stimulation with MDA-MB-231 tumor
cells. For analysis, triplicate wells were pooled and the proliferation of live (PI-),
CD8+ T-cells analyzed. Numbers above each ram indicate the number of cell
divisions the proliferating subset underwent, and the fraction of T-cells in each gate
that underwent =4/3/2/1 cell divisions is provided next to each histogram. (D)
ELISA for IL-2 production by R12 ROR1-chimeric receptor T-cells after a 24-hour
co-culture with MDA-MB-231 in plain medium, and after addition of an antibody
cocktail blocking of the NKG2D pathway [anti-NKG2D (clone 1D11), anti-MICA/B
(clone 6D4) and anti-ULBP] or matched isotype control mAbs. O.D. of 0.6
corresponds to approximately 1900 pg/ml.
Figure 12. Effect of extracellular spacer length on recognition and
triggering of tumor cell lysis by CD8+ human T cells that express a HER2-
specific chimeric receptor. A.) Depiction of Herceptin Fab epitope location on
tumor cell membrane al e on human HER2, B.) ural formats of
Herceptin scFv CAR spacer length variants as –T2A- linked polypeptides with the
carboxyl EGFRt marker transmembrane protein, C.) Western blot detection of short,
medium, and long spacer Herceptin-CAR variant expression in human CD8+ CTL’s,
D.) Flow cytometric detection of EGFRt by transduced human CD8+ CTL’s
transduced with Herceptin CAR variants then immunomagnetically purified by
Herceptin-biotin, anti-biotin microbeads, E.) ct cytolytic on by T cells
transduced to s the Herceptin CAR variants (short – S; medium – M; and long
– L) against HER2+ Med411FH and D283 human medulloblastoma cell lines (D341
is a HER2- control medulloblastoma cell line, inset flow plots are tumor target lines
stained with anti-HER2 specific mAb). Green=full IgG4 (Long Spacer,?),
Blue=IgG4hinge:CH3(Medium Spacer;?), Red=IgG4hinge only (Short Spacer;¦).
Figure 13: CD19-chimeric receptor vectors and tion of CD19-
chimeric receptor T cells.
(A) Design of lentiviral transgene inserts encoding a panel of pecific
chimeric receptors that differ in extracellular spacer length and intracellular costimulation.
Each chimeric receptor d the CD19-specific single chain variable
fragment derived from the FMC63 mAb in a VL-VH orientation, an IgG4-derived
spacer domain of Hinge-CH2-CH3 (long spacer, 229 AA) or Hinge only (short
, 12 AA), and a signaling module containing CD3? with CD28 or 4-1BB alone
or in tandem. Each chimeric receptor cassette contains a truncated EGFR marker
d downstream of a cleavable 2A element. (B, C) Polyclonal T cell lines
modified with each of the himeric receptor constructs were prepared from
purified CD8+ CD45RO+ CD62L+ central memory T cells (TCM) of normal donors.
Following lentiviral transduction, ene-positive T cells in each cell line were
purified using the tEGFR marker and expanded for in vitro and in vivo experiments.
(D) MFI after staining for the tEGFR marker shows equivalent transgene expression
in T cells modified with each of the CD19-chimeric receptors.
Figure 14: In vitro cytotoxicity, cytokine production, and proliferation
of T cells modified with distinct himeric receptors. (A) tic activity
of T cells expressing the various CD19-chimeric ors against CD19+ and
control target cells. (B) Multiplex cytokine assay of supernatants obtained after 24
hours from triplicate co-cultures of T cells expressing the various CD19-chimeric
receptors and K562 cells ected with CD19, and CD19+ Raji cells. (C)
Comparison of cytokine production by T cells expressing the various CD19-
chimeric receptors. Multiplex cytokine data from 6 independent experiments were
normalized (cytokine release by CD19-chimeric receptor ‘short/CD28’ CTL = 1)
and analyzed by Student’s t-test. (D) CFSE dye dilution was used to measure
proliferation of CD19-chimeric receptor T cells 72 hours after stimulation with
K562/CD19 (upper panel) and CD19+ Raji tumor cells (lower panel) without
on of ous cytokines. For analysis, triplicate wells were pooled and the
proliferation of live (PI-), CD8+ T cells analyzed. s above each histogram
indicate the number of cell divisions the proliferating subset underwent, and the
fraction of T cells in each gate that underwent =4/3/2/1 cell divisions is ed in
the upper left of each plot. (E) PI staining was performed at the end of a 72-hour coculture
of T cells expressing the various CD19-chimeric receptors with Raji tumor
cells. The percentage of PI+ cells within in ic receptor T cell line (CD3+) is
provided in each histogram.
Figure 15: CD19-chimeric receptor T cells with a short extracellular
spacer domain eradicate Raji tumors in NOD/SCID mice. (A) Cohorts of mice
were inoculated with fluc via tail vein injection, and T cells transduced with
CD19-chimeric receptors containing long and short spacer domains or with tEGFR
alone were administered 2 and 9 days after tumor inoculation by tail vein injection.
Tumor progression and bution was evaluated by serial bioluminescence
imaging after injection of luciferin substrate. (B) Serial inescence imaging of
tumor in cohorts of mice either treated with T cells expressing CD19-chimeric
receptors with short spacer (‘short/CD28’ and ‘short/4-1BB’) and long spacer
(‘long/CD28’ and ‘long/4-1BB’) domains, with T cells transduced with the tEGFR
control , or ted. Each diagram representing cohorts of mice treated with
CD19-chimeric receptor or tEGFR transduced T cells also shows the mean of tumor
progression in untreated mice for comparison (red triangles). (C) -Meier
analyses of survival of untreated mice and mice treated with T cells expressing
himeric receptors with short spacer (‘short/CD28’ and /4-1BB’), long
spacer (‘long/CD28’ and ‘long/4-1BB’) domains, and with control tEGFR.
Statistical analyses were performed using the log-rank test. The data shown in B and
C are representative of results obtained in 3 independent experiments.
Figure 16: CD19-chimeric receptor T cells with a short spacer (short/4-
1BB) eradicate established Raji tumors in NSG mice in a dose-dependent
manner. (A) Mice were ated with Raji-ffluc via tail vein injection and tumor
engraftment confirmed by bioluminescence imaging on day 6. On day 7, mice
received a single i.v. injection of s doses of T cells transduced with the CD19-
chimeric receptor ‘short/4-1BB’ or with the tEGFR-control lentivirus. (B, C) Dose
ent anti-tumor efficacy of T cells expressing the CD19-chimeric receptor
‘short/4-1BB’. A control cohort of mice received a single high dose of T cells
modified with tEGFR alone. (D) Persistence of CD19-chimeric receptor T cells
following adoptive transfer into NSG/Raji mice. Flow cytometric analysis of
peripheral blood (eye bleeds) in the cohort of mice treated with 2.5x106 CD19-
chimeric or ‘short/4-1BB’ T cells. The frequency of CD8+ tEGFR+ T cells is
shown as percentage of live peripheral blood cells.
Figure 17: T cells expressing CD19-chimeric receptors with a short
spacer and either CD28 or 4-1BB are more effective against established
lymphoma than those expressing CD19-chimeric receptors with a long spacer.
(A) NSG mice were inoculated with Raji-ffluc on day 0, and treated on day 7 with
one dose of 2.5x106 CD19 chimeric receptor T cells expressing short or long spacer
and either CD28 or 4-1BB costimulatory domain. (B) Kaplan-Meier analyses of
survival of mice in each of the treatment groups. tical analyses were performed
using the nk test. (C) Bioluminescence imaging of cohorts of mice treated with
T cells sing CD19-chimeric receptors with short spacers (‘short/CD28’ and
‘short/4-1BB’), and long spacers (‘long/CD28 and ‘long/4-1BB’). The mean tumor
burden observed in untreated mice at each time point is shown in each diagram for
comparison (triangles). (D) In vivo persistence of T cells expressing himeric
receptor with short spacer domain is enhanced compared to T cells expressing
CD19-chimeric receptors with long spacer domain. The ncy of CD8+ tEGFR+
T cells in the peripheral blood ed at day 3 and 10 after transfer was determined
by flow cytometry and is shown as percentage of live (PI-) peripheral blood cells.
Statistical analyses were performed by Student’s t-test. The data shown in B-D are
entative for results obtained in 3 independent experiments.
Figure 18: sing chimeric receptor T cell dose or augmenting
costimulatory signaling does not improve the anti-tumor efficacy of CD19-
chimeric receptors with a long spacer domain against established lymphoma.
(A) Cytolytic activity of T cells expressing ‘long/CD28’, ‘long/4-1BB’ and
‘long/CD28_4-1BB’ CD19 chimeric receptors against CD19+ and l target
cells. (B) Multiplex cytokine assay of supernatant ed after 24 hours from
triplicate co-cultures of K562/CD19 and Raji tumor cells with T cells expressing the
various himeric receptors. (C) Evaluation of proliferation of CD19-chimeric
receptor T cells 72 hours after stimulation with CD19+ tumor cells (K562/CD19 –
left panel; Raji – right panel) by CFSE dye dilution. For analysis, triplicate wells
were pooled and the proliferation of live (PI-) CD8+ T cells analyzed. s
above each histogram indicate the number of cell divisions the proliferating subset
underwent, and the fraction of T cells in each gate that underwent =4/3/2/1 cell
divisions is provided in the upper left of each plot. (D) Kaplan-Meier analyses of
survival of mice treated with T cells expressing CD19-chimeric receptors with short
(‘short/CD28’) and long spacer domain (‘long/CD28’ and ‘long/CD28_4-1BB’), or
T cells ed with a tEGFR-encoding control lentiviral vector. Statistical
analyses were performed using the log-rank test. (E) Bioluminescence imaging of
cohorts of mice treated with T cells expressing CD19-chimeric receptors with short
spacer (‘short/CD28’), and long spacers (‘long/CD28 and ‘long/CD28_4-1BB’).
Diagrams show mean tumor progression in untreated mice for comparison (red
triangles). (F) In vivo persistence of T cells expressing the various CD19-chimeric
receptors. The frequency of CD8+ tEGFR+ T cells in the peripheral blood obtained at
day 3 and 10 after transfer was determined by flow cytometry and is shown as
percentage of live (PI-) peripheral blood cells. Statistical analyses were med
by t’s t-test.
Figure 19: CD19-chimeric receptor T cells with a long spacer domain
are activated by tumor in vivo but fail to increase in cell number. (A)
Expression of CD69 and CD25 on T cells modified with each CD19-chimeric
receptor prior to transfer into NSG/Raji mice. (B) Cohorts of mice were inoculated
with Raji-ffluc tumor cells and 7 days later received CFSE-labeled CD19-chimeric
or transduced or control T cells. Bone marrow and spleens were harvested
from subgroups of mice 24 and 72 hours after T cell administration. (C, D)
Multiparameter flow cytometric analysis of bone marrow clear cells
obtained 24 hours (C) and 72 hours (D) after T cell transfer. Dot plots show anti
CD3 and anti CD45 ng after gating on PI- cells to detect viable human T cells.
The CD3- CD45+ gate contains Raji tumor cells. Expression of CD25 and CD69 on
live (PI-) CD3+ CD45+ T cells is shown in the histograms. (E) Frequency of CD3+
CD45+ T cells in s obtained 24 and 72 hours after T cell transfer. Dot plots are
gated on live PI- splenocytes and the percentage of CD3+ CD45+ T cells is shown in
each plot. (F) PI ng of bone marrow and splenocytes hours after T cell transfer
into NSG/Raji mice. The numbers in the histograms indicate the percentage of PI+
cells within the CD3+ population. (G) Bioluminescence g of s of mice
treated with T cells expressing CD19-chimeric receptors with short spacer
t/CD28’ and ‘short/4-1BB’), long spacers (‘long/CD28 and ‘long/4-1BB’), or
control T cells.
Figure 20: T cells expressing CD19 chimeric receptors with 4-1BB and
CD3zeta and a modified IgG4-Fc hinge exhibit superior in vitro and in vivo function
compared to T cells expressing CD19 chimeric receptors with 4-1BB and CD3zeta and
a CD8 alpha hinge.A. Cytolytic activity of CD19 chimeric receptor modified T-cells with
IgG4 Fc hinge, CD8 alpha hinge and control T cells against Cr51-labeled K562 cells
transfected with CD19, Raji ma cells that express CD19, and K562 control T cells.
Lysis is shown at ent E/T ratios in a 4 hour Cr51 release assay. B. eron gamma
production by 5x104 T cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge or
CD8 alpha hinge after a 24-hour coculture with Raji tumor cells. O.D. of 1 ponds to
~500 pg/ml of interferon gamma. C. CFSE dye dilution assay to measure proliferation of T
cells expressing a CD19 chimeric receptor with an IgG4 Fc hinge or CD8 alpha hinge and T
cells that express tEGFR alone (control) after 72 hours coculture with CD19 positive Raji
lymphoma cells. Numbers above each histogram indicate the number of cell divisions the
proliferating cell subset underwent. The fraction of T cells in each gate that underwent
>3/2/1 cell divisions is ed next to the plot. D. In vivo antitumor activity of T cells
expressing a CD19 chimeric receptor with an IgG4 Fc hinge (group 1) or CD8 alpha hinge
(group 2) and T cells that express tEGFR alone (group 3) in NSG mice inoculated with Raji
tumor cells expressing firefly luciferase (ffluc). Mice were imaged 17 days after tumor
inoculation and 10 days after T cell inoculation. The data shows greater tumor burden in
mice treated with control tEGFR T cells (group 3) or with CD19 chimeric receptor CD8
alpha hinge T cells (group 2) compared with mice treated with CD19 ic receptor
IgG4 Fc hinge T cells (group 1).
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which the invention pertains.
“About” as used herein when referring to a measurable value is meant to
encompass ions of ±20% or ±10%, more preferably ±5%, even more
preferably ±l%, and still more preferably ±0.1 % from the specified value.
ation", as used herein, refers to the state of a T cell that has been
sufficiently stimulated to induce able cellular proliferation, cytokine
production or expression of cell surface markers such as CD69 and CD25, or
detectable effector functions.
ation Induced cell death” as used herein refers to a state of a T cell that
is activated but is not able to proliferate for more than 2 generations and exhibits
markers of apoptosis.
"Antigen" or "Ag" as used herein refers to a molecule that es an
immune response. This immune response may involve either antibody production, or
the tion of specific immunologically-competent cells, or both. It is readily
apparent that an antigen can be generated synthesized, produced recombinantly or
can be derived from a biological sample. Such a biological sample can include, but
is not limited to a tissue sample, a tumor , a cell or a biological fluid.
"Anti-tumor effect" as used , refers to a biological effect, which can be
manifested by a decrease in tumor volume, a decrease in the number of tumor cells,
a decrease in the number of metastases, an increase in life expectancy, or a decrease
of various physiological symptoms associated with the ous condition. An
"anti-tumor effect" can also be manifested by a decrease in ence or an increase
in the time before recurrence.
“Chimeric receptor” as used herein refers to a synthetically designed receptor
comprising a ligand binding domain of an antibody or other protein sequence that
binds to a molecule associated with the disease or disorder and is linked via a spacer
domain to one ore more intracellular signaling domains of a T cell or other
receptors, such as a costimulatory domain.
"Co-stimulatory domain," as the term is used herein refers to a signaling
moiety that provides to T cells a signal which, in addition to the primary signal
provided by for instance the CD3 zeta chain of the 3 complex, es a T
cell response, including, but not limited to, activation, proliferation, differentiation,
cytokine secretion, and the like. A co-stimulatory domain can include all or a portion
of, but is not d to, CD27, CD28, 4-1BB, OX40, CD30, CD40, , ICOS,
lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds with CD83. In embodiments, the costimulatory
domain is an intracellular signaling domain that interacts with other
intracellular mediators to mediate a cell response ing activation, proliferation,
differentiation and cytokine secretion, and the like.
"Coding for" are used herein refers to the property of specific sequences of
nucleotides in a cleotide, such as a gene, a cDNA, or an mRNA, to serve as
templates for synthesis of other macromolecules such as a defined sequence of
amino acids. Thus, a gene codes for a protein if transcription and translation of
mRNA corresponding to that gene produces the protein in a cell or other biological
system. A "nucleic acid sequence coding for a polypeptide" includes all nucleotide
sequences that are degenerate versions of each other and that code for the same
amino acid sequence.
“Cytotoxic T lymphocyte “(CTL) as used herein refers to a T lymphocyte
that expresses CD8 on the surface thereof (i.e., a CD8+ T cell). In some
embodiments such cells are preferably "memory" T cells (TM cells) that are antigenexperienced.
"Central memory" T cell (or "TCM") as used herein refers to an antigen
experienced CTL that ses CD62L or CCR-7 and CD45RO on the surface
thereof, and does not express or has decreased expression of CD45RA as compared
to naive cells. In embodiments, l memory cells are ve for expression of
CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and have decreased expression
of CD54RA as compared to naïve cells.
"Effector memory" T cell (or "TEM") as used herein refers to an antigen
experienced T cell that does not express or has decreased expression of CD62L on
the surface thereof as compared to central memory cells, and does not express or has
sed expression of CD45RA as compared to naïve cell. In embodiments,
effector memory cells are negative for expression of CD62L andCCR7, compared
to naïve cells or central memory cells, and have variable expression of CD28 and
CD45RA.
“Naïve “ T cells as used herein refers to a non antigen enced T
cyte that expresses CD62L and CD45RA, and does not express - as
compared to central or effector memory cells. In some embodiments, naïve CD8+ T
lymphocytes are characterized by the expression of phenotypic markers of naïve T
cells including CD62L, CCR7, CD28, CD127, and CD45RA.
“Effector “ “TE” T cells as used herein refers to a antigen experienced
cytotoxic T lymphocyte cells that do not s or have decreased expression of
CD62L, CCR7, CD28, and are positive for me B and in as compared to
central memory or naïve T cells.
"Enriched" and "depleted" as used herein to describe amounts of cell types in
a mixture refers to the subjecting of the mixture of the cells to a process or step
which results in an increase in the number of the "enriched" type and a decrease in
the number of the ted" cells. Thus, depending upon the source of the original
population of cells subjected to the enriching s, a e or composition may
contain about 60, 70, 80, 90, 95, or 99 percent or more (in number or count) of the
"enriched" cells and about 40, 30, 20, 10, 5 or 1 percent or less (in number or count)
of the "depleted" cells.
“Epitope” as used herein refers to a part of an antigen or molecule that is
recognized by the immune system including antibodies, T cells, and/ or B cells.
Epitopes usually have at least 7 amino acids and can be linear or conformational.
"Isolated," when used to describe the various polypeptides disclosed herein,
means polypeptide or nucleic acid that has been identified and separated and/or
recovered from a component of its natural environment. Preferably, the isolated
polypeptide or nucleic acid is free of association with all components with which it
is naturally associated. Contaminant components of its l nment are
materials that would typically interfere with diagnostic or therapeutic uses for the
polypeptide or nucleic acid, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes.
“Intracellular signaling domain” as used herein refers to all or a n of
one or more domains of a molecule (here the chimeric receptor molecule) that
provides for activation of a lymphocyte. Intracellular domains of such molecules
mediate a signal by interacting with cellular mediators to result in proliferation,
differentiation, activation and other effector functions. In embodiments, such
les include all or portions of CD28, CD3, 4-1BB, and combinations f.
“Ligand” as used herein refers to a substance that binds ically to
another substance to form a complex. Example of ligands include epitopes on
antigens, molecules that bind to receptors, substrates, tors, es, and
activators. “Ligand binding domain” as used herein refers to substance or portion of
a substance that binds to a ligand. Examples of ligand binding domains include
antigen binding ns of antibodies, ellular s of receptors, and active
sites of enzymes.
"Operably linked" as used herein refers to functional linkage between a
regulatory sequence and a heterologous c acid sequence resulting in
expression of the latter. For example, a first nucleic acid sequence is operably linked
with a second nucleic acid sequence when the first nucleic acid sequence is placed in
a functional onship with the second nucleic acid sequence. For instance, a
promoter is operably linked to a coding sequence if the er affects the
transcription or expression of the coding sequence. Generally, operably linked DNA
sequences are contiguous and, where necessary to join two n coding regions,
in the same reading frame.
"Percent (%) amino acid sequence identity" with respect to the chimeric
or polypeptide sequences identified herein is defined as the percentage of
amino acid residues in a candidate sequence that are identical with the amino acid
residues in the reference sequence for each of the ligand binding domain, spacer,
transmembrane domain, and/or the lymphocyte activating , after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum t
sequence identity, and not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that are within the skill in the art,
for instance, using ly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including any algorithms
needed to achieve maximal alignment over the full-length of the sequences being
compared. For example, % amino acid sequence identity values generated using the
WU-BLAST-2 computer program [Altschul et al., Methods in logy,
266:460-480 (1996)] uses l search parameters, most of which are set to the
default values. Those that are not set to default values (i.e., the adjustable
ters) are set with the following values: overlap span=1, overlap
fraction=0.125, word threshold (T)=11 and scoring matrix=BLOSUM62. A %
amino acid ce identity value is determined by dividing (a) the number of
matching identical amino acid residues n the each or all of the polypeptide
amino acid sequence of the reference chimeric or sequence provided in Table
2 and the comparison amino acid ce of interest as determined by WU-
2 by (b) the total number of amino acid residues of the polypeptide of
interest.
"Chimeric receptor variant polynucleotide" or "chimeric or variant
nucleic acid sequence" as used herein refers to a ptide-encoding nucleic acid
molecule as defined below having at least about 80% nucleic acid sequence identity
with the polynucleotide acid sequence shown in Table 1 or a specifically derived
fragment thereof, such as polynucleotide coding for an n binding domain, a
polynucleotide encoding a spacer domain, a cleotide coding for a
transmembrane domain and/ or a cleotide coding for a lymphocyte
stimulatory domain. Ordinarily, a chimeric or variant of polynucleotide or
fragment thereof will have at least about 80% nucleic acid sequence identity, more
preferably at least about 81% nucleic acid sequence identity, more preferably at least
about 82% nucleic acid sequence identity, more preferably at least about 83%
nucleic acid sequence identity, more preferably at least about 84% nucleic acid
sequence identity, more preferably at least about 85% nucleic acid sequence identity,
more preferably at least about 86% nucleic acid sequence identity, more preferably
at least about 87% nucleic acid sequence identity, more preferably at least about
88% nucleic acid sequence identity, more ably at least about 89% nucleic acid
sequence identity, more preferably at least about 90% nucleic acid sequence identity,
more preferably at least about 91% nucleic acid sequence identity, more preferably
at least about 92% nucleic acid sequence identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid
ce identity, more preferably at least about 95% nucleic acid sequence identity,
more preferably at least about 96% nucleic acid sequence ty, more preferably
at least about 97% nucleic acid sequence identity, more ably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence ty with the nucleic acid sequence as shown in Table or a
derived fragment thereof. Variants do not encompass the native nucleotide sequence.
In this regard, due to the degeneracy of the c code, one of ordinary skill in the
art will immediately ize that a large number of chimeric receptor variant
polynucleotides having at least about 80% c acid sequence identity to the
nucleotide sequence of Table 1 will encode a polypeptide having an amino acid
sequence which is identical to the amino acid sequence of Table 2.
antially purified" refers to a molecule that is essentially free of other
molecule types or a cell that is essentially free of other cell types. A substantially
purified cell also refers to a cell, which has been separated from other cell types with
which it is normally associated in its naturally occurring state. In some instances, a
population of substantially ed cells refers to a homogenous population of cells.
“Not substantially found” when used in reference the presence of a tumor
antigen or other les on normal cells refers to the percentage of a normal cell
type that has the antigen or molecule, and / or the density of the antigen on the cells.
In ments, not substantially found means that the antigen or molecule is found
on less than 50% of normal cell type and/or at a 50% less density as compared to the
amount of cells or n found on a tumor cell or other diseased cell.
"T cells" or "T lymphocytes" as used herein may be from any mammalian,
ably primate, species, including monkeys, dogs, and humans. In some
embodiments the T cells are allogeneic (from the same species but different donor)
as the recipient subject; in some embodiments the T cells are autologous (the donor
and the recipient are the same); in some embodiments the T cells arc syngeneic (the
donor and the recipients are different but are identical twins).
