U.S. patent application number 14/410659 was filed with the patent office on 2015-07-23 for toxicity management for anti-tumor activity of cars.
This patent application is currently assigned to The Children's Hospital of Philadelphai. The applicant listed for this patent is The Children's Hospital of Philadelphia, The Trustees of the University of Pennsylvania. Invention is credited to Stephan Grupp, Carl H. June, Michael D. Kalos, Bruce L. Levine.
Application Number | 20150202286 14/410659 |
Document ID | / |
Family ID | 49916566 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202286 |
Kind Code |
A1 |
June; Carl H. ; et
al. |
July 23, 2015 |
Toxicity Management for Anti-Tumor Activity of CARs
Abstract
The present invention provides compositions and methods for
treating cancer in a patient. In one embodiment, the method
comprises a first-line therapy comprising administering to a
patient in need thereof a genetically modified T cell expressing a
CAR wherein the CAR comprises an antigen binding domain, a
transmembrane domain, a costimulatory signaling region, and a CD3
zeta signaling domain and monitoring the levels of cytokines in the
patient post T cell infusion to determine the type of second-line
of therapy appropriate for treating the patient as a consequence of
the presence of the CAR T cell in the patient.
Inventors: |
June; Carl H.; (Merion
Station, PA) ; Levine; Bruce L.; (Cherry Hill,
NJ) ; Kalos; Michael D.; (Philadelphia, PA) ;
Grupp; Stephan; (Havertown, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania
The Children's Hospital of Philadelphia |
Philadelphia
Philadelphia |
PA
PA |
US
US |
|
|
Assignee: |
The Children's Hospital of
Philadelphai
Philadelphia
PA
|
Family ID: |
49916566 |
Appl. No.: |
14/410659 |
Filed: |
July 12, 2013 |
PCT Filed: |
July 12, 2013 |
PCT NO: |
PCT/US2013/050267 |
371 Date: |
December 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61671482 |
Jul 13, 2012 |
|
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|
61782982 |
Mar 14, 2013 |
|
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Current U.S.
Class: |
424/133.1 ;
424/93.21 |
Current CPC
Class: |
A61K 35/00 20130101;
A61K 2039/505 20130101; C12Q 1/6886 20130101; A61P 35/00 20180101;
G01N 2333/52 20130101; A61K 48/0083 20130101; A61P 39/02 20180101;
A61P 37/06 20180101; A61P 35/02 20180101; G01N 2800/52 20130101;
A61K 31/7088 20130101; A61K 2039/515 20130101; A61K 45/06 20130101;
G01N 33/6803 20130101; A61K 31/713 20130101; A61P 35/04 20180101;
A61K 39/3955 20130101; A61P 43/00 20180101; A61K 31/7088 20130101;
A61K 2300/00 20130101; A61K 31/713 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; G01N 33/68 20060101
G01N033/68; A61K 35/00 20060101 A61K035/00; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating a patient having a disease, disorder or
condition associated with an elevated expression of a tumor
antigen, the method comprising administering a first-line therapy
and a second-line therapy to a patient in need thereof, wherein the
first line therapy comprises administering to the patient an
effective amount of a cell genetically modified to express a CAR,
wherein the CAR comprises an antigen binding domain, a
transmembrane domain, and an intracellular signaling domain.
2. The method of claim 1, wherein following the administration of
the first-line therapy, cytokine levels in the patient are
monitored to determine the appropriate type of second-line therapy
to be administered to the patient and the appropriate second-line
therapy is administered to the patient in need thereof.
3. The method of claim 2, wherein an increase in the level of a
cytokine identifies a type of cytokine inhibitory therapy to be
administered to the patient in need thereof.
4. The method of claim 3, wherein the cytokine is selected from the
group consisting of IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8,
IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha.,
IFN-.gamma., MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF,
G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF,
HGF, FGF-.beta., CD40, CD40L, ferritin, and any combination
thereof.
5. The method of claim 3, wherein the cytokine inhibitory therapy
is selected from the group consisting of a small interfering RNA
(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an
expression vector encoding a transdominant negative mutant, an
antibody, a peptide, a small molecule, a cytokine inhibitory drug,
and any combination thereof.
6. The method of claim 2, wherein the cytokine levels are monitored
by detecting the protein level of the cytokine in a biological
sample from the patient.
7. The method of claim 2, wherein the cytokine levels are monitored
by detecting the nucleic acid level of the cytokine in a biological
sample from the patient.
8. A method of reducing or avoiding an adverse effect associated
with the administration of a cell genetically modified to express a
CAR, wherein the CAR comprises an antigen binding domain, a
transmembrane domain, and an intracellular signaling domain, the
method comprising monitoring the levels of a cytokine in a patient
to determine the appropriate type of cytokine therapy to be
administered to the patient and administering the appropriate
cytokine therapy to the patient.
9. The method of claim 8, wherein an increase in the level of a
cytokine identifies a type of cytokine inhibitory therapy to be
administered to the patient.
10. The method of claim 9, wherein the cytokine is selected from
the group consisting of IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8,
IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha.,
IFN-.gamma., MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF,
G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF,
HGF, FGF-.beta., CD40, CD40L, ferritin, and any combination
thereof.
11. The method of claim 9, wherein the cytokine inhibitory therapy
is selected from the group consisting of a small interfering RNA
(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an
expression vector encoding a transdominant negative mutant, an
intracellular antibody, a peptide, a small molecule, a cytokine
inhibitory drug, and any combination thereof.
12. The method of claim 8, wherein the cytokine levels are
monitored by detecting the protein level of the cytokine in a
biological sample from the patient.
13. The method of claim 8, wherein the cytokine levels are
monitored by detecting the nucleic acid level of the cytokine in a
biological sample from the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/671,482, filed on Jul. 13, 2012 and U.S.
Provisional Patent Application No. 61/782,982, filed on Mar. 14,
2013, each of which application is hereby incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Patients with relapsed and chemotherapy-refractory acute
lymphocytic leukemia (ALL) have a poor prognosis despite the use of
aggressive therapies such as allogeneic hematopoietic stem cell
transplantation (Barrett et al., 1994, N Engl J Med 331:1253-8;
Gokbuget et al., 2012, Blood 120:2032-41) and bi-specific CD19
antibody fragments (Bargou et al., 2008, Science 321:974-7).
Chimeric antigen receptor modified T cells targeting
lineage-specific antigens CD19 and CD20 have been reported to be
effective in adults with CLL and B-cell lymphomas (Till et al.,
2008, Blood 112:2261-71; Kochenderfer et al., 2010, Blood
116:4099-102; Brentjens et al., 2011, Blood 118:4817-28; Porter et
al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science
Translational Medicine 3:95ra73; Savoldo et al., 2011, J Clin
Invest 121:1822-5). However, the effects of CAR T cells on ALL
blasts, a more immature leukemia with a more rapid progression,
have not been fully investigated.
[0003] Delayed onset of the tumor lysis syndrome and cytokine
secretion, combined with vigorous in vivo chimeric antigen receptor
T-cell expansion has been reported (Porter et al., 2011, N Engl J
Med 365:725-33; Kalos et al., 2011, Science Translational Medicine
3:95ra73). However, the effects of cytokine secretion and disorders
associated with in vivo chimeric antigen recept T-cell expansion
have not been fully investigated.
[0004] Thus, there is an urgent need in the art for compositions
and methods for treatment of cancer using CARs and addressing
toxicity of the CARs. The present invention addresses this
need.
SUMMARY OF THE INVENTION
[0005] The invention provides a method of treating a patient having
a disease, disorder or condition associated with an elevated
expression of a tumor antigen. In one embodiment, the method
comprises administering a first-line therapy and a second-line
therapy to a patient in need thereof, wherein the first line
therapy comprises administering to the patient an effective amount
of a cell genetically modified to express a CAR, wherein the CAR
comprises an antigen binding domain, a transmembrane domain, and an
intracellular signaling domain.
[0006] In one embodiment, following the administration of the
first-line therapy, cytokine levels in the patient are monitored to
determine the appropriate type of second-line therapy to be
administered to the patient and the appropriate second-line therapy
is administered to the patient in need thereof.
[0007] In one embodiment, an increase in the level of a cytokine
identifies a type of cytokine inhibitory therapy to be administered
to the patient in need thereof.
[0008] In one embodiment, the cytokine is selected from the group
consisting of IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10,
IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha.,
IFN-.gamma., MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF,
G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF,
HGF, FGF-.beta., CD40, CD40L, ferritin, and any combination
thereof.
[0009] In one embodiment, the cytokine inhibitory therapy is
selected from the group consisting of a small interfering RNA
(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an
expression vector encoding a transdominant negative mutant, an
antibody, a peptide, a small molecule, a cytokine inhibitory drug,
and any combination thereof.
[0010] In one embodiment, the cytokine levels are monitored by
detecting the protein level of the cytokine in a biological sample
from the patient.
[0011] In one embodiment, the cytokine levels are monitored by
detecting the nucleic acid level of the cytokine in a biological
sample from the patient.
[0012] The invention provides a method of reducing or avoiding an
adverse effect associated with the administration of a cell
genetically modified to express a CAR, wherein the CAR comprises an
antigen binding domain, a transmembrane domain, and an
intracellular signaling domain, the method comprising monitoring
the levels of a cytokine in a patient to determine the appropriate
type of cytokine therapy to be administered to the patient and
administering the appropriate cytokine therapy to the patient.
[0013] In one embodiment, an increase in the level of a cytokine
identifies a type of cytokine inhibitory therapy to be administered
to the patient.
[0014] In one embodiment, the cytokine is selected from the group
consisting of IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10,
IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha.,
IFN-.gamma., MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF,
G-CSF, CXCL9, CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF,
HGF, FGF-.beta., CD40, CD40L, ferritin, and any combination
thereof.
[0015] In one embodiment, the cytokine inhibitory therapy is
selected from the group consisting of a small interfering RNA
(siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an
expression vector encoding a transdominant negative mutant, an
intracellular antibody, a peptide, a small molecule, a cytokine
inhibitory drug, and any combination thereof.
[0016] In one embodiment, the cytokine levels are monitored by
detecting the protein level of the cytokine in a biological sample
from the patient.
[0017] In one embodiment, the cytokine levels are monitored by
detecting the nucleic acid level of the cytokine in a biological
sample from the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0019] FIG. 1 is an image demonstrating serum cytokine levels in
four different patients. All patients exhibited cytokine release,
including IL-6.
[0020] FIG. 2 is an image depicting serum cytokines plotted in a
representative patient. The patient was critically ill on days 5 to
7, and only began to improve following tocilizumab
administration.
[0021] FIG. 3 is an image demonstrating that antibody interventions
do not impact CART 19 cellular functionality as measured for
markers of T cell activity (perforin and IFN-.gamma.).
[0022] FIG. 4, comprising FIGS. 4A through 4C, is a series of
images depicting clinical responses. FIG. 4A shows two children
with multiply-relapsed chemotherapy-refractory CD19+ B cell
precursor acute lymphoblastic leukemia who were treated with CTL019
cells, infused on Day 0. Changes in serum lactate dehydrogenase
(LDH) and body temperature after CTL019 infusion, with maximum
temperature per 24 hour period demarcated with circles. CHOP-100
was given methylprednisolone starting on day 5 at 2 mg/kg/day,
tapered to off by day 12. On the morning of day 7, etanercept was
given 0.8 mg/kg.times.1. At 6 pm in the evening of day 7,
tocilizumab 8 mg/kg.times.1 was administered. A transient
improvement in pyrexia occurred with administration of
corticosteroids on day 5 in CHOP-100, with complete resolution of
fevers occurring after administration of cytokine-directed therapy
consisting of etanercept and tocilizumab on day 8. FIG. 4B shows
serum cytokines and inflammatory markers measured at frequent time
points after CTL019 infusion. Cytokine values are shown using a
semi-logarithmic plot with fold-change from baseline. Baseline (Day
0 pre-infusion) values (pg/ml serum) for each analyte were
(CHOP-100, CHOP-101): IL1-.beta.: (0.9, 0.2); IL-6: (4.3, 1.9);
TNF-.alpha.: (1.5, 0.4); IL2R.alpha.: (418.8, 205.7); IL-2: (0.7,
0.4); IL-10 (9.9, 2.3); IL1R.alpha.: (43.9, 27.9). Both patients
developed pronounced elevations in a number of cytokines and
cytokine receptors, including soluble interleukin 1A and 2 receptor
(IL-1RA and IL-2R), interleukins 2, 6 and 10 (IL-2, IL-6 and
IL-10), tumor necrosis factor-.alpha. (TNF-.alpha.), and
interferon-.gamma. (INF-.gamma.). FIG. 4C shows changes in
circulating absolute neutrophil count (ANC), absolute lymphocyte
count (ALC) and white blood cell (WBC) count. Of note, the increase
in the ALC was primarily composed of activated CT019 T
lymphocytes.
