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. Author manuscript; available in PMC: 2015 Dec 31.
Published in final edited form as: Discov Med. 2010 Apr;9(47):277–288.

Adoptive Immunotherapy for B-cell Malignancies with Autologous Chimeric Antigen Receptor Modified Tumor Targeted T Cells

Jae H Park 1, Renier J Brentjens 2
PMCID: PMC4697441  NIHMSID: NIHMS746465  PMID: 20423671

Abstract

Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

Introduction

B-cell malignancies represent a heterogeneous group of hematological cancers with variable clinical histories and prognoses. Most subtypes are initially responsive to combination chemotherapy regimens but many patients relapse. Currently, most patients remain incurable and the prognosis of patients with refractory or relapsed disease is poor. Clinical studies have demonstrated that B-cell malignancies are susceptible to immunotherapeutic approaches including antibody-based therapy (e.g., rituximab, alemtuzumab) and allogeneic hematopoietic stem cell transplantation (HSCT). A major benefit of allogeneic HSCT is the potential for a graft-versus-tumor (GVT) effect mediated by infused donor T-cells present in the stem cell harvest specific to allogeneic antigens expressed by the host tumor cells. Evidence suggestive of a donor T cell GVT effect in B-cell malignancies includes the lower relapse rates and long-term remissions after HSCT in relapsed or refractory lymphomas (Khouri et al., 2008; Sorror et al., 2008; Bishop et al., 2008; Mandigers et al., 2003; Marks et al., 2002), chronic lymphocytic leukemia (Dregger et al., 2008; Toze et al., 2005; Farina et al., 2009; Gribben et al., 2005; Schetelig et al., 2008; Ritgen et al., 2008), and acute lymphoblastic leukemia (Hahn et al., 2006; Thiebaut et al., 2000; Thomas et al., 2004; Yanada et al., 2006). However, the presumed immunotherapeutic benefits of treatment with HSCT is limited by the overall restricted number of patients eligible for this therapeutic approach due to age constraints, availability of compatible donors, and significant transplant-related morbidity and mortality from acute and chronic graft-versus-host disease (GvHD). For these reasons, there is an urgent need for the development of more specific and less-toxic forms of immune-based cellular therapies to treat B cell malignancies.

Genetically Targeted T Cells: Chimeric Antigen Receptors

Autologous T cells may be genetically modified to target tumor-associated antigens (TAA). One way of genetically modifying T cells to target specific TAA is by gene transfer of cloned T-cell receptors (TCRs) derived from T-cell clones specific to tumor antigens. This approach has been shown to be feasible in clinical trials of metastatic melanoma (Johnson et al., 2009; Morgan et al., 2006), but published data utilizing this approach in the setting of B-cell malignancies is limited (Dossett et al., 2009; Xue et al., 2005). Moreover, because the TCR gene transfer approach can only recognize TAAs as peptides that are processed and presented by human leukocyte antigen (HLA) molecules, specificity of the TCR is restricted to specific patient HLA phenotypes and therefore lacks universal applicability. In addition, many tumor cells down-regulate HLA molecules and/or have dysfunctional antigen-presenting machinery so that the targeted TAA-derived peptides are often not adequately presented on the targeted tumor cell surface (Khong et al., 2002; Gottschalk et al., 2001).

One way to circumvent the limitations of TCR gene transfer is the use of chimeric antigen receptors (CARs). CARs are composed of a single-chain variable-fragment (scFv) antibody specific to TAA, fused to a transmembrane (TM) domain, which is further fused to a T-cell signaling moiety, most commonly either the CD3ζ or Fc receptor γ cytoplasmic signaling domains. The CAR approach has several advantages over TCR gene-modified T cells: 1) CAR recognition of the target antigen is HLA-independent, and is therefore applicable to patients of all HLA types and is furthermore unaffected by HLA down-regulation on the tumor cells; 2) CARs function in both CD4+ and CD8+ T cells, enabling both helper and cytotoxic tumor-targeted effector function; and 3) CARs can be further modified to overcome the lack of co-stimulatory ligands on tumor cells to enhance modified T cell anti-tumor efficacy as described below (Brentjens et al., 2009; Chekmasova et al., 2010).

