CAR-modified anti-CD19 T cells for the treatment of B-cell malignancies: rules of the road.

Review

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Nyu Medical Center on 11/29/13 For personal use only.

CAR-modified anti-CD19 T cells for the treatment of B-cell malignancies: rules of the road 1.

Introduction

2.

Pre-infusion

3.

Peri-infusion

4.

Post-infusion

5.

Conclusions

6.

Expert opinion

Saar Gill & David L Porter† University of Pennsylvania, Abramson Cancer Center, Perelman School of Medicine, Division of Hematology-Oncology, Department of Medicine, Philadelphia, PA, USA

Introduction: Malignancies of the B lymphocyte or its precursor include B-cell non-Hodgkin lymphoma as well as chronic and acute lymphoid leukemias. These are among the most common hematologic malignancies and many patients with B-cell malignancies are incurable. Although most patients initially respond to first-line treatment, relapse is frequent and is associated with a poor prognosis. T cells that are genetically engineered to express chimeric antigen receptors (CARs) recognizing the B-cell-associated molecule CD19 have emerged as a potentially potent and exciting therapeutic modality in recent years. Areas covered: This review explores the current peer-reviewed publications in the field and a discussion of expert opinion. Expert opinion: Genetic engineering of T cells has become clinically feasible and appears to be safe. Here we provide an insight into the process of patient selection, engineered T-cell production, infusion procedure, expected toxicities and efficacy of this exciting approach as it is practiced in the treatment of B-cell malignancies. Anti-CD19-redirected T cells likely represent the vanguard of an exciting new approach to treating cancer. Keywords: B-cell acute lymphoblastic leukemia, B-cell lymphoma, CD19-redirected T cells, chimeric antigen receptors Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

B-cell malignancies comprise indolent and aggressive non-Hodgkin lymphomas (NHL) as well as acute and chronic lymphoid leukemias (ALL and CLL, respectively). Annual incidence in the USA per 100,000 population is 16.6 (B-cell NHL), 4.3 (CLL) and 1.7 (ALL) (http://seer.cancer.gov/data, accessed 8/24/13). These disorders typically respond to initial chemotherapy but have a variable propensity to relapse [1,2]. Relapse can sometimes be successfully treated with hematopoietic stem cell transplantation (HCT). However, HCT is largely non-specific and associated with extensive morbidity and even mortality. Therapies that target only the malignant cells can limit non-specific toxicity and may not only be safer anti-cancer agents, but may be more effective. There are clear prognostic factors that help the treating physician to decide which particular patient will ultimately fail therapy and succumb to the disease. Although a full discussion of these risk factors is beyond the scope of this review, they include a combination of clinical factors such as disease bulk and degree of response or refractoriness to first-line therapy, patient age and performance status, as well as biological factors such as histological grade, cytogenetic or molecular characteristics of the cancer. These observations indicate that some patients likely to do poorly with standard therapy can be prospectively identified and may be considered for enrolment in clinical trials of novel therapies. 10.1517/14712598.2014.860442 © 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

1

S. Gill & D. L. Porter

Article highlights. . .

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In the last few years, genetic modification of T cells for the treatment of cancer has become a reality. The field has been significantly advanced by the unusual situation whereby ablation of antigen-expressing normal cells (CD19) is well tolerated, allowing the relatively safe targeting of CD19 in patients. Several different institutions have produced exciting results, demonstrating the feasibility and effectiveness of this approach. Considerable variations in gene engineering and T-cell production approaches between institutions have likely led to the wide differences in reported outcomes. In some cases, marked potency of the T cells has to be balanced against the potential for toxicity relating to cytokine release, macrophage activation. Ongoing preclinical research and careful correlative analyses from currently accruing clinical trials will be essential to further enhance our understanding of the requirements for persistence of T cells, thus improving efficacy.

This box summarizes key points contained in the article.

Virtually all B-cell malignancies (with the exception of some very immature acute lymphoid leukemias) express the antigen CD19 on their surface [3]. CD19 is an ideal target antigen. It is present on normal and malignant B cells as well as on follicular dendritic cells, and lacks homology to other known proteins [3,4]. Its function is thought to be in establishing the threshold for B-cell receptor (BCR)-dependent and -independent signaling, playing a role in modulating the balance between antigen-induced response and tolerance induction [5]. During B-cell development, CD19 is first seen on the cell surface during immunoglobulin gene rearrangement in pre-B cells and is lost during plasma cell differentiation [3,6]. It is important to note that although CD19 is seen on some normal plasma cells, it is lacking in most plasma cell myelomas [7]. Apart from its roles in B-cell activation, CD19 signaling is thought to play a role in B-cell maturation in the bone marrow [8]. Studies of mice genetically deficient in CD19 show the expected reduction in circulating and splenic B cells but no effect on the number of B-cell precursors in the marrow [4]; the remaining B cells in these mice show reduced antigen-specific proliferation and impaired humoral immunity. Of most relevance, a recently described syndrome of immunodeficiency due to mutations in the CD19 gene shows that patients with this disorder have impaired antigeninduced BCR response, poor antibody response to some vaccinations and increased susceptibility to infection [9]. Nonetheless, extensive clinical experience with the antiCD20 monoclonal antibody rituximab shows that profound, long-term B-cell depletion is generally well-tolerated [10]. Of the novel agents investigated in recent years, three broad classes have capitalized on the ubiquity of CD19 expression in B-cell malignancies. These include monoclonal antibodies 2