[0023] FIG. 5, comprising FIGS. 5A through 5D, is a series of
imaged depicting expansion and visualization of CTL019 cells in
peripheral blood, bone marrow and CSF. FIG. 5A shows flow
cytometric analysis of peripheral blood stained with antibodies to
detect CD3 and the anti-CD19 CAR. Depicted are the percent of CD3
cells expressing the CAR in CHOP-100 and CHOP-101. FIG. 5B shows
the presence of CTL019 T cells in peripheral blood, bone marrow,
and CSF by quantitative real-time PCR. Genomic DNA was isolated
from whole blood, bone marrow aspirates and CSF collected at serial
time points before and after CTL019 infusion. FIG. 5C shows flow
cytometric detection of CTL019 cells in CSF collected from CHOP-100
and CHOP-101. FIG. 5D shows images of activated large granular
lymphocytes in Wright-stained smears of the peripheral blood and
cytospins of the CSF.
[0024] FIG. 6 is an image showing CD19 expression at baseline and
at relapse in CHOP-101. Bone marrow samples from CHOP-101 were
obtained prior to CTL019 infusion and at time of relapse 2 months
later. Mononuclear cells isolated from marrow samples were stained
for CD45, CD34 and CD19 and analyzed on an Accuri C6 flow
cytometer. After gating on live cells, the blast gate (CD45+SSC
low) was subgated on CD34+ cells and histograms generated for CD19
expression. Division line represents threshold for the same gating
on isotype controls. Pre-therapy blasts have a range of
distribution of CD19, with a small population of very dim staining
cells seen as the tail of the left histogram at 102 on the X-axis.
The relapse sample does not have any CD19 positive blasts. Analysis
of CD19 expression on the pre-treatment blast population revealed a
small population of CD19 dim or negative cells. The mean
fluorescence intensity (MFI) of this small population of cells was
187 (left panel), similar to the MFI of the anti-CD19-stained
relapsed blast cells (201, right panel). Pre-therapy marrow sample
was hypocellular with 10% blasts and relapse marrowsample was
normocellular with 68% blasts, accounting for differences in events
available for acquisition.
[0025] FIG. 7 is an image showing induction of remission in bone
marrow in CHOP-101 on day+23 after CTL019 infusion. Clinical
immunophenotyping report for CHOP-101 at baseline (Top panel) and
at day+23 (Bottom panel). Cells were stained for CD10, CD19, CD20,
CD34, CD38 and CD58. Flow cytometry was done after lysis of the red
blood cells. The report on day+23 indicated that the white blood
cells consisted of 42.0% lymphocytes, 6.0% monocytes, 50.3% myeloid
forms, 0.17% myeloid blasts and no viable lymphoid progenitors.
There was no convincing immunophenotypic evidence of residual
precursor B cell lymphoblastic leukemia/lymphoma by flow cytometry.
Essentially no viable B cells were identified.
[0026] FIG. 8 is an image depicting in vivo expansion and
persistence of CTL019 cells in blood. The number of white blood
cells (WBC), CD3+ T cells, and CTL019 cells in blood is shown for
CHOP-100 and CHOP-101. Cell numbers are shown on a semi-logarithmic
plot.
[0027] FIG. 9, comprising FIGS. 9A and 9B, is a series of images
demonstrating that subjects had an elimination of CD19 positive
cells in bone marrow and blood within 1 month after CTL019
infusion. FIG. 9A shows persistent B cell aplasia in CHOP-100. The
top panel shows a predominant population of leukemic blast cells in
bone marrow aspirated from CHOP-100 expressing CD19 and CD20 on
day+6. This population is absent at day+23 and 6 months. FIG. 9B
shows B cell aplasia and emergence of CD19 escape variant cells in
CHOP-101. Flow cytometric analysis of bone marrow aspirates from
CHOP-101 stained with anti-CD45, CD34 and CD19. In the bottom row,
side scatter and the CD45 dim positive cells were used to identify
leukemic cells that express variable amounts of CD34 and CD19 at
baseline. Only CD19 negative blasts were detected on day 64.
Numerical values in the top panel represent the fraction of the
total leukocytes represented in each quadrant. Numerical values in
the lower panel represent the percentage from the total leukocytes
represented in the CD45dim/SS low gate.
[0028] FIG. 10 is a graph depicting the levels of ferritin present
in the patient following receipt of CAR T cells.
[0029] FIG. 11 is a graph depicting the levels of myoglobin present
in the patient following receipt of CAR T cells.
[0030] FIG. 12 is a graph depicting the levels of plasminogen
activator inhibitor-1 (PAI-1) present in the patient following
receipt of CART cells.
DETAILED DESCRIPTION
[0031] The invention relates to compositions and methods for
treating cancer including but not limited to hematologic
malignancies and solid tumors. The invention also encompasses
methods of treating and preventing certain types of cancer,
including primary and metastatic cancer, as well as cancers that
are refractory or resistant to conventional chemotherapy. The
methods comprise administering to a patient in need of such
treatment or prevention a therapeutically or prophylactically
effective amount of a T cell transduced to express a chimeric
antigen receptor (CAR). CARs are molecules that combine
antibody-based specificity for a desired antigen (e.g., tumor
antigen) with a T cell receptor-activating intracellular domain to
generate a chimeric protein that exhibits a specific anti-tumor
cellular immune activity.
[0032] As part of the overall treatment regimen, the invention
encompasses methods of managing certain cancers (e.g., preventing
or prolonging their recurrence, or lengthening the time of
remission) by evaluating the profile of soluble factors in patients
post T cell infusion. Preferably, the profile of soluble factors
includes evaluation of a cytokine profile. When the cytokine
profile indicates an increase in a particular cytokine post T cell
infusion compared to pre T cell infusion, a skilled artisan can
elect to administer to the patient in need of such management an
effective amount of a cytokine inhibitory compound or a
pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,
clathrate, or pro drug thereof to manage the elevated levels of the
cytokine post T cell infusion.
[0033] The present invention is partly based on the discovery that
the identify of a unique combination of factors whose modulation
from baseline or pre-existing levels at baseline can help track T
cell activation, target activity, and potential harmful side
effects following CAR T cell infusion in order to help manage the
treatment of the cancer. Exemplary factors include but are not
limited to IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12,
IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha., IFN-.gamma.,
MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF, G-CSF, CXCL9,
CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-.beta.,
CD40, CD40L, ferritin, and the like.
[0034] The present invention relates to a strategy of adoptive cell
transfer of T cells transduced to express a chimeric antigen
receptor (CAR) in combination with toxicity management, where a
profile of soluble factors from a post T cell infusion patient is
generated and a therapy directed against the elevated soluble
factor is carried out in order to treat the cancer. For example,
generating a real time soluble factor profile allows for
intervention of the elevated soluble factors with the appropriate
inhibitor in order to bring the levels down to normal levels.
[0035] In one embodiment, the CAR of the invention comprises an
extracellular domain having an antigen recognition domain that
targets a desired antigen, a transmembrane domain, and a
cytoplasmic domain. The invention is not limited to a specific CAR.
Rather, any CAR that targets a desired antigen can be used in the
present invention. Compositions and methods of making CARs have
been described in PCT/US11/64191, which is incorporated by
reference in its entirety herein.
[0036] In some embodiments of any of the methods above, the methods
result in a measurable reduction in tumor size or evidence of
disease or disease progression, complete response, partial
response, stable disease, increase or elongation of progression
free survival, increase or elongation of overall survival, or
reduction in toxicity.
DEFINITIONS
[0037] 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. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0038] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0039] The articles "a" and "an" are used herein to refer to one or
to more than one (i. e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0040] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, in some instances
.+-.5%, in some instances .+-.1%, and in some instances .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0041] "Activation," as used herein, refers to the state of a T
cell that has been sufficiently stimulated to induce detectable
cellular proliferation. Activation can also be associated with
induced cytokine production, and detectable effector functions. The
term "activated T cells" refers to, among other things, T cells
that are undergoing cell division.
[0042] "Activators" or "agonists" of a soluble factor are used
herein to refer to molecules of agents capable of activating or
increasing the levels of the soluble factor. Activators are
compounds that increase, promote, induce activation, activate, or
upregulate the activity or expression of soluble factor, e.g.,
agonists. Assays for detecting activators include, e.g., expressing
the soluble factor in vitro, in cells, or cell membranes, applying
putative agonist compounds, and then determining the functional
effects on activity of the soluble factor, as described elsewhere
herein.
[0043] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins. Antibodies are often tetramers
of immunoglobulin molecules. The antibodies in the present
invention may exist in a variety of forms including, for example,
polyclonal antibodies, monoclonal antibodies, Fv, Fab and
F(ab).sub.2, as well as single chain antibodies and humanized
antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al.,
1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,
N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0044] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, scFv antibodies, and multispecific
antibodies formed from antibody fragments.
[0045] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. Furthermore, one skilled in the art will understand that an
antigen need not be encoded solely by a full length nucleotide
sequence of a gene. It is readily apparent that the present
invention includes, but is not limited to, the use of partial
nucleotide sequences of more than one gene and that these
nucleotide sequences are arranged in various combinations to elicit
the desired immune response. Moreover, a skilled artisan will
understand that an antigen need not be encoded by a "gene" at all.
It is readily apparent that an antigen can be generated synthesized
or can be derived from a biological sample. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a biological fluid.
[0046] The term "auto-antigen" means, in accordance with the
present invention, any self-antigen which is recognized by the
immune system as if it were foreign. Auto-antigens comprise, but
are not limited to, cellular proteins, phosphoproteins, cellular
surface proteins, cellular lipids, nucleic acids, glycoproteins,
including cell surface receptors.
[0047] The term "autoimmune disease" as used herein is defined as a
disorder that results from an autoimmune response. An autoimmune
disease is the result of an inappropriate and excessive response to
a self-antigen. Examples of autoimmune diseases include but are not
limited to, Addision's disease, alopecia greata, ankylosing
spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's
disease, diabetes (Type I), dystrophic epidermolysis bullosa,
epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr
syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus
erythematosus, multiple sclerosis, myasthenia gravis, pemphigus
vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,
sarcoidosis, scleroderma, Sjogren's syndrome,
spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,
pernicious anemia, ulcerative colitis, among others.
[0048] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0049] "Allogeneic" refers to a graft derived from a different
animal of the same species.
[0050] "Xenogeneic" refers to a graft derived from an animal of a
different species.
[0051] The term "cancer" as used herein is defined as disease
characterized by the rapid and uncontrolled growth of aberrant
cells. Cancer cells can spread locally or through the bloodstream
and lymphatic system to other parts of the body. Examples of
various cancers include but are not limited to, breast cancer,
prostate cancer, ovarian cancer, cervical cancer, skin cancer,
pancreatic cancer, colorectal cancer, renal cancer, liver cancer,
brain cancer, lymphoma, leukemia, lung cancer and the like.
[0052] As used herein, by "combination therapy" is meant that a
first agent is administered in conjunction with another agent. "In
conjunction with" refers to administration of one treatment
modality in addition to another treatment modality. As such, "in
conjunction with" refers to administration of one treatment
modality before, during, or after delivery of the other treatment
modality to the individual. Such combinations are considered to be
part of a single treatment regimen or regime.
[0053] As used herein, the term "concurrent administration" means
that the administration of the first therapy and that of a second
therapy in a combination therapy overlap with each other.
[0054] "Co-stimulatory ligand," as the term is used herein,
includes a molecule on an antigen presenting cell (e.g., an aAPC,
dendritic cell, B cell, and the like) that specifically binds a
cognate co-stimulatory molecule on a T cell, thereby providing a
signal which, in addition to the primary signal provided by, for
instance, binding of a TCR/CD3 complex with an MHC molecule loaded
with peptide, mediates a T cell response, including, but not
limited to, proliferation, activation, differentiation, and the
like. A co-stimulatory ligand can include, but is not limited to,
CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,
inducible costimulatory ligand (ICOS-L), intercellular adhesion
molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,
lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or
antibody that binds Toll ligand receptor and a ligand that
specifically binds with B7-H3. A co-stimulatory ligand also
encompasses, inter alia, an antibody that specifically binds with a
co-stimulatory molecule present on a T cell, such as, but not
limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3, and a ligand that specifically binds with CD83.
[0055] A "co-stimulatory molecule" refers to the cognate binding
partner on a T cell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the T cell,
such as, but not limited to, proliferation. Co-stimulatory
molecules include, but are not limited to an MHC class I molecule,
BTLA and a Toll ligand receptor.
[0056] A "co-stimulatory signal," as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to T cell proliferation and/or upregulation or
downregulation of key molecules.
[0057] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0058] An "effective amount" as used herein, means an amount which
provides a therapeutic or prophylactic benefit.
[0059] As used herein "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0060] As used herein, the term "exogenous" refers to any material
introduced to an organism, cell, tissue or system that was produced
outside the organism, cell, tissue or system.
[0061] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter.
[0062] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses (e.g.,
lentiviruses, retroviruses, adenoviruses, and adeno-associated
viruses) that incorporate the recombinant polynucleotide.
