Batlevi Rev Immunotherapy

REVIEWS
Novel immunotherapies in lymphoid

malignancies
Connie Lee Batlevi1, Eri Matsuki1, Renier J. Brentjens2 and Anas Younes1
Abstract | The success of the anti‑CD20 monoclonal antibody rituximab in the treatment of
lymphoid malignancies provided proof-of-principle for exploiting the immune system
therapeutically. Since the FDA approval of rituximab in 1997, several novel strategies that harness
the ability of T cells to target cancer cells have emerged. Reflecting on the promising clinical
efficacy of these novel immunotherapy approaches, the FDA has recently granted ‘breakthrough’
designation to three novel treatments with distinct mechanisms. First, chimeric antigen receptor
(CAR)-T‑cell therapy is promising for the treatment of adult and paediatric relapsed and/or
refractory acute lymphoblastic leukaemia (ALL). Second, blinatumomab, a bispecific T‑cell engager
(BiTE®) antibody, is now approved for the treatment of adults with Philadelphia-chromosome-
negative relapsed and/or refractory B‑precursor ALL. Finally, the monoclonal antibody nivolumab,
which targets the PD‑1 immune-checkpoint receptor with high affinity, is used for the treatment of
Hodgkin lymphoma following treatment failure with autologous-stem-cell transplantation and
brentuximab vedotin. Herein, we review the background and development of these three distinct
immunotherapy platforms, address the scientific advances in understanding the mechanism of
action of each therapy, and assess the current clinical knowledge of their efficacy and safety. We
also discuss future strategies to improve these immunotherapies through enhanced engineering,
biomarker selection, and mechanism-based combination regimens.
The concept of immunotherapy for treating cancer of cell-based therapy directed at TAAs expressed on the
emerged almost a century ago; the graft-versus-tumour tumour-cell surface, typically CD19 in B‑cell malignan-
effect following allogeneic haematopoietic-stem-cell cies (BOX 1). Antibody-based therapies include a variety of
transplantation (HSCT) was one of the first examples of immune-checkpoint-inhibitor-based therapies that either
immunotherapy 1. Furthermore, the success of rituximab block anergic signals from tumour cells, or enhance T‑cell
in treating lymphoid malignancies provided proof-of- activation directly. Bispecific T‑cell engagers (BiTE®)
principle for exploiting the immune system in a target- direct T cells to target TAAs (FIG. 1).
specific manner 2–4. With improved technology and a The three distinct classes of drugs, CAR T cells,
better understanding of immune-regulatory mechanisms, bispecific antibodies and immune-checkpoint inhibi-
cancer immunotherapy is rapidly evolving to exploit the tors, have been granted ‘breakthrough’ designation by the
therapeutic value of activating autologous T cells. US FDA; one such agent, the BiTE® blinatumomab, has
The types of immunotherapy available for haemato already received approval by the FDA for the treatment of
1
Lymphoma Service, logical malignancies range from cell-based to antibody- Philadelphia-chromosome (Ph)-negative relapsed and/or
Department of Medicine,
Memorial Sloan Kettering
based therapies. Early attempts with cell-based therapies refractory B‑precursor ALL (B-ALL). Each treatment
Cancer Center. focused on the adoptive transfer of cytotoxic T lympho- approach is based on unique platforms that will probably
2
Leukemia Service, cytes (CTLs) that targeted tumour-associated antigens encourage development of further therapeutic agents in
Department of Medicine, (TAAs). The success of this approach using WT‑1‑specific the future. In this article, we review these platforms, and
Memorial Sloan Kettering
and Epstein–Barr virus (EBV)-specific CTLs has been discuss the emerging clinical activity and unique toxicity.
Cancer Center, 1275 York
Avenue, Box 330, New York, reported for various lymphoproliferative disorders,
NY 10065, USA. including acute lymphoblastic leukaemia (ALL), Engineered CAR T cells
Correspondence to A.Y. Hodgkin lymphoma (HL), and post-transplantation CAR T cells are autologous T lymphocytes that are
younesa@mskcc.org lymphoproliferative disorder (PTLD)5–9. The excitement genetically engineered to express the binding site of
doi:10.1038/nrclinonc.2015.187 of cell-based therapy was followed by the use of engi- specific antibodies, thereby directing the autologous
Published online 3 Nov 2015 neered chimeric antigen receptor (CAR) T cells, a type polyclonal T cells to bind a specific TAA. The construct
NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 25
© 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS
Key points Cancer Research Center (Seattle, WA, USA) use a lenti

viral transfection system25–27. The MD Anderson Cancer
• Immunotherapies that activate T‑cell responses against tumour cells have been Center (Houston, TX) continues to develop the Sleeping
successful in the treatment of lymphoid malignancies Beauty transposon system, which combines the advan-
• Second-generation chimeric antigen receptor (CAR) T cells targeting tages of viruses and naked DNA. The advantages and
CD19‑expressing B cells have shown promise in B‑lymphoid malignancies limitations of each approach have not been fully eluci-
• Both CAR-T‑cell therapy and blinatumomab produce adverse effects related to T‑cell dated at this point; however, potential differences include
activation, in the form of cytokine-release syndrome and central-nervous-system- the expression level of CARs, persistence of cells, safety
related symptoms (including the potential for carcinogenesis), manu
• Immune-checkpoint inhibitors demonstrated significant clinical activity against facturing efficacy, and costs. Lentiviral and retroviral
Hodgkin lymphoma delivery could potentially result in integration of the
• Further understanding of each of these treatment modalities will establish the role of CAR construct proximally to growth-promoting genes,
immunotherapy as a key component in the management of lymphoid malignancies leading to malignant transformation. In the cumulative
experience of using viral-based CAR T cells, insertional
mutagenesis has not been reported. Transposon systems
is composed of a single-chain variable fragment (scFv) have a lower risk of insertional mutagenesis, but CAR
of an antibody fused to the activating intracellular- transgene expression is much lower with this approach.
signalling domain of the T‑cell receptor (TCR), typi The antigen-binding domain varies between the differ-
cally the ζ signalling domain (FIG. 2a)10–12. Polyclonal CAR ent CAR constructs, with most researchers using either
T cells recognize their target antigen through the anti- the mouse hybridoma derived FMC63 or SJ25C1 CD19
body domain resulting in T‑cell activation independent scFv constructs28,29. Both constructs have been developed
of major histocompatibility complex (MHC) presenta- to target CD19‑positive cells of haematological malig-
tion13. The scFvs are constructed by cloning the heavy nancies, and have shown efficacy in various in vitro and
and light chain variable regions of an antigen-specific in vivo models. However, whether differences in CAR
monoclonal antibody, separated by a short peptide constructs or the inherent design of CAR T cells pro-
linker, into a single polypeptide14–16. DNA encoding this vides advantages over other immunotherapies remains
construct can be transduced ex vivo using transfection, unclear, as these approaches have not been compared in
gamma retroviral or lentiviral recombinant vectors, or a randomized controlled trials21,30,31.
transposon system17–22. Various CAR-T‑cell constructs CAR T cells can be further modified to increase
exist with distinct scFvs and signalling domains (FIG. 2b). their efficacy and durability by the incorporation of co-
Knowledge of CD19‑directed CAR T cells is more estab- stimulatory domains (FIG. 2c). Clinical studies of first-
lished than that of other forms, with published studies generation CAR T cells that were generated to treat B‑cell
from the Memorial Sloan–Kettering Cancer Center malignancies (by targeting CD20 or CD19 antigens)
(MSKCC; New York, NY, USA), the University of demonstrated the feasibility of this approach; however,
Pennsylvania (UPenn; Philadelphia, PA, USA), and the these engineered cells lacked significant antitumour
National Cancer Institute (NCI; Bethesda, MA, USA). activity, probably because of inadequate CAR-T‑cell
CAR‑T‑cell constructs from the MSKCC, the NCI, and persistence32,33. Second-generation and third-generation
also the Baylor College of Medicine (Houston, TX, USA) CAR designs incorporated one or two co-stimulatory
share a common gammaretroviral vector and a CD28 signalling domains. Second-generation receptors are
signalling domain20,23,24. By comparison, constructs capable of delivering both a primary activation signal
developed at the City of Hope Comprehensive Cancer through the TCR ζ‑chain as well as a co‑stimulatory signal
Center (Duarte, CA, USA), UPenn, and Fred Hutchinson through the CD28 or 4‑1BB domains in the cytoplasmic
tail34,35. Clinical studies showed that second-generation
CARs resulted in improved in vivo expansion and per-
Box 1 | Glossary of terms sistence of the transfected T cells24. Third-generation
CARs contain two co-stimulatory domains, with the first
• Chimeric antigen receptor T cells: engineered
consisting of a CD28 or 4‑1BB domain and the second
receptors with specificity of a monoclonal antibody
grafted onto a T cell
provided by other molecules, such as OX40, CD28, or
4‑1BB36–39. Fourth-generation ‘armoured CAR’ T cells
• Bispecific monoclonal antibodies: fusion proteins
are engineered to additionally express cytokines or co-
composed of fragments of two different monoclonal
antibodies and therefore binds two different antigens stimulatory ligands, which aim to enhance expansion and
longevity of the CAR T cells40. Additional innovations in
• Immune-checkpoint receptors: cell-surface molecules
expressed by T cells or normal tissue that helps the technology include introduction of a suicide-gene
maintain self-tolerance and control the intensity and system, which can be activated to control the expansion
duration of an immune response of CAR T cells and thereby minimize excessive toxicity 41.
• Overall response rate: reduction in tumour burden The efficacy of CAR-T‑cell therapy can be improved by
meeting criteria for complete or partial responses modulating homing mechanisms through expression
• Complete response: disappearance of all target lesions of chemokine receptors, such as CCR4 or CXCR2, on
the modified T cells, or by including lymphodepleting
• Partial response: at least a 50% reduction in the sum of
longest diameters for all target lesions chemotherapy 42,43. Preconditioning lymphodepleting
therapy decreases antigen load by reducing the number
26 | JANUARY 2016 | VOLUME 13 www.nature.com/nrclinonc

