Download A Primer on Immuno-Oncology Part 2

A Primer on Immuno-Oncology Part 2 - Targeted
Redirection of T Cells
Soo Kim, Mark Osterman, Shailesh Maingi
Introduction
In addition to anticancer vaccines, cytokines, and
immune checkpoint inhibitors, bispecific T cell
engagers (BiTE) antibody (Ab) constructs, and
chimeric antigen receptor T cells (CAR-T cells)
represent alternative methods for harnessing the
immune system to fight cancer. Immune checkpoint
inhibitors, BiTEs, and CAR-T cells all utilize T cells
in their fight against cancer, but in differing ways.
Whereas immune checkpoint inhibitors amplify
antigen-specific T cell response, BiTEs provide assistance in increasing T cell activity, and CAR-T cell
therapy augments the capabilities of T cells. Both
BiTEs and CAR-T cells provide targeted redirection
of T cells against highly expressed tumor associated
antigens (TAA). BiTEs have enjoyed some clinical
success, with the recent approvals of catumaxomab
and blinatumomab. The field of CAR-T has also
grown significantly in recent years, there have been
over 800 publications on CAR-T cells within the past
five years and many clinical trials showing success,
particularly for B cell malignancies[5]. These therapies offer novel treatment options, but have yet to be
fully vetted for safety and efficacy. This report will
provide a brief overview of BiTEs, as well as a more
comprehensive review of CAR-T cell therapy.
This report is part two of two of Kineticos’ series of
research reports on cancer immunotherapies.
Bispecific T-Cell Engagers (BiTE)
Antibody Constructs
Bispecific T-cell engager antibody constructs (BiTEs)
are a type of bispecific Ab which are engineered
by combining two scFv domains of different Abs
onto one polypeptide chain, with one scFv directed
against the CD3 epitope of the T cell receptor (TCR)
complex expressed on T cells, and the other scFv directed against a tumor-associated antigen (TAA)[6].
By simultaneously binding to both a T cell and cancer cell, BiTEs form an “immunological synapse[7],”
which results in target cell dependent polyclonal
T cell activation and proliferation, followed by the
release of IL-2 and IFNγ to induce the proliferation
1
and expansion of T cells at the tumor site[8], as well
as the release of perforin and granzyme from the T
cell to ultimately cause the destruction of the target
cancer cell by apoptosis[5]. Perforin assembles into
multimeric pores in the tumor cell membrane, which
enables granzyme to enter into the target cancer cell.
The “immunological synapse” formed by BiTEs are
indistinguishable from those formed during the natural cytotoxic T cell recognition process[7]. CD8+
and CD4+ TEM cells are the primary T cell subsets
responsible for mediating the cytotoxic activity of
BiTEs due to their large number of perforin and
granzyme filled vesicles, as well as
“[CAR-T cell] therapies offer novel
treatment options, but have yet to be fully
vetted for safety and efficacy.”
their high capacity for proliferation[9]. It should be
noted that BiTE-induced T cell activation, cytokine
release, proliferation, and redirected lysis are target
antigen-dependent and do not rely on the specificity of the TCR; thus, BiTEs are able to engage any
T cell, regardless of the specificity of its TCR[9].
Furthermore, the size of the “synapse,” the distance
between the anti-CD3 and anti-TAA moieties of the
BiTE, determines the cytotoxicity of the BiTE as
the apoptotic proteins perforin and granzyme must
passively diffuse across the “synapse” from the T
cell into the tumor cell[10]. Decreasing the size of
the “synapse,” and thus the distance between the T
cell and the tumor cell, results in increased cytotoxic
activity. BiTEs are also able to induce T cells to
enter into a “serial killing” mode, in which one T cell
is able to sequentially induce apoptosis in multiple
target cancer cells: it has been observed that, over
a 9h period, one T cell was able to kill up to five
target cancer cells[11]. Thus the formation of BiTEinduced “immunological synapses” are significantly
more efficient than that of naturally occurring immunological synapses between TCRs and peptide MHC
complexes[9].
BiTEs may be able to circumvent certain common tu-
A Primer on Immuno-Oncology Part 2
mor escape mechanisms such as loss of MHC Class
I molecules or impaired antigen processing and/or
transport; however, research into methods to increase
the efficacy of BiTEs is ongoing and includes target
antigen loss, expression of immune checkpoints (e..g
PD1 or CTLA4), methods to increase T cell infiltration into the tumor, and the immunosuppressive
tumor microenvironment. A combinatorial approach
such as the use of BiTEs with immune checkpoint
inhibitors may synergize and increase the efficacy of
treatment[9]. BiTEs may have difficulty in reducing
the size of a large tumor mass; however, BiTE treatment may be applied in a minimal residual disease or
metastatic disease setting in order to kill the remaining population of cancer (stem) cells. In this setting,
a combinatorial approach such as the addition of
immune checkpoint inhibitors would be particularly
useful to overcome the immunosuppressive tumor
microenvironment[9].
Blinatumomab, an anti-CD19 BiTE, has demonstrated clinical efficacy in Philadelphia chromosomenegative relapsed or refractory acute lymphoblastic
leukemia, and received conditional approval by the
FDA in 2014. Other BiTEs being developed in clinical trials include MT112, MT111, MED-565, AMG330, AMG-110, AMG-211, and AMG-212.
BsAb
Blinatumomab
Sponsor
Amgen
NationalCancer
Institute
Chimeric Antigen Receptor T Cells
(CAR-T Cells)
CAR Structure
Another strategy for redirecting T cells against cancer cells is to engineer T cells to express a chimeric
antigen receptor (CAR). CARs are composed of: 1)
the scFv of a mAb fused to 2) a hinge/spacer region
fused to 3) a transmembrane (TM) domain and 4) an
intracellular signaling domain (ICD) which is able to
activate the T cell.
