From www.bloodjournal.org by guest on December 23, 2014. For personal use only.

Review Series
ANTIBODY DERIVATIVES AS NEW THERAPEUTICS FOR HEMATOLOGIC
MALIGNANCIES

Antibody-modied T cells: CARs take the front seat for

hematologic malignancies
Marcela V. Maus,1,2 Stephan A. Grupp,1,3,4 David L. Porter,1,2 and Carl H. June1,5
1
Abramson Cancer Center, 2Department of Medicine, and 3Division of Oncology, Department of Pediatrics, Perelman School of Medicine at the University of
Pennsylvania, Philadelphia, PA; 4Division of Oncology, Childrens Hospital of Philadelphia, Philadelphia, PA; and 5Department of Pathology and Laboratory
Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA

T cells redirected to specific antigen targets with engineered chimeric antigen

receptors (CARs) are emerging as powerful therapies in hematologic malignancies.
Various CAR designs, manufacturing processes, and study populations, among other
variables, have been tested and reported in

over 10 clinical trials. Here, we review and

compare the results of the reported clinical
trials and discuss the progress and key
emerging factors that may play a role in
effecting tumor responses. We also discuss
the outlook for CAR T-cell therapies, including managing toxicities and expanding

the availability of personalized cell therapy

as a promising approach to all hematologic
malignancies. Many questions remain in the
field of CAR T cells directed to hematologic
malignancies, but the encouraging response
rates pave a wide road for future investigation. (Blood. 2014;123(17):2625-2635)

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.

BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17

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.

Anatomy of CARs and CAR T-cell products

CARs are synthetic, engineered receptors that can target surface
molecules in their native conformation.2 Unlike TCRs, CARs engage molecular structures independent of antigen processing by the
target cell and independent of MHC. CARs typically engage the
target via a single-chain variable fragment (scFv) derived from an
antibody, although natural ligands have also been used.3 Individual
scFvs targeting a surface molecule are either derived from murine or
humanized antibodies or synthesized and screened via phage display
libraries.4 Unlike TCRs, where a narrow range of afnity dictates the
activation and specicity of the T cell, CARs typically have a much
higher and perhaps broader range of afnities that will engage the
target without necessarily encountering cross-reactivity issues. Preclinical data suggest that the spatial location of epitope binding has
a bigger effect on CAR activity than variation in afnity.5 The length,
exibility, and origin of the hinge domain is also an important
variable in the design of CARs.6-8 A major challenge to the eld is
that it is currently necessary to empirically test these design variables
The online version of this article contains a data supplement.
2014 by The American Society of Hematology

2625

From www.bloodjournal.org by guest on December 23, 2014. For personal use only.
2626

MAUS et al

BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17

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.

as there are no general rules guiding CAR design for target

molecules.
The generations of CARs typically refer to the intracellular
signaling domains. First-generation CARs include only CD3z as
an intracellular signaling domain, whereas second-generation
CARs include a single costimulatory domain derived from either
CD28 or 4-1BB; third-generation CARs include two costimulatory domains, such as CD28, 4-1BB, and other costimulatory
molecules (Figure 2). The hinge and transmembrane domains are
probably the least commented on aspect of CAR design, though
they may make important contributions to the interaction with
antigen, assembly of the immunologic synapse, and association
of the CAR with other proteins necessary to transduce a robust
activation signal. Most investigators are using the hinge and
transmembrane domains of CD8a or CD28; hinge domains derived
from Fc regions have also been investigated and modied in length6
and have also been reported to engage Fc receptors and activate
innate immune cells.7,8
Investigators in the eld are using a variety of methods to introduce their CAR constructs into T cells.9 Each has advantages and
disadvantages with regard to cost, safety, and level of expression.
Nonviral-based DNA transfection was initially used because of cost
and the low risk of insertional mutagenesis. This method requires
long-term culture and antibiotic selection due to the relative inefciency
of gene transfer. The long-term culture may be detrimental to the

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

Figure 2. Chimeric antigen receptors. CARs target

surface antigens in an MHC-independent fashion and
consist of an ectodomain, hinge domain, transmembrane domain, and endodomain. The initial trials tested
first-generation CARs that have a single cytoplasmic
domain. Current trials are testing second- and thirdgeneration CARs that have combinations of signaling
domains.