Modes of the Disclosure
Described herein are chimeric receptor nucleic acids, and vectors and host
cells including such nucleic acids. The chimeric receptor nucleic acid comprises a
number of modular components that can be excised and replaced with other
components in order to customize the chimeric receptor for a ic target
molecule. The disclosure describes that one of the r components is the
spacer component. It has been surprisingly found that the length of the spacer
region that is presumed not to have ing capability affects the in vivo
efficacy of the T cells modified to express the chimeric receptor and needs to be
customized for individual target molecules for enhanced therapeutic activity.
In one aspect, methods and nucleic acid constructs are described to design
a chimeric receptor that has improved tumor recognition, sed T cell
proliferation and/or ne production in response to the ligand as compared to
a reference chimeric receptor. In embodiments, a library of nucleic acids is
described, wherein each nucleic acid codes for a spacer region that differs from
the others in ce and length. Each of the nucleic acids can then be used to
form a chimeric receptor nucleic acid construct that can be tested in vivo (in an
animal model) and/or in vitro so that a spacer can be selected that es for
improved tumor recognition, increased T cell proliferation and/or cytokine
production in response to the ligand.
In embodiments, a chimeric receptor nucleic acid comprises a
polynucleotide coding for a ligand binding domain, wherein the ligand is a tumor
or viral specific antigen or molecule, a polynucleotide coding for a customized
ptide spacer, wherein the spacer provides for enhanced T cell proliferation;
a polynucleotide coding for a transmembrane ; and a polynucleotide
coding for one or more intracellular signaling domains. In embodiments, a long
spacer is employed if the e of the target molecule is membrane proximal on
the target cell and a short spacer is ed if the epitope of the target molecule
is membrane distal on the target cell.
The design of a chimeric receptor can be customized depending on the
type of tumor or virus, the target antigen or molecule present on the tumor, the
affinity of the antibody for the target le, the ility needed for the
antigen binding domain, and/or the ellular signaling domain. In
embodiments, a number of chimeric receptor constructs are tested in vitro and in
in vivo models to determine the ability of T cells modified with the receptor to
kill tumor cells in immunodeficient mice and to proliferate and persist after
adoptive transfer. In ments, a chimeric receptor is selected that provides
for capability of at least 30% of the cells to proliferate through at least two
generations in vitro and/or within 72 hours after introduction in vivo. In
embodiments, a chimeric receptor is not selected that results in greater than 50%
of the cells undergoing activation induced cell death (AICD) within 72 hours in
vivo in immunodeficient mice, and fails to eradicate tumor cells.
Depending on r the target molecule is present on a subject’s tumor
cells, the chimeric receptor includes a ligand binding domain that specifically binds
to that target le. In embodiments, a subject’s tumor cells are characterized for
cell surface tumor molecules. The target molecule may be selected based on a
determination of its presence on a particular subject’s tumor cells. In embodiments,
a target molecule is selected that is a cell surface molecule found inantly on
tumor cells and not found on normal tissues to any substantial degree. In
embodiments, an dy is selected to bind to an epitope on the targeted cell
surface molecule. In some cases, the e is characterized with respect to its
proximity to the cell membrane. An epitope is characterized as proximal to the
membrane when it is predicted or known by structural analysis to reside closer to the
target cell membrane than ative epitopes that are predicted or known by
structural analysis to reside a greater distance from the target cell membrane. In
embodiments, the affinity of the antibody from which the scFV is constructed is
compared by binding assays, and dies with different affinities are examined in
chimeric receptor formats expressed in T cells to determine which affinity confers
optimal tumor recognition, based on superior cytotoxicity of target cells, and/or T
cell cytokine production and proliferation.
In addition, the spacer region of the chimeric receptor may be varied to
optimize T cell recognition of the ligand on the target cell. In embodiments, when an
dy binds to an epitope on the target cell that is very proximal to the
membrane, a spacer is selected that is longer than about 15 amino acids. For
example, in ments, if the e or portion thereof on the target antigen is in
the first 100 amino acids of the linear sequence of the extracellular domain adjacent
to the transmembrane domain, a long spacer region may be selected. In
ments, when an antibody binds to an epitope on the target cell that is distal to
the membrane, a spacer is selected that is about 119 or 15 amino acids or less. For
example, in embodiments, when the epitope or portion thereof is found in the 150
amino acids of the linear ce of the extracellular domain from the terminus, a
short or inetermediate spacer may be utilized. In embodiments, a spacer comprises
an amino acid sequence X1PPX2P.
A variety of ations of primary and costimulatory intracellular
signaling domain may be employed to enhance the in vivo efficacy of the chimeric
receptor. In embodiments, different constructs of the chimeric receptor can be tested
in an in vivo animal model to determine efficacy for tumor killing. In embodiments,
a costimulatory intracellular signaling domain is selected from the group consisting
of CD28 and modified versions thereof, 4-1BB and modified ns thereof and
combinations f. Other costimulatory domains, such as OX40 may be
incorporated.
CD8+ central memory T cells have an intrinsic programming that allows
them to persist for extended periods after administration, which makes them a
red subset of CD8+ T cells for immunotherapy. In ments, CD19
specific chimeric receptor modified cytotoxic T cells prepared from sort purified
CD8+ central memory T cells are administered in the ce or absence of CD4+
CD19 specific chimeric receptor -modified T cells. In ments, tumor-specific
CD4+ T cells exert anti-tumor reactivity and provide help to tumor-specific CD8+ T
cells in vitro and in vivo. In a specific embodiment, tumor-specific CD4+ T cells or
CD4+ T cells selected from the naïve or the central memory subsets are utilized
alone or in combination with CD8+ TCM.
Nucleic Acids, Vectors, and polypeptides
Also described is a chimeric receptor nucleic acid useful for transforming or
transducing lymphocytes for use in adoptive therapy. In embodiments, the
nucleic acid contains a number of modular components that provide for easy
substitution of elements of the nucleic acid. While not meant to limit the scope of
the sure, it is believed that the chimeric receptor for each tumor antigen is
desirably customized in terms of components in order to provide for in vivo efficacy
and efficient expression in mammalian cells. For example, in a specific embodiment,
for efficacy of a chimeric receptor comprising a scFV that binds to a ROR1 epitope
located in the membrane distal Ig/Frizzled domain, a spacer that is about 15 amino
acids or less is employed. In another specific embodiment, for efficacy of a chimeric
receptor comprising a scFV that binds to a ROR1 epitope located in the membrane
proximal Kringle domain, a spacer that is longer than 15 amino acids is employed.
In r embodiment, for efficacy of a chimeric receptor comprising a scFV that
binds to CD19, a spacer that is 15 amino acids or less is employed.
In embodiments, an isolated chimeric receptor nucleic acid comprises a
cleotide coding for a ligand g domain, wherein the target molecule is a
tumor specific n, a polynucleotide coding for a ptide spacer wherein the
polypeptide spacer is about 229 amino acids or less; a polynucleotide coding for a
embrane domain; and a polynucleotide coding for an intracellular signaling
. In embodiments, an expression vector comprises a chimeric nucleic acid as
described herein. Polypeptides encoded by all of or a portion of the chimeric
receptor nucleic acids are also included herein.
Ligand binding domain
In embodiments, the ic receptor nucleic acid comprises a
cleotide coding for a ligand binding domain. In embodiments, the ligand
binding domain ically binds to a tumor or viral specific antigen. In
embodiments, the ligand binding domain is an antibody or fragment thereof. A
nucleic acid sequence coding for an antibody or antibody fragment can readily be
determined. In a specific embodiment, the polynucleotide codes for a single chain
Fv that specifically binds CD19. In other specific embodiments, the polynucleotide
codes for a single chain Fv that ically binds ROR1. The sequences of these
antibodies are known to or can readily be determined by those of skill in the art.
Tumor antigens are ns that are produced by tumor cells that elicit an
immune response. The selection of the ligand binding domain described herein will
depend on the type of cancer to be d, and may target tumor antigens or other
tumor cell e molecules. A tumor sample from a subject may be characterized
for the presence of certain biomarkers or cell surface markers. For example, breast
cancer cells from a subject may be positive or negative for each of Her2Neu,
Estrogen receptor, and/or the Progesterone or. A tumor antigen or cell surface
molecule is selected that is found on the individual subject’s tumor cells. Tumor
antigens and cell surface molecules are well known in the art and include, for
example, carcinoembryonic antigen (CEA), prostate specific n, PSMA,
Her2/neu, estrogen receptor, progesterone or, ephrinB2, CD19, CD20, CD22,
CD23, CD123, CS-1, ROR1, mesothelin, c-Met, GD-2, and MAGE A3 TCR. In
embodiments a target molecule is a cell surface molecule that is found on tumor
cells and is not substantially found on normal tissues, or restricted in its expression
to non-vital normal s.
Other target molecules include but are not limited to antigens derived from
infectious pathogens such as HIV (human immunodeficiency virus), HBV (hepatitis
B , HPV (human papilloma virus) and Hepatitis C virus.
In one embodiment, the target molecule on the tumor ses one or more
epitopes associated with a malignant tumor. Malignant tumors express a number of
proteins that can serve as target antigens for T cell receptor or chimeric receptor
mediated recognition. Other target molecules belong to the group of cell
transformation-related molecules such as the oncogene HER-2/Neu/ErbB2. In
embodiments, the tumor antigen is selectively expressed or overexpressed on the
tumor cells as compared to control cells of the same tissue type. In other
embodiments, the tumor antigen is a cell surface polypeptide.
Once a tumor cell surface molecule that might be targeted with a chimeric
or is identified, an epitope of the target molecule is selected and characterized.
In embodiments, an epitope is selected that is proximal to the tumor cell membrane.
In other embodiments, an epitope is selected that is distal to the tumor cell
membrane. An epitope is characterized as proximal to the ne when it is
predicted or known by structural analysis to reside closer to the target cell ne
than alternative epitopes that are predicted or known by structural analysis to reside
a greater distance from the target cell membrane.
dies that specifically bind a tumor cell surface molecule can be
prepared using methods of obtaining monoclonal antibodies, methods of phage
display, methods to generate human or humanized antibodies, or methods using a
transgenic animal or plant engineered to e human antibodies. Phage display
libraries of partially or fully synthetic antibodies are available and can be ed
for an antibody or fragment thereof that can bind to the target molecule. Phage
y libraries of human antibodies are also available. In ments, antibodies
specifically bind to a tumor cell surface molecule and do not cross react with
nonspecific components such as bovine serum n or other unrelated antigens.
Once identified, the amino acid ce or polynucleotide sequence coding for the
antibody can be isolated and/or determined.
Antibodies or antigen binding fragments include all or a n of
polyclonal antibodies, a monoclonal antibody, a human antibody, a humanized
antibody, a synthetic antibody, a chimeric dy, a bispecific antibody, a
minibody, and a linear antibody. Antibody fragments" comprise a portion of an
intact antibody, preferably the antigen binding or le region of the intact
antibody and can readily be prepared. Examples of antibody fragments include Fab,
Fab', 2, and Fv nts; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody fragments.
In embodiments, a number of different dies that bind to a particular
tumor cell surface molecules can be isolated and characterized. In embodiments, the
antibodies are characterized based on epitope specificity of the targeted molecule. In
addition, in some cases, dies that bind to the same e can be selected
based on the affinity of the antibody for that epitope. In embodiments, an dy
has an affinity of at least 1 mM, and preferably <50 nM. In embodiments, an
antibody is selected that has a higher affinity for the epitope as compared to other
antibodies. For example, an antibody is selected that has at least a 2 fold, at least a 5
fold, at least a 10 fold, at least a 20 fold, at least a 30 fold, at least a 40 fold, or at
least a 50 fold greater affinity than a reference antibody that binds to the same
In embodiments, target molecules are selected from the group consisting of
CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, mesothelin, Her2, c-Met, PSMA,
GD-2, MAGE A3 TCR and combinations thereof.
In specific ments, the target antigen is CD19. A number of antibodies
specific for CD19 are known to those of skill in the art and can be readily
characterized for sequence, epitope binding, and affinity. In a specific embodiment,
the chimeric receptor uct includes a scFV sequence from FMC63 antibody. In
other embodiments, the scFV is a human or humanized ScFv comprising a variable
light chain comprising a CDRL1 sequence of SKYLN, CDRL2 sequence
of SRLHSGV, and a CDRL3 sequence of GNTLPYTFG. In other embodiments, the
scFV is a human or humanized ScFv comprising a variable heavy chain comprising
CDRH1 sequence of DYGVS , CDRH2 sequence of VIWGSETTYYNSALKS, and
a CDRH3 sequence of YAMDYWG. The disclosure also contemplates variable
regions that have at least 90% amino acid sequence identity to that of the scFv for
FMC63 and that have at least the same affinity for CD19. In embodiments, the
chimeric receptor has a short or intermediate spacer of 119 amino acids or less, or 12
amino acids or less. In a specific embodiment, the spacer is 12 amino acid or less
and has a sequence of SEQ ID NO:4.
In embodiments, CDR regions are found within antibody regions as
numbered by Kabat as follows: for the light chain; CDRL1 amino acids 24-
34;CDRL2 amino acids 50-56; CDRL3 at amino acids 89-97; for the heavy chain at
CDRH1 at amino acids 31-35; CDRH2 at amino acids 50-65; and for CDRH3 at
amino acids 95-102. CDR regions in dies can be readily determined.
In specific embodiments, the target antigen is ROR1. A number of dies
specific for ROR1 are known to those of skill in the art and can be readily
characterized for sequence, epitope binding, and affinity. In a specific embodiment,
the ic receptor construct es a scFV sequence from R12 dy. In
other embodiments, the scFV is a human or zed ScFv comprising a le
light chain comprising a CDRL1 sequence of SAYYM, CDRL2 sequence
of TIYPSSG, and a CDRL3 sequence of ADRATYFCA. In other embodiments, the
scFV is a human or humanized ScFv comprising a variable heavy chain comprising
CDRH1 sequence of DTIDWY, CDRH2 sequence of VQSDGSYTKRPGVPDR,
and a CDRH3 sequence of YIGGYVFG. The disclosure also contemplates variable
regions that have at least 90% amino acid sequence identity to that of the scFv for
R12 and that have at least the same affinity for ROR1. In ments, the chimeric
receptor has a short or intermediate spacer of 119 amino acids or less, or 12 amino
acids or less. In a specific embodiment, the spacer is 12 amino acid or less and has a
sequence of SEQ ID NO:4.
In specific embodiments, the target antigen is ROR1. A number of antibodies
specific for ROR1 are known to those of skill in the art and can be readily
characterized for sequence, epitope binding, and affinity. In a specific embodiment,
the chimeric receptor construct includes a scFV sequence from R11 antibody. In
other embodiments, the scFV is a human or humanized ScFv sing a variable
light chain comprising a CDRL1 sequence of SGSDINDYPIS, CDRL2 ce of
INSGGST, and a CDRL3 sequence of YFCARGYS. In other embodiments, the
scFV is a human or humanized ScFv comprising a variable heavy chain comprising
CDRH1 sequence of SNLAW, CDRH2 sequence of RASNLASGVPSRFSGS, and a
CDRH3 sequence of NVSYRTSF. The disclosure also contemplates variable
regions that have at least 90% amino acid sequence identity to that of the scFv for
R11 and that have at least the same affinity for ROR1. In embodiments, the chimeric
receptor has a long spacer of 229 amino acids or less. In a specific embodiment, the
spacer is 229 amino acids and has a ce of SEQ ID NO:50.
In specific embodiments, the target antigen is Her2. A number of antibodies
specific for Her2 are known to those of skill in the art and can be readily
characterized for sequence, epitope binding, and affinity. In a specific ment,
the chimeric receptor construct includes a scFV sequence from Herceptin antibody.
In other embodiments, the scFV is a human or humanized ScFv comprising a
variable light chain comprising a CDRL1 sequence, CDRL2 sequence and a CDRL3
sequence of the Herceptin antibody. In other embodiments, the scFV is a human or
humanized ScFv comprising a variable heavy chain comprising CDRH1 sequence,
CDRH2, and a CDRH3 sequence of Herceptin. The CDR sequences can readily be
determined from the amino acid sequence of Herceptin. The disclosure also
plates variable regions that have at least 90% amino acid sequence identity to
that of the scFv for Herceptin and that have at least the same affinity for Her2. In
embodiments, the chimeric receptor has a long spacer of 229 amino acids or less. In
a specific ment, the spacer is 229 amino acids and has a ce of SEQ ID
NO:50.
In embodiments, a polynucleotide coding for a ligand binding domain is
operably linked to a polynucleotide coding for a spacer . In embodiments, the
polynucleotide coding for a ligand binding domain may also have one or more
ction enzyme sites at the 5’ and/or 3’ ends of the coding sequence in order to
provide for easy excision and replacement of the polynucleotide with another
polynucleotide coding for a ligand binding domain coding for a different antigen or
that has different binding characteristics. For e, a restriction site, NheI, is
encoded upstream of the leader sequence; and a 3’ RsrII located within the hinge
region allows subcloning of any desirable scFv into a chimeric or vector. In
embodiments, the polynucleotide is codon optimized for expression in mammalian
cells.
In ments, the polynucleotide coding for a ligand binding domain is
operably linked to a signal peptide. In embodiments the signal peptide is a signal
peptide for granulocyte colony stimulating factor. cleotides coding for other
signal peptides such as CD8 alpha can be utilized.
In embodiments, the polynucleotide coding for a ligand g domain is
operably linked to a promoter. A promoter is selected that es for expression of
the chimeric antigen receptor in a mammalian cell. In a specific embodiment the
promoter is the elongation growth factor promoter (EF-1). Another example of a
suitable promoter is the ate early cytomegalovirus (CMV) promoter
sequence. However, other constitutive promoter sequences may also be used,
including, but not limited to the simian virus 40 (SV 40) early promoter, mouse
mammary tumor virus (MMTV), human immunodeficiency virus (HlV) long
terminal repeat (LTR) promoter, MuMoLV promoter, an avian leukemia virus
promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus
promoter, as well as human gene promoters such as, but not limited to, the actin
promoter, the myosin promoter, the hemoglobin er, and the creatine kinase
promoter. Inducible promoters are also contemplated. Examples of inducible
promoters include, but are not limited to a metallothionine promoter, a
glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
A specific embodiment of a polynucleotide coding for a ligand binding
domain is shown in Table 1 as the scFv from an antibody that specifically binds
CD19, such as FMC63. A cleotide ng for a flexible linker including
the amino acids GSTSGSGKPGSGEGSTKG (SEQ ID NO:36)separates the VH and
VL chains in the scFV. The amino acid sequence of the scFv including the linker is
shown in Table 2.(SEQ ID NO:11) Other argeting antibodies such as SJ25C1
and HD37 are known. (SJ25C1: Bejcek et al. Cancer Res 2005, PMID 7538901;
HD37: Pezutto et al. JI 1987, PMID 2437199).
Spacer
In embodiments, the ic receptor nucleic acid comprises a
polynucleotide coding for a spacer region. It has been surprisingly found that the
length of the spacer region that is ed not to have signaling capability affects
the in vivo efficacy of the T cells modified to express the chimeric receptor and
needs to be customized for individual target molecules for optimal tumor or target
cell recognition. In ments, the chimeric receptor nucleic acid comprises a
polynucleotide coding for a izable spacer region selected from a library of
polynucleotides coding for spacer regions. In embodiments, a spacer length is
selected based upon the location of the epitope, affinity of the antibody for the
epitope, and/or the y of the T cells expressing the chimeric receptor to
proliferate in vitro and/or in vivo in response to antigen recognition.
lly a spacer region is found between the ligand binding domain and
the transmembrane domain of the chimeric receptor. In embodiments, a spacer
region provides for flexibility of the ligand binding domain, allows for high
sion levels in lymphocytes. A CD19-specific chimeric or having a
spacer domain of about 229 amino acids had less antitumor activity than a CD19-
specific chimeric receptor with a short spacer region comprised of the modified
IgG4 hinge only. Other chimeric receptors, such as those constructed from the R12
or 2A2 scFvs also require a short spacer for optimal triggering of T cell effector
functions, while a chimeric receptor constructed with the R11 ROR1 scFv requires a
long spacer domain of about 229 amino acids for tumor recognition.
In embodiments, a spacer region has at least about 10 to 229 amino acids,
about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino
acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75
amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30
amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and
including any integer between the endpoints of any of the listed ranges. In
embodiments, a spacer region has about 12 amino acids or less, about 119 amino
acids or less, or about 229 amino acids or less.
In some embodiments, the spacer region is derived from a hinge region of an
immunoglobulin like molecule. In embodiments, a spacer region comprises all or a
portion of the hinge region from a human IgG1, human IgG2, a human IgG3, or a
human IgG4, and may contain one or more amino acid substitutions. Exemplary
sequences of the hinge regions are provided in Table 8. In embodiments, a portion
of the hinge region includes the upper hinge amino acids found between the variable
heavy chain and the core, and the core hinge amino acids including a oline
region. Typically, the upper hinge region has about 3 to 10 amino acids. In some
cases, the spacer region comprises an amino acid sequence of X1PPX2P(SEQ ID
NO:1). In embodiments, X1 is a ne, glycine, or arginine and X2 is a cysteine or
a threonine.
In embodiments, hinge region sequences can be modified in one or more
amino acids in order to avoid undesirable structural interactions such as
dimerization. In a specific embodiment, the spacer region comprises a portion of a
modified human hinge region from IgG4, for example, as shown in Table 2 or Table
8(SEQ ID . A representative of a polynucleotide coding for a portion of a
modified IgG4 hinge region is ed in Table 1. (SEQ ID NO:4)In embodiments,
a hinge region can have at least about 90%, 92%, 95%, or 100% sequence identity
with a hinge region amino acid sequence identified in Table 2 or Table 8. In a
ic embodiment, a n of a human hinge region from IgG4 has an amino
acid substitution in the core amino acids from CPSP to CPPC.
In some embodiments, all or a n of the hinge region is ed with
one or more domains of a constant region of an immunoglobulin. For example, a
portion of a hinge region can be combined with all or a n of a CH2 or CH3
domain or variant thereof. In embodiments, the spacer region does not include the
47-48 amino acid hinge region sequence from CD8apha or the spacer region
consisting of an extracellular portion of the CD28 molecule.
In embodiments, a short spacer region has about 12 amino acids or less and
comprises all or a portion of a IgG4 hinge region sequence or variant thereof, an
intermediate spacer region has about 119 amino acids or less and comprises all or a
portion of a IgG4 hinge region sequence and a CH3 region or variant thereof, and a
long spacer has about 229 amino acids or less and comprises all or a portion of a
IgG4 hinge region sequence , a CH2 region, and a CH3 region or variant thereof.
A polynucleotide coding for a spacer region can be readily prepared by
synthetic or inant methods from the amino acid sequence. In embodiments, a
polynucleotide coding for a spacer region is operably linked to a polynucleotide
coding for a embrane region. In embodiments, the polynucleotide coding for
the spacer region may also have one or more restriction enzyme sites at the 5’ and/or
3’ ends of the coding ce in order to provide for easy excision and replacement
of the polynucleotide with another polynucleotide coding for a different spacer
region. In embodiments, the polynucleotide coding for the spacer region is codon
optimized for expression in mammalian cells.
In embodiments, a library of polynucleotides, each coding for different
spacer region is described. In an ment, the spacer region is selected from the
group consisting of a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 or
portion thereof, a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in
combination with all or a portion of a CH2 region or variant thereof, a hinge region
ce from IgG1, IgG2, IgG3, or IgG4 in combination with all or a n of a
CH3 region or variant thereof, and a hinge region sequence from IgG1, IgG2, IgG3,
or IgG4 in combination with all or a portion of a CH2 region or variant thereof, and
a CH3 region or variant thereof. In embodiments, a short spacer region is a modified
IgG4 hinge sequence(SEQ ID NO:4) having 12 amino acids or less, an intermediate
ce is a IgG4 hinge sequence with a CH3 sequence having 119 amino acids or
less(SEQ ID NO:49); or a IgG4 hinge ce with a CH2 and CH3 region having
229 amino acids or less (SEQ ID NO:50)
In embodiments, a method of selecting a spacer region for a chimeric
receptor is described herein. Surprisingly some chimeric receptor constructs,
although effective to activate T cells and direct their killing of tumor cells in vitro,
were not ive in vivo. In addition, the side effect profile of the chimeric receptor
modified T cells can be such as to result in more cells undergoing activation induced
cell death or causing an se in in vivo cytokines. In embodiments, a method
comprises providing a plurality of chimeric receptor nucleic acids, wherein the
chimeric receptor nucleic acids differ only in the spacer region; introducing each of
the chimeric receptor nucleic acids into a separate T lymphocyte population;
expanding each separate lymphocyte population in vitro, and introducing each
lymphocyte population into an animal bearing a tumor to determine the anti-tumor
efficacy of each of the chimeric receptors when expressed in T cells, and ing a
chimeric receptor that provides anti-tumor efficacy as compared to each of the other
separate lymphocyte populations modified with each of the other chimeric receptors.