The scFv of a CAR directs CAR-T cells to tumor
cells expressing the target neoantigen. Whereas T
cell receptors traditionally recognize antigens on
MHC Class I molecules, a CAR is able to recognize
antigens in an MHC-independent manner[3]. In
addition, CARs are able to recognize a wide variety
of targets, including proteins, carbohydrates, gangliosides, proteoglycans, etc.[1]. The affinity of the scFv
for its target may affect CAR-T cell function; lowaffinity scFvs were shown to be specific for and supported killing of tumor cells expressing high levels
of antigen, but did not kill normal cells expressing
low or normal levels of antigen[13]. This suggests
that scFv affinity may be optimized based on antigen
expression on tumor vs. healthy tissues to increase
safety and targeting efficiency.
Targets
CD3×CD19
ClinicalTrialIdentifier
ApprovedinUSA
PhaseI,NCT00274742
PhaseII,NCT01207388
PhaseII,NCT01209286
PhaseI,NCT02568553
Diseases
ALL
RelapsedNHL
BcellALL
Relapsed/refractoryALL
NHL
PhaseII,NCT02143414
CD3×EpCAM
PhaseIII,NCT02003222
Completedphase
I,NCT00635596
AdultB-ALLwith
t(9;22)(q34;q11.2);BCR-ABL1;
untreatedadultALL
BCR-ABL-negativeBlineageALL
Solidtumors
Solitomab(MT110,
AMG110)
Amgen
AMG330
MT112
(BAY2010112)
MT111(MEDI-565)
Amgen
Bayer
CD33×CD3
PSMA×CD3
PhaseI,NCT02520427
PhaseI,NCT01723475
Relapsed/refractoryAML
Prostaticneoplasms
MedImmune
CEA×CD3
Completedphase
I,NCT01284231
PhaseI,NCT01723475
Completedphase
I,NCT01284231
Gastrointestinaladenocarcinomas
BAY2010112
MEDI-565
Bayer
MedImmune
CD3×PSMA
CEA×CD3
T able 1. BiTEs in Clinical Trials[12].
2
Prostaticneoplasms
Gastrointestinaladenocarcinomas
A Primer on Immuno-Oncology Part 2
The scFv of CARs are typically derived from murine
immunoglobulins; this may result in an immune response, and binding of human anti-mouse Abs to the
CAR may block antigen recognition by the CAR[1].
The spacer/hinge domain of a CAR joins the scFv to
the T cell membrane and is commonly derived from
IgG subclasses or CD8; IgG1 is most commonly
used, although not all CAR-T cells utilize a spacer/
hinge domain[1]. Membrane-proximal epitope
binding requires the use of a spacer/hinge domain;
however, they have been found to reduce CAR-T
cell function when the epitope is expressed near the
amino terminal portion of the target antigen[14]. The
spacer/hinge domain of a CAR affects CAR function; the distance between the CAR-T cell and the
cancer cell is determined by the position of the target
epitope and the length of the spacer region. This
distance affects tumor recognition and T cell cytokine production and proliferation, as well as synapse
formation between the CAR-T cell and the target
cancer cell[15].
The transmembrane (TM) domain affects CAR
expression on the cell surface, using a CD28 TM domain has been shown to result in high expression of
the CAR as compared to the CD3ζ TM domain[16].
There have been multiple iterations of improved
CAR design based on upgrading the intracellular
signaling domain (ICD). First generation CARs possessed only the ICD of the CD3ζ chain and lacked
in vivo efficacy. This led to the creation of second
and third generation CARs, which added on single
or multiple co-stimulatory molecules, respectively,
such as OX40, CD28ζ or 4-1BBζ to improve CAR
signaling, cytokine production, and T cell proliferation, differentiation, and survival[17]. Different costimulatory domains may be utilized for to achieve
different results; the use of CARs with CD28ζ
demonstrated increased IL-2 production and cytotoxicity, whereas CARS containing 4-1BBζ demonstrated improved persistence in vitro[18, 19]. A CAR
containing CD28 and OX40 demonstrated increased
survival of the CAR-T cells, as compared to a CART cell containing only CD28[20]. ICOS is a costimulatory receptor involved in TH17 polarization;
integration of ICOS into the ICD of a CAR resulted
in increased in vivo persistence of TH17-polarized
CAR-T cells, compared to CAR-T cells containing
CD28 or 4-1BB[21]. Duong et al. assessed the antitumor activity of various CAR-T cells with differing
3
costimulatory domains, and found that using DAP10
and CD27 with CD3ζ produced CAR-T cells with the
greatest antitumor activity[22].
Each component of the CAR plays a unique role
which affects the performance of the CAR and the
CAR-T cells Different indications may require their
own unique combination of scFv, hinge, transmembrane, ICD, and co-stimulatory domains to achieve
efficacy[23].
Figure 1. Evolution of CAR Signaling Capacities[4]. First generation CARs
transmitted activating signals only via ITAM-bearing signaling chains like
CD3ζ or FcεRIγ, licensing the engrafted T cells to eliminate tumor cells.
Second generation CARs contain an additional costimulatory domain (CM
I), predominantly the CD28 domain. Signaling through these costimulatory domain leads to enhanced proliferation, cytokine secretion, and renders
engrafted T cells resistant to immunosuppression and induction of AICD.
Recent developments fused the intracellular part of a second costimulatory
molecule (CM II) in addition to CD28 and ITAM-bearing signaling chains,
thus generating tripartite signaling CARs. T cells engrafted with third generation CARs seem to have superior qualities regarding effector functions and in
vivo persistence.
In addition to engineering the “right” CAR, efficient
delivery and persistent expression of the CAR is
crucial for achieving clinical efficacy. The T cell
population must be isolated, stimulated, expanded,
transduced with the CAR, and then reintroduced into
the patient, ideally following lymphodepletion preconditioning in order to maximize CAR T-cell expansion and engraftment[3].