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

Review of clinical data with CAR T cells in

hematologic malignancies
There are 14 publications reporting clinical trials of CAR T cells in
hematologic malignancies. All but one of these focused on B-cell
malignancies by targeting CD19 or CD20; the other focused on acute
myeloid leukemia (AML) by targeting Lewis-Y antigen. The CAR
design, manufacturing process, and results are summarized in Table 1.
Each group has designed slightly different protocols, and they
vary with regard to design of the CAR, expression of the CAR on the
T cells, T-cell culture conditions, lymphodepleting strategy, cytokine
support for the infused T cells, disease targeted, and timing of CAR
T-cell infusion with regard to standard therapy such as bone marrow
transplantation. Although the number of variables across trials makes
for challenging comparisons, together the clinical data suggest that
the most effective CAR T cells exhibit high levels of CAR expression
before infusion and expand and persist in vivo, ie, engraft, for at
least several weeks. Not surprisingly, the most effective CAR T-cell
products are also associated with on-target toxicity. The details of
trials conducted at the Fred Hutchinson Cancer Research Center, City
of Hope, Baylor College of Medicine, MD Anderson Cancer Center,
National Cancer Institute, Memorial Sloan-Kettering Cancer Center
(MSKCC), the University of Pennsylvania, and the University of
Melbourne are found in supplemental Materials.19-41
Lessons learned from clinical experience

The results of these clinical trials point to several key factors

that may have an impact on the efcacy of CAR-modied T cells
in hematologic malignancies. One is that the disease under study
may be differentially susceptible to CAR-mediated T-cell killing;

for example, although all express CD19, it appears that acute

lymphocytic leukemia (ALL) has a higher response rate than chronic
lymphocytic leukemia (CLL) or indolent lymphomas, with an
impressive (;80%) response rate across CAR designs, trial designs,
and institutions. This could be true for several reasons, including host
T-cell defects in lymphomas such as those described in CLL
patients,42,43 inhibitory effects of the tumor microenvironment,44,45
the length and nature of prior treatments, the age of the patient, and the
robustness and composition of the T cells in the starting product and in
the infused product. At the level of product characterization, the
relative importance of the CD4:CD8 ratio or the proportion of
regulatory T cells (Tregs) in the nal product is not clear, and it is not
known if the T-cell product can be improved upon by selection or
graft engineering. Characterization of the tumor microenvironment,
and in particular the inhibitory factors that may affect or abrogate
CAR T-cell lytic functions, will be even more complex to sort out.
Both gene expression proling and ow cytometric analysis of
recovered T cells postinfusion show that infused CAR T cells express
PD134,46 and are susceptible to PD1/PD-L1 interactions. Encouragingly, based on preclinical animal modeling,47 checkpoint blockade
is already being tested in clinical trials in combination with CAR
T cells. Several investigators have also aimed to address the question
of whether or which lymphodepletion strategy to use and whether
supporting the infused T cells with systemically administered cytokines will improve their expansion or persistence; although both
strategies appear to be improve T-cell engraftment, they may confound interpretation of efcacy and toxicity, and neither appears to
be universally required for CAR T-cellmediated responses.
At least a few key characteristics of efcacious CAR T-cell products
have emerged. One is that expression of the CAR at the cell surface
seems to be required for efcacy; another is that in vivo detection of the
CAR T-cell product in the blood is a sign of adequate engraftment and
that engraftment is required for responses. Detection of the CAR
transgene by polymerase chain reaction does not inform about the
surface expression of the CAR, which is the only form that matters for
efcacy. Thus, the availability of reagents to specically detect CARs
at the cell surface by ow cytometry is crucial to understand the
activity and engraftment of CAR T cells. In addition, it is not yet clear if
there is a relationship between the dose of CAR T cells administered
and the level of engraftment that is achieved, ie, the in vivo dose. When
CAR T cells expand efciently, very low doses of CAR T cells can still

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

107 CAR1 cells/kg

cyclophosphamide

None or 1.5 g/m2

1.46 3 105 to 1.6 3

2 d)

CD3/28

cells/kg (split over

16 d

0.4-3 3 107 CAR1

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)

Day 28 after ASCT

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

OKT3 1 IL-2; 1 3 108/m2 to 3.3 3

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

8 with CLL, 1 with

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

and 1 ALL with

1-3 wk, only 1 CLL

detectable CAR at

2 CLL patients had

CD3z)

mg at 2 wk in CD28-

(peak 1286 copies/

detectable by PCR

than 6 mo, only

persisted longer

CD28-CD3z

infusions

after 3 of the 7

PCR only at 1 wk

Detectable cells by

detected by PCR

wk with IL-2; only

1-3 wk alone, 5-9

Persistence (peak
and duration)

aplasia)

remission (4 B cell

6 objective

aplasia

lasted 3-6 mo)