[0063] "Homologous" refers to the sequence similarity or sequence
identity between two polypeptides or between two nucleic acid
molecules. When a position in both of the two compared sequences is
occupied by the same base or amino acid monomer subunit, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
the molecules are homologous at that position. The percent of
homology between two sequences is a function of the number of
matching or homologous positions shared by the two sequences
divided by the number of positions compared.times.100. For example,
if 6 of 10 of the positions in two sequences are matched or
homologous then the two sequences are 60% homologous. By way of
example, the DNA sequences ATTGCC and TATGGC share 50% homology.
Generally, a comparison is made when two sequences are aligned to
give maximum homology.
[0064] The term "immunoglobulin" or "Ig," as used herein, is
defined as a class of proteins, which function as antibodies.
Antibodies expressed by B cells are sometimes referred to as the
BCR (B cell receptor) or antigen receptor. The five members
included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
IgA is the primary antibody that is present in body secretions,
such as saliva, tears, breast milk, gastrointestinal secretions and
mucus secretions of the respiratory and genitourinary tracts. IgG
is the most common circulating antibody. IgM is the main
immunoglobulin produced in the primary immune response in most
subjects. It is the most efficient immunoglobulin in agglutination,
complement fixation, and other antibody responses, and is important
in defense against bacteria and viruses. IgD is the immunoglobulin
that has no known antibody function, but may serve as an antigen
receptor. IgE is the immunoglobulin that mediates immediate
hypersensitivity by causing release of mediators from mast cells
and basophils upon exposure to allergen.
[0065] As used herein, the term "immune response" includes T cell
mediated and/or B cell mediated immune responses. Exemplary immune
responses include T cell responses, e.g., cytokine production and
cellular cytotoxicity. In addition, the term immune response
includes immune responses that are indirectly effected by T cell
activation, e.g., antibody production (humoral responses) and
activation of cytokine responsive cells, e.g., macrophages Immune
cells involved in the immune response include lymphocytes, such as
B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen
presenting cells (e.g., professional antigen presenting cells such
as dendritic cells, macrophages, B lymphocytes, Langerhans cells,
and non-professional antigen presenting cells such as
keratinocytes, endothelial cells, astrocytes, fibroblasts,
oligodendrocytes); natural killer cells; myeloid cells, such as
macrophages, eosinophils, mast cells, basophils, and
granulocytes.
[0066] "Inhibitors" or "antagonists" of a soluble factor are used
herein to refer to molecules of agents capable of inhibiting,
inactivating or reducing the levels of the soluble factor.
Inhibitors are compounds that, e.g., bind to, partially or totally
block activity, decrease, prevent, delay activation, inactivate,
desensitize, or down regulate the activity or expression of soluble
factor, e.g., antagonists. Inhibitors include polypeptide
inhibitors, such as antibodies, soluble receptors and the like, as
well as nucleic acid inhibitors such as siRNA or antisense RNA,
genetically modified versions of the soluble factor, e.g., versions
with altered activity, as well as naturally occurring and synthetic
soluble factor antagonists, small chemical molecules and the like.
Assays for detecting inhibitors include, e.g., expressing the
soluble factor in vitro, in cells, or cell membranes, applying
putative antagonist compounds, and then determining the functional
effects on activity of the soluble factor, as described elsewhere
herein.
[0067] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of the
compositions and methods of the invention. The instructional
material of the kit of the invention may, for example, be affixed
to a container which contains the nucleic acid, peptide, and/or
composition of the invention or be shipped together with a
container which contains the nucleic acid, peptide, and/or
composition. Alternatively, the instructional material may be
shipped separately from the container with the intention that the
instructional material and the compound be used cooperatively by
the recipient.
[0068] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0069] A "lentivirus" as used herein refers to a genus of the
Retroviridae family. Lentiviruses are unique among the retroviruses
in being able to infect non-dividing cells; they can deliver a
significant amount of genetic information into the DNA of the host
cell, so they are one of the most efficient methods of a gene
delivery vector. HIV, SIV, and FIV are all examples of
lentiviruses. Vectors derived from lentiviruses offer the means to
achieve significant levels of gene transfer in vivo.
[0070] The phrase "level of a soluble factor" in a biological
sample as used herein typically refers to the amount of protein,
protein fragment or peptide levels of the soluble factor that is
present in a biological sample. A "level of a soluble factor" need
not be quantified, but can simply be detected, e.g., a subjective,
visual detection by a human, with or without comparison to a level
from a control sample or a level expected of a control sample.
[0071] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the level of a response in a
subject compared with the level of a response in the subject in the
absence of a treatment or compound, and/or compared with the level
of a response in an otherwise identical but untreated subject. The
term encompasses perturbing and/or affecting a native signal or
response thereby mediating a beneficial therapeutic response in a
subject, preferably, a human.
[0072] "Parenteral" administration of an immunogenic composition
includes, e.g., subcutaneous (s.c.), intravenous (i.v.),
intramuscular (i.m.), or intrasternal injection, or infusion
techniques.
[0073] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0074] The term "simultaneous administration," as used herein,
means that a first therapy and second therapy in a combination
therapy are administered with a time separation of no more than
about 15 minutes, such as no more than about any of 10, 5, or 1
minutes. When the first and second therapies are administered
simultaneously, the first and second therapies may be contained in
the same composition (e.g., a composition comprising both a first
and second therapy) or in separate compositions (e.g., a first
therapy in one composition and a second therapy is contained in
another composition).
[0075] The term "simultaneous administration," as used herein,
means that a first therapy and second therapy in a combination
therapy are administered with a time separation of no more than
about 15 minutes, such as no more than about any of 10, 5, or 1
minutes. When the first and second therapies are administered
simultaneously, the first and second therapies may be contained in
the same composition (e.g., a composition comprising both a first
and second therapy) or in separate compositions (e.g., a first
therapy in one composition and a second therapy is contained in
another composition).
[0076] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific. In some instances, the
terms "specific binding" or "specifically binding," can be used in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, to mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A," the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0077] By the term "stimulation," is meant a primary response
induced by binding of a stimulatory molecule (e.g., a TCR/CD3
complex) with its cognate ligand thereby mediating a signal
transduction event, such as, but not limited to, signal
transduction via the TCR/CD3 complex. Stimulation can mediate
altered expression of certain molecules, such as downregulation of
TGF-.beta., and/or reorganization of cytoskeletal structures, and
the like.
[0078] A "stimulatory molecule," as the term is used herein, means
a molecule on a T cell that specifically binds with a cognate
stimulatory ligand present on an antigen presenting cell.
[0079] A "stimulatory ligand," as used herein, means a ligand that
when present on an antigen presenting cell (e.g., an aAPC, a
dendritic cell, a B-cell, and the like) can specifically bind with
a cognate binding partner (referred to herein as a "stimulatory
molecule") on a T cell, thereby mediating a primary response by the
T cell, including, but not limited to, activation, initiation of an
immune response, proliferation, and the like. Stimulatory ligands
are well-known in the art and encompass, inter alia, an MHC Class I
molecule loaded with a peptide, an anti-CD3 antibody, a
superagonist anti-CD28 antibody, and a superagonist anti-CD2
antibody.
[0080] The term "subject" is intended to include living organisms
in which an immune response can be elicited (e.g., mammals).
Examples of subjects include humans, dogs, cats, mice, rats, and
transgenic species thereof.
[0081] As used herein, a "substantially purified" cell is 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 purified cells refers to a homogenous population of
cells. In other instances, this term refers simply to cell that
have been separated from the cells with which they are naturally
associated in their natural state. In some embodiments, the cells
are cultured in vitro. In other embodiments, the cells are not
cultured in vitro.
[0082] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
[0083] The term "therapeutically effective amount" refers to the
amount of the subject compound that will elicit the biological or
medical response of a tissue, system, or subject that is being
sought by the researcher, veterinarian, medical doctor or other
clinician. The term "therapeutically effective amount" includes
that amount of a compound that, when administered, is sufficient to
prevent development of, or alleviate to some extent, one or more of
the signs or symptoms of the disorder or disease being treated. The
therapeutically effective amount will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the subject to be treated.
[0084] A "transplant," as used herein, refers to cells, tissue, or
an organ that is introduced into an individual. The source of the
transplanted material can be cultured cells, cells from another
individual, or cells from the same individual (e.g., after the
cells are cultured in vitro). Exemplary organ transplants are
kidney, liver, heart, lung, and pancreas.
[0085] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0086] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process by which exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one which has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0087] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
DESCRIPTION
[0088] The present invention provides compositions and methods for
treating cancer in a patient. In one embodiment, the treatment
method comprises a first-line of therapy comprising administering
the CAR of the invention into the patient to induce an anti-tumor
immune response and monitoring the levels of soluble factors in the
patient post T cell infusion to determine the type of second-line
of therapy appropriate to treat the patient as a consequence of the
first-line of therapy.
[0089] In one embodiment, the second-line of therapy comprises
evaluating the profile of soluble factors in a patient following
receipt of an infusion of the appropriate CAR T (referred elsewhere
herein as "post T cell infusion") where when the soluble factor
profile indicates an increase in a particular soluble factor post T
cell infusion compared to pre T cell infusion, a skilled artisan
can elect to administer to the patient in need of an effective
amount of a soluble factor inhibitory compound in order to manage
the elevated levels of the soluble factor post T cell infusion.
Accordingly, the second-line of therapy in one embodiment includes
administering a type of soluble factor inhibitory therapy to manage
the elevated levels of certain soluble factor s resulting from the
first-line of therapy of using CART cells.
[0090] In yet another embodiment, the second-line of therapy
relating to administering a soluble factor inhibitory compound to
the patient can be combined with other conventionally therapies
used to treat, prevent or manage diseases or disorders associated
with, or characterized by, undesired angiogenesis. Examples of such
conventional therapies include, but are not limited to, surgery,
chemotherapy, radiation therapy, hormonal therapy, biological
therapy and immunotherapy.
[0091] In one embodiment, the CAR of the invention can be
engineered to comprise an extracellular domain having an antigen
binding domain that targets tumor antigen fused to an intracellular
signaling domain of the T cell antigen receptor complex zeta chain
(e.g., CD3 zeta). An exemplary tumor antigen B cell antigen is CD19
because this antigen is expressed on malignant B cells. However,
the invention is not limited to targeting CD19. Rather, the
invention includes any tumor antigen binding moiety. The antigen
binding moiety is preferably fused with an intracellular domain
from one or more of a costimulatory molecule and a zeta chain.
Preferably, the antigen binding moiety is fused with one or more
intracellular domains selected from the group of a CD137 (4-1BB)
signaling domain, a CD28 signaling domain, a CD3zeta signal domain,
and any combination thereof.
[0092] In one embodiment, the CAR of the invention comprises a
CD137 (4-1BB) signaling domain. This is because the present
invention is partly based on the discovery that CAR-mediated T-cell
responses can be further enhanced with the addition of
costimulatory domains. For example, inclusion of the CD137 (4-1BB)
signaling domain significantly increased CAR mediated activity and
in vivo persistence of CAR T cells compared to an otherwise
identical CAR T cell not engineered to express CD137 (4-1BB).
However, the invention is not limited to a specific CAR. Rather,
any CAR that targets a tumor antigen can be used in the present
invention. Compositions and methods of making and using CARs have
been described in PCT/US11/64191, which is incorporated by
reference in its entirety herein.
Methods
[0093] The treatment regimen of the invention result in a
measurable reduction in tumor size or evidence of disease or
disease progression, complete response, partial response, stable
disease, increase or elongation of progression free survival,
increase or elongation of overall survival, or reduction in
toxicity.
[0094] As part of the overall treatment regimen, the invention
encompasses a first-line and a second-line therapy, wherein the
first-line therapy comprises administering a CAR T cell of the
invention to the patient in need thereof. The treatment regimen of
the invention allows for the management of the cancer and treatment
thereof by evaluating the soluble factor profile in patients post T
cell infusion. An appropriate second-line therapy comprises
administering an appropriate soluble factor inhibitor to the
patient in order to reduce the elevated levels of the soluble
factor resulting from the first-line therapy. In some instances,
the appropriate second-line therapy comprises administering an
appropriate soluble factor activator to the patient in order to
increase the suppressed levels of the soluble factor resulting from
the first-line therapy.
[0095] In one embodiment, an appropriate second-line therapy
comprises administering an appropriate cytokine inhibitor to the
patient in order to reduce the elevated levels of the cytokine
resulting from the first-line therapy. In some instances, the
appropriate second-line therapy comprises administering an
appropriate cytokine activator to the patient in order to increase
the suppressed levels of the cytokine resulting from the first-line
therapy.
[0096] In one embodiment, differential levels are over expression
(high expression) or under expression (low expression) as compared
to the expression level of a normal or control cell, a given
patient population, or with an internal control. In some
embodiments, levels are compared between the patient and a normal
individual, between the patient post T cell infusion and pre T cell
infusion, or between the patient post T cell infusion at a first
time point and a second time point.
