Chimeric Antigen Receptor T Cells and Hematopoietic Cell Transplantation: How Not to Put the CART Before the Horse

Abstract

Hematopoietic cell transplantation (HCT) remains an important and potentially curative option in most hematological malignancies. As a form of immunotherapy, allogeneic HCT offers the potential for durable remissions but is limited by transplant related morbidity and mortality due to organ toxicity, infection and graft versus host disease. The recent positive outcomes of chimeric antigen receptor T (CART) cell therapy in B cell malignancies may herald a paradigm shift in the management of these disorders and perhaps other hematological malignancies. Clinical trials will now need to address the relative roles of CART cells and HCT in the context of transplant-eligible patients. In this review we summarize the state of the art of the development of CART cell therapy for leukemia, lymphoma and myeloma and discuss our perspective of how CART cell therapy can be applied in the context of HCT.

BIOLOGY OF CHIMERIC ANTIGEN RECEPTOR T CELLS

A chimeric antigen receptor (CAR) is a synthetic protein that is composed of three distinct domains (Figure 1), which are transcribed and translated in toto from the introduced genetic material.^{1, 2} The antigen-recognition domain is usually derived from a single chain variable fragment (scFv) based on the light and heavy chains of a monoclonal antibody against the antigen of interest (eg, CD19). Upon binding antigen, the scFv that is linked by a hinge and spacer region to a transmembrane domain transmits signal to the intracellular signaling domain(s). The hinge is typically derived from the CD8 or IgG4 molecules and may contain a spacer of variable length.³ The hinge and spacer play a role in dimerization of the scFv and have been shown to impact optimal CAR function.^{3, 4} The transmembrane domain is typically a portion of the CD8 or CD28 molecule and is required for appropriate cell-surface expression of the CAR.⁵ The intracellular domain typically contains the CD3zeta chain that serves as a signaling domain with or without additional costimulatory signaling domains.^{4, 6} The first generation of CART cells contained the CD3z signaling domain without additional co-stimulatory molecules.² While these 1^st generation CARTs were able to specifically target antigen, they had very modest clinical activity and poor in vivo persistence.⁷ Provision of a costimulatory signal within the integral CAR structure enhances T cell activation and effector functions.⁸ These CARs are referred to as second generation CARs. Examples of such costimulatory molecules are CD28^{4, 6}, CD137^{4, 6}, CD134⁹, CD2¹⁰, CD27¹¹, or ICOS¹². These costimulatory molecules have different biological functions and therefore may result in CART cells of somewhat diverse functional capacity. For example CD28 co-stimulated CARTs result in initially potent effector functions but the in vivo persistence of these cells appears inferior to that of CD137 (41BB) co-stimulated T cells.^{4, 6} The inclusion of the ICOS molecule appears to drive TH1/TH17 differentiation.¹²

Figure 1.

Composition of a chimeric antigen receptor (CAR). A CAR is composed of an extracellular single chain variable fragment, linked to a transmembrane domain (CD8 or CD28) through a hinge (CD8 or IgG4), one or more intra-cellular costimulatory molecules (41BB, CD28, CD27, ICOS, or OX40), and CD3z signaling molecule.

Most clinical trials to date use second generation CARs with CD28 or CD137 co-stimulation.^13–17 Third and fourth generation CAR constructs are in development and contain more than one costimulatory molecule with or without a suicide switch. While most of these constructs are still in the preclinical stage, at least one group has begun to evaluate fourth generation CARs.¹⁸ Other interesting developments have included provision of activating signals in trans (i.e. not included within the basic CAR construct but rather co-expressed within the same T cell using a bi-cistronic expression vector).⁶ The optimal structure of the CAR remains an area of active investigation and it is possible that different targets or diseases would be best treated with different CAR constructs.

GENERATION OF CART CELLS

The construction, culture conditions, T cell selection, stimulation and gene delivery method vary between centers, and have been reviewed elsewhere, ^{1, 19–21} and will be only briefly discussed here. In principle, the process of CART manufacturing involves these steps:^{19, 20, 22} (1) patients undergo leukapheresis (2) T cells may be enriched from peripheral blood mononuclear cells (PBMC) using magnetic or mechanical techniques, (3) T cells are stimulated in culture using beads, cytokines or artificial antigen presenting cells, (4) the CAR transgene is introduced into the T cells and (5) the T cells are further expanded in culture over several days to weeks; (6) the CART cells are re-infused into patients (often after lymphodepleting chemotherapy), where they are intended to proliferate, traffic to tumor sites, recognize their target antigen, and release cytotoxic molecules resulting in tumor death.^{1, 19, 23}