19% CAR1 T cells,

(peak at day 13,

cytometry and PCR

Detectable by flow

CAR1 by flow

.20% of the T cells

persisted for 6 mo);

copies/mg and

declined to 10-1000

PBMCs at day 20,

mg of DNA in

.100 000 copies/

and PCR (peak

flow cytometry

cells detectable by

2 CR, 1 PR; 3 B-cell In vivo expansion of

aplasia (ALL)

in LAD, 1 B-cell

1 death, 1 reduction

2 with SD; both NHL

after ASCT

2 maintained CR

PR, 4 SD; all in NHL

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

Table 1. CAR T-cell trials in hematologic malignancies

From www.bloodjournal.org by guest on December 23, 2014. For personal use only.
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;

2 PD, 6 SD, 1 PR, 1

cells/kg (allogeneic

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

at day 12) and PCR

cytometry (2%-7%

Detectable by flow

blood by PCR only

derived)
None

day 21)
Detectable for
counts; 2/6 with

decreased B-cell

4 of 8 patients with

(allogeneic donor

8 patients post-allo

108 total T cells/m2

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,

PBMCs at day ;10)

1 fludarabine

(AML)

2 SD, 1 transient

copies/ug of DNA in

(peak .10 000

(.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,

1.5 3 107 - 1.2 3

cells/kg

5 enrolled, 4 treated

PCR
In vivo expansion of

lasted 3-8 wk) and

relapsed; transient
B-cell aplasia

40% CAR1 T cells,

4 went to allo, 1

cytometry (peak at

Detectable by flow

9-12 mo

PCR only, lasted

Peak at 1 23% by

with frank disease;

MRD2, including 2

5 converted to

B-cell aplasia)

with delayed PR (no

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

allo and post-

1 with

2 (ALL; one post-

treated (ALL)

14 enrolled, 5

(2 MCL, 1 FL)

cyclophosphamide/

None

None

Responses

4 enrolled, 3 treated No progression in 2

Number of
patients

107 CAR1 cells/kg

at day 21

cyclophosphamide

1.5-3 g/m2

at day 22

14 d

SC IL-2 3

Cytokine
support

1.4 3 106 and 1.2 3

cells/kg

OKT3 1 IL-2; 1.4-9.2 3 106 CAR1

beads; 10 d

CD3/28

beads; 14 d

1.5-3 3 106 CAR1

. 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

BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17

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)

From www.bloodjournal.org by guest on December 23, 2014. For personal use only.
ANTIBODY-MODIFIED T CELLS
2629

CLL (randomized to 1 of 2 doses)

CLL

ALL

Auto-HSCT for NHL followed by T-cell

CD19

CD19

CD19

CD19

CLL (residual disease following upfront

CD19

NHL, CLL

ALL, CLL, NHL

ALL, CLL, NHL postallo-HSCT (prophylaxis

CD19

CD19

B-cell malignancies postallo-HSCT

B-cell malignancies postauto-HSCT

Pediatric leukemia and lymphoma

CLL, small lymphocytic lymphoma; MCL,

CD19

CD19

CD19

CD19

CD19

ALL, DLBCL, MCL, NHL, CLL relapsed

CD19/EGFRt

ALL, CLL, NHL

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

CD137-CD3z and CD3z

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

CD284-1BB-CD3z and CD28-

CD28-CD3z

CD28-CD3z

CD28-CD3z

CD28-CD3z

CD28-CD3z

CD28-CD3z; 4-1BBCD3z

Phase/ID

Sponsor

NCT01864889

1/2/

NCT01195480

1/2/

NCT01475058

Chinese PLA General Hospital

University College, London

Center

Fred Hutchinson Cancer Research

Center
1/2/

Fred Hutchinson Cancer Research

1/2/

Seattle Childrens Hospital

City of Hope

City of Hope

National Cancer Institute

National Cancer Institute

National Cancer Institute

MD Anderson Cancer Center

MD Anderson Cancer Center

MD Anderson Cancer Center

MD Anderson Cancer Center

Baylor College of Medicine

Baylor College of Medicine

Baylor College of Medicine

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

from donor; 2nd cohort adds vaccination with

CD19 CAR-transduced EBV-specific CTLs

CD62L1 TCM

Donor-derived, CMV- or EBV-specific

suicide system)

TCM-enriched T cells (cetuximab as possible

TCM-enriched CD81 T cells

T cells from donor

IL-2

IL-2

Low- and high-dose cohorts with and without

Donor derived

(CMV, EBV, and adenovirus) from donor

CD19 CAR-transduced trivirus-specific CTLs

T cells)

Ipilimumab in low-grade disease (2 wk after

2 CARs at the same time

from donor

CD19 CAR-transduced EBV-specific CTLs

T cells from donor

Cell type/selection/drug combination

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.

