U.S. patent application number 15/296666 was filed with the patent office on 2017-10-12 for chimeric antigen receptor-expressing t cells as anti-cancer therapeutics.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Haiyan Chu, Yong Gu Lee, Philip S. Low.
Application Number | 20170290900 15/296666 |
Document ID | / |
Family ID | 50979254 |
Filed Date | 2017-10-12 |
United States Patent
Application |
20170290900 |
Kind Code |
A1 |
Low; Philip S. ; et
al. |
October 12, 2017 |
CHIMERIC ANTIGEN RECEPTOR-EXPRESSING T CELLS AS ANTI-CANCER
THERAPEUTICS
Abstract
Cytotoxic lymphocytes expressing chimeric antigen receptors
(CAR) that target and bind small conjugate molecules (SCM) are
disclosed, as well as methods of using the cells and the SCMs in
the treatment of cancer.
Inventors: |
Low; Philip S.; (West
Lafayette, IN) ; Chu; Haiyan; (West Lafayette,
IN) ; Lee; Yong Gu; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Family ID: |
50979254 |
Appl. No.: |
15/296666 |
Filed: |
October 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14654227 |
Jun 19, 2015 |
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PCT/US2013/076986 |
Dec 20, 2013 |
|
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15296666 |
|
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61740384 |
Dec 20, 2012 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61P 35/00 20180101; A61K 35/17 20130101; A61K 2039/5156 20130101;
A61K 47/551 20170801; A61K 2039/5158 20130101; A61K 2039/585
20130101; A61K 47/60 20170801; A61K 47/555 20170801; A61K 47/545
20170801; A61K 47/6901 20170801; A61K 39/0013 20130101; A61K
39/0011 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. A two component cancer therapeutic comprising: (a) a small
conjugate molecule (SCM) comprising a targeted moiety conjugated to
a tumor receptor ligand via a linker, wherein the tumor receptor
ligand is folate, the targeted moiety is fluorescein isothiocyanate
(FITC), and the linker is ethylenediamine; and (b) a chimeric
antigen receptor (CAR)-expressing cytotoxic lymphocyte, wherein the
CAR is a fusion protein comprising a recognition region, a
co-stimulation domain and an activation signaling domain, and
wherein the CAR has binding specificity for the targeted moiety or
can be bound by the targeted moiety.
2.-7. (canceled)
8. The two component cancer therapeutic of claim 1, wherein the
recognition region of the CAR is a single chain fragment variable
(scFv) region of an anti-FITC antibody.
9. The two component cancer therapeutic of claim 1, wherein the
co-stimulation domain of the CAR is chosen from CD28, CD 137
(4-1BB), and CD 134 (OX40).
10. The two component cancer therapeutic of claim 1, wherein the
activation signaling domain of the CAR is a T cell CD3.zeta.
chain.
11. The two component cancer therapeutic of claim 1, wherein the
recognition region is a single chain fragment variable (scFv)
region of an anti-FITC antibody, wherein the co-stimulation domain
is CD137 (4-1BB), and wherein the activation signaling domain is a
T cell CD3.zeta. chain.
12.-48. (canceled)
49. A two component cancer therapeutic comprising: (a) a small
conjugate molecule (SCM) comprising a targeted moiety conjugated to
a tumor receptor ligand via a linker, wherein the targeted moiety
is fluorescein isothiocyanate (FITC), the tumor receptor ligand is
folate, and the linker is ethylenediamine and; (b) a chimeric
antigen receptor (CAR)-expressing cytotoxic lymphocyte, wherein the
CAR is a fusion protein comprising a recognition region, a
co-stimulation domain and an activation signaling domain, and
wherein the recognition region is a single chain fragment variable
(scFv) region of an anti-FITC antibody, the co-stimulation domain
is CD137 (4-1BB), and the activation signaling domain is a T cell
CD3.zeta. chain; and wherein the CAR has binding specificity for
the targeted moiety or can be bound by the targeted moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/654,227, filed on Jun. 19, 2015, which is a
national stage entry under 35 U.S.C. .sctn.371(b) of PCT
International Application No. PCT/US2013/076986, filed on Dec. 20,
2013, which claims priority under 35 U.S.C .sctn.119(e) to U.S.
Provisional Application Ser. No. 61/740,384, filed on Dec. 20,
2012, each of which is incorporated herein by reference.
BACKGROUND OF INVENTION
[0002] Immunotherapy based on adoptive transfer of lymphocytes
(e.g., T cells) into a patient can play an important role in
eliminating cancer. Among many different types of immunotherapeutic
agents, one of the most promising therapeutic methods being
developed is T cells expressing Chimeric Antigen Receptors (CAR).
CARs are genetically engineered receptors that are designed to
target a specific antigen of a selected tumor [1]. For example, T
cells that have cytotoxic activity are transfected with and grown
to express CARs such that T cells expressing CARs can target and
kill tumors via tumor-associated antigens.
[0003] First generation CARs are composed of two main regions.
First, a recognition region, e.g., a single chain fragment variable
(scFv) region derived from a tumor-targeted antibody, is used to
recognize and bind tumor-associated antigens. Second, an activation
signaling domain, e.g., the CD3.zeta. chain of T cells, serves as a
T cell activation signal in CARs [2]. Although T cells transduced
to express such constructs showed positive results in vitro, they
have been found to have limited performance in eliminating tumor
cells in clinical trials. The main limitation has been the relative
inability to prolong and expand the T cells population and achieve
sustained antitumor effects in vivo.
[0004] To address these problems, a co-stimulation domain (e.g.
CD137, CD28 or CD134) is included in second generation CARs to
achieve full, prolonged activation of T cells. Addition of a
co-stimulation domain enhances the in vivo proliferation and
survival of T cells containing CARs, and initial clinical data have
shown that such constructs are a promising therapeutic agent in the
treatment of tumors [3].
[0005] Although use of CAR-expressing T cells as an
immunotherapeutic agent shows promise, there remain several
challenges to overcome in order to achieve significant clinical
outcomes. First, `off-target` toxicities may result due to the fact
that it is difficult to target only cancer cells via
tumor-associated antigens because in many cases normal cells also
express the tumor-associated antigen. For example, CD19 is a
tumor-associated antigen that is expressed on malignant B cells.
CARs containing anti-CD19 antibody were generated and used treated
to patients. Although remission of malignant B cells was found,
normal B cells were depleted in the patients as well because normal
B cells also express CD19 [4]. Another example pertains to carbonic
anhydrase IX (CAIX) which is overexpressed in clear cell renal
carcinoma. Liver toxicity was found in subjects of the first
clinical trial using CAR-targeting CAIX, likely due to the fact
that CAIX is also expressed in bile duct epithelial cells and as
such, the T cells targeted normal tissue as well [5].
[0006] Second, `unregulated CAR activity` may be found where the
rapid eradication of cancer cells by CARs induces a constellation
of metabolic disturbances, called tumor lysis syndrome or a
cytokine storm, which can be a fatal consequence to patients [1, 4,
6]. This is a result because transduced T cells expressing CARs
cannot be easily regulated. Once transduced T cells are infused to
patients, it is currently very difficult to regulate or control the
activation of the cells.
[0007] Therefore, while CAR-expressing T cells show great promise
as a tool in the treatment of cancer, the next generation of the
CAR system is needed that provides reduced off-target toxicity and
greater control of activation. The present invention is directed to
this and other important ends.
BRIEF SUMMARY OF INVENTION
[0008] The invention relates to a Chimeric Antigen Receptor (CAR)
system and methods for using the system in the treatment of
subjects with cancer. The CAR system of the present invention
includes cytotoxic lymphocytes expressing CARs that target a moiety
that is not produced or expressed by cells of the subject being
treated. This CAR system thus allows for focused targeting of the
cytotoxic lymphocytes to target cells, such as cancer cells. The
targeted moiety is part of a small conjugate molecule (SCM) that
also comprises a ligand of a tumor cell receptor. Administration of
a SCM along with the CAR-expressing cytotoxic lymphocytes results
in the targeting of the cytotoxic lymphocyte response to only those
cells expressing the tumor receptor to which the SCM is bound.
[0009] In a first embodiment, the invention is directed to
CAR-expressing cytotoxic lymphocytes. The CAR is a fusion protein
comprising a recognition region, at least one co-stimulation
domain, and an activation signaling domain. The CAR has binding
specificity for a selected targeted moiety or can be bound by a
targeted moiety.
[0010] In certain aspects of this embodiment, the recognition
region of the CAR is a single chain fragment variable (scFv) region
of an antibody with binding specificity for the targeted moiety. In
a particular aspect, the recognition region of the CAR is a single
chain fragment variable (scFv) region of an anti-FITC antibody.
[0011] In certain aspects of this embodiment, the co-stimulation
domain of the CAR is selected from the group consisting of CD28,
CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).
[0012] In certain aspects of this embodiment, the activation
signaling domain of the CAR is the T cell CD3.zeta. chain or Fc
receptor .gamma..
[0013] In certain aspects of this embodiment, the cytotoxic
lymphocytes are one or more of cytotoxic T cells, natural killer
(NK) cells, and lymphokine-activated killer (LAK) cells.