Animal models of ent tumors are known. Anti-tumor efficacy can be
measured by identifying a decrease in tumor volume, by determining animal death,
persistence of the genetically modified T cells in vivo, activation of genetically
modified T cells (for example, by detecting an increase in expression of CD25
and/CD69), and/or proliferation of genetically modified T cells in vivo. In an
ment, a chimeric or is selected that provides for the best umor
efficacy in vivo as determined by one or more of these parameters. Lack of antitumor
efficacy can be ined by lack of persistence of the genetically modified
lymphocytes in vivo, animal death, an increase in apoptosis as measured by an
increase in induction of caspase -3, and/or a decrease in proliferation of genetically
modified lymphocytes.
In other ments, a method for selecting a spacer comprises selecting an
e of a target molecule and characterizing the location of the epitope with
t to the cell membrane, selecting a spacer region that is long or short
depending on the location of the e with respect to the cell membrane, selecting
an dy or fragment thereof that has an affinity for the epitope that is higher or
lower as compared to a reference antibody, and determining whether the chimeric
receptor construct provides for ed T cell proliferation or cytokine production
in vitro and/or in vivo.
In some embodiments, if the target epitope or portion f is located
proximal to the ne it is located in the first 100 amino acids of the linear
sequence of the ellular domain adjacent to the transmembrane domain. If the
epitope is d proximal to the membrane, a long spacer (e.g. 229 amino acids or
less and greater than 119 amino acids) is selected. In some embodiments, if the
target epitope is located distal to the ne, it is located in the first 150 amino
acids of the linear sequence of the extracellular domain terminus. If the epitope is
located distal to the membrane, an intermediate or short spacer is selected (e.g. 119
amino acids or less or 12-15 amino acids or less). atively, whether the epitope
is proximal or distal to the membrane can be determined by modeling of the three
dimensional structure or based on analysis of the crystal structure,
In some embodiments, a chimeric receptor is selected that provides for at
least 30% of the cells proliferating through two generations in vitro and/or in vivo.
In other embodiments a chimeric or is not selected if it results in at least 50%
of the cells undergoing activation induced cell death in 72 hours. In embodiments, a
short spacer (e.g. 15 amino acids or less) is selected if the epitope is distal to the
membrane. In embodiments, a long spacer (e.g. 229 amino acid or less and greater
than 119 amino acids) is selected if the epitope is proximal to the membrane.
In embodiments, providing a plurality of chimeric receptor nucleic acids,
wherein the chimeric receptor nucleic acids differ only in the spacer region
comprises providing a chimeric receptor construct comprising a polynucleotide
coding for a ligand binding domain, n the ligand is a tumor specific antigen,
viral antigen, or any other molecule expressed on a target cell population that is
le to mediate recognition and elimination by a lymphocyte; a polynucleotide
coding for a first polypeptide spacer having a d restriction site at the 5’ and 3’
end of the coding sequence for the first polypeptide spacer; a polynucleotide coding
for a transmembrane domain; and a polynucleotide coding for one or more
intracellular signaling domains.
In embodiments, a method further comprises providing one or more
polynucleotides, each encoding a different spacer region. In embodiments, the
different spacer regions are selected from the group consisting of a hinge region
sequence from IgG1, IgG2, IgG3, or IgG4 or variant thereof or portion thereof, a
hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in combination with all or a
portion of a CH2 region or variant thereof, a hinge region sequence from IgG1,
IgG2, IgG3, or IgG4 in combination with all or a portion of a CH3 region or variant
thereof, and a hinge region sequence from IgG1, IgG2, IgG3, or IgG4 in
combination with all or a portion of a CH2 region or variant thereof and a CH3
region or variant thereof. In embodiments, CH2 or CH3 regions may be modified by
one or more deletions or amino acid substitutions in order to provide for expression
in lymphocytes and/or in order to minimize interactions with other molecules. In
embodiments, a portion of a hinge region comprises at least the upper amino acids
and the core sequence. In embodiments, a hinge region comprises the sequence
In embodiments, a method r comprises replacing the polynucleotide
coding for the spacer region with a polynucleotide encoding a different spacer
region to form a ic receptor nucleic acid with a different spacer . The
method can be repeated to form any number of chimeric receptor nucleic acids,
each differing in the spacer region. In ments, the chimeric receptor nucleic
acids differ from one another only in the spacer region.
embrane domain
In embodiments, the chimeric receptor nucleic acid comprises a
polynucleotide coding for a transmembrane domain. The transmembrane domain
provides for anchoring of the chimeric receptor in the membrane.
In an embodiment, the transmembrane domain that naturally is associated
with one of the domains in the chimeric receptor is used. In some cases, the
transmembrane domain can be selected or modified by amino acid substitution to
avoid binding of such domains to the transmembrane domains of the same or
different surface membrane ns to minimize interactions with other members of
the receptor complex.
The transmembrane domain may be d either from a natural or a
synthetic source. When the source is natural, the domain may be derived from any
ne-bound or transmembrane n. embrane regions comprise at
least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell
receptor, CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22; CD33, CD37, CD64,
CD80, CD86, CD134, CD137 and CD154. In a specific embodiment, the
embrane domain comprises the amino acid sequence of the CD28
transmembrane domain as shown in Table 2. A representative polynucleotide
sequence coding for the CD28 transmembrane domain is shown in Table 1(SEQ ID
NO:5).
A transmembrane domain may be synthetic or a variant of a naturally
ing transmembrane . In embodiments, synthetic or variant
transmembrane domains comprise predominantly hydrophobic residues such as
leucine and valine. In embodiments, a transmembrane domain can have at least
about 80%, 85%, 90%, 95%, or 100% amino acid sequence ty with a
transmembrane domain as shown in Table 2 or Table 6. Variant transmembrane
domains preferably have a hydrophobic score of at least 50 as calculated by Kyte
Doolittle.
A polynucleotide coding for a transmembrane domain can be y
prepared by synthetic or recombinant s. In ments, a polynucleotide
coding for a transmembrane domain is operably linked to a polynucleotide coding
for a intracellular signaling region. In embodiments, the polynucleotide coding for a
transmembrane domain may also have one or more restriction enzyme sites at the 5’
and/or 3’ ends of the coding sequence in order to provide for easy excision and
replacement of the polynucleotide coding for a transmembrane domain with another
polynucleotide coding for a different transmembrane domain. In embodiments, the
polynucleotide coding for a transmembrane domain is codon optimized for
expression in mammalian cells.
ellular signaling domain
In embodiments, the chimeric receptor nucleic acid comprises a
polynucleotide coding for an intracellular signaling domain. The intracellular
signaling domain es for activation of one function of the transduced cell
expressing the chimeric receptor upon binding to the ligand expressed on tumor
cells. In embodiments, the intracellular signaling domain contains one or more
intracellular signaling domains. In embodiments, the intracellular signaling domain
is a n of and/or a variant of an intracellular ing domain that provides for
activation of at least one function of the transduced cell.
Examples of ellular signaling domains for use in a chimeric receptor of
the disclosure include the cytoplasmic sequences of the CD3 zeta chain, and/or coreceptors
that act in t to initiate signal transduction following chimeric
or engagement, as well as any derivative or variant of these sequences and any
synthetic sequence that has the same functional lity. T cell activation can be
said to be mediated by two distinct classes of cytoplasmic signaling sequence: those
that initiate antigen-dependent primary activation and provide a T cell receptor like
signal (primary cytoplasmic signaling sequences) and those that act in an antigenindependent
manner to provide a secondary or co-stimulatory signal (secondary
cytoplasmic signaling sequences). Primary cytoplasmic ing sequences that act
in a atory manner may contain signaling motifs which are known as receptor
tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary
asmic signaling sequences include those derived from CD3 zeta, FcR gamma,
CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In
embodiments, the primary ing intracellular domain can have at least about
80%, 85%, 90%, or 95% sequence identity to CD3zeta having a sequence ed
in Table 2. In embodiments variants, of CD3 zeta retain at least one, two, three or
all ITAM regions as shown in Table 7.
In a red embodiment, the intracellular signaling domain of the chimeric
receptor can be designed to se the CD3-zeta signaling domain by itself or
combined with any other desired cytoplasmic domain(s). For example, the
intracellular signaling domain of the chimeric receptor can comprise a CD3zeta
chain and a costimulatory signaling region.
The costimulatory signaling region refers to a portion of the chimeric
receptor comprising the intracellular domain of a costimulatory molecule. A
costimulatory le is a cell surface molecule other than an antigen receptor or
their ligands that is required for a response of lymphocytes to an antigen. es
of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40,
cyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds with CD83. In embodiments, the
costimulatory signaling domain can have at least about 80%, 85%, 90%, or 95%
amino acid sequence identity to the intracellular domain of CD28 as shown in Table
or to 4-1BB having a sequence provided in Table 2. In an embodiment, a variant
of the CD28 intracellular domain comprises an amino acid substitution at positions
186-187, n LL is substituted with GG.
The intracellular signaling sequences of the chimeric receptor may be linked
to each other in a random or specified order. ally, a short oligo- or
ptide linker, preferably between 2 and 10 amino acids in length may form the
linkage. In one embodiment, the intracellular signaling domains comprises all or a
portion of the signaling domain of CD3-zeta or variant f and all or a portion of
the signaling domain of CD28 or a t thereof. In r embodiment, the
ellular signaling domain comprises all or a portion of the signaling domain of
CD3-zeta or variant thereof and all or a portion of the signaling domain of 4-lBB or
variant f. In yet another embodiment, the intracellular signaling domain
comprises all or a portion of the signaling domain of CD3-zeta or variant thereof, all
or a portion of the signaling domain of CD28 or variant thereof, and all or a portion
of the signaling domain of 4-lBB or variant thereof. In a specific embodiment, the
amino acid sequence of the intracellular signaling domain comprising a variant of
CD3zeta and a portion of the 4-1BB intracellular signaling domain is provided in
Table 2. A representative nucleic acid sequence is provided in Table 1(SEQ ID
NO:6; SEQ ID NO:7).
In an embodiment, a polynucleotide coding for an intracellular signaling
domain comprises a 4-1BB intracellular domain linked to a portion of a CD3zeta
domain. In other embodiments, a 4-1BB intracellular domain and a CD28
intracellular domain are linked to a portion of a CD3 zeta domain.
A polynucleotide coding for an intracellular signaling domain can be readily
prepared by synthetic or inant methods from the amino acid sequence. In
embodiments, the cleotide coding for an intracellular signaling domain may
also have one or more restriction enzyme sites at the 5’ and/or 3’ ends of the coding
sequence in order to provide for easy excision and replacement of the polynucleotide
coding for an intracellular signaling domain with another polynucleotide coding for
a different intracellular signaling domain. In ments, the polynucleotide
coding for an intracellular signaling domain is codon optimized for sion in
mammalian cells.
Marker sequences
In embodiments, the chimeric receptor nucleic acid ally further
comprises a polynucleotide sequence coding for a marker sequence. A marker
sequence can provide for selection of transduced cells, and identification of
transduced cells. In embodiments, the marker sequence is operably linked to a
polynucleotide sequence coding for a linker ce. In embodiments, the linker
ce is a cleavable linker sequence.
A number of different marker sequences can be employed. Typically a
marker sequence has a functional characteristic that allows for selection of
transduced cells and/or detection of transduced cells. In ments, the marker
sequence is ible with transduction of human lymphocytes.
The positive selectable marker may be a gene, which upon being introduced
into the host cell, expresses a dominant phenotype permitting positive selection of
cells carrying the gene. Genes of this type are known in the art, and include, inter
alia, hygromycin-B phosphotransferase gene (hph) which confers resistance to
hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5
which codes for resistance to the otic G418, the dihydrofolate reductase
(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance
(MDR) gene.
In an ment, a chimeric receptor nucleic acid further ses a
polynucleotide coding for a marker sequence. In an embodiment, the marker
sequence is a ted epidermal growth factor receptor as shown in Table 2. An
exemplary polynucleotide for the truncated epidermal growth factor receptor is
shown in Table 1. (SEQ ID NO:9)In embodiments, the polynucleotide coding for the
marker ce is operably linked to a polynucleotide coding for a linker sequence.
In a specific embodiment, the linker sequence is a cleavable linker sequence T2A as
shown in Table 2. An exemplary polynucleotide sequence coding for the T2A linker
is ed in Table 1.(SEQ ID NO:8)
A polynucleotide coding for marker sequence can be readily prepared by
synthetic or recombinant methods from the amino acid sequence. In embodiments a
polynucleotide coding for a marker sequence is operably linked to a polynucleotide
coding for an intracellular signaling domain. In embodiments, the polynucleotide
coding for a marker ce may also have one or more restriction enzyme sites at
the 5’ and/or 3’ ends of the coding sequence in order to provide for easy excision
and replacement of the polynucleotide coding for a marker sequence with another
polynucleotide coding for a different marker sequence. In embodiments, the
polynucleotide coding for a marker sequence is codon optimized for expression in
mammalian cells.
Vectors, Cells and Methods of transducing cells
Selection and Sorting of T cyte populations
The compositions described herein provide for CD4+ and/or CD8+ T
lymphocytes. T lymphocytes can be collected in accordance with known techniques
and enriched or depleted by known techniques such as affinity binding to antibodies
such as flow cytometry and/or immunomagnetic selection. After ment and/or
depletion steps, in vitro expansion of the desired T lymphocytes can be carried out in
accordance with known techniques ding but not limited to those described in
US Patent No. 6,040,177 to Riddell et al.), or variations thereof that will be apparent
to those skilled in the art. In embodiments, the T cells are gous T cells
obtained from the patient.
For example, the d T cell tion or subpopulation may be
expanded by adding an initial T lymphocyte tion to a culture medium in vitro,
and then adding to the culture medium feeder cells, such as non-dividing eral
blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells
contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T
lymphocyte in the initial population to be expanded); and incubating the culture (e.g.
for a time sufficient to expand the numbers of T cells). The non-dividing feeder cells
can se gamma-irradiated PBMC feeder cells. In some ments, the
PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to
prevent cell division. The order of addition of the T cells and feeder cells to the
culture media can be reversed if desired. The culture can typically be incubated
under conditions of temperature and the like that are le for the growth of T
lymphocytes. For the growth of human T lymphocytes, for example, the temperature
will generally be at least about 25 degrees Celsius, preferably at least about 30
degrees, more preferably about 37 degrees.
The T lymphocytes expanded include CD8+ xic T lymphocytes (CTL)
and CD4+ helper T lymphocytes that may be specific for an antigen present on a
human tumor or a pathogen.
Optionally, the expansion method may further comprise the step of adding
non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can
be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL
feeder cells may be provided in any suitable amount, such as a ratio of LCL feeder
cells to initial T lymphocytes of at least about 10:1.
Optionally, the expansion method may further comprise the step of adding
anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a tration
of at least about 0.5 . ally, the expansion method may further comprise
the step of adding IL-2 and/or IL-15 to the culture medium (e.g., wherein the
concentration of IL-2 is at least about 10 units/m1).
After isolation of T lymphocytes both cytotoxic and helper T lymphocytes
can be sorted into naïve, memory, and effector T cell subpopulations either before or
after expansion.
CD8+ cells can be obtained by using standard methods. In some
embodiments, CD8+ cells are further sorted into naïve, central memory, and effector
memory cells by identifying cell surface antigens that are associated with each of
those types of CD8+ cells. In embodiments, memory T cells are t in both
CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC are
sorted into CD62L-CD8+ and CD62L+CD8+ fractions after ng with D8
and anti-CD62L antibodies. In some embodiments, the expression of phenotypic
s of l memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and
CD127 and are negative or low for granzyme B. In some embodiments, central
memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments,
effector TE are negative for CD62L, CCR7, CD28, and CD127, and positive for
granzyme B and perforin. In some ments, naïve CD8+ T lymphocytes are
characterized by the expression of phenotypic s of naïve T cells including
CD62L, CCR7, CD28, CD3, CD127, and CD45RA.
Whether a cell or cell population is positive for a particular cell surface
marker can be determined by flow cytometry using staining with a specific antibody
for the surface marker and an isotype matched control antibody. A cell population
negative for a marker refers to the absence of significant staining of the cell
population with the specific antibody above the isotype control, positive refers to
uniform ng of the cell tion above the isotype control. In some
embodiments, a decrease in expression of one or markers refers to loss of 1 log10 in
the mean fluorescence intensity and/or decrease of percentage of cells that exhibit
the marker of at least about 20% of the cells, 25% of-the cells, 30% of the cells, 35%
of the cells, 40% of the cells, 45% of the cells, 50% of the cells, 55% of the cells,
60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the
cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells and
any % between 20 and 100% when compared to a reference cell population. In
some embodiments, a cell population positive for one or markers refers to a
tage of cells that exhibit the marker of at least about 50% of the cells, 55% of
the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80%
of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the
cells and any % between 50 and 100% when compared to a reference cell
population.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells
by identifying cell populations that have cell surface antigens. CD4+ lymphocytes
can be obtained by standard methods. In some embodiments, naïve CD4+ T
lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some
embodiments, central memory CD4+ cells are CD62L+ and +. In some
embodiments, effector CD4+ cells are CD62L- and CD45RO-.
In embodiments, populations of CD4+ and CD8+ that are antigen specific
can be obtained by stimulating naïve or antigen specific T cytes with n.
For e, antigen-specific T cell lines or clones can be generated to
Cytomegalovirus antigens by isolating T cells from infected subjects and stimulating
the cells in vitro with the same antigen. Naïve T cells may also be used. Any
number of antigens from tumor cells may be utilized as targets to elicit T cell
responses. In some embodiments, the adoptive cellular immunotherapy
compositions are useful in the ent of a disease or disorder including a solid
tumor, hematologic malignancy, breast cancer or melanoma.
Modification of T lymphocyte populations
In some embodiments it may be desired to introduce functional genes into
the T cells to be used in therapy in accordance with the present disclosure.
For example, the introduced gene or genes may improve the efficacy of therapy by
ing the ity and/or function of transferred T cells; or they may provide a
genetic marker to permit selection and/or evaluation of in vivo survival or migration;
or they may incorporate functions that improve the safety of immunotherapy, for
example, by making the cell susceptible to negative selection in vivo as described by
Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene
Therapy 3:319-338 (1992); see also the publications of 91/08442 and
94/05601 by Lupton et al. describing the use of tional selectable
fusion genes derived from fusing a dominant ve selectable marker with a
negative selectable marker. This can be carried out in accordance with known
techniques (see, e.g., US Patent No. 6,040,177 to Riddell et al. at columns 14-17) or
variations thereof that will be apparent to those skilled in the art based upon the
present disclosure.
In embodiments, T cells are modified with chimeric receptors as described
herein. In some embodiments, the T cells are obtained from the subject to be
treated, in other embodiments, the lymphocytes are obtained from allogeneic human
donors, ably healthy human donors.
In some embodiments, chimeric receptors comprise a ligand binding domain
that specifically binds to a tumor cell surface molecule, a polypeptide spacer region,
a transmembrane domain and an intracellular signaling domain as described herein.
In embodiments, the ligand binding domain is a single-chain dy fragment
(scFv) that is derived from the variable heavy (VH) and variable light (VL) chains of
a monoclonal antibody (mAb). Costimulatory signals can also be provided through
the chimeric receptor by fusing the ulatory domain of CD28 and/or 4-1BB to
the CD3? chain. Chimeric receptors are specific for cell surface molecules
independent from HLA, thus overcoming the limitations of TCR-recognition
including HLA-restriction and low levels of HLA-expression on tumor cells.
Chimeric receptors can be constructed with a specificity for any cell surface
marker by ing n binding fragments or dy variable domains of, for
example, antibody molecules. The n binding molecules can be linked to one or
more cell signaling modules. In embodiments, cell signaling modules include CD3
transmembrane domain, CD3 intracellular signaling domains, and CD28
transmembrane domains. In ments, the intracellular signaling domain
comprises a CD28 embrane and signaling domain linked to a CD3 zeta
intracellular domain. In some embodiments, a chimeric receptor can also include a
transduction marker such as tEGFR.
In embodiments, the same or a different chimeric receptor can be introduced
into each of population of CD4+ and CD8+ T lymphocytes. In embodiments, the
ic receptor in each of these populations has a ligand binding domain that
specifically binds to the same ligand on the tumor or infected cell. The cellular
signaling modules can differ. In embodiments, the intracellular signaling domain of
the CD8+ cytotoxic T cells is the same as the intracellular signaling domain of the
CD4+ helper T cells. In other embodiments, the intracellular signaling domain of the
CD8+ xic T cells is different than the intracellular signaling domain of the
CD4+ helper T cells.
In embodiments each of the CD4 or CD8 T lymphocytes can be sorted in to
naïve, central memory, effector memory or effector cells prior to transduction as
described herein. In alternative ments, each of the CD4 or CD8 T
lymphocytes can be sorted in to naïve, central memory, effector memory, or effector
cells after transduction.
Various transduction ques have been developed which utilize
inant ious virus particles for gene delivery. This ents a currently
preferred approach to the transduction of T lymphocytes described herein. The viral
vectors which have been used in this way include virus vectors derived from simian
virus 40, adenoviruses, adeno-associated virus (AAV), lentiviral vectors, and
retroviruses. Thus, gene transfer and expression methods are numerous but
essentially function to uce and express genetic material in mammalian cells.
Several of the above techniques have been used to transduce hematopoietic or
lymphoid cells, including calcium ate transfection, protoplast fusion,
electroporation, and infection with recombinant adenovirus, associated virus
and retrovirus vectors. Primary T lymphocytes have been successfully transduced by
electroporation and by retroviral or iral infection.
Retroviral and iral vectors provide a highly ent method for gene
transfer into eukaryotic cells. Moreover, retroviral or lentiviral integration takes
place in a controlled fashion and results in the stable integration of one or a few
copies of the new genetic information per cell.
It is contemplated that overexpression of a stimulatory factor (for example, a
lymphokine or a cytokine) may be toxic to the treated dual. Therefore, it is
within the scope of the present disclosure to include gene segments that cause the T
cells described herein to be susceptible to ve selection in vivo. By ''negative
selection" is meant that the infused cell can be eliminated as a result of a change in
the in vivo condition of the individual. The negative selectable phenotype may result
from the insertion of a gene that confers sensitivity to an stered agent, for
example, a compound. Negative selectable genes are known in the art, and include,
inter alia the following: the Herpes simplex virus type I thymidine kinase (HSV-I
TK) gene, which confers ganciclovir sensitivity; the cellular hypoxanthine
phosphribosyltransferase (HPRT) gene, the cellular adenine
oribosyltransferase (APRT) gene, and bacterial cytosine deaminase,
In some embodiments it may be useful to include in the T cells a positive
marker that enables the selection of cells of the negative selectable phenotype in
vitro. The ve selectable marker may be a gene that upon being introduced into
the host cell expresses a dominant phenotype permitting positive selection of cells
carrying the gene. Genes of this type are known in the art, and include, inter alia,
hygromycin-B phosphotransferase gene (hph) which s resistance to
hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5
which codes for resistance to the antibiotic G418, the dihydrofolate reductase
(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance
(MDR) gene.
A variety of methods can be employed for transducing T lymphocytes, as is
well known in the art. In embodiments, transduction is carried out using lentiviral
vectors.