CAR-T Cell Production
Methods for introducing the CAR transgene into
T cells include nonviral gene transfer for DNA
plasmids, in vitro transcribed mRNA species, or
viral-mediated transduction. Initially, nonviral DNA
transfection was used to produce CAR-T cells due to
its low immunogenicity and low risk of insertional
mutagenesis. However, cells produced using this
A Primer on Immuno-Oncology Part 2
method were short-lived[1]. Now, almost all studies of CAR-T cells have utilized retroviral vectors
such as gamma-retroviral and lentiviral vectors to
introduce the CAR gene into T cells[24]. Both of
these methods are able to efficiently and permanently
transduce T cells, and lentiviral vectors are also able
to transduce nondividing cells. T cells are usually
activated with either anti-CD28 Ab, Ab costimulation, or coculture with peripheral blood
for the treatment of acute lymphoblastic leukemia
(ALL), chronic lymphoblastic leukemia (CLL), multiple myeloma (MM), and lymphoma[1].
Trials using CAR-T cell therapy have had differing
outcomes which were affected by the CAR-T cell
strategy used, but the exact effect these differences
had remain unclear. Differences include vector design (e.g. the use of a CD28/CD3ζ signaling domain
vs. a 4-1BB/CD3ζ signaling domain), the manufacturing process, conditioning chemotherapy strategy,
the timing of CAR-T cell infusion, and CAR-T cell
dosage and derivation[26]. For example, a trial
targeting Her-2 failed due to “on-target, off-tumor”
“The most studied and successful target for
CAR-T cell therapy has been CD19. CD19
is an ideal target because it is expressed
by most B cell malignancies, but not in
normal tissues...”
Figure 2. Overview of CAR Adoptive Cell Transfer[3]. T cells are collected via apheresis, expanded
ex vivo and genetically modified to express a desired CAR construct before they are infused back into
the patient.
mononuclear cells (PBMCs) derived from antilogous patients or donors[1]. IL-2 is typically used
to induce T cell expansion ex vivo, however, other
cytokines such as IL-7, IL-15, or IL-21 may be used
to modify the T cells for the desired effector function.Research has shown that the addition of IL-7 and
IL-15 resulted in CAR-T cells with a more favorable
T memory stem cell phenotype[25]. After removing
and selection for a subpopulation of T cells from the
patient, expanding the cells, and transducing them
with a CAR gene, the cells are infused back into the
patient. The dosage of cells given to cancer patients
has typically been up to 10^11 T cells.
CAR-T Cell Targets
The most studied and successful target for CAR-T
cell therapy has been CD19. CD19 is an ideal target
because it is expressed by most B cell malignancies, but not in normal tissues and has been targeted
4
effects caused by low-level expression of Her-2 on
normal lung epithelium; in addition, the high dose
of CAR-T cells may have precluded a potential
therapeutic window[27]. Another trial targeting
Her-2 (for a different indication) used a different
scFv, administered a lower dose of CAR-T cells, and
did not co-administer IL-2 or use lymphodepleting
chemotherapy prior to transfusion. Patients in this
study did not experience any toxicity, and some patients exhibited stable disease or partial response[28].
These two trials show the importance of mAb selection, CAR generation, target distribution, and host
preconditioning[27].
“Armored” CAR-T Cells
CAR-T cells may lose their cytotoxic ability in the
immunosuppressive tumor microenvironment, much
like endogenous T cells. This has been shown in
vivo; injection of CAR-T cells into mice bearing
large solid tumors resulted in the upregulation of the
T cell inhibitory enzymes diacylglycerol
A Primer on Immuno-Oncology Part 2
kinase and SHP-1, as well as upregulation of immune
checkpoint receptors such as PD1, ultimately leading
to an inability to kill the tumor[29]. Modifying CART cells to express immune-modulatory proteins, such
as cytokines and ligands, represents one strategy to
overcome the immunosuppressive tumor microenvironment. One such method is to fuse the PD-1 extracellular receptor domain to the CD28 intracellular signaling domain. CAR-T cells may also be engineered
to constitutively express stimulatory ligands such as
CD40L or 4-1BBL. CD40L enhances the immunogenicity of CD40+ tumor cells, and stimulates dendritic
cells[30]; 4-1BBL binds to T cell co-stimulatory
receptors and stimulates the transduced cells, as well
as adjacent T cells[31]. One other method includes
engineering CAR-T cells to secrete pro-inflammatory
cytokines such as IL-2, IL-15, and IL-12[32]. Hoyos
et al. found that CAR-T cells expressing IL-15 and a
suicide gene had increased proliferation, reduced PD-1
expression, and resulted in increased antitumor activity in a mouse model of B-cell lymphoma[33]. While
systemic administration of IL-12 has been shown to be
toxic, local administration via CAR-T cells engineered
to secrete IL-12 may provide the beneficial effects
of IL-12 without systemic toxicity[32]. Localized
administration of IL-12 via CAR-T cells has been
shown to remove the need for preconditioning chemotherapy, enhance CAR-T cell persistence, provide
resistance to Treg cell and MDSC mediated inhibition,
and increase antitumor efficacy[34-36]. A clinical trial
(NCT02498912) using CAR-T cells which secrete
IL-12 for the treatment of ovarian cancer is currently
ongoing.
Key Companies and Collaborations in the CAR-T Cell Therapy
Space[37]
Table 2 shows a list of companies and assets in the
CAR-T cell space. Major players are discussed below.
Novartis
In 2015, Novartis and UPenn showed success in a trial
of CTL019, a CAR-T cell therapy targeting CD19, for
the treatment of acute lymphoblastic leukemia (ALL).
“...local administration (of IL-12) via CAR-T cells engineered to
secrete IL-12 may provide the beneficial effects of IL-12 without
systemic toxicity.”