[0014] In a particular aspect of this embodiment, the recognition
region is a single chain fragment variable (scFv) region of an
anti-FITC antibody, the co-stimulation domain is CD137 (4-1BB), and
the activation signaling domain is the T cell CD3.zeta. chain.
[0015] In certain aspects of this embodiment, the targeted moiety
is a molecule selected from the group consisting of
2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin,
digoxigenin, fluorescein, fluorescein isothiocyanate (FITC),
NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl
ester (TFP), a knottin, a centyrin, and a DARPin.
[0016] In certain aspects of this embodiment, the binding
specificity of the CAR for the targeted moiety has an affinity of
at least about 100 pM.
[0017] In a second embodiment, the invention is directed to small
conjugate molecules (SCM) comprising a targeted moiety conjugated
to a tumor receptor ligand, wherein the tumor receptor ligand is
folate, 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA),
or cholecystokinin 2 receptor (CCK2R) ligand.
[0018] Targeted moieties that may be used in the SCMs of the
invention include, but are not limited to, 2,4-dinitrophenol (DNP),
2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein,
fluorescein isothiocyanate (FITC), NHS-fluorescein,
pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), a
knottin, a centyrin, and a DARPin.
[0019] In certain aspects of this embodiment, the targeted moiety
and the ligand are conjugated via a linker domain. Linker domains
include, but are not limited to, polyethylene glycol (PEG),
polyproline, a hydrophilic amino acid, a sugar, an unnatural
peptideoglycan, polyvinylpyrrolidone, and pluronic F-127. In a
particular aspect, the linker domain is (PEG).sub.12.
[0020] In certain aspects of this embodiment, the targeted moiety
is FITC.
[0021] In certain aspects of this embodiment, the targeted moiety
is FITC and the linker is (PEG).sub.12.
[0022] In particular aspects of this embodiment, the SCM is
FITC-folate, FITC-DUPA, FITC-CCK2R ligand,
FITC-(PEG).sub.12-folate, FITC-(PEG).sub.12-DUPA, or
FITC-(PEG).sub.12-CCK2R ligand.
[0023] In a third embodiment, the invention is directed to a two
component cancer therapeutic comprising:
[0024] (a) a small conjugate molecule (SCM) comprising a targeted
moiety conjugated to a tumor receptor ligand, wherein the tumor
receptor ligand is folate, DUPA, or CCK2R ligand; and
[0025] (b) chimeric antigen receptor (CAR)-expressing cytotoxic
lymphocytes, wherein the CAR is a fusion protein comprising a
recognition region, a co-stimulation domain and an activation
signaling domain, and wherein the CAR has binding specificity for
the targeted moiety.
[0026] Targeted moieties that may be used in the SCMs include, but
are not limited to, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol
(TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate
(FITC), NHS-fluorescein, pentafluorophenyl ester (PFP),
tetrafluorophenyl ester (TFP), a knottin, a centyrin, and a
DARPin.
[0027] In certain aspects of this embodiment, the targeted moiety
and the ligand are conjugated via a linker domain. Linker domains
include, but are not limited to, polyethylene glycol (PEG),
polyproline, a hydrophilic amino acid, a sugar, an unnatural
peptideoglycan, polyvinylpyrrolidone, and pluronic F-127. In a
particular aspect, the linker domain is (PEG).sub.12.
[0028] In certain aspects of this embodiment, the targeted moiety
is FITC.
[0029] In certain aspects of this embodiment, the targeted moiety
is FITC and the linker is (PEG).sub.12.
[0030] In particular aspects of this embodiment, the SCM is
FITC-folate, FITC-DUPA, FITC-CCK2R ligand,
FITC-(PEG).sub.12-folate, FITC-(PEG).sub.12-DUPA, or
FITC-(PEG).sub.12-CCK2R ligand.
[0031] In certain aspects of this embodiment, the recognition
region of the CAR is a single chain fragment variable (scFv) region
of an antibody with binding specificity for the targeted moiety. In
a particular aspect, the recognition region of the CAR is a single
chain fragment variable (scFv) region of an anti-FITC antibody.
[0032] In certain aspects of this embodiment, the co-stimulation
domain of the CAR is CD28, CD137 (4-1BB), CD134 (OX40), or CD278
(ICOS).
[0033] In certain aspects of this embodiment, the activation
signaling domain of the CAR is the T cell CD3.zeta. chain or Fc
receptor .gamma..
[0034] In certain aspects of this embodiment, the cytotoxic
lymphocytes are one or more of cytotoxic T cells, natural killer
(NK) cells, and lymphokine-activated killer (LAK) cells.
[0035] In a particular aspect of this embodiment, the recognition
region is a single chain fragment variable (scFv) region of an
anti-FITC antibody, the co-stimulation domain is CD137 (4-1BB), and
the activation signaling domain is the T cell CD3.zeta. chain.
[0036] In certain aspects of this embodiment, the binding
specificity of the CAR for the targeted moiety is an affinity of at
least about 100 pM.
[0037] In a fourth embodiment, the invention is directed to a
method of treating cancer in a subject. In a first aspect the
method comprises:
[0038] (a) culturing a population of cytotoxic lymphocytes under
conditions promoting activation;
[0039] (b) transfecting the lymphocyte population of (a) with a
vector encoding a chimeric antigen receptor (CAR), wherein the CAR
is a fusion protein comprising a recognition region, a
co-stimulation domain and an activation signaling domain;
[0040] (c) administering a therapeutically effective number of the
transfected lymphocytes of (b) to a subject having cancer; and
[0041] (d) administering a small conjugate molecule (SCM)
comprising a targeted moiety conjugated to a tumor receptor ligand
to the subject, wherein the ligand is recognized and bound by a
receptor on the surface of a cell of the cancer, and wherein the
CAR has binding specificity for the targeted moiety or can be bound
by the targeted moiety;
[0042] thereby treating cancer in a subject.
[0043] In a related embodiment the method comprises:
[0044] (a) culturing a population of cytotoxic lymphocytes under
conditions promoting activation;
[0045] (b) transfecting the lymphocyte population of (a) with a
vector encoding a chimeric antigen receptor (CAR), wherein the CAR
is a fusion protein comprising a recognition region, a
co-stimulation domain and an activation signaling domain;
[0046] (c) administering a small conjugate molecule (SCM)
comprising a targeted moiety conjugated to a tumor receptor ligand
to a subject having cancer, wherein the ligand is recognized and
bound by a receptor on the surface of a cell of the cancer; and
[0047] (d) administering a therapeutically effective number of the
transfected T cells of (b) to the subject, and wherein the CAR has
binding specificity for the targeted moiety or can be bound by the
targeted moiety;
[0048] thereby treating cancer in a subject.
[0049] In a further related embodiment the method comprises:
[0050] (a) culturing a population of cytotoxic lymphocytes under
conditions promoting activation;
[0051] (b) transfecting the lymphocytes population of (a) with a
vector encoding a chimeric antigen receptor (CAR), wherein the CAR
is a fusion protein comprising a recognition region, a
co-stimulation domain and an activation signaling domain, and
wherein the CAR has binding specificity for a targeted moiety or
can be bound by the targeted moiety;
[0052] (c) incubating the lymphocytes of (b) with a small conjugate
molecule (SCM) comprising a targeted moiety conjugated to a tumor
receptor ligand;
[0053] (d) administering a therapeutically effective number of the
transfected lymphocytes of (c) to a subject having cancer;
[0054] thereby treating cancer in a subject.
[0055] In these three related embodiments the cytotoxic lymphocytes
may be autologous or heterologous cells, with respect to the
subject being treated, or a combination of both.
[0056] In these three related embodiments the culturing conditions
of (a) may comprise culturing the population of lymphocytes in the
presence of anti-CD3 antibodies or anti-CD28 antibodies, or
both.
[0057] In certain aspects of these three related embodiments, the
recognition region of the CAR is a single chain fragment variable
(scFv) region of an antibody with binding specificity for the
targeted moiety. In a particular aspect, the recognition region of
the CAR is a single chain fragment variable (scFv) region of an
anti-FITC antibody.
[0058] In certain aspects of these three related embodiments, the
co-stimulation domain of the CAR is CD28, CD137 (4-1BB), CD134
(OX40), or CD278 (ICOS).
[0059] In certain aspects of these three related embodiments, the
activation signaling domain of the CAR is the T cell CD3.zeta.
chain or Fc receptor .gamma..
[0060] In certain aspects of these three related embodiments, the
cytotoxic lymphocytes are one or more of cytotoxic T cells, natural
killer (NK) cells, and lymphokine-activated killer (LAK) cells.
[0061] In certain aspects of these three related embodiments, the
recognition region is a single chain fragment variable (scFv)
region of an anti-FITC antibody, the co-stimulation domain is CD137
(4-1BB), and the activation signaling domain is the T cell
CD3.zeta. chain.
[0062] In certain aspects of these three related embodiments, the
targeted moiety is a molecule selected from the group consisting of
2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin,
digoxigenin, fluorescein, fluorescein isothiocyanate (FITC),
NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl
ester (TFP), a knottin, a centyrin, and a DARPin. In a particular
aspect, the targeted moiety is FITC.