(including those that carry a toxic payload, so-called antibody-drug conjugates or ADC) [11,12], bispecific antibodies that recruit effector T cells to interact with B cells (so-called BiTE, exemplified by blinatumumab) [13] and adoptive transfer of genetically engineered T cells that are redirected to target CD19 by expressing a chimeric antigen receptor or CAR (we will refer to these as CART cells in this review). Adoptive cellular therapy with T cells was first reported in patients more than 20 years ago, and initially relied on the infusion of high numbers of ex vivo expanded lymphocytes including tumor-infiltrating lymphocytes (TIL), bulk lymphocyte populations or tumor antigen-specific clones [14-18]. In combination with the proven track record of HSCT to treat hematologic malignancy [19] and supported by the reliance of cancer on inducing immunosuppression [20], these observations indicated that the immune system could be reliably harnessed to treat malignancy if only we could learn some basic ‘road rules’. The first of these rules is to find a suitable cancer-specific target. Having identified CD19 as a suitable target for anti B-cell immunotherapy, the second rule is to identify a potent mechanism to bring effector T cells to the proximity of cells bearing CD19, and induce the T cells to respond to these targets. One way to do this is by transferring to T cells genetic material encoding an antigen receptor to CD19, thus constructing CART cells [21]. The CAR is constructed from a targeting moiety, a transmembrane domain for stable cell surface expression with a hinge to provide flexibility, and signaling molecules to induce intracellular pathways (Figure 1). Typically, the targeting moiety is composed of a single chain variable fragment (scFv) derived from the fused variable heavy and light chains of an antibody that could be generated to any cell-surface antigen, including protein, glycoprotein, carbohydrate or glycolipid. Thus, a novel entity is created that combines the high affinity and specificity of an antibody for a cell-surface target, with the multiple effector functions of T cells. Hence, unlike the ‘native’ recognition of T cells via the T-cell receptor, there is no need for antigen processing, presentation in the MHC groove, or MHC restriction [22]. The hinge region may be derived from a CD8 or IgG4 molecule, and intracellular signaling is typically transduced via the CD3z chain. Different groups have employed either no co-stimulation or diverse co-stimulation strategies, that include provision of CD28 or 4-1BB signaling downstream of the CD3z chain [23-27]. First-generation CAR technology employed no co-stimulatory signal and was compromised by the induction of activation-induced apoptosis or anergy in the stimulated T cells [28]. Second generation CAR technology includes the provision of a ‘signal 2’ from CD28, 4-1BB (CD137) or OX-40 molecules and most of the published clinical trials of CAR-modified T cells have employed this approach [29-33]. Finally, third-generation CARs employ more than one co-stimulatory molecule and the feasibility of this approach has been demonstrated clinically [34-36].

Expert Opin. Biol. Ther. (2013) 14(2)

CAR-modified anti-CD19 T cells for the treatment of B-cell malignancies: rules of the road

Antigen-targeting moiety

VL

VH

Linker/ hinge region

CD8α IgG4

Stimulation/ Co-simulation intracellular signal transduction molecule(s)

4-1BB/ CD28/ OX-40

CD3-ζ


Figure 1. Schematic diagram of the structure of a typical chimeric antigen receptor.

Although the technology for transducing T cells with CAR has been available since the late 1980s [21], it is important to note that the first successful clinical result in CD19-expressing malignancies was published only recently and thus patient numbers and follow-up are relatively limited [24]. In this review, we will describe the ‘rules of the road’ for CAR therapy using the paradigm of anti-CD19 CART cells based on our experience and illustrated by the relevant published clinical trials and by important preclinical studies. In Table 1, we present salient details on the 36 patients that have been treated on clinical trials and whose results are available in the peer-reviewed literature. In this Expert Opinion article, we present the process of designing, enrolling, treating and following patients on CART19 trials in a simple chronological order.

2.