CAR Design: First, Second-, and Third-generation CARs

For optimal activation and proliferation, T cells require both TCR engagement and signaling (termed “signal 1″), as well as co-stimulatory signaling through co-stimulatory receptors on T cells binding to cognate ligands expressed either by the target tumor cell or professional antigen-presenting cells (termed “signal 2″). Initial “first-generation” CARs were constructed through the fusion of a scFv-based TAA binding domain to an inert TM domain (e.g., the CD8 transmembrane domain), fused to a cytoplasmic signaling domain typically derived from the CD3-ζ or Fc receptor γ chains. The resulting CAR, when expressed by a T cell, engages the targeted antigen and delivers a “signal 1″ to the T cells, rendering tumor targeted T cells susceptible to anergy or apoptosis (Gimmi et al., 1993; Jenkins et al., 1990) resulting in truncated in vivo persistence due to the lack of T cell co-stimulation (i.e., “signal 2″) (Brentjens et al., 2003).

To overcome the lack of T cell co-stimulation, first-generation CARs have been further modified by incorporating the cytoplasmic signaling domains of T cell co-stimulatory receptors into the CAR (Figure 1). To date, investigators have constructed and tested “second-generation” CARs containing co-stimulatory cytoplasmic signaling domains derived from the T cell co-stimulatory receptors CD28, 4-1BB (CD137), and OX40 (CD134). Several groups subsequently confirmed that these second-generation CARs, when expressed in T cells, upon activation with targeted antigen, resulted in: 1) enhanced IL-2 production; 2) superior in vitro antigen-dependent proliferation; and 3) T cell up-regulation of the anti-apoptotic proteins (e.g., Bcl-XL) (Finney et al., 2004; Pule et al., 2005; Carpenito et al., 2009; Hombach et al., 2001).

Figure 1.

Figure 1

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Structure of chimeric antigen receptors (CARs). First-generation CARs are typically composed of a single fragment length antibody (scFv) containing the heavy (VH) and light chain (VL) variable regions specific to a TAA, fused to an inert TM domain of the CD8, fused to a cytoplasmic signaling domain of the T cell receptor (TCR) (most commonly the TCR ζ chain). Second-generation CARs include a co-stimulatory signaling domain (CD28, 4-1BB, or OX40), while third-generation CARs contain tandem cytoplasmic signaling domains from 2 co-stimulatory receptors (i.e., CD28-4-1BB and CD28-OX40).

More recently, several investigators have constructed and tested “third-generation” CARs containing tandem cytoplasmic signaling domains from two co-stimulatory receptors (i.e., CD28-4-1BB or CD28-OX40) (Figure 1) demonstrating a potential enhanced T cell signaling capacity when compared to second generation CARs (Kochenderfer et al., 2009; Milone et al., 2009; Wang et al., 2007).

Target Antigens for Immunotherapy of B-cell Malignancies

B-cell malignancies express several B cell specific TAAs that may serve as potential targets for CAR-mediated adoptive immunotherapy including CD19 and CD20. CD19 and CD20 are B cell specific antigens variably expressed on developing and mature B cells, are significantly absent on pluripotent hematopoietic stem cells, and are highly retained over the process of neoplastic transformation and therefore are stably expressed by most B-cell malignancies. For these reasons, CD19 and CD20 antigens represent attractive targets for adoptive immunotherapy of B cell cancers, and most of the preclinical and clinical studies using CAR modified T cells to date have focused on these target antigens.