[0063] In certain aspects of these three related embodiments, the
ligand is folate, DUPA, CCK2R ligand.
[0064] In certain aspects of these three related embodiments, the
targeted moiety and the ligand are conjugated via a linker domain.
The linker domain may be, for example, polyethylene glycol (PEG),
polyproline, a hydrophilic amino acid, a sugar, an unnatural
peptideoglycan, polyvinylpyrrolidone, or pluronic F-127.
[0065] In certain aspects of these three related embodiments, the
targeted moiety is FITC and the linker is (PEG).sub.12.
[0066] In certain aspects of these three related embodiments, the
vector is a lentivirus vector.
[0067] In certain aspects of these three related embodiments, the
binding specificity of the CAR for the targeted moiety is an
affinity of at least about 100 pM.
[0068] In certain aspects of these three related embodiments, the
subject is a human.
[0069] In certain embodiments of these three related embodiments,
the cancer is one or more of a cancer of the brain, thyroid, lung,
pancreas, kidney, stomach, gastrointestinal stroma, endometrium,
breast, cervix, ovary, colon, prostate, leukemias, lymphomas, other
blood-related cancers or head and neck cancer.
[0070] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described herein, which form the subject of the claims of
the invention. It should be appreciated by those skilled in the art
that any conception and specific embodiment disclosed herein may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that any description, figure, example, etc. is
provided for the purpose of illustration and description only and
is by no means intended to define the limits the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0071] FIG. 1A is a schematic showing the CAR4-1BBZ construct.
[0072] FIG. 1B-1, FIG. 1B-2, and FIG. 1C show the transduction
efficiency into T cells of the CAR4-1BBZ construct. 96 h after
transduction, the expression of CAR4-1BBZ was identified through
copGFP expression by flow cytometry. As shown in the figures,
approximately 30% of transduced T cells expressed CAR4-1BBZ.
[0073] FIG. 1D shows the expression of CAR4-1BBZ on transduced T
cells using confocal microscopy. copGFP was expressed on positive
transduced T cells containing CAR4-1BBZ (arrows). However, copGFP
expression was not detected on non-transduced T cells that do not
express CAR4-1BBZ.
[0074] FIG. 2 shows binding of FITC-Folate and
FITC-(PEG).sub.12-Folate conjugates to CAR-transduced T cells. FIG.
2 shows confocal microscopy of copGFP expression in transduced T
cells containing CAR 4-1BBZ (top row) and FITC-folate binding on
CAR-1BBZ transduced T cells (middle row). The bottom row shows the
same view in the absence of fluorescence.
[0075] FIGS. 3A-D show the binding ability of FITC-Folate
conjugates to cancer cells via florescence microscopy. FIG. 3A
shows the binding of FITC-Folate (EC17) conjugates to KB cancer
cells. FIG. 3B shows the binding of FITC-Folate (EC17) conjugates
to L1210A cancer cells. FIG. 3C shows the binding of
FITC-(PEG).sub.12-Folate conjugates to KB cancer cells.
[0076] FIG. 3D shows the binding of FITC-(PEG).sub.12-Folate
conjugates to L1210A cancer cells. The competitor was 50.times.
excess folate acid.
[0077] FIGS. 4A-B show the results of assays to determine whether
CAR-expressing T cells are cytotoxic to cancer cells in the
presence or absence of FITC-Folate conjugates using KB cells (FIG.
4A) or L1210A cells (FIG. 4B).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0078] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found, for example, in Benjamin Lewin, Genes VII,
published by Oxford University Press, 2000 (ISBN 019879276X);
Kendrew et al. (eds.); The Encyclopedia of Molecular Biology,
published by Blackwell Publishers, 1994 (ISBN 0632021829); and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by Wiley, John & Sons,
Inc., 1995 (ISBN 0471186341); and other similar technical
references.
[0079] As used herein, "a" or "an" may mean one or more. As used
herein when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more. Furthermore, unless
otherwise required by context, singular terms include pluralities
and plural terms include the singular.
[0080] As used herein, "about" refers to a numeric value,
including, for example, whole numbers, fractions, and percentages,
whether or not explicitly indicated. The term "about" generally
refers to a range of numerical values (e.g., +/-5-10% of the
recited value) that one of ordinary skill in the art would consider
equivalent to the recited value (e.g., having the same function or
result). In some instances, the term "about" may include numerical
values that are rounded to the nearest significant figure.
[0081] As used herein, "treat" and all its forms and tenses
(including, for example, treat, treating, treated, and treatment)
refer to both therapeutic treatment and prophylactic or
preventative treatment.
II. The Present Invention
[0082] The present invention is directed to a CAR system for use in
the treatment of subjects with cancer. The CAR system of the
present invention (e.g., cytotoxic lymphocytes expressing novel
CARs and cognate small conjugate molecules (SCM)) makes use of CARs
that target a moiety that is not produced or expressed by cells of
the subject being treated. This CAR system thus allows for focused
targeting of the cytotoxic lymphocytes to target cells, such as
cancer cells. The targeted moiety is part of a small conjugate
molecule (SCM) that also comprises a ligand of a tumor cell
receptor. Because small organic molecules are typically used as the
targeted moiety, clearance of the SCM from the bloodstream can be
achieved within about 20 minutes. By administration of a SCM along
with the CAR-expressing cytotoxic lymphocytes, the lymphocyte
response can be targeted to only those cells expressing the tumor
receptor, thereby reducing off-target toxicity, and the activation
of lymphocytes can be more easily controlled due to the rapid
clearance of the SCM. As an added advantage, the CAR-expressing
lymphocytes can be used as a "universal" cytotoxic cell to target a
wide variety of tumors without the need to prepare separate CAR
constructs. The targeted moiety recognized by the CAR may also
remain constant. It is only the ligand portion of the SCM that
needs to be altered to allow the system to target cancer cells of
different identity.
[0083] One embodiment of the invention provides an illustration of
this novel CAR system. In this embodiment, and as a first
component, a SCM is prepared that comprises FITC linked to a ligand
of a selected tumor cell receptor. As a second component, cytotoxic
T cells are transduced to express a CAR that comprises anti-FITC
scFv. This CAR thus targets Fluorescein Isothiocyanate (FITC)
instead of a tumor-associated antigen that might also be expressed
by healthy, non-target cells. The two components are administered
to a subject having cancer and the FITC-SCM (first component) is
bound by the target tumor cells (through binding of the ligand
portion of the molecule to cognate tumor cell receptor). The FITC
portion of the SCM is then recognized and bound by the anti-FITC
CAR expressed by the T cells (second component). Upon FITC binding,
the anti-FITC CAR-expressing T cells are activated and the tumor
cell is killed. As will be apparent to the skilled artisan, the
cytotoxic T cells cannot kill cells with first binding to a tumor
cell. As it will be further apparent, T cells will not bind to
non-target cells because the recognition region of the CAR will
only recognize and bind FITC, which is not produce or expressed by
cells of the subject. The SCM thus acts as a bridge between the
cytotoxic T cells and the target tumor cells. As long as the
targeted moiety of the SCM is a moiety not found in the host, the
activity of the T cells can be limited to the target cells.
Further, the activation of the CAR-expressing T cells can be
regulated by limiting the amount of SCM administered to a subject,
for example, by manipulating infusion of the small conjugate
molecule if a side effect is detected. Thus, the CAR system of the
present invention overcomes problems associated with conventional
CAR therapy.
Small Conjugate Molecules (SCM)
[0084] The CAR system of the present invention utilizes small
conjugate molecules (SCMs) as the bridge between cytotoxic
lymphocytes and targeted cancer cells. The SCMs are conjugates
comprising a targeted moiety on one end of the molecule and a tumor
receptor ligand on the other, optionally connected by a bridge
domain. The targeted moiety is a molecule that is recognized by a
CAR of a transduced lymphocyte or that can bind to a region of the
CAR. The identity of the targeted moiety is limited only in that it
must be a molecule that can be recognized and bound by CAR
expressed by a lymphocyte, or recognized and bind the CAR itself,
in both cases preferably with specificity, and that it have a
relatively low molecular weight. Exemplary targeted moieties are
haptens that can be recognized and bound by CARs and include small
molecular weight organic molecules such as DNP (2,4-dinitrophenol),
TNP (2,4,6-trinitrophenol), biotin, and digoxigenin, along with
fluorescein and derivatives thereof, including FITC (fluorescein
isothiocyanate), NHS-fluorescein, and pentafluorophenyl ester (PFP)
and tetrafluorophenyl ester (TFP) derivatives. Suitable targeted
moieties that themselves bind to one or more regions of a CAR
include knottins [16], centyrins and DARPins [7].