REVIEWS
CAR construct 19,26, or at some centres, electroporation

is used to introduce a transposon or plasmid to the
PD1 TCR
Native
activated T cells22,33. Finally, the T cells are expanded
T cell 1,000‑fold via co-stimulation with CD3 and CD2817,47.
CTLA-4 CAR-T‑cell doses that range from 1.5 × 10 6/kg to
3 × 107/kg are achieved over a culture period of 1–2 weeks.
Engineered
T cell
BiTE® Immune This product contains a mixture of CD4‑positive and
CD3 PD1 checkpoint CD8‑positive T cells as well as regulatory and memory
inhibitors
TCR
T cells, at varying ratios. CAR T cells are infused into
the patient over 1–2 days as single or split doses — the
latter is preferred for safety and monitoring of immedi-
ate toxicities. Inpatient admission is commonly required
CAR to monitor for CAR-T‑cell-related toxicities including
MHC I/II PD-L1 cytokine-release syndrome (CRS) and central nervous
CD19 PD-L2 system (CNS) toxicities. The duration of admission is
variable and can be as short as 5–10 days, with discharge
dictated by the patient’s clinical condition. In vivo, CAR
Naked
mAb T cells expand 1,000–10,000‑fold with T‑cell persis-
CD19 tence of weeks to years, although prolonged T‑cell
CD20 Malignant persistence is found only in a minority of patients. In
CD22 cell the following section, we review the published clinical
ADC trial data on the use of CAR T cells in patients with
lymphoid malignancies.
Clinical data with CAR T cells

Acute lymphoblastic leukaemia. To date, CAR‑T‑cell
therapies have been most efficacious in patients with
Figure 1 | Mechanisms of action of immunotherapy modalities.
Nature ReviewsNative T cells
| Clinical can
Oncology B‑cell ALL (TABLE 1). At the MSKCC, most patients
recognize tumour-specific antigens in an MHC-dependent manner. The T cells also treated with CAR T cells have been adults, whereas
require co-stimulation for activation. Upon antigen recognition, without co-stimulatory
other groups treated both paediatric and adult patients.
signal, or with the stimulation of inhibitory molecules, such as through the PD‑1–PD‑L1
Despite differences in the CAR constructs used, condi-
axis, the T cells can be induced to anergy or become exhausted. Immune-checkpoint
inhibitors can block the inhibitory signal of T cells to avert T cells from anergy. BiTE® tioning regimens, infused T‑cell doses and patient popu-
antibodies bring T cells and malignant cells into close proximity through dual antigen lations, three published studies reported similar complete
binding, and can induce T‑cell activation without co-stimulatory signals. T‑cells can also response (CR) rates of 70–90%44,48–52. Investigators at the
be engineered to express CARs to recognize cell-surface molecules independent of MSKCC published the first report of CAR‑T‑cell ther-
MHC. Later-generation CARs have both TCR and co-stimulatory signalling components, apy in patients with ALL using a second- generation
thereby activating the T cells without additional co-stimulatory signal. Abbreviations: CD19‑targeted CAR T cell with both a TCR zeta-chain
ADC, antibody–drug conjugate; BiTE®, bispecific T‑cell engager antibody; CAR, chimeric and CD28 signalling domain (19‑28z CAR T-cell). One
antigen receptor; CTLA‑4, cytotoxic T‑lymphocyte-associated protein 4; mAb, patient was treated with 19‑28z‑CAR T cells after sec-
monoclonal antibody; MHC, major histocompatibility complex; PD‑1, programmed cell
ond remission presented with prolonged B‑cell aplasia
death protein 1; PD‑L1, programmed cell death 1 ligand 1; TCR, T‑cell receptor.
while waiting for allogeneic HSCT44. The patient was
successfully treated with allogeneic HSCT, but died unex-
pectedly of pulmonary embolism 2 months post HSCT
of tumour cells, and also depletes immunosuppressive while in complete remission from ALL49. A follow-up
cells in the tumour microenvironment, which promotes study with 19‑28z‑CAR T cells demonstrated a high
pro-survival cytokine signals that lead to expansion and response rate, whereby all five treated patients achieved
persistence of CAR T cells. Clinical studies in lymphoid remission and tested negative for minimal residual dis-
malignancies have focused on second-generation CAR ease (MRD)48. On the basis of data from 22 evaluable
T cells that target CD19‑expressing B‑cell malignancies; patients treated at MSKCC, the median overall survival
however, the use of different CAR constructs and trans- after 19‑28z‑CAR-T-cell therapy was 9 months49,50. The
fection methods between clinical t rials hampers the ability number of treated patients is small, with limited follow
to compare results across d
ifferent groups (FIG. 2b)22,44–46. up; however, the response rate and survival rates have
Generation of clinical-grade CAR T cells begins with generated a considerable excitement, considering that
apheresis of a patient’s peripheral blood mononuclear more than half of the patients had undergone multiple
cells (PBMCs) for ex vivo transduction and expansion. lines of treatment before CAR-T‑cell therapy53–55. At
Apheresis occurs over 1–2 days and the product is fro- UPenn, 30 patients with ALL who were treated with CAR
zen until the time of transduction. Isolated PBMCs are T cells demonstrated 6‑month event-free-survival (EFS)
then thawed and the T cells are activated and selected and overall-survival rates of 67% and 78%, respectively51.
by incubation with anti‑CD3 and anti‑CD28 para Outcomes of 20 patients treated at the NCI revealed a
magnetic beads. The activated T cells are then trans- leukaemia-free survival rate of 79% at 5 months52. At all
duced with retroviral or lentiviral vectors carrying the three centres, CAR T cells have been given successfully

REVIEWS
a B-cell receptor (BCR) T-cell receptor (TCR) Chimeric antigen receptor (CAR)
• Antigen recognition by BCR
• Signalling through TCR,
independent of MHC
VH VH MHC I/II
VL VL
Antigen binding VH VH
scFV VL VL
CD3 CD3 Hinge
Transmembrane
βα αβ δε εγ
CD79 CD79 ζζ
CD3
Signalling/
co-stimulation CD3ζ
Signalling Signalling Signalling
b 19-28z 19-28z 19-BBz

FMC63 SJ25C1 FMC63
VH VL VH VL VH VL
CD28 CD28 4-1BB/CD137
CD3ζ CD3ζ CD3ζ
Institute Transfection Hinge/ Institute Transfection Hinge/ Institute Transfection Hinge/

transmembrane transmembrane transmembrane
NCI Retrovirus CD28 MSKCC Retrovirus CD28 U Penn (CTL019) Lentivirus CD8
Baylor Retrovirus IgG-CD28 Fred Hutchinson Lentivirus IgG1-CD4
City of hope Lentivirus IgG4-Fc
MDACC Sleeping beauty IgG4-Fc
Fred Hutchinson Lentivirus IgG1-CD4
c 1st generation 2nd generation 3rd generation 4th generation

Dual signalling Multiple signalling (armoured)
scFV
Antigen
binding VH VL VH VL VH VL VH VL
Cytokines Co-
stimulatory
Hinge ligand
Trans-
membrane
Co-stimulatory-1
Signalling/ (CD28, 4-1BB/CD137)
co-stimulation CD3ζ
Co-stimulatory-2
CD3ζ CD3ζ
CD3ζ • Cytokine transgene