[0097] In one embodiment, the invention includes evaluating
differential levels of one or more cytokines to generate a cytokine
profile in a patient post T cell infusion in order to determine the
type of cytokine therapy to be applied to the patient for the
purpose of regulating the cytokine level back to normal levels. The
invention may therefore be applied to identify cytokine levels
elevated as a result of the presence of the CART cells of the
invention in the patient, which allows the specialized treatment of
the patient with cytokine inhibitors to decrease the elevated
levels of the cytokine. In another embodiment, invention may be
applied to identify cytokine levels decreased as a result of the
presence of the CART cells of the invention in the patient, which
allows the specialized treatment of the patient with cytokine
activators to increase the diminished levels of the cytokine.
[0098] In one embodiment, cytokines levels that are elevated as a
result of receiving a CART cell infusion include but are not
limited to IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12,
IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha., IFN-.gamma.,
MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF, G-CSF, CXCL9,
CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-.beta.,
CD40, CD40L, ferritin, and the like. However, the invention should
not be limited to these listed cytokines. Rather, the invention
includes any cytokine identified to be elevated in a patient as a
result of receiving a CAR T cell infusion.
[0099] In one embodiment, cytokines levels that are decreased as a
result of receiving a CART cell infusion include but are not
limited to IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12,
IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN-.alpha., IFN-.gamma.,
MIP-1.alpha., MIP-1.beta., MCP-1, TNF.alpha., GM-CSF, G-CSF, CXCL9,
CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-.beta.,
CD40, CD40L, ferritin, and the like. However, the invention should
not be limited to these listed cytokines. Rather, the invention
includes any cytokine identified to be decreased in a patient as a
result of receiving a CAR T cell infusion.
Detecting a Cytokine and Treatment Thereof
[0100] Although this section describes detection of a cytokine and
treatment thereof as part of the second-line therapy, the invention
encompasses detection of any soluble factor and treatment thereof
as part of the second-line therapy. Therefore, the description in
the context of a "cytokine" can equally be applied to a "soluble
factor."
[0101] In one embodiment, as part of the second-line therapy, the
invention includes methods of detecting levels of a cytokine in a
patient that has received infusion of a CART cell of the invention.
In some embodiments, the presence or level of a cytokine can be
used to select a candidate treatment. In some other embodiments,
the presence or levels of the cytokine can be used to determine the
success during the course of or after treatment of the first-line,
second-line, or both the first and second-line of therapy.
[0102] Biological samples in which the cytokine can be detected
include, for example, serum. In some embodiments, biological
samples include a tissue biopsy which may or may not have a liquid
component.
[0103] Immunoassays can be used to qualitatively or quantitatively
analyze the cytokine levels in a biological sample. A general
overview of the applicable technology can be found in a number of
readily available manuals, e.g., Harlow & Lane, Cold Spring
Harbor Laboratory Press, Using Antibodies: A Laboratory Manual
(1999).
[0104] In addition to using immunoassays to detect the levels of
cytokines in a biological sample from a patient, assessment of
cytokine expression and levels can be made based on the level of
gene expression of the particular cytokines. RNA hybridization
techniques for determining the presence and/or level of mRNA
expression are well known to those of skill in the art and can be
used to assess the presence or level of gene expression of the
cytokine of interest.
[0105] In some embodiments, the methods of the present invention
utilize selective binding partners of the cytokine to identify the
presence or determine the levels of the cytokine in a biological
sample. The selective binding partner to be used with the methods
and kits of the present invention can be, for instance, an
antibody. In some aspects, monoclonal antibodies to the particular
cytokine can be used. In some other aspects, polyclonal antibodies
to the particular cytokine can be employed to practice the methods
and in the kits of the present invention.
[0106] Commercial antibodies to the cytokine are available and can
be used with the methods and kits of the present invention. It is
well known to those of skill in the art that the type, source and
other aspects of an antibody to be used is a consideration to be
made in light of the assay in which the antibody is used. In some
instances, antibodies that will recognize its antigen target on a
Western blot might not applicable to an ELISA or ELISpot assay and
vice versa.
[0107] In some embodiments, the antibodies to be used for the
assays of the present invention can be produced using techniques
for producing monoclonal or polyclonal antibodies that are well
known in the art (see, e.g., Coligan, Current Protocols in
Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal
Antibodies: Principles and Practice (2d ed. 1986); and Kohler &
Milstein, Nature 256:495-497 (1975). Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors, as well as
preparation of polyclonal and monoclonal antibodies by immunizing
rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281
(1989); Ward et al., Nature 341:544-546 (1989)). Such antibodies
can be used for therapeutic and diagnostic applications, e.g., in
the treatment and/or detection of any of the specific
cytokine-associated diseases or conditions described herein.
[0108] Detection methods employing immunoassays are particularly
suitable for practice at the point of patient care. Such methods
allow for immediate diagnosis and/or prognostic evaluation of the
patient. Point of care diagnostic systems are described, e.g., in
U.S. Pat. No. 6,267,722 which is incorporated herein by reference.
Other immunoassay formats are also available such that an
evaluation of the biological sample can be performed without having
to send the sample to a laboratory for evaluation. Typically these
assays are formatted as solid assays where a reagent, e.g., an
antibody is used to detect the cytokine. Exemplary test devices
suitable for use with immunoassays such as assays of the present
invention are described, for example, in U.S. Pat. Nos. 7,189,522;
6,818,455 and 6,656,745.
[0109] In some aspects, the present invention provides methods for
detection of polynucleotide sequences which code for the cytokine
in a biological sample. As noted above, a "biological sample"
refers to a cell or population of cells or a quantity of tissue or
fluid from a patient. Most often, the sample has been removed from
a patient, but the term "biological sample" can also refer to cells
or tissue analyzed in vivo, i.e., without removal from the patient.
Typically, a "biological sample" will contain cells from the
patient, but the term can also refer to noncellular biological
material.
[0110] In one embodiment, amplification-based assays are used to
measure the level of a desired cytokine. In such an assay, nucleic
acid sequences of the desired cytokine act as a template in an
amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
In a quantitative amplification, the amount of amplification
product will be proportional to the amount of template in the
original sample. Comparison to appropriate controls provides a
measure of the copy number of the cytokine associated gene. Methods
of quantitative amplification are well known to those of skill in
the art. Detailed protocols for quantitative PCR are provided,
e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y.). RT-PCR methods are well
known to those of skill (see, e.g., Ausubel et al., supra). In some
embodiments, quantitative RT-PCR, e.g., a TaqMan.TM. assay, is
used, thereby allowing the comparison of the level of mRNA in a
sample with a control sample or value. The known nucleic acid
sequences for a desired cytokine are sufficient to enable one of
skill to routinely select primers to amplify any portion of the
gene. Suitable primers for amplification of specific sequences can
be designed using principles well known in the art (see, e.g.,
Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual
(1995)).
[0111] In some embodiments, hybridization based assays can be used
to detect the amount of a desired cytokine in the cells of a
biological sample. Such assays include dot blot analysis of RNA as
well as other assays, e.g., fluorescent in situ hybridization,
which is performed on samples that comprise cells. Other
hybridization assays are readily available in the art.
[0112] In numerous embodiments of the present invention, the level
and/or presence of a cytokine polynucleotide or polypeptide will be
detected in a biological sample, thereby detecting the differential
expression of the cytokine to generate a cytokine profile from a
biological sample derived from a patient infused with a CAR T cell
of the invention compared to the control biological sample.
[0113] The amount of a cytokine polynucleotide or polypeptide
detected in the biological sample indicates the presence of a
cytokine to generate a cytokine profile for the purpose of
classifying the patient for the appropriate cytokine treatment. For
example, when the cytokine profile indicates an increase in a
particular cytokine post T cell infusion compared to control (e.g.,
pre T cell infusion), a skilled artisan can elect to administer to
the patient in need of such management an effective amount of a
cytokine inhibitory compound. Alternatively, when the cytokine
profile indicates a decrease in a particular cytokine post T cell
infusion compared to control (e.g., pre T cell infusion), a skilled
artisan can elect to administer to the patient in need of such
management an effective amount of a cytokine activator
compound.
[0114] In some embodiments, the difference in cytokine levels
between the post T cell infusion sample and the control sample and
be at least about 0.5, 1.0, 1.5, 2, 5, 10, 100, 200, 500, 1000
fold.
[0115] The present methods can also be used to assess the efficacy
of a course of treatment. For example, in a post T cell infusion
patient containing an elevated amount of a cytokine IL-6, the
efficacy of an anti-IL-6 treatment can be assessed by monitoring,
over time, IL-6. For example, a reduction in IL-6 polynucleotide or
polypeptide levels in a biological sample taken from a patient
following a treatment, compared to a level in a sample taken from
the mammal before, or earlier in, the treatment, indicates
efficacious treatment.
[0116] In one embodiment, a treatment regimen can be based on
neutralizing the elevated cytokine. For example, antagonists of a
cytokine can be selected for treatment. Antibodies are an example
of a suitable antagonist and include mouse antibodies, chimeric
antibodies, humanized antibodies, and human antibodies or fragments
thereof. Chimeric antibodies are antibodies whose light and heavy
chain genes have been constructed, typically by genetic
engineering, from immunoglobulin gene segments belonging to
different species (see, e.g., Boyce et al., Annals of Oncology
14:520-535 (2003)). For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments. A typical chimeric antibody is thus a hybrid
protein consisting of the V or antigen-binding domain from a mouse
antibody and the C or effector regions from a human antibody.
[0117] Humanized antibodies have variable region framework residues
substantially from a human antibody (termed an acceptor antibody)
and complementarity determining regions substantially from a
mouse-antibody, (referred to as the donor immunoglobulin). See
Queen et al., Proc. NatL. Acad. Sci. USA 86:10029-10033 (1989) and
WO 90/07861, U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,693,761, U.S.
Pat. No. 5,585,089, U.S. Pat. No. 5,530,101 and Winter, U.S. Pat.
No. 5,225,539. The constant region(s), if present, are also
substantially or entirely from a human immunoglobulin. Antibodies
can be obtained by conventional hybridoma approaches, phage display
(see, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO
92/01047), use of transgenic mice with human immune systems
(Lonberg et al., WO93/12227 (1993)), among other sources. Nucleic
acids encoding immunoglobulin chains can be obtained from
hybridomas or cell lines producing antibodies, or based on
immunoglobulin nucleic acid or amino acid sequences in the
published literature.
[0118] Other antagonists of a desired cytokine can also be used for
treatment purposes. For example, a class of antagonists that can be
used for the purposes of the present invention, are the soluble
forms of the receptors for the cytokine. By way of merely
illustrative purposes, an IL-6 antagonist is an anti-IL-6 antibody
that specifically binds to IL-6. A specific antibody has the
ability to inhibit or antagonize the action of IL-6 systemically.
In some embodiments, the antibody binds IL-6 and prevents it from
interacting with or activating its receptors (e.g. IL-6Ra or
IL-6R.beta.). In some embodiments, the activity of IL-6 can be
antagonized by using an antagonist to the interleukin-6 receptors
(IL-6R). U.S. Application number 2006251653 describes methods for
treating interleukin-6 related disease and discloses a number of
interleukin-6 antagonists including, for example, humanized
anti-IL-6R antibodies and chimeric anti-IL-6R antibodies. In some
embodiments, an IL-6 or IL-6R derivative can be used to block and
antagonize the interaction between IL-6/IL-6R.
[0119] The invention is not limited to the cytokines and their
corresponding activators and inhibitors described herein. Rather,
the invention includes the used of any cytokine activator and/or
inhibitor that is used in the art to modulate the cytokine. This is
because the invention is based on managing cancer treatment in a
patient receiving infusion of CAR T cells of the invention wherein
the infused CAR T cells result in increase and decrease levels of
various cytokines. One skilled in the art based on the disclosure
presented herein that differential expression levels of a cytokine
in a post T cell infusion sample compared to a control sample can
be targeted for treatment for have the cytokine level be increased
or decreased to normal levels.
Therapeutic Application
[0120] The present invention encompasses a cell (e.g., T cell)
transduced with a lentiviral vector (LV). For example, the LV
encodes a CAR that combines an antigen recognition domain of a
specific antibody with an intracellular domain of CD3-zeta, CD28,
4-1BB, or any combinations thereof. Therefore, in some instances,
the transduced T cell can elicit a CAR-mediated T-cell
response.
[0121] The invention provides the use of a CAR to redirect the
specificity of a primary T cell to a tumor antigen. Thus, the
present invention also provides a method for stimulating a T
cell-mediated immune response to a target cell population or tissue
in a mammal comprising the step of administering to the mammal a T
cell that expresses a CAR, wherein the CAR comprises a binding
moiety that specifically interacts with a predetermined target, a
zeta chain portion comprising for example the intracellular domain
of human CD3zeta, and a costimulatory signaling region.
[0122] In one embodiment, the present invention includes a type of
cellular therapy where T cells are genetically modified to express
a CAR and the CAR T cell is infused to a recipient in need thereof.