Pre-infusion

Patient selection Experimental therapies, as promising as they may seem, are generally first offered to patients who have failed or not tolerated standard therapies. However, within the spectrum of disease bulk and especially in the context of relatively slowly growing malignancies such as CLL or the indolent B-cell NHL, it may be possible to plan to treat patients at higher or lower burdens of disease. A fascinating aspect of the new paradigm of cancer therapy illustrated by anti-CD19 CART cells is that CAR-modified T cells are not inert drug: they act upon their target and are acted upon by it, unlike the case with conventional anti-neoplastic therapy. Specifically, target exposure induces proliferation in T cells, upon which they gain further effector function. Hence in the context of CART cell therapy, the presence of extensive tumor burden could have a stimulating effect on the ability of the T cells to proliferate and release effector cytokines, as is the case with viral infections [37]. Indeed, in our studies some of the most notable responses have occurred in patients with bulky disease [38] and in other studies the degree of increase in serum cytokines correlates with tumor burden [39]. At this time, it is not definitively known whether tumor bulk enhances or limits response to CART therapy. 2.1

Another consideration in patients who have been heavily pre-treated, is that the patient’s circulating T cells may be quantitatively or functionally diminished. This is a particular problem in patients treated with lympholytic drugs such as steroids or monoclonal antibodies such as alemtuzumab. Thus, prior to enrolling the patient on a CAR clinical trial it is important that functional T cells are available for manipulation. Thus, the situation may arise in which a patient who presents for treatment cannot mobilize a sufficient number of cells and is thus ineligible for treatment. This situation is a reflection of the fact that CART cell therapy is truly personalized therapy; a personalized product must be made for each individual. The only clinical scenario that resembles this is where a patient in need of autologous stem cell transplantation cannot undergo the procedure if he or she is unable to mobilize sufficient stem cells. One possible way around this dilemma is the use of allogeneic modified T cells. To date, this approach has been taken only in patients who have relapsed with a B-cell malignancy after a prior allogeneic stem cell transplant [40], although several clinical trials are currently recruiting for this indication (Table 2). This approach is potentially limited by the risk of graft-versus-host disease (GVHD) occurring when donor T cells that are modified to express a CAR, but that still express their native T-cell receptor, are stimulated to recognize host antigens. Despite this theoretical risk, GVHD after infusion of allogeneic CAR19 cells has not yet been reported [40,41]. Many other factors go into patient selection for CART therapy. We have treated patients between age 1 and 78, and age does not appear to be relevant within reasonable boundaries. However, there may be significant toxicity associated with CART cell proliferation and cell killing; it is important that patients have adequate organ function and physiologic reserve. Most clinical trials have very specific selection criteria (e.g., see NCT01029366 on clinicaltrials.gov) that would need to be carefully considered when trying to expand these therapies for more general use. Whether CAR-modified T cells behave differently based on characteristics of the patient or malignancy is at present unknown.


3

4

Disease state

Lymphodepletion

Total T-cell dose


Relapsed

Relapsed

PentoCy

BR

CLL (62)

CLL (61)

18

19

Relapsed, progressive

Relapsed Relapsed

Cy

Cy

Cy Cy

No

No No No No

1.4 109 CAR cells

cells cells cells cells

No

CAR CAR CAR CAR

7.6 108 CAR cells

109 109 109 109 No No

4 108 CAR cells 4 108 CAR cells

2.5 1.2 1.1 3.2

No

Fever

Fever Fever fever Fever, ARF, hypotension Fever Fever, hypotension Fever

Fever, hypotension Fever, dyspnea, cardiac dysfunction TLS, fever, transaminitis

No

1.5 105/kg CAR cells

NR NR

No No

No

NR NR NR NR

No No No No

1.0 107/kg CAR cells

Lymphopenia

IL-2

SD

SD

PR (delayed) PD

PD PD PD NE (Died)

CR

PR

CR

PD PD

SD PD SD PD

NR

NE

Best disease response

1 mo

2 mo*

4 mo

9 NA

NA NA NA NA

10*

7

11*

1.5 0.5

10 1 3 1.5

5

1

weeks weeks weeks weeks

30

20

8 8

35 NR NR 1

> 24 weeks

> 24 weeks

> 24 weeks

6 weeks 6 weeks

6 6 6 6

1

1

Time to Time to CAR response relapse/ persistence Progression (days) (months)

*Indicates ongoing response at time of report. z This patient was treated twice. AlloHCT: Allogeneic hematopoietic cell transplantation; B: Bendamustine; BR: Bendamustine and rituximab; CLL: Chronic lymphoid leukemia; CR: Complete response; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; FluCy: Fludarabine and cyclophosphamide; NE: Non-evaluable; NR: No response; PentoCy: Pentostatin and cyclophosphamide; PR: Partial response; SD: Stable disease; SMZL: Splenic marginal zone lymphoma; VP16: Etoposide.