Gene delivery of the CAR into T cells

Several methods for gene transfer into the T cells have been used by different investigators and centers. Each has its own advantages and disadvantages. Retroviral and lentiviral vectors rely on the propensity of these viruses to permanently integrate into the host genome. Clinical grade viral vector production is costly, labor-intensive and time-consuming. However, it does lead to high transduction efficiency of over 50%, and has been utilized in most clinical trials to date.^13–17 While insertional mutagenesis and subsequent leukemia development was reported following retroviral transduction of hematopoietic stem cells,²⁴ retroviral or lentiviral transduction of primary T cells has been shown to be safe after several decades (and several thousand patient-years) of follow up. ²⁵

To obviate the disadvantages of viral gene transfer methods, alternative non-viral vectors have been used. The most commonly used non-viral vectors are those based on the transposon/transposase system such as Sleeping Beauty.²⁶ This system has been shown to result in sustained transgenic expression of CARs on T cells.²⁷ CD19 redirected CARTs using the sleeping beauty system have resulted in clinical activity in early phase clinical trials. ²⁸ Notably, the prolonged expansion protocols required to manufacture acceptable numbers of gene-modified cells for infusion typically result in a terminally differentiated T cell phenotype ²⁹ while recent data indicate that a less differentiated phenotype may be desirable for generating an optimal anti-tumor effect. ²⁹ Another novel approach is the electroporation of T lymphocytes with in vitro transcribed RNA, as optimized and practiced at our institution.^{30, 31} This approach relies on the transfer of mRNA into T cells, resulting in high but transient expression of the CAR on T cell surface that lasts for a few days. In essence, this technique results in “biodegradable” CART cells. This provides a significant safety advantage and could be particularly useful when off target expression and toxicity is a concern, such as in acute myeloid leukemia (AML) or in solid tumors.^{32, 33} A clinical trial of RNA modified mesothelin directed CART cells in mesothelin positive solid tumors was recently reported and confirmed the safety and feasibility of this approach. ³⁴ RNA-electroporation is being used in a phase I clinical trial of CD123 directed CART cells in relapsed-refractory AML (NCT02623582).

T cell expansion and culture conditions

Variable procedures and processes of T cell stimulation and expansion ex-vivo are being used by different centers. Characteristics of the final T cell product depend on this process and play an important role in the activity and persistence of CART cells in vivo. The anti-CD3/CD28 beads, the anti CD3 antibody OKT3, or artificial antigen presenting cells are commonly used to stimulate T cells, usually with some degree of cytokine support in culture (IL-2, IL-7, or IL-15).^{35, 36} The bioreactor culture systems are more compatible with large scale clinical production and are under investigation to potentially commercialize CART cell therapy.³⁷ Despite the central role of T cell expansion technique in the subsequent function of the infused product, the optimal clinical procedure for T cell expansion has not been rigorously investigated or compared.

T cell Selection

The final T cell composition is likely to be an important variable in the ultimate success of CART cell therapy. Preclinical work suggests that adoptive transfer of less differentiated T cells will result in better anti-tumor activity when compared with more differentiated cells.^{29, 38, 39} CD4 or CD8 CART cells derived from naive T cells or central memory T cells have more potent anti-tumor activity in vivo compared to effector T cells.⁴⁰ This observation led to a recently published clinical trial using a manufacturing strategy that attempted to enrich for CD8+ central memory CART cells in a 1:1 ratio with CD4+ CART cells; while this approach is feasible and the rationale is attractive, it remains to be seen whether it is superior to more “traditional” methods of T cell manufacturing.^41–43 Other T cell subsets such as Th17 cells have demonstrated superior proliferation, persistence, and anti- tumor activity compared to Th1 cells in preclinical models, and thus further nuances in the T cell manufacturing process may ultimately be shown to exhibit desirable features such differential cytokine production or expansion characteristics.⁴⁴ It is noteworthy however, that to date, the most potent and durable clinical responses have been obtained when bulk populations of CD8 and CD4 are used. ^13–16

CLINICAL APPLICATIONS OF CART CELL THERAPY

The first report, in 2010, of a patient with follicular lymphoma treated with autologous CART19 followed by disease regression and B cell aplasia lasting 39 weeks provided proof of concept that CART cells modified to target an antigen are able to exhibit anti-tumor activity and could potentially represent a valid approach in B cell malignancies.⁴⁵ This was followed by further reports of dramatic responses, in-vivo persistence, and remission of bulky disease in three patients treated with CART19 for refractory chronic lymphocytic leukemia (CLL).^{46, 47} Subsequent clinical experience of CART cell therapy reported to date has been with CD19 directed CARTs in B cell malignancies (CLL, acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL)).^{13–17, 42, 48, 49} Other B cell antigens have been targeted by CART cells, including CD20⁵⁰ and CD22⁵¹. Promising early data of BCMA directed CART cells in multiple myeloma were reported at the 2015 annual meeting of the American Society of Hematology.⁵² In Hodgkin Lymphoma, clinical trials of CD30 directed CARTs have been enrolling for few years and outcomes of a small number of patients were recently reported⁵³ and trials targeting AML with CD33 and CD123 are just beginning. ^54,55