[0085] The tumor receptor ligands that comprise the SCMs of the
present invention are molecules recognized and bound by receptors
expressed by target tumor cells, typically expressed on the surface
of the tumor cells. Suitable ligands include: 1) DUPA
(DUPA-(99m)Tc), a ligand bound by PSMA-positive human prostate
cancer cells with nanomolar affinity (K.sub.D=14 nM; [8]); 2) CCK2R
ligand, a ligand bound by CCK2R-positive cancer cells (e.g.,
cancers of the thyroid, lung, pancreas, ovary, brain, stomach,
gastrointestinal stroma, and colon; [9]); 3) folate, a ligand bound
by the folate receptor on cells of cancers that include cancers of
the ovary, cervix, endometrium, lung, kidney, brain, breast, colon,
and head and neck cancers [10].
[0086] The targeted moiety and the ligand can be directly
conjugated through such means as reaction between the
isothiocyanate group of FITC and free amine group of small ligands
(e.g. folate, DUPA and CCK2R ligand). However, the use of a linking
domain to connect the two molecules can be helpful as it can
provide flexibility and stability to the SMC depending on the
identity of the components comprising the SCM. Examples of suitable
linking domains include: 1) polyethylene glycol (PEG); 2)
polyproline; 3) hydrophilic amino acids; 4) sugars; 5) unnatural
peptideoglycans; 6) polyvinylpyrrolidone; 7) pluronic F-127.
Linkers lengths that are suitable include, but are not limited to,
linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39 or 40, or more atoms.
[0087] While the affinity at which the ligands and cancer cell
receptors bind can vary, and in some cases low affinity binding may
be preferable (such as about 1 .mu.M), the binding affinity of the
ligands and cancer cell receptors will generally be at least about
100 .mu.M, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM
or 10 pM, even more preferably at least about 100 pM.
[0088] The skilled artisan will understand and recognize that
various means can be used to prepare SMCs comprised of a targeted
moiety, a linking domain, and a ligand. Examples are provided in
the Examples included herein.
[0089] Prior to being administered to a subject, the SCMs are
prepared in a pharmaceutically acceptable formulation. Such
formulations may contain a pharmaceutically acceptable carrier or
diluent.
[0090] Exemplary SCMs included within the scope of the invention
include the following molecules.
##STR00001## ##STR00002##
Chimeric Antigen Receptors (CARs)
[0091] The CAR system of the present invention also utilizes
cytotoxic lymphocytes engineered to express chimeric antigen
receptors (CARs) that recognize and bind the targeting moiety of
the SCMs. The CARs used in the CAR system comprise three domains.
The first domain is the recognition region which, as the name
suggests, recognizes and binds the targeting moiety. The second
domain is the co-stimulation domain which enhances the
proliferation and survival of the lymphocytes. The third domain is
the activation signaling domain which is a cytotoxic lymphocyte
activation signal. The three domains, together in the form of a
fusion protein, comprise the CARs of the present invention.
[0092] As suggested above, the recognition region is the portion of
a CAR that recognizes and binds a targeting moiety. The recognition
regions comprising the CARs of the present invention are single
chain fragment variable (scFv) regions of antibodies that bind the
targeted moiety. Preferably, the scFv regions bind the targeted
moiety with specificity. The identity of the antibody used in the
production of the recognition region is limited only in that it
binds the targeted moiety of the SCM. Thus, as non-limiting
examples, scFv regions of antibodies that bind one of the following
targeted moieties are included within the scope of the invention:
DUPA, CCK2R ligand, folate. The scFv regions can be prepared from
(i) antibodies known in the art that bind a targeted moiety, (ii)
antibodies newly prepared using a selected targeted moiety as a
hapten, and (iii) sequence variants derived from the scFv regions
of such antibodies, e.g., scFv regions having at least about 80%
sequence identity to the amino acid sequence of the scFv region
from which they are derived. The use of unaltered (i.e., full size)
antibodies, such as IgG, IgM, IgA, IgD or IgE, in the CAR or as the
CAR is excluded from the scope of the invention.
[0093] The co-stimulation domain serves to enhance the
proliferation and survival of the cytotoxic lymphocytes upon
binding of the CAR to a targeted moiety. The identity of the
co-stimulation domain is limited only in that it has the ability to
enhance cellular proliferation and survival activation upon binding
of the targeted moiety by the CAR. Suitable co-stimulation domains
include: 1) CD28 [11]; 2) CD137 (4-1BB), a member of the tumor
necrosis factor (TNF) receptor family [12]; 3) CD134 (OX40), a
member of the TNFR-superfamily of receptors [13]; 4) CD278 (ICOS),
a CD28-superfamily co-stimulatory molecule expressed on activated T
cells [14]. The skilled artisan will understand that sequence
variants of these noted co-stimulation domains can be used without
adversely impacting the invention, where the variants have the same
or similar activity as the domain on which they are modeled. Such
variants will have at least about 80% sequence identity to the
amino acid sequence of the domain from which they are derived.
[0094] In some embodiments of the invention, the CAR constructs
comprise two co-stimulation domains. While the particular
combinations include all possible variations of the four noted
domains, specific examples include: 1) CD28+CD137 (4-1BB) and 2)
CD28+CD134 (OX40).
[0095] The activation signaling domain serves to activate cytotoxic
lymphocytes upon binding of the CAR to a targeted moiety. The
identity of the activation signaling domain is limited only in that
it has the ability to induce activation of the selected cytotoxic
lymphocyte upon binding of the targeted moiety by the CAR. Suitable
activation signaling domains include the T cell CD3.zeta. chain and
Fc receptor .gamma.. The skilled artisan will understand that
sequence variants of these noted activation signaling domains can
be used without adversely impacting the invention, where the
variants have the same or similar activity as the domain on which
they are modeled. Such variants will have at least about 80%
sequence identity to the amino acid sequence of the domain from
which they are derived.
[0096] Constructs encoding the CARs of the invention are prepared
through genetic engineering. As an example, a plasmid or viral
expression vector can be prepared that encodes a fusion protein
comprising a recognition region, one or more co-stimulation
domains, and an activation signaling domain, in frame and linked in
a 5' to 3' direction. However, the CARs of the present invention
are not limited in this arrangement and other arrangements are
acceptable and include: (i) a recognition region, an activation
signaling domain, and one or more co-stimulation domains, and (ii)
a recognition region, a co-stimulation domain, and an activation
signaling domain, linked in a 5' to 3' direction. It will be
understood that because the recognition region must be free to bind
the targeted moiety, the placement of the recognition region in the
fusion protein will generally be such that display of the region on
the exterior of the cell is achieved. In the same manner, because
the co-stimulation and activation signaling domains serve to induce
activity and proliferation of the cytotoxic lymphocytes, the
constructs will generally encode a fusion protein that displays
these two domains in the interior of the cell.
[0097] The CARs may include additional elements, such a signal
peptide to ensure proper export of the fusion protein to the cells
surface, a transmembrane domain to ensure the fusion protein is
maintained as an integral membrane protein, and a hinge domain that
imparts flexibility to the recognition region and allows strong
binding to the targeted moiety.
[0098] An example of an exemplary CAR of the present invention is
shown in FIG. 1A where the fusion protein is encoded by a
lentivirus expression vector and where "SP" is a signal peptide,
the CAR is an anti-FITC CAR, a CD8.alpha. hinge is present, a
transmembrane domain is present ("TM"), the co-stimulation domain
is 4-1BB, and the activation signaling domain is CD3. The sequence
of the CAR-encoding vector is provided as SEQ ID NO:1.
[0099] In addition to the use of plasmid and viral vectors,
cytotoxic lymphocytes can be engineered to express CARs of the
invention through retrovirus, lentivirus (viral mediated CAR gene
delivery system), sleeping beauty, and piggyback
(transposon/transposase systems that include a non-viral mediated
CAR gene delivery system).
[0100] While the affinity at which the CARs, expressed by the
cytotoxic lymphocytes, bind to the targeted moiety can vary, and in
some cases low affinity binding may be preferable (such as about 50
nM), the binding affinity of the CARs to the targeted ligand will
generally be at least about 100 nM, 1 pM, or 10 pM, preferably at
least about 100 pM, 1 fM or 10 fM, even more preferably at least
about 100 fM.
CAR-Expressing Cytotoxic Lymphocytes
[0101] The cells used in the CAR system of the present invention
are cytotoxic lymphocytes selected from (i) cytotoxic T cells (also
variously known as cytotoxic T lymphocytes, CTLs, T killer cells,
cytolytic T cells, CD8.sup.+ T cells, and killer T cells), natural
killer (NK) cells, and lymphokine-activated killer (LAK) cells.
Upon activation, each of these cytotoxic lymphocytes triggers the
destruction of target tumor cells. For example, cytotoxic T cells
trigger the destruction of target tumor cells by either or both of
the following means. First, upon activation T cells release
cytotoxins such as perforin, granzymes, and granulysin. Perforin
and granulysin create pores in the target cell, and granzymes enter
the cell and trigger a caspase cascade in the cytoplasm that
induces apoptosis (programmed cell death) of the cell. Second,
apoptosis can be induced via Fas-Fas ligand interaction between the
T cells and target tumor cells.
[0102] The cytotoxic lymphocytes will preferably be autologous
cells, although heterologous cells can also be used, such as when
the subject being treated using the CAR system of the invention has
received high-dose chemotherapy or radiation treatment to destroy
the subject's immune system. Under such circumstances, allogenic
cells can be used.