The infused cell is able to kill tumor cells in the recipient.
Unlike antibody therapies, CAR T cells are able to replicate in
vivo resulting in long-term persistence that can lead to sustained
tumor control.
[0123] In one embodiment, the CAR T cells of the invention can
undergo robust in vivo T cell expansion and can persist for an
extended amount of time. In another embodiment, the CAR T cells of
the invention evolve into specific memory T cells that can be
reactivated to inhibit any additional tumor formation or growth.
For example, it was unexpected that the CART 19 cells of the
invention can undergo robust in vivo T cell expansion and persist
at high levels for an extended amount of time in blood and bone
marrow and form specific memory T cells. Without wishing to be
bound by any particular theory, CAR T cells may differentiate in
vivo into a central memory-like state upon encounter and subsequent
elimination of target cells expressing the surrogate antigen.
[0124] Without wishing to be bound by any particular theory, the
anti-tumor immunity response elicited by the CAR-modified T cells
may be an active or a passive immune response. In addition, the CAR
mediated immune response may be part of an adoptive immunotherapy
approach in which CAR-modified T cells induce an immune response
specific to the antigen binding moiety in the CAR. For example, a
CART19 cells elicits an immune response specific against cells
expressing CD19.
[0125] While the data disclosed herein specifically disclose
lentiviral vector comprising anti-CD19 scFv derived from FMC63
murine monoclonal antibody, human CD8.alpha. hinge and
transmembrane domain, and human 4-1BB and CD3zeta signaling
domains, the invention should be construed to include any number of
variations for each of the components of the construct as described
elsewhere herein. That is, the invention includes the use of any
antigen binding moiety in the CAR to generate a CAR-mediated T-cell
response specific to the antigen binding moiety. For example, the
antigen binding moiety in the CAR of the invention can target a
tumor antigen for the purposes of treat cancer.
[0126] Cancers that may be treated include tumors that are not
vascularized, or not yet substantially vascularized, as well as
vascularized tumors. The cancers may comprise non-solid tumors
(such as hematological tumors, for example, leukemias and
lymphomas) or may comprise solid tumors. Types of cancers to be
treated with the CARs of the invention include, but are not limited
to, carcinoma, blastoma, and sarcoma, and certain leukemia or
lymphoid malignancies, benign and malignant tumors, and
malignancies e.g., sarcomas, carcinomas, and melanomas. Adult
tumors/cancers and pediatric tumors/cancers are also included.
[0127] Hematologic cancers are cancers of the blood or bone marrow.
Examples of hematological (or hematogenous) cancers include
leukemias, including acute leukemias (such as acute lymphocytic
leukemia, acute myelocytic leukemia, acute myelogenous leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (such as chronic myelocytic
(granulocytic) leukemia, chronic myelogenous leukemia, and chronic
lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's
disease, non-Hodgkin's lymphoma (indolent and high grade forms),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, myelodysplastic syndrome, hairy cell leukemia and
myelodysplasia.
[0128] Solid tumors are abnormal masses of tissue that usually do
not contain cysts or liquid areas. Solid tumors can be benign or
malignant. Different types of solid tumors are named for the type
of cells that form them (such as sarcomas, carcinomas, and
lymphomas). Examples of solid tumors, such as sarcomas and
carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, lymphoid malignancy, pancreatic cancer, breast
cancer, lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma,
melanoma, and CNS tumors (such as a glioma (such as brainstem
glioma and mixed gliomas), glioblastoma (also known as glioblastoma
multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma,
Schwannoma craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
neuroblastoma, retinoblastoma and brain metastases).
[0129] In one embodiment, the antigen bind moiety portion of the
CAR of the invention is designed to treat a particular cancer. For
example, the CAR designed to target CD19 can be used to treat
cancers and disorders including but are not limited to pre-B ALL
(pediatric indication), adult ALL, mantle cell lymphoma, diffuse
large B-cell lymphoma, salvage post allogenic bone marrow
transplantation, and the like.
[0130] In another embodiment, the CAR can be designed to target
CD22 to treat diffuse large B-cell lymphoma.
[0131] In one embodiment, cancers and disorders include but are not
limited to pre-B ALL (pediatric indication), adult ALL, mantle cell
lymphoma, diffuse large B-cell lymphoma, salvage post allogenic
bone marrow transplantation, and the like can be treated using a
combination of CARs that target CD19, CD20, CD22, and ROR1.
[0132] In one embodiment, the CAR can be designed to target
mesothelin to treat mesothelioma, pancreatic cancer, ovarian
cancer, and the like.
[0133] In one embodiment, the CAR can be designed to target
CD33/IL3Ra to treat acute myelogenous leukemia and the like.
[0134] In one embodiment, the CAR can be designed to target c-Met
to treat triple negative breast cancer, non-small cell lung cancer,
and the like.
[0135] In one embodiment, the CAR can be designed to target PSMA to
treat prostate cancer and the like.
[0136] In one embodiment, the CAR can be designed to target
Glycolipid F77 to treat prostate cancer and the like.
[0137] In one embodiment, the CAR can be designed to target
EGFRvIII to treat gliobastoma and the like.
[0138] In one embodiment, the CAR can be designed to target GD-2 to
treat neuroblastoma, melanoma, and the like.
[0139] In one embodiment, the CAR can be designed to target
NY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, and the like.
[0140] In one embodiment, the CAR can be designed to target MAGE A3
TCR to treat myeloma, sarcoma, melanoma, and the like.
[0141] However, the invention should not be construed to be limited
to solely to the antigen targets and diseases disclosed herein.
Rather, the invention should be construed to include any antigenic
target that is associated with a disease where a CAR can be used to
treat the disease.
[0142] The CAR-modified T cells of the invention may also serve as
a type of vaccine for ex vivo immunization and/or in vivo therapy
in a mammal Preferably, the mammal is a human.
[0143] With respect to ex vivo immunization, at least one of the
following occurs in vitro prior to administering the cell into a
mammal: i) expansion of the cells, ii) introducing a nucleic acid
encoding a CAR to the cells, and/or iii) cryopreservation of the
cells.
[0144] Ex vivo procedures are well known in the art and are
discussed more fully below. Briefly, cells are isolated from a
mammal (preferably a human) and genetically modified (i.e.,
transduced or transfected in vitro) with a vector expressing a CAR
disclosed herein. The CAR-modified cell can be administered to a
mammalian recipient to provide a therapeutic benefit. The mammalian
recipient may be a human and the CAR-modified cell can be
autologous with respect to the recipient. Alternatively, the cells
can be allogeneic, syngeneic or xenogeneic with respect to the
recipient.
[0145] The procedure for ex vivo expansion of hematopoietic stem
and progenitor cells is described in U.S. Pat. No. 5,199,942,
incorporated herein by reference, can be applied to the cells of
the present invention. Other suitable methods are known in the art,
therefore the present invention is not limited to any particular
method of ex vivo expansion of the cells. Briefly, ex vivo culture
and expansion of T cells comprises: (1) collecting CD34+
hematopoietic stem and progenitor cells from a mammal from
peripheral blood harvest or bone marrow explants; and (2) expanding
such cells ex vivo. In addition to the cellular growth factors
described in U.S. Pat. No. 5,199,942, other factors such as flt3-L,
IL-1, IL-3 and c-kit ligand, can be used for culturing and
expansion of the cells.
[0146] In addition to using a cell-based vaccine in terms of ex
vivo immunization, the present invention also provides compositions
and methods for in vivo immunization to elicit an immune response
directed against an antigen in a patient.
[0147] Generally, the cells activated and expanded as described
herein may be utilized in the treatment and prevention of diseases
that arise in individuals who are immunocompromised. In particular,
the CAR-modified T cells of the invention are used in the treatment
of CCL. In certain embodiments, the cells of the invention are used
in the treatment of patients at risk for developing CCL. Thus, the
present invention provides methods for the treatment or prevention
of CCL comprising administering to a subject in need thereof, a
therapeutically effective amount of the CAR-modified T cells of the
invention.
[0148] The CAR-modified T cells of the present invention may be
administered either alone, or as a pharmaceutical composition in
combination with diluents and/or with other components such as IL-2
or other cytokines or cell populations. Briefly, pharmaceutical
compositions of the present invention may comprise a target cell
population as described herein, in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers such as
neutral buffered saline, phosphate buffered saline and the like;
carbohydrates such as glucose, mannose, sucrose or dextrans,
mannitol; proteins; polypeptides or amino acids such as glycine;
antioxidants; chelating agents such as EDTA or glutathione;
adjuvants (e.g., aluminum hydroxide); and preservatives.
Compositions of the present invention are preferably formulated for
intravenous administration.
[0149] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease, although
appropriate dosages may be determined by clinical trials.
[0150] When "an immunologically effective amount", "an anti-tumor
effective amount", "an tumor-inhibiting effective amount", or
"therapeutic amount" is indicated, the precise amount of the
compositions of the present invention to be administered can be
determined by a physician with consideration of individual
differences in age, weight, tumor size, extent of infection or
metastasis, and condition of the patient (subject). It can
generally be stated that a pharmaceutical composition comprising
the T cells described herein may be administered at a dosage of
10.sup.4 to 10.sup.9 cells/kg body weight, preferably 10.sup.5 to
10.sup.6 cells/kg body weight, including all integer values within
those ranges. T cell compositions may also be administered multiple
times at these dosages. The cells can be administered by using
infusion techniques that are commonly known in immunotherapy (see,
e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The
optimal dosage and treatment regime for a particular patient can
readily be determined by one skilled in the art of medicine by
monitoring the patient for signs of disease and adjusting the
treatment accordingly.
[0151] In certain embodiments, it may be desired to administer
activated T cells to a subject and then subsequently redraw blood
(or have an apheresis performed), activate T cells therefrom
according to the present invention, and reinfuse the patient with
these activated and expanded T cells. This process can be carried
out multiple times every few weeks. In certain embodiments, T cells
can be activated from blood draws of from 10 cc to 400 cc. In
certain embodiments, T cells are activated from blood draws of 20
cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not
to be bound by theory, using this multiple blood draw/multiple
reinfusion protocol may serve to select out certain populations of
T cells.
[0152] The administration of the subject compositions may be
carried out in any convenient manner, including by aerosol
inhalation, injection, ingestion, transfusion, implantation or
transplantation. The compositions described herein may be
administered to a patient subcutaneously, intradermally,
intratumorally, intranodally, intramedullary, intramuscularly, by
intravenous (i.v.) injection, or intraperitoneally. In one
embodiment, the T cell compositions of the present invention are
administered to a patient by intradermal or subcutaneous injection.
In another embodiment, the T cell compositions of the present
invention are preferably administered by i.v. injection. The
compositions of T cells may be injected directly into a tumor,
lymph node, or site of infection.
[0153] In certain embodiments of the present invention, cells
activated and expanded using the methods described herein, or other
methods known in the art where T cells are expanded to therapeutic
levels, are administered to a patient in conjunction with (e.g.,
before, simultaneously or following) any number of relevant
treatment modalities, including but not limited to treatment with
agents such as antiviral therapy, cidofovir and interleukin-2,
Cytarabine (also known as ARA-C) or natalizumab treatment for MS
patients or efalizumab treatment for psoriasis patients or other
treatments for PML patients. In further embodiments, the T cells of
the invention may be used in combination with chemotherapy,
radiation, immunosuppressive agents, such as cyclosporin,
azathioprine, methotrexate, mycophenolate, and FK506, antibodies,
or other immunoablative agents such as CAM PATH, anti-CD3
antibodies or other antibody therapies, cytoxin, fludaribine,
cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,
FR901228, cytokines, and irradiation. These drugs inhibit either
the calcium dependent phosphatase calcineurin (cyclosporine and
FK506) or inhibit the p70S6 kinase that is important for growth
factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815,
1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al.,
Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the
cell compositions of the present invention are administered to a
patient in conjunction with (e.g., before, simultaneously or
following) bone marrow transplantation, T cell ablative therapy
using either chemotherapy agents such as, fludarabine,
external-beam radiation therapy (XRT), cyclophosphamide, or
antibodies such as OKT3 or CAMPATH. In another embodiment, the cell
compositions of the present invention are administered following
B-cell ablative therapy such as agents that react with CD20, e.g.,
Rituxan. For example, in one embodiment, subjects may undergo
standard treatment with high dose chemotherapy followed by
peripheral blood stem cell transplantation. In certain embodiments,
following the transplant, subjects receive an infusion of the
expanded immune cells of the present invention. In an additional
embodiment, expanded cells are administered before or following
surgery.
[0154] The dosage of the above treatments to be administered to a
patient will vary with the precise nature of the condition being
treated and the recipient of the treatment. The scaling of dosages
for human administration can be performed according to art-accepted
practices. The dose for CAMPATH, for example, will generally be in
the range 1 to about 100 mg for an adult patient, usually
administered daily for a period between 1 and 30 days. The
preferred daily dose is 1 to 10 mg per day although in some
instances larger doses of up to 40 mg per day may be used
(described in U.S. Pat. No. 6,120,766).