Significantly, efficient T cell targeting of the CD19 and CD20 antigens would predictably result in prolonged or permanent B-cell aplasias and subsequently impairment of humoral immunity since normal B cells also express these antigens. While this potential treatment-related side effect may be clinically managed with periodic prophylactic infusions of human immunoglobulin in treated patients, several investigators have studied the feasibility to targeting alternative TAAs expressed more selectively by B-cell malignant cells. To this end, Vera et al. developed a CAR directed against the κ light chain of human immunoglobulin thereby sparing normal B cells which express the λ light chain thus preserving a population of normal B cells. The authors demonstrate that CAR+ T cells targeted to the κ light chain effectively lysed κ+ tumor cell lines and primary tumor cells both in vitro and in vivo while λ+ normal B cells remain unaffected (Vera et al., 2006). Alternatively, James et al. developed a CAR targeted to the CD22 B cell specific antigen, an antigen preferentially expressed by B cell lymphoma cells when compared to normal B cells (James et al., 2008). Due to the limited published literature on selective light chain and CD22 targeting, further discussion in this review will be focused on the development of adoptive immunotherapy using CAR modified T cells targeted to the CD19 and CD20 antigens.

CD19-targeted T cells: Preclinical Studies

First-generation CD19-targeted CARs

In our laboratory we have constructed a first generation CD19-targeted CAR (termed 19z1). A single infusion of human T cells transduced to express the 19z1 CAR into SCID-Beige mice eradicates established systemic Raji tumors, a human Burkitt’s lymphoma cell line (Brentjens et al., 2003). Notably, similar therapy failed to fully eradicate systemic NALM-6 tumors, a human pre-B cell ALL cell line which, in contrast to Raji tumor cells, lacks expression of the CD80 and CD86 co-stimulatory ligands. To further elucidate the role of in vivo co-stimulation, we genetically modified NALM-6 tumors to express CD80 and were able to successfully eradicate disease in a subset of 19z1 CAR+ T cell treated mice. These findings suggested a limitation of first-generation CARs and the requirement of in vivo co-stimulation for effective T cell mediated tumor eradication.

Cooper et al. similarly developed a CD19-targeted first generation CAR, which, when expressed in human T cells, mediated antigen-specific tumor lysis of CD19+ tumor cell lines and primary B-ALL blasts in vitro as well as subsequent modified T cell proliferation (Cooper et al., 2003). Furthermore, these CD19-targeted T cells demonstrated efficient in vivo anti-tumor efficacy in immune compromised NOD/SCID mice bearing established subcutaneous (s.c.) CD19+ human lymphoma tumors (Cooper et al., 2004).

Cheadle et al. transduced PBMC from nine patients with Non-Hodgkin’s lymphoma (NHL) with a different first-generation CD19-targeted CAR, demonstrating effective lysis of both the CD19+ Raji cells as well as autologous malignant B cells derived from patients’ lymph node biopsies (Cheadle et al., 2005). Furthermore, mouse T cells modified to express this CD19-targeted CAR successfully eradicated established s.c. B-cell lymphoma xenografts in SCID/Beige mice (Cheadle et al., 2009).

Second-generation CD19-targeted CARs

Kowolik et al. directly compared the in vitro and in vivo biology of first- and second-generation CD19-targeted CARs (Kowolik et al., 2006). In vitro, T cells expressing a second-generation CD19-targeted CAR, containing the CD28 cytoplasmic signaling domain, demonstrated a 10-fold higher proliferative response when co-cultured with CD19+ target cells, a 33-fold higher IL-2 production, and a 15-fold increased expression of Bcl-XL after stimulation when compared to T cells which expressed a first generation CD19 targeted CAR. In vivo, T cells expressing this second-generation CAR, when compared to T cells expressing the first generation CAR, persisted longer (50 vs. 6 days) and displayed superior anti-tumor efficacy in NOD/SCID mice with established intraperitoneal (i.p.) CD19+ CD80/86+ Daudi tumor cells (a human Burkitt’s lymphoma cell line).

Similarly, Loskog et al. directly compared first and second-generation CD19-targeted CARs. In this in vitro study, the second-generation CAR+ T cells showed a statistically higher antigen-specific proliferative response and persistent cytolytic activity against CD19+ CD80/86+ Daudi cells, even in the presence of immune suppressive regulatory T cells or in the context of the inhibitory IL-10 and TGF-β cytokines (Loskog et al., 2006).