CLL (68) CLL (68)

16 17

retroviral SJ25C1 anti-CD19 scFv-CD28-CD3z, [27] CLL (51) Relapsed No CLL (72) Relapsed No CLL (73) Relapsed No CLL (69) Relapsed Cy

CLL (64)

11

MSKCC, 12 13 14 15

CLL (77)

10

Lymphopenia

IL-2

CAR % Exogenous Toxicities cytokines

City of hope, retroviral FMC63 anti-CD19 scFv-CD3z with thymidine kinase suicide gene [24] NR 1 FL (NR) Refractory Flu after 108/m2 (#1) dose #1 109/m2 (#2, #3) 2 109/m2 (#4, #5) NR 2 FL (NR) Refractory Flu after 108/m2 (#1) 109/m2 (#2, #3) dose #1 2 109/m2 (#4) Baylor, retroviral FMC63 anti-CD19 scFv-CD3z and anti-CD19 scFv-CD28-CD3z [23] 3 SLL (53) Relapsed No 2 107/m2 20 -- 60 20 -- 60 4 FL/DLBCL (56) Relapsed No 2 107/m2 20 -- 60 5 DLBCL (46) Relapsed No 1 108/m2 20 -- 60 6 DLBCL (57) Relapsed No 1 108/m2 Refractory 20 -- 60 7 FL/DLBCL Relapsed No 2 108/m2 20 -- 60 8 DLBCL Relapsed No 2 108/m2 University of Pennsylvania, lentiviral FMC63 anti-CD19 scFv-41BB-CD3z [25,38] 9 CLL (65) Relapsed B 1.6 107/kg CAR cells

Patients Disease (age)

Table 1. Clinical trials with anti-CD19 T-cell therapy.



CLL (54)

CLL (57)

CLL (61)

Follicular (63) Relapsed

26

27

28


29

45

53 50 30 51

71

CyFlu D-7 to D-1 2.5 107/kg

CyFlu D-7 to D-1 2.0 107/kg CyFlu D-7 to D-1 0.6 107/kg CyFlu D-7 to D-1 5.5 107/kg CyFlu D-7 to D-1 5.4 107/kg

CyFlu D-7 to D-1 4.2 107/kg

No

IL-2

IL-2

IL-2

IL-2

IL-2

IL-2

IL-2 IL-2

Cytopenias, fever Fatigue, zoster Bacteremia, pneumonia, stroke Hypotension, ARF, hypoxemia, capillary leak, hyperbilirubinemia Diarrhea, fatigue Fever, fatigue, hypotension Hypotension, capillary leak Obtundation, ARF, capillary leak, hyperbilirubinemia Hypotension, obtundation, ARF, capillary leak

CR (molecular)

PR

PR

PR

SD

PR

CR

PR NE

PR

Diarrhea, hypo- NE (alloHCT tension, at 8 weeks) neutropenia


9*

8*

7*

7

6

12

15*

18* NE (died, influenza)

7

NE

180*

132*

132*

20

20

20

20

20 20

6 weeks 20

15



University of Pennsylvania, lentiviral FMC63 anti-CD19 scFv-41BB-CD3z [40] 30 ALL (7) Relapsed, No 1.2 107/kg CAR refractory

Relapsed

Relapsed

Relapsed

Relapsed

SMZL (55)

25

Relapsed

Relapsed Relapsed

CLL (61)

FL (48) FL (48)

24

22b 23

IL-2

No


63 65

1.8 108 CAR cells

Total T-cell dose

CyFlu D-7 to D-1 2.1 107/kg CyFlu D-7 to D-1 0.5 107/kg

Cy

Lymphodepletion

64

ALL (67)

Relapsed, progressive Relapsed

Disease state

21 ALL (48) Relapsed NA NCI, retroviral FMC63 anti-CD19 scFv-CD28-CD3z, [42], FL (47) Relapsed CyFlu D-7 to D-1 0.5 107/kg 22az

20


Table 1. Clinical trials with anti-CD19 T-cell therapy (continued).



5

6 No

No

No

No

3.2 108 CAR

2.9 108 CAR

1.4 108 CAR

ALL (59)

ALL (58)

ALL (23)

34

35


36

Cy

Cy

Cy

Neutropenia, diarrhea, hypotension Febrile neutropenia, hypotension Fatigue, febrile neutropenia, hypotension, seizure Hypoxia, altered mental state, febrile neutropenia, hypoxia, seizure Fever

MRD-

MRD-

MRD-

MRD-

MRD-

NA (alloHCT) 28

NA (alloHCT) 35

70

D30

CR and MRDby D8

NA (alloHCT) 55

NA (alloHCT) 47

CR D11, 1.5 (relapse 47 MRD- D59 with CD19* disease)

D28

2 (relapse with CD19disease)



MRD+

Refractory

Refractory

Cy

3.2 108 CAR

MRD-

ALL (56)

No


Fever, respiratory failure, hypotension Fever, myalgias, CR (MRD+) confusion


33

1.4 106/kg CAR of donor origin

Total T-cell dose

No

Lymphodepletion

1.8 108 CAR

ALL (10)

Disease state

Relapsed Cy, VP16 refractory post-alloHCT and blinatumumab MSKCC, retroviral SJ25C1 anti-CD19 scFv-CD28-CD3z, [39] 32 ALL (66) Relapsed, MRD+ Cy

31


Table 1. Clinical trials with anti-CD19 T-cell therapy (continued).