5
They reported a 92% remission rate, with a six-month
duration of response of 76% and six-month event-free
survival rate of 70%. In another study for diffuse large
B-cell lymphoma (DLBCL) and follicular lymphoma,
they reported an overall response rate at 3 months of
47% for DLBCL and 73% for follicular lymphoma.
Novartis plans on filing a marketing application for
CTL019 for the treatment of pediatric relapsed/refractory ALL in 2017.
In early September 2016, Novartis closed down its
Cell and Gene Therapy Unit. However, Novartis
released a statement saying that they will continue to
develop their CAR-T cell therapies and plans to submit CTL019 for FDA approval to treat relapsed/refractory B-cell ALL early this year. In addition, Novartis
stated that they planned to submit KTE-C19 for EMA
approval in 2017 as well, having already received the
Agency’s PRIME (Priority Medicines) designation
which may speed up the time needed for review and
approval.
Juno Therapeutics
Juno, in partnership with Memorial Sloan Kettering, reported positive results from their trial of their
CD19 targeted CAR-T cell therapy, JCAR015, for
the treatment of adult relapsed/refractory ALL. They
reported an 82% complete response, and minimal
residual disease-negative complete response in 83%
of those patients. JCAR014 also had positive results, with a 93% complete response rate in patients
receiving monotherapy, and 100% complete response
rate in patients who received JCAR014 with fludarabine. JCAR015 was in a Phase II clinical trial with
cyclophosphamide preconditioning for the treatment
of all – however, recently, Juno halted this trial due
to several patient deaths from cerebral edema. The
trial had been previously halted by the FDA due to
patient deaths, but was resumed with a modified
preconditioning regimen which Juno asserted would
minimize patient deaths. This most recent hold on the
trial was imposed by Juno themselves, rather than by
the FDA. In March 2017, Juno announced that they
were permanently halting all future development of
JCAR015, and will instead focus on developoing their
other CAR-T cell therapy products such as JCAR016,
JCAR018, and JCAR014, which are being developed
for other blood cancers such as acute myeloid leukemia and chronic lymphocytic leukemia. This puts
Juno behind rivals Kite Pharma and Novartis.
A Primer on Immuno-Oncology Part 2
Table 2. Clinical-Stage CAR-T Projects with Commercial Licensees (Excluding China)[38].
Company
Aurora
Autolus
AcademicCenter
BaylorCollegeofMedicine
UniversityCollege,London
ProjectName
AU105
1RG-CART
Antigen
Her2
GD2
Co-stim
CD28
CD28
Transfection
retrovirus
unknown
ScFv
murine
unknown
Bellicum
BaylorCollegeofMedicine
BPX-601
PSCA
retrovirus
murine
BluebirdBio
&Celgene
BluebirdBio
BaylorCollegeofMedicine
bb2121
BCMA
none
(1st-gen)
4-1BB
lentivirus
murine
UppsalaUniversity
CD19
unknown
BaylorCollegeofMedicine
CD30
CD28&
4-1BB
CD28
retrovirus
BluebirdBio
3rd-gen
CD19CAR
CD30.CAR
murine
Cellectis,Pfizer&
Servier
CellularTherapeutics
Ltd
CellularTherapeutics
Ltd
Juno
Juno
UniversityCollege,London
UCART19
CD19
4-1BB
gammaretro-virus
lentivirus
ChristieHospitalNHS
FoundationTrust
CancerResearchUK
aCD19z
CD19
retrovirus
murine
anti-CEAMFEz
CEA
unknown
murine
FredHutchinson&NCI
MemorialSloanKettering
JCAR014
JCAR015
CD19
CD19
none
(1st-gen)
none
(1st-gen)
4-1BB
CD28
murine
murine
Juno
SeattleChildren’sHospital
JCAR017
CD19
4-1BB
lentivirus
gamma-
retrovirus
lentivirus
Juno,viaOpusBio
Juno
NCI
SeattleChildren’sHospital
JCAR018
JCAR023
4-1BB
4-1BB
lentivirus
lentivirus
human
murine
Juno
MemorialSloanKettering
JCAR020
CD22
L1CAM
(=CD171)
MUC16
CD28
Juno
JCAR024
ROR1
4-1BB
fully
human
rabbit
KTE-C19
CD19
CD28
EGFRvIII
CD28
LeucidBio
King'sCollegeLondon
CityofHopeMedical
Center
CityofHopeMedical
Center
UniversityofPennsylvania
ErbB
dimers
CD123
CD28
Mustang
(FortressBio)
Mustang
(FortressBio)
Novartis
Anti-EGFRvIII
CAR
LEU-001
(T1E28z)
MB-102
CD28
gamma-
retrovirus
gamma-
retrovirus
gamma-
retrovirus
lentivirus
murine
KitePharma
FredHutchinsonCancer
Center
ZeligEshhar(Cabaret
Biotech)
NCI
gamma-
retrovirus
retrovirus
MB-101
IL13Rα2
4-1BB
lentivirus
murine
CART-BCMA
BCMA
4-1BB
lentivirus
murine
Novartis
Novartis
Novartis
Novartis
UniversityofPennsylvania
UniversityofPennsylvania
UniversityofPennsylvania
UniversityofPennsylvania
CTL019
CTL119
CART22cells
CART-
EGFRvIII
CART-meso
CD19CAR
CD19
CD19
CD22
EGFRvIII
4-1BB
4-1BB
4-1BB
4-1BB
lentivirus
lentivirus
lentivirus
lentivirus
murine
humanised
murine
murine
mesothelin 4-1BB
CD19
CD28
lentivirus
Sleeping
Beauty
murine
murine
KitePharma
6
Novartis
UniversityofPennsylvania
Ziopharm,Intrexon& MDAndersonCancer
MerckKGaA
Center
murine
murine
murine
murine
murine
A Primer on Immuno-Oncology Part 2
In May 2015, Juno partnered with Editas to utilize
their CRISPR/Cas9 technology to develop Juno’s
CAR-T cell therapies. Juno paid Editas an up-front
payment of $25M, with additional milestone payments. In April 2016, Celgene partnered with Juno to
develop JCAR015, JCAR014, and JCAR017. Celgene paid $150M in an up-front payment to Juno, plus
an $864M equity stake. The deal gave Celgene commercial rights outside of North America and China.