All of the above studies tested the function of the second-generation CARs against human tumor cell lines which expressed the CD80 and CD86 co-stimulatory ligands. Based on our previous observation that first-generation 19z1 CAR+ T cells were unable to eradicate systemic NALM-6 tumor lacking CD80/86 expression, we tested the second-generation CAR with CD28 signaling domain (19-28z) against CD19+ CD80/86− ALL tumor cells (NALM-6), demonstrating successful eradication of systemic tumor in a subset of treated SCID-Beige mice (Brentjens et al., 2007).

Alternative second-generation CARs targeted to CD19 incorporating the cytoplasmic signaling domain of 4-1BB (CD137) or OX40 (CD134) co-stimulatory receptors (Figure 1) have produced mixed results. Imai et al. reported superior in vitro proliferative response and cytolytic activity of T cells expressing the second-generation CAR containing the 4-1BB cytoplasmic domain when compared to T cells expressing a first generation CAR (Imai et al., 2004). We have previously reported on a series of second-generation CARs containing the cytoplasmic signaling domains of CD28 (19-28z), 4-1BB (19-BBz), or OX40 (19-OX40z). Of these CARs, we found that only the inclusion of the CD28 molecule (19-28z) enhanced both T-cell proliferation and cytokine secretion in vitro in the absence of exogenous costimulatory ligands when compared to T cells expressing the first generation 19z1 CAR (Brentjens et al., 2007). Discrepancies between our finding and those published by others demonstrating enhanced co-stimulation in second-generation CARs bearing the cytoplasmic signaling domains of the 4-1BB and OX-40 receptors (Imai et al., 2004; Finney et al., 2004) may be due to both differences in the epitopes on the CD19 antigen recognized by these CARs and CAR construction, implying a critical role of extracellular CAR design to signaling function.

Third-generation CD19-targeted CARs

Somewhat unexpectedly, Kochenderfer et al. reported lower in vitro IFNγ and IL-2 cytokine secretion by T cells modified to express a third generation CD19-targeted CAR containing the CD28 and 4-1BB signaling domains in tandem when compared to T cells modified to express a second-generation CAR containing the CD28 signaling domain alone (Kochenderfer et al., 2009). This discrepancy may be due in part to the observed lower expression of the third generation CAR when compared to the second generation CAR. However, due to the fact that these CARs contained different extracellular hinge regions, interpretation of these studies is somewhat limited with respect to the assessment of the apparent inhibitory biologic function of the 4-1BB cytoplasmic domain incorporated into this third generation CAR.

Somewhat consistent with these in vitro findings, Milone et al. demonstrated that T cells expressing a third-generation CD19 targeted CAR containing the CD28 and 4-1BB signaling domains did not have a superior in vitro proliferative response when compared to T cells expressing the first-generation CAR (Milone et al., 2009). However, in vivo, treatment of NOD/SCID mice bearing systemic primary human pre-B cell ALL xenografts with T cells modified to express this third-generation CAR significantly enhanced survival when compared to mice treated with T cells expressing either the first- or second-generation CD19-targeted CARs.

In summary, based on preclinical studies, it seems clear that second-generation CD19-targeted CARs are superior to first generation CARs with respect to signaling and anti-tumor efficacy when expressed by T cells. However, whether third generation CD19-targeted CARs further enhance or impede T cell anti-tumor function remains unclear and will require further pre-clinical study. All published preclinical studies of CD19-targeted CARs are summarized in Table 1a.

Table l.