CART cells for Chronic Lymphocytic Leukemia

Our group reported dramatic responses following treatment with CART19 in three patients with relapsed refractory CLL in 2011.^{47, 56} In those reports, CARTs expanded several hundred fold in-vivo, trafficked to tumor sites, produced cytokines, and resulted in elimination of bulky tumor masses. Each CART cell or its progeny were capable of killing thousands of tumor cells and CART cells differentiated into memory T cells that persisted up to four years in some patients; the finding of long-term B cell aplasia in these patients suggests these persisting cells remain functional.⁵⁶ This pilot study included a total of 14 CLL patients treated at the University of Pennsylvania, the overall response rate (ORR) was 57% (8/14) with 28.5% (4/14) achieving a minimal residual disease (MRD) negative complete response (CR). Additionally, investigators from Penn reported a phase II dose finding study in which 26 patients with relapsed CLL were assigned to receive 5x10⁷ (n=13) vs. 5x10⁸ (n=13) CART19 cells following lymphodepleting chemotherapy. Of the 23 evaluable patients, 9 achieved objective response, including CR in 5 patients.¹⁴ Similar outcomes were reported by the National Cancer Institute (NCI) ¹⁷ and the Fred Hutchinson Cancer Research Center (FHCRC) (Table 1).⁴² The identification of a group of non-responders or partial responders in CLL¹⁴ highlights the need for a deep mechanistic understanding of the anti-tumor activity of CART cell and the CART cell-tumor interaction. The lower CR rates could be related to the heavily pretreated group of patients, prior use of T cell depleting/toxic chemotherapeutic agents, immunosuppressive microenvironment, or dysfunctional T cells in CLL. ^{14, 57} Nevertheless, these results show that CART19 is a useful and promising approach for patients with relapsed/refractory CLL and further development and optimization of this therapy for patients with CLL is warranted.

Table 1.

Summary of the reported clinical trials of CART cell therapy in hematological malignancies.