[0103] The cytotoxic lymphocytes can be isolated from peripheral
blood using techniques well known in the art, include Ficoll
density gradient centrifugation followed by negative selection to
remove undesired cells.
[0104] Cytotoxic lymphocytes can be engineered to express CAR
constructs by transfecting a population of lymphocytes with an
expression vector encoding the CAR construct. Appropriates means
for preparing a transduced population of lymphocytes expressing a
selected CAR construct will be well known to the skilled artisan,
and includes retrovirus, lentivirus (viral mediated CAR gene
delivery system), sleeping beauty, and piggyback
(transposon/transposase systems that include a non-viral mediated
CAR gene delivery system), to name a few examples.
[0105] Transduced cytotoxic lymphocytes are grown in conditions
that are suitable for a population of cells that will be introduced
into a subject such as a human Specific considerations include the
use of culture media that lacks any animal products, such as bovine
serum. Other considerations include sterilized-condition to avoid
contamination of bacteria, fungi and mycoplasma.
[0106] Prior to being administered to a subject, the cells are
pelleted, washed, and resuspended in a pharmaceutically acceptable
carrier or diluent. Exemplary formulations comprising
CAR-expressing cytotoxic lymphocytes include formulations
comprising the cells in sterile 290 mOsm saline, infusible
cryomedia (containing Plasma-Lyte A, dextrose, sodium chloride
injection, human serum albumin and DMSO), 0.9% NaCl with 2% human
serum albumin or any other sterile 290 mOsm infusible
materials.
Methods of Treatment
[0107] The CAR system of the present invention can be used in the
treatment of a subject having cancer. The methods of treatment
encompassed by the invention generally includes the steps of (i)
obtaining a population of autologous or heterologous cytotoxic
lymphocytes, (ii) culturing the lymphocytes under conditions that
promote the activation of the cells, (iii) transfecting the
lymphocytes with an expression vector encoding a CAR, (iv)
administering a formulation comprising the transfected lymphocytes
to a subject having cancer, and (v) administering a formulation
comprising SCM to the subject.
[0108] The invention also includes variations on this theme such,
as administering the formulation comprising SMC to the subject
before the formulation comprising the transfected lymphocytes, or
at the same time as the formulation comprising the transfected
lymphocytes. A further variation includes culturing the formulation
comprising the transfected lymphocytes with the SCM prior to
administration to the subject.
[0109] The population of cytotoxic lymphocytes can be obtained from
a subject by means well known in the art. For example, cytotoxic T
cells can be obtained by collecting peripheral blood from the
subject, subjecting the blood to Ficoll density gradient
centrifugation, and then using a negative T cell isolation kit
(such as EasySep.TM. T Cell Isolation Kit) to isolate a population
of cytotoxic T cells from the blood. While the population of
cytotoxic lymphocytes need not be pure and may contain other blood
cells such as T cells, monocytes, macrophages, natural killer cells
and B cells, depending of the population being collected,
preferably the population comprises at least about 90% of the
selected cell type. In particular aspects, the population comprises
at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
even 100% of the selected cell type. As indicated earlier, the
population of cells may come from the subject to be treated, from
one or more different subjects, or the population may be a
combination of cells from the subject to be treated and one or more
different subjects.
[0110] After the population of cytotoxic lymphocytes is obtained,
the cells are cultured under conditions that promote the activation
of the cells. The culture conditions will be such that the cells
can be administered to a subject without concern for reactivity
against components of the culture. For example, when the population
will be administered to a human, the culture conditions will not
include bovine serum products, such as bovine serum albumin. The
activation of the lymphocytes in the culture can be achieved by
introducing known activators into the culture, such as anti-CD3
antibodies in the case of cytotoxic T cells. Other suitable
activators include anti-CD28 antibodies. The population of
lymphocytes will generally be cultured under conditions promoting
activation for about 1 to 4 days. The appropriate level of cellular
activation can be determined by cell size, proliferation rate or
activation markers by flow cytometry.
[0111] After the population of cytotoxic lymphocytes has been
cultured under conditions promoting activation, the cells are
transfected with an expression vector encoding a CAR. Such vectors
are described above, along with suitable means of transfection.
After transfection, the resulting population of cells can be
immediately administered to a subject or the cells can be culture
for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18 or more days, or between about 5 and 12 days,
between about 6 and 13 days, between about 7 and 14 days, or
between about 8 and 15 days, for example, to allow time for the
cells to recover from the transfection. Suitable culture conditions
with be the same as those conditions under which the cells were
culture while activation was being promoted, either with or without
the agent that was used to promote activation and expansion.
[0112] When the transfected cells are ready a formulation
comprising the cells is prepared and administered to a subject
having cancer. Prior to administration, the population of cells can
be washed and resuspended in a pharmaceutically acceptable carrier
or diluent to form the formulation. Such carriers and diluents
include, but are not limited to, sterile 290 mOsm saline, infusible
cryomedia (containing Plasma-Lyte A, dextrose, sodium chloride
injection, human serum albumin and DMSO), 0.9% NaCl with 2% human
serum albumin or any other sterile 290 mOsm infusible materials.
Alternatively, depending on the identity of the culture media used
in the previous step, the cells can be administered in the culture
media as the formulation, or concentrated and resuspended in the
culture media before administration. The formulation can be
administered to the subject via suitable means, such as parenteral
administration, e.g., intradermally, subcutaneously,
intramuscularly, intraperitoneally, intravenously, or
intrathecally.
[0113] The total number of cells and the concentration of cells in
the formulation administered to a subject will vary depending on a
number of factors including the type of cytotoxic lymphocytes being
used, the binding specificity of the CAR, the identity of the
targeted moiety and the ligand, the identity of the cancer or tumor
to be treated, the location in the subject of the cancer or tumor,
the means used to administer the formulations to the subject, and
the health, age and weight of the subject being treated. However,
suitable formulations comprising transduced lymphocytes include
those having a volume of between about 5 ml and 200 ml, containing
from about 1.times.10.sup.5 to 1.times.10.sup.15 transduced cells.
Typical formulations comprise a volume of between about 10 ml and
125 ml, containing from about 1.times.10.sup.7 to 1.times.10.sup.10
transduced cells. An exemplary formulation comprises about
1.times.10.sup.9 transduced cells in a volume of about 100 ml.
[0114] The final step in the method is the administration of a
formulation comprising SCM to the subject. As described above, the
SCM will be prepared in a formulation appropriate for the subject
receiving the molecules. The concentration of SCM in a SCM
formulation will vary depending on factors that include the binding
specificity of the CAR, the identity of the targeted moiety and the
ligand, the identity of the cancer or tumor to be treated, the
location in the subject of the cancer or tumor, the means used to
administer the formulations to the subject, and the health, age and
weight of the subject being treated. However, suitable formulations
comprising SCM include those having a volume of between about 1 ml
and 50 ml and contain between about 20 ug/kg body weight and 3
mg/kg body weight SCM. Typical formulations comprise a volume of
between about 5 ml and 20 ml and contain between about 0.2 mg/kg
body weight and 0.4 mg/kg body weight SCM. An exemplary formulation
comprises about 50 ug/kg body weight SCM in a volume of about 10
ml.
[0115] The timing between the administration of transduced
lymphocyte formulation and the SCM formation may range widely
depending on factors that include the type of cytotoxic lymphocytes
being used, the binding specificity of the CAR, the identity of the
targeted moiety and the ligand, the identity of the cancer or tumor
to be treated, the location in the subject of the cancer or tumor,
the means used to administer the formulations to the subject, and
the health, age and weight of the subject being treated. Indeed,
the SCM formation may be administered prior to, simultaneous with,
or after the lymphocyte formulation. In general, the SCM formation
will be administered after the lymphocyte formulation, such as
within 3, 6, 9, 12, 15, 18, 21, or 24 hours, or within 0.5, 1, 1.5,
2, 2.5, 3, 4 5, 6, 7, 8, 9, 10 or more days. When the SCM formation
is administered before the lymphocyte formulation, the lymphocyte
formulation will generally be administered within about 0.25, 0.5,
0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours. When the
SCM formation and the lymphocyte formulation are added
simultaneously, it is preferable that the formations are not
combined and thus administered separately to the subject.
[0116] Depending on the cancer being treatment the step of
administering the lymphocyte formulation, or the step of
administering the SCM formulation, or both, can be repeated one or
more times. The particular number and order of the steps is not
limited as the attending physician may find that a method can be
practiced to the advantage of the subject using one or more of the
following methodologies, or others not named here: (i)
administering the lymphocyte formulation (A) followed by the SCM
formulation (B), i.e., A then B; (ii) B then A; (iii) A then B then
A then B; (iv) A then B then A; (v) B then A then B then A; (vi) A
then A then B; (vii) B then A then A; (vii) B then B then A.
[0117] The formulations can be administered as single continuous
doses, or they can be divided and administered as a multiple-dose
regimen depending on the reaction (i.e., side effects) of the
patient to the formulations.