a. Preclinical Studies for CD19-Targeled CAR
CAR Design Receptor Type Lymphodepleting
Therapy		 In Vivo Study Cell Lines Used
for In Vivo Study		 Reference
First Generation CAR scFv-CD3ζ - +, SCID/Beige i.v. Raji Brentjens et al., 2003
scFv-CD3ζ - - - Cooper et al., 2003
scFv-CD3ζ - +, NOD/SCID s.c. Daudi Cooper et al., 2004
scFv-CD3ζ - - - Cheadle et al., 2005
scFv-CD3ζ +, Cy i.p. 200mg/kg +, SCID/Beige i.v. Raji Cheadle et al., 2008
scFv-CD3ζ* -
+, Cy i.p. 200mg/kg		 +, SCID/Beige
+, Balb/c		 s.c. A20 Cheadle et al., 2009
scFv-CD3ζ +, 6Gy TBI or Cy +, Balb/c i.v. A20 Cheadle et al, 2010
Second-Generation CAR scFv-CD3ζ
scFv-4-lBB-CD3ζ		 - - - Imai et al., 2004
scFv-CD28-CD3ζ - - - Loskog et al., 2006
scFv-CD3ζ
scFv-CD28-CD3ζ		 - +, NOD/SCID i.p. Daudi Kowolik et al., 2006
scFv-CD28-CD3ζ
scFv-4-lBB-CD3ζ
scFv-OX40-CD3ζ		 - +, SCID/Beige i.v. NALM-6 Brentjens et al., 2007
Third-Generation CAR scFv-CD3ζ
scFv-CD28-CD3ζ
scFv-4-lBB-CD3ζ
scFv-CD28-4-lBB-CD3Cζ		 - +, NOD/SCID i.v. primary human pre-B ALL Milone et al., 2009
scFv-CD28-CD3ζ
scFv-CD28-4-IBB-CD3ζ		 - - - Kochenderfer et al., 2009
b. Preclinical Studies for CD20-Targeted CAR
CAR Design Receptor Type Lymphodepleting
Therapy		 In Vivo Study Cell Lines Used for
In Vivo Study		 Reference
First-Generation CAR scFv-CD3ζ - - - Jensen et al., 1998
scFv-CD3ζ - - - Jensen et al., 2003
scFv-CD3ζ - - - Wang et al., 2003
scFv-CD3ζ - +, SCID/Beige i.v. Burkitt’s lymphoma Chen et al., 2004
(mCD20) scFv-CD3ζ Anti-hCD20 Ab +, Balb/c i.v. BM 185 James et al., 2009
Second-Generation CAR scFv-CD3ζ
scFv-CD28-CD3ζ
scFv-CD28-4-1BB-CD3ζ		 - - - Wang et al., 2007
scFv-CD28-CD3ζ - - - Yu et al., 2008
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Cell lines: Daudi: human Burkitt’s lymphoma; Raji: human Burkitt’s lymphoma; A20: mouse B-cell lymphoma; NALM-6: human pre-B cell.

EL4: mouse thymoma; BM 185: mouse pre-B cell lymphoma.

CD20-specific T cells: Preclinical Studies

First-generation CD20-targeted CARs

Jensen et al. initially constructed a first-generation CAR targeted to CD20, and demonstrated antigen-specific IL-2 production in CAR+ Jurkat cells and in vitro cytotoxicity against the human lymphoma cell lines (Jensen et al., 1998). These results were confirmed in studies with human T cells wherein first-generation CAR+ T cells conferred antigen-dependent IFNγ secretion after expansion, and in vitro lysis of CD20+ lymphoma cell lines (Jensen et al., 2003) and tumor cells isolated from patients with a variety of lymphoid B cell malignancies (Wang et al., 2004).

Chen et al. reported on a CD20-targeted first-generation CAR, which, when expressed in T cells, delayed progression of systemic CD20+ Burkitt’s lymphoma xenografts in SCID/Beige mice as evidenced by slower onset of hind limb paralysis (HLP) in 4 out of 5 treated mice (median 84 days) while all 7 untreated mice developed HLP by day 27 (Chen et al., 2004). One treated mouse did not develop HLP when followed up to 125 days, consistent with complete tumor eradication by these CD20-targeted CAR+ T cells.