• Co-stimulatory ligand
transgene
Nature Reviews | Clinical Oncology


REVIEWS
even in a post-allogeneic-HSCT setting, without the achieved a CR in the bone marrow, but had progressive
induction of graft-versus-host-disease (GVHD)49,51,52. disease in the lymph nodes, and three patients achieved a
In the aforementioned studies, persistence of CAR PR59. These early results published in abstract form sug-
T cells varied considerably. CAR-T‑cell expansion in vivo gested that CAR T cells might be more effective against
peaks at approximately 14 days post-infusion 49,51,52. CLL cells residing in the bone marrow compared with
CD28‑based constructs typically persist for 2–3 months, disease in the lymph nodes.
whereas 4‑1BB‑based constructs can persist beyond A pilot study at UPenn, in 14 patients with relapsed
2 years in a small subset of patients49,51,52. Disease relapses and/or refractory CLL, demonstrated an overall
can be associated with a lack of CAR-T‑cell persistence response rate (ORR) of 57%, with three outcomes fully
and immune escape via a CD19‑negative malignant published and the remainder presented in abstracts45,60,61.
clone, although complete remissions lasting longer than Preconditioning chemotherapy varied and included
1 year have been noted in patients even when CAR fludarabine, pentostatin, cyclophosphamide, or benda-
T cells could not be detected beyond 2 months after mustine. In this study, CAR T cells were administered
infusion49,51,52. The optimal length of CAR-T‑cell persis- over 3 days. Six patients had detectable CAR T cells for
tence remains unknown. Future development of CAR at least 5 months, and some were detectable 3 years after
T cells for the treatment of patients with B‑ALL will infusion45,60,61. A subsequent phase II study in patients
include administration of donor-derived CAR T cells with relapsed and/or refractory CLL, with data pub-
after an allogenic HSCT as maintenance therapy or lished in abstract form62, confirmed the initial results,
salvage therapy 56,57. although the ORR was slightly lower at 35% among 23
evaluated patients; T‑cell persistence in these patients
Chronic lymphocytic leukaemia. The role of CAR has not been reported.
T cells in the treatment of chronic lymphoc ytic leu- Investigators at the NCI treated four patients with
kaemia (CLL) is evolving. Initial studies at the MSKCC CLL with preconditioning fludarabine and cyclophos-
in heavily pretreated patients with relapsed/refractory phamide before CAR-T‑cell infusion, and the treatment
and bulky disease who were treated with CAR T cells was supplemented with IL‑2 to promote T‑cell expansion.
without preconditioning chemotherapy demonstrated This approach resulted in an ORR of 75%63. IL‑2 admin-
no responses44. Follow-up studies that incorporated istration was associated with more prominent toxicities,
cyclophosphamide conditioning demonstrated better such as hypotension, fevers, fatigue, renal failure, and
results, with two of four patients achieving stable dis- obtundation, that can overlap with symptoms of CRS.
ease (SD), one patient achieving a CR and one achieving Elimination of IL‑2 in subsequent studies resulted in
a partial response (PR)44,58. Currently, CAR T cells are similar efficacy, with an ORR of 100% in four patients
being studied as consolidative therapy for patients with (three patients with CR, and one patient with a PR)64. The
MRD following frontline chemotherapy with pento duration of response (DoR) ranged from 4–22 months.
statin, cyclophosphamide and rituximab (PCR)59. Of
seven patients, one patient achieved a CR, two patients Non-Hodgkin lymphoma. The cumulative experience of
CAR-T‑cell therapy in patients with non-Hodgkin lym-
phoma (NHL) is predominantly generated in patients
◀ Figure 2 | General structure of CAR. a | CARs are created by the fusion of a with diffuse large-B‑cell lymphoma (DLBCL) or follicular
tumour-specific scFv antibody to either the TCR-associated CD3ζ signalling domain or lymphoma (FL). The NCI investigators first reported a PR
another intracellular signalling domains from co-stimulatory protein receptors. The scFvs lasting 32 weeks in a patient with FL46. The same group
are constructed by cloning the heavy and light chain variable regions of a later published results from four patients with indolent
tumour-specific mAb, separated by a short peptide linker, into a single polypeptide. This lymphoma: three patients with FL and one patient with
structure allows CARs to have the tumour specificity of BCR, and to activate T cells splenic marginal zone B‑cell lymphoma (SMZL)63. Three
through TCR independently of MHC. CARs can recognize various cell-surface molecules, patients were evaluated for response, all of whom achieved
including proteins, carbohydrate, and glycolipid structures. b | Structure of a PR63. In a subsequent study in patients with chemo-
first-generation to fourth-generation CARs. The first-generation CAR contains one
therapy-refractory DLBCL, four out of seven patients
intracellular signalling domain, typically with the CD3ζ signalling domain to allow for
achieved a CR, two achieved a PR, and one achieved SD64.
TCR signalling. The second-generation CARs have two intracellular signalling domains:
a co-stimulatory domain comprising either a CD28 or a 4‑1BB signalling domain, coupled In addition, one patient with low-grade NHL achieved
with a CD3ζ signalling domain. This arrangement enables T-cell activation and a CR and another patient with SMZL achieved a PR64.
proliferation upon antigen recognition by the scFv region of the CAR. The The DoR assessed in six patients was >12 months. The
third-generation CARs have two co-stimulatory domains and a CD3ζ signalling domain. NCI group’s current approach of using a reduced dose
The first co-stimulatory domain is either a CD28 or a 4‑1BB domain, with the second of fludarabine and cyclophosphamide to minimize toxi
co-stimulatory domain consisting of either a CD28, a 4‑1BB or a OX40 domain. city was reported to result in an ORR of 66.7%, with five
Fourth-generation ‘armoured CAR T cells’ combine a second-generation CAR with the of eight patients responding — one with a CR and four
addition of various genes, including cytokine and co-stimulatory ligands, to enhance with a PR65. In a proof-of-concept study using donor-
the tumoricidal effect of the CAR T cells. c | Common second-generation CAR T cells.
lymphocyte-derived CAR T cells, the NCI group treated
Each academic centre has developed and studied slightly different CAR constructs.
patients with B‑cell malignancies who relapsed after allo-
Abbreviations: Baylor, Baylor College of Medicine; BCR, B-cell receptor; CAR, chimeric
antigen receptor; mAb, monoclonal antibody; MDACC, MD Anderson Cancer Center; geneic HSCT; the patients were infused with CAR T cells
MHC, major histocompatibility complex; NCI, National Cancer Institute; Fred generated from the PBMC of their allogeneic-stem-cell
Hutchinson, Fred Hutchinson Cancer Research Center; scFv, single‑chain variable donor57. Remarkably, no increase of GVHD was seen, and
fragment; TCR, T-cell receptor; UPenn, University of Pennsylvania. among 10 treated patients, one patient with CLL achieved