[0118] The types of cancers which may be treated using the methods
of the invention will be governed based on the identity of the
ligand used in the SCM. When the ligands defined above are used
(i.e., DUPA, CCK2R ligand, folate) cancers that may be treated
using the CAR system and methods of the present invention generally
include solid tumors, and more specifically include prostate cancer
adenocarcinoma, hepatoma, colorectal liver metastasis, and cancers
of neuroendocrine origin.
[0119] In each of the embodiments and aspects of the invention, the
subject is a human, a non-human primate, bird, horse, cow, goat,
sheep, a companion animal, such as a dog, cat or rodent, or other
mammal.
III. Examples
1. Generation of CAR4-1BBZ and Transduction of Mouse T Cells to
Express CAR
[0120] To generate modified T cells containing CAR that target
cancer cells by FITC-ligand small molecule conjugates, CAR
constructs were designed and generated as shown in FIG. 1.
A) Generation of Chimeric Antigen Receptor (CAR) in Lentiviral
Vector
[0121] An overlap PCR method was used to generate CAR constructs
comprising scFv against FITC (CAR4-1BBZ). scFV against FITC, 4M5.3
(Kd=270 fM, 762 bp) derived from anti-fluorescence (4-4-20)
antibody, was synthesized based on a previous report [15]. As shown
in FIG. 1, sequence encoding the mouse CD8.alpha. signal peptide
(SP, 81 bp), the hinge and transmembrane region (207 bp), the
cytoplasmic domain of 4-1BB (CD137, 144 bp) and the CD3 chain (339
bp) were fused with the anti-FITC scFV by overlapping PCR. The
resulting CAR construct (CAR4-1BBZ) (1533 bp; SEQ ID NO:2) was
inserted into Nhel/Notl cleaved lentiviral expression vector
pCDH-EF1-MCS-BGH-PGK-GFP-T2A-Puro (System Biosciences, Mountain
View, Calif.). CAR4-1BBZ expression is regulated by EFla promoter
in the lentiviral vector. The sequence of CAR constructs in
lentiviral vector (CAR4-1BBZ) was confirmed by DNA sequencing
(Purdue Genomic Core Facility) and is provided in SEQ ID NO:1.
copGFP expression encoded in the lentiviral vector was monitored to
identify CAR4-1BBZ expression (FIG. 1D).
B) Isolation and Transduction of Mouse T Cells
[0122] T cells were isolated from mouse spleen or peripheral blood.
To isolate T cells, mouse splenocytes and peripheral blood
mononuclear cells (PBMC) were isolated by Ficoll density gradient
centrifugation (GE Healthcare Lifesciences). After washing away
remaining Ficoll solution, T cells were isolated by EasySep.TM.
Mouse T Cell Isolation Kit (STEM CELL technologies). Purified T
cells are cultured in RPMI 1640 with 10% heat inactivated fetal
bovine serum (FBS), 1% penicillin and streptomycin sulfate, 10 mM
HEPES. To prepare lentiviral virus containing CAR4-1BBZ, 293TN
packaging cell line was co-transfected with CAR4-1BBZ lentiviral
vector and packaging plasmids. After 48 and 72 hours transfection,
supernatants containing CAR4-1BBZ lentivirus were harvested and
virus particles were concentrated for transduction. For
transduction of mouse T cells, isolated T cells were activated with
Dynabeads coupled with anti-CD3/CD28 antibodies (Life Technologies)
for 12-24 hours in the presence of mouse IL-2 (50 units/nil), then
infected with lentiviral expression vector containing CAR4-1BBZ.
Mouse IL-2 (50 units/ml) was provided every other day. After 96
hours, cells were harvested and the expression of CAR on transduced
T cells was identified by flow cytometry. As shown in FIG. 1B-1,
FIG. 1B-2, and FIG. 1C, approximately 30% of transduced T cells
expressed CAR4-1BBZ.
C) Flow Cytometry
[0123] The expression of CAR4-1BBZ on transduced T cells was
determined by flow cytometry. Since lentiviral expression backbone
also encodes copGFP expression, CAR4-1BBZ transduced T cells were
verified by copGFP expression. FIG. 1D shows the expression of
CAR4-1BBZ on transduced T cells. Transduced T cells were further
confirmed by confocal microscope. copGFP was expressed on positive
transduced T cells containing CAR4-1BBZ (arrows). However, copGFP
expression was not detected on non-transduced T cells that do not
express CAR4-1BBZ. Data was analyzed with FlowJo software.
[0124] Based on these result, it was evident that that the
CAR4-1BBZ constructs was successfully transduced to mouse T cells
and that the transduced T cells express CAR4-1BBZ.
D) Cell Culture
[0125] Two cancer cell lines were used in this study: L1210A and
KB. L1210A is a murine leukemia cell line. KB is a human epidermoid
carcinoma cell line. Both of them have higher folate receptor
expression on cell surface.
[0126] L1210A and KB cells were cultured in folic acid-deficient
RPMI medium, 10% of heat inactivated fetal bovine serum (FBS), 1%
penicillin and streptomycin sulfate were included in the culture
media.
2. Generation of Small Conjugate Molecules Comprising FITC and
Ligands
A) Synthesis of FITC-Folate
[0127] Folate-.gamma.-ethylenediamine was coupled to FITC isomer I
(Sigma-Aldrich, St. Louis, Mo.) in anhydrous dimethylsulfoxide in
the presence of tetramethylguanidine and diisopropylamine. The
crude product was loaded onto an Xterra RP18 preparative HPLC
column (Waters) and eluted with gradient conditions starting with
99% 5 mM sodium phosphate (mobile phase A, pH7.4) and 1%
acetonitrile (mobile phase B) and reaching 90% A and 10% B in 10
min at a flow rate of 20 mL/min. Under these conditions, the
folate-FITC main peak typically eluted at 27-50 min. The quality of
folate-FITC fraction was monitored by analytical reverse-phase HPLC
with a UV detector. Fractions with greater than 98.0% purity (LCMS)
were lyophilized to obtain the final folate-FITC product.
##STR00003##
B) Synthesis of FITC-(PEG).sub.12-Folate
[0128] Universal PEG Nova Tag.TM. resin (0.2 g) was loaded into a
peptide synthesis vessel and washed with i-PrOH (3.times.10 mL),
followed by DMF (3.times.10 mL). Fmoc deprotection was carried out
using 20% piperidine in DMF (3.times.10 mL). Kaiser tests were
performed to assess reaction completion. To the vessel was then
introduced a solution of Fmoc-Glu-(O-t-Bu)-OH (23.5 mg) in DMF,
i-Pr.sub.2NEt (4 equiv), and PyBOP (2 equiv). Fmoc deprotection was
carried out using 20% piperidine in DMF (3.times.10 mL). To the
vessel was then introduced a solution of N10-TFA-Pte-OH (22.5 mg),
DMF, i-Pr.sub.2NEt (4 equiv), and PyBOP (2 equiv). Argon was
bubbled for 2 h, and resin was washed with DMF (3.times.3 mL) and
i-PrOH(3.times.3 mL). After swelling the resin in DCM, a solution
of 1M HOBT in DCM/TFE (1:1) (2.times.3 mL) was added for removal of
Mmt group. Argon was bubbled for 1 h, the solvent was removed and
resin was washed with DMF (3.times.3 mL) and i-PrOH(3.times.3 mL).
After swelling the resin in DMF, a solution of
Fmoc-NH-(PEG).sub.12-COOH (46.3 mg) in DMF, i-Pr.sub.2NEt (4
equiv), and PyBOP (2 equiv) was added. Argon was bubbled for 2 h,
and resin was washed with DMF (3.times.3 mL) and i-PrOH (3.times.3
mL). Fmoc deprotection was carried out using 20% piperidine in DMF
(3.times.10 mL). Kaiser tests were performed to assess reaction
completion. To the vessel was then introduced a solution of FITC
(21.4 mg) in DMF, i-Pr.sub.2NEt (4 equiv), then Argon was bubbled
for 2 h, and resin was washed with DMF (3.times.3 mL) and
i-PrOH(3.times.3 mL). Then to the vessel was added 2%
NH.sub.2NH.sub.2 in DMF (2.times.2 mL). Final compound was cleaved
from resin using a TFA:H.sub.2O:TIS (95:2.5:2.5) and concentrated
under vacuum. The concentrated product was precipitated in
Et.sub.2O and dried under vacuum. The crude product was purified by
using preparative RP-HPLC (mobile phase: A=10 mM ammonium acetate
pH=7, B=ACN; method: 0% B to 30% B in 30 min at 13 mL/min) The pure
fractions were pooled and freeze-dried, furnishing the
FITC-(PEG).sub.12-Folate.