Second- and third-generation CD20-targeted CARs

While there are no published in vivo preclinical studies using the CD20-targeted second- and third-generation CARs, several groups have reported superior in vitro activity of these CARs when compared to first-generation CARs. Wang et al. constructed a second-generation CAR containing the CD28 molecule and a third-generation CAR containing the CD28 in tandem with 4-1BB (Wang et al., 2007). In this study, T cells expressing either the second- or third-generation CAR exhibited a significantly superior proliferation, IFNγ production, and in vitro cytotoxicity compared with the T cells expressing the first-generation CAR. Yu et al. confirmed the effective in vitro cytotoxicity of a second-generation CD20-targted CAR against human Burkitt’s lymphoma cell lines (Daudi and Raji), but did not compare its function to a first-generation CD20-targeted CAR (Yu et al., 2008). All published preclinical studies of CD20-targeted CARs are summarized in Table 1b.

Optimizing the CAR Function: Modulating the Host Environment

Other than incorporating co-stimulatory signaling molecules, another way to enhance anti-tumor efficacy and lead to successful adoptive T cell therapy is preconditioning lymphodepleting therapy prior to adoptive T cell transfer. It has been reported that preconditioning of the recipient host with lymphodepleting chemotherapy improves the engraftment potential of adoptively transferred CD19- and CD20-targeted CAR+ T cells (Cheadle et al., 2009). The most commonly used agent to induce lymphodepletion in adoptive immunotherapy for B-cell malignancies is cyclophosphamide (Cy). Cy has been shown to enhance the engraftment of adoptively transferred T cells by 1) inducing homeostatic proliferation of T cells potentially through the removal of lymphocytes that compete for homeostatic cytokines (e.g., IL-7, IL-15, IL-21) known to induce T cell proliferation and maturation (Klebanoff et al., 2005); 2) decreasing the number of inhibitory regulatory T cells in tumor microenvironment (Liu et al., 2010); and 3) enhancing the activation and function of antigen presenting cells by triggering tumor cell death and antigen release (Chernysheva et al., 2002).

In the setting of adoptive therapy of B-cell malignancies by CAR+ T cells, the role of Cy-induced lymphodepletion was first studied in immunodeficient mice. Cheadle et al. pretreated SCID/Beige mice bearing systemic Burkitt’s lymphoma xenografts with i.p. Cy 200mg/kg (5 days after tumor injection) followed by the infusion of first-generation CD19-targeted CAR+ T cells demonstrating a 50% long-term survival (followed up to day 100) while all mice treated with T cells alone succumbed to tumor by day 60 (Cheadle et al., 2008). However, the mechanism by which Cy enhanced the anti-tumor efficacy of the modified T cells in this model remains unclear, as no increase in the number of T cells was detected in bone marrow, blood, or spleen 6 hours after systemic injection. In immune-competent mice, Cy preconditioning therapy was shown to be necessary for the CAR+ T cell engraftment by the same group of investigators (Cheadle et al., 2009). In this study, Balb/c mice were preconditioned with i.p. Cy 200mg/kg or saline on day −2, followed by s.c. injection of syngeneic A20 murine lymphoma cells modified to express human CD19 (hCD19) on day −1. Twenty-four hours later, mice received an i.v. infusion of T cells expressing the first-generation hCD19-targeted CAR. The in vivo engraftment and overall survival of the modified T cells were significantly enhanced in Cy-conditioned mice when compared to mice treated without prior preconditioning. Similarly, in a syngeneic immune-competent mouse model developed in our laboratory, we have found that preconditioning chemotherapy with Cy was critical for the adoptively transferred CD19-targeted T-cell anti-tumor efficacy (manuscript in preparation).