REVIEWS
Table 1 | Clinical efficacy of second generation CAR-T‑cell therapy

Disease and Number Conditioning therapy Infused CAR T-cell Response rate Survival
treating of dose outcomes
ORR CR PR SD
institute patients
(%) (%) (%) (%)
ALL
MSKCC44,48–50 22 CY (1.5–3.0 g/m2) 1–3 × 106/kg NA 91 NA NA Median OS:
(16* + 6‡) 9 months
UPenn51 30* FLU (30 mg/m2 × 4 days)/CY (500 mg/m2 × 2 days): 13, 0.76–14.96 × 106/kg NA 90 NA NA NA
FLU (30 mg/m2 × 4 days)/CY (300 mg/m2 × 2 days): 2,
CY (440 mg/m2 × 2 days)/VP (100 mg/m2 × 2 days): 5,
CVAD (CY 300 mg/m2 q12h × 3 days, vincristine 2 mg
day 3, doxorubicin 50 mg/m2 day 3): 2, CY (300 mg/m2
q12h × 3 days or 1,000 mg/m2 × 1 day): 3, clofarabine
30 mg/m2 × 5 days: 1; VP (150 mg/m2 × 1 day)/Ara-C
(300 mg/m2 × 1 day): 1
None: 3
NCI 52
20* FLU (25 mg/m2 × 3 days)/CY (900 mg/m2 × 1 day) 1 or 3 × 106/kg NA 70 NA 15 RFS:
78.8% at
4.8 months
Fred 7‡ Lymphodepleting chemotherapy 2 × 105/kg, 2 × 106/kg, NA 71.4 NA NA NA
Hutchinson88 or 2 × 107/kg
CLL
UPenn45,60,61 14 FLU (30 mg/m2 × 3 days)/CY (300 mg/m2 × 3 days): 3, 0.14–5.9 × 108 57.1 21.4 35.7 NA NA
(3* + 11‡) pentostatin/CY§: 5, bendamustine§: 6
UPenn62 23‡ Lymphodepleting chemotherapy 5 × 107 or 5 × 108 39 22 17 NA NA
NCI63 4* FLU (25 mg/m2 × 5 days)/CY (60 mg/kg × 2 days) + i.v. 0.3–3 × 107/kg 75 25 50 25 NA
IL‑2 following CAR-T-cell infusion
NCI64 4* FLU (25 mg/m2 × 5 days)/CY (60 or 120 mg/kg × 2 days) 1–5 × 106/kg 100 75 25 NA NA
MSKCC 44,58
10 None: 4, CY‑conditioning (1.5 or 3 g/m ): 4,
2
0.4–1.0 × 10 /kg
7
20 10 10 20 NA
(8* + 2‡) BR (rituximab 375 mg/m2 × 1 day, bendamustine
90 mg/m2 × 2 days): 2
MSKCC59 7‡ PCR|| × 6 cycles, CY (600 mg/m2) 3–30 × 106/kg 57.2 14.3 42.9 NR NA
B-NHL
NCI63 4* FLU (25 mg/m2 × 5 days)/CY (60 mg/kg × 2 days) + i.v. 0.3–3 × 107/kg 100 0 100 0 NA
IL‑2 following CAR‑T cell infusion
NCI64 11* FLU (25 mg/m2 × 5 days)/CY (60 or 120 mg/kg × 2 days) 1–5 × 106/kg 88.9 55.6 33.3 11.1 NA
NCI 65
9‡
FLU (30 mg/m × 3 days)/CY (300 mg/m × 3 days)
2 2
1 × 10 /kg
6
66.7 11.1 55.6 0 NA
MSKCC67 6‡ BEAM conditioning and autologous SCT 5–10 × 106/kg 100 100 0 0 NA
UPenn 66
8‡
EPOCH, CY, bendamustine, FLU/CY §
3.7–8.9 × 10 /kg
6
50 37.5 12.5 0 NA
(median 5.8 × 106/kg)
Fred 9‡ Lymphodepleting chemotherapy 2 × 105/kg, 2 × 106/kg, 66.7 11.1 55.6 NA NA
Hutchinson88 or 2 × 107/kg
*In published report. ‡In reported abstract. §Doses unknown. ||PCR is pentostatin 4 mg/m2 day 1, cyclophosphamide 600 mg/m2 day 1, rituximab 375 mg/m2 day 1.
Abbreviations: ALL, acute lymphocytic leukaemia; BEAM, BCNU (carmustine) + etoposide + cytarabine + melphalan; B-NHL, B-cell non-Hodgkin lymphoma; CAR,
chimeric antigen receptor; CLL, chronic lymphocytic leukaemia; CR, complete response; CVAD, cyclophosphamide + vincristine + doxorubicin + dexamethasone;
CY, cyclophosphamide; EPOCH, etoposide + vincristine + doxorubicin + cyclophosphamide + prednisone; FLU, fludarabine; Fred Hutchinson, Fred Hutchinson Cancer
Research Center; i.v., intravenous; MSKCC, Memorial Sloan Kettering Cancer Center; NCI, National Cancer Institute; NA, not applicable; ORR, overall response rate;
OS, overall survival; PR, partial response; RFS, relapse-free survival; SD, stable disease; UPenn, University of Pennsylvania; VP, etoposide.
a CR and one patient with mantle-cell lymphoma (MCL) 23 patients had been enrolled, and eight were evalu
achieved a PR57. The omission of preconditioning chemo- able for treatment response — six patients with DLBCL
therapy might have contributed to the lack of significant and two patients with FL. The ORR at 3 months was
clinical responses in this study. Nevertheless, the results 50%, with three CR noted (in two patients with DLBCL
successfully demonstrated the safety of donor-derived and one patient with FL) and one PR in a patient with
CAR T cells, infused as donor leukocyte infusions in a FL66. Four patients with DLBCL had disease progression
post-allogeneic-transplant setting. before or at initial response assessment 66. In a different
UPenn reported preliminary phase II data, in abstract strategy reported by the MSKCC group, CAR T cells was
form, for patients with B‑cell malignancies treated with used as a consolidative therapy after autologous HSCT
CAR-T‑cell-based therapy 66. At the time of reporting, for patients with relapsed DLBCL, in a phase I study that

REVIEWS
enrolled high-risk patients with bone-marrow involve- lymphocytosis involving CAR-T‑cell penetration of the
ment at relapse or PET-positive disease after second-line CNS, and the infused CAR-T‑cell dose.
chemotherapy 67. CAR T cells are infused following con- Common to all B‑cell-directed therapies, B‑cell
ditioning with high-dose chemotherapy and autologous- aplasia has been noted in CAR-T‑cell therapy owing
stem-cell infusion. Six patients have been treated on this to depletion of endogenous CD19 B lymphocytes. The
study, with all patients achieving and maintaining a CR duration of B‑cell aplasia and its association with the
at a median follow up of 6 months67. persistence of CAR T cells in vivo ranges from days to
years48,52,72. Intravenous immunoglobulin supplementa-
Toxicities. Adverse events associated with CAR-T‑cell tion can help reduce the risk of opportunistic infection
therapy do not necessarily correlate with the infused cell in patients who develop this condition; however, strat
dose or timing, but are instead associated with the expan- egies to restore endogenous B-cell populations might be
sion or the persistence of the cells52,68. The most-notable part of future investigations.
toxicities related to CAR T cells are CRS, encephalopathy,
and B‑cell aplasia. The frequency and severity of each Future perspectives
symptom varies greatly among different studies: CRS has Collective evidence on the use of CAR T cells indicate
been reported in 18–100% of patients, with severe CRS the following: first, second-generation CAR T cell are
noted in 27–53% of patients; encephalopathy in 25–47% more efficient than first-generation CAR T cells; second,
of patients; and B‑cell aplasia, an expected on-target event, lymphodepletion by preconditioning before CAR-T‑cell
has been reported in 86–100% of patients immediately infusion seems necessary for successful treatment out-
after lymphodepleting chemotherapy and CAR-T‑cell come and is associated with improved CAR-T-cell persis-
infusion49,51,52,64. B‑cells commonly recovered within tence; third, CRS is more frequently observed in patients
6 months after aplasia, but a small number of patients with a high tumour burden, but can be managed with
had B‑cell aplasia persisting for more than 1 year49,51,52,64. tocilizumab as well as steroids. Incorporating tumour-
CRS occurs as CAR T cells expand and induce the reducing chemotherapy as well as lymphodepleting
expression of cytokines by cells in the reactive tumour chemotherapy before CAR-T‑cell infusion might improve
microenvironment, which can initiate a cascade of the safety and efficacy profile of this treatment by redu
cytokine release49,69. The incidence and severity of CRS cing the numbers of reactive inflammatory cells in the
is variable after CAR-T-cell infusion, ranging from tumour microenvironment and creating a niche for
life-threatening fulminant CRS necessitating intensive- CAR‑T‑cell expansion and subsequent persistence50,51,73.
care monitoring, to laboratory-diagnosed CRS with CAR-T‑cell therapy is a promising approach, especi
no overt clinical symptoms. Typically, patients experi ally for transplant ineligible patients. Several challenges
ence fever, tachycardia, hypotension, capillary-leak must be addressed, however, before CAR T cells are
syndrome, and/or respiratory-distress syndrome, widely adopted in clinical practice. Identifying an ideal
within the first 3 weeks of cell infusion68,69. A milieu of dose of CAR T cells is difficult because in vivo expan-
cytokines is released in patients who develop this con- sion of the cells is highly variable, and might predispose
dition, and these include IL‑6, IFNγ, and IL‑1045,49,52. to inconsistency of response and unpredictable toxicity.
Laboratory manifestations of macrophage-activation At present, management of immune-related t oxicities
syndrome, including cytopenias, elevated C‑reactive can be challenging. New methods to increase the safety
protein (CRP) levels, marked hyperferritinaemia, and of therapy are being evaluated and include the intro-
decreased fibrinogen concentrations, have been noted70. duction of a suicide gene via Herpes simplex virus
Elevated serum CRP levels, in conjunction with clini- thymidine kinase and inducible caspase 9 (iCasp9),
cal symptoms, seems to be a biomarker of severe CRS49. or targetable cell-surface proteins, such as truncated
Algorithms developed to aid the clinical management EGFR or CD20 (REFS 41,74,75).
of CRS include administration of steroids and the With the success of CD19‑targeted CAR T cells, tar-
IL‑6‑receptor-blocking antibody, tocilizumab49,68; how- geting of other cell-specific TAAs is being explored76–78.
ever, steroids blunt the function of CAR T cells, whereas Selective targeting of κ or λ light chain might reduce the
long-term impact of tocilizumab on CAR-T‑cell function incidence of B‑cell aplasia and result in reduction of pro-
is unknown. In some series, the presence and severity longed hypogammaglobulinaemia79. CD30‑targeted and
of CRS correlated with the extent of disease burden at CD123‑targeted CAR T cells are also being explored for
time of infusion49,52, although this observation needs to potential use in treating HL80,81. Finally, incorporating
be confirmed in ongoing studies in which CAR T cells chemokine or cytokine expression into the CAR-T‑cell
are infused during states of minimal disease. construct might improve delivery and trafficking of the
Encephalopathy, although closely associated with cells to the tumour 42,43,82,83. Combination treatment with
CRS, is considered a distinct entity with an incidence small-molecule inhibitors, such as ibrutinib or lenalido
as high as 50% in patients treated with CAR T cells64,71. mide, or with immune-checkpoint inhibitors can be
Symptoms range widely, from mild confusion to obtun- explored to improve CAR-T‑cell activation or suppress
dation, aphasia, and seizures. Cerebral spinal fluid the endogenous T‑cell-inhibitory microenvironment,
(CSF) lymphoc ytosis is occasionally noted52. While which might enhance treatment efficacy 84–86.
most symptoms are reversible, the aetiology of these Relapses following treatment with CAR T cells are
symptoms remains unclear. Many theories have been typically associated with a lack of T‑cell persistence
proposed, including associations with cytokine release, or the development of a CD19‑negative tumour-cell

REVIEWS
Bispecific T-cell engager Dual affinity retargeting Tetravalent tandem diabody