Treatment of Cytokine Release Syndrome (CRS)
[0155] The invention is based partly on the discovery that in vivo
proliferation of CART19 cells and the potent anti-tumor activity
associated therewith is also associated with CRS, leading to
hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage
Activation Syndrome (MAS). Without wishing to be bound by any
particular theory, it is believed that that MAS/HLH is a unique
biomarker that is associated with and may be required for CART19
potent anti-tumor activity.
[0156] Accordingly, the invention provides a first-line of therapy
comprising administering the CAR of the invention into the patient
and a second-line of therapy comprising administering a type of
therapy to manage the elevated levels of certain soluble factors
resulting from the first-line of therapy of using CAR T cells.
[0157] In one embodiment, the second-line of therapy comprises
compositions and methods for the treatment of CRS. Symptoms of CRS
include high fevers, nausea, transient hypotension, hypoxia, and
the like. The present invention is based on the observation that
CART19 cells induced elevated levels of soluble factors in the
patient including but is not limited to IFN-.gamma., TNF.alpha.,
IL-2 and IL-6. Therefore, the second-line of therapy comprises
compounds and methods for neutralizing the effects against the
elevated cytokines resulting from the administration of the CART19
cells. In one embodiment, the neutralizing agents are capable of
counteracting undesired concerted burst of cytokine
expression/activity and, thus, are useful for the prevention,
amelioration and treatment of CRS associated with CART19
therapy.
[0158] In one embodiment, the treatment of CRS is performed around
day 10-12 post-infusion of CART19 cells.
[0159] In one embodiment, the second-line of therapy comprises
administering a steroid to the patient. In another embodiment, the
second-line of therapy comprises administering one of more of a
steroid, an inhibitor of TNF.alpha., and an inhibitor of IL-6. An
example of a TNF.alpha. inhibitor is entanercept. An example of an
IL-6 inhibitor is Tocilizumab (toc).
Experimental Examples
[0160] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0161] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1
Cytokine Therapy in Combination with CAR T Cell Infusion
[0162] The results presented herein demonstrate that patients
following infusion of CART cells exhibit differential expression
levels of various cytokines. In some instances, the elevated levels
of some cytokines are a result of the toxicity of the infused CAR T
cells (FIG. 1). It was observed that tocilizumab (anti-IL6) can
ameliorate the toxicity of CARs and seemingly preserve antitumor
effects in 2 of 2 patients (FIG. 2). Without wishing to be bound by
any particular theory, it is believed that anakinra and other
reagents that block IL-1 may also be useful in this regard. The
data presented herein also demonstrates that IL-1 is elevated in
patients, and this may lead to the later rise in IL-6. Anakinra is
an IL-1Ra recombinant protein which binds to the IL1 receptors and
blocks both IL-1 alpha and beta signaling. Anakinra has a short 1/2
life. There is an advantage to use Anakinra to start treating
patients since both IL-1 alpha and beta would be blocked, and also
relieve the cytokine storm and keep the anti-tumor effect.
[0163] It was also observed that antibody interventions did not
impart CART19 cellular functionality as measured by Perforin and
IFN-.gamma. (FIG. 3).
Example 2
CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells Induce a
Cytokine Release Syndrome (CRS) and Induction of Treatable
Macrophage Activation Syndrome (MAS) that can be Managed by the
IL-6 Antagonist Tocilizumab (Toc)
[0164] Infusion of CART19 cells results in 100 to 100,000.times. in
vivo proliferation, tumor lysis syndrome followed by durable
antitumor activity, and prolonged persistence in patients with B
cell tumors. The results presented herein demonstrate that in vivo
proliferation of CART19 cells and potent anti-tumor activity
therefrom is associated with CRS, leading to hemophagocytic
lymphohistiocytosis (HLH), also termed MAS. Without wishing to be
bound by any particular theory, it is believed that MAS/HLH is a
unique biomarker that is associated with and may be required for
potent anti-tumor activity.
[0165] Autologous T cells were lentivirally transduced with a CAR
composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded
ex-vivo with anti-CD3/anti-CD28 beads, and then infused into ALL or
CLL patients with persistent disease after 2-8 prior treatments.
CART19 anti ALL activity was also modeled in a xenograft mouse
model with high level of human ALL/human T cell engraftment and
simultaneous detection of CAR T cells and ALL using 2-color
bioluminescent imaging.
[0166] The results presented herein provides updated results of 10
patients who received CART19 cells, including 9 patients with CLL
and 1 pediatric patient with relapsed refractory ALL. 6/9 evaluable
patient s had a complete recovery (CR) or partial recovery (PR),
including 4 sustained CRs. While there was no acute infusional
toxicity, all responding patients also developed CRS. All had high
fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded
peak blood expression of CART19 cells, and then increased in
intensity until the CART19 cell peak (D10-31 after infusion). The
ALL patient experienced the most significant toxicity, with grade 4
hypotension and respiratory failure. Steroid therapy on D6 resulted
in no improvement. On D9, noting high levels of TNF.alpha. and IL-6
(peak increases above baseline: IFN.gamma. at 6040x; IL-6 at 988x;
IL-2R at 56x, IL-2 at 163x and TNF.alpha. at 17x), TNF.alpha. and
IL-6 antagonists (entanercept and toc) were administered. This
resulted in resolution of fever and hypotension within 12 hr and a
rapid wean from ventilator support to room air. These interventions
had no apparent impact on CART19 cell expansion or efficacy: peak
of CART cells (2539 CAR+ cells/uL; 77% of CD3 cells by flow)
occurred on D11, and D23 bone marrow showed CR with negative
minimal residual disease (MRD), compared to her initial on-study
marrow which showed 65% blasts. Although she had no history of CNS
ALL, spinal fluid showed detectable CART19 cells (21 lymphs/mcL;
78% CAR+). At 4 mo post infusion, this patient remained in CR, with
17 CART19 cells/uL in the blood and 31% CAR+CD3 cells in the
marrow.
[0167] Clinical assessment of subsequent responding patients shows
all had evidence of MAS/HLH including dramatic elevations of
ferritin and histologic evidence of HLH. Peak ferritin levels range
from 44,000 to 605,000, preceding and continuing with peak T cell
proliferation. Other consistent findings include rapid onset
hepatosplenomegaly unrelated to disease and moderate DIC.
[0168] Subsequently, 3 CLL patients have also been treated with
toc, also with prompt and striking resolution of high fevers,
hypotension and hypoxia. One patient received toc on D10 and
achieved a CR accompanied by CART19 expansion. Another patient had
rapid resolution of CRS following toc administration on day 9 and
follow up for response is too short. A 3rd CLL patient received toc
on D3 for early fevers and had no CART-19 proliferation and no
response.
[0169] To model the timing of cytokine blockade, xenografts using
bioluminescent primary pediatric ALL were established and then
treated with extra cells from the clinical manufacture. The CART19
cells proliferated and resulted in prolonged survival. Cytokine
blockade prior to T cell infusion with toc and/or etanercept
abrogated disease control with less in vivo proliferation of
infused CART19 cells, confirming the result seen in the one patient
given early toc (D3).
[0170] CART19 T cells can produce massive in-vivo expansion,
long-term persistence, and anti-tumor efficacy, but can also induce
significant CRS with features suggestive of MAS/HLH that responds
rapidly to cytokine blockade. Given prior to initiation of
significant CART19 proliferation, blockade of TNF.alpha. and/or
IL-6 may interfere with proliferation and effector function, but if
given at a point where cell proliferation is underway, toc may
ameliorate the symptoms that have been observed that correlate with
robust clinical responses.
Example 3
Remission of all by Chimeric Antigen Receptor-Expressing T
Cells
[0171] The results presented herein demonstrate that CAR T cells
have clinical activity in acute lymphocytic leukemia (ALL).
Briefly, two pediatric patients with relapsed and refractory pre-B
cell ALL were treated with 10.sup.6 to 10.sup.7/kg T cell
transduced with anti-CD19 antibody and a T-cell signaling molecule
(CTL019 CAR T cells; also referred to as CART19). The CTL019 T
cells expanded more than 1000-fold in both patients, and trafficked
to bone marrow. In addition, the CAR T cells were able to cross the
blood brain barrier and persisted at high levels for at least 6
months, as measured in the cerebral spinal fluid. Eight severe
adverse events were noted. Both patients developed a cytokine
release syndrome (CRS) and B cell aplasia. In one child, the CRS
was severe and cytokine blockade with etanercept and tocilizumab
was effective in reversing the syndrome, and yet did not prevent
CAR T cell expansion and anti-leukemic efficacy. Complete remission
was observed in both patients, and is ongoing in one patient at 9
months after treatment. The other patient relapsed with blast cells
that no longer express CD19 approximately 2 months after
treatment.
[0172] The results presented herein demonstrate that CAR modified T
cells are capable of killing even aggressive treatment refractory
acute leukemia cells in vivo. The emergence of tumor cells that no
longer express the target indicates a need to target other
molecules in addition to CD19 in some patients with ALL.
[0173] The in vivo expansion and robust anti-leukemic effects of
CTL019 (CART19) cells in 3 patients with CLL as been reported
(Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011,
Science Translational Medicine 3:95ra73). CTL019 is a CAR that
includes a CD137 (4-1BB) signaling domain and is expressed using
lentiviral vector technology (Milone et al., 1009, Mol Ther
17:1453-64). The results presented herein demonstrate the use of
CTL019 in 2 pediatric patients with refractory and relapsed ALL.
Both patients had remission of leukemia, accompanied by robust
expansion of CTL019 in vivo with trafficking to marrow and the CNS.
The anti-leukemic effects were potent since one patient had
chemotherapy refractory disease precluding allogeneic donor stem
cell transplantation and the other patient relapsed after
allogeneic cord blood transplantation and was resistant to
blinatumomab (chimeric bispecific anti-CD3 and anti-CD19)
therapy.
[0174] The materials and methods employed in these experiments are
now described.
Materials and Methods
[0175] Cart 19
[0176] CTL019 (CART19) production has been previously reported
(Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011,
Science Translational Medicine 3:95ra73). CTL019 was detected and
quantified in patient specimens as previously reported (Porter et
al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011, Science
Translational Medicine 3:95ra73).
[0177] Sample Draws and Processing
[0178] Samples (peripheral blood, bone marrow) were collected in
lavender top (K2EDTA,) or red top (no additive) vacutainer tubes
(Becton Dickinson). Lavender top tubes were delivered to the
laboratory within 2 hours of draw, or shipped overnight at room
temperature in insulated containers essentially as described (Olson
et al., 2011, J Transl Med 9:26) prior to processing. Samples were
processed within 30 minutes of receipt according to established
laboratory SOP. Peripheral blood and marrow mononuclear cells were
purified, processed, and stored in liquid nitrogen as described
(Kalos et al., 2011, Science Translational Medicine 3:95ra73). Red
top tubes were processed within 2 hours of draw including
coagulation time; serum isolated by centrifugation, aliquoted in
single use 100 .mu.L aliquots and stored at -80.degree. C. CSF was
delivered to the laboratory within 30 minutes of aspiration and
cells in CSF were collected by centrifugation of CSF fluid and
processed for DNA and flow cytometry.
[0179] Q-PCR Analysis
[0180] Whole-blood or marrow samples were collected in lavender top
(K2EDTA) BD vacutainer tubes (Becton Dickinson). Genomic DNA was
isolated directly from whole-blood and Q-PCR analysis on genomic
DNA samples was performed in bulk using ABI Taqman technology and a
validated assay to detect the integrated CD19 CAR transgene
sequence as described (Kalos et al., 2011, Science Translational
Medicine 3:95ra73) using 200 ng genomic DNA per time-point for
peripheral blood and marrow samples, and 18-21.7 ng genomic DNA per
time-point for CSF samples. To determine copy number per unit DNA,
an 8-point standard curve was generated consisting of 5 to 10.sup.6
copies CTL019 lentivirus plasmid spiked into 100 ng non-transduced
control genomic DNA. Each data-point (sample, standard curve) was
evaluated in triplicate with a positive Ct value in 3/3 replicates
with % CV less than 0.95% for all quantifiable values. A parallel
amplification reaction to control for the quality of interrogated
DNA was performed using 20 ng input genomic DNA from peripheral
blood and marrow (2-4.3 ng for CSF samples), and a primer/probe
combination specific for non-transcribed genomic sequence upstream
of the CDKN1A gene as described (Kalos et al., 2011, Science
Translational Medicine 3:95ra73). These amplification reactions
generated a correction factor (CF) to correct for calculated versus
actual DNA input. Copies of transgene per microgram DNA were
calculated according to the formula: copies calculated from CTL019
standard curve per input DNA (ng).times.CF.times.1000 ng. Accuracy
of this assay was determined by the ability to quantify marking of
the infused cell product by Q-PCR. These blinded determinations
generated Q-PCR and flow marking values of 11.1% and 11.6%,
respectively, for the CHOP-100 and 20.0% and 14.4%, respectively,
marking for the CHOP-101 infusion products.