Pediatric ALL postallo-HSCT

CD19

postallo-HSCT

Relapse/refractory CLL, NHL, or ALL

Pediatric ALL

CD19

CD19/EGFRt

infusion (day 2 or 3)

Auto-HSCT for NHL followed by T-cell

infusion (day 2 or 3)

Auto-HSCT for NHL followed by T-cell

CD19/EGFRt

B-cell malignancies relapsed postallo-HSCT

CD19

Lentivirus
Retrovirus/

4-1BBCD3z

4-1BBCD3z

4-1BBCD3z

CAR signaling domain

MAUS et al

CD19

follicular lymphoma, large-cell lymphoma

CLL

Leukemia/lymphoma postcord blood HSCT

CD19

or therapy)

Pediatric relapsed B-cell ALL

CD19

CD19

pentostatin/cyclophosphamide/rituximab)

Relapsed ALL postallo-HSCT

CD19

infusion

ALL (postallo-HSCT)

CD19

Lentivirus

Lentivirus

CD191 malignancies

CD19

Gene transfer
Lentivirus

Cancers

Pediatric B-cell leukemia and lymphoma

2630

CD19

Antigen

Table 2. Ongoing CAR T-cell trials in hematologic malignancies

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BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17

From www.bloodjournal.org by guest on December 23, 2014. For personal use only.

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

Chinese PLA General Hospital

1/2/

NCT01886976

CD137-CD3z and CD3z

Retrovirus
Relapsed and/or chemotherapy resistant
CD138

multiple myeloma

Chinese PLA General Hospital

1/2/

NCT01864902

Retrovirus
Relapsed/refractory CD331 AML
CD33

CD137-CD3z and CD3z

Chinese PLA General Hospital

NCT01735604

1/2/
4-1BBCD3z
Retrovirus
CD201 leukemia and lymphoma
chain

CD20

Baylor College of Medicine

1/NCT00881920
Ig k light

Lymphoma, myeloma, leukemia

Retrovirus

CD28-CD3z

University of Cologne

Peter MacCullum Cancer Centre,

CD30 CAR-transduced EBV-specific CTLs

Baylor College of Medicine

1/NCT01645293
CD28-CD3z
CD30

Retrovirus

NHL, HL

Mycosis fungoides/CTCL

CD30

Retrovirus

CD28-CD3z

1/NCT01192464

Cell type/selection/drug combination

Sponsor

Baylor College of Medicine

Phase/ID

1/NCT01316146

CAR signaling domain

CD28-CD3z
Retrovirus
NHL, HL

Gene transfer
Cancers
Antigen

CD30

Table 2. (continued)

ANTIBODY-MODIFIED T CELLS

41

78

Reference

BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17

2631

exert dramatic effects and control tumor33; given the complexity of

manufacturing CAR T cells, the ability to administer low doses of an
effective product is very appealing.
Some degree of persistent engraftment is also required, although
the length of this persistence has not been established. We hypothesize
that for CAR T cells to be able to replace allogeneic transplantation as
denitive therapy, persistence for at least several months will probably
be required based on the kinetics of tumor clearance that we have
observed.37 If, on the other hand, CAR T cells are only to serve as
a bridge to follow on therapy with allogeneic transplant, then they
may only need to persist for a few weeks until conditioning for the
transplant begins. The question of whether CAR T cells are on par with
the efcacy of transplant would best be answered in randomized trials.
However, short of this, we also anticipate many recipients of CAR
T cells will not eligible for transplant or have comorbidities or only
suboptimal donors available such that the transplant is impractical.
Long-term follow up of these patients will provide critical information
about the durability of CAR T-cellinduced remissions. Alternatively,
the answer may come from patients who have relapsed after an
allogeneic transplant, for whom a second transplant may not be
possible or efcacious; these patients may benet from CAR T-cell
products that potentially offer durable remissions. The use of CARmodied donor T cells to treat relapsed ALL is being tested by our
group and will hopefully be studied in a multisite trial. In addition,
multisite trials are underway in collaboration with Novartis, and
a dual-center grant-funded trial between MSKCC and the University
of Pennsylvania involves a competitive repopulation study design
where T cells transduced with Penn vector and MSKCC vector are
infused simultaneously into each patient.