(BiTE®) (DART) (TandAb®)
CD19 CD3
CD19 CD3 CD3 CD19
CD19 CD3
• Single polypeptide chain • Two polypeptide chains • Single polypeptide chain

• Interchain disulfide bridge • Chain dimerization
VL VH VL VH VL VH VH VL VH VL
Disulfide
bridge
VL VH VL VH VL VH
Figure 3 | Structure of different types of T‑cell-engaging antibodies. BiTE® are constructed of single polypeptide
Natureare
chain that consists of two VL and VH pairs that recognize CD3 and CD19, respectively. DARTs Reviews | Clinical
constructed Oncology
of two
separate, but paired, polypeptide chains, each comprising VL and VH regions that recognize different cell-surface
molecules; the two polypeptide chains dimerize and are linked by interchain disulphide bridge, forming two functional
VL–VH pairs that each comprise a VL from one polypeptide and a VH from the other. TandAb® are constructed of dimerized
single polypeptide chains; each chain contains two different VL regions and two different VH regions, which upon
dimerization, form four antigen-recognition sites for two different antigen (two VL–VH pairs; targeting CD19 and CD3 in
this case). DARTs and TandAb® have longer half-life compared to BiTE® due to their structure. Abbreviations: BiTE®,
bispecific T‑cell engagers; DART, dual affinity retargeting antibody; TandAb®, tetravalent tandem diabody; VH, antibody
heavy-chain variable region; VL, antibody light-chain variable region.
clone51,52. Strategies to increase the efficacy of CAR treated patients across multiple centres with detailed
T‑cells through modification of CAR constructs, such toxicity assessment will be essential in guiding the
as the use of third-generation and fourth-generation clinical development of this novel treatment strategy.
armoured constructs, are being evaluated40. Another
alternative approach is to infuse patients with poly Bispecific antibodies and derivatives
specific CAR T cells that targets multiple cell-surface Bispecific antibodies and subsequent derivatives have
proteins to prevent immune escape. Methods to increase been developed through protein engineering of the
persistence of CAR T cells to promote treatment effi- antibody backbone to increase valency, which facilitates
cacy include using allogeneic virus-specific T cells and engagement of the immune system. The initial develop
a combination of CD8‑positive central memory T cells ment of bispecific-antibody constructs faced many
and CD4‑positive T cells 27,87,88. Off-the-shelf CAR challenges, including immunogenicity of the product,
T cells that are matched to the recipient by HLA typing insufficient clinical activity, and difficulties in large-scale
can reduce time and resource constraints of using CAR production. Novel platforms are being developed for the
T cells89. Other groups have explored the possibility treatment of lymphoid malignancies. Blinatumomab
of downregulation of TCR to make a f oundation for (BLINCYTO®, Amgen), a first-in-class bispecific T‑cell
universal T‑cell-based immunotherapy 90. engager (BiTE®), is a 55 kDa molecule composed of two
On the basis of promising clinical results, multiple scFv, one targeting CD19 and one against CD3, joined
pharmaceutical companies (such as Novartis, Juno by a glycine–serine 5‑amino-acid non-immunogenic
Therapeutics, Cellular Biomedicine Group, Bellicum, linker manufactured by recombinant engineering 92,93.
Celgene/Bluebird, Kite Pharma/Amgen, Cellectis/ The molecule has high affinity for both CD19 and
Servier/Pfizer, Opus Bio, TheraVectys) are developing CD3, with an equilibrium dissociation constant (KD)
large-scale clinical-grade production of CAR T cells91. of 10–9 M and 10–7 M, respectively 94. Similar platforms
The participation of pharmaceutical companies is criti include bivalent bifunctional dual affinity retarget-
cal for success; however, the treatment is unlikely to be ing antibodies (DARTs), tetravalent bifunctional tandem
standardized in the near future owing to patent issues. antibodies, and trispecific antibodies (FIG. 3).
Identification of a lead CAR-T‑cell construct is unlikely Blinatumomab is the first drug in the bispecific anti-
in the absence of head-to-head trials that directly com- body class to be approved by the FDA. The recombi-
pare each construct and each method in specific dis- nant protein engages T cells via the anti‑CD3 arm and
ease settings. Results of larger studies of homogenously creates a structurally normal immune synapse that

REVIEWS
targets CD1995. The functional immune synapse results with eradication of disease in the bone marrow of
in IL‑2‑independent polyclonal T‑cell activation and patients with NHL in a phase I study106. Sixteen out
apoptotic cell death of target cells96–98. Except for naive of 20 evaluable patients (80%) achieved MRD-negativity,
T cells, all CD4‑positive and CD8‑positive T‑cell popu- all within the first treatment cycle105. At a median follow
lations were found to proliferate and engage in cell lysis99. up of 33 months, 12 patients remained in CR, resulting
CTLs activated by blinatumomab upregulated perforin in a haematological relapse-free survival (RFS) rate of
and granzyme synthesis, with eventual creation of a per- 61%105. As a result of this response, 9 patients proceeded
forin pore at the immune synapse and discharge of toxic to receive allogeneic HSCT105. Among the 11 patients
secretory proteins that induced apoptosis99. At doses who did not receive allogeneic HSCT, five relapses
greater than 5 μg/m2 per day, CD19‑positive cells in the occurred, all within 7 months of treatment105. Overall,
peripheral blood underwent apoptosis98. Calcium che- six patients relapsed after achieving a CR, characterized
lators, which inhibit T‑cell signalling and the assembly as CD19‑negative relapses in two patients and relapse
of functional perforin pores, and perforin inhibitors can in sites of immune privilege (CNS and testis) in another
inhibit blinatumomab‑induced cell lysis100. two patients105.
Blinatumomab has a short half-life of less than 2 h101 Following these promising results, two larger phase II
due to its small molecular weight and the lack of a con- studies were conducted to include ALL patients with
stant Fc domain that functions to stabilize antibody haematological relapse (TABLE 2)107,108. The first study var-
reserves in the body. Accordingly, this agent is admin- ied from the phase I study in terms of the preconditioning
istered by continuous intravenous infusion (CIV). regimen used and deployed step-up dosing of blinatu-
Early dose-escalation trials in patients with NHL and momab from 5–30 μg/m2 per day to optimize outcome
CLL, in which blinatumomab was administered as a while minimizing occurrence of CRS and neurological
2 h or 4 h intravenous infusion at doses ranging from toxicities107. In total, 25 of 36 patients (69%) achieved
0.75–13 μg/m2 up to three times weekly, observed no a CR or CRh (CR with a partial recovery of peripheral
objective responses 102. Conversely, adverse events, blood counts); the median RFS was 7.6 months, and the
especially neurological events such as aphasia, ataxia, median overall survival was 9.8 months107. The relapsed
disorientation, and seizures, were reported and resulted cases included both CD19‑positive and CD19‑negative
in treatment discontinuation in 12 patients102. As a result, phenotypes 107. A larger multic entre study 108, 189
all three short-term infusion trials were terminated early. patients with Ph-negative relapsed/refractory B‑ALL
Subsequent studies were designed to lengthen the mode were treated with 9 μg per day of blinatumomab for the
of administration to CIV, which increased exposure to first week, and at an escalated dose of 28 μg per day for
the drug and heightened treatment efficacy to an ORR of weeks 2–4 by CIV. Preconditioning with dexametha-
69% in a phase I study in patients with relapsed and/or sone was required for these patients with high-volume
refractory NHLs of various histology 103. disease; of these, 81 (43%) achieved a CR or CRh (CR
33%, CRh 10%) within the first two treatment cycles.
Clinical data with blinatumomab The median overall survival and RFS were 6.1 months
Acute lymphoblastic leukaemia. CD19 is highly expressed after a median follow-up of 9.8 months, and 5.9 months
by B-ALL, and can be targeted by blinatumomab104. after a median follow-up of 8.9 months, respectively.
In a phase II study105, 21 patients with MRD-positive Patients with less than 50% blasts in their bone marrow
ALL were treated with blinatumomab at 15 μg/m2 per at baseline evaluation had a higher CR or CRh rate. No
day for 4 weeks per cycle — a dose that was associated other subgroup had a differential response to treatment.
Table 2 | Clinical efficacy of blinatumomab

Disease Number Treatment schedule Response rate Relapse- Median
of free overall
ORR (%) CR (%) PR (%) SD (%)
patients survival survival
MRD-positive 20* 15 μg/m2 per day continuous i.v. × 4 weeks NA 80 NA NA 61% at NA
ALL105,106 every 6‑week cycle 33 months
ALL107 36* 5 μg/m2 and 15 μg/m2 per day (week 1, NA 69 NA NA Median of 9.8 months
and thereafter until 4 weeks, respectively) 7.6 months
ALL108 189* 9 μg and 28 μg per day (week 1, and NA 43 NA NA Median of 6.1 months
thereafter, respectively) continuous i.v. 5.9 months
× 4 weeks every 6‑week cycle
B-NHL (FL, 35‡ 60 μg/m2 per day continuous i.v. 69 37 32 NA NA NA
MCL, DLBCL)103
DLBCL109 21‡ Cohort I and III: 9 μg, 28 μg, and 112 μg 43 19 23.8 NA NA NA
per day (week 1, week 2, and thereafter,
respectively), cohort II: 112 μg per day
× 8 weeks
*In published report. ‡In reported abstract. Abbreviations: ALL, acute lymphocytic leukaemia; B-NHL, B-cell non-Hodgkin lymphoma; CR, complete response;
DLBCL, diffuse large-B-cell lymphoma; FL, follicular lymphoma; i.v., intravenous; MCL, mantle-cell lymphoma; MRD, minimal residual disease; NR, not applicable
or available; ORR, overall response rate; PR, partial response; SD, stable disease.

