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ANTIBODY DERIVATIVES AS NEW THERAPEUTICS FOR HEMATOLOGIC
MALIGNANCIES
Introduction
Immune-based therapies for cancer have the tantalizing possibility of
effecting long-term durable remissions and perhaps even offering the
possibility of a cure. This is the basis for the US Food and Drug
administration (FDA) approval of interleukin-2 (IL-2) in melanoma;
more recent immune therapies that are FDA-approved treatments for
cancer involve checkpoint blockade, which is a form of releasing the
brakes on tumor-specic T cells and allowing them to persist and
expand in vivo, leading to control or regression of cancer. Adoptive
T-cell therapy also offers this possibility but has thus far been limited
in application to those patients with melanoma who have adequate
culture and expansion of isolated tumor-inltrating lymphocytes.1
The main barriers to this approach have been the difculty in culturing
and manufacturing of tumor-inltrating lymphocytes, immune tolerance to self-antigens, and the requirement for major histocompatibility
complex (MHC) presentation of antigens (Figure 1).
The infusion of gene-modied T cells directed to specic target
antigens offers the same possibilities of long-term disease control
and has the added benet of the rapid onset of action that is usually
seen with cytotoxic chemotherapy or with targeted therapies. In
particular, T cells modied to express antibody-based chimeric antigen receptors circumvent both immune tolerance of the T-cell
repertoire and MHC restriction. Furthermore, advances in the culture
process and molecular and virology techniques used to introduce
novel genes into T cells have made the manufacturing of genemodied peripheral bloodderived T cells relatively straightforward.
In the last 5 years, chimeric antigen receptor (CAR)-redirected
T cells have emerged from the bench and made splashy headlines in
the clinical setting at a number of academic institutions. It is not
surprising that CAR T cells directed to hematologic malignancies
have been the rst ones tested, given the extent of the known surface
antigens expressed on hematologic cells, the relative ease of sampling
tumor, and the natural preference of T-cell homing to hematologic
Submitted November 7, 2013; accepted December 19, 2013. Prepublished
online as Blood First Edition paper, February 27, 2014; DOI 10.1182/blood2013-11-492231.
organs such as the blood, bone marrow, and lymph nodes. Here, we
will introduce the various CAR designs that have been tested
clinically, the results from a series of clinical trials testing CAR
T cells, and an overview and comparison of the manufacturing
processes used. We will also discuss the emerging toxicity proles
and management strategies and future outlook of CAR T-cell
therapies. We limit our discussion to CAR T cells in hematologic
malignancies and will not cover CARs that have been tested in
solid tumors or engineered T-cell receptors (TCRs) that have
been tested in any setting.
2625
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2626
MAUS et al
Figure 1. Therapeutic approaches to overcome immune tolerance to tumors. Cytokines and vaccines can be used to augment natural T-cell responses to tumor.
Antibodies targeting negative regulatory molecules such as programmed death 1 (PD-1) and cytotoxic T-cell lymphocyte-associated antigen 4 (CTLA-4) can be infused to
release the brakes on natural T cells responsive to tumor. Chemotherapy can reduce immune suppressive cells such as Tregs and myeloid-derived suppressor cells (MDSC)
in addition to its direct effect on the tumor cells. Adoptive T-cell transfer strategies using clonally expanded cytotoxic T cells or T cells engineered to express TCRs or CARs
are being tested.
activity and persistence of the infused cells, and the antibioticresistance gene products may render them immunogenic. Transposonbased systems can integrate transgenes more efciently than plasmids
that do not contain an integrating element.10,11 Sleeping Beauty was
shown to provide efcient stable gene transfer and sustained transgene
expression in multiple cell types, including T cells,12 but the culture
time remains relatively prolonged. This system13 is now entering
clinical trials as an approach to engineer T cells.
Most investigators have been using g retroviruses; these are
relatively easy to produce, they efciently and permanently transduce T cells, and they have preliminarily proven safe from an
integration standpoint in primary human T cells.14 One potential
disadvantage of retrovirus as the vehicle is the potential for
silencing of expression of the CAR based on silencing of the long
terminal repeats; in the right context, this could be an advantage of
CAR-based therapies if they are to be used as a bridge to another
denitive treatment such as allogeneic bone marrow transplant.