##STR00004##
C) Synthesis of FITC-(PEG).sub.12-DUPA
[0129] Synthesis of DUPA-(PEG).sub.12-EDA: 1,2-Diaminoethane
trityl-resin (0.025 g) was loaded into a peptide synthesis vessel
and washed with i-PrOH (3.times.10 mL), followed by DMF (3.times.10
mL). To the vessel was then introduced a solution of
Fmoc-NH-(PEG).sub.12-COOH (42.8 mg) in DMF, i-Pr.sub.2NEt (2.5
equiv), and PyBOP (2.5 equiv). The resulting solution was bubbled
with Ar for 1 h, the coupling solution was drained, and the resin
washed with DMF (3.times.10 mL) and i-PrOH (3.times.10 mL). Kaiser
tests were performed to assess reaction completion. Fmoc
deprotection was carried out using 20% piperidine in DMF
(3.times.10 mL). This procedure was repeated to complete the all
coupling steps (2.times.1.5 equiv of Fmoc-Phe-OH and 1.5 equiv of
8-aminooctanoic acid and 1.2 equiv of DUPA were used on each of
their respective coupling steps). After the DUPA coupling, the
resin was washed with DMF (3.times.10 mL) and i-PrOH (3.times.10
mL) and dried under reduced pressure. The peptide was cleaved from
the resin in the peptide synthesis vessel using a cleavage mixture
consisting of 95% CF.sub.3CO.sub.2H, 2.5% H.sub.2O, and 2.5%
triisopropylsilane. Fifteen milliliters of the cleavage mixture was
added to the peptide synthesis vessel, and the reaction was bubbled
under Ar for 15 min. The resin was treated with two additional 10
mL quantities of the cleavage mixture for 5 min each. The cleavage
mixture was concentrated to ca. 5 mL, and ethyl ether was added to
induce precipitation. The precipitate was collected by
centrifugation, washed with ethyl ether three times, and dried
under high vacuum, resulting in the recovery of
DUPA-(PEG).sub.12-EDA as crude material.
##STR00005##
[0130] Synthesis of FITC-(PEG).sub.12-DUPA:
[0131] To a stirred solution of the crude DUPA-(PEG).sub.12-EDA (10
mg) and FITC (5.6 mg) in dimethylsulfoxide (DMSO, 1 mL) was added
Pr.sub.2NEt (5 equiv) at room temperature and stirring continued
for 6 hr under argon. The reaction was monitored by LCMS and
purified by preparative HPLC (mobile phase: A=10 mM ammonium
acetate pH=7, B=ACN; method: 0% B to 50% B in 30 min at 13 mL/min).
The pure fractions were pooled and freeze-dried, furnishing the
FITC-(PEG).sub.12-DUPA.
##STR00006##
D) Synthesis of FITC-(PEG).sub.12-CCK2R Ligand (Z360)
[0132] Synthesis of CCK2R Ligand-(PEG).sub.12-EDA:
[0133] 1,2-Diaminoethane trityl-resin (0.025 g) was loaded into a
peptide synthesis vessel and washed with i-PrOH (3.times.10 mL),
followed by DMF (3.times.10 mL). To the vessel was then introduced
a solution of Fmoc-NH-(PEG).sub.12-COOH (42.8 mg) in DMF,
i-Pr.sub.2NEt (2 equiv), and PyBOP (1 equiv). The resulting
solution was bubbled with Ar for 1 h, the coupling solution was
drained, and the resin washed with DMF (3.times.10 mL) and i-PrOH
(3.times.10 mL). Kaiser tests were performed to assess reaction
completion. Fmoc deprotection was carried out using 20% piperidine
in DMF (3.times.10 mL). Then added Z360 (10 mg) in DMF,
i-Pr.sub.2NEt (2.5 equiv), and PyBOP (2.5 equiv). After the Z360
coupling, the resin was washed with DMF (3.times.10 mL) and i-PrOH
(3.times.10 mL) and dried under reduced pressure. The peptide was
cleaved from the resin in the peptide synthesis vessel using a
cleavage mixture consisting of 95% CF.sub.3CO.sub.2H, 2.5%
H.sub.2O, and 2.5% triisopropylsilane. Fifteen milliliters of the
cleavage mixture was added to the peptide synthesis vessel, and the
reaction was bubbled under Ar for 15 min. The resin was treated
with two additional 10 mL quantities of the cleavage mixture for 5
min each. The cleavage mixture was concentrated to ca. 5 mL, and
ethyl ether was added to induce precipitation. The precipitate was
collected by centrifugation, washed with ethyl ether three times,
and dried under high vacuum, resulting in the recovery of crude
material.
##STR00007##
[0134] Synthesis of FITC-(PEG).sub.12-CCK2R Ligand:
[0135] To a stirred solution of the crude CCK2R
ligand-(PEG).sub.12-EDA (10 mg) and FITC (6 mg) in
dimethylsulfoxide (DMSO, 1 mL) was added Pr.sub.2NEt (5 equiv) at
room temperature and stirring continued for 6 hr under argon. The
reaction was monitored by LCMS and purified by preparative HPLC
(mobile phase: A=10 mM ammonium acetate pH=7, B=ACN; method: 0% B
to 50% B in 30 min at 13 mL/min) The pure fractions were pooled and
freeze-dried, furnishing the FITC-(PEG).sub.12-CCK2R ligand.
##STR00008##
3. Binding of FITC-Folate and FITC-(PEG).sub.12-Folate Conjugates
to Transduced T Cells Through Anti-FITC scFV in CAR4-1BBZ
[0136] To examine the ability of the transduced T cells containing
CAR4-1BBZ to bind the FITC-Folate conjugates, binding assays with
the FITC-Folate conjugate and the FITC-(PEG).sub.12-Folate
conjugate were performed. Since the excitation wavelength of FITC
in the FITC-Folate conjugates overlapped with copGFP in CAR4-1BB
transduced T cells, it was difficult to distinguish between FITC
binding and copGFP expression. Therefore, an anti-Folate Acid (FA)
monoclonal antibody and a fluorephore (excitation 640 nm)-labeled
anti-mouse IgG antibody were utilized.
[0137] First, the transduced T cells were incubated with
FITC-Folate or FITC-(PEG).sub.12-Folate conjugates at room
temperature for 1 hour. After washing with 1.times.PBS, transduced
T cells were incubated with anti-FA monoclonal antibody (1:15
dilution) for 1 hour. After another washing with 1.times.PBS,
transduced T cells further were incubated with fluorophore
(excitation 640 nm) labeled anti-mouse IgG antibody (1:50
dilution). Finally, unbound antibodies were washed away and a
confocal microscope was used to confirm the FITC-Folate and
FITC-(PEG).sub.12-Folate conjugates binding ability. All data was
analyzed by Olympus Fluoview software. As shown in FIG. 2, copGFP
expression was observed on the transduced T cells containing CAR
4-1BBZ (top row), but not in non-transduced T cells. As shown in
the middle panel, FITC-folate binding was observed on CAR-1BBZ
transduced T cells, but not on non-transduced T cells. More
importantly, only those transduced T cells showing copGFP
expression (top row) also show FITC-Folate conjugates binding
(middle row). By confocal microscopy, it was confirmed that
FITC-Folate and FITC-(PEG).sub.12-Folate conjugates were
successfully bound to anti-FITC in transduced T cells, but not to
non-transduced T cells. Simultaneously, binding of FITC-Folate and
FITC-(PEG).sub.12-Folate conjugates (arrows) were also detected on
the CAR4-1BBZ transduced T cells. Based on this data, it was
concluded that transduced T cells express CAR4-1BBZ, and that
FITC-Folate conjugates can bind to transduced T cells through
anti-FITC scFv expressed by CAR4-1BBZ transduction.
4. The Binding of FITC-Folate Conjugates to FR Positive Cancer
Cells
[0138] The ability of the FITC-Folate and FITC-(PEG).sub.12-Folate
conjugates to bind folate receptor (FR) positive cancer cells was
next investigated. FR positive cells lines L1210A (Mouse
lymphocytic leukemia) and KB (Mouth epidermal carcinoma), which are
FR positive cell lines, were used to test the binding affinity of
the FITC-Folate conjugates. Briefly, 3-4.times.10.sup.4 cancer
cells were prepared to perform binding affinity assays with
FITC-Folate and FITC-(PEG).sub.12-Folate conjugates. With two
different concentrations (e.g. 20 nM, 70 nM), the two FITC-Folate
conjugates were incubated with cancer cells at room temperature for
1 hour. Since both FITC-Folate conjugates have fluorescence, the
binding ability of FITC-Folate conjugates to cancer cells can be
detected by florescence microscopy. After two washes with 1.times.
phosphate buffered saline (PBS) to remove all unbound compound, the
cancer cells were observed by confocal microscope. In order to
specify whether FITC-Folate conjugates bind to cancer cells via FR
on the surface of cancer cells, Folate Acid was also incubated with
cancer cells as a competition compound. As shown in FIGS. 3A, B, C,
and D, the FITC-Folate conjugates can bind to both cancer cells
(KB, L1210A) via FR on the cell surface. As shown in competition
panel, addition of 50.times. excess folate acid blocked FITC-Folate
binding with these cancer cells, which indicates the specific
binding is between folate acid and FR on cell surface.
[0139] As shown in FIG. 3A, the FITC-Folate conjugate was
internalized into KB cell cytoplasm, caused by FR mediated
endocytosis. However, as shown in FIG. 3C, the
FITC-(PEG).sub.12-Folate conjugate, which has a (PEG).sub.12 linker
between Folate and FITC, stayed on the extracellular surface of the
KB cells.