Target	Center	Target	#	CAR	Vector	Disease	Results	Role of HCT	Chemo	Notes
Chronic Lymphocytic Leukemia	FHCRC41, 42	CD19	6	41BB-ζ	LV	CLL	CR: 3/6 (50%), PR: 1/6 (17%)	N/A	Two cohorts: CY and FLU/CY	FLU/CY improved persistence and CR
	MDACC28	CD19	2	28-ζ	SB	CLL	Responses not reported for each disease type	N/A	N/A
	NCI 17	CD19	4	28-ζ	RV	CLL	CR 3/4 (75%), PR 1/4 (25%)	None	FLU/CY
	NCI63, 82	CD19	5	28-ζ	RV	CLL post allo	1/5 CR, 1/5 PR	Allo-CARTs to treat relapses post HCT	No chemo	No GVHD
	UPenn 14	CD19	14	41BB-ζ	LV	CLL	CR 4/14 (28.5%), PR 4/14 (28.5%)	No relapses if patients achieve CR	FLU/CY (3) Pento/CY (5) Benda (6)	Two developed CD19 negative Richter
Acute Lymphoid Leukemia	China (multi- center)18	CD19	50	28-41BB- 27-ζ		ALL	CR 94.3% when blasts <50%, CR 66.7% when blasts >50%	N/A	Variable
	MDACC28	CD19	42	28-ζ	SB	Adjuvant post- transplant (10) Relapse (8)	Adjuvant trial: CR 3/10 (30%) Relapse trial: CR3/13 (23% - for all diseases)	N/A	N/A
	MSKCC 16	CD19	46	28-ζ	RV	Adult ALL	83% CR rate	12 patients underwent HCT	CY	No differences in outcomes whether or not HCT was done
	NCI 83	CD19	21	28-ζ	RV	children and young adult ALL	CR (ITT): 61%, OS 51% at 6m, DFS: 78.8% at 6m. CR: 13/16 (81%) in low burden (<25% blasts), 10/22 (46%) in high burden.	17/20 patients with MRD negative disease went to HCT.	FLU/CY	No relapses after HCT, 2/3 patients with no HCT relapsed with CD19 negative disease
	NCI63, 82	CD19	5	28-ζ	RV	ALL relapse post- transplant	4/5 patients with MRD negative CR	Allogeneic CARTs to treat relapses post HCT	No chemo	No GVHD
	Swedish (two- center)99	CD19	2	28- 41BB-ζ	RV	ALL	CR: 6/11 (55%) (for all diseases)	N/A	Flu/Cy	One patient developed CD19 negative relapse
	UPenn/ CHOP 59	CD19	53	41BB-ζ	LV	Children and young adults ALL	CR:50/53 (94%), EFS 70% at 6m and 45% at 12m. OS is 78% at 12m. 20 relapsed, 13/20 (65%) CD19 neg disease. CART persisted 3–39 m.	6/29 patients in CR received HCT	FLU/CY	Of the 20 relapsed patients, 3 had HCT after CART19
Non Hodgkin Lymphoma	City of Hope43, 66	CD19	16	NHL1: ζ NHL2:28-ζ	LV	NHL (adjuvant after HCT)	NHL1, CR:5/8 (63%), PFS:50% at 2y NHL2,CR:8/8(100%),PFS:1 00% at6m	CARTs were given after auto-HCT	High-dose chemo and HCT	NHL1:CD8+Tcm NHL2:Tcm (CD4 & CD8)
	FHCRC41, 42	CD19	28	41BB-ζ	LV	NHL	CY cohort: CR 1/12 (8%), PR 5/12(42%) FLU/CY:CR 5/12(42%),PR 3/12 (25%)	N/A	Two cohorts: CY and FLU/CY	FLU/CY improved persistence and CR
	MDACC28	CD19	17	28-ζ	SB	NHL (n=17),	Adjuvant trial, CR4/5 (80%) Relapse Trials, CR3/13 (23% - for all diseases)	Allogeneic CARTs to treat relapse post HCT	N/A	No GVHD
	MSKCC67	CD19	8	28-ζ	RV	NHL after HCT	5/8 CR, 2/8 PD, 1/8 NRM	CARTs were given after auto-HCT	High-dose chemo and HCT
	NCI 17	CD19	15	28-ζ	RV	Lymphoma, adults	85.6% CR in DLBCL, 100% in indolent lymphoma	None	FLU/CY
	NCI63, 82	CD19	10	28-ζ	RV	MCL (n=5) DLBCL (n=5)	MCL:PR (1/5) DLBCL: CR (1/5)	Allogeneic CARTs to treat relapses post HCT	No chemo	No GVHD
	Swedish (two- center)99	CD19	9	28-41BB-ζ	RV	Lymphoma	CR: 6/11 (55%) (for all diseases)	N/A	Flu/Cy	One patient developed CD19 negative relapse
	UPenn 65	CD19	38	41BB-ζ	LV	Adult NHL (DLBCL 19, 8FL, 2 MCL)	CR: 54% in DLBCL, 100% in FL, 50% in MCL. PFS: 62% at 10m (DLBCL 54%; FL 100%)	N/A	Benda (6), CY(11), FluCy(1), EPOCH(3), RadCY(3)
	NCI51	CD22	6	41BB-ζ	LV	R/R ALL, children and young adults	MRD negative CR:1/6 (33%), SD 2/6 (67%)	N/A	FLU-CY	5 patients had a CD19 negative relapse at enrolment
	FHCRC50	CD20	4	28- 41BB-ζ	LV	NHL	PR 1/4 (25%), 2/4 (50%) remained in remission	N/A	CY	CART20 was used in consolidation or treat residual disease
Multiple Myeloma	UPenn68	CD19	1	41BB-ζ	LV	Multiple Myeloma	Stringent CR	CARTs were given after auto-HCT	High-dose chemo and HCT	Rationale is to target myeloma stem cell
	NCI52	BCMA	11	28-ζ	RV	R/R MM (median regimens 7)	1/6 on dose 2 VGPR, 2/6 on dose level 2 SD. 1/2 highest dose CR	N/A	Flu-cy	Improved responses in higher dose cohort
Acute Myeloid Leukemia	Peter MacCallum Cancer Centre100	LeY	4	28-ζ	RV	R/R AML	Transient cytogenetic CR 1/5(20%), transient reduction in blasts 1/5(20%), SD 2/5(40%)	None	FLU-CY	CARTs were detected in sites of extramedullary disease
	PLA general hospital54	CD33	1	41BB-ζ	RV	R/R AML	Transient reduction in blasts	None	No Chemo	Patient developed CRS and transient hyperbilirubinemia
	Zhejiang University55	CD123	1	28-41BB- 27-ζ	LV	R/R AML	Transient reduction in blasts	None	CY	Patient developed CRS
Hodgkin Lymphoma	Baylor53	CD30	9	28-ζ	RV	7 HL, 2 ALCL	CR:1/9 (11%), PR:1/9(11%), SD:4/9(44%), PD:3/9 (33%)	N/A	No chemo	None

FHCRC=Fred Hutchinson Cancer Research Center, MDACC=MD Anderson Cancer Center, MSKCC=Memorial Sloan Kettering Cancer Center, NCI=National Cancer Institute, UPenn=University of Pennsylvania, CHOP=Children’s Hospital of Philadelphia. LV=lentiviral, RV=retroviral, SB=sleeping beauty, CLL=chronic lymphocytic leukemia, ALL=acute lymphoblastic leukemia, NHL=non-Hodgkin lymphoma, DLBCL=diffuse large B cell lymphoma, MCL=mantle cell lymphoma, FL=follicular lymphoma, AML=acute myeloid leukemia, HL=Hodgkin lymphoma, MM = multiple myeloma, ALCL=anaplastic T cell lymphoma, R/R=relapsed refractory, CR=complete response, PR=partial response, SD=stable disease, PD=progressive disease, VGPR=very good partial response, EFS=event free survival, PFS=progression free survival, DFS=disease free survival, MRD=minimal residual disease, OS=overall survival, CART=chimeric antigen receptor T cells, HCT= hematopoietic cell transplantation, FLU=fludarabine, CY= cyclophosphamide, NRM=non-relapse mortality, Tcm=central memory T cells, GVHD=graft versus host disease, CRS=cytokine release syndrome.