Lentiviral vectors also efciently and permanently transduce T cells
but are more expensive to manufacture; they are also potentially safer
than retrovirus based on integration preferences15 examined in hematopoietic stem cells, though it is not clear that this applies to primary
human T cells. Use of specic promoters in combination with lentiviral
transduction has enabled sustained surface expression of CARs on
transduced T cells; this likely extends the survival of functional CAR
T cells in vivo.
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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17
ANTIBODY-MODIFIED T CELLS
2627
The methods and duration of T-cell culture used in the manufacturing of CAR T cells may also be an important variable in the
composition of the nal CAR T-cell product. Generally, the options
have involved combinations of TCR stimulation through antibodies
and supportive cytokines or articial antigen-presenting cells that are
either cell based or bead based.16 Culture conditions that combine
costimulation and various cytokines support the maintenance of a
central memory phenotype.17,18
CD19
CD19
CD19
CD19
CD28
CD8-CD8
CD28
IgG-CD28
CD3z
CD28 and
CD3z
4-1BB and
CD3z
CD28 and
CD28-CD3z
CD3z and
CD3z
none
None
None
None
for CD20,
24 d
OKT3 1 IL-2;
10 d
beads;
0.3-3 3 10
CAR1 cells/kg
pentostatin/
as tolerated
32, fludarabine 25
mg/m2 3 5
every 8 h
IV IL-2
None
None
None
IL-2
patients
last 4
SC IL-2 in
Cytokine
support
cyclophosphamide
60 mg/kg
cyclophosphamide
Bendamustine or
cyclophosphamide
2 d)
CD3/28
16 d
2 3 109/m2
beads;
CD3/28
6-18 d
None
OKT3 1 IL-2;
2 3 107/m2 to
CD19
fludarabine for
109/m2 (3
infusions)
or fludarabine
Cyclophosphamide
108/m2 to 2 3
2-5 d apart)
109/m2 (3 infusions
Lymphodepletion
feeders; 3 mo
LCL
irradiated
IgG-CD4
or hygromycin/
2-4 mo
HSV-tk (CD19)
CD20
Dose
or
SV40-neomycin
Culture
CD19
CD3z
Other genes
OKT3 1 IL-2 1
IgG-CD4
Signaling
domain
Neomycin (CD20)
CD20
scFv
MZL)
8 (3 FL, 4 CLL, 1
3 (CLL)
ALL
6 (NHL)
4 (2 FL, 2 DLBCL)
MCL)
7 (indolent and
Number of
patients
marrow at 2-3 wk
cells by PCR of
vector copies/100
wk by IHC; 0.02-1
persistence at 6-8
detectable CAR at
CD3z)
mg at 2 wk in CD28-
detectable by PCR
persisted longer
CD28-CD3z
infusions
after 3 of the 7
PCR only at 1 wk
Detectable cells by
detected by PCR
Persistence (peak
and duration)
aplasia)
remission (4 B cell
6 objective
aplasia
Detectable by flow
CAR1 by flow
copies/mg and
declined to 10-1000
mg of DNA in
flow cytometry
cells detectable by
aplasia (ALL)
in LAD, 1 B-cell
1 death, 1 reduction
after ASCT
2 maintained CR
2 maintained CR, 1
Responses
MAUS et al
allo, allogeneic hematopoietic stem cell transplantation; ASCT, autologous stem cell transplantation; CR, complete response; DLBCL, diffuse large B-cell lymphoma; DLI, donor lymphocyte infusion; FL, follicular lymphoma; GVHD, graftversus-host disease; HSV-tk, herpes simplex virus thymidine kinase; IHC, immunohistochemistry; IV, intravenous; MCL, mantle cell lymphoma; LAD, lymphadenopathy; LCL, lymphoblastoid cell line; MRD, minimal residual disease; PBMC,
peripheral blood mononuclear cell; PCR, polymerase chain reaction; PD, progressive disease; PR, partial response; qPCR, quantitative polymerase chain reaction; SC, subcutaneous; SD, stable disease.