In embodiments, CD4+ and CD8+ cells each can separately be modified with
an sion vector encoding a chimeric receptor to form defined populations. In
embodiments, these cells are then further sorted into subpopulations of naïve, central
memory and effector cells as described above by sorting for cell surface ns
unique to each of those cell populations. In addition, CD4+ or CD8+ cell
tions may be selected by their cytokine profile or erative activities. For
example, CD4+ T lymphocytes that have ed production of cytokines such as
IL-2, IL-4, IL-10, TNFa, and IFN? as compared to sham transduced cells or
transduced CD8+ cells when stimulated with antigen can be selected. In other
embodiments, naïve or central memory CD4+ T cells that have enhanced production
of IL-2 and/or TNFa are selected. Likewise, CD8+ cells that have enhanced IFN?
production are selected as ed to sham transduced CD8+ cells.
In embodiments, CD4+ and lls that proliferate in response to n
or tumor targets are selected. For example, CD4+ cells that proliferate vigorously
when stimulated with antigen or tumor targets as compared to sham uced cells,
or CD8+ transduced cells are selected. In some embodiments, CD4+ and CD8+ cells
are selected that are xic for antigen g cells. In embodiments, CD4+ are
expected to be weakly cytotoxic as compared to CD8+ cells.
In a preferred embodiment, transduced lymphocytes, such as CD8+ central
memory cells, are selected that provide for tumor cell killing in vivo using an animal
model established for the particular type of cancer. Such animal models are known
to those of skill in the art and exclude human beings. As described herein, not all
chimeric or ucts transduced into lymphocytes confer the ability to kill
tumor cells in vivo despite the ability to become activated and kill tumor cells in
vitro. In particular, for some target molecules T cells having chimeric receptor
constructs with a long spacer region were less ive at killing tumor cells in vivo
as compared to T cells having a chimeric or with short spacer region. For other
target molecules, T cells having chimeric receptor constructs with a short spacer
region were less effective at killing tumor cells in vivo as compared to T cells
having chimeric receptors with a long spacer .
In yet other embodiments, transduced chimeric receptor expressing T cells
are selected that can persist in vivo using an animal model established for the
particular type of cancer. In embodiments, transduced chimeric receptor CD8+
central memory cells with a short spacer region have been shown to persist in vivo
after introduction into the animal for about 3 day or more, 10 days or more, 20 days
or more, 30 days or more, 40 days or more, or 50 days or more.
The sure contemplates that combinations of CD4+ and CD8+ T cells
will be utilized in the compositions. In one embodiment, combinations of chimeric
receptor transduced CD4+ cells can be combined with chimeric receptor transduced
CD8+ cells of the same ligand specificity or combined with CD8+ T cells that are
specific for a distinct tumor ligand. In other embodiments, chimeric receptor
transduced CD8+ cells are combined with chimeric receptor transduced CD4+ cells
specific for a different ligand expressed on the tumor. In yet another ment,
chimeric receptor ed CD4+ and CD8+ cells are ed. In embodiments
CD8+ and CD4+ cells can be combined in different ratios for example, a 1:1 ratio of
CD8+ and CD4+, a ratio of 10:1 of CD8+ to CD4+, or a ratio of 100:1 of CD8+ to
CD4+. In embodiments, the combined tion is tested for cell proliferation in
vitro and/or in vivo, and the ratio of cells that provides for proliferation of cells is
selected.
As described herein, the disclosure contemplates that CD4+ and CD8+ cells
can be r separated into subpopulations, such as naïve, central memory, and
effector memory cell populations. As described herein, in some ments, naïve
CD4+ cells are CD45RO-, CD45RA+, CD62L+, CD4+ ve T cells. In some
embodiments, central memory CD4+ cells are CD62L positive and CD45RO
positive. In some embodiments, effector CD4+ cells are CD62L negative and
CD45RO positive. Each of these populations may be independently modified with a
chimeric receptor.
As described herein, in embodiments, memory T cells are present in both
CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC are
sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8
and D62L antibodies. In some embodiments, the expression of phenotypic
markers of central memory T cells (TCM) include CD62L, CCR7, CD28, CD3, and
CD127 and are negative or low for granzyme B. In some embodiments, central
memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments,
effector T cells (TE) are negative for CD62L, CCR7, CD28, and CD127, and positive
for granzyme B and perforin. In some embodiments, naïve CD8+ T lymphocytes are
characterized by CD8+, CD62L+, CD45RO+, CCR7+, CD28+ CD127+, and
CD45RO+. Each of these populations may be independently modified with a
chimeric or .
After transduction and/or selection for chimeric receptor bearing cells, the
cell populations are preferably expanded in vitro until a sufficient number of cells
are obtained to provide for at least one infusion into a human t, typically
around 104 cells/kg to 109 cells/kg In embodiments, the transduced cells are cultured
in the presence of antigen bearing cells, anti CD3, anti CD28, and IL 2, IL-7, IL 15,
IL-21 and combinations thereof.
Each of the subpopulations of CD4+ and CD8+ cells can be combined with
one another. In a ic embodiment, modified naïve or central memory CD4+
cells are combined with modified central memory CD8+ T cells to provide a
synergistic cytotoxic effect on antigen bearing cells, such as tumor cells.
Compositions
Also described is an adoptive cellular immunotherapy ition
comprising a genetically modified T cyte cell preparation as described
herein.
In embodiments, the T lymphocyte cell preparation comprises CD4 + T cells
that have a chimeric receptor comprising an ellular antibody variable domain
ic for a ligand associated with the disease or disorder, a customizable spacer
region, a embrane domain, and an intracellular signaling domain of a T cell
or or other ors as described herein. In other embodiments, an adoptive
cellular immunotherapy composition further comprises a chimeric receptor modified
tumor-specific CD8+ cytotoxic T lymphocyte cell preparation that provides a
cellular immune response, wherein the cytotoxic T lymphocyte cell preparation
comprises CD8+ T cells that have a ic or comprising an extracellular
single chain antibody specific for a ligand associated with the disease or disorder, a
customizable spacer region, a transmembrane domain, and an intracellular signaling
domain of a T cell receptor as described . In embodiments, the chimeric
receptor modified T cell population of the disclosure can persist in vivo for at least
about 3 days or longer.
In some embodiments, an adoptive cellular therapy composition
comprises a chimeric receptor modified tumor-specific CD8+ cytotoxic T
lymphocyte cell preparation that provides a cellular immune response, wherein the
xic T lymphocyte cell preparation comprises CD8+ T cells that have a
chimeric receptor comprising an extracellular single chain antibody specific for a
ligand associated with the disease or disorder, a customizable spacer region, a
transmembrane domain, and an intracellular signaling domain of a T cell receptor, in
combination with an antigen-reactive ic receptor modified naïve CD4+ T
helper cell derived from CD45RO- CD62L+ CD4+ T cells, and a pharmaceutically
acceptable carrier.
In other embodiments, an adoptive cellular immunotherapy composition
ses an antigen specific CD8+ cytotoxic T lymphocyte cell preparation that
provides a cellular immune response derived from the patient combined with an
antigen-reactive chimeric receptor modified naïve CD4+ T helper cell that ts
the CD8+ immune response, wherein the helper T lymphocyte cell preparation
comprises CD4 + T cells that have a chimeric receptor comprising an extracellular
antibody le domain specific for the antigen associated with the disease or
disorder, a customizable spacer , a transmembrane domain, and an
intracellular signaling domain of a T cell receptor.
In a r embodiment, an adoptive cellular immunotherapy ition
comprises an antigen-reactive chimeric receptor modified naïve CD4+ T helper cell
that ts the CD8+ immune response, wherein the helper T lymphocyte cell
preparation comprises CD4 + T cells that have a chimeric or comprising an
extracellular dy variable domain ic for a ligand associated with a disease
or disorder, a customizable spacer region, a transmembrane domain, and an
intracellular signaling domain of a T cell receptor.
In embodiments, the CD4+ T helper lymphocyte cell is selected from the
group consisting of naïve CD4+ T cells, central memory CD4+ T cells, effector
memory CD4+ T cells, or bulk CD4+ T cells. In some embodiments, CD4+ helper
lymphocyte cell is a naïve CD4+ T cell, wherein the naïve CD4+ T cell comprises a
CD45RO-, CD45RA+, CD62L+ CD4+ T cell. In embodiments, the CD8+ T
cytotoxic lymphocyte cell is selected from the group consisting of naïve CD8+ T
cells, central memory CD8+ T cells, effector memory CD8+ T cells or bulk CD8+ T
cells. In some embodiments, the CD8+ cytotoxic T lymphocyte cell is a central
memory T cell wherein the central memory T cell comprises a CD45RO+, CD62L+,
CD8+ T cell. In yet other embodiments, the CD8+ cytotoxic T lymphocyte cell is a
central memory T cell and the CD4+ helper T lymphocyte cell is a naïve or central
memory CD4+ T cell.
Methods
Also described are methods of making adoptive immunotherapy
compositions and uses or methods of using these compositions for performing
cellular immunotherapy in a subject having a disease or er. In embodiments,
the chimeric receptor modified T cells as bed herein are able to persist in vivo
for at least 3 days, or at least 10 days. In embodiments, the chimeric receptor
modified T cells as described herein can proliferate in vivo through at least 2, or at
least 3 generations as determined by CFSE dye dilution. Proliferation and
persistence of the chimeric receptor modified T cells can be determined by using an
animal model of the disease or disorder and administering the cells and determining
persistence and/ or proliferative capacity of the transferred cells. In other
embodiments, eration and activation can be tested in vitro by going through
multiple cycles of activation with antigen bearing cells.
In embodiments, a method of manufacturing the compositions comprises
obtaining a ed naïve CD4+ T helper cell, n the ed helper T
lymphocyte cell preparation comprises CD4+ T cells that have a chimeric receptor
comprising a ligand binding domain specific for a tumor cell surface molecule, a
customized spacer domain, a transmembrane domain, and an intracellular signaling
domain as described .
In another embodiment, a method further comprises obtaining a modified
CD8+ cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cell
ation comprises CD8+ cells that have a chimeric receptor comprising a ligand
binding domain specific for a tumor cell surface molecule, a ized spacer
, a transmembrane domain, and an intracellular ing domain as
described herein.
In another embodiment, a method ses obtaining a modified CD8+
cytotoxic T cell, wherein the modified cytotoxic T lymphocyte cell preparation
comprises CD8+ T cells that have a chimeric receptor comprising a ligand binding
domain specific for a tumor cell e molecule, a customized spacer domain, a
transmembrane domain, and an intracellular signaling domain as described herein,
and further comprising combining the modified CD8+ cytotoxic T cells with a
CD4+ helper cell lymphocyte cell preparation.
The preparation of the CD4+ and CD8+ cells that are modified with a
chimeric or has been described above as well as in the examples. Antigen
specific T lymphocytes can be obtained from a patient having the disease or disorder
or can be prepared by in vitro ation of T lymphocytes in the presence of
n. Subpopulations of CD4+ and CD8+ T lymphocytes that are not selected for
antigen specificity can also be isolated as described herein and combined in the
methods of manufacturing. In ments, the combination of cell populations can
be evaluated for uniformity of cell e makers, the ability to proliferate through
at least two generations, to have a uniform cell differentiation status. Quality control
can be performed by coculturing an cell line expressing the target ligand with
chimeric receptor modified T cells to ine if the chimeric receptor modified T
cells recognize the cell line using cytotoxicity, proliferation, or cytokine production
assays that are known in the field. Cell differentiation status and cell surface markers
on the chimeric receptor ed T cells can be determined by flow cytometry. In
embodiments, the markers and cell differentiation status on the CD8+ cells include
CD3, CD8, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4, CD45RO, and
CD45RA. In embodiments, the markers and the cell differentiation status on the
CD4+ cells include CD3, CD4, CD62L, CD28, CD27, CD69, CD25, PD-1, CTLA-4
CD45RO, and CD45RA.
In embodiments, a method of selecting a spacer region for a chimeric
receptor is described . Surprisingly some chimeric receptor constructs,
although effective to activate T cells in vitro, were not effective in vivo. In
embodiments, a method comprises providing a plurality of chimeric receptor nucleic
acids, wherein the chimeric receptor nucleic acids differ only in the spacer region;
introducing each of the chimeric receptor nucleic acids into a separate T
lymphocyte population; expanding each separate lymphocyte population in vitro,
and introducing each lymphocyte population into an animal bearing a tumor to
determine the anti-tumor cy of each of the chimeric receptor modified T cells,
and ing a chimeric receptor that provides anti-tumor efficacy as ed to
each of the other separate lymphocyte populations modified with each of the other
chimeric receptor ed T cells.
Animal models of different tumors are known. Anti-tumor efficacy can be
measured by identifying a decrease in tumor , by determining animal death,
persistence of the genetically modified T cells in vivo, activation of cally
modified T cells (for example, by detecting an increase in sion of CD25
and/CD69), and/or eration of genetically modified T cells in vivo. In an
embodiment, a chimeric receptor is selected that es for the best anti-tumor
efficacy in vivo as ined by one or more of these parameters. Lack of antitumor
efficacy can be determined by lack of persistence of the genetically modified
lymphocytes in vivo, animal death, an increase in apoptosis as ed by an
increase in induction of caspase -3, and/or a decrease in proliferation of genetically
modified lymphocytes.
In embodiments, providing a plurality of chimeric receptor c acids,
wherein the chimeric receptor nucleic acids differ only in the spacer region
comprises providing a ic receptor construct comprising a polynucleotide
coding for a ligand binding , n the ligand is a tumor specific antigen,
viral antigen, or any other molecule expressed on a target cell population that is
suitable to mediate ition and elimination by a lymphocyte; a polynucleotide
coding for a first polypeptide spacer having a defined restriction site at the 5’ and 3’
end of the coding sequence for the first polypeptide spacer; a polynucleotide coding
for a transmembrane domain; and a polynucleotide coding for an intracellular
signaling domain.
Also described are methods of performing cellular immunotherapy in a
subject having a disease or disorder comprising: administering a composition of
lymphocytes expressing a chimeric receptor as bed herein. In other
embodiments, a method comprises administering to the subject a genetically
modified cytotoxic T lymphocyte cell preparation that provides a ar immune
response, wherein the cytotoxic T lymphocyte cell preparation comprises CD8 + T
cells that have a chimeric or comprising a ligand binding domain ic for a
tumor cell surface molecule, a customized spacer domain, a transmembrane domain,
and an intracellular ing domain as described herein, and a genetically modified
helper T lymphocyte cell preparation that elicits direct tumor ition and
augments the genetically ed cytotoxic T lymphocyte cell preparations ability
to mediate a cellular immune response, wherein the helper T lymphocyte cell
preparation comprises CD4+ T cells that have a chimeric receptor comprising a
ligand binding domain ic for a tumor cell surface molecule, a customized
spacer domain, a transmembrane domain, and an intracellular signaling domain as
described herein.
While not limiting the scope of the disclosure, it is believed by selecting the
chimeric receptor modified T cell population that can persist and proliferate in vivo
prior to administration may result in the ability to use a lower dose of T cells and
provide more uniform therapeutic activity. In embodiments, the dose of T cells can
be reduced at least 10%, 20%, or 30% or r. Reduction in the dose of T cells
may be beneficial to reduce the risk or tumor lysis syndrome and cytokine storm.
In another embodiment, a method of performing ar immunotherapy in
subject having a disease or disorder comprises: stering to the subject a
genetically modified helper T lymphocyte cell ation, wherein the modified
helper T lymphocyte cell preparation comprises CD4+ T cells that have a chimeric
receptor comprising a ligand binding domain specific for a tumor cell surface
molecule, a customized spacer domain, a transmembrane domain, and an
intracellular signaling domain as described herein. In an embodiments, the method
further comprises administering to the t a genetically modified cytotoxic T
lymphocyte cell preparation, wherein the modified cytotoxic T lymphocyte cell
preparation comprises CD8+ cells that have a chimeric receptor sing a ligand
binding domain specific for a tumor cell e molecule, a customized spacer
domain, a transmembrane domain, and an intracellular signaling domain as
bed herein.
Another embodiment describes a method of performing cellular
immunotherapy in a subject having a disease or disorder comprising: ing a
biological sample of the subject for the presence of a target molecule associated with
the disease or er and administering the ve immunotherapy compositions
described herein, n the chimeric receptor specifically binds to the target
molecule.
In some embodiments, the CD4+ T helper lymphocyte cell is selected prior
to introduction of the chimeric receptor from the group consisting of naïve CD4+ T
cells, central memory CD4+ T cells, effector memory CD4+ T cells or bulk CD4+ T
cells. In a specific embodiment, CD4+ helper lymphocyte cell is a naïve CD4+ T
cell, wherein the naïve CD4+ T cell ses a CD45RO-, CD45RA+, CD62L+
CD4+ T cell. In yet other embodiments, the CD8+ T cytotoxic lymphocyte cell is
selected prior to introduction of the chimeric or from the group consisting of
naïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells
or bulk CD8+ T cells. In a specific embodiment, the CD8+ cytotoxic T lymphocyte
cell is a central memory T cell wherein the central memory T cell comprises a
CD45RO+, CD62L+, CD8+ T cell. In a specific embodiment, the CD8+ cytotoxic T
lymphocyte cell is a central memory T cell and the CD4+ helper T lymphocyte cell
is a naïve CD4+ T cell.
In embodiments, the CD8+ T cell and the CD4+ T cell are both genetically
modified with a chimeric receptor comprising an dy heavy chain domain that
specifically binds a tumor-specific cell surface molecule. In other ments, the
ellular ing domain of the CD8 xic T cells is the same as the
ellular ing domain of the CD4 helper T cells. In yet other embodiments,
the intracellular signaling domain of the CD8 cytotoxic T cells is different than the
intracellular signaling domain of the CD4 helper T cells.
Subjects that can be treated by the methods described herein are, in general,
human and other primate subjects, such as s and apes for veterinary
medicine purposes. The subjects can be male or female and can be any suitable age,
ing , juvenile, adolescent, adult, and geriatric subjects.
The methods are useful in the treatment of, for example, hematologic
malignancy, melanoma, breast cancer, and other epithelial malignancies or solid
tumors. In some embodiments, the molecule associated with the disease or disorder
is selected from the group consisting of orphan tyrosine kinase receptor ROR1,
Her2, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen.
Subjects that can be d include subjects afflicted with cancer, including
but not limited to colon, lung, liver, breast, renal, prostate, ovarian, skin (including
melanoma), bone, and brain cancer, etc. In some ments the tumor associated
antigens or molecules are known, such as melanoma, breast cancer, squamous cell
carcinoma, colon cancer, leukemia, myeloma, and prostate . In other
embodiments the tumor associated molecules can be targeted with genetically
modified T cells expressing an engineered chimeric receptor. Examples include but
are not limited to B cell lymphoma, breast cancer, prostate cancer, and leukemia.
Cells prepared as described above can be utilized in methods and
compositions for adoptive immunotherapy in accordance with known ques, or
variations thereof that will be apparent to those skilled in the art based on the instant
disclosure.
In some embodiments, the cells are formulated by first harvesting them from
their culture medium, and then washing and concentrating the cells in a medium and
container system suitable for administration (a "pharmaceutically acceptable"
carrier) in a treatment-effective amount. le infusion medium can be any
isotonic medium formulation, typically normal , Normosol R (Abbott) or
Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be
utilized. The infusion medium can be supplemented with human serum albumin,
fetal bovine serum or other human serum components.
A treatment effective amount of cells in the ition is at least 2 cell
subsets (for example, 1 CD8+ central memory T cell subset and 1 CD4+ helper T
cell subset) or is more lly greater than 102 cells, and up to 106, up to and
including 108 or 109 cells and can be more than 1010 cells. The number of cells will
depend upon the te use for which the composition is intended as will the type
of cells included therein. For example, if cells that are specific for a particular
antigen are desired, then the population will contain greater than 70%, generally
greater than 80%, 85% and 90-95% of such cells. For uses bed herein, the cells
are generally in a volume of a liter or less, can be 500 mls or less, even 250 mls or
100 mls or less. Hence the density of the desired cells is typically greater than 104
cells/m1 and lly is greater than 107 cells/ml, generally 108 cells/ml or greater.
The clinically relevant number of immune cells can be apportioned into multiple
ons that cumulatively equal or exceed 106, 107, 108, 108, 109, 1010 or 1011 cells.
In some embodiments, the lymphocytes described herein may be used to
confer immunity to duals. By "immunity" is meant a lessening of one or more
physical symptoms associated with a se to infection by a pathogen, or to a
tumor, to which the lymphocyte response is directed. The amount of cells
administered is usually in the range present in normal individuals with immunity to
the pathogen. Thus, the cells are usually administered by infusion, with each
infusion in a range of from 2 cells, up to at least 106 to 3x1010 cells, preferably in the
range of at least 107 to 109 cells. The T cells may be administered by a single
infusion, or by le infusions over a range of time. However, since different
duals are expected to vary in responsiveness, the type and amount of cells
infused, as well as the number of infusions and the time range over which multiple
infusions are given are determined by the attending physician, and can be
determined by routine examination. The generation of sufficient levels of T
lymphocytes (including cytotoxic T lymphocytes and/or helper T lymphocytes) is
readily achievable using the rapid expansion method described herein, as
exemplified herein. See, e.g., US Patent No. 6,040,177 to Riddell et al. at column 17.
In embodiments, the composition as described herein are administered
intravenously, intraperitoneally, intratumorly, into the bone marrow, into the lymph
node, and /or into cerebrospinal fluid. In embodiments, the chimeric receptor
engineered compositions are delivered to the site of the tumor. atively, the
compositions as described herein can be combined with a compound that targets the
cells to the tumor or the immune system compartments and avoid sites such as the
lung.
In embodiments, the compositions as described herein are administered with
herapeutic agents and/or immunosuppressants. In an embodiment, a patient is
first d with a herapeutic agent that inhibits or destroys other immune
cells followed by the compositions described herein. In some cases, chemotherapy
may be avoided entirely.
The t invention is illustrated further in the examples set forth below.
EXPERIMENTAL
Example I. Customizing spacer domain length and scFv affinity for optimal
recognition of ROR1 with chimeric receptor modified T cells
We constructed chimeric receptors ic for the ROR1 molecule that is
expressed on a large number of human malignancies including chronic cytic
leukemia, mantle cell lymphoma, acute lymphoblastic leukemia, and breast, lung
prostate, pancreas and ovarian cancer. The ROR1 chimeric ors were designed
from ROR1 ic scFVs with different affinities and containing extracellular
IgG4-Fc spacer domains of different lengths. The ability of T-cells expressing each
ROR-1 specific chimeric receptor to recognize ROR1+ hematopoietic and epithelial
tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into
immunodeficient mice was analyzed.
Materials and Methods
Human subjects
Peripheral blood mononuclear cells (PBMC) were obtained from healthy
donors and patients after written informed consent on research protocols ed
by the Institutional Review Board of the Fred nson Cancer Research Center
(FHCRC).
Cell lines
The K562, Raji, JeKo-1, MDA-MB-231, MDA-MB-468, and 293T cell lines
were obtained from the American Type Culture Collection. Dr. Edus H. Warren
(FHCRC) kindly ed the renal cell cancer lines FARP, TREP and RWL.
K562/ROR1 and Raji/ROR1 were ted by lentiviral transduction with the full-
length ROR1-gene. To derive JeKo-1/ffluc, native JeKo-1 cells were transduced
with a lentiviral vector encoding the firefly luciferase (ffluc)-gene upstream of a T2A
sequence and eGFP. The uced JeKo-1 cells were sorted for eGFP expression,
and expanded for in vivo experiments.
phenotyping
PBMC and cell lines were stained with the ing conjugated mAbs:
CD3, CD4, CD5, CD8, CD19, CD28, CD45RO, CD62L, CD314 (NKG2D),
MICA/B and matched isotype controls (BD Biosciences). Propidium iodide (PI)
staining was performed for live/dead cell discrimination. Cell surface expression of
ROR1 was analyzed using a polyclonal goat anti-human-ROR1 antibody (R&D
Systems).
Surface expression of 2A2 ROR1chimeric receptor was analyzed using a
polyclonal goat anti-mouse-IgG dy (Fab-specific) (Jackson ImmunoResearch).
Flow analyses were done on a FACSCanto®, sort-purifications on a FACSAriaII®
n Dickinson) and data ed using FlowJo® software (Treestar).