CLL

B-ALL

B-cell malignancy

B-cell malignancy B-cell malignancy B-cell malignancy

CD19+ ALL

MSKCC

MSKCC

FHCRC

MDACC NCI Children’s Hospital of Philadelphia Seattle Children’s

*Based on a search of clinicaltrials.gov 9/1/2013.

MSKCC Beijing FHCRC

CLL/SLL Aggressive B-NHL, relapsed/ refractory B-ALL B-cell malignancy B-cell malignancy

1 -- 26 yo

B-cell NHL

COH

Penn MSKCC

1 -- 65 1 -- 30 yo 1 -- 21 yo

B-cell malignancy B-ALL B-ALL B-cell malignancy

Penn Penn MSKCC NCI

< 26 yo 5 -- 90 yo > 18 yo

> 18 yo 18 -- 70 yo

18 -- 75

< 19 yo

> 18 yo

> 18 yo

> 18 yo > 18 yo > 18 yo 18 -- 75

scFv-41BB-CD3z scFv-41BB-CD3z scFv-CD28-CD3z scFv-CD28-CD3z

Anti-CD19 scFv-CD28-CD3z Anti-CD19 scFv-41BB-CD3z

Anti-CD19 scFv-41BB-CD3z Anti-CD19 scFv-CD28-CD3z

Anti-CD19 scFv-CD28-CD3z Anti-CD19 scFv-41BB-CD3z

Anti-CD19 scFv-CD28-CD3z


Anti-CD19 Anti-CD19 Anti-CD19 Anti-CD19


18 -- 68 18 -- 65

B-cell malignancy B-cell lymphoma

NCI MDACC

scFv-CD28-CD3z scFv-CD28-CD3z scFv-CD28-CD3z scFv-CD28-CD3z

Anti-CD19 Anti-CD19 Anti-CD19 Anti-CD19

CAR construct

> 18 yo Any Any Any

CLL B-cell malignancy B-cell malignancy B-cell malignancy

MSKCC BCM BCM BCM

Patient population

Disease

Center

Table 2. Currently recruiting anti-CD19 T-cell therapy trials*.

LV

RV

LV RV

RV LV

RV

RV

LV LV RV RV

RV

RV RV RV RV

Gene transfer

Autologous Autologous Autologous

Autologous Autologous

Autologous

Autologous, central memory CD8+ T cells Autologous, consolidation after chemo Allogeneic donorderived EBV CTL Allogeneic donorderived CD8+ central memory viral-specific CTL Allogeneic T cells Autologous Autologous

Autologous Autologous Autologous Allogeneic donorderived trivirusspecific CTL Autologous Autologous (after autologous HCT) Autologous Allogeneic Autologous Allogeneic

Cellular product

EGFR+ construct (may allow deletion) 2 dose level comparison After autologous SCT

After alloHCT

After alloHCT

After alloHCT

Upfront therapy

Active GVHD not allowed

With IL-2 With or without IL-2

Dose-escalation With ipilimumab Dose escalation After alloHCT

Notes


NCT01860937 NCT01864889 NCT01865617

NCT01747486 NCT01840566

NCT01683279

NCT01497184 NCT01593696 NCT01626495

NCT01475058

NCT01430390

NCT01416974

NCT01318317

NCT01029366 NA NCT01044069 NCT01087294

NCT00924326 NCT00968760

NCT00466531 NCT00586391 NCT00608270 NCT00840853

Clinicaltrials.gov identifier


7



2.2

T-cell expansion

T cells are harvested from the patient via peripheral blood leukapheresis without prior cytokine stimulation. Approaches for ex vivo T-cell expansion vary between centers, and include artificial antigen-presenting cell using magnetic beads coated with anti-CD3 and anti-CD28 antibodies with or without cytokines such as IL-2 to enhance T-cell proliferation [38]; alternatively T cells might be grown in the presence of soluble anti-CD3 antibodies alone [24,42]. T cells are grown under Good Manufacturing Practice in dedicated facilities, and must meet strict release criteria, which include CAR expression by flow cytometry, average vector copy number by PCR, absence of replication-competent virus and negative microbial and endotoxin testing. Within the first few days of the T-cell expansion culture, the genetic material encoding the CAR is introduced (more on this below). The degree to which T cells expand (generally reported in absolute numbers or in population doublings), the transduction efficiency (percent of T cells bearing the introduced genetic material, often 50% or less), viability and procedure and criteria for terminating the culture and freezing of the cell product, are subject to Standard Operating Procedures in each center, and are not standardized between institutions. These discrepancies may have an as-yet unappreciated role in the downstream behavior of the cell product. Similarly, the input as well as the final Tcell phenotype varies from center to center and patient to patient in terms of CD4 and CD8 numbers, memory or effector phenotype and other parameters. While preclinical investigations indicate variously that naı¨ve, central memory, Th17 or memory stem cell populations evince more potent anti-tumor effect in vivo, this point remains a subject of active investigation [43-47]. Several of the currently recruiting clinical trials are employing T-cell products enriched for CD8 or for central memory cells (Table 2). Selection of a CAR construct A crucial consideration is the selection of the individual components that go to making a CAR construct. With one notable exception, there have not been direct head-to-head comparisons of different CAR constructs in the same patient or even in the same trial [23]. Thus to date, different groups employ different single chain variable fragments as the targeting moieties of their CAR, and these are placed in different orientations (light-to-heavy or heavy-to-light chains direction), followed by different lengths of spacer or hinge regions, followed by different signal transduction moieties (generally the CD3z chain). The optimal co-stimulatory molecule is not known, with some centers using CD28 and others 4-1BB (CD137). ‘Third--generation’ CARs utilizing more than one co-stimulatory molecule have been reported [36]. These differences in co-stimulatory molecule utilization likely result from conclusions derived from preclinical investigations, usually in vitro, in which the construct producing the optimal desired effect (cytotoxicity, cytokine production or 2.3