[0181] Soluble Factor Analysis
[0182] Whole blood was collected in red top (no additive) BD
vacutainer tubes (Becton Dickinson), processed to obtain serum
using established laboratory SOP, aliquoted for single use and
stored at -80.degree. C. Quantification of soluble cytokine factors
was performed using Luminex bead array technology and kits
purchased from Life technologies (Invitrogen). Assays were
performed as per the manufacturer protocol with a 9 point standard
curve generated using a 3-fold dilution series. The 2 external
standard points were evaluated in duplicate and the 5 internal
standards in singlicate; all samples were evaluated in duplicate at
1:2 dilution; calculated % CV for the duplicate measures were less
than 15%. Data were acquired on a FlexMAP-3D by percent and
analyzed using XPonent 4.0 software and 5-parameter logistic
regression analysis. Standard curve quantification ranges were
determined by the 80-120% (observed/expected value) range. Reported
values included those within the standard curve range and those
calculated by the logistic regression analysis.
[0183] Antibody Reagents
[0184] The following antibodies were used for these studies:
MDA-CAR (Jena and Cooper, 2013, L. Anti-idiotype antibody for CD19.
PlosONE 2013; in press), a murine antibody to CD19 CAR conjugated
to Alexa647. Antibodies for multi-parametric immunophenotyping: T
cell detection panels: anti-CD3-FITC, anti-CD8-PE,
anti-CD14-PE-Cy7, anti-CD16-PE-Cy7, anti-CD19-PE-Cy7
anti-CD16-PE-Cy7. B cell detection panels: anti-CD20-FITC,
anti-CD45-PE, anti-CD45-APC, anti-CD19-PE-Cy7, anti-CD19-PE,
anti-CD34-PCP-e710 and anti CD34-APC were procured from
e-Biosciences.
[0185] Multi-Parameter Flow Cytometry
[0186] Cells were evaluated by flow cytometry directly after
Ficoll-Paque processing, with the exception of the CHOP-101
baseline sample which was evaluated immediately after thaw of a
cryopreserved sample. Multi-parametric immunophenotyping for
peripheral blood and marrow samples was performed using
approximately 0.2-0.5.times.10.sup.6 total cells per condition
(depending on cell yield in samples), and for CSF samples using
trace amounts of cells collected following centrifugation of CSF
fluid, and using fluorescence minus one (FMO) stains as described
in the text. Cells were stained in 100 .mu.L PBS for 30 minutes on
ice using antibody and reagent concentrations recommended by the
manufacturer, washed, and resuspended in 0.5% paraformaldehyde and
acquired using an Accuri C6 cytometer equipped with a Blue (488)
and Red (633 nm) laser. Accuri files were exported in FCS file
format and analyzed using FlowJo software (Version 9.5.3,
Treestar). Compensation values were established using single
antibody stains and BD compensation beads (Becton Dickinson) and
were calculated by the software. The gating strategy for T cells
was as follows: Live cells (FSC/SSC)>dump channel
(CD14+CD16+CD19-PECy7) vs CD3+>CD3+. The general gating strategy
for B cells was as follows: Live cells (FSC/SSC)>SSC low
events>CD19+. More gating details for the CHOP-100 and CHOP-101
samples are described in the individual Figures.
[0187] Molecular MRD Analysis
[0188] Molecular MRD analysis was performed by Adaptive
Biotechnologies (Seattle, Wash.) and high-throughput
next-generation sequencing of the BCR IGH CDR3 region using the
Illumina HiSeq/MiSeq platform-based immunoSEQ assay (Larimore et
al., 2012, J Immunol 189:3221-30). For these analyses, 701-6,000 ng
(approximately 111,000-950,000 genome equivalents) of genomic DNA
isolated from whole blood or marrow samples obtained from patients
were subjected to combined multiplex PCR and sequencing followed by
algorithmic analyses to quantify individual IGH CDR3 sequences in
samples. Parallel amplifications and sequencing of the TCRB CDR3
region (Robins et al., 2009, Blood 114:4099-107) in each sample
were done to assess quality of DNA samples. For each patient, the
IGH CDR3 nucleotide sequences assayed from samples of different
time points were aligned using EMBL-EBI multiple sequence alignment
tool (Goujon et al., 2010, Nucleic Acids Res 38:W695-9; Sievers et
al., 2011, Mol Syst Biol 7:539). The dominant clone from the
earliest time-point sample was bioinformatically tracked across the
assayed IGH CDR3 sequences in the following time-point samples to
identify presence of sequences with 95% or greater pair-wise
sequence identity. The total sequencing reads for those sequences
similar to the dominant clone are reported for each time-point.
[0189] The results of the experiments are now described.
Case Reports
[0190] CHOP-100 was a 7 yo girl in her second recurrence of ALL.
She was diagnosed 2 years prior and achieved a minimal residual
disease (MRD) negative remission, relapsing 17 months after
diagnosis. She re-entered remission after reinduction chemotherapy
but recurred 4 months later, after which she did not respond to
furtheclofaribine/etoposide/cyclophosphamide. Her karyotype at
baseline was 48,XX,del(9)(p21.3),+11,del(14)(q2?q24),+16/46,XX[4].
Peripheral blood mononuclear cells (PBMC) were collected by
apheresis before the intensive chemotherapy, anticipating that
there may be insufficient circulating T cells available for cell
manufacturing after such intensive treatment. This patient was
infused with CTL019 cells that had been anti-CD3/CD28 expanded and
lentivirally transduced to express the anti-CD19 CAR in a total
dose of 3.8.times.10.sup.8 cells/kg (1.2.times.10.sup.7 CTL019
cells/kg) given over 3 consecutive days as previously described
(Porter et al., 2011, N Engl J Med 365:725-33; Kalos et al., 2011,
Science Translational Medicine 3:95ra73). She did not receive
lymphodepleting chemotherapy before her CTL019 infusions, with the
most recent cytotoxic therapy given 6 weeks before CTL019 infusion.
No immediate infusional toxicities were noted, but she was
hospitalized for low-grade fevers which progressed to high fevers
by day 4, and on day 5 the patient was transferred to the pediatric
ICU (CHOP-100, FIG. 4A). This was followed by rapid progression to
significant respiratory and cardiovascular compromise requiring
mechanical ventilation and blood pressure support.
[0191] The second ALL patient was a 10 yo girl (CHOP-101) who had
experienced her second relapse after a 4/6 matched unrelated
umbilical cord transplant 28 months after diagnosis and 10 months
before CTL019 infusion. She had experienced graft vs. host disease
(GVHD) after her transplant, which resolved with treatment; she was
off immunosuppression at the time of her relapse. She did not
subsequently re-enter remission in spite of multiple cytotoxic and
biologic therapies. Her baseline karyotype was 46 XX, del(1)(p13),
t(2;9)(q?21;q?21), t(3;17)(p24;q23), del(6)(q16q21),
del(9)(q13q22), der(16)t(1;?;16)(p13;?p13.3)[9],//46, Xy[1]. Before
PBMC collection, she was treated with two cycles of blinatumomab
(Bargou et al., 2008, Science 321:974-7) with no response. Her
peripheral blood cells were 68% donor origin at the time of PBMC
collection. CTL019 T cells were manufactured and infused as a total
dose of 10.sup.7 cells/kg (1.4.times.10.sup.6 CTL019 cells/kg) in a
single dose, after etoposide/cyclophosphamide chemotherapy given
for lymphodepletion the week before. Her bone marrow on the day
before CTL019 infusion was replaced by a population of
CD19+/CD34+ALL cells, with variable expression of CD19 by standard
clinical flow cytometry (FIG. 7). She had no immediate infusional
toxicities, but developed a fever on D+6 and was admitted to the
hospital. She experienced no cardiopulmonary toxicities, and did
not receive glucocorticoids or anti-cytokine therapy. CHOP-101
experienced fever of unknown origin, suspected to be due to
cytokine release (FIG. 4B), myalgias and two days of confusion
(grade 3), which spontaneously resolved. She had no evidence of
GVHD after the infusion of the CTL019 cells. Though these cells had
been collected from the patient, they were largely of donor (cord
blood) origin.
Induction of Remission in Both Subjects
[0192] Both subjects had an increase in circulating lymphocytes and
neutrophils in the 2 weeks following CTL019 infusion, as shown by
plots depicting total WBC, ALC, and ANC relative to timing of
CTL019 infusion (FIG. 4C). Most of the lymphocytes were comprised
of T cells that expressed the chimeric antigen receptor (FIG. 8),
shown in more detail in FIG. 5. In both subjects, high-grade
non-infectious fevers were documented, followed by elevations of
LDH (FIG. 4A). The elevations of LDH and high grade fevers were
similar to those previously described in CLL patients after CTL019
infusion (.Porter et al., 2011, N Engl J Med 365:725-33; Kalos et
al., 2011, Science Translational Medicine 3:95ra73). Approximately
one month after infusion, MRD negative (<0.01%) morphologic
remission of leukemia was achieved in both subjects (Table 1).
[0193] The clinical remission in CHOP-100 was associated with a
deep molecular remission that has persisted for at least 9 months
as of January 2013 (Table 1). High-throughput DNA sequencing of the
IGH locus revealed a pronounced decrease in total IGH reads at D+23
in the blood and marrow of CHOP-100. The malignant clone was not
detected in the blood or marrow in more than 1 million cell
equivalents that were sequenced at D+180. In contrast, T-cell
receptor sequences were readily detected in blood and marrow,
indicating the integrity of the DNA tested at all timepoints.
TABLE-US-00001 TABLE 1 Induction of molecular remission in blood
and bone marrow of CHOP-100 and 101 Number of Tumor input genomes
Total IGH clone Timepoint (cell Total TCR.beta. Total IGH unique
Dominant frequency Patient Tissue (day) equivalents) reads reads
reads clone reads (%) CHP959-100 Blood -1 111,340 525,717 189 6 185
97.88 28 218,210 1,651,129 0 0 0 0.00 87 288,152 1,416,378 0 0 0
0.00 180 420,571 1,276,098 6 2 0 0.00 Marrow -1 317,460 348,687
59,791 318 59,774 99.97 23 362,819 1,712,507 37 2 33 89.19 87
645,333 425,128 10 1 10 100.00 180 952,381 800,670 45 7 0 0.00
CHP959-101 Blood -1 152,584 1,873,116 38,170 52 30,425 79.71 23
417,371 1,462,911 92 5 18 19.60 Marrow -1 158,730 2,417,992 68,368
65 50,887 74.43 25 305,067 1,978,600 1,414 11 946 66.90 60 916,571
N/A 530,833 206 363,736 68.90 Molecular analysis of minimal
residual disease was performed on DNA isolated from whole blood or
marrow
Toxicity of CTL019
[0194] Grade 3 and 4 adverse events are summarized in Table 2.
Acute toxicity was observed in both patients that consisted of
fever, and a cytokine release syndrome (CRS) that evolved into a
macrophage activation syndrome (MAS). Both patients were monitored
and given prophylaxis for tumor lysis syndrome. Both experienced
substantial elevations of LDH, the causes of which were likely
multifactorial but could have included tumor lysis syndrome. Each
uric acid value in CHOP-100 was either below normal or in the
normal range, and she received allopurinol only on days 5-6.
CHOP-101 received prophylactic allopurinol on days 0-14 and had
abnormal uric acid values of 4.8-5.7 on days 8-10, consistent with
mild tumor lysis syndrome.
TABLE-US-00002 TABLE 2 Adverse events (grade 3 and 4) in CHOP-100
and CHOP-101 AE Category AE Toxicity AE Grade AE Description
Duration CHP953-100 INFECTION Febrile 3 Febrile neutropenia.
Temperature: Peak temperature 7 days neutropenia 40.7.degree. C.
resolved day 7 after administration of tocilizumab. CARDIAC
Hypotension 4 Shock requiring pressor support. Off all pressor 4
days at grade 4, off GENERAL support on day 7 other than weaning
dobutamine all pressors by day 12 VASCULAR Acute vascular 4
Life-threatening; pressor support or respiratory see above leak
syndrome support indicated PULMONARY/ Adult Respiratory 4 Present,
incubation indicated. Chest X-ray cleared on 12 days UPPER Distress
Syndrome day 2. RESPIRATORY (ARDS) CHP953-101 INFECTION Febrile 3
Febrile neutropenia. Peak temperature 40.3.degree. C. 6 days
neutropenia resolved day 6 NEUROLOGY Encephalopathy 3 Parents
reported confusion. MRI was normal. 3 days METABOLIC/ elevated AST
4 Peak AST value: 1068 (Grade 4) 1 day at grade 4 LABORATORY
METABOLIC/ elevated AST 4 Peak AST value: 748 (Grade 4) 1 day at
grade 4 LABORATORY Adverse events were graded according to Common
Terminology Criteria for Adverse Events 3.0
[0195] In CHOP-100, glucocorticoids were administered on D+5 with a
brief response in the fever curve but without remission of
hypotension. A single course of anti-cytokine therapy consisting of
etanercept and tocilizumab was given on D+8 and was followed by
rapid clinical effects: within hours she defervesced, was weaned
off vasoactive medications and ventilatory support as her clinical
and radiologic ARDS resolved. She did not have laboratory evidence
of a tumor lysis syndrome; however, biochemical evidence of MAS was
noted with elevation of ferritin to 45,529 ng/dl on D+11,
coagulopathy with elevated d-dimer and hypofibrinogenemia,
hepatosplenomegaly, elevation of transaminases, elevated LDH (FIG.