More recently, a group of investigators have studied the engraftment and anti-tumor efficacy of syngeneic CD19- and CD20-targeted CAR modified T cells in immune-competent mice where normal B cells express high levels of CD19 and CD20. In these studies, Cheadle et al. demonstrated a shortened in vivo survival of the mouse CD19 (mCD19)-targeted first-generation CAR+ T cells in a Cy-conditioned syngeneic immune-competent mouse that correlated with a resurgence of circulating normal mCD19+ B cells. The suggestion from this observation was that the mCD19-targeted CAR+ T cells were targeting natural B cells, and that, after a period of time, these T cells most likely die out through excessive antigen stimulation from the regenerating pool of B cells as the lymphodepleting effect of Cy ended (Cheadle et al., 2010). James et al. reported a similar finding, demonstrating human CD20 (hCD20) expression on normal B cells in hCD20 transgenic immune-competent mice profoundly impaired the hCD20-targeted CAR, resulting in T-cell deletion and limited trafficking of targeted T cells to the tumor site. Subsequently, a selective depletion of natural hCD20+mCD20+ B cells in hCD20 transgenic immune-competent mice permitted the trafficking of the adoptively transferred first-generation mouse CD20 (mCD20)-targeted CAR+ T cells to the tumor site, and enhanced survival and function of the mCD20-targeted T cells (James et al., 2009). Taken together, these studies emphasize the importance of clinically relevant syngeneic animal models with normal tissue (i.e., B cell) expression of the target antigen.

Clinical Trials

Promising preclinical data utilizing CD19- and CD20-targeted CAR+ T cells has recently resulted in a robust translation of this technology to the clinical setting by multiple investigators. Currently, there are 10 active phase I clinical trials targeting CD19 (Table 2a), and 2 active clinical trials targeting CD20 (Table 2b). These trials vary widely with respect to CAR design, means of gene transfer, ex vivo T-cell expansion, cell dose, targeted malignancy, and whether or not patients receive prior lymphodepleting chemotherapy. The relevance of these multiple variables on clinical outcome and other secondary endpoints (i.e., modified T cell persistence) remains to be seen and will ultimately require careful comparisons of results of completed trials.

Table 2.

a. Summary of Actively Recruiting Clinical Trials with CD19-Targeted CAR
Clinical Trial
Identifier		 CAR Construct Patient Population T Cell Dose Accompanying
Lymphodepleting Cytotoxic
Therapy		 Additional
Immunotherapy		 Trial Site
NCT00466531 ScFv-CD28-CD3ζ Chemo-refractory CLL Dose 1: 1×l07 cells/kg
Dose 2: 3×l07 cells/kg
Dose 3: 1×108 cells/kg		 Cy None US, New York
NCT01044069 ScFv-CD28-CD3ζ Chemo-refractory ALL Dose 1: 1×l07 cells/kg
Dose 2: 3×l07 cells/kg
Dose 3: 1×108 cells/kg		 Cy None US, New York
NCT00586391 ScFv-CD3ζ
ScFv-CD28-CD3ζ		 Relapsed or refractory low-intermediate grade NHL, CLL Dose 1: 2×l07 cells/m2
Dose 2: 1×108 cells/m2
Dose 3: 2×l08 cells/m2 		None None US, Texas
NCT008912I5 ScFv- CD3ζ
ScFv-41BB-CD3ζ		 Chemo-refractory B-cell leukemia/lymphoma (ALL, CLL, FL, MCL, DLBCL) 2 infusions of 2×109 – 5×1010 cells total None None US, Pennsylvania
NCT01029366 ScFv-CD3ζ
ScFv-41BB-CD3ζ		 Chemo-refractory B-cell leukemia/lymphoma (ALL, CLL, FL, MCL, DLBCL N/A None None US, Pennsylvania
NCT00608270 ScFv-CD3ζ
ScFv-CD28-CD3ζ		 Relapsed or refractory low-intermediate grade NHL, CLL N/A None None US, Texas
NCT00182650 ScFv-CD3ζ Relapsed or refractory FL N/A Fludarabine + Rituximab Low dose s.c. 1L-2 US, California
NCT00840853 Allogeneic multivirus-specific T cells with ScFv-CD3ζ Relapsed ALL following HSCT Dose 1: 1.5×107/m2
Dose 2: 4.5×107/m2
Dose 3: 1.2×108/m2 		None None US, Texas
NCT00924326 ScFv-CD3ζ CDl9-expressing B cell malignancy of any type N/A Fludarabine + Cy i.v. IL-2 US, NIH
N/A ScFv-CD3ζ NHL Cy UK
b. Summary of Actively Recruiting Clinical Trials with CD20-Targeted CAR
Clinical Trial
Identifier		 CAR Construct Patient Population T Cell
Dose		 Accompanying
Lymphodepleting
Cytotoxic Therapy		 Additional
Immunotherapy		 Trial Site
NCT00012207 ScFv-CD3ζ Relapsed or refractory indolent NHL N/A Oral Cy, ural prednisone, IV vincristine SC IL-2 for 14 days US, California
NCT00621452 ScFv-CD28-CD137ζ Relapsed or refractory MCL or indolent NHL N/A IV Cy SC IL-2 for 14 days US, California
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CLL: chronic lymphocytic leukemia; ALL: acute lymphocytic leukemia; NHL: non-Hodgkin’s lymphoma; FL: follicular lymphoma; MCL: mantle cell lymphoma; DLBCL: diffuse large B cell lymphoma; HSCT: hematopoietic stem cell transplant; Cy: cyclophosphamide; SC: subcutaneous; IV: intravenous.