Gammaretrovirus
Gammaretrovirus
30
27, 28
Gammaretrovirus
21
Lentivirus
Electroporation
20
33, 34
Electroporation
Vector
81
Reference
2628
Hinge/
transmembrane
domain
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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17
Gammaretrovirus
Lentivirus
Gammaretrovirus
Gammaretrovirus
Gammaretrovirus
32
37
41
23
29
CD19
CD19
Lewis-
CD19
CD19
CD20
scFv
CD28
IgG-CD28
CD8-CD28
CD8-CD8
CD28
IgG1-CD4
Hinge/
transmembrane
domain
CD3z
CD28 and
CD3z
CD28 and
CD3z
CD28 and
CD3z
4-1BB and
CD3z
CD28 and
CD3z
4-1BB and
CD28 and
Signaling
domain
None
None
None
none
none
SV40-neomycinR
Other genes
8d
transient B-cell
aplasia in 3 patients
allo, post-DLI (4
CLL, 2 DLBCL, 4
MCL)
donor derived)
CR; no GVHD;
10 patients post-
DNA)
None
no GVHD
OKT3 1 IL-2; 0.4-7.8 3 106 CAR1
(4.2-53 copies/mg
objective response;
median 8 wk in
cells/mL) for ,1 mo
(peak at 40 CAR1
cytometry (2%-7%
Detectable by flow
derived)
None
day 21)
Detectable for
counts; 2/6 with
decreased B-cell
4 of 8 patients with
(allogeneic donor
8 patients post-allo
None
copies/1000 cells at
remission
with in vivo
expansion (1100
1 transient
None
Up to 10 mo by
qPCR, 1 patient
cytogenetic
reduction in blasts,
1 fludarabine
(AML)
2 SD, 1 transient
copies/ug of DNA in
(.70% CAR1
T cells) and PCR
aplasia
flow cytometry
cells detectable by
both with B-cell
negative relapse;
cyclophosphamide
recovery from
2 CR; 1 durable .
18 mo, 1 with CD19-
EBV-LCLs,
cells/kg
5 enrolled, 4 treated
PCR
In vivo expansion of
cytometry (peak at
Detectable by flow
9-12 mo
Peak at 1 23% by
MRD2, including 2
5 converted to
B-cell aplasia)
patients, 1 patient
Persistence (peak
and duration)
IL-2; 5-6 wk
Ad.pp65,
12 d
At bone marrow
None
blinatumomab)
etoposide; 1 with
none
1 with
treated (ALL)
14 enrolled, 5
(2 MCL, 1 FL)
cyclophosphamide/
None
None
Responses
Number of
patients
at day 21
cyclophosphamide
1.5-3 g/m2
at day 22
14 d
SC IL-2 3
Cytokine
support
cells/kg
beads; 10 d
CD3/28
beads; 14 d
. 69 d
CD3/28
1 g/m2
1 3 108 - 3.3 3
109/m2 (3 infusions)
OKT3 1 IL-2;
cyclophosphamide
Lymphodepletion
Dose
Culture
allo, allogeneic hematopoietic stem cell transplantation; ASCT, autologous stem cell transplantation; CR, complete response; DLBCL, diffuse large B-cell lymphoma; DLI, donor lymphocyte infusion; FL, follicular lymphoma; GVHD, graftversus-host disease; HSV-tk, herpes simplex virus thymidine kinase; IHC, immunohistochemistry; IV, intravenous; MCL, mantle cell lymphoma; LAD, lymphadenopathy; LCL, lymphoblastoid cell line; MRD, minimal residual disease; PBMC,
peripheral blood mononuclear cell; PCR, polymerase chain reaction; PD, progressive disease; PR, partial response; qPCR, quantitative polymerase chain reaction; SC, subcutaneous; SD, stable disease.