CART cells for Acute Lymphoblastic Leukemia

In relapsed or refractory ALL, unprecedentedly high CR rates have been reported by different centers. ^{13, 15, 16, 18} Despite significant differences in CAR construct and gene delivery methods, these studies collectively showed that autologous CART19 cells generated from these patients were able to achieve complete remission rates of approximately 70–90% in these patients with advanced disease. Many of these responses were durable even though in some cases patients did not have a follow up allogeneic hematopoietic cell transplantation (HCT) (Table 1).^{18,13, 15, 16, 58}

Investigators at the Children’s Hospital of Philadelphia (CHOP) updated their results recently.^{13, 59} In 53 pediatric patients treated with CART19, the CR rate was 94% and the 12-month disease free survival (DFS) and overall survival (OS) were 45% and 78%, respectively. CART19 persisted between 3 and 39 months. Twenty patients relapsed and 13 out of these 20 patients (65%) relapsed with CD19 negative relapse.⁵⁹ Similar outcomes were reported in adult ALL.^{60, 61} Lee and colleagues from the NCI treated 21 children and young adults with refractory ALL.¹⁵ The six-month OS and DFS were 78.8% and 51%, respectively. No relapses were observed after allogeneic transplantation in their study.⁶² The Memorial Sloan Kettering Cancer Center (MSKCC) group reported a CR rate of 83% in 46 adult ALL patients.⁵⁸ Of these, 12 patients underwent allo-HCT. In another report, split dosing of CART19 dose in 12 adult patients with ALL resulted in an ORR of 84%. One important observation from this study was that fractionation of CART19 did was used as an adaptive approach allowing intra-patient dose modification (de-escalation) at the development of early signs of cytokine release syndrome (CRS).⁶⁰ This did not impair CART19 activity.

A multicenter clinical trial was conducted in china and included 50 patients with ALL. High CR rates of 94.3% were reported when the blasts were <50% at baseline compared to 66.7% when baseline blasts were >50%.¹⁸ The NCI recently reported their results of allogeneic CART19 in patients that relapsed after allo-HCT. These CARTs were manufactured from the original donors. Four of the 5 ALL patients treated with this approach achieved a MRD negative relapse.⁶³

CART cells are capable of trafficking to the central nervous system (CNS) and are effective in clearing CNS disease.^{13, 62, 64} For example, patients treated at the NCI had evidence of CART19 trafficking to the CNS based on cerebrospinal fluid analysis.³⁸ Furthermore, the two patients with known CNS involvement had clearance of the disease after CART19.¹⁵ In 98% of children treated at the University of Pennsylvania, CART cells were detected in spinal fluid analysis. Out of 12 patients with established CNS disease, all developed remission (including negative spinal fluid analysis) after CART19. Four of these 12 patients relapsed with BM disease and none relapsed with CNS disease.⁶⁴

CART cells for Non-Hodgkin Lymphoma

Investigators from the NCI reported the activity of CART19 in 15 patients with non-Hodgkin lymphoma using a CD28 co-stimulated CAR construct. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had CLL. In patients with DLBCL, a CR rate of 57% was reported and a CR rate of 100% was reported in indolent lymphoma. ¹⁷

Similar results were reported by the groups at the University of Pennsylvania and the FHCRC. Schuster and colleagues reported 38 adults with relapsed/refractory NHL treated with CTL019. Of 22 evaluable patients, 15 achieved a response. This included 7 of 13 patients with DLBCL and 7 of 7 with follicular lymphoma.⁶⁵ The FHCRC trial included 28 patients in two cohorts; cohort 1 received cyclophosphamide lymphodepleting chemotherapy and cohort 2 received fludarabine in combination with cyclophosphamide (FluCy). The DFS of the patients receiving FluCy was superior to that of patients receiving Cyclophosphamide, possibly related to the observation of superior T cell engraftment and persistence in the FluCy group.⁴² Two groups have used CART19 as a consolidation therapy after high dose chemotherapy and autologous HCT, as a strategy to target MRD. The high dose conditioning chemotherapy and intensive lymphodepletion could provide an ideal environment for CART cell persistence. Clinical trials were done in non-Hodgkin lymphoma by the groups at MSKCC and the City of Hope.⁶⁶ The City of Hope group recently reported the results of two safety studies. The first study (NHL1) included engineered T cell central memory-derived product using a first generation CAR19 construct. In the second study (NHL2), CART cells were manufactured from CD4+ and CD8+ T cell central memory phenotype using a CD28 co-stimulated CAR construct. Of the 8 patients treated on the second study (NHL2), 6 were progression-free at one year.⁴³ Similar results are reported by the MSKCC group.⁶⁷ Longer follow up will be needed to understand the role of CART19 in inducing or maintaining these remissions. A strategy of post autologous HCT CART19 infusion could be a very attractive approach for patients undergoing autologous HCT with active lymphoma.