8

proliferation, but rarely all three) is selected for further study [33]. Finally, the method by which the genetic material is introduced into the T cells varies by center. Most groups utilize retroviral transduction [42,48] or lentiviral transduction [25] although DNA has also been transferred by electroporation [49] or by transposon [50]. Viral transduction leads to permanent integration into the T-cell genome, associated in theory with a risk of genotoxicity, but in practice the longterm safety of gamma retroviral transfer into T cells has been well demonstrated [51,52]; viral work is however clearly associated with increased regulatory hurdles and costs related to the production process [53]. Where transient expression of the chimeric construct is desired, T cells may be electroporated with in vitro-transcribed mRNA, leading to expression of the encoded protein for up to a week [54]. This approach appears to be cost-effective and efficient and is being trialed at our institution [22]. It is as yet unknown whether factors intrinsic to the scFv are important, and the two scFv employed in the antiCD19 studies have been derived from clones FMC63 [24,25,42] or SJ25C1 [27]. To date, all scFv used in clinical trials have been derived from murine anti-human antibodies, leading to the theoretical risk of inducing an anti-CAR immune response. Although in one report the persistence of CAR T cells was possibly curtailed by an anti-transgene immune response, this was likely related to the thymidine kinase suicide switch in the construct [24] and most trials have not identified significant anti-CAR immunity. Engineered T cells retain functionality of their native T-cell receptor, a fact that has been utilized in preclinical studies where virus-specific T cells were modified to express CAR [55,56]. The possible advantage of this approach is that when taken from an allogenic or third-party donor, virusspecific T cells should not induce GVHD after transfer from an allogeneic donor, and can furthermore receive costimulation by viruses latent in the host. Clinical trials investigating this approach are in progress (Table 2) and one has recently been published [41]. 3.

Peri-infusion

We believe that for maximal efficacy, CART cells must not only proliferate, but persist. In our trials, the degree of in vivo expansion seems to correlate with response. No patient without significant CART cell expansion responded (unpublished data). Furthermore, it is likely that persistence is important for long-term disease control, and trials with disappointing efficacy generally also report limited T-cell persistence (Table 1). Many factors intrinsic to the CAR molecule are critical to direct proper T-cell activation and expansion, and may promote antigen-dependent as well as antigen-independent Tcell survival. However, patient-specific factors likely also influence CART cell activity. Preclinical studies indicate that one of the requirements for T-cell proliferation, expansion




and persistence after adoptive transfer is the degree of lymphodepletion [48,57] and this has also been explored in patients [27,58,59]. This is generally achieved by the administration of chemotherapy or less commonly radiation. Lymphodepletion is thought to enhance T-cell persistence by creating ‘homeostatic space’, increasing the production and availability of homeostatic cytokines such as IL-15 and IL-7 and by depleting the population of endogenous suppressive regulatory T cells [58,60,61]. Clinical trials of CART cell for B-cell malignancies employ different drugs and doses (Table 1), and the optimal schedule and degree of lymphopenia is as yet unknown. Indeed, the first trials with anti-CD19 CAR-modified T cells did not employ lymphodepletion and results were disappointing [23,24]. There have been no direct comparisons with or without lymphodepletion, but a single institution study showed enhanced persistence and improved outcomes when patients received lymphodepletion compared with an earlier cohort of patients undergoing no lymphodepletion [27], and probably T cells should be infused soon after the lymphodepletion regimen as recent data suggest that Tcell infusion in the first few days after conditioning leads to superior reconstitution [62,63]. As with any adoptive cellular therapy such as the infusion of platelets, red blood cells or stem cells, there is the potential for infusion-associated reactions. Where reported, acute infusion reactions associated with CART19 therapy have been mild [24,25,27,42]. Patients typically receive standard premedications and then observed for several hours after each infusion, if treatment is initiated in the outpatient setting. The administration of exogenous cytokines remains controversial. There is a long history of IL-2 use for the in vivo expansion of transferred or endogenous lymphocytes [64], however IL-2 may impair the formation of memory and also increases the number of regulatory T cells with a potential deleterious effect on anti-tumor immunity [65]. 4.