Electroporation
Vector
19
Reference
Table 1. (continued)
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ANTIBODY-MODIFIED T CELLS
2629
CLL
ALL
CD19
CD19
CD19
CD19
CD19
NHL, CLL
CD19
CD19
CD19
CD19
CD19
CD19
CD19
CD19/EGFRt
CD19
Retrovirus
Retrovirus
Lentivirus
Lentivirus
Lentivirus
Lentivirus
Lentivirus
Retrovirus
Retrovirus
Retrovirus
Transposon
Transposon
Transposon
Transposon
Retrovirus
Retrovirus
Retrovirus
Retrovirus
Retrovirus
Retrovirus
Retrovirus
lentivirus
4-1BBCD3z
CD3z
CD28-CD3z
CD3z
CD28-CD3z
CD28-CD3z
CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z
CD28-CD3z; 4-1BBCD3z
Phase/ID
Sponsor
NCT01864889
1/2/
NCT01195480
1/2/
NCT01475058
Center
Center
1/2/
1/2/
City of Hope
City of Hope
MSKCC
MSKCC
MSKCC
MSKCC
MSKCC
MSKCC/University of Pennsylvania
University of Pennsylvania
University of Pennsylvania
University of Pennsylvania
CHOP/University of Pennsylvania
NCT01865617
1/NCT01683279
1/NCT01815749
NCT01318317
1/2/
1/NCT01087294
NCT00924326
1/2/
1/NCT01593696
1/NCT00968760
1/NCT01497184
1/NCT01362452
1/NCT01653717
NCT00840853
1/2/
1/NCT00586391
1/NCT01853631
1/NCT01860937
1/NCT1416974
1/NCT01430390
1/NCT01840566
1/NCT01044069
NCT00466531
1/2/
II/NCT01747486
01551043
1/NCT
1/NCT01029366
1/NCT01626495
irradiated EBV-LCL
CD62L1 TCM
suicide system)
IL-2
IL-2
Donor derived
T cells)
from donor
29
23
30
32
34
37
Reference
Allo-HSCT, allogeneic hematopoietic stem cell transplantation; auto-HSCT, autologous hematopoietic stem cell transplantation; CHOP, Childrens Hospital of Philadelphia; CMV, cytomegalovirus; CTCL, cutaneous T-cell lymphoma;
CTLs, cytotoxic T lymphocytes; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; HL, Hodgkin lymphoma; Ig, immunoglobulin; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma.
CD19
postallo-HSCT
Pediatric ALL
CD19
CD19/EGFRt
infusion (day 2 or 3)
infusion (day 2 or 3)
CD19/EGFRt
CD19
Lentivirus
Retrovirus/
4-1BBCD3z
4-1BBCD3z
4-1BBCD3z
MAUS et al
CD19
CLL
CD19
or therapy)
CD19
CD19
pentostatin/cyclophosphamide/rituximab)
CD19
infusion
ALL (postallo-HSCT)
CD19
Lentivirus
Lentivirus
CD191 malignancies
CD19
Gene transfer
Lentivirus
Cancers
2630
CD19
Antigen
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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17
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Retrovirus
AML, MDS, multiple myeloma
Lewis-Y
Australia
Allo-HSCT, allogeneic hematopoietic stem cell transplantation; auto-HSCT, autologous hematopoietic stem cell transplantation; CHOP, Childrens Hospital of Philadelphia; CMV, cytomegalovirus; CTCL, cutaneous T-cell lymphoma;
CTLs, cytotoxic T lymphocytes; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; HL, Hodgkin lymphoma; Ig, immunoglobulin; MCL, mantle cell lymphoma; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma.
AntiLewis-Y-CD28-CD3z
1/NCT01716364
NCT01886976
multiple myeloma
NCT01864902
Retrovirus
Relapsed/refractory CD331 AML
CD33
1/2/
4-1BBCD3z
Retrovirus
CD201 leukemia and lymphoma
chain
CD20
Retrovirus
CD28-CD3z
University of Cologne
1/NCT01645293
CD28-CD3z
CD30
Retrovirus
NHL, HL
Mycosis fungoides/CTCL
CD30
Retrovirus
CD28-CD3z
1/NCT01192464
Phase/ID
1/NCT01316146
CD28-CD3z
Retrovirus
NHL, HL
Gene transfer
Cancers
Antigen
CD30
Table 2. (continued)
ANTIBODY-MODIFIED T CELLS
41
78
Reference
2631
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2632
MAUS et al
universal T-cell products from allogeneic donors, based on knockdown of the HLA genes coupled with enforced expression of
nonclassical HLA molecules to avoid natural killer (NK) cellmediated
recognition and lysis.56,57 In addition, it may not be necessary to use
T cells with integrated CAR transgenes, because transient expression
of CARs with RNA transfection has also been effective in preclinical
models.58 Similarly, because the cumulative experience with retroviral
or lentiviral transduced T cells demonstrates a notable lack of generation of replication-competent retroviruses,59,60 it is possible that the
testing requirements for this will be relaxed, which would make manufacturing less expensive and shorten the time from completion of
culture to potential infusion, which is an important consideration in
patients with aggressive malignancies.60 As CAR T cells enter the
mainstream of the therapeutic armamentarium for hematologic malignancies, more streamlined and centralized manufacturing will need to
be established, with shorter times in culture.61 In addition, the use of
serum-free medium will be mandatory, because projections indicate
an insufcient world supply that will peak in a few years, a situation
similar to the concept of peak oil production.62
Defining the active ingredient and cell type
Future outlook
Manufacturing
Aside from dening the optimal cell product, there are 2 main hurdles
in broadening the use of CAR-directed T cells beyond B-cell
malignancies: target discovery and manufacturing on a wide scale.