[0140] From this data it was concluded that the FITC-Folate and
FITC-(PEG).sub.12-Folate conjugates can bind to cancer cells
through FR on the surface of cancer cells. It was also found that
the PEG linker between Folate and FITC can prohibit the
internalization of FITC-Folate conjugate. Surface localization of
FITC-Folate conjugate would help increase the chance for cancer
cells to be recognized by transduced T cell containing CAR4-1BBZ.
If FITC-Folate conjugates are internalized via FR mediated
endocytosis, transduced T cells cannot target cancer cells through
FITC-Folate conjugates.
5. Cytotoxicity of Anti-FITC CAR-Modified T Cells Against Folate
Receptor Positive (FR+) Cancer Cells
[0141] In order to explore whether CAR-expressing T cells can kill
cancer cells, .sup.51Chromium release assays were conducted. In
this assay, cancer cells are labeled with .sup.51Cr. When cancer
cells are lysed, .sup.51Cr would be released from cancer cells to
supernatant. By measuring .sup.51Cr in supernatant, the number of
cancer cells killed can be determined.
[0142] First, target cancer cells L1210A were incubated in 50 .mu.L
growth medium containing 50 .mu.Ci .sup.51Chromium to get labeled.
After washing with PBS X3, L1210A cells were resuspended and
incubated at 37.degree. C. for 1 hour in the growth medium
containing 70 nM FITC-Folate conjugate or FITC-(PEG).sub.12-Folate
conjugate. After washing away excess FITC conjugates,
5.times.10.sup.3 target cancer cells were added in each well of
96-well plates. When adhesive KB cells were used as the target,
they were treated similar as L1210A cells, except KB cells were
grown in 96-well plates for 24 hours before .sup.51Cr labeling and
FITC-Folate conjugates binding were performed in 96-well plates.
Effector T cells were then harvested, resuspended in growth media
and added to the wells containing target cancer cells. The effector
T cell to target cancer cell ratio was 20:1. After incubation for
4-10 hours, 20 .mu.L aliquots of cell-free supernatant were
harvested and .sup.51Cr in the supernatants was measured with a
scintillation counter or a .gamma.-counter.
[0143] Percent specific cytolysis was calculated using following
formula:
% cytolysis=(Experimental .sup.51Cr release-control .sup.51Cr
release)/(Maximum .sup.51Cr release-control .sup.51Cr
release).times.100
[0144] Control wells contained only target cancer cells, effector T
cells were never added in these wells. Maximum .sup.51Cr release
was determined by measuring .sup.51Cr release from labeled target
cells treated with 2.5% SDS to lyse all cells.
[0145] As shown in FIG. 4A, in the absence of FITC-Folate
conjugates, CAR-expressing T cells showed negligible activity
(.about.5%) on target KB cell cytolysis. In the presence of
FITC-Folate conjugate, CAR-expressing T cells showed .about.18%
cancer cell cytolysis. It is implicated that FITC-Folate conjugate
acts as a bridge to redirect anti-FITC CAR-expressing T cells to
FR+KB cells. Furthermore, in the presence of
FITC-(PEG).sub.12-Folate conjugate, which has a .about.40 .ANG.
(PEG).sub.12 spacer between FITC and Folate molecules, anti-FITC
CAR-expressing T cells showed much higher activity (.about.39%) on
cancer cell cytolysis. With .about.40 .ANG. distance between FITC
and Folate, this conjugate can redirect CAR-expressing T cells to
FR+KB cells much better (39% vs. 18%). As the non-specific
cytotoxicity control, unmodified T cells showed only 5-10%
cytolysis. The existence or absence of the FITC-Folate conjugates
did not show any effect on unmodified T cell cytotoxicity against
FR+KB cells.
[0146] FIG. 4B shows cytotoxicity of anti-FITC CAR modified T cells
against cancer cell line L1210A. In the absence of FITC-Folate
conjugates, CAR-expressing T cells showed negligible activity
(2-3%) on target L1210A cell cytolysis. In the presence of
FITC-Folate conjugate, CAR-expressing T cells showed .about.29%
cancer cell cytolysis. It is implicated that FITC-Folate conjugate
acts as a bridge to redirect anti-FITC CAR-expressing T cells to
FR+L1210A cells. Furthermore, in the presence of the
FITC-(PEG).sub.12-Folate conjugate, which has a .about.40 .ANG.
(PEG).sub.12 spacer between FITC and Folate molecules, anti-FITC
CAR-expressing T cells showed much higher activity (.about.51%) on
FR+L1210A cytolysis. With .about.40 .ANG. distance between FITC and
Folate, this conjugate can redirect CAR-expressing T cells to
FR+L1210A much better (51% vs. 29%). As the non-specific
cytotoxicity control, unmodified T cells showed only 5-10%
cytolysis as expected.
[0147] From this data it was concluded that anti-FITC
CAR-expressing T cells do not have innate cytotoxicity against FR+
cancer cells. However, in the presence of the FITC-Folate
conjugate, anti-FITC CAR-expressing T cells are redirected to FR+
cancer cells and cause specific cytolysis, and the activation of
CAR-expressing T cells can be regulated by controlling of the
addition of FITC-Folate conjugates. Further, the
FITC-(PEG).sub.12-Folate conjugate, which has a .about.40 .ANG.
spacer between the FITC and the Folate molecules, can redirect
anti-FITC CAR-expressing T cells to FR+ cancer cells much better
than the conjugate without the spacer.
[0148] While the invention has been described with reference to
certain particular embodiments thereof, those skilled in the art
will appreciate that various modifications may be made without
departing from the spirit and scope of the invention. The scope of
the appended claims is not to be limited to the specific
embodiments described.
REFERENCES
[0149] All patents and publications mentioned in this specification
are indicative of the level of skill of those skilled in the art to
which the invention pertains. Each cited patent and publication is
incorporated herein by reference in its entirety. All of the
following references have been cited in this application: [0150] 1.
Sadelain, M. et al., The basic principles of chimeric antigen
receptor design. Cancer Discovery. 2013. 3(4):388-98. [0151] 2.
Cartellieri, M. et al., Chimeric antigen receptor-engineered T
cells for immunotherapy of cancer. J Biomedicine and Biotechnology.
2010. Article ID 956304, 13 pages. [0152] 3. Urba, W. J. et al.,
Redirecting T cells. N Engl J Med. 2011. 365:8. [0153] 4. Porter,
D. L. et al., Chimeric antigen receptor-modified T cells in chronic
lymphoid leukemia. N Engl J Med. 2011. 365:725-33. [0154] 5. Cor,
H. J. et al., Treatment of Metastatic Renal Cell Carcinoma With
Autologous T-Lymphocytes Genetically Retargeted Against Carbonic
Anhydrase IX: First Clinical Experience. J Clin Oncol. 2006.
24(13):e20-2. [0155] 6. Kochenderfer, J. N. et al., B-cell
depletion and remissions of malignancy along with
cytokine-assoicated toxicity in clinical trial of anti-CD19
chimeric antigen receptor transduced T cells. Blood. 2012.
119(12):2790-20. [0156] 7. Reichert, J. M. Day 1, Emerging
Disruptive Technologies and Cutting-Edge Analytical Techniques.
MAbs 2009. 1(3):190-209. [0157] 8. Kularatne, S. A. et al.,
Prostate-specific membrane antigen targeted imaging and therapy of
prostate cancer using a PSMA inhibitor as a homing ligand. Mol
Pharm. 2009. 6(3):780-9. [0158] 9. Wayua. C. et al., Evaluation of
a Cholecystokinin 2 Receptor-Targeted Near-Infrared Dye for
Fluorescence-Guided Surgery of Cancer. Molecular Pharmaceutics.
2013. (ePublication). [0159] 10. Sega, E. I. et al., Tumor
detection using folate receptor-targeted imaging agents. Cancer
Metastasis Rev. 2008. 27(4):655-64. [0160] 11. Alvarez-Vallina, L.
et al., Antigen-specific targeting of CD28-mediated T cell
co-stimulation using chimeric single-chain antibody variable
fragment-CD28 receptors. Eur J Immunol. 1996. 26(10):2304-9. [0161]
12. Imai, C. et al., Chimeric receptors with 4-1BB signaling
capacity provoke potent cytotoxicity against acute lymphoblastic
leukemia. Leukemia. 2004. 18:676-84. [0162] 13. Latza, U. et al.,
The human OX40 homolog: cDNA structure expression and chromosomal
assignment of the ACT35 antigen. Eur. J. Immunol. 1994. 24:677.
[0163] 14. Hutloff, A. et al., ICOS is an inducible T-cell
costimulator structurally and functionally related to CD28. Nature.
1999. 397:263. [0164] 15. Orr, B. A., et al., Rapid Method for
Measuring ScFv Thermal Stability by Yeast Surface Display.
Biotechnol Prog. 2003. 19(2):631-8. [0165] 16. Kolmar, H. et al.,
Alternative binding proteins: biological activity and therapeutic
potential of cysteine-knot miniproteins. The FEBS Journal. 2008.
275(11):26684-90.
* * * * *