Toxicities and management: from CRS,

CNS, and MAS/hemophagocytic
lymphohistiocytosis to B-cell aplasia
Given the nding that, in most cases, cytokine release syndrome (CRS)
seems to correlate with antitumor activity, one question that has emerged
is the degree to which the innate immune system contributes to antitumor
efcacy. In addition, we have shown that CRS is often accompanied by
a macrophage activation syndrome (MAS), which may be driven in part
by high levels of IL-6.37 Although it is straightforward to hypothesize
that CAR T cells directly kill tumor cells, it is not entirely clear which cell
type produces the vast majority of the cytokines, particularly IL-6
(which our work has demonstrated may be key to the toxicity
response),37,51 and whether blockade of cytokines with anticytokine therapy such as tocilizumab or general immune suppression
with corticosteroids affects the antitumor response. It is possible that
the IL-6 is produced by the dying B cells, dying tumor cells, or
activated macrophages that are recruited to digest lysed tumor cells.
Does interruption of the cytokine cascade lead to interruption of
the antitumor effect? This remains an unanswered question and has
direct clinical impact for patients and physicians deciding on when
to abort the CRS. Furthermore, although, in our experience, most
responding patients have some degree of CRS, it is not yet clear
whether the severity of CRS or macrophage activation syndrome
(MAS) is related to antitumor efcacy. The severity of CRS does
appear to be related to the tumor burden. If engagement of the innate
immune system contributes to the mechanism of action, this could
bode well for the use of CAR T cells in solid tumors, where T cells
may not preferentially home to and persist at the sites of tumors as
efciently as they do in hematologic malignancies.

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MAUS et al

Several patients in CD19-CAR trials across institutions have

experienced obtundation, seizures, aphasia, and mental status changes,
which have all been reversible. Some of these may be related to CRS,
but whether this results from systemic cytokines crossing the bloodbrain barrier and engaging cytokine receptors in the brain or from direct
cytokine production in the central nervous system (CNS) is not clear.
Many of these patients develop MAS, and MAS is often associated
with neurologic toxicity.38-40 In addition, we have unexpectedly found
CAR T cells in the cerebrospinal uid of asymptomatic patients, even
when there is no evidence of CD191 disease there. It is possible that
the hyperthermia and IL-6 release during CRS enhances trafcking
of CAR T cells to the cerebrospinal uid in an antigen-independent
mechanism.48 It is also possible that there is some cross-reactivity or asyet-undetected expression of CD19 in the brain. Blinatumomab, a type
of bispecic T-cellengaging antibody (BiTE) that is a fusion protein
between an anti-CD19 scFv and an anti-CD3 scFv, also has neurologic
toxicity and seizures as its dose-limiting toxicity, even though it does
not appear to control CNS disease. It is interesting that blinatumomab
has also been shown to cause MAS.49 Optimistically, CAR T cells may
provide a way of controlling occult or frank CNS malignancy without
chemotherapy or radiotherapy.
B-cell aplasia is an expected on-target result of CD19-directed
therapies and has served as useful surrogate to determine the persistence and effectiveness of CD19-directed CAR T cells. Fortunately,
B-cell aplasia is a manageable disorder; patients may be infused with
g-globulin as replacement therapy, though this could become an
expensive and difcult treatment to implement across all diseases
that may be eventually treated with CAR T cells. Persistent B-cell
aplasia could also result in an increased risk of infection even with
replacement therapy. In an ideal setting, the CAR T cells would
persist long enough to mediate denitive control of disease but then
allow for recovery of normal B-cell and plasma cell recovery such
that patients could be revaccinated.
As more patients are treated with CAR T cells directed to CD19,
clinician investigators will need to establish straightforward algorithms for management of toxicities, including the optimal timing
and dose of administration of cytokine blockade, corticosteroids, and
immunoglobulin replacement.
Because gene-modied T cells are emerging as powerful therapies
capable of effecting dramatic antitumor responses as well as signicant
toxicities,50 strategies to incorporate suicide genes or abortive mechanisms may become necessary. This is especially true as CAR-directed
T cells are further engineered to express cytokines and adjuvants51,52
that could act in trans to amplify inammatory cascades, or as CAR
T cells directed to antigens with more widespread expression are
tested. However, suicide systems are still difcult to implement in all
CAR T-cell trials, because many of the suicide systems are immunogenic (eg, herpes simplex virus thymidine kinase) or require intravenous administration of the suicide-inducing prodrug.53 Alternatively,
altering the homing of T cells via enforced expression of chemokine
receptors54 or pharmacologic blockade of chemokine receptors55 may
be a strategy to both enhance efcacy and alleviate toxicity.