Post-infusion

Toxicity Successful T-cell expansion and engagement of target are generally associated with a systemic inflammatory response, which usually occurs within 1 -- 3 weeks of infusion [38-40,42]. High fevers are common, and patients may develop hypotension, pulmonary infiltrates or capillary leak (Table 1). This is thought to be related to the release of cytokines from the proliferating T cells, including TNF-a, IFN-g, IL-2 and IL-6, among others [27,38,40,42]. Interestingly, similar reactions have been reported after administration of the bispecific antiCD19 antibody blinatumumab [66]. Delayed but massive tumor lysis has been reported several weeks after CARmodified anti-CD19 T-cell therapy [25] coincident with Tcell proliferation. Highlighting the paradigm shift away from conventional anti-neoplastic chemotherapy, it is becoming apparent that the maximal effect of the intervention may at times be delayed; although not routinely reported and 4.1

subject to the dictates of clinical trial follow-up schemata, some patients clearly have deepening responses over time (Table 1). This observation has important implications for patient follow-up and response assessment. Laboratory abnormalities associated with severe cytokine release syndrome include features of macrophage activation, including cytopenias, marked hyperferritinemia and decreased fibrinogen concentrations [22]. While laboratory abnormalities can be quite profound, it is usually the presence of clinical complications that spurs the need for intervention; in our experience, steroids are variably effective in controlling the exuberant inflammatory reaction. Where severe, additional therapies such as anti-cytokine agents tociluzumab (NB) anti-IL6receptor (IL-6R) or etanercept (anti-TNF-a) may be effective [40]. As anticipated from their mechanism of action, anti-CD19 CAR T-cell infusion leads to profound B-cell aplasia, and may also deplete normal plasma cells [38]. T-cell persistence is associated with prolonged absence of circulating B cells and hence a theoretical immunodeficiency, however in practice opportunistic infections or an increase in the expected infection rate have not been reported, and patients routinely receive intravenous gamma globulin repletion [25,27,42].

Efficacy Response rates vary widely between centers (Table 1), and may reflect differences in any of the parameters described above. With this proviso, out of the 36 patients reported in the literature, 18 achieved any degree of response and 10 achieved a complete response. A clinical trial has been designed to directly study the results of infusion of two different CAR constructs into each patient, and the results of this trial will likely be of great consequence for the design of future iterations of anti-CD19 CAR therapy. Results of published trials indicate that reduction and elimination of disease can be seen in peripheral blood, marrow as well as in bulky lymphadenopathy, and tends to occur within the first few weeks after T-cell infusion. Recently published results indicate that CAR T cells can penetrate the CNS and are seen in the CSF [40]. This result is of particular importance given the predilection of some B-cell malignancies for involvement of the CNS [67], and may reflect a compromised blood-brain barrier due to inflammation or prior chemotherapy, or indeed a physiologic property of activated, antigen-specific T cells; we are not aware of any studies looking specifically at the migration properties and homing markers on CAR T cells although this would like be an important point to investigate in future. Some recipients of CART cells develop as yet unexplained neurotoxicity with confusion, somnolence and even seizures; this may be mediated by a direct toxic effect of CART cells within the CNS or alternatively by inflammatory cytokines secreted during the CRS (unpublished data). Therefore, CNS infiltration with CART cells will need to be monitored and studied carefully. However, this also suggests 4.2


9



that while as yet unproven, CART cells may be able to eliminate extramedullary disease where present. A theoretical advantage of CD19 as a target is that CD19-bearing cells are constantly produced in the bone marrow, hence possibly providing ongoing, low-level antigenic stimulation to the infused CART cells to aid in their persistence, a similar concept to booster vaccination. Whether this is in fact the case has not yet been experimentally demonstrated. As anticipated, the absence of normal CD19-expressing B cells in the blood serves as a surrogate marker for persistence of an anti-CD19 effect [42,68]. Where patients relapse after prior allogeneic HCT, allogeneic donor-type, host-derived T cells modified to express the CAR construct have been used safely without GVHD [40,41]. Alternative mechanisms to allow safe transfer of allogeneic T cells by generating virus-specific T cells have been pioneered and are being investigated in currently accruing clinical trials (Table 2) [51,69]. Effector cells other than T cells include natural killer (NK) cells or cytokine-induced killer (CIK) cells do not cause GVHD and could prove to be useful agents in the allogeneic setting. Preclinical studies support the potential of this approach and a clinical trial investigating this approach has been initiated [70]. Relapse Too few patients have been treated with CART cells to determine the durability of responses. At the University of Pennsylvania, we have observed ongoing remissions beyond 3 years in CLL and beyond 1 year in ALL (unpublished data) and relapses are uncommon. Whereas disease progression after a partial response is to be expected, particularly in the setting of limited T-cell persistence, antigen-loss relapses have been reported after potent CD19-directed T-cell as well as bispecific antibody therapy with blinatumumab [13,40]. These likely reflect the outgrowth of a minor CD19-negative clone that was present at diagnosis and expanded under potent selective pressure. These observations highlight a main limitation of such potent antigen-specific immunotherapy and suggest that future iterations of this modality might incorporate multi-antigen-specific T cells, perhaps by infusing different T-cell products specific for different B-cell antigens [71]. Treatment of larger numbers of patients, and further follow-up is needed to determine the likelihood of relapse and factors that may contribute to disease recurrence, such as the presence of residual disease and treatment, loss of CART cell persistence, antigen loss or other factors. 4.3