After the most recent halt of Juno’s JCAR015 trial in
ALL due to patient deaths, shares of Juno’s stock have
plummeted by ~30%.
Kite Pharma
Kite has a cooperative research and development
agreement with the NCI surgery branch to develop
KTE-C19, their CD19 targeted CAR-T cell therapy
product. In their phase I trial (ZUMA-1) of KTE-C19
for the treatment of DLBCL, they reported an overall
response rate of 71% after treatment with KTE-C19.
Furthermore, treatment durability was higher than
expected, the number of patients in complete remission remained unchanged from 3mo to 6mo poststudy. KTE-C19 is currently in Phase II clinical trials,
and has received Breakthrough Therapy Designation
and Orphan Drug status from the U.S. Food and Drug
Administration for the treatment of patients with
chemorefractory DLBCL, primary mediastinal B-cell
lymphoma, and transformed follicular lymphoma. The
European Medicines Agency has also granted KTEC19 access to regulatory support under its Priority
Medicines (PRIME) initiative for the treatment of
DLBCL and Orphan Drug Designation for various
hematological indications. Unlike the Juno trials,
Kite has not seen any patient deaths from cerebral
edema, but one patient did die from KTE-C19 related
cytokine release syndrome and another from a fungal
infection unrelated to KTE-C19. In December 2016,
Kite initiated a rolling submission of the US FDA
Biologics License Application (BLA) for KTE-C19,
“In March 2017, Juno announced that they were permanently
halting all future development of JCAR015, and will instead
focus on developoing their other CAR-T cell therapy products
such as JCAR016, JCAR018, and JCAR014, which are being
developed for other blood cancers such as acute myeloid
leukemia and chronic lymphocytic leukemia. This puts Juno
behind rivals Kite Pharma and Novartis.”
7
and announced the US Adopted Name for KTE-C19,
“axicabtagene ciloleucel”.
Other notable collaborations with Kite include Adimab
for the discovery and optimization of fully humanized
Abs against CAR targets, Alpine Immune Sciences
to use their AIS Transmembrane Immunomodulatory
Protein Technology for Kite’s CAR-T cell program,
Amgen to utilize their oncology targets, Cell Design
Labs to use their suicide switch in CAR-T cells,
Genentech for a clinical trial utilizing both KTE-C19
CAR-T cell product and atezolizumab (Genentech’s
anti-PDL1 Ab), the Leukemia and Lymphoma Society
to develop KTE-C19, and the Tel-Aviv Sourasky
Medical Center to develop CAR-T cell products.
Bluebird Bio
Bluebird and Celgene, along with Baylor College of
Medicine, entered into a collaboration to treat multiple
myeloma using CAR-T cells by targeting the B-cell
maturation antigen (BCMA), which is expressed on
the surface of most multiple myeloma cells.
Celgene and Bluebird reached a deal wherein Celgene agreed to pay $225M in milestone payments
to Bluebird, and Celgene has an option to exclusive
rights to any BCMA related CAR-T cell therapies. In
May 2015, Bluebird entered into an exclusive license
agreement with Five Prime to research, develop, and
commercialize Juno’s CAR-T cell therapies using Five
Prime’s proprietary lentiviral gene therapy platform
and proprietary human Abs to an undisclosed cancer target for hematological malignancies and solid
tumors. Five Prime received $1.5M up-front, with
subsequent milestone payments totaling up to over
$130M per licensed product, as well as tiered royalties to Five Prime. In December 2015, ViroMed and
Bluebird entered into an exclusive license agreement
to research, develop, and commercialize CAR-T cell
therapies using ViroMed’s proprietary humanized
Ab to an undisclosed cancer target for solid tumors.
ViroMed received an up-front payment of $1M with
milestone payments of up to $48M and tiered royalties.
Bluebird started their first CAR-T clinical trial for
bb2121 (NCT02658929) targeting BCMA for the
treatment of multiple myeloma in February 2016.
Preliminary results showed an overall response rate
of 78% with no grade 3 or higher neurotoxicities or
cytokine release syndrome resulting from treatment.
A Primer on Immuno-Oncology Part 2
Cellectis
In 2014, Pfizer and Cellectis entered into a global
strategic collaboration to develop Cellectis’ CART cell therapies. Cellectis received an up-front
payment of $80M and up to $185M in milestone
payments, as well as tiered royalties. The deal gave
Pfizer exclusive rights to pursue development and
commercialization of CAR-T cell therapies directed
towards 15 targets selected by Pfizer. In 2015, Pfizer
obtained the rights to Cellectis’ UCART19 CAR-T
cell therapy through an intermediary, Servier. Servier paid Cellectis $38M in an up-front payment, with
up to $300M in milestone payments, for global rights
to UCART19. Shortly after the deal was reached,
Servier sold the US rights to Pfizer for an undisclosed sum. UCART19 is an off-the-shelf CAR-T
cell therapy which uses allogeneic cells rather than
the patient’s own cells, Cellectis is the only company
to utilize this allogeneic approach.
Challenges
Immunogenicity
CAR-T cells may elicit severe toxicities; thus, safety
is a major concern. CARs have mostly been derived
from mouse Abs, and both Ab and T cell responses
against CARs have been reported in clinical trials[39]. In order to minimize the immunogenicity
of CAR-T cells, one may utilize humanized or fully
human Abs[40].