NHL: non-Hodgkin’s lymphoma; MCL: mantle cell lymphoma; Cy: cyclophosphamide; IV: intravenous; SC: subcutaneous.

While many of these trials have recently opened, one clinical trial has been completed and results from this trial have been published. Till et al. reported the results of a multi-institutional phase I clinical trial wherein 7 patients with relapsed or refractory indolent B cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a first-generation CD20-specific CAR followed by low-dose subcutaneous IL-2 injection (Till et al., 2008). In this study, patients were allowed to receive cytoreductive chemotherapy at the discretion of referring physician but required a 4-week interval between chemotherapy and T-cell infusion. Subsequently, patients received 3 infusions of autologous CD20-specific T cells 2 to 5 days apart in escalating doses (108 cells/m2, 109 cells/m2, and 3.3 × 109 cells/m2) followed by 14 days of s.c. low-dose (500,000 IU/m2) IL-2 injections twice daily. Modified T cells persisted in peripheral blood for 5–9 weeks when given with IL-2. Of the 7 treated patients, 2 maintained a previous complete remission, 1 achieved a partial remission, and 4 had stable disease. No grade 3 or 4 toxicities were seen, and no adverse events attributable to the T-cell infusions were observed. While clinical benefit was modest, this trial demonstrated the safety and feasibility of adoptive immunotherapy with genetically engineered T cells. The authors suggested several possible explanations for the limited therapeutic anti-tumor activity in this trial, including insufficient numbers of surviving modified T cells, CD20 antigen competition from native B cells, ineffective localization of T cells to tumor sites, lack of co-stimulatory signaling capacity from the first generation CAR construct, and suboptimal in vitro cytotoxicity of these cells.

Conclusion

As evidenced by the growing number of clinical trials utilizing B-cell tumor targeted CAR-modified T cells, adoptive cellular therapy is of increasing interest in the treatment of B-cell malignancies. CD19 and CD20 are attractive tumor-associated target antigens for the CAR approach, and multiple preclinical in vivo studies have demonstrated the efficacy and feasibility of this approach. More importantly, CD19 and CD20 targeted CAR modified T cells are now being utilized in multiple clinical trials conducted at multiple institutions. While currently open clinical trials vary with respect to overall design, it seems reasonable to assume that upon completion, data generated from these multiple trials will collectively offer investigators better insight into the in vivo biology of these modified T cells, and give guidance to the design of future generation clinical trials to ultimately optimize anti-tumor efficacy.

Acknowledgements

Supported by CA13873801, The Damon Runyon Clinical Investigator Award, The Translational and Integrative Medicine Fund Research Grant (MSKCC), The Annual Terry Fox Run for Cancer Research (New York, NY) organized by the Canada Club of New York, Kate’s Team, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Cancer Foundation for Research and the Experimental Therapeutics Center of MSKCC, The Geoffrey Beene Cancer Foundation, and The Bocina Cancer Research Fund. The authors thank Dr. Michel Sadelain for critical review of the manuscript.

Contributor Information

Jae H Park, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Address: 1275 York Avenue, New York, New York, 10065, United States.

Renier J Brentjens, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Address: New York, New York, United States.

References

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