4C), and elevated triglycerides, as well as a cytokine profile
consistent with MAS. Her ferritin decreased to 2,368 by D+26 and
the clinical and laboratory abnormalities of MAS resolved.
[0196] In CHOP-101, although there was no direct evidence of a
tumor lysis syndrome other than fever and changes in LDH (FIG. 4C),
she also developed features of MAS with elevations in ferritin to
33,360 on D+7, peaking at 74,899 on day 11, transaminases that
reached grade 4 for 1 day, and an elevated d-dimer in serum. These
biochemical changes were reversible, as transaminases improved to
grade 1 and the ferritin decreased to 3,894 by D+21. She was
discharged from the hospital on day D+16.
[0197] Both subjects developed prominent elevations in a number of
cytokines and cytokine receptors in the serum (FIG. 1B). In both
patients, elevations in interferon-.gamma. and IL-6 were most
prominent. These observations are similar to the pattern observed
previously in patients with CLL who also experienced remission of
leukemia after CTL019 infusion (Kalos et al., 2011, Science
Translational Medicine 3:95ra73). The peak cytokine elevations were
temporally correlated with systemic inflammation as judged by
changes in core body temperature (FIG. 4C).
In Vivo Expansion of CTL019 in Patients with ALL
[0198] The fraction of CTL019 T cells in circulation progressively
increased in vivo to 72% (CHOP-100) and 34% (CHOP-101) of T cells
(FIG. 5A). The initial transduction efficiency was 11.6% and 14.4%
in the T cells infused in CHOP-100 and -101, respectively. Given
that the total ALC increased substantially in both patients (FIG.
4C), and that the frequency of CTL019 cells progressively increased
in vivo from the baseline frequency (FIG. 8), there was a robust
and selective expansion of CTL019 cells in both patients. The
selective increase in T cells expressing CTL019 in both patients is
consistent with an anti-leukemic mechanism involving CD19-driven
expansion, and with the subsequent elimination of cells that
express CD19 in both patients (FIG. 6 and FIG. 9).
[0199] Molecular deep sequence analysis of TCRs in the peripheral
blood and marrow samples in CHOP-100 obtained at D+23, when >65%
of CD3+ cells in peripheral blood and marrow were shown to be
CTL019+ by flow cytometry, revealed the absence of a dominant T
cell TCR clonotype in either compartment, with the 10 most abundant
T cells present at frequencies between 0.18-0.7% in bone marrow and
0.19 to 0.8% in peripheral blood. Six of the 10 dominant clones
were shared between the two compartments. In addition both CD4 and
CD8 CAR T cells are present. Thus, the CART cells appear to
proliferate after CD19-stimulated expansion, and not by TCR signals
or clone-specific events such as activation by integration of the
lentivirus.
Trafficking and Morphology of CTL019 CAR T Cells in Marrow and
CNS
[0200] CTL019 cells expanded more than 1000-fold in the peripheral
blood and bone marrow (FIG. 5). The frequency of CTL019 cells
increased to more than 10% of circulating T cells by D+20 in both
subjects (FIG. 8), with the absolute magnitude of CTL019 expansion
similar to that observed in patients with CLL (Kalos et al., 2011,
Science Translational Medicine 3:95ra73). Unexpectedly, cells in
the CSF also showed a high degree of CTL019 gene marking and also
persisted at high frequency out to 6 months (FIG. 5B). The
trafficking of CTL019 cells to the CSF was surprising given that
neither patient had detectable CNS leukemia by cytospin at the time
of infusion or at the 1 month post-treatment evaluation.
Furthermore, prior reports of CAR therapy for B cell malignancies
have not observed trafficking of CAR T cells to the CNS (Till et
al., 2008, Blood 112:2261-71; Brentjens et al., 2011, Blood
118:4817-28; Savoldo et al., 2011, J Clin Invest 121:1822-5; Jensen
et al., 2010, Biol Blood Marrow Transplant 16:1245-56; Till et al.,
2012, Blood 119:3940-50; Kochenderfer et al., 2012, Blood
119:2709-20). The morphology of the lymphocytes in blood and CSF is
shown for CHOP-100 and 101 in FIG. 5D. Since >70% of lymphocytes
in circulation on D+10 were CTL019 cells (FIGS. 5A and 5B), most of
the large granular lymphocytes shown in the left panel of FIG. 5D
are likely CTL019 cells. Similarly, since many lymphocytes in the
CSF obtained from CHOP-101 on D+23 were CTL019 cells (FIGS. 5B and
5C), the cytospin of CSF lymphocytes in FIG. 5D most likely
represents the morphology of CTL019 cells in vivo that have
trafficked to the CNS.
Induction of B Cell Aplasia
[0201] Both subjects had an elimination of CD19 positive cells in
bone marrow and blood within 1 month after CTL019 infusion (FIG. 6,
and FIG. 9). In CHOP-100, a large proportion of cells remaining in
the marrow at D+6 after infusion were CD19+CD20+ leukemic blast
cells. This population of cells was not detectable by D+23, an
effect that is maintained beyond 9 months in this patient (FIG.
9A). Given that CHOP-100 did not have chemotherapy in the 6 weeks
preceding CTL019 infusion, this indicates that CTL019 cells were
sufficient to ablate normal and leukemic B cells in this case.
Emergence of CD19 Escape Variant in CHOP-101
[0202] CHOP-101 experienced a clinical relapse apparent in the
peripheral blood at 2 months after CTL019 infusion, as evidenced by
the reappearance of blast cells in the circulation. These cells
were CD45dim positive, CD34 positive and did not express CD19 (FIG.
6). The absence of the original dominant CD34dim+CD34+CD19dim+
cells is consistent with a potent anti-leukemic selective pressure
of the CTL019 CART cells directed to CD19 (FIG. 9B). Deep IGH
sequencing revealed the presence of the malignant clone in
peripheral blood and marrow as early as D+23 (Table 1), despite a
clinical assessment of MRD negativity by flow cytometry at this
timepoint (FIG. 7). In addition, deep sequencing of material
obtained at clinical relapse revealed that the
CD45dimCD34+CD19-cells are clonally related to the initial dominant
CD45dim+CD34+CD19dim+ cells, since they share the same IGH
sequence.
Remission of ALL by Chimeric Antigen Receptor-Expressing T
Cells
[0203] The results presented herein demonstrate the induction of
remission of relapsed and refractory leukemia in the first two
patients treated on this protocol. Remission has been sustained in
one subject and was accompanied by relapse due to the emergence of
CD19 negative blasts in the other subject. Genetically modified
CTL019 cells trafficked to the CNS at high levels in both patients.
Cytokine elevations were observed that were on target, reversible,
and temporally accompanied by elimination of blast cells that
expressed CD19 in both subjects. The induction of complete
remission in refractory CD19 positive ALL following infusion of CAR
T cells is encouraging, particularly given the low frequency of
remissions following the infusion of allogeneic donor lymphocyte
infusions that do not express CARs (Kolb et al., 1995, Blood
86:2041-50; Collins et al., 1997, J Clin Oncol 15:433-44; Collins
et al., 2000, Bone Marrow Transplant 26(5):511-6). Deep sequencing
technology indicated that the CTL019 CAR infusion was associated
with a sustained 5-log reduction in the frequency of malignant B
cells in CHOP-100, further indicating potent antitumor effects in
chemotherapy-refractory leukemia.
[0204] The unfortunate emergence of CD19-negative blast cells in
one subject is consistent with previous reports that document the
existence of CD19-negative precursor cells in some cases of ALL
(Hotfilder et al., 2005, Cancer Research 65:1442-9; le Viseur et
al., 2008, Cancer Cell 14:47-58). It is possible that the
coinfusion of CAR T cells redirected to novel specificities in
addition to CD19 might decrease the likelihood of this event. Thus
far, relapse with CD19-negative escape cells in adults with CLL
after treatment with CTL019 cells have not been observed (Kalos et
al., 2011, Science Translational Medicine 3:95ra73), suggesting
that this issue may be specific for a subset of acute leukemias.
The induction of remission in CHOP-100 did not require concomitant
chemotherapy, and is consistent with a previous report showing that
remissions in CLL could be delayed for several weeks following
chemotherapy (Porter et al., 2011, N Engl J Med 365:725-33). Thus,
concomitant administration of cytotoxic chemotherapy may not be
necessary for CAR-mediated antitumor effects.
[0205] Both pediatric ALL patients experienced substantial toxicity
after CTL019 infusion. The induction of B-cell aplasia was
observed, and indicates that the CART cells can function in the
setting of relapsed acute leukemia. Both patients have also
developed clinical and laboratory evidence of cytokine release
syndrome and macrophage activation syndrome within a week of
infusion. The cytokine profile observed in these patients is
similar to prior reports of cytokine patterns in children with
hemaphagocytosis and macrophage activation syndrome or
hemophagocytic lymphohistiocytosis (Tang et al., 2008, Br J
Haematol 143:84-91; Behrens et al., 2011, J Clin Invest
121(6):2264-77). Macrophage activation syndrome is characterized by
hyperinflammation with prolonged fever, hepatosplenomegaly, and
cytopenias. Laboratory findings characteristic of this syndrome are
elevated ferritin, triglycerides, transaminases, bilirubin (mostly
conjugated) and soluble interleukin-2 receptor .alpha.-chain, and
decreased fibrinogen (Janka et al., 2012, Annu Rev Med 63:233-46).
Recent studies indicate that tocilizumab (anti-IL6) has promise for
glucocorticoid resistant GVHD (Drobyski et al., 2011, Biol Blood
Marrow Transplant 17(12):1862-8; Le Huu et al., 2012, J Invest
Dermatol 132(12):2752-61; Tawara et al., 2011, Clinical Cancer
Research 17:77-88), and the results presented herein are consistent
with these data.
[0206] The vigorous in vivo expansion of CTL019, persistent B-cell
aplasia and prominent anti-leukemia activity imply substantial and
sustained effector functions of the CTL019 cells in pediatric
patients with advanced ALL. The high efficiency of trafficking of
CAR T cells to the CNS is encouraging as a mechanism for
surveillance to prevent relapse in a sanctuary site such as the CNS
(Pullen et al., 1993, J Clin Oncol 11(5):839-49), and supports the
testing of CAR T-cell-directed therapies for CNS lymphomas and
primary CNS malignancies. With the exception of B-cell aplasia, the
duration of which is currently undefined, it is believed that the
use of immune-based therapies such as CTL019 may have a favorable
profile of long-term adverse effects compared to the high doses of
chemotherapy and radiation that are employed as the current
standard of care for most cases of pediatric leukemia
(Garcia-Manero and Thomas, 2001, Hematol Oncol Clin North Am
15(1):163-205).
Induction of Complete Remissions of ALL by Chimeric Antigen
Receptor-Expressing T Cells
[0207] Tocilizumab (anti-IL6) has promise for glucocorticoid
resistant GVHD, and the results presented herein are consistent
with these data. Further, it is interesting to note that in CHOP
100, the CRS manifesting as high fever, hypotension and multi-organ
failure was resistant to the high doses of glucocorticoids
administered over the previous 2 days before cytokine directed
therapy. Finally, in CHOP-100 the biphasic changes in IL-1.beta.,
IL-1RA and IL-2 shown in FIG. 4B may have been related to
cytokine-directed therapy with etanercept and tocilizumab.
[0208] The induction of remission in a patient refractory to
blinatumomab therapy further highlights the potency of CTL019
cells. The high efficiency of trafficking of CAR T cells to the CNS
is encouraging as a mechanism for surveillance to prevent relapse
in a sanctuary site such as the CNS, and supports the testing of
CAR T-cell-directed therapies for CNS lymphomas and primary CNS
malignancies. Neither patient has experienced cognitive effects
that might be ascribed to the trafficking of T cells to the
CNS.
Example 4
Summary Information
[0209] Various markers were measured in patients receiving CAR T
cells. As a non-limiting example, Ferritin, Myoglobin, and
plasminogen activator inhibitor-1 (PAI-1) were measure; see FIGS.
10, 11 and 12, respectively. Elevated levels of these markers
correlated with outcome. Patients designated as -01, -03, -09, -100
and -101 were classified as complete responders. Patients
designated as -02, -05, -10 (second infusion and response around
D70) and -12 were classified as partial responders. Patient
designated as -06, -07 and -14 were classified as
non-responders.
[0210] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
* * * * *