REVIEWS
Non-Hodgkin lymphoma. Patients with relapsed/refrac- Future perspectives

tory indolent lymphoma were treated with CIV of blina- Blinatumomab was approved for the treatment of
tumomab at 0.5–90 μg/m2 per day, with the maximum relapsed and/or refractory B‑ALL by the FDA in
tolerated dose identified as 60 μg/m2 per day98. The December 2014. The cumbersome administration sched-
study subsequently included patients with DLBCL, and ule is driving ongoing improvements of this treatment
76 patients in total received treatment. The histologi- platform. The BiTE® concept relies on a scFv linked by
cal subtypes were defined as 37% FL, 32% MCL, 18% a short flexible polypeptide linker allowing the VH and
DLBCL, and 13% other indolent lymphomas103. Clinical VL domains to interact preferentially with more-distant
responses were seen above doses of 15 μg/m2 per day. molecules. Manipulation of the structure, valency, and
Among the patients treated at 60 μg/m2 per day (n = 35), stability, via Fc expression or protein conjugation through
the ORR across NHL subtype was 69%, with a CR rate of protein engineering, have generated therapies such as
37% (TABLE 2), and a median DoR of 404 days103. Patients DARTs and tandem antibody-based therapies.
with FL had the highest ORR at 80%, followed by 71% DARTs are encoded by two paired polypeptides, each
in patients with MCL, and 55% in those with DLBCL103. composed of the VL of the scFV targeting one antigen
A phase II study of blinatumomab is currently enrolling in tandem with the VH of the scFV targeting the other
patients with relapsed/refractory DLBCL109; patients will antigen, connected by a shorter linker that does not allow
be treated at either a weekly step-up dose of 9 μg per day, for intrachain interaction (FIG. 3)113. A covalent disulphide
28 μg per day, and 112 μg per day or a fixed-dose of 112 μg bond formed between cysteines at the C‑termini of the
per day for 8 weeks, and all patients will receive dexameth- polypeptides stabilizes the complex of the two Fv chains.
asone as prophylaxis for CRS. To date, 25 patients have In comparison to BiTE®, DARTs have longer in vitro half-
been enrolled, and the ORR among 21 evaluable patients life, while maintaining effective cytotoxicity and capacity
was 43%, including four CRs (19%), and five PRs (23.8%). of B‑cell lysis114. Tandem diabodies (TandAb®) are com-
posed of four variable domains expressed in one long
Toxicity. Most adverse events experienced in the phase I polypeptide, which are connected by linkers of varying
study of blinatumomab were mild-to-moderate, and were lengths that direct complementary dimerization to form
reversible; however, neurological toxicities that included tetravalent bispecific antibodies or diabody folding to
headache, tremor, aphasia, ataxia, disorientation and form bivalent bispecific antibodies115. The efficiency of
seizure, as well as infection associated with leukopenia TandAb® formation is dependent on the linker length, but
and neutropenia, raise concern102. Subsequent studies this structure offers the potential for higher avidity from
explored various CIV dosing schedules and precon- bivalency for each target. Production strategies include
ditioning strategies to minimize toxicities. Fewer than bacterial expression systems that require a refolding step
10% of patients in each study experienced grade ≥3 CRS. to generate functional molecules, or mammalian recom-
Neurological events were reversible, and were managed binant protein expression, which doesn’t require this addi-
with either dexamethasone administration or treatment tional step but is more costly. Methods to further stabilize
interruption in a limited number of cases105–108. Overall, diabodies (such as DARTs and TandAb®) include covalent
10% of patients discontinued blinatumomab treatment linkage, PEGylation, N‑glycosylation, introduction of an
owing to therapy-related toxicities105–108. Nevertheless, Fc fragment, or direct fusion to albumin116,117.
caution and awareness of neurological toxicity remains The design of bispecific antibodies requires a bal-
imperative in the management of these patients. Steroids ance of features that include bioavailability, target
can limit T‑cell proliferation, which is a concern in the affinity, stability, and efficacy. Attempts to modify the
case of treatment with CAR T cells, but dexamethasone bispecific-antibody platforms should improve bioavail-
showed no impact on treatment efficacy of blinatu- ability and mitigate the inconvenient continuous dos-
momab108. Other common adverse effects seen in the ing requirement (FIG. 3). Incorporation of an Fc domain
phase II studies of blinatumomab were fever, fatigue, greatly increases bioavailability, but might hinder pro-
headache, tremor, leukopenia, hypokalemia, decrease of duction. Increasing valency, as in TandAb®, potentially
blood immunoglobulin, febrile neutropenia, and anae- improves the affinity and stability of bispecific anti-
mia105–109; severe adverse events included infections, and bodies. However, because affinity, stability and efficacy
CNS and psychiatric disorders. Transient B‑cell apla- are independent parameters, efficacy may not be evi-
sia and resulting hypogammaglobulinaemia have been dent until late stages of clinical development. Clinical
reported in patients treated with blinatumomab110. developments incorporating these novel platforms are
Predictive markers of neurological adverse events of under way, and include a TandAb® that recognizes
blinatumomab have been explored. A low B cell:T cell CD30 and CD16A and is being tested in patients with
ratio in the peripheral blood was associated with an HL (NCT01221571)118.
increased risk of CNS toxicity in patients with NHL111.
Presumably, circulating B‑cells act as a sink to stimulate Immune-checkpoint inhibitors
T‑cell proliferation in the peripheral blood before the The immune-checkpoint axis serves to maintain self-
drug triggered a similar response in the CNS102. Patients tolerance and prevent autoimmunity 119. The immune
with high B cell:T cell ratio were successfully treated with synapse formed between antigen-presenting cells (APCs)
a fixed dose of 60 μg/m2 per day without major CNS com- and T cells is controlled by many co-stimulatory and
plications, whereas patients with a lower B cell:T cell ratio inhibitory interactions that modulate the intensity and
benefited from step-up dosing of blinatumomab111,112. duration of T‑cell responses initiated through the TCR

REVIEWS
a Activating receptors Inhibitory receptors Figure 4 | The immune-checkpoint axis that serves to
maintain self-tolerance and prevent autoimmunity.
Agonist • Ipilimumab
• Dacetuzumab a | Components of the immune synapse. T cells
CD40L CTLA-4 • Tremelimumab
• Chi Lob 7/4 recognize antigens presented on the MHC by the TCR.
• CP-870893 αPD-1 The fate of T cells upon antigen recognition is
Antagonist OX40 • Nivolumab determined by the additional ligand–receptor
• Lucatumumab • Pembrolizumab
PD-1 • Pidilizumab interactions between the T cells and APCs (or tumour
T cell
• MED16469 4-1BB • AMP-224 cells). The co-stimulatory signals activated via CD28,
(CD137) αPD-L1 4‑1BB (CD137), OX40, and CD27 promote activation of
• Urelumab • Avelumab
(BMS-663513) T cells, whereas those sent via CTLA‑4 and PD‑1
CD27 TIM3 (MSB0010718C) decrease T‑cell activation. Various treatment modalities
• PF-05082566 • MEDI4736
• MEDL3280A are being developed to modulate these signals.
• Varlilumab CD28
(CDX-1127) LAG3 • BMS-936559 Antagonistic antibodies have been developed that
TCR target co‑stimulatory signals delivered via OX40–
Agonistic antibodies Blocking antibodies
OX40L, 4‑1BB (CD137)–4‑1BBL (CD137L), and CD27–
b CD70 interaction. Both agonistic and antagonistic
Tumour site Activated T cell antibodies that target the CD40–CD40L interaction are
in development. Immune-checkpoint inhibitors target
the inhibitory signals transduced through the PD‑1–
Tumour cell
PD‑L1 axis and CTLA‑4 interactions. Molecules engaged
Activation in co-stimulatory signalling are coloured in pink, and
TCR signal those involved in inhibitory signalling are coloured in
PD-1 red. b | Mechanism of T‑cell activation at the tumour site
SHP2 AKT PI3K and the lymph node. APCs take up TAAs at the site of
tumour. The APCs migrate to the lymph node, where
PD-L1/L2 PD-1 Inhibitory signal they present the TAA to naive (inactive) T cells. The
specific T cells that recognize the TAA are activated
(primed) via TCR-mediated signalling as well as
co-stimulation through CD28 and CD80 and/or CD86
interactions. T‑cell activation is interrupted when
CTLA‑4 is mobilized to the cell surface from intracellular
stores and competes with CD28 for interaction with
Lymph node Naive T-cell
CD80 and CD86. The activated (primed) T cells circulate
to the peripheral tissues and organs, and will be
reactivated upon re-challenge with the TAA at the
Antigen- tumour site. Activation of T cells in the periphery is
presenting cell decreased upon expression of PD‑1 on the surface of
CD80/86 CTLA-4 Inhibitory signal activated T cells after its transcriptional activation and
CTLA-4
engagement with its ligand PD‑L1/PD‑L2 that can be
Activation signal expressed on the tumour cells or on other immune cells
in the tumour microenvironment. Abbreviations:
APC, antigen-presenting cell; CTLA‑4, cytotoxic
CD80/86 CD28 Co-stimulatory T‑lymphocyte-associated protein 4; MHC, major
signal histocompatibility complex; PD‑1, programmed cell
death protein 1; PD‑L1, programmed cell death 1
ligand 1; PD‑L2, programmed cell death 1 ligand 2;
TAA, tumour-associated antigen; TCR, T‑cell receptor.
Nature Reviews | Clinical Oncology