Increasing Engraftment and Survival
Infused CAR-T cells must survive and also possibly proliferate in order to exert therapeutic efficacy
and disease clearance. Choosing a specific population of T cells for transducing the CAR gene may
offer certain benefits; CD19-specific CD8+ central
memory T-enriched cells expressing naïve markers
such as CD62L may have improved engraftment as
compared to effector/more differentiated T cells[41].
Alternatively, using cytokines such as IL-12 may
increase T cell proliferation and survival. CAR-T
cells engineered to express IL-12 were shown to
retain a central memory effector phenotype and had
increased antitumor activity[42]. Similarly, CAR-T
cells expressing IL-15 were shown to have increased
persistence and no loss of antitumor activity[33].
Solid Tumors
Although CAR-T cell therapy was first utilized to
8
treat solid tumors, most of the successes of CAR-T
cell therapy has been from the treatment of hematological malignancies[43]. Trials using first
generation CAR-T cells for the treatment of solid
tumors mostly failed to achieve an effective antitumor response[1]. However, HER2.CD28ζ CAR-T
cells have showed some success in clinical trials for
HER2+ sarcoma: four patients experienced disease
stabilization for 12wks – 14mos[28]. Challenges
for the treatment of solid tumors using CAR-T cell
therapy includes inefficient tumor targeting, the absence of unique TAA, the immunosuppressive tumor
microenvironment, and limited persistence of CAR-T
cells[1]. Approaches for addressing these challenges,
and others, are discussed below.
Specificity
One major concern of CAR-T cell therapy is “ontarget, off-tumor” toxicity[1]. This refers to CAR-T
cells targeting normal, healthy tissues which express
low or normal levels of the target antigen, resulting
in immune-mediated destruction of healthy tissues.
One such example is the development of B-cell
aplasia in patients receiving CD-19 targeted CAR-T
cells[44]. Mutated tumor antigens may be targeted
to minimize “on-target, off-tumor” toxicity as they
are expressed only on cancer cells; these include
EGFRvIII, EphA2 epitopes, and tumor-specific
glycosylation patterns of MUC-1[43]. Recently, researchers at the University of Pennsylvania have targeted truncated carbohydrate molecules on MUC-1
protein, which is not expressed on healthy tissue, but
is expressed on cancer cell’s surface of many types
of solid tumors and leukemias. Their efforts showed
antitumor efficacy in a mouse model of pancreatic
cancer and leukemia.
“One major concern of CAR-T cell
therapy is “on-target, off-tumor” toxicity.
This refers to CAR-T cells targeting
normal, healthy tissues which express
low or normal levels of the target
antigen, resulting in immune-mediated
destruction of healthy tissues.”
A Primer on Immuno-Oncology Part 2
Another strategy to increase the specificity of CART cells is to engineer CAR-T cells to express additional receptors. Chemokines have been shown
to play a role in influencing lymphocyte migration;
thus engineering T cells to express certain chemokine
receptors may alter the migration pattern of the T cells
so that they traffic to those tumor chemokine receptors[1]. CAR-T cells specific for CD30 (Hodgkin’s
Lymphoma) were engineered to express CCR4, resulting in increased localization to the tumor site[45].
Similarly, in a strategy targeting the tumor stroma,
CAR-T cells have also been engineered to express
CCR2b for neuroblastoma[46] and VEGFR2 which is
expressed in tumor blood vessels[35]. Targeting the
tumor stroma via targets such as VEGFR2 or FAP has
shown antitumor efficacy[43]. In addition, inducing
expression of heparanase in CAR-T cells has been
shown to improve their ability to degrade the extracellular matrix of tumors, and increase their infiltration
into tumors without affecting CAR-T cells’ viability,
expansion, or effector function while increasing antitumor efficacy[47].
Recent research has shown that bispecific CARs,
which require binding to two distinct antigens, may
have increased safety while maintaining antitumor
efficacy[48]. In this strategy, the activating and costimulatory domains are separated onto two separate
CARs located on one T cell. Binding to just one of
the target antigens does not result in activation of the
CAR-T cell; rather, the CAR-T cell must bind to both
target antigens in order to become activated. In a
similar strategy used to increase the safety of CART cells, two CARs are again used, with one CAR
specific for an antigen expressed by normal tissues and
conjugated to a suppressive domain. Binding of this
inhibitory, bispecific CAR (iCAR to normal, healthy
tissue results in inhibition of T cell function, and thus
increased safety[49].
Local delivery may also be utilized to deliver CAR-T
cells to tumors. Intra-tumoral delivery of CAR-T cells
targeted towards ErbB for head and neck squamous
cell carcinoma is currently in Phase I clinical trial.
Ovarian cancer and malignant pleural mesothelioma
may also be candidates for local delivery of CAR-T
cells as they possess a propensity for localized dissemination within peritoneal and pleural cavities[43].
Immunosuppressive Tumor Microenvironment
Figure 4. Schematic Representation of the Chimeric Antigen Receptor (CAR) Structure[1]. A) CAR
T-cells redirected for universal cytokine killing (TRUCKs) employ a vector encoding the CAR
construct that also possesses a cytokine expression cassette. These cytokines such as IL-12 can effectively recruit other components of the immune system to enhance the antitumor immune response
toward those cancer cells that are invisible to CAR T-cells. B) To increase the specificity of the CAR
T-cells, T-cell signal 1 is separated from signal 2. Both target antigens that are expressed on tumor
cells must be engaged to deliver signals 1 and 2 and fully activate CAR T-cells. Normal cells that
express only one of two antigens do not signal T-cells sufficiently to accomplish full activation. C)
A CAR that delivers a dominant inhibitory signal such as PD-1 and CTLA-4 is coexpressed with a
CAR capable of full T-cell activation. Engaging both antigens on normal cells could inhibit T-cell
function, whereas encountering only the activating ligand on tumor cells generates a sustained T-cell
response. CAR = chimeric antigen receptor; CCR = chimeric costimulatory receptor; iCAR = inhibitory CAR.