Vector construction and preparation of chimeric receptor encoding lentivirus
ROR1-specific and CD19-specific chimeric receptors were ucted using VL
and VH chain segments of the 2A2, R12, and R11 mAbs (ROR1) and FMC63 mAb
(CD19). (Variable region ces for R11 and R12 are provided in Yang et al,
Plos One 6(6):e21018, June 15, 2011) Each scFV was linked by a (G4S)3(SEQ ID
NO:12) peptide to a spacer domain derived from IgG4-Fc (Uniprot Database:
P01861,SEQ ID NO:13) sing either ‘Hinge-CH2-CH3’ (229 AA, SEQ ID
NO:), ‘Hinge-CH3’ (119 AA,SEQ ID NO:) or ‘Hinge’ only (12 AA,SEQ. ID NO:4)
sequences (Figure 1). All spacers contained a S?P substitution within the ‘Hinge’
domain located at position 108 of the native IgG4-Fc protein, and were linked to the
27 AA transmembrane domain of human CD28 (Uniprot: P10747, SEQ ID NO:14)
and to a signaling module comprising either (i) the 41 AA cytoplasmic domain of
human CD28 with an LL?GG substitution located at positions 186-187 of the
native CD28 protein (SEQ ID or (ii) the 42 AA cytoplasmic domain of
human 4-1BB (Uniprot: Q07011, SEQ ID NO:15), each of which was linked to the
112 AA cytoplasmic domain of m 3 of human CD3? (Uniprot: P20963, SEQ
ID NO;16). The construct encoded a T2A ribosomal skip t (SEQ ID
NO:8))and a tEGFR sequence (SEQ ID NO:9) downstream of the chimeric receptor.
Codon-optimized nucleotide sequences encoding each transgene were synthesized
(Life Technologies) and cloned into the epHIV7 lentiviral vector
ROR1-chimeric or, CD19-chimeric receptor or tEGFR-encoding
lentiviruses were produced in 293T cells using the packaging vectors pCHGP-2,
pCMV-Rev2 and pCMV-G, and Calphos® transfection reagent (Clontech).
tion of T-cell lines expressing ROR1 and CD19-chimeric receptors
CD8+ CD45RO+ CD62L+ central memory T-cells (TCM) or bulk CD4+ T-
cells were sorted from PBMC of normal donors, activated with anti-CD3/CD28
beads (Life Technologies), and transduced on day 3 after activation by
centrifugation at 800 g for 45 min at 32ºC with lentiviral supernatant (MOI = 3)
supplemented with 1 µg/mL polybrene (Millipore). T-cells were expanded in RPMI
with 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin (CTL
), supplemented with recombinant human IL-2 to a final concentration of 50
U/mL. The tEGFR+ subset of each T-cell line was enriched by immunomagnetic
selection with biotin-conjugated GFR mAb (ImClone Systems) and
streptavidin-beads (Miltenyi). ROR1-chimeric receptor and tEGFR control T-cells
were expanded using a rapid expansion protocol (Riddell SR, Greenberg PD,The use
of anti-CD3 and anti-CD28 monoclonal dies to clone and expand human
antigen-specific T cells J Immunol Methods. 1990;128(2):189-201. Epub
1990/04/17.), and himeric or modified T-cells were expanded by
stimulation with irradiated (8,000 rad) B-LCL at a T-cell:LCL ratio of 1:7. T-cells
were cultured in CTL medium with 50 U/mL IL-2.
Cytotoxicity, ne secretion and proliferation assays
Target cells were labeled with 51Cr (PerkinElmer), washed and incubated in
triplicate at 1-2x103 cells/well with effector chimeric receptor modified T-cells at
various effector to target (E:T) ratios. Supernatants were harvested for ?-counting
after a 4-hour incubation and specific lysis calculated using the standard formula.
For analysis of cytokine ion, 5x104 T-cells were plated in triplicate with target
cells at an E:T ratio of 1:1 ry CLL), 2:1 (Raji/ROR1; JeKo-1), 4:1
(K562/ROR1, K562/CD19 and K562) or 10:1 (MDA-MB-231), and IFN-?, TNF-a
and IL-2 measured by ELISA or multiplex cytokine immunoassay (Luminex) in
supernatant removed after 24-h incubation. In experiments blocking NKG2D
signaling, anti-NKG2D (clone 1D11), anti-MICA/B (clone 6D4, all from BD) and
anti-ULBP (kindly provided by Dr. Veronika Groh, FHCRC) were used at saturating
concentrations. For analysis of proliferation, s were labeled with 0.2 µM
carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in
triplicate with stimulator cells in medium without exogenous cytokines. After 72-h
incubation, cells were labeled with anti-CD8 mAb and PI, and analyzed by flow
cytometry to assess cell division of live CD8+ T-cells.
Experiments in NOD/SCID/?c-/- (NSG) mice
The Institutional Animal Chimeric receptor and Use Committee approved all
mouse experiments. Six- to 8-week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
(NSG) mice were obtained from the Jackson Laboratory or bred in-house. Mice
were injected with 0.5x106 JeKo-1/ffluc tumor cells via tail vein and received a
uent tail vein ion of chimeric receptor-modified or control s.
For bioluminescence imaging of tumor growth, mice received intraperitoneal
injections of luciferin substrate (Caliper Life Sciences) resuspended in PBS (15 µg/g
body weight). Mice were anesthetized with isoflurane and imaged using an n
IVIS Imaging System (Caliper) 10, 12 and 14 minutes after the injection of luciferin
in small binning mode at an acquisition time of 1 s to 1 min to obtain unsaturated
images. rase activity was analyzed using Living Image Software er) and
the photon flux ed within s of interest that encompassed the entire body
or the thorax of each individual mouse.
Statistical es
Statistical analyses were performed using Prism Software (GraphPad®).
Student’s t-test was performed as a two-sided paired test with a confidence interval
of 95% and results with a p-value of p<0.05 were considered icant. Statistical
analysis of survival were done by nk testing and results with a p-value of
p<0.05 considered significant.
Results
Truncating the spacer domain of the 2A2 himeric receptor confers
superior recognition of ROR1+ tumors
We previously reported the design of a ROR1-specific chimeric receptor
using the 2A2 scFV, which binds to an epitope in the NH2-terminal, membrane
distal Ig-like/Frizzled portion of ROR1-1. The initial 2A2 ROR1-chimeric receptor
had a long 229 AA spacer that included the ‘Hinge-CH2-CH3’ region of IgG4-Fc,
and orated CD28 costimulatory and CD3? signaling domains (Hudecek M et
al. Blood, 2010). This chimeric receptor red specific recognition of ROR1+
tumors, but we hypothesized that because of the membrane distal location of the
ROR1 epitope, truncating the spacer domain might enhance tumor recognition and
T-cell signaling. Therefore, we constructed 2 additional chimeric receptors in which
the IgG4-Fc spacer domain was tially deleted to derive ‘Hinge-CH3’ (119
AA, ediate), and ‘Hinge-only’ (12 AA, short) variants. Each of the new
receptors contained the identical 2A2 scFV, and CD28 and CD3? signaling modules.
The transgene cassette included a truncated EGFR ) to serve as a
transduction, selection and in vivo tracking marker for chimeric receptor-modified
T-cells.
We transduced purified CD8+ TCM with the 2A2 ROR1-chimeric receptors
containing full length or ted IgG4-Fc spacers, and with a tEGFR control
vector. The mean transduction efficiency was 15% (range 9-22%), and transgenepositive
s were enriched to uniform purity (>90%) on day 10 by selection for
tEGFR expression, and expanded (Figure 2A). Surface expression of each of the
chimeric receptors was confirmed by staining with F(ab)-specific antibodies (Figure
2A).
Analysis of the in vitro function of CD8+ T-cells modified to express each of
the 2A2 ROR1-chimeric ors demonstrated that each receptor conferred
specific lysis of JeKo-1 MCL and primary CLL cells that naturally express ROR1,
and of K562 cells that had been uced with ROR1, but did not confer
ition of control ROR1- targets (Figure 2B). T-cells expressing the short
‘Hinge-only’ 2A2 ROR1-chimeric receptor had maximum cytolytic activity, and a
hierarchy >intermediate>>long) of tumor lysis was y evident against all
ROR1+ tumor targets (Figure 2B), illustrating the importance of spacer domain
length on the recognition of ROR1+ tumor cells.
umor efficacy of adoptive T-cell therapy correlates with proliferation
and survival of transferred T-cells, which could be altered by signaling through the
chimeric receptor. We used CFSE dilution assays to analyze proliferation of T-cells
modified with each of the 2A2 ROR1-chimeric ors after engagement of
Raji/ROR1 or CLL, and found that the short spacer construct ed the greatest
T-cell proliferation following ation (Figure 2C). To ensure that the enhanced
proliferation was not associated with greater activation d cell death (AICD),
we also analyzed the proportion of 2A2 ROR 1 chimeric receptor modified T-cells
that stained with propidium iodide (PI) after stimulation with Raji/ROR1 and JeKo-
1 tumor cells. We detected a much lower frequency of PI+ CD8+ T-cells in the T-cell
line modified with the short (Raji/ROR1: 17.2%/JeKo-1: 20.2%) compared to the
intermediate (41.6%/42.4%) and long (44.5%/48.5%) spacers.
Quantitative analysis of cytokine production in response to stimulation with
Raji/ROR1 and primary CLL cells showed production of IFN-?, TNF-a and IL-2 by
T-cells expressing each of the 2A2 ROR1 chimeric receptors. As observed in
cytotoxicity assays, the short spacer construct was superior in mediating cytokine
secretion after tumor recognition (Figure 2D). Thus, this analysis shows that
truncating the extracellular IgG4-Fc spacer domain of the 2A2 himeric
receptor leads to a significant increase in cytotoxicity, eration and in vitro
effector ons after tumor recognition.
The R11 scFv that is specific for a membrane proximal epitope in the ROR1
Kringle domain requires a long extracellular spacer domain.
We transduced purified CD8+ T cells with ROR1-chimeric receptors
containing the R11 scFv that is specific for the Kringle domain of ROR1 and
containing full length or truncated IgG4-Fc spacers (CH3 and hinge only). The
transduction efficiency with each of the short (IgG4 hinge only), intermediate (IgG4
CH3), and long (IgG4 hinge/CH2/CH3) s was comparable (45-51%) as
measured by EGFR expression. (Figure 3A). T cells transduced with each of the
vectors were assayed for cytolysis (Figure 3 B), proliferation (Figure 3C), and
cytokine production e 3D) in response to leukemia or lymphoma cells that did
or did not express ROR1. As shown, only T cells transduced with the R11 chimeric
receptor containing a long spacer sequence were able to efficiently recognize
ROR1+ tumors and mediate effector ons.
ROR1 chimeric ors derived from a mAb R12 with higher affinity than 2A2
mediate superior anti-tumor reactivity
We next examined whether sing the affinity of the scFV used to
construct the ROR1 chimeric receptor might influence tumor ition and T-cell
function. We generated ROR1-specific chimeric ors from the mAb R12 that
like 2A2, binds to an epitope in the NH2-terminal Ig/Frizzled domain of ROR1 but
with >50-fold higher monovalent binding ty.
R12 ROR1 chimeric receptors were constructed with both long and short
IgG4-Fc spacers to determine whether the optimal spacer design for this higher
affinity scFV differed from that for a lower affinity scFV. We found that similar to
2A2, the short spacer R12 ROR1 chimeric receptor conferred improved cytolytic
activity, cytokine secretion and proliferation (data not shown), suggesting that the
shorter spacer length provides superior spatial ment of the T-cell and ROR1+
target cell for T-cell activation.
We then designed R12 and 2A2 ROR1 chimeric receptors that contained an
optimal (short) extracellular spacer, and either a CD28 or 4-1BB costimulatory
domain in tandem with CD3? (4 ucts) for comparison (Figure 4A.B). These
ROR1-chimeric receptor constructs were expressed in purified CD8+ TCM of healthy
, and we confirmed equivalent transgene expression by tEGFR staining
(Figure 5A). T-cells modified with each of the 2A2 and R12 ROR1-chimeric
receptors specifically lysed K562/ROR1 and Raji/ROR1 tumor cells with
approximately equivalent efficiency e 5B). However, analysis of cytokine
production showed that the high affinity R12 ROR1 chimeric receptors that
contained CD28 or 4-1BB red significantly higher IFN-?, TNF-a and IL-2
production compared to the corresponding 2A2 constructs (Figure 5C). We found
that T-cells expressing ic receptors with a CD28 costimulatory domain
produced more IFN-?, TNF-a and IL-2 compared to those with 4-1BB.
Experiments to analyze the proliferation of ROR1 chimeric receptor T-cells
showed a higher percentage of proliferating T-cells and a higher number of cell
divisions in T-cells expressing the high affinity R12 ROR1 chimeric ors with
CD28 and 4-1BB domain ed to T-cells expressing the respective 2A2
counterparts (Figure 4D). There was more vigorous proliferation in T-cells that
expressed chimeric receptors with a CD28 domain, consistent with higher IL-2
production induced by these receptors. There was a lower frequency of AICD as
ed by PI staining in T-cell lines modified with R12 compared to 2A2 ROR1-
chimeric receptors after stimulation with Raji/ROR1 and JeKo-1 tumor cells
respectively (R12: 5.6%/6.9% vs. 2A2: 10%/9.65%). T-cell lines that expressed
chimeric receptors with a CD28 domain also had lower AICD compared to 4-1BB in
response to Raji/ROR1 and JeKo-1 tumor cells tively (R12: 16.4%/18.4% vs.
2A2 38.1%/39.6%).
To ine if the enhanced function observed with R12 ROR1 chimeric
receptors in CD8+ T-cells extended to CD4+ T-cells, we uced bulk CD4+ T-
cells with the 2A2 and R12 ROR1 chimeric receptors containing the short spacer
and CD28 costimulatory domain. In se to Raji/ROR1+ tumor cells, CD4+ T-
cells that sed the high affinity R12 scFV produced higher levels of IFN-?,
TNF-a, IL-2, IL-4, and IL-10, and underwent greater proliferation than CD4+ T-cells
that expressed 2A2 (Figure 5A,B). Both cytokine production and proliferation was
superior in CD4+ compared to CD8+ T-cells modified with the same ROR1 chimeric
receptors. In summary, our data demonstrate that tailoring both the length of the
non-signaling extracellular chimeric receptor spacer domain and scFV affinity are
independent parameters that affect the function of ROR1-chimeric receptor T-cells.
D8+ s ed with a high affinity ROR1 chimeric receptor have
comparable activity to a CD19 chimeric receptor against primary CLL in vitro
ROR1 and CD19 are both uniformly sed on all primary CLL (Figure
6A), however the absolute number of ROR1-molecules per tumor cell is estimated to
be 10-fold lower than that of CD19, which has been successfully targeted in clinical
trials with CD19 ic receptor T-cells. We compared recognition of primary
CLL by CD8+ T-cells expressing the optimized R12 and 2A2 ROR1 chimeric
receptors, and a CD19 chimeric receptor derived from the FMC63 scFV.
We used purified CD8+ TCM for ic receptor-modification to provide a
uniform cell product and each chimeric receptor contained a short IgG4-Fc ‘Hingeonly
’ spacer and 4-1BB costimulatory domain. We med our CD19 chimeric
receptor (IgG4 Hinge) was at least as and more effective in recognizing CD19+
tumors as a CD19 chimeric receptor with CD8a Hinge spacer and 4-1BB
costimulatory domain that is being used in ongoing clinical trials. (Figure 20). T
cells expressing CD19 chimeric receptors with 4-1BB and CD3zeta and a ed
IgG4-Fc hinge exhibit superior in vitro and in vivo function compared to T cells
expressing CD19 chimeric ors with 4-1BB and CD3zeta and a CD8 alpha
hinge. In Figure 20D, in vivo antitumor activity of T cells expressing a CD19
chimeric receptor with an IgG4 Fc hinge (group 1) or CD8 alpha hinge (group 2)
and T cells that express tEGFR alone (group 3) in NSG mice inoculated with Raji
tumor cells sing firefly luciferase (ffluc) were compared. Mice were imaged
17 days after tumor inoculation and 10 days after T cell inoculation. The data shows
greater tumor burden in mice treated with control tEGFR T cells (group 3) or with
CD19 chimeric receptor CD8 alpha hinge T cells (group 2) compared with mice
treated with CD19 ic receptor IgG4 Fc hinge T cells (group 1).
The cytolytic activity of R12 ROR1 chimeric receptor T-cells against
primary tumor cells from multiple CLL ts (n=4) was higher compared to T-
cells ed with the lower affinity 2A2 ROR1 chimeric receptor, and equivalent
to the lysis observed with CD19 chimeric receptor T-cells (Figure 6B). Multiplex
cytokine is showed nearly equivalent production of IFN-? and TNF-a, but less
IL-2 production by CD8+ T-cells sing the R12 ROR1 compared with those
expressing the himeric receptor after co-culture with primary CLL (Figure
6C). 2A2 ROR1 chimeric or T-cells produced lower amounts of all cytokines
than R12 ROR1 chimeric receptor T-cells as noted previously. Cytokine production
by all of the chimeric receptor-transduced T-cells after stimulation with CLL was
substantially less than with Raji/ROR1, which unlike CLL expresses both CD80 and
CD86 that can engage CD28 expressed on chimeric receptor T-cells (Figure 6A, C).
We observed less proliferation of T-cells expressing the R12 and 2A2 ROR1
chimeric receptor compared to the CD19 chimeric receptor after stimulation with
CLL (CD19>R12>2A2) (Figure 6D). We hypothesized that proliferation of CD8+
ROR1 chimeric or T-cells in response to CLL may be augmented in the
presence of chimeric or-modified CD4+ T-cells because of their higher
secretion of IL-2 compared to CD8+ TCM e 4A; Figure 8A). To test this
possibility, we med in vitro co-culture experiments where CD4+ and CD8 TCM
were separately modified with the R12 ROR1, 2A2 ROR1 and CD19 chimeric
ors respectively, enriched for chimeric receptor expression, and combined at a
1:1 ratio to ensure equivalent proportions of CD8+ and CD4+ T-cells modified with
each of the vectors. These cells were CFSE-labeled and stimulated with primary
CLL. We observed a dramatic increase in proliferation of CD8+ R12 ROR1 chimeric
receptor T-cells after addition of chimeric or-transduced, but not untransduced
CD4+ T-cells (Figure 8B). Notably, when provided with lp, we ed
equivalent proliferation of R12 ROR1 and CD19 chimeric receptor CD8+ T-cells in
response to CLL, whereas proliferation of CD8+ s sing the lower
affinity 2A2 ROR1 chimeric receptor remained less. Collectively, our data show that
the high ty R12 ROR1 chimeric receptor confers superior reactivity compared
to 2A2 t primary CLL cells in vitro.
ROR1-chimeric receptor T-cells mediate in vivo anti-tumor activity in a mouse
model of systemic mantle cell lymphoma
It remained uncertain whether the superior in vitro activity of T-cells
modified with the higher affinity R12 chimeric receptor would translate into
improved anti-tumor activity in vivo, and how targeting ROR1 would compare to
targeting CD19. To address these questions, we inoculated cohorts of
immunodeficient NSG mice with the human MCL line JeKo-1/ffluc by tail vein
injection, and seven days later when tumor was disseminated, d the mice with a
single enous dose of R12 ROR1, 2A2 ROR1 or CD19 chimeric receptor CD8+
T-cells. Control mice were treated with tEGFR T-cells or untreated. All chimeric
receptors had the optimal short spacer and the 4-1BB costimulatory .
Untreated NSG/JeKo-1 mice developed a rapidly progressive systemic lymphoma
necessitating euthanasia approximately 4 weeks after tumor inoculation (Figure 9A-
C).
We observed tumor regression and improved survival in all mice treated with
R12 ROR1, 2A2 ROR1 and CD19 chimeric receptor T-cells. Mice treated with R12
ROR1 chimeric receptor T-cells had a superior anti-tumor response and survival
compared to mice treated with 2A2 ROR1 chimeric receptor T-cells (p<0.01), and
comparable anti-tumor activity to mice treated with CD19 chimeric receptor T-cells
(Figure 9A-C).
We ed the frequency of chimeric receptor T-cells in the peripheral
blood following adoptive transfer and detected higher numbers of tEGFR+ T-cells in
mice treated with the R12 ROR1 chimeric receptor compared to the 2A2 ROR1
chimeric receptor, suggesting more vigorous proliferation in vivo improved tumor
l. To m this, we administered abeled CD19 chimeric receptor,
R12 and 2A2 ROR1 chimeric receptor T-cells to cohorts of NSG mice bearing
JeKo-1/ffluc, and ed T-cell proliferation in the peripheral blood, bone marrow
and spleen 72 hours after transfer. A higher percentage of the R12 and CD19
chimeric receptor T-cells proliferated and underwent a greater number of cell
divisions compared to 2A2 ROR1 chimeric receptor T-cells (Figure 9D). The JeKo-
1 tumor eventually recurred in all mice treated with ROR1 or CD19 chimeric
receptor T-cells e 9A-C). Tumor recurrence was not a result of the selection of
ROR1 or CD19 loss variants, as recurrent tumors were positive for both molecules.
For comparison, we analyzed anti-tumor efficacy of CD19 chimeric receptor
T-cells in NSG mice engrafted with Raji tumors and observed complete tumor
eradication, indicating the recurrence of JeKo-1 reflects difficulty eradicating this
tumor (data not shown). In summary, this data is the first to show that ROR1
chimeric or T-cells have anti-tumor efficacy in vivo, and suggest that for B-
cell malignancies, an zed ROR1 chimeric receptor such as R12 may be
effective and spare normal CD19+ B-cells that lack ROR1 expression.
T-cells expressing the R12 ROR1 chimeric or have superior vity
compared to 2A2 against ROR1+ epithelial tumor cells
ROR1 has been ed on many epithelial tumors, although it is unknown
whether ROR1 expression is sufficient for ition by ROR1 chimeric or
T-cells. Using flow cytometry, we confirmed ROR1 expression on breast cancer
lines MDA-MB-231 and 468, and on the renal cell carcinoma lines FARP, TREP,
and RWL (Figure 10A). We then analyzed tumor recognition by CD8+ T-cells
transduced with the R12 ROR1 chimeric receptors with the optimal short spacer and
4-1BB domain, and observed efficient recognition of MDA-MB-231, -
468, FARP, TREP and RWL (Figure 11A). We analyzed cytokine secretion and
eration of T-cells modified with the R12 and 2A2 ROR1-chimeric receptors
after co-culture with MDA-MB-231, and observed greater cytokine production and
proliferation with the R12 ROR1 ic receptor (Figure 11 B, C). Similar to
what we observed with ROR1+ B cell malignancies, the superior activation of R12
ROR1 chimeric receptor T cells after stimulation with MDA-MB-231 was not
associated with sed AICD (R12: 9.8% vs. 2A2: .
Discussion
ROR1 has attracted interest as a potential target for cancer immunotherapy
due to its expression on the surface of many B-lymphoid and epithelial cancers,
including subsets of lung, colorectal and renal cell cancer. We previously showed
that CLL and MCL were specifically recognized by s modified to express a
ROR1-specific ic receptor (Hudecek M, et al. Blood. 2010;116(22):4532-41.
Epub 2010/08/13). The design and function of ROR1-chimeric receptors has been
improved through modification of the extracellular spacer domain and deriving the
chimeric receptor from a scFV of higher affinity, and demonstrate that T-cells
modified with designed ROR1 chimeric receptors have in vivo activity against
ROR1+ B-cell lymphoma and in vitro ty against a wide range of epithelial
tumors.
We compared the function of T-cells modified with ROR1 ic
receptors derived from the 2A2 mAb that contained either the original long IgG4-Fc
‘Hinge-CH2-CH3’ spacer that we have shown enables high level cell surface
sion, or truncated intermediate ‘Hinge-CH3’ and short ‘Hinge-only’ spacer
variants. We ved the 12 AA Hinge domain in our short spacer construct based
on prior data that a flexible spacer was required for separating the scFV from the T-
cell membrane and allowing n recognition on tumor cells ( Fitzer-Attas CJ, et
al.,Harnessing Syk family tyrosine kinases as signaling domains for chimeric single
chain of the variable domain receptors: optimal design for T cell activation. J
Immunol. 1998;160(1):145-54. Epub 4/29.)