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

Although CAR T cells have been developed entirely in the academic

setting, the recent FDA publication of a Draft Guidance for Industry
on early phase trials of cell and gene therapies63 indicates a shift
of this concept from a fringe research topic to acceptance as a
mainstream investigational product. One mandate of the guidance is
for sponsors to attempt to dene the active ingredient in the cell or
gene therapy product. For gene-modied T cells, there are multiple
factors that contribute to the denition of the active ingredient:
optimal vector, culture conditions, CAR design, cell type, and dose
of that cell type. The simplest way to dene the active ingredient at
the moment is the number of CAR1 cells. However, the precise type
of cell that is transduced may be important in dening the active
ingredient as well. For example, it may be that only the central
memory CD81 T cells contribute to engraftment and therefore are
the only cell type that contributes to the active ingredient.
Most investigators have focused on peripheral bloodderived
T cells and subsets of these such as virus-specic T cells, central
memory T cells, or cord bloodderived T cells.22 However, there is
also potential for the use of multilineage effector cells when hematopoietic stem cells or other precursor cells are used as the starting
cell type.64-66 Dening the phenotype or active ingredient of these
cell types will be even more challenging than T cells. Finally, NK
cells can also be powerful effector cells, and some investigators have
transduced NK cells with second-generation CARs.67 Although
technically outside the scope of this review, BiKEs (bispecic
killer engagers) engage NK cells with bispecic antibodies to the
target antigen (CD33) and the NK activator CD1668; these are a
corollary to BiTEs, which are bispecic engagers of T cells, like
blinatumomab; both of these engage the immune system with an
antibody-like structure.
New targets

Future outlook
Manufacturing

All investigators involved in CAR T-cell trials are acutely aware of

the technical, regulatory, and nancial challenges in manufacturing
single-patient product lots. One potential solution is to generate

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

encouraging results with either antibody-drug conjugates or naked

antibodies such as CS1-elotuzumab72 and CD38-daratumomab.73
In AML, there are currently no open trials of CAR-modied
T cells in the United States, though there are trials open in China and
in Australia targeting CD33 and Lewis-Y, respectively (Table 2).
Based on the clinical experience with the anti-CD33 drug conjugate
gemtuzumab ozogamicin (Mylotarg), a CD33-directed CAR may
also be evaluated, and preclinical data with a CD33/CD3 BiTE is
encouraging.74 Preclinical data of CD123-directed CARs have been
reported by 2 groups.75,76 A CD44v6 CAR has also been tested in in
vitro and in xenogeneic models of AML and myeloma77; these investigators found that in vitro activation with anti-CD3/28 beads and
culture in IL-7 and IL-15 were necessary for antitumor efcacy in vivo.
Baylor has 2 open clinical trials targeting CD30 in Hodgkin
disease, and the University of Cologne plans to treat patients with
mycosis fungoides with CD30 CAR T cells encoding a CD28
domain with a deleted lck binding moiety, based on preclinical
data that this reduces Tregs in the tumor microenvironment.78
There are also open trials targeting the immunoglobulin G k light
chain in B-cell malignancies including lymphoma, myeloma, and
leukemia; attractive aspects of this approach are the suitability of
targeting myeloma and that theoretically, on-target toxicity will
be a more limited B-cell aplasia than what is seen with CD19
CARs that target all B cells. Finally, although no CD19-negative
escape variants of CLL have been described, other potential
targets for CLL are CD23 and R0R1, and preclinical data are
promising.79,80

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

the pharmaceutical and biotechnology industries as well as

cooperative group networks. This is an exciting time in the
treatment strategies for all hematologic malignancies; a decade ago,
few would have predicted that the promises of gene-modied
cell therapies would be delivered by CAR T cells directed to
aggressive hematologic malignancies such as adult and pediatric
B-cell ALL.

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: [email protected].

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2014 123: 2625-2635

doi:10.1182/blood-2013-11-492231 originally published
online February 27, 2014

Antibody-modified T cells: CARs take the front seat for hematologic

malignancies
Marcela V. Maus, Stephan A. Grupp, David L. Porter and Carl H. June

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