9
After CAR-T cells are infused into the patient, they
must infiltrate the tumor and the tumor microenvironment in order to exert their therapeutic effect.
The tumor microenvironment possesses a variety of
immunosuppressive factors used to defeat the host’s
immune system response to the tumor and can limit
the efficacy of CAR-T cell therapy. Thus, researchers
have explored different methods for overcoming the
immunosuppressive tumor microenvironment. One
such method is engineering CAR-T cells to express a
dominant-negative form of TGFβ receptor in order to
overcome the inhibitory effects of TGFβ[50]. CAR-T
cells have also been engineered to target NKG2D in
order to recognize NKG2D ligands expressed by immunosuppressive cells[1]. Granulocyte-macrophage
colony-stimulating factor (GM-CSF) has been shown
to be secreted at high levels by tumor cells in vivo
and is implicated in myeloid-derived suppressor cells’
(MDSCs) recruitment. MDSCs have been shown to
expand in response to liver metastases and inhibit the
antitumor efficacy of CAR-T cells; thus,
A Primer on Immuno-Oncology Part 2
neutralization of GM-CSF may prevent MDSC expansion and increase the antitumor efficacy of CAR-T
cells[43].
An alternative strategy is to use combinatorial therapy,
CAR-T cell therapy may be used in conjunction with
immune checkpoint inhibitors such as a PD-1 Ab[51]
or a chimeric PD1-CD28 receptor, which can convert
PD-L1 to a ligand, which transmits a CD28 costimulatory signal to CD8+ T cells, thus enhancing their
antitumor activity[52]. Conditioning chemotherapy
(e.g. fludarabine and cyclophosphamide) may also
be used to achieve resistance to immunosuppressive
factors such as indoleamine 2,3-dioxygenase[53]. In
addition, “armored” CAR-T cells expressing cytokines
such as IL-2, IL-12, IL-15 or TGFβ may also be used,
this strategy has been discussed above.
Toxicity
CAR-T cell therapy may result in cytokine release
syndrome (CRS); this is caused by the production
of pro-inflammatory cytokines such as IL-6, TNFα,
and IFNγ by large numbers of activated T cells and
is characterized by high fever, hypotension, hypoxia,
and potentially results in organ failure[1]. Inhibition
of IL-6 using the IL-6 receptor antagonist tocilizumab
has been used to control severe CRS without affecting CAR-T cell therapy efficacy[54]. The severity of
CRS may directly correlate with the tumor burden at
the time of therapy; thus, treating patients with preinfusion conditioning chemotherapy combined with
intensive chemotherapy to reduce the tumor burden
may significantly reduce the risk of severe CRS[55].
On-target, off-tumor toxicity and strategies for mitigating such toxicity has been discussed above. The use of
safety switches or suicide genes to avoid T cell mediated toxicity is discussed below.
Safety Switches
In the event of T-cell mediated toxicity, elimination of
the CAR-T cells from the circulation would be useful.
For this purpose, safety switches may be engineered
into the CAR-T cells. Early approaches used expression of a protein which metabolized an inactive
prodrug into a cytotoxic metabolite to kill the CAR-T
cells. In a later approach, a membrane bound protein
was expressed on the surface of CAR-T cells, and
an Ab against that protein was used to deplete cells
expressing that protein[56].
Recently, the inducible caspase-9 (iCasp9) system has
10
been utilized for this purpose. This system uses an
incomplete pro-apoptotic caspase-9 lacking its caspase
recruitment domain fused to a mutated peptide derived
from FKBP12 protein. When the mutated FKBP12
domain interacts with a specific small molecule,
it induces dimerization of the fusion protein and,
subsequently, activates caspase-9 induced apoptosis[57]. Wu et al. presented another approach in which
they produced a dissociated CAR, with one protein
possessing the antigen binding and transmembrane
domains, and another protein with the intracellular
signaling domains[58]. Both of the subunits also pos-
“CAR-T cell therapy may result in cytokine
release syndrome (CRS); this is caused
by the production of pro-inflammatory
cytokines such as IL-6, TNFα, and IFNγ
by large numbers of activated T cells and
is characterized by high fever, hypotension,
hypoxia, and potentially results in organ
failure.”
sess a heterodimerization module which assembles the
CAR into one fully functional unit in the presence of a
specific small molecule; thus this CAR-T cell requires
both antigen binding and the dimerizing agent to be
present in order to activate the T cell, allowing for
both real-time and dose-dependent control of CAR-T
cell activity[56].
Choosing the Right Therapy
As tumor cells evolve, they are eliminated from the
body by immune surveillance; in particular, by T cells
responding to tumor neoantigen-derived peptides
presented by MHCs. Certain types of tumors, such as
melanoma or NSCLC, which have a high frequency
of somatic mutations leading to an increased presentation of neoantigens, and may be more likely to escape
immune surveillance via co-evolution in the immunosuppressive tumor microenvironment[32]. In addition,
the immunosuppressive tumor microenvironment
inhibits CAR-T cell function, as discussed above. In
this type of environment, inducing an immune-mediated antitumor response may best be achieved through
the use of immune-modulating mAbs such as anti-PD1
A Primer on Immuno-Oncology Part 2
or anti-CTLA4 checkpoint inhibitors[32]. In tumors
with low antigen presentation and a non-immunosuppressive tumor microenvironment, immune modulating mAbs may not be sufficient and instead, CAR-T
cells may represent a more efficacious option. In
tumors with a phenotype in between these extremes,
a combinatorial approach may be most efficacious.
In a preclinical model of sarcoma and breast cancer,
PD-1 blockade alone or CAR-T cell therapy alone had
significantly less antitumor response as compared to a
combinatorial approach utilizing both strategies[51].