Our studies with the 2A2 ROR1 chimeric receptor show that T-cell cytokine
secretion and proliferation after tumor cell recognition are superior with the
intermediate and short spacer constructs compared to the long spacer construct.
Staining with (ab) Abs showed equivalent ic receptor expression of all
three ors, demonstrating the improved T-cell function with the short spacer
chimeric receptor was not due to differences in chimeric receptor density. This data
supports the principle that the design of extracellular spacers should be tailored for
each target molecule and e.
The affinity of the scFV selected for designing a chimeric receptor is an
additional parameter that could affect T-cell recognition. We generated and
characterized a panel of ROR1-specific mAbs of different affinities and selected the
R12 mAb, which recognizes an epitope in the Ig-like/Frizzled region as 2A2. R12
has a higher ty for ROR1-protein due to a much slower dissociation. The R12
chimeric receptor, like the 2A2 chimeric receptor conferred optimal T-cell
recognition and function when designed with a short extracellular spacer. A direct
comparison of proliferation and cytokine production after tumor engagement by T-
cells modified with the 2A2 and R12 chimeric ors demonstrated that the R12
chimeric receptor derived from the higher affinity mAb was superior. We were
concerned that the slower dissociation of R12 from ROR1 could prolong T-cell
activation and confer an increased susceptibility to AICD. However, we detected a
lower rate of AICD in T-cells modified with the R12 ROR1-chimeric receptor
compared to 2A2, demonstrating that the sed affinity of R12 had no
detrimental effect on T-cell survival in our preclinical models.
ROR1 has a ial advantage over CD19 as a target for CLL and MCL
since it is not expressed on normal mature naïve and memory B-cells. However,
there is a lower number of ROR1 molecules on B-cell tumors compared with CD19
and it is uncertain if an optimized ROR1 chimeric receptor would be as effective as
a CD19 chimeric receptor similar in design to those being used in the clinic.
Unfortunately, B-cell tumor xenograft models used usly in NSG mice to
evaluate the function of CD19 chimeric receptor T-cells including Raji, Daudi and
Nalm-6, are not derived from CLL or MCL and do not constitutively express ROR1.
Thus, to compare targeting CD19 and ROR1 in vivo, we used the JeKo-1 MCL cell
line, which naturally expresses both CD19 and ROR1 and engrafts in NSG mice. To
make our model clinically relevant, we inoculated JeKo-1 lymphoma cells
intravenously to generate systemic tumors, and treated mice with T-cell products of
uniform consistency once tumors were ished. We found that T-cells expressing
the high affinity R12 chimeric or conferred lent anti-tumor activity in
vivo as CD19 chimeric receptor T-cells. Consistent with our in vitro analysis, the
R12 ROR1 chimeric receptor also mediated superior activity in vivo compared to the
optimal 2A2 ROR1-chimeric receptor. These results should be reted usly
since murine tumor models may not predict the efficacy of adoptive therapy in
clinical gs. However, the results suggest that ROR1 warrants consideration as
an alternative to CD19, or to provide an additional target to minimize the potential
for CD19 loss variants to emerge.
ROR1 appears to play a decisive role in survival of some epithelial tumors.
Thus, an advantage of ing ROR1 is that a single chimeric or may be
useful to treat patients with a large number of hematopoietic and non-hematopoietic
tumors.
Our data shows for the first time that s that express a designed ROR1
chimeric receptor efficiently recognize epithelial cancers in vitro. Cytokine secretion
and T-cell eration induced by ROR1+ breast cancer cells were higher than that
induced by leukemia cells, despite the absence of the CD80/86 costimulatory ligand.
The studies reported here demonstrate that the design of the extracellular spacer
domain and chimeric receptor affinity are parameters that can be modulated to
enhance the recognition of ROR1+ hematologic and epithelial tumors in vitro and in
vivo by ROR1-chimeric receptor ed T-cells. The pment of ROR1-
chimeric receptors with enhanced tumor reactivity provides the opportunity for
clinical applications in a variety of human cancers.
Example 2
Effect of ellular spacer domain length on triggering of tumor cell lysis
with a Her2-specific chimeric receptor that recognizes an e located
proximal to the tumor cell membrane.
The effect of CAR spacer length on recognition and ring of tumor cell
recognition by CD8+ human T lymphocytes that expressed a HER2-specific
chimeric receptor was examined using similar methods to those described above for
ROR1. HER2-specific chimeric receptors were constructed using VL and VH chain
segments of a HER2-specific mAb that recognized a membrane proximal epitope on
HER2 (Figure 12A), and the scFVs were linked to IgG4 hinge/CH2/CH3, IgG4
hinge/CH3, and IgG4 hinge only extracellular spacer domains and to the CD28
transmembrane domain, 4-1BB and CD3 zeta signaling domains (Figure 12B).
y CD8+ T cells were transduced with each of the HER2 chimeric receptors
and selected for sion of the EGFR transducton marker (Figure 12D).
Expression of the HER2 chimeric receptors and the size of each receptor was
confirmed by Western Blot (Figure 12C). The T cells were then ed with anti
CD3 mAb and feeder cells and examined for their ability to recognize HER2+ tumor
cells. As observed with the R11 ROR 1 specific chimeric receptor, the HER2
ic receptor that contained a long extracellular spacer domain conferred
superior T cell recognition of HER2+ tumor cells (Figure 12E).
Discussion
This example of the effect of extracellular spacer length on chimeric receptor
modified T cell recognition of tumor cells used a chimeric receptor comprising a
scFv built from the VH+L sequences of the Herceptin chimeric mAb. Studies by Cho
et al (Nature 421:756, 2003) localized to e location of tin to a
membrane proximal location on the HER2 (ERRB2) extracellular domain e
12A). Based on our understanding of the structure of human IgG4 hinge:Fc
variants (Figure 12B), we hypothesize that a membrane proximal location of the
targeting e on an extracellular tumor cell antigen would best recognized by
effector T cells that express a chimeric receptor encoding a long spacer. Our data
demonstrating a gradient of cytolytic activity from near back ground activity by T
cells expressing a short spacer Herceptin chimeric receptor, to intermediate activity
by T cells expressing a medium length spacer chimeric receptor, and maximal lysis
by T cells that expressed the long spacer chimeric receptor. Thus, the extracellular
spacer has definitive effects on tumor recognition by T cells, and this data provides
further t for the need to tailor chimeric receptor design based on epitope
location of tumor expressed target molecules.
Example 3 –
Customizing spacer length and sequence for optimal recognition and in vivo
efficacy of CD19 with chimeric receptor modified T cells.
Materials and Methods
Human subjects
Blood samples were obtained from y donors who provided written
ed t to participate in research protocols approved by the Institutional
Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). eral
blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-
Hypaque (Sigma, St.Louis, MO), and cryopreserved in RPMI, 20% human serum
and 10% dimethyl sulfoxide.
Cell lines
The K562, Raji, JeKo-1, and 293T cell lines were obtained from the
American Type e Collection (Manassas, VA) and cultured as directed. A
lentivirus encoding the ffluc-gene am of a T2A sequence and eGFP was
produced in 293T cells and used to transduce Raji and JeKo-1 tumor cells. Raji, and
JeKo-1 cells were expanded after lentiviral transduction and the eGFP positive
subset sort-purified.
Immunophenotyping
PBMC and T-cell lines were stained with one or more of the following
conjugated monoclonal antibodies: CD3, CD4, CD8, CD25, CD45RA, CD45RO,
CD62L, CD69 and d isotype controls (BD Biosciences). Staining with
propidium iodide (PI, BD Biosciences) was med for ead cell
discrimination as directed by the manufacturer. Flow analyses were done on a
FACSCanto, sort-purifications on a FACSAriaII (Becton son) and data
analyzed using FlowJo software (Treestar).
Vector construction and preparation of CD19 chimeric receptor encoding
lentivirus
CD19 specific chimeric ors were ucted using: (1) the VL and VH
chain segments of the CD19-specific mAb FMC63 (SEQ ID NO:3), linked by a
(G4S)3 linker (SEQ ID NO:12)peptide (VL-linker-VH); (2) a spacer domain derived
from IgG4-Fc (Uniprot Database: P01861, (SEQ ID NO:13)) comprising either the
Hinge-CH2- CH3 portion (229 AA, (SEQ ID NO:)) or Hinge only (12 AA; (SEQ ID
NO:4)). Both spacers contained a S ? P tution within the Hinge domain
located at position 108 of the native IgG4-Fc protein; the 27 AA embrane
domain of human CD28 (Uniprot Database: P10747, (SEQ ID NO:14)); (4) a
signaling module comprising either (i) the 41 AA cytoplasmic domain of human
CD28 with an LL ? GG substitution located at position 186-187 of the native CD28
protein (SEQ ID NO:14) ; and/or (ii) the 42 AA cytoplasmic domain of human 4-
1BB (Uniprot se: Q07011, (SEQ ID NO:15)); linked to (iii) the 112 AA
cytoplasmic domain of isoform 3 of human CD3? (Uniprot Database: P20963, (SEQ
ID ); the self cleaving T2A sequence (SEQ ID NO:8); and (6) a truncated
epidermal growth factor receptor (EGFR)sequence (SEQ ID NO:9).
Codon-optimized nucleotide sequences encoding each trans gene were
synthesized (LifeTechnologies, Carlsbad, CA) and cloned into the epHIV7 lentiviral
vector using NheI and Not1 restriction sites. The epHIV7 lentiviral vector had been
derived from the pHIV7 vector by replacing the cytomegalovirus promoter of
pHIV7 with an EF-1 promoter.
CD19 chimeric receptor or tEGFR-encoding lentivirus was produced in 293T
cells co-transfected with the iral vector and the packaging vectors pCHGP-2,
pCMV-Rev2 and pCMV-G using Calphos transfection t (Clontech). Medium
was changed 16 h after transfection, and lentivirus collected after 24, 48 and 72 h.
Generation of T -cell lines expressing the CD19 chimeric receptors
Sort-purified CD8+ CD45RA- CD45RO+ CD62L + central memory T -cells
(TCM) of normal donors were activated with anti-CD3/ CD28 beads (Life
Technologies) according to the manufacturer's instructions, and transduced with
lentiviral supernatant (MOI = 3) supplemented with 1 µg/mL polybrene (Millipore)
on day 3 after activation by fugation at 2,100 rpm for 45 min at 32°C. T cells
were expanded in RPMI, 10% human serum, 2 mM L-glutamine and 1 % penicillinstreptomycin
(CTL medium), supplemented with recombinant human (rh) lL-2 to a
final concentration of 50 U/mL every 48 h. After expansion, an aliquot of each
transduced T cell line was d with biotin-conjugated anti-EGFR (epithelial
growth factor receptor) antibody and streptavidin-beads nyi), and tEGFR+ T
cells isolated by immunomagnetic selection.
The tEGFR+ T-cell subset was then stimulated with irradiated (8,000 rad)
TM EBV-LCL at a T cell: LCL ratio of 1 :7, and ed for 8 days in CTL
medium with addition of 50 U/mL rh IL-2 every 48 h.
um release, cytokine secretion and CFSE proliferation assays
Target cells were labeled with 51Cr (PerkinElmer) overnight, washed and
incubated in triplicate at 1-2x l03 cells/well with effector T cells at various effector
to target (E:T) ratios. Supernatants were harvested for ? counting after a 4-hour
tion and specific lysis calculated using the standard formula. For analyses of
cytokine ion, target and effector cells were plated in cate wells at an E:T
ratio of 2: 1 (Raji) or 4: 1 (K562/CDI9 and K562), and INF-?, TNF-a, IL-2, IL-4,
IL-6 and IL-10 measured by multiplex cytokine immunoassay ex) in
supernatant d after a 24-hour incubation.
For analysis of proliferation, T cells were labeled with 0.2 µM
carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in
triplicate wells with stimulator cells at a ratio of 2: 1 (Raji) or 4: 1 (K562/CD19 and
K562) in CTL medium without exogenous cytokines. After 72 h of incubation, cells
were labeled with anti-CD3 mAb and propidium iodide (PI) to exclude dead cells
from analysis. Samples were ed by flow cytometry and cell division of live
CD3+ T-cells assessed by CFSE dilution.
Experiments in NOD/SCID and NOD/SCID/?c-/- (NSG) mice
All mouse experiments were approved by the FRCRC Institutional Animal
Chimeric receptore and Use Committee. Six- to 8-week old female NOD.CBI7-
cid /J (NOD/SCID) and NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ (NSG) mice were
obtained from the Jackson Laboratory or bred in-house (FRCRC. Mice were injected
enously (i. v.) with 0.5 x l06 Raji-ffluc tumor cells via tail vein injection, and
received injections of ic receptor-modified T cells, l T cells, or PBS via
tail vein ion as indicated.
For bioluminescence imaging, mice received intraperitoneal (i.p.) injections
of freshly prepared luciferin substrate (Caliper Life Sciences, MA) resuspended in
PBS (15 µg/g body ) and were then anesthetized with isoflurane in an
induction chamber. After induction of deep esia, mice were imaged using an
n IVIS In Vivo Imaging System (Caliper Life Sciences, MA) at 10, 12 and
14 minutes post i.p. injection of luciferin at an acquisition time of 1 second to 1
minute in small binning mode to obtain unsaturated images. Luciferase activity was
analyzed using Living Image Software (Caliper Life Sciences, MA) and the photon
flux analyzed within regions of interest that encompassed the entire body of each
individual mouse.
Statistical analyses
Statistical analyses were performed using Prism Software (GraphPad, CA).
Student's t-test was performed as a two-sided test with a confidence interval of 95%
and results considered significant with a p-value of p<0.05. Statistical analysis of
survival were done by Log-rank testing and results considered significant with a pvalue
of p<0.05.
Results
Preparation of polyclonal CD8+ TCM-derived cell lines that express CD19 chimeric
receptors with long and short extracellular spacers
We constructed individual lentiviral s encoding a panel of codon-
zed CD 19 chimeric receptor genes to examine the influence of extracellular
spacer length on the in vitro function and in vivo antitumor activity of CD19
chimeric receptor-modified T cells. Each chimeric receptor was comprised of a
single chain variable fragment ponding to the sequence of the CDl9-specific
mAb FMC63 (scFv: , a spacer derived from IgG4-Fc including either the
-CH2-CH3' domain (229 AA, long spacer) or the 'Hinge' domain only (12
AA, short spacer), and a signaling module of CD3? with membrane proximal CD28
or 4-1 BB costimulatory domains, either alone or in tandem (Figure 13A). The
transgene cassette included a truncated EGFR ) downstream from the
chimeric receptor gene and separated by a cleavable T2A element, to serve as a
transduction, selection and in vivo tracking marker for chimeric or-modified T
cells.
We isolated a CD8+ CD45RO+ CD62L+ central memory T cell (TCM) cell
population by cell sorting from the blood of normal donors for transduction and
expansion, because of the superior y of TCM to persist in vivo after adoptive
transfer. CD8+ T cells were stimulated with anti CD3/28 beads, transduced with
each of the lentiviral vectors, and expanded in culture for 18 days before being used
for in vitro and in vivo experiments. (Figure 13B) Similar transduction efficiencies
were achieved with each of the lentiviral vectors (mean 25%) and transgene-positive
T cells were enriched to uniform purity by immunomagnetic selection using a
biotinylated anti-EGFR mAb and streptavidin beads. Following tEGFR-enrichment,
each of the CD19 chimeric receptor T cell lines were expanded by a single
stimulation with CD19+B-LCL, without apparent differences in in vitro growth
cs between T cell lines expressing the various CD 19 chimeric receptor
constructs. After expansion, the tEGFR marker was expressed at lent levels
on >90% of the T cells transduced with each of the vectors (Figure 13C).
CD19 chimeric receptors with long and short extracellular spacer domain confer
specific anti-tumor vity in vitro
We compared the effector function of TCM-derived T cell lines ed to express
CD19 ic ors with CD28 and 4-1BB costimulatory signaling moieties,
and either a short ('short/CD28';'short/4-1BB') or long ('long/CD28'; 4-1BB')
extracellular spacer domain respectively. T cells expressing each of the 4 CD19
chimeric receptor constructs conferred specific cytolytic activity against CD19+ Raji
and JeKo-l lymphoma cells, and against K562 cells that had been stably transfected
with CD19, but not native CD19- K562 cells (Figure 14A). Quantitative es of
cytokine production in response to stimulation with K562/CD19 or Raji tumor cells
by multiplex ne assay (Luminex) showed tion of IFN-?, TNF-a, IL-2,
IL-4, IL-6, and IL-10 by T cells expressing each of the CD19 chimeric receptors
(Figure 14B). T cells expressing CD19 chimeric receptors with a CD28
costimulatory domain produced significantly higher levels of IFN-?, TNF-a, IL-2
and IL-10 ed to the ponding constructs with a 4-1BB costimulatory
domain (Figure 14B, C). There was significantly higher IFN-y production and
significantly less IL-4 production by T cells expressing the CD19 'long/CD28'
chimeric receptor compared with those expressing the 'short/CD28' chimeric
or. Amongst the CD19 chimeric receptors with 4-1BB costimulatory signaling
module, we detected significantly higher levels of IFN-?, TNF -a, IL-2, IL-4, and
IL-10 secretion in T cells expressing the construct with the short spacer domain
e 14B, C).
We used CFSE dye dilution to analyze proliferation of T cells ed with
each of the CD 19 chimeric receptors after engagement of CD 19+ tumor cells.
Specific and vigorous eration of each of the CD19 chimeric receptor T cell
lines was observed 72 hours following stimulation with either K562/CD19 or Raji.
The average number of cell divisions was higher for CD19 chimeric receptor T cells
with a CD28 costimulatory domain compared to those with 4-1BB, consistent with
greater IL-2 production by T cells expressing a CD28 containing chimeric or
(Figure 14B-D). We also analyzed the proportion of chimeric receptor T cells that
underwent activation induced cell death after stimulation with K562/CD19 and Raji
tumor cells at the end of the 72-hours by costaining the culture with CD3+ and PI.
We detected a higher frequency of CD3+ CD8+ PI+ T cells in the CD 19 chimeric
receptor cell line 'long/4-1 BB', but few PI+ cells were observed with the other
CD19 chimeric receptors. (Figure 14E).
This analysis of in vitro effector functions was consistent with prior s
that have compared CD28 and 4-1BB costimulatory domains, and did not reveal
differences in T cell function that would suggest that a particular CD19 chimeric
receptor construct from this panel would lack anti-tumor efficacy in vivo.
T cells expressing CDI9 chimeric ors with short extracellular spacer
domains but not long extracellular spacer domains eradicate Raji tumors in
immunodeficient mouse models
We next evaluated the in vivo antitumor efficacy of T cells ed with
each of the CD19 chimeric receptors in immunodeficient (NOD/SCID) mice
engrafted with y luciferase transfected Raji cells (Raji-ffluc), which enables
sequential quantitative analyses of tumor burden and distribution using
bioluminescence imaging. NOD/SCID mice inoculated with 0.5x106 Raji-ffluc cells
via tail vein injection ped disseminated ma, which if untreated led to
hind limb paralysis after approximately 3.5 weeks, itating euthanasia. Tumor
bearing mice were treated with 2 doses of CD8+ TCM-derived T cells modified with
each of the CD19 chimeric receptors or with a tEGFR control vector administered
on day 2 and day 9 after tumor inoculation (Figure 15A).
Surprisingly, only T cells modified to express CD19 chimeric receptors with
short extracellular spacer domain ('short/CD28' and 'short/4-1BB') eradicated Raji
tumors in this model, whereas mice treated with T cells expressing CD19 chimeric
ors with long spacer ('long/CD28' and 'long/4-1BB') developed systemic
lymphoma and hind limb paralysis with nearly identical kinetics as untreated mice
or mice treated with control tEGFR+ T cells (Figure 15B, C). The striking difference
in antitumor activity between CD19 chimeric receptors with short and long spacer
s was highly icant and reproducible in multiple experiments with
chimeric receptor T cell lines generated from 3 different normal donors.
The NOD/SCID lymphoma model may be imal for ting antitumor
activity in a clinical setting because of the short interval between tumor
inoculation and T cell administration and the greater resistance to engraftment of
human cells compared to more immunodeficient mouse strains such as
NOD/SCID/?c-/- (NSG). Thus, we evaluated antitumor activity of adoptive y
in a more clinically relevant model in which Raji-ffluc lymphoma was ished in
NSG mice, and the CD19 chimeric receptor T cells were administered after 7 days
when the tumor was readily detectable in the bone marrow by bioluminescence
g (Figure 16A). We performed initial dose titration experiments to determine
the minimal dose of T cells uced with the CD19 'short/4-1BB' ic
receptor that was required for eradication of established Raji tumors. A single dose
of 2.5x106 T cells expressing CD19-chimeric receptor 'short/4-1BB' promoted
complete regression of established Raji tumors and resulted in long-term tumor-free
survival in 100% of mice (Figure 16B,C). At the 2.5x106 dose level, the T-cells were
easily detected in the peripheral blood of NSG mice for at least 3 weeks following
adoptive transfer and tumor eradication. Thus, this model enabled comparative
studies both of antitumor activity and persistence of T cells modified with each of
the CD19-chimeric receptors in our panel (Figure 16D).
We then treated cohorts of NSG mice that were engrafted with Raji
lymphoma with PBS alone, with a single dose of 6 T cells expressing each of
the CD19 chimeric receptors or with T cells modified with a tEGFR encoding
control vector (Figure 17A). In this model of established lymphoma, T cells
expressing CD19 chimeric receptors with a short extracellular spacer domain and
either 4- 1BB or CD28 costimulatory domains (‘short/CD28' and 'short/4-1BB')
mediated complete tumor regression over 7-10 days and all mice survived tumor
free for >56 days. By contrast, mice treated with T cells modified to express CD19
chimeric receptors with a long spacer domain ('long/CD28' and 'long/4-1BB')
exhibited tumor progression and had to be sacrificed at a similar time as mice that
had received control tEGFR T cells (Figure 17B, C). The lack of in vivo mor
activity of the chimeric receptor constructs with long spacers was unexpected given
the ability of T cells expressing these constructs to lyse tumor cells in vitro, and the
enhanced IL-2 production and proliferation after ment of T cells expressing
the 'long/CD28' CD19 ic receptor compared to the 4-1BB constructs.
To provide insight into the basis for the lack of efficacy, we performed
sequential flow cytometry on peripheral blood samples of mice at intervals after the
T cell infusion. All mice treated with T cells expressing the 'short/CD28' and
'short/4-1BB' CD19 chimeric receptors had significantly higher levels of erred
T cells in the blood at all time points after adoptive transfer, ed to mice
treated with T cells that expressed ponding CD19 chimeric receptors with long
extracellular spacer (p<0.01) (Figure 17D). We did not observe significant
differences in T-cell persistence in the peripheral blood of mice that had received T
cells expressing CD19 ic receptors with CD28 or 4-1BB co-stimulatory
domains and short spacer s (Figure 17D).
The in vivo anti-tumor efficacy of CD19 chimeric ors with long spacers is
not improved by increasing T cell dose or providing an additional costimulatory
domain
The lack of in vivo anti-tumor efficacy and the lower level of persisting
chimeric receptor T cells in mice treated with T cells ed with CD19 chimeric
receptors with long spacer domains ted that efficacy might be improved by
sing the chimeric receptor T cell dose or by including both CD28 and 4- IBB
domains into the chimeric receptor to augment costimulatory signaling. To evaluate
this possibility we modified CD8+ TCM with 'long/CD28', 'short CD28', and
'long/CD28_ 4-1BB' CD19 chimeric or vectors and med that the
D28_ 4-1BB' CD19 ic receptor conferred specific lysis and cytokine
production in vitro after recognition of CD19+ target cells (Figure 18A-C).
tent with previous studies of CD19 chimeric receptors, the level of cytokine
production and proliferation in vitro in T cells expressing the CD28_ 4-IBB' CDI9
chimeric receptor was inferior compared to the identical construct with CD28 alone,
and superior to T cells sing the 'long 4-IBB' CD19 chimeric receptor (Figure
18B, C).