Several investigators are investigating new targets for myeloma,
including BCMA,69 CD70,70 CD74,71 CD38, CD138, and CS1.67
Some of these targets are purely based on differential expression of
the target in myeloma plasma cells, whereas others are based on
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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17
ANTIBODY-MODIFIED T CELLS
Conclusions
In the case of CD19-directed CAR T cells, multisite trials are in the
planning stages for several groups and now include involvement of
2633
Acknowledgments
The authors thank Anne Chew and Bruce Levine for constructive
comments.
This work was supported in part by grants from the National
Institutes of Health, National Cancer Institute (K08 CA166039, 5R01
CA165206, and R01 CA120409), the Pennsylvania Department of
Health, Cookies for Kids Cancer, and the American Cancer and
Leukemia and Lymphoma Societies (7000-02).
Authorship
Contribution: M.V.M. and C.H.J. wrote the manuscript, and S.A.G.
and D.L.P. edited the manuscript.
Conict-of-interest disclosure: M.V.M., D.L.P., and C.H.J. have
patents in the eld of adoptive immunotherapy. All authors have
sponsored research grant support from Novartis.
Correspondence: Marcela Maus, 3400 Civic Center Blvd,
8th Floor, Philadelphia, PA, 19104-5156; e-mail: marcela.maus@
uphs.upenn.edu; and Carl June, 3400 Civic Center Blvd, 8th Floor,
Philadelphia, PA, 19104-5156; e-mail: cjune@exchange.upenn.edu.
References
1. Rosenberg SA, Yang JC, Sherry RM, et al.
Durable complete responses in heavily pretreated
patients with metastatic melanoma using T-cell
transfer immunotherapy. Clin Cancer Res. 2011;
17(13):4550-4557.
2. Eshhar Z, Waks T, Bendavid A, Schindler DG.
Functional expression of chimeric receptor genes
in human T cells. J Immunol Methods. 2001;
248(1-2):67-76.
3. Hegde M, Corder A, Chow KK, et al.
Combinational targeting offsets antigen escape
and enhances effector functions of adoptively
transferred T cells in glioblastoma. Mol Ther.
2013;21(11):2087-2101.
4. Sadelain M, Brentjens R, Riviere
` I. The basic
principles of chimeric antigen receptor design.
Cancer Discov. 2013;3(4):388-398.
5. Haso W, Lee DW, Shah NN, et al. Anti-CD22chimeric antigen receptors targeting B-cell
precursor acute lymphoblastic leukemia. Blood.
2013;121(7):1165-1174.
6. Hudecek M, Lupo-Stanghellini MT, Kosasih PL,
et al. Receptor affinity and extracellular domain
modifications affect tumor recognition by ROR1specific chimeric antigen receptor T cells. Clin
Cancer Res. 2013;19(12):3153-3164.
7. Hombach A, Hombach AA, Abken H. Adoptive
immunotherapy with genetically engineered
T cells: modification of the IgG1 Fc spacer
domain in the extracellular moiety of chimeric
antigen receptors avoids off-target activation
From www.bloodjournal.org by guest on December 23, 2014. For personal use only.
2634
MAUS et al
an
P, Liu D, et al. The
lymph node microenvironment promotes B-cell
receptor signaling, NF-kappaB activation, and
tumor proliferation in chronic lymphocytic
leukemia. Blood. 2011;117(2):563-574.
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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17
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