(FIG. 4a).
Tumour and/or nontumour cells from the sur- Although CTLA‑4 and PD‑1 both exhibit inhibitory
rounding microenvironment commonly overexpress effects on tumour immunity, their mechanisms of action
inhibitory proteins that suppress T‑cell-effector functions, are distinct (FIG. 4b). CTLA‑4 is predominantly expressed
such as cytotoxic T‑lymphocyte-associated-protein 4 on T cells in the lymph nodes, where the cells are initially
(CTLA‑4) and programmed cell-death protein 1 (PD‑1), activated by APCs. Activation of T cells via TCR signal-
leading to immune escape of the tumour 119–122. The dis- ling and CD28 co‑stimulation mobilizes the intracellular
covery that inhibition of CTLA‑4 dampened tumour pool of CTLA‑4 to relocate to the cell surface124, where
growth in mouse models provided the first clue that CTLA‑4 can bind to its ligands CD80 and CD86 and
modulation of immune checkpoints might be a viable mediate signalling that terminates CD28 co-stimulation
therapeutic strategy 123. Since this discovery, blockade of and T‑cell activation125,126. By contrast, PD‑1 predom-
CTLA‑4 and PD‑1 pathways has changed the manage- inantly functions in activated T cells in the periphery
ment of several solid cancers. Antagonistic antibodies upon recognition of its ligands on tumour cells (or other
targeting immune-inhibitory signals and agonist anti- host cell types); activation of T cells leads to transcrip-
bodies targeting immune-activating signals are currently tional activation of PD‑1 and its expression on the cell
being explored for the treatment of cancer, including surface, and subsequent engagement of PD‑1 with its
lymphoid malignancies. ligands PD‑L1 or PD‑L2 antagonizes PI3K activity,

REVIEWS
leading to the blockade of T‑cell activation (FIG. 4b)127. lymphoma (n = 23), including 13 patients with mycosis
In mice, phenotypes resulting from inactivation of PD‑1 fungoides and five patients with peripheral T‑cell lym-
or its ligands are usually mild, consisting of late-onset, phoma (PTCL)145. The ORR for these patients was 17%,
organ-specific inflammation128–130. By contrast, CTLA‑4 with four patients (17%) achieving a PR — two patients
knockout mice succumb to a lethal multiorgan lym- with mycosis fungoides and two with PTCL145. A phase I
phoproliferative disease131,132. These phenotypic differ- study of pembrolizumab in patients with NHL is currently
ences possibly correlate with the range of toxicities that ongoing (NCT01953692)146,147.
are seen clinically with the anti-CTLA‑4 and anti-PD‑1
blocking antibodies. Hodgkin lymphoma. Phase I studies of both nivolumab
Targeting the PD‑1–PD‑L1 axis in patients with and pembrolizumab have demonstrated exciting results in
haematological malignancies has attracted attention patients with HL (TABLE 3). Nivolumab given at 1 mg/kg or
because of the frequent expression of PD‑L1 and/or 3 mg/kg in weeks 1 and 4, and then every 2 weeks there-
PD‑L2 in various lymphoid malignancies133–135. PD‑L1 after until disease progression showed an ORR of 87% in
is expressed on haematopoietic cells, such as T cells, 23 patients: six (26%) had a CR, 14 (61%) had a PR145,148.
B cells, macrophages, natural killer cells, and dendritic Additionally, three (13%) patients had SD148. The progres-
cells, as well as on nonhaematopoietic cells136. The mech- sion-free survival rate at 24 weeks was 86%148. The patients
anisms that lead to PD‑L1 and/or PD‑L2 overexpression included in this study were heavily pretreated with 87%
in patients with haematological malignancies are varied, having received three or more prior treatments, 78% had
including genetic alterations or activation of JAK/STAT received brentuximab vedotin, and 78% had undergone
signalling pathways in some cases, as well as in response autologous HSCT148. Nivolumab was active in all patients
to cytokine stimuli, such as IFNγ133,137–139. PD‑L1 is fre- regardless of their prior treatment status148,149. In a sep-
quently expressed in Hodgkin Reed–Sternberg cells arate phase I study, pembrolizumab was also evaluated
through chromosome translocation, gene amplifica- for efficacy and safety in patients with relapsed/refractory
tion, and EBV-related mechanism133,137,140. In the set- HL150. All patients had prior exposure to brentuximab
ting of NHL, PD‑L1 is expressed in various histologies vedotin, and 69% of the patients had received autolo-
including DLBCL, primary mediastinal large-B‑cell gous HSCT150. Among 29 evaluable patients, the ORR
lymphoma (PMBL), and anaplastic large-cell lym- was 63%, including six patients with CR, and 13 patients
phoma134,135. In patients with PMBL, gene fusions place with PR150. Six patients obtained SD150. With a median
the genes encoding PD‑1 ligands under the regulation of observation period of 153 days, the median DoR had not
MHC class II transactivator (CIITA), resulting in their been reached (range 1–185 days)150. Both of these studies
aberrant expression. In patients with HL, amplification have evaluated the expression of PD‑L1 and/or PD‑L2 in
of chromosome 9p23–24 (where the genes encoding the tumour cells in patients with available samples, and
PD‑L1 and PD‑L2 reside) is frequently observed140,141. showed PD‑L1 and/or PD‑L2 expression by the malignant
At the present time, clinical experience with immune- Reed–Sternberg cells in all cases148,150.
checkpoint inhibition in patients with lymphoma is Other immune-checkpoint-targeting antibodies in
limited to antibodies targeting PD‑1 (pidilizumab, development include anti-PD‑L1 and anti‑4‑1BB anti-
pembrolizumab, nivolumab) and CTLA‑4 (ipili- bodies. Anti-PD‑L1 antibodies have been developed
mumab); however, interest in targeting components of and are currently being tested in patients with various
the co-stimulatory pathway, such as 4‑1BB and OX40, is cancers including NHL (NCT02220842)151,152. Urelumab
growing. Indeed, agonist antibodies against 4‑1BB and is an antibody to 4‑1BB that is currently being evalu-
OX40 are in v arious stages of clinical development. ated in a clinical trial in combination with rituximab
(NCT01775631)153. Ipilimumab has been evaluated
Clinical data on immune-checkpoint inhibition for efficacy in a pilot study in patients with NHL who
Non-Hodgkin lymphoma. Pidilizumab was the first PD‑1 relapsed following allogeneic HSCT154. The treatment
blocking antibody to be tested in patients with lymphoid was relatively safely administered, with no cases of
malignancies. Data from phase I and phase II studies of grade 3 or 4 GVHD observed following ipilimumab
pidilizumab in patients with DLBCL, and in combination treatment, although two patients developed organ-
with rituximab in patients with relapsed/refractory FL, specific immune-related adverse events, both involv-
showed promising results142–144; however, pidilizumab is ing the lung 154. Two patients (14.3%) achieved CR to
considered to have low specificity for PD‑1. Nivolumab ipilimumab and another two patients had SD154.
and pembrolizumab are also being evaluated in patients
with various haematological malignancies, including Toxicity. Toxicities related to immune-checkpoint inhib-
NHL. In a recent phase I study of nivolumab (TABLE 3), itors are typically immune-related and include pneumo-
31 patients with B‑cell lymphoma were included145. nitis, colitis, hepatitis, hypophysitis, and thyroiditis155.
Among 29 patients, excluding two patients with PMBL, The frequency and severity of these adverse effects vary
eight patients (28%) had an objective response, including depending on the antibody, pathway, and disease. On the
three patients with a CR and five patients with a PR145. The basis of the abundant experience of immune-checkpoint
response rate was highest in patients with FL, in whom inhibitors in solid tumours, the occurrence of grade 3–4
the ORR was 40%145. Patients with DLBCL had an ORR immune-related adverse events is approximately 20% with
of 36%, including two patients with a CR and two patients ipilimumab, compared with 5–10% with nivolumab or
with a PR145. This study also included patients with T‑cell pembrolizumab156. In general, PD‑1 blockade is associated