Groups of NSG mice with established Raji tumors were then treated with a
high dose of T cells (10 x106 ) T cells expressing the 'long/CD28' CD19 chimeric
receptor, the 'long/CD28_ 4-IBB' CDI9 chimeric receptor, the /CD28' CD19-
chimeric receptor, and tEGFR alone. Tumor burden was measured by
bioluminescence imaging and serial flow cytometric analyses of peripheral blood
samples performed to ine the frequency of transferred T cells. Consistent
with the results of our prior experiments using much lower doses of T cells, Raji
tumors were completely eradicated in mice treated with T cells expressing the
/CD28' CD19-chimeric receptor. However, even with a 4-fold higher T cell
dose, treatment with T cells expressing the 'long/CD28' CD19 chimeric receptor or
the 'long/CD28_ 4-1BB' CD19 chimeric receptor did not provide a discernible
mor effect (Figure 18D,E).
Thus, increasing the chimeric receptor T cell dose and adding a 4-1BB
costimulatory domain to CD19 chimeric receptors failed to overcome the negative
impact of the longer spacer domain on mor activity in vivo. Thus, in this
model, anti-tumor reactivity of CD19 chimeric receptors is dictated to a great extent
by the length of the extracellular spacer domain, and not by the intracellular
costimulatory signaling modules.
T cells modified with CD19 chimeric receptors that possess long extracellular
s undergo activation induced cell death in vivo
We sought to determine potential mechanisms underlying the inferior in vivo
antitumor activity of T cells that express CD19 chimeric receptors with long spacer
domains. Because lower numbers of transferred T cells modified to express CD19
chimeric ors with long spacer domains were present in the blood, we
ered the possibility that the T cells were not efficiently activated by tumor
cells in vivo or sely, that they ent activation induced T cell death in
vivo. Therefore, we labeled CD19 chimeric or modified and corresponding
control T cells with CFSE and administered these T cells to tumor bearing NSG/Raji
mice to examine activation, proliferation and survival of T cells ed with each
of the CD19 chimeric or constructs at tumor sites in vivo (Figure 19A). At the
end of their in vitro expansion and immediately prior to CFSE labeling and infusion
into NSG mice g established Raji , T cells transduced with each of the
CD19 chimeric receptors expressed low levels of the activation markers CD69 and
CD25 e 19B).
Bone marrow was obtained from subgroups of mice 24 and 72 hours after the
T cell infusion to examine the frequency, activation and proliferation of transferred
T cells. At 24 hours, tumor cells (CD45+ CD3-) were present in the bone marrow in
all treatment groups and a large fraction of chimeric receptor T cells, but not control
T cells, had upregulated CD69 and CD25. There was no measurable dilution of
CFSE in the transferred chimeric or T cells. (Figure 19C) Both CD69 and
CD25 were sed in a higher proportion of T cells modified with 'long spacer'
CD19 chimeric ors, suggesting these cells may have received a stronger
stimulus compared to T cells with 'short spacer' CD19 chimeric receptors (Figure
__C). Despite evidence of T cell activation at 24 hours there were significantly
lower numbers of chimeric receptor T cells in the bone marrow of mice treated with
T cells modified with the CD28 and 4-IBB 'long spacer' constructs compared to
those modified with the CD28 and 4-IBB 'short spacer' constructs, or with the
control tEGFR vector (Figure 19C, E).
At 72 hours after T cell transfer, T cells expressing the 'short/CD28' and
'short/4-lBB' CD19 chimeric receptors had increased 3 to > 10 fold in frequency in
the bone marrow and , and had undergone several cell divisions (Figure
19D,E). Control tEGFR+ T cells remained present in the bone marrow and spleen at
72 hours at a level similar to that observed at 24 hours, and had not divided as
measured by CFSE dilution. By contrast, the numbers of T cells expressing the
'long/CD28' and 'long/4-IBB' CD19 chimeric receptors had not increased in the bone
marrow and spleen. (Figure 19D, E) Consistent with lower cell numbers, analysis of
CFSE staining in viable PI- 'long/CD28' and 'long/4-IBB' CDl9 chimeric receptor T
cells demonstrated these cells had undergone a much lower number of cell divisions
compared with 'short/CD28' and 'short/4-IBB' CDl9 chimeric receptor T cells.
(Figure en the flow data was analyzed to include PI+ T cells, we detected a
much higher frequency of PI+ CD3+ T cells in bone marrow and spleen of mice that
received CD19 chimeric receptor T cells with 'long spacer' domains, demonstrating
that a significant proportion of T cells, e being activated by tumor in vivo had
undergone cell death (Figure 19F). Consistent with the bioluminescence imaging,
CD45+ CD3- Raji tumor cells were present in greater numbers in the bone marrow
of mice d with T cells expressing CD19 ic receptors with long spacer
domains or expressing tEGFR only ed to mice treated with CD19 chimeric
receptors with short spacer domains (Figure 19D,E, G).
Collectively, the data provides evidence that CD19 chimeric receptors with
long extracellular spacer domain, despite mediating equivalent or superior effector
function in vitro and recognizing tumor in vivo, induce a high level of activation
induced cell death in vivo and fail to eradicate established lymphoma.
Discussion
Chimeric receptors are artificial receptors that include an ellular
antigen-binding scFv, a spacer domain that provides separation of the scFv from the
cell membrane and an intracellular signaling module that mediates T cell activation.
Chimeric ors that contain a scFv derived from the CD19-specific FMC63 mAb
studied here, have advanced to testing in clinical trials in patients with B-cell
malignancies. Antitumor activity and T cell persistence have varied substantially in
different . Each of these clinical trials differed in potentially critical variables,
including different gene transfer vectors, cell culture methodologies, and
ioning regimens prior to CD19 ic receptor T cell transfer.
We examined the possibility that the extracellular spacer domain of CD19
chimeric receptors may be an important inant of anti-tumor activity in vivo,
independent of the costimulatory signaling provided by the chimeric receptor. We
derived spacer domains from IgG4-Fc, which enables high levels of chimeric
receptor cell surface expression and is less likely to e recognition by innate
immune cells compared to other IgG isotypes. We used the IgG4 ‘Hinge-CH2-CH3’
in the design of the long (229 AA) spacer constructs and the IgG4 ‘Hinge’ domain in
our short (12 AA) spacer ic receptors. To compare the dual chimeric
receptor constructs, we used purified (>90%) chimeric receptor positive CD8+ TCM–
d T cells to remove differences in the ar composition and transduction
frequency as a potential source of bias in the is of in vitro and in vivo
function. CD8+ TCM have been shown to have superior traits for adoptive
immunotherapy, compared with other more prevalent T cell subsets in blood that
t poorly and are ineffective in tumor therapy. The CD19 ic receptor T
cells were generated using a standardized culture protocol that is similar to that used
to derive chimeric receptor T cells for clinical trials. Our data show that CD19
chimeric receptors with a short IgG4 ‘Hinge’ spacer conferred potent anti-tumor
reactivity in vitro and in vivo, whereas corresponding CD19 chimeric receptors with
a long spacer of IgG4 ‘Hinge-CH2-CH3’, despite equivalent or superior reactivity in
vitro, failed to confer significant anti-tumor effects in murine lymphoma models.
singly, the length of the spacer domain proved to be a decisive element for in
vivo mor activity, and the lack of efficacy of the ‘long spacer’ chimeric
receptor could not be overcome by increasing the T cell dose.
We also observed major ences in cytokine secretion and proliferation in
vitro between T cells expressing CD19 chimeric receptors containing CD28 and 4-
1BB costimulatory domains, with CD28 augmenting secretion of IFN-?, IL-2, and
TNF-a compared with 4-1BB. CD19 chimeric receptors that possessed a tandem
CD28_4-1BB also produced higher levels of these nes compared to chimeric
receptors encoding 4-1BB only. However, our data shows that these ences in in
vitro function were not predictive of in vivo anti-tumor efficacy, since CD19
chimeric receptors with either CD28 or 4-1BB costimulatory domain and a short
spacer were similarly effective at eradicating advanced established Raji tumors in
NSG mice. In contrast, CD19 chimeric receptors with suboptimal spacer length and
CD28, 4-1BB, or both costimulatory domains, despite conferring similar in vitro
function as the identical chimeric receptor construct with a short spacer domain,
lacked significant anti-tumor activity in vivo, demonstrating the contribution of
spacer length to in vivo function of chimeric receptor T cells.
Our studies provide insight into the mechanism sible for the lack of in
vivo efficacy of CD19 chimeric receptors with long spacer domains. T cells
expressing CD19 chimeric receptors with both long and short spacer s could
be detected in the bone marrow and spleen after adoptive transfer into NSG mice
bearing ished Raji lymphoma, and the majority were activated as demonstrated
by upregulation of CD25 and CD69. However, T cells modified to express a CD19
chimeric receptor with a long spacer domain exhibited a steep decline in cell
number, in contrast to the marked in vivo expansion of T cells expressing CD19
chimeric receptors with a short spacer domain. The e in T cell number was a
consequence of much higher levels of cell death in the first 72 hours after adoptive
transfer compared with T cells with short spacer domains, and control T cells that
did not s a CD19 chimeric receptor. Collectively, these data te that
recognition of tumor cells in vivo resulted in death of T cells sing CD19-
chimeric receptors with long spacer domains. A r mechanism may explain the
short duration and low levels of T cell persistence in the clinical trials that employed
long spacer CD19-chimeric receptors (14).
The studies ed here are the first to show that the spacer domains of
CD19 ic ors that lack intrinsic signaling ties have dramatic
effects on in vivo antitumor activity independent of costimulatory signaling, and
identify the importance of analyzing the optimal composition of this region in the
design of chimeric receptors for clinical applications.
The term “comprising” as used in this specification and claims means
“consisting at least in part of”. When interpreting statements in this specification,
and claims which include the term “comprising”, it is to be understood that other
features that are additional to the features prefaced by this term in each statement or
claim may also be present. Related terms such as “comprise” and “comprised” are
to be interpreted in similar .
In this specification where reference has been made to patent specifications,
other external documents, or other sources of information, this is generally for the
purpose of providing a context for discussing the features of the invention. Unless
specifically stated ise, reference to such external documents is not to be
construed as an admission that such documents, or such sources of information, in
any jurisdiction, are prior art, or form part of the common general knowledge in the
art.
In the description in this specification reference may be made to subject
matter that is not within the scope of the claims of the t application. That
subject matter should be readily identifiable by a person skilled in the art and may
assist in putting into practice the invention as defined in the claims of this
application.
The foregoing is illustrative of the present invention, and is not to be
construed as limiting thereof. The ion is defined by the following claims, with
lents of the claims to be included therein. All references and documents
referred to herein are hereby incorporated by reference.
Table 1
Sequence of D19 short spacer chimeric or
GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt
Atgctgctgctggtgaccagcctgctgctgtgcgagctgccccaccccgcctttctgctgatcccc
(GMCSFRss) (SEQ ID NO:2)
Gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccggg
ccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctac
cacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacagcctgac
catctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggc
acaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcacc
aagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgca
ccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatg
gctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaag
gacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgc
caagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc
(CD19scFv) (SEQ ID NO:3)
Gaatctaagtacggaccgccctgccccccttgccct (IgG4hinge) (SEQ ID NO:4)
Atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatctt
ttgggtg (CD28tm-)(SEQ ID NO:5)
Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagg
aagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (41BB) (SEQ ID
NO:6)
Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctga
acctgggcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagc
ggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcg
agatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgcca
ccaaggatacctacgacgccctgcacatgcaggccctgcccccaagg (CD3Zeta)- (SEQ ID NO:7)
Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg
(T2A) (SEQ ID NO:9)
Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtg
taacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgc
acctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggat
ccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaac
aggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctctt
gcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgataattt
caggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaacc
aaaattataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccga
gggctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggac
aagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtg
tcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattga
cggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgca
gacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgt
ccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccc
tggggatcggcctcttcatgtga (EGFRt) (SEQ ID NO:9)
Table 2
GMCSFRss
DNA: ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC
AA: M L L L V T S L L L C E L P H P A
CD19scFv
DNA: TTTCTGCTGATCCCC:GACATCCAGATGACCCAGACCACCTCCAGCCTGAGC
AA: F L L I P D I Q M T Q T T S S L S
DNA: GCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATC
AA: A S L G D R V T I S C R A S Q D I
DNA: AGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTG
AA: S K Y L N W Y Q Q K P D G T V K L
DNA: CTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGC
AA: L I Y H T S R L H S G V P S R F S
DNA: GGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAG
AA: G S G S G T D Y S L T I S N L E Q
DNA: GAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACC
AA: E D I A T Y F C Q Q G N T L P Y T
DNA: TTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGC
AA: F G G G T K L E I T G S T S G S G
DNA: AAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAA
AA: K P G S G E G S T K G E V K L Q E
DNA: AGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACC
AA: S G P G L V A P S Q S L S V T C T
DNA: GTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCC
AA: V S G V S L P D Y G V S W I R Q P
DNA: CCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACC
AA: P R K G L E W L G V I W G S E T T
40 DNA: TACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGC
AA: Y Y N S A L K S R L T I I K D N S
DNA: AAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCC
AA: K S Q V F L K M N S L Q T D D T A
DNA: ATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGAC
AA: I Y Y C A K H Y Y Y G G S Y A M D
IgG4hinge
50 DNA: TACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC:GAGAGCAAGTACGGA
AA: Y W G Q G T S V T V S S E S K Y G
CD28tm
DNA: TGCCCCCCTTGCCCT:ATGTTCTGGGTGCTGGTGGTGGTCGGAGGC
55 AA: P P C P P C P M F W V L V V V G G
DNA: GTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGG
AA: V L A C Y S L L V T V A F I I F W
41BB
DNA: GTG:AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATG
AA: V K R G R K K L L Y I F K Q P F M
DNA: GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA
AA: R P V Q T T Q E E D G C S C R F P
CD3Zeta
DNA: GAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAG:TTCAGCAGAAGCGCC
AA: E E E E G G C E L R V K F S R S A
DNA: GACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC
AA: D A P A Y Q Q G Q N Q L Y N E L N
DNA: CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC
AA: L G R R E E Y D V L D K R R G R D
DNA: CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT
AA: P E M G G K P R R K N P Q E G L Y
DNA: AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG
AA: N E L Q K D K M A E A Y S E I G M
DNA: AAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTG
AA: K G E R R R G K G H D G L Y Q G L
DNA: TCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCC
AA: S T A T K D T Y D A L H M Q A L P
DNA: CCAAGG:CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGT
AA: P R L E G G G E G R G S L L T C G
EGFRt
DNA: GACGTGGAGGAGAATCCCGGCCCTAGG:ATGCTTCTCCTGGTGACAAGCCTT
AA: D V E E N P G P R M L L L V T S L
DNA: CTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTG
AA: L L C E L P H P A F L L I P R K V
DNA: TGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCT
AA: C N G I G I G E F K D S L S I N A
DNA: ACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCAC
45 AA: T N I K H F K N C T S I S G D L H
DNA: ATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTG
AA: I L P V A F R G D S F T H T P P L
DNA: GATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTT
AA: D P Q E L D I L K T V K E I T G F
DNA: TTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAG
AA: L L I Q A W P E N R T D L H A F E
DNA: AACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT
AA: N L E I I R G R T K Q H G Q F S L
DNA: GTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG
AA: A V V S L N I T S L G L R S L K E
DNA: ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCA
AA: I S D G D V I I S G N K N L C Y A
DNA: AATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAA
AA: N T I N W K K L F G T S G Q K T K
DNA: ATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGC
AA: I I S N R G E N S C K A T G Q V C
DNA: CATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGC
AA: H A L C S P E G C W G P E P R D C
DNA: GTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAAC
AA: V S C R N V S R G R E C V D K C N
DNA: CTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAG
AA: L L E G E P R E F V E N S E C I Q
DNA: TGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG
AA: C H P E C L P Q A M N I T C T G R
DNA: GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGC
AA: G P D N C I Q C A H Y I D G P H C
DNA: GTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGG
AA: V K T C P A G V M G E N N T L V W
DNA: AAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACC
AA: K Y A D A G H V C H L C H P N C T
DNA: TACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAG
45 AA: Y G C T G P G L E G C P T N G P K
DNA: ATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTG
AA: I P S I A T G M V G A L L L L L V
50 DNA: GTGGCCCTGGGGATCGGCCTCTTCATGTGA (SEQ ID NO:10)
AA: V A L G I G L F M * (SEQ ID NO:11)
Table 3
ZXR-014 Nucleotide and amino acid sequences (map of sections)
GMCSFRss: -2149
CD19scFv: nt2150-2884
Igg4Hinge: nt2885-2920
CD28tm: nt2921-3004
41BB: nt3005-3130
Zeta: -3466
T2A: nt3467-3538
EGFRt: -4612
Primers for sequencing:
Oligo name Sequence Region
oJ02649 ATCAAAAGAATAGACCGAGATAGGGT pre-U5(SEQ ID NO:22)
oJ02648 CCGTACCTTTAAGACCAATGACTTAC SEQ ID NO:23)
oJ02650 TTGAGAGTTTTCGCCCCG mid-Ampr(SEQ ID NO:24)
oJ02651 AATAGACAGATCGCTGAGATAGGT post-Ampr(SEQ ID NO:25)
oJ02652 TCCGGTAAGCGG CoE1 ori(SEQ ID NO:26)
oJ02653 CGACCAGCAACCATAGTCC SV40(SEQ ID NO:27)
oJ02654 TAGCGGTTTGACTCACGG CMV(SEQ ID NO:28)
oJ02655 GCAGGGAGCTAGAACGATTC psi(SEQ ID NO:29)
oJ02656 ATTGTCTGGTATAGTGCAGCAG RRE(SEQ ID NO:30)
oJ02657 TCGCAACGGGTTTGCC EF1p(SEQ ID NO:31)
oJ02658 AGGAAGATATCGCCACCTACT CD19Rop(SEQ ID NO:32)
oJ02601 CGGGTGAAGTTCAGCAGAAG Zeta(SEQ ID NO:33)
oJ02735 ACTGTGTTTGCTGACGCAAC WPRE(SEQ ID NO:34)
ATGCTTCTCCTGGTGACAAG EGFRt(SEQ ID NO:35)
Table 4 Uniprot P0861 IgG4-Fc(SEQ ID NO:13)
20 30 40 50 60
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS
70 80 90 100 110 120
GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES PSCP APEFLGGPSV
130 140 150 160 170 180
FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY
190 200 210 220 230 240
RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK
250 260 270 280 290 300
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS YSRL TVDKSRWQEG
310 320
NVFSCSVMHE ALHNHYTQKS LSLSLGK
1-98 CH1
99-110 Hinge
0 CH2
221-327 CH3
Position 108 S?P
Table 5 Uniprot P10747 CD28(SEQ ID NO:14)
20 30 40 50 60
MLRLLLALNL FPSIQVTGNK PMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD
70 80 90 100 110 120
SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY MYPP
130 140 150 160 170 180
PYLDNEKSNG GKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR
190 200 210 220
SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS
1-18 signal peptide
19-152 extracellular domain
153-179 transmembrane domain
180-220 intracellular domain
Position 186-187 LL?GG
Table 6 Uniprot Q07011 4-1BB(SEQ ID NO:15)
20 30 40 50 60
MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CDNN RNQICSPCPP NSFSSAGGQR
70 80 90 100 110 120
TCDICRQCKG VFRTRKECSS TSNAECDCTP AGCS MCEQDCKQGQ ELTKKGCKDC
130 140 150 160 170 180
CFGTFNDQKR GICRPWTNCS LVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE
190 200 210 220 230 240
PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG
CSCRFPEEEE GGCEL
1-23 signal peptide
24-186 extracellular domain
187-213 transmembrane domain
214-255 intracellular domain
Table 7 Uniprot P20963 human CD3? isoform 3 (SEQ ID NO:16)
20 30 40 50 60
MKWKALFTAA ILQAQLPITE AQSFGLLDPK GILF IYGVILTALF LRVKFSRSAD
70 80 90 100 110 120
APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA
130 140 150 160
EAYSEIGMKG ERRRGKGHDG TATK DTYDALHMQA LPPR
1-21 signal peptide
22-30 extracellular
31-51 transmembrane
52-164 intracellular domain
61-89 ITAM1
100-128 ITAM2
131-159 ITAM3
Table 8 ary Hinge region Sequences
Human IgG1 EPKSCDKTHTCPPCP (SEQ ID NO:17)
Human IgG2 ERKCCVECPPCP (SEQ ID NO:18)
Human IgG3 ELKTPLGDTHTCPRCP (EPKSCDTPPPCPRCP)3 (SEQ ID NO:19)
Human IgG4 ESKYGPPCPSCP (SEQ ID NO:20)
Modified Human IgG4 ESKYGPPCPPCP (SEQ ID NO:21)
Modified Human IgG4 YGPPCPPCP (SEQ ID NO:51)
Modified Human IgG4 KYGPPCPPCP (SEQ ID NO:52)
Modified Human IgG4 EVVKYGPPCPPCP (SEQ ID NO:53)
Table 9
R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:37)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
AGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
CACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
AGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
40 CCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TACAGATCCAAGCTGTGACCGGCGCCTACG
GCTAGC GAATTCCTCGAGGCC
ACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC
45 TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA
CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC
TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC
ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG
GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA
GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG
ACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA
GCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG
GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC
ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA
GCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT
ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC
GACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC
TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC
GTGACCGGCGAGTCTAAG
IgG4 spacer
TA CGGACCGCCCTGCCCCCCTTGCCCT
GAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG
AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT
GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG
TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC
AAGGCCAAG
GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG
ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC
GTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT
GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG
AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG
4-1BB
ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3 zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
GGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
40 CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
GATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CAGGCCCTGCCCCCAAGG
45 CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
tEGFR
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
CTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
40 CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
45 CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
40 GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
45 TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 10
Leader _R12- Hinge-CH2-CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:38)
Leader
MLLLVTSLLLCELPHPAFLLIP
R12 scFv
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG
WVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI
WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD
TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE
ADYYCGADYIGGYVFGGGTQLTVTG
Hinge Spacer
ESKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHN
EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
CD28
MFWVLVVVGGVLACYSLLVTVAFIIFWV
4-1BB
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3 zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
LEGGGEGRGSLLTCGDVEENPGPR
tEGFR
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT
KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK
TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL
LLLVVALGIGLFM
Table 11
R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:39)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
TCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
ACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TCCAAGCTGTGACCGGCGCCTACG
GCTAGCGAATTCCTCGAGGCC
R12 ScFv
45 CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC
TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA
CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC
TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC
ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG
GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA
GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG
GCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA
GCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG
GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC
ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA
GCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT
ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC
GACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC
TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC
GTGACCGGCGAGTCTAAG
Hinge Spacer
TA GCCCTGCCCCCCTTGCCCT
GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG
ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT
GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG
AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG
4-1BB
ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CAGGCCCTGCCCCCAAGG
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
40 tEGFR
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
45 CTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
TCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
40 CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
TAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
45 ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
TCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
40 TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
45 CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 12
Leader _R12- Hinge- CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:40)
Leader
MLLLVTSLLLCELPHPAFLLIP
R12 scFV
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG
KTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI
WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD
TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE
ADYIGGYVFGGGTQLTVTG
Hinge Spacer
ESKYGPPCPPCP
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
CD28tm
MFWVLVVVGGVLACYSLLVTVAFIIFWV
4-1BB
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3 zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
LEGGGEGRGSLLTCGDVEENPGPR
tEGFR
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT
KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK
TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL
LLLVVALGIGLFM
Table 13
R12 short spacer CAR: PJ_R12-Hinge-41BB-Z-T2A-tEGFR (SEQ ID NO:41)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
40 GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TACAGATCCAAGCTGTGACCGGCGCCTACG
GCTAGF
R12 scFV
CTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC
45 TTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACA
CCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCC
TACTACATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCC
ACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCACCTGGGTGAACGGACG
GTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACA
GCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCG
ACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTA
GCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTG
GTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATC
ACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCA
GCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT
ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCC
TACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTAC
TGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACC
GTGACCGGCGAGTCTAAG
Hinge/Spacer
TA CGGACCGCCCTGCCCCCCTTGCCCT
4-1BB
ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3 zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CAGGCCCTGCCCCCAAGG
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
tEGFR
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
ATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
40 TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
ATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
45 CAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
CCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
TGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
GCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
40 AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
45 CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
CCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
TTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
40 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
45 TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 14
Leader _R12 - CD28tm/41BB-Z-T2A-tEGFR(SEQ ID NO:42)
Leader
MLLLVTSLLLCELPHPAFLLIP
scFv R12
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSG
KTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNI
WGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTD
TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDE
ADYYCGADYIGGYVFGGGTQLTVTG
Hinge/spacer
ESKYGPPCPPCP
CD28tm
VVGGVLACYSLLVTVAFIIFWV
4-1BB
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
LEGGGEGRGSLLTCGDVEENPGPR
tEGFR
SLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT
KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK
TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL
LLLVVALGIGLFM
Table 15
R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:43)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
TCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
ACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TACAGATCCAAGCTGTGACCGGCGCCTACG
GCTAGC
scFv R12
45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC
CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG
GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC
AACGACTACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG
ATCGGCTTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC
CGGTTCACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT
GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA
CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG
AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA
CCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT
GCCAGGCCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC
CCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG
CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC
GGCGTGCAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA
CGTGTCCTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA
Hinge/Spacer
TA CGGACCGCCCTGCCCCCCTTGCCCT
GCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG
GAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT
GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG
TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC
AAGGCCAAG
GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG
ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT
GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG
AGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG
4-1BB
TGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
40 CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CAGGCCCTGCCCCCAAGG
45 CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
tEGFR
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
ATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
40 CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
45 AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
40 ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
45 TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
GGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 16
Leader _R11- Hinge-CH2-CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:44)
Leader
MLLLVTSLLLCELPHPAFLLIP
R11 scFv
QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW
YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT
ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ
KPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV
SYRTSFGGGTEVVVK
Hinge/Spacer
ESKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
CD28tm
MFWVLVVVGGVLACYSLLVTVAFIIFWV
4-1BB
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
LEGGGEGRGSLLTCGDVEENPGPR
tEGFR
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT
KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK
TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL
LLLVVALGIGLFM
Table 17
R11 ediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR (SEQ ID NO:45)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
CATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
AGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
ACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TACAGATCCAAGCTGTGACCGGCGCCTACG
GCTAGC
R11 scFV
45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC
CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG
GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC
AACGACTACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG
ATCGGCTTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC
CGGTTCACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT
GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA
CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG
AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA
CCCAGACCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT
GCCAGGCCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC
GGCCAGCCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG
CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC
CAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA
CGTGTCCTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA
Hinge/spacer
TAGGACCGCCCTGC CCCCCTTGCCCT
GCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAG
GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTG
AGCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGT
GCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGG
TGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGC
AAGGCCAAG
CH3
GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG
ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT
GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG
TGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG
4-1BB
ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
40 ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CTGCCCCCAAGG
45 T2A
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
tEGFR
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
ACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCGGCCGCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
TGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
GGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
40 CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
GAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
45 AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
ACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
40 ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
TTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
45 TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
CCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 18
Leader _R11- Hinge-CH3- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:46)
Leader
MLLLVTSLLLCELPHPAFLLIP
scFV R11
QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW
YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT
ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ
TLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV
SYRTSFGGGTEVVVK
Hinge/spacer
ESKYGPPCPPCP
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
CD28tm
MFWVLVVVGGVLACYSLLVTVAFIIFWV
4-1BB
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
LEGGGEGRGSLLTCGDVEENPGPRM
tEGFR
LLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
DSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTK
QHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT
KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEG
EPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG
ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLL
LLVVALGIGLFM
Table 19
R11 short spacer CAR: - 41BB-Z-T2A-tEGFR(SEQ ID NO:47)
GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT
TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG
TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
TCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAG
CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGG
GCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGA
GGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGA
AAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAG
TATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAA
CATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACA
GAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTG
AGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA
AGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGA
CACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCA
AATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGT
AGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGA
AGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATC
AAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGGCAAA
GAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC
TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTG
ACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTG
CTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATC
AAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACA
GCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT
TGGATCTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTG
GGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTAC
AGGGACAGCAGAGATCCAGTTTGGGGATCAATTGCATGAAGAATCTGCTTAGG
GTTAGGCGTTTTGCGCTGCTTCGCGAGGATCTGCGATCGCTCCGGTGCCCGTCA
GTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG
GCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT
GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT
GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
CTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA
GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCC
TCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC
40 CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT
GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGT
TACAGATCCAAGCTGTGACCGGCGCCTACG
GCTAGC
scFV R11
45 GAATTCGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCC
CACCCCGCCTTTCTGCTGATCCCCCAGAGCGTGAAAGAGTCCGAGGGCGACCTG
GTCACACCAGCCGGCAACCTGACCCTGACCTGTACCGCCAGCGGCAGCGACATC
AACGACTACCCCATCTCTTGGGTCCGCCAGGCTCCTGGCAAGGGACTGGAATGG
TTCATCAACAGCGGCGGCAGCACTTGGTACGCCAGCTGGGTCAAAGGC
CGGTTCACCATCAGCCGGACCAGCACCACCGTGGACCTGAAGATGACAAGCCT
GACCACCGACGACACCGCCACCTACTTTTGCGCCAGAGGCTACAGCACCTACTA
CGGCGACTTCAACATCTGGGGCCCTGGCACCCTGGTCACAATCTCTAGCGGCGG
AGGCGGCAGCGGAGGTGGAGGAAGTGGCGGCGGAGGATCCGAGCTGGTCATGA
CCCAGACCCCCAGCAGCACATCTGGCGCCGTGGGCGGCACCGTGACCATCAATT
GCCAGGCCAGCCAGAGCATCGACAGCAACCTGGCCTGGTTCCAGCAGAAGCCC
CCCCCCACCCTGCTGATCTACAGAGCCTCCAACCTGGCCAGCGGCGTG
CCAAGCAGATTCAGCGGCAGCAGATCTGGCACCGAGTACACCCTGACCATCTCC
GGCGTGCAGAGAGAGGACGCCGCTACCTATTACTGCCTGGGCGGCGTGGGCAA
CGTGTCCTACAGAACCAGCTTCGGCGGAGGTACTGAGGTGGTCGTCAAA
Hinge/spacer
TACGGACCGCCCTGCCCCCCTTGCCCT
GGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATG
ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGT
GGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACG
TGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG
4-1BB
ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTG
GTGACAGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTG
TTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG
CD3zeta
CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAA
TCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGG
ATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTA
CAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGC
CTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATG
CAGGCCCTGCCCCCAAGG
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGA
GGAGAATCCCGGCCCTAGG
tEGFR
40 ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
TCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA
GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATA
CTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA
45 CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCT
TTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTC
TTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA
TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA
CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATA
AGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTT
GTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG
GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG
AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGC
CTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC
CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGA
GTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGT
GTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA
AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGG
GGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA
GCTCTAGACCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAATC
AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC
TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT
CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAT
GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCT
GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG
ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTG
CCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT
CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCT
GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCT
TCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTC
AGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGTCGACTAG
CCGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT
AAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATC
TGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCT
CTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT
GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC
AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGAATTCGATATCAAGCTTAT
CGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGT
ACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAA
TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGG
CGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT
AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAAT
CAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT
CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
40 GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG
GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG
ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGA
GCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCAC
CGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGG
45 AAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT
CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG
AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
ACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG
GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG
AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA
GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT
GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA
TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACC
ACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG
AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT
CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG
GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT
GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTT
GGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT
TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT
TCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCA
CTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCTCGAGGTCGAGA
TCCGGTCGACCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
40 CCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA
TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAG
GCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTCGACGGTATCGATTGGCTCA
TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
45 TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT
CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC
AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC
ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC
ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT
ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGAATTCGGAGTGGCG
AGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTG
Table 20
Leader _R11- Hinge- CD28tm/41BB-Z-T2A-tEGFR (SEQ ID NO:48)
Leader
MLLLVTSLLLCELPHPAFLLIP
ScFv R11
QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTW
YASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWGPGTLVT
ISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQ
KPGQPPTLLIYRASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNV
SYRTSFGGGTEVVVK
Spacer/Hinge
ESKYGPPCPPCP
CD28tm
MFWVLVVVGGVLACYSLLVTVAFIIFWV
4-1BB
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3zeta
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
GRGSLLTCGDVEENPGPR
tEGFR
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRT
KQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK
RGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALL
LLLVVALGIGLFM
Table 21
Intermediate Spacer (SEQ ID NO:49)
Hinge/spacer
ESKYGPPCPPCP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
Long spacer (SEQ ID NO:50)
Hinge
ESKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK
Table 22 Her2 uct-short spacer (SEQ Id No:54 )
GMCSFss-Her2scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt
Leader
Atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatccca
Her2scFV
gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgccgtgccagtcaggatgtg
aatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatccttcctctactctggagtccct
tctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagacttcgcaacttattactgtcag
caacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggcggtggctccgggggcg
gatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgtttgtc
agcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggcctggaatgggttgcaag
gatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacacatccaaaaacacagcct
acctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacggcttctatgctatggacta
ctggggtcaaggaaccctggtcaccgtctcgagt
Hinge spacer
Gagagcaagtacggaccgccctgccccccttgccct
CD28tm
atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg
4-1BB
ggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagc
tgccgatttccagaagaagaagaaggaggatgtgaactg
CD3 zeta
Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag
aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag
gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg
gcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc
caagg
Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg
tEGFR
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt
attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc
tgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc
acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc
aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga
tgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa
ttataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc
ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc
40 aagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg
ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa
acaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca
ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg
gccctggggatcggcctcttcatgtga
Table 23
Her2 construct-intermediate spacer (SEQ Id No:55 )
Leader
Atgcttctcctggtgacaagccttctgctctgtgagttaccacaccca
Her2scFv
Gcattcctcctgatcccagatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgc
cgtgccagtcaggatgtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatcct
tcctctactctggagtcccttctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagactt
cgcaacttattactgtcagcaacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggc
ggtggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagg
gggctcactccgtttgtcctgtgcagcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggc
ctggaatgggttgcaaggatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacac
atccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacgg
cttctatgctatggactactggggtcaaggaaccctggtcaccgtctcgagt
Hinge spacer
GagagcaagtacggaccgccctgccccccttgccctGgccagcctagagaaccccaggtgtacaccctgcctcccagccagga
agagatgaccaagaaccaggtgtccctgacctgcctggtcaaaggcttctaccccagcgatatcgccgtggaatgggagagcaac
ggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggcagcttcttcctgtactcccggctgaccgtgg
acaagagccggtggcaggaaggcaacgtcttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccc
tgagcctgagcctgggcaag
CD28tm
Atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg
4-1BB
ggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagc
tgccgatttccagaagaagaagaaggaggatgtgaactg
CD3 zeta
Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag
aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag
gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg
gcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc
caagg
Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg
tEGFR
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt
attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc
tggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc
acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc
aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga
40 tgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa
ttataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc
ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc
gtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg
ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa
45 acaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca
ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg
gccctggggatcggcctcttcatgtga
Table 24
Her2 construct-long spacer (SEQ Id No:56 )
leader
ctcctggtgacaagccttctgctctgtgagttaccacaccca
Her2scFV
gcattcctcctgatcccagatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgcc
gtgccagtcaggatgtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatcctt
cctctactctggagtcccttctcgcttctctggttccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagactt
cgcaacttattactgtcagcaacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggc
ggtggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagg
gggctcactccgtttgtcctgtgcagcttctggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggc
ctggaatgggttgcaaggatttatcctacgaatggttatactagatatgccgatagcgtcaagggccgtttcactataagcgcagacac
atccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacgg
cttctatgctatggactactggggtcaaggaaccctggtcaccgtctcgagt
long spacer
gagagcaagtacggaccgccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagcc
caaggacaccctgatgatcagccggacccccgaggtgacctgcgtggtggtggacgtgagccaggaagatcccgaggtccagtt
caattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggt
gtctgtgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaagtgcaaggtgtccaacaagggcctgcccagcagc
atcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacaccctgcctccctcccaggaagagatgacc
aagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcct
gagaacaactacaagaccacccctcccgtgctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagcc
aggaaggcaacgtctttagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtc
cctgggcaag
CD28tm
atgttctgggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg
4-1BB
aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagct
gccgatttccagaagaagaagaaggaggatgtgaactg
CD3zeta
Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcag
aagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccag
gaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggg
gcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccc
caagg
Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg
tEGFR
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggt
40 attggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcc
tgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatc
acagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggacc
aagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggaga
tgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaa
45 ttataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc
ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagcc
aagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacgg
ggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaa
ccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggcca
ggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtg
gccctggggatcggcctcttcatgtga
Claims (57)
1. A chimeric receptor, the chimeric or comprising: (a) an extracellular domain consisting of: 5 an extracellular ligand binding domain consisting of a single chain variable fragment (scFv) that binds to a CD19, wherein the scFv comprises a le light chain (VL) domain comprising a CDRL1, a CDRL2, and a CDRL3 of the amino acid sequence encoded by SEQ ID NO:3, and a variable heavy chain (VH) domain comprising a CDRH1, a CDRH2, and a CDRH3 of the amino acid sequence encoded by SEQ ID NO:3; and 10 an extracellular polypeptide spacer that is about 15 amino acids or less in length and comprises an amino acid sequence of P, wherein X1 is a cysteine, glycine, or arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); (b) a transmembrane domain; and (c) an intracellular signaling domain that comprises a CD3? signaling domain 15 and a costimulatory domain.
2. A chimeric receptor, the chimeric receptor comprising: (a) a single chain le fragment (scFv) that binds to CD19, wherein the scFv comprises: 20 the amino acid sequence encoded by SEQ ID NO:3; (b) a transmembrane domain; (c) a polypeptide spacer located between the scFv and the transmembrane domain, wherein the polypeptide spacer is about 15 amino acids or less in length and comprises an amino acid ce of X1PPX2P, wherein X1 is a cysteine, glycine, or 25 arginine and X2 is a cysteine or a threonine (SEQ ID NO:1); and (d) an intracellular ing domain that comprises a CD3? signaling domain and a costimulatory domain.
3. The chimeric receptor of claim 1 or 2, wherein the polypeptide spacer comprises a human IgG1, IgG2, or IgG4 hinge region or a modified version of a human IgG1, IgG2, or IgG4 hinge region.
4. The chimeric receptor of any one of claims 1-3, wherein the polypeptide 5 spacer comprises the amino acid ce set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
5. The chimeric receptor of any one of claims 1-3, wherein the ptide spacer comprises a modified human IgG4 hinge region.
6. The chimeric receptor of any one of claims 1-5, wherein the polypeptide 10 spacer comprises the amino acid sequence set forth in SEQ ID NO:51.
7. The chimeric receptor of any one of claims 1-6, wherein the polypeptide spacer is 10 to 15 amino acids in length.
8. The chimeric receptor of any one of claims 1-7, wherein the polypeptide spacer is about 12 amino acids in length. 15
9. The chimeric receptor of any one of claims 1-8, n the polypeptide spacer consists of the amino acid sequence set forth in SEQ ID NO:21.
10. The chimeric or of any one of claims 1 and 3-9, wherein the VL domain comprises the VL domain present in the amino acid sequence encoded by SEQ ID NO:3; and the VH domain comprises the VH domain present in the amino acid sequence 20 encoded by SEQ ID NO:3.
11. The chimeric receptor of any one of claims 1 and 3-10, wherein the VL domain comprises a CDRL1 sequence of RASQDISKYLN, a CDRL2 sequence of SRLHSGV, and a CDRL3 ce of GNTLPYTFG, and the VH domain comprises a CDRH1 sequence of DYGVS, a CDRH2 sequence of VIWGSETTYYNSALKS, and a CDRH3 sequence of YAMDYWG.
12. The chimeric receptor of any one of claims 1-11, wherein the VH domain and the VL domain are separated by a peptide linker. 5
13. The chimeric receptor of claim 12, wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:36.
14. The chimeric or of claim 12 or 13, wherein the scFv has a VL-linker-VH orientation.
15. The chimeric or of any one of claims 1-14, n the 10 transmembrane domain comprises a transmembrane domain of a CD8 or of a CD28.
16. The chimeric receptor of any one of claims 1-15, n the transmembrane domain comprises a transmembrane domain of a CD28 comprising the amino acid sequence encoded by SEQ ID NO:5, or the amino acid sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV. 15
17. The chimeric or of any one of claims 1-16, wherein the costimulatory domain comprises: the signaling domain of a 4-1BB or a modified version thereof; or the signaling domain of a CD28 or a modified version thereof.
18. The chimeric receptor of claim 17, wherein the 4-1BB signaling domain 20 comprises amino acids 214-255 of SEQ ID NO:15 or ses the amino acid sequence encoded by SEQ ID NO:6.
19. The chimeric receptor of claim 17, wherein the CD28 signaling domain comprises amino acids 180-220 of SEQ ID NO:14 or comprises a modified n thereof comprising an LL ? GG substitution located at positions 186-187 of SEQ ID NO:14.
20. The chimeric receptor of any one of claims 1-19, wherein the CD3? 5 signaling domain ses the amino acid sequence encoded by SEQ ID NO:7.
21. The chimeric receptor of any one of claims 1, 3-18 and 20, wherein: the VL domain comprises the VL domain present in the amino acid sequence encoded by SEQ ID NO:3; and the VH domain comprises the VH domain present in the amino acid sequence encoded by SEQ ID NO:3; 10 the ptide spacer comprises the amino acid sequence set forth in SEQ ID NO:51; the transmembrane domain ses the amino acid sequence encoded by SEQ ID NO:5; and the ellular signaling domain comprises the amino acid sequence encoded by 15 SEQ ID NO:6 and the amino acid sequence encoded by of SEQ ID NO:7.
22. The chimeric receptor of any one of claims 1, 3-18, 20 and 21, wherein: the VL domain comprises the VL domain present in the amino acid sequence encoded by SEQ ID NO:3; and the VH domain comprises the VH domain t in the amino acid sequence encoded by SEQ ID NO:3; 20 the ptide spacer consists of the amino acid sequence encoded by SEQ ID NO:4 or consists of the amino acid sequence set forth in SEQ ID NO:21; the transmembrane domain comprises the amino acid sequence encoded by SEQ ID NO:5; and the intracellular signaling domain comprises the amino acid sequence encoded by 25 SEQ ID NO:6 and the amino acid sequence encoded by of SEQ ID NO:7.
23. The chimeric receptor of any one of claims 1, 3, 7, 8, 10, 18 and 20, wherein: the scFv has a VL-linker-VH orientation; the polypeptide spacer comprises a modified IgG4 hinge ; the transmembrane domain ses the amino acid sequence encoded by SEQ ID NO:5; and 5 the intracellular signaling domain comprises the amino acid sequence encoded by SEQ ID NO:6 and the amino acid sequence encoded by SEQ ID NO:7.
24. The chimeric receptor of any one of claims 1 and 3-23, wherein the scFv is encoded by the sequence set forth in SEQ ID NO:3.
25. The chimeric receptor of any one of claims 1-18 and 20-24, wherein the 10 chimeric receptor is encoded by a nucleic acid that comprises: (a) the sequence set forth in SEQ ID NO:3 encoding the scFv; (b) the ce set forth in SEQ ID NO:4 encoding the polypeptide spacer; (c) the sequence set forth in SEQ ID NO:5 encoding the embrane domain; 15 (d) the sequence set forth in SEQ ID NO:6 encoding the costimulatory domain; (e) the sequence set forth in SEQ ID NO:7 encoding the CD3? signaling domain.
26. The chimeric receptor of any one of claims 1-18 and 20-25, wherein the 20 chimeric receptor is encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:10.
27. A chimeric or encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:10.
28. A ic receptor, wherein the ic receptor comprises the sequence 25 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGK PGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE WLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIF WVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA 5 YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
29. An isolated host cell, comprising the ic receptor of any one of claims 1-28.
30. The host cell of claim 29, wherein the host cell is an immune cell. 10
31. The host cell of claim 29 or 30, wherein the host cell is a lymphocyte that is CD45RA-, CD45RO+, and CD62L+.
32. The host cell of any of claims 29-31, wherein the host cell is a T cell.
33. The host cell of any one of claims 29-32, wherein the host cell is a CD8+ T cell or a CD4+ T cell. 15
34. The host cell of any one of claims 29-33, wherein the host cell is a CD8+ T cell selected from the group consisting of a naïve CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell and a bulk CD8+ T cell.
35. The host cell of claim 34, wherein the CD8+ T cell is a l memory CD8+ T cell, wherein the central memory CD8+ T cell is CD45RO+ and CD62L+ . 20
36. The host cell of any one of claims 29-33, wherein the host cell is a CD4+ T cell selected from the group consisting of a naïve CD4+ T cell, a l memory CD4+ T cell, an effector memory CD4+ T cell, and a bulk CD4+ T cell.
37. The host cell of claim 36, wherein the CD4+ T cell is a naïve CD4+ T cell, wherein the naïve CD4+ T cell is CD45RA+, CD62L+, and -.
38. A composition, comprising the host cell of any one of claims 29-37, in a pharmaceutically acceptable excipient. 5
39. The composition of claim 38, wherein the composition comprises a CD4+ T cell and/or a CD8+ T cell.
40. The composition of claim 39, wherein the composition comprises CD4+ T cells and CD8+ T cells and the CD4+ T cells and the CD8+ T cells are present at about a 1:1 ratio in the composition. 10
41. Use of a pharmaceutical composition, comprising the ic or of any one of claims 1-28, the host cell of any one of claims 29-37, or the composition of any one of claims 38-40 in the manufacture of a medicament for the treatment of a tumor expressing CD19.
42. Use of a population of immune cells comprising the chimeric receptor of 15 any one of claims 1-28 in the manufacture of a medicament for the ent of a tumor expressing CD19.
43. The use of claim 42, wherein the population of immune cells comprises CD8+ T cells.
44. The use of claim 43, wherein the population of immune cells further 20 comprises CD4+ T cells.
45. Use of a population of CD8+ T cells comprising the chimeric or of any one of claims 1-28 in the manufacture of a medicament for the treatment of a tumor expressing CD19.
46. The use of claim 45, wherein the tion of CD8+ T cells is for use in combination with a population of CD4+ T cells comprising the chimeric receptor of any one of claims 1-28.
47. Use of a tion of CD4+ T cells comprising the chimeric receptor of 5 any one of claims 1-28 in the manufacture of a medicament for the treatment of a tumor expressing CD19.
48. The use of claim 47, n the population of CD4+ T cells is for use in combination with a population of CD8+ T cells the chimeric receptor of any one of claims 1-28. 10
49. Use of a combination comprising a population of CD8+ T cells comprising the chimeric receptor of any one of claims 1-28 and a population of CD4+ T cells comprising the chimeric receptor of any one of claims 1-28 in the manufacture of a medicament for the treatment of a tumor expressing CD19.
50. The use of any one of claims 46, 48 and 49, wherein the population of 15 CD8+ T cells and the tion of CD4+ T cells is for use at a ratio of about 1:1 CD8+ T cells to CD4+ T cells.
51. The use of any one of claims 46 and 48-50, wherein the population of CD8+ T cells and the population of CD4+ T cells express the same chimeric receptor.
52. The use of any one of claims 41-51, n the tumor is a lymphoma or a 20 leukemia.
53. The use of any one of claims 41-52, wherein the tumor is a leukemia that is a chronic lymphocytic leukemia (CLL).
54. A chimeric receptor as claimed in any one of claims 1-28 substantially as herein described and with reference to any example thereof.
55. An isolated host cell as claimed in any one of claims 29-37 ntially as herein described and with reference to any example thereof 5
56. A ition as claimed in any one of claims 38-40 substantially as herein described and with reference to any example thereof.
57. A use as claimed in any one of claims 41-53 substantially as herein described and with reference to any example thereof. PCT/U
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261691117P | 2012-08-20 | 2012-08-20 | |
US61/691,117 | 2012-08-20 | ||
NZ738636A NZ738636B2 (en) | 2012-08-20 | 2013-08-20 | Method and compositions for cellular immunotherapy |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ745372A NZ745372A (en) | 2020-09-25 |
NZ745372B2 true NZ745372B2 (en) | 2021-01-06 |
Family
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