CART cells for Multiple Myeloma

Different approaches have been used to apply CART cell therapy to multiple myeloma. CART19 as a consolidation therapy after high dose melphalan and autologous HCT has been used at the University of Pennsylvania.⁶⁸ In a patient with high risk multiple myeloma, this approach led to a stringent CR and no measurable serum or urine monoclonal protein at 12 months of follow up. The rationale of using CART19 in multiple myeloma is to target the rare CD19 positive myeloma tumor-initiating cells. ⁶⁸

An approach addressing a common myeloma surface antigen, BCMA, was reported by investigators from the NCI in abstract form at the 2015 annual meeting of the American Society of Hematology. During a pilot dose-escalation study, stringent CR was reported in patients treated on the high dose level.⁵² Thus, the use BCMA CAR for relapsed/refractory MM seems very promising and will likely find an important place in the burgeoning field of novel therapies for this poor-prognosis disease.

CART cells for Acute Myeloid Leukemia

One of the major challenges of CART cell therapy is to extend its use and applications beyond B cell neoplasms. Unlike CD19, most myeloid markers are shared between malignant and normal hematopoietic cells.^{32, 69} Therefore, targeting myeloid antigens with a CART cell is expected to cause myelotoxicity. In fact, CD123 and CD33 directed CART cell therapy has resulted in potent anti-leukemic activity, but this is associated with significant myelotoxicity in preclinical models.^{32, 69–71} This represents a challenge when using a permanently transduced CART cells that could result in long term myeloablation. However, the potent anti-leukemic and myeloablative activity makes it appealing to use CART cell therapy as a novel cellular conditioning regimen prior to allogeneic transplantation.^{32, 69} In this setting, the effect of CART cells will need to be terminated prior to infusion of the donor graft in order to prevent graft rejection. Termination can be accomplished by using RNA electroporated T cells or the incorporation of suicide gene or depletion mechanism in the construct, such as the inducible caspase 9 gene⁷², surface EGFR molecule⁷³, or CD20^{74, 75} molecule. Figure 3 is a hypothetical schema of how CART cells may be used in myeloid neoplasms as part of pre-transplant conditioning regimen.

Figure 3.

Schema of how myeloid chimeric antigen receptor T cell therapy can be used in a novel conditioning regimen as a way to induce anti-leukemic activity and myeloablation.

CART cells as a bridge to transplantation

Allo-HCT is largely ineffective for patients with refractory or active disease at the time of transplant. CART cells may be an effective way to induce remission and serve as a “bridge to transplant” that potentially could improve outcome of HCT for these patients. As an example, 17 out of 20 pediatric patients at the NIH that achieved MRD negative disease received allogeneic transplantation with no subsequent relapses observed. ¹⁵ Many other patients with refractory ALL were able to undergo successful allo-HCT at other centers as well^{13, 16, 58} as highlighted in Table 1. It is important to highlight that we do not yet know if going into transplant with a CR induced with CART cells in previously refractory patients carries the same prognostic significance of CR induced by conventional chemotherapy.

CART cells instead of allogeneic transplantation

Allo-HCT has well known acute and long-term complications, and even long-term survivors who have no other typical side effects have accelerated ageing.⁷⁶ The question whether consolidation allogeneic transplantation is required to maintain remissions induced by CART19 in ALL or CLL remains largely unanswered. No relapses were observed in CLL patients achieving a CR after 4-1BB co-stimulated CART19 therapy at the University of Pennsylvania, suggesting that in this group of patients a consolidation allo-HCT might not be required.^{14, 77} This group of patients is identified by having prolonged persistence of CART cells and prolonged (likely longer than 6 months) B cell aplasia. Similarly, of the 53 ALL patients treated at CHOP, only 6 received consolidation allo-HCT and some patients remain in durable remissions.⁵⁹ A total of 20 relapses were reported and three of these relapses were after transplantation, and thus relapses were not less frequent in patients undergoing allo-HCT.⁵⁹ The observation of relapses despite allo-HCT questions the appropriate timing of transplantation after CART19 in this setting. As loss of CART cell persistence or loss of B cell aplasia appears to presage relapse, a reasonable strategy would be to closely monitor patients and transplant at early signs of loss of CART cells or B-cell recovery. The group at MSKCC analyzed the outcomes of adult ALL patients treated with CART-19 based on whether or not a follow up allo-HCT was performed. Based on the updated results, 13 of 37 patients underwent allo-HCT after achieving a CR. The 6-month OS did not differ significantly between patients who underwent allo-HCT (79%) and those who did not (80%). ⁵⁸ Given this was not a randomized trial, it is impossible to ascertain whether patients that were thought to be at higher risk of relapse were amongst those who were treated with a consolidative allo-HCT.