However, it should be noted that there is an increased
risk of toxicity when utilizing a combinatorial approach such as this. There is currently an ongoing
clinical trial using a combination of CTLA-4 mAb and
CAR-T cell therapy (NCT00586391).
Regulatory Considerations[59]
CAR-T cells are unique in that they are somatic cells,
and thus fall under the guidelines for cell therapy;
however, they are also genetically modified, and thus
also fall under the guidelines of gene therapy products. Different nations have their own guidelines for
clinical trials utilizing CAR-T cells, and herein we
will discuss the US FDA and the EU EMA.
The US FDA classifies CAR-T cells as 351 biological products regulated under “Considerations for the
design of early-phase clinical trials of cellular and
gene therapy products” and “Guidance for industry:
preclinical assessment of investigational cellular
and gene therapy products” (Nov. 2013, OCTGT
CBER). CAR-T cell therapies should meet the CMC
criteria under the “Guidance for FDA reviewers and
sponsors: content and review of chemistry, manufacturing, and control (CMC) information for human
“CAR-T cells are unique in that they are somatic cells, and
thus fall under the guidelines for cell therapy; however, they
are also genetically modified, and thus also fall under the
guidelines of gene therapy products.”
somatic cell therapy investigational new drug (IND)
applications” (Apr. 2008) and “Guidance for FDA
reviewers and sponsors: content and review of chemistry, manufacturing, and control (CMC) information
for human gene therapy investigational new drug
11
applications (INDs).”
The EU EMA issued a guideline, “Guideline on
quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells”
(2012). The guideline focuses on the quality, safety,
and efficacy requirements of genetically modified
cells to be used as a medicinal product. The manufacture of CAR-T cells is regulated by the regulation on advanced therapy medicinal products No
1394/2007 and guidance on quality, preclinical and
clinical aspects of gene transfer medicinal products
(CPMP/BWP/3088/99).
Patent Disputes
CAR-T cell therapies have been in development for
decades, primarily in academic centers. However,
now that the technology has begun to reach the commercialization stage, patent issues have arisen, although several disputes have recently been resolved.
Juno settled a dispute with UPenn and Novartis in
2015 over a patent licensed from St. Jude Children’s
Research Hospital. The settlement gave Juno and St.
Jude’s $12.25M, plus future milestone payments and
royalty payments on relevant CAR-T cell products.
Conclusion
While CAR-T cell therapy is an exciting and growing
field, challenges associated with CAR-T cell therapies, such as safety and toxicity, must be addressed
prior to commercialization. Some strategies to mitigate these challenges, such as bispecific CAR-T cells
or armored CAR-T cells, have been discussed in this
report. In addition, before CAR-T cell therapies can
reach the market, patent disputes over CAR-T cell
therapy products must be resolved and may require
cross-licensing deals between academic groups and
corporations, as companies in the CAR-T cell space
often own technologies for just one part of CAR-T
cell therapies (such as gene editing technology or a
suicide switch). This hints at the possibility, or even
the necessity, of more deals between pharma and
biotech companies in this space. Mark Osterman,
Senior VP at Kineticos, recently published a Kineticos Insight regarding the movement of innovation
away from big pharma to smaller biotech/pharma
companies. Similarly, companies in the CAR-T cell
space may follow such a trend, with smaller biotech
companies that possess the rights for one component of CAR-T cell therapies being acquired by or
A Primer on Immuno-Oncology Part 2
licensing the rights to big pharma, who is then able
to further develop and commercialize the technology. Such a trend may foster increased innovation by
encouraging the growth and conception of smaller,
novel, and innovative biotech companies, ultimately
leading to improved therapies.
12
A Primer on Immuno-Oncology Part 2
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15
A Primer on Immuno-Oncology Part 2
Authors
Soo Kim, PhD
Analyst
Kineticos
Mark Osternan
Senior Vice President
Kineticos
Shailesh Maingi
Founder and CEO
Kineticos
Dr. Kim recently graduated
from the Eshelman School of
Pharmacy, Division of Molecular
Pharmaceutics at UNC–Chapel
Hill. His research involved a
novel approach to formulating
exosomes, nano-sized extracellular
vesicles used in intracellular
communication, to deliver poorly
soluble anticancer drugs for the
treatment of pulmonary metastases.
Mr. Osterman adds more than
25 years of experience in the
biopharmaceutical industry to the
team.
Mr. Maingi founded Kineticos
Life Sciences in 2012 and
develops long-term and day-to-day
execution strategies.
Mr. Osterman held roles in
sales, clinical, and marketing at
GlaxoSmithKline before moving
to Johnson & Johnson, where he
launched their stem cell internal
venture and was responsible for
the cardiovascular, pulmonary,
metabolic, and mimetibody
therapeutic areas in the Global
Biologic Strategic Marketing.
Mr. Maingi serves on the
Board of Directors at Gallus
Biopharmaceutical and he
serves on the advisory boards of
Novolipid and Enci Therapeutics.
Prior to earning his PhD, Dr. Kim
received a BS in Biology from
UNC–Chapel Hill.
Mr. Osterman earned a dual BA
in Business Management and
Economics from North Carolina
State University.
3005 Carrington Mill Blvd
Suite 510
Morrisville, NC 27560
Office: 919.678.3206
Email: [email protected]
www.kineticos.com
16
Prior to founding Kineticos Life
Sciences, Mr. Maingi was a senior
executive at Cardinal Health/
PTS and Sigma-Aldrich/Supelco,
where he held leadership roles in
strategy, business development,
and marketing.
About Kineticos
Kineticos is a specialized management consulting
firm serving the life science industry. The firm
is focused on identifying opportunities to drive
strategic growth and achieve operational excellence
for it clients.
Through its three practice areas - Biopharmaceutical,
Biopharmaceutical Services, and Diagnostics Kineticos has experience working with companies
across the life science industry ecosystem.