REVIEWS
Table 3 | Clinical efficacy of immune-checkpoint inhibitors

Drug Number Treatment schedule Response rate Median Survival outcomes
(manufacturer) of duration of
ORR CR PR SD
and disease patients response
(%) (%) (%) (%)
(range)
Nivolumab (BMS, USA)
B-NHL145* 31‡ 1 mg/kg or 3 mg/kg week 1, week 4, 26 10 16 52 NA NA
and every 2 weeks thereafter
DLBCL145* 11‡ 1 mg/kg or 3 mg/kg week 1, week 4, 36 18 18 27 22 weeks NA
and every 2 weeks thereafter (6–77 weeks)
Follicular 10‡ 1 mg/kg or 3 mg/kg week 1, week 4, 40 10 30 60 Not reached NA
lymphoma145* and every 2 weeks thereafter
(27–82 weeks)
T-NHL145 23‡ 3 mg/kg week 1, week 4, and every 17 0 17 43 NA NA
2 weeks thereafter
Hodgkin 23§ 1 mg/kg or 3 mg/kg week 1 and 4, 87 26 61 13 NA PFS: 86% at 24 weeks
lymphoma145,148 and every 2 weeks thereafter
OS: median not reached
Pembrolizumab (Merck, USA)
Hodgkin 29‡ 10 mg/kg every 2 weeks 66 21 45 21 Not reached NA
lymphoma150 (1–185 days)
Ipilimumab (BMS, USA)
B-NHL154 18 3 mg/kg → 1 mg/kg × 3 doses 11.1 5.6 5.6 NA NA NA
(or 3 mg/kg × 4 doses in 6 patients)
Hodgkin lymphoma 14§ 0.1–3.0 mg/kg 14.3 14.3 0 14.3 NA NA
(post allo SCT)172
*Comprises DLBCL, follicular lymphoma, primary mediastinal B-cell lymphoma, and other B-cell lymphomas; data from this study for patients with DLBCL and follicular
lymphoma are shown separately in the following two rows. ‡In reported abstract. §In published report. Abbreviations: BMS, Bristol-Myers Squibb; B-NHL, B-cell
non-Hodgkin lymphoma; CR, complete response; DLBCL, diffuse large-B-cell lymphoma; NR, not applicable or available; ORR, overall response rate; PR, partial response;
SD, stable disease; T-NHL, T-cell non-Hodgkin lymphoma.
with fewer and less-severe toxicities compared with in three (10%) patients. Three patients experienced four
CTLA‑4 blockade. Combination immune-checkpoint grade 3 treatment-related toxic effects, which included
blockade with nivolumab and ipilimumab substantially axillary pain, hypoxia, joint swelling, and pneumonitis150.
increased the occurrence of grade ≥3 treatment-related
adverse events to over 50%, with 45% patients not com- Future perspectives
pleting all doses of therapy157. Immune-related toxici- Targeting the PD‑1–PD‑L1 axis in patients with lymphoid
ties can be managed with immune-modulating agents, malignancies is a promising treatment strategy, especially
including corticosteroids and infliximab. An algorithm for for those with HL. Larger-scale studies are necessary to
managing CTLA‑4 blockade has been developed to ease confirm the efficacy of these drugs. Phase II studies of
the difficulties in managing these patients and a similar nivolumab as a single agent against relapsed and/or refrac-
approach is used for anti-PD‑1 agents158. tory FL, DLBCL, and Hodgkin lymphoma are ongoing
Although experience in patients with lymphoid (NCT02038946, NCT02038933, NCT02181738)160–162.
malignancies is limited, immune-related toxicities of The observed responses are rarely CRs supporting the
immune-checkpoint therapy seem to be similar to those rational combination of immune-checkpoint therapies
observed in patients with solid tumours. Pidilizumab with other agents to improve the quality of response and
therapy in patients with haematological cancer resulted in response duration. Several combination studies are cur-
no immune-related adverse events, and the most frequent rently being conducted, including combinations with
grade 3 to 4 adverse effects were neutropenia (19%) and other immune-checkpoint inhibitors, such as ipilimumab
thrombocytopenia (8%), which might have been related (NCT01592370)163.
to preceding autologous HSCT142–144. Among patients Substantial effort has been invested in finding predict
with lymphoma treated with nivolumab, the most- ive biomarkers of response to these agents. Experience
common adverse events were rash (22%), decreased with solid tumours indicates the utility of PD‑L1 expres-
platelet count (17%), fatigue (13%), and pneumonitis sion, mutational load, and T‑cell infiltration of the
(11%); drug-related grade ≥3 toxicities, including acute tumours as potential predictive factors of response164–168;
respiratory distress syndrome, pneumonitis, and sepsis, however, the utility of these biomarkers have not been
were observed in 21% of patients148,159. Clinical experience validated in lymphoid malignancies. Of note, the base-
of pembrolizumab in patients with lymphoma is currently line landscape of somatic mutations in lymphoid malig-
limited to a small number of patients with relapsed HL. nancies remain low compared with solid tumours,
The most frequent adverse events seen in this population and the frequent loss of MHC class I/II expression in
were hypothyroidism and pneumonitis, both observed HL poses an interesting question as to the mechanism

REVIEWS
of T‑cell activation in these tumours. Further efforts In addition to further exploration of efficacy, we
should be made to understand the biology underlying will need to understand in granular detail the mech-
these responses169–171. anism of actions of each treatment modality to better
manage and sequence each treatment option for our
Conclusions patients. Thus far, head-to-head comparison studies
We are entering an exciting era of immunotherapies for have not been performed, which precludes compari
lymphoid malignancies. Promising results with CAR sons between treatment modalities. Each platform
T cells, bispecific antibodies and their derivatives, and has its own strengths and weaknesses. For example,
immune-checkpoint blockade have been demonstrated, the similar mechanism of action of blinatumomab
and without doubt, immunotherapies will become one and CD19‑targeted CAR T cells present a similar toxi
of the central components of treatment strategies in lym- city profile. CIV administration of blinatumomab is
phoid malignancies, especially in the relapsed and/or inconvenient, although the short half-life of this agent
refractory setting. Despite the excitement, several issues is advantageous in that it enables rapid titration of the
remain to be overcome, including technical engineer- drug to minimize toxicity. The in vivo persistence and
ing, especially of CAR-T‑cell therapies and bispecific expansion of CAR T cell results in a variable dose–effect
antibodies. Compared with the astounding result of relationship across patients; however, the longevity of
both CAR-T‑cell therapy and bispecific antibodies in the T cells might provide long-term disease control.
the treatment of ALL, the results seen in patients with Anti-PD‑1 antibodies have shown remarkable efficacy
NHL and CLL are somewhat less striking but remain against HL, but combination treatments will be needed
promising; this inconsistency might in part be due to to improve CR rates. The results of ongoing and future
the immunosuppressive microenvironment associated studies will enable us to understand the differential use
with these tumours, although further investigation is of these treatments as a single or a combined modality
necessary to explain this difference in efficacy. that improves the prognosis of patients.
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REVIEWS

Novel immunotherapies in lymphoid

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 25

© 2015 Macmillan Publishers Limited. All rights reserved

Key points Cancer Research Center (Seattle, WA, USA) use a lenti­

26 | JANUARY 2016 | VOLUME 13 www.nature.com/nrclinonc

© 2015 Macmillan Publishers Limited. All rights reserved

CAR construct 19,26, or at some centres, electroporation

Clinical data with CAR T cells

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 27

© 2015 Macmillan Publishers Limited. All rights reserved

CD3 CD3 Hinge

Signalling Signalling Signalling

b 19-28z 19-28z 19-BBz

CD28 CD28 4-1BB/CD137

CD3ζ CD3ζ CD3ζ

Institute Transfection Hinge/ Institute Transfection Hinge/ Institute Transfection Hinge/

c 1st generation 2nd generation 3rd generation 4th generation

CD3ζ • Cytokine transgene

Nature Reviews | Clinical Oncology

© 2015 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 29

© 2015 Macmillan Publishers Limited. All rights reserved

Table 1 | Clinical efficacy of second generation CAR-T‑cell therapy

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© 2015 Macmillan Publishers Limited. All rights reserved

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 31

© 2015 Macmillan Publishers Limited. All rights reserved

Bispeciﬁc T-cell engager Dual aﬃnity retargeting Tetravalent tandem diabody

• Single polypeptide chain • Two polypeptide chains • Single polypeptide chain

32 | JANUARY 2016 | VOLUME 13 www.nature.com/nrclinonc

© 2015 Macmillan Publishers Limited. All rights reserved

Table 2 | Clinical efficacy of blinatumomab

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 13 | JANUARY 2016 | 33

© 2015 Macmillan Publishers Limited. All rights reserved

Non-Hodgkin lymphoma. Patients with relapsed/refrac- Future perspectives

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© 2015 Macmillan Publishers Limited. All rights reserved

Nature Reviews | Clinical Oncology

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Table 3 | Clinical efficacy of immune-checkpoint inhibitors

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© 2015 Macmillan Publishers Limited. All rights reserved

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© 2015 Macmillan Publishers Limited. All rights reserved

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Key points Cancer Research Center (Seattle, WA, USA) use a lenti