Thus, it is highly likely that prolonged persistence of CART cells is needed to achieve deep remissions and prevent relapses, by generating protracted immunosurveillance. Based on data gleaned from multiple different trials from different groups, it appears that CART19 cells generated using 4-1BB co-stimulation and a lentiviral vector¹⁴ can persist for longer periods of time compared to CART19 cells costimulated with CD28 and transduced using a retroviral vector. However, direct comparisons have not been made. Recently, preclinical work was undertaken to understand the differences between these 4-1BB and CD28 costimulation. It was noted by several independent laboratories that inclusion of CD28 molecule in CART cell structure leads to constitutive stimulation, growth and proliferation⁷⁸ and that the incorporation of 4-1BB ameliorates exhaustion induced by this continuous signaling of CART cells. ^{79, 80} It is certainly possible that in some situations the high short-term potency leading to deep remissions may be sufficient to make up for the relative lack of long-term persistence.¹⁶ While persistence appears critical for the long-term success of CART19 therapy, it is still not known what the optimal persistence should be.

CART cells after allogeneic transplantation

For patients who relapse with ALL after allo-HCT, options are extremely limited and prognosis is poor. DLI for relapsed ALL post transplantation results in a CR rate of less than 10% and most remissions are transient.⁸¹

Clinical trials conducted at the University of Pennsylvania and CHOP suggests that CART19 in pediatric ALL yield similar response rates when used before or after allogeneic transplantation. The CR rate was 89% in patients that relapsed post allo-HCT and 100% in those with no prior allo-HCT and there was no difference in DFS between the two groups. ⁵⁹ In adult ALL, the MSKCC group reported similar results. Of the 46 patients, 28 had no prior allo-HCT and 18 had a relapse post allo-HCT. The CR rate after CART19 was 79% with no prior allo-HCT and 91% for patients with relapsed disease post allo-HCT. These observations collectively suggest that prior allo-HCT does not impact outcomes following CART19 therapy.

In this setting, whether to manufacture CART cells from patients (autologous CART) or from their original donors (allogeneic CART) remains an area of investigation. Using cells from the original transplant donor would overcome the technical challenges of T cell expansion and CART cell manufacturing in patients with active disease and multiple prior treatments. In fact, 20 patients were treated by using cells collected from the original transplant donor by the investigators from the NCI. Results were generally similar to using autologous CART cells (or donor derived cells collected from fully engrafted relapsed patients after transplant). Of the 5 patients with ALL, 4 developed MRD negative disease. One of five CLL patients developed a CR and one developed a PR and responses were also noted in lymphoma. It is important to note that none of these patients developed graft versus host disease (GVHD).^{63, 82} Similar results were reported by the group at MD Anderson and the City of Hope (Table 1). This validates the safety of donor derived CART cells for relapse after allo-HCT, as none of the clinical trials reported GVHD using this approach. This approach is a very attractive way to overcome the even more difficult barriers that confront clinicians trying to keep acute leukemia at bay while attempting to manufacture autologous CART cells.

LIMITATIONS TO CART CELL THERAPY

CART cell manufacturing processes

The process of T cell culture and CART cell manufacture is one of the major technical issues in the field of CART cell therapy and needs to be carried out in a GMP certified laboratory setting. The use of artificial antigen presenting cells is associated with longer T cell culture time. These protocols are more expensive and less practical. A more efficient method of T-cell activation and expansion is via the use of anti-CD3/anti-CD28 monoclonal Ab coated beads.^{47, 56} This enables the simultaneous selection and activation of T cells and results in relatively short expansion duration of about 10 days. Further fractionating of cell subsets can be performed via immunomagnetic separation. This allows for T cell selection or depletion and has been used in clinical trials at the FHCRC.⁴² This process adds to the complexity and expense of an already involved manufacturing process. An additional aspect to T cell manufacturing from patients with heavily pretreated disease or who recently received chemotherapy is the presence of quantitatively or qualitatively reduced T cell numbers in the patient at the time of apheresis. Therefore, some current protocols mandate a minimum “wash-out” period from prior immunosuppressant (including steroids) or chemotherapy treatment. This may be particularly challenging in patients relapsing after allogeneic transplantation with active GVHD.

The time needed to manufacture the cells (or to ship the pheresis product to a specialized center) is another significant limitation to the delivery of CART cell therapy. Patients with active disease need to be able to wait for few weeks and might require some form of active therapy to prevent disease progression in the meantime. Furthermore, the inclusion of only patients whose disease can be stabilized while CART cells are manufactured may result in a selection bias in these clinical trials; this could be remedied by reporting results as intention-to-treat rather than per-protocol assessments.