5.

Conclusions

Genetic engineering of T cells using retroviral or lentiviral approaches is feasible and safe, and clinical grade reagents can be produced in a reasonable and clinically useful time frame for the treatment of patients with both indolent and aggressive malignancies. Response rates vary between centers and probably relate to patient heterogeneity as well as to 10

important unresolved issues of CAR design and transgenic T-cell production. The main weakness to date therefore relates the lack of standardization and the absence of experimentally proven optimal approaches to the various components of the CAR T-cell treatment. The field will therefore gain significantly from more cooperation between the leading centers in order to identify the optimal ingredients to successful CAR therapy. For example, is there an optimal epitope to target on CD19 (and by extension, an optimal clone from which the scFv is derived)? What is the best method of gene transfer -- lentiviral or retroviral transduction, transposon or mRNA electroporation? Which co-stimulatory molecule(s) provide the best proliferation and activation signals? Novel treatment-related toxicities, cytokine release syndrome and macrophage activation syndrome, likely originate from a potent activation of the infused T cells and their cytokine-mediated interaction with endogenous macrophages. Early data suggest that some measures of CRS/MAS are indicative of a productive immune response of CART against their cognate target and predictive of a good response. However, the pathophysiology and optimal management of these manifestations remain opaque and will require intensive study. Targeting of B-cell malignancies with CAR-modified T cells may be but the lowest hanging fruit among malignancies, due to the specificity of expression of CD19 to B cells and the relative safety of long-term depletion of B cells. However, conclusions gleaned from a thorough investigation of CART-19 treatment should be generalizable to other malignancies once other appropriate CAR targets are identified. 6.

Expert opinion

Anti-CD19 CAR-modified T-cell therapy represents arguably the most successful implementation of genetic engineering to date. The exciting early-phase clinical trials currently in progress bring fresh insights almost monthly, and more are planned (Table 2). Ultimately, the field will benefit from direct comparisons of products, in order to identify the optimal patient population, disease state, conditioning regimens, dose and characteristics of the vector and infused T cells. Targeting of CD19 will likely be a useful paradigm for the treatment of other malignancies, where access of T cells to the tumor microenvironment may be more difficult (some solid tumors) or where ablation of normal cells expressing the target antigen is more difficult to manage (most other malignancies). In order to forestall antigen-loss relapses, treatment of B-cell malignancies could also be enhanced by sequential or simultaneous administration of T cells targeted to additional B-cell antigens, such as CD20, ROR1 or the light chain [24,49,72,73], a similar paradigm to the early days of anti-neoplastic chemotherapy. Other improvements to the basic CAR concept could include the provision of autocrine cytokines or other proliferative signals to drive T-cell proliferation, as has been shown to be feasible in preclinical studies [48,74]; any enhancement in efficacy would however have




to be weighed up against the risk of producing unrestrained T-cell proliferation or potent cytokine release syndrome. Although prolonged B-cell depletion appears to be well tolerated for a few years, we do not yet know whether there is an upper limit to this apparent safety, and we should keep an open mind about new manifestations of immunosuppression or autoimmunity in patients who experience truly long-term B-cell aplasia. With this in mind, the development of constructs that incorporate a suicide gene or other modalities to delete the infused T cells at-will would provide an important safety feature. This has been shown to be feasible in the setting of allogeneic haploidentical transplantation [75]. The improvement of existing techniques or implementation of new techniques that do not rely on retroviral or lentiviral transduction would have the advantage of less onerous and expensive production and regulatory requirements [22,50], thus facilitating the provision of this exciting treatment modality to more patients these have not been tested in any detail. These recent exciting findings in the field are likely to be followed by scientific, clinical, regulatory developments that will Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Affiliation

Saar Gill MD PhD & David L Porter† MD † Author for correspondence University of Pennsylvania, Abramson Cancer Center, Perelman School of Medicine, Division of Hematology-Oncology, Department of Medicine, Philadelphia, PA 19106, USA E-mail: [email protected]

13

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