Toxicities

Almost all patients treated with CART19 developed B cell aplasia, an expected on-target off-tumor toxicity. These patients are typically managed with replacement intravenous immunoglobulin therapy. ^13–16 Several unique toxicities following CART cell therapy have emerged and continue to present diagnostic and management challenges. These toxicities include; CRS, macrophage activation syndrome (MAS), and neurotoxicity. ^{13, 14, 16, 17, 83} A novel observation after CART cell therapy across several clinical trials is the development of the CRS; a syndrome characterized by high grade fevers, hypotension, capillary leak, and respiratory distress that can be life threatening. CRS is usually accompanied by the MAS, which is characterized by clinical and biochemical findings of hemophagocytic lymphohistiocytosis (fevers, pancytopenia, splenomegaly, hemophagocytosis and extreme elevations of ferritin and C-reactive protein). The development of CRS strongly correlates with response rates.^13–15

At the University of Pennsylvania/CHOP trial, 48 of the 53 pediatric ALL patients treated with CART19 developed some degree of CRS.⁵⁹ It was also noted that severe CRS began earlier than mild CRS (median onset is 1 vs 4 days after CART cell infusion). Similarly, the NCI reported CRS in 15 of the 20 children treated with CART19, with a median onset of 4 days.¹⁵ In adults, severe CRS was observed in 11 of the 46 adults treated with CART19 at MSKCC⁵⁸ (three patients had refractory grade 5 CRS) and in 11 of 12 adult ALL treated at the University of Pennsylvania (3 developed refractory CRS despite treatment and died in the context of refractory concomitant infections). These patients with severe CRS in both the MSKCC and University of Pennsylvania trials had higher disease burden and received higher cell dose compared to other patients that developed CRS.

Collectively, these reports showed that CRS symptoms start with the onset of CART cell expansion and correlate with high disease burden, possibly related to increased T cell activation and more cytokine production. This is associated with elevation of C-reactive protein and marked elevation of IL-6, interferon-γ and other cytokines. ^13–15

Clinical trials have also shown that the use of steroids with or without the anti-IL6 receptor antibody tocilizumab can reverse these syndromes. However, the early introduction of immunosuppressive medications could potentially limit the benefit of immunotherapy. Therefore, clinical severity grading of the disease and management algorithms based on severity were developed.⁶² In efforts to develop a predictive model for the development of CRS, comprehensive cytokine profiling was done on 51 patients treated with CART19. It was noted that early elevation of three cytokines after CART19 was strongly predictive of the later development of severe CRS. ⁸⁴ We speculate that this observation could pave the way to a risk-adapted pre-emptive treatment approach where by anti-cytokine therapy such as tocilizumab is applied before the patient develops severe CRS.

In most clinical trials, CART19 therapy was also associated with unexplained neurotoxicity that is mostly reversible. This presents as generalized encephalopathy, obtundation, aphasia and focal neurological deficits have also been reported.^{13, 17} These symptoms develop during CRS and are similar to the neurotoxicity seen after blinatumumab.⁸⁵ Therefore, it is unclear whether they are related to T cell activation, cytokine production or to CD19 targeting. Grade 3–4 neurological toxicities were reported in 13 of 46 patients treated with CART19 in the MSKCC trial.⁵⁸ Neurotoxicity was also reported in 6/20 pediatric ALL patients treated at the NIH¹⁵ and 6/14 CLL patients treated at the University of Pennsylvania. ¹⁴ A correlation between the presence of CART cells in the cerebrospinal fluid and neurotoxicity was not observed. More recently, unpublished reports of lethal cerebral edema occurred in adult ALL patients conditioned with FluCy and infused with a CD28 containing CAR (clinical trial NCT02535364). Similar events apparently did not occur using this CAR in patients conditioned with only cyclophosphamide suggesting the conditioning regimen, or degree of lymphodepletion is a critical component effecting toxicity.

CD19 negative relapse

More than 50% of relapses after CART19 occur in the setting of detectable CART cells where the disease relapses with a CD19-negative phenotype.^{13, 15} This represents an example of an escape mechanism of tumor cells under selective and powerful immune pressure exerted by CART cells. The pathogenesis is unclear but one explanation may be pre-existing splice variants of CD19 where cells that express CD19 isoforms lacking the CART-triggering domain are selected.⁸⁶ Options are very limited in this situation. A handful of these patients were treated with CD22 CART cells with variable responses.⁵¹ Current efforts are underway to prevent CD19 negative relapse in preclinical models, by targeting multiple antigens.^{87, 88}

IMPROVING CART CELL THERAPY

Universal CART cells

A major limitation of CART cell therapy is that products are made individually for each patient. In addition to being labor intensive and time consuming, T cell production from heavily pretreated patients can be technically challenging and time consuming in a group of patients who may have rapid progression of disease. Using allogeneic, third party donor derived CART cells is an attractive way to generate “off-the-shelf” product. However, two problems should be overcome in order to accomplish this; CART cell rejection by the host due to recognition of donor major histocompatibility complex (MHC) by recipient T cell receptor (TCR), and GVHD caused by recognition of recipient MHC by donor TCR.

The most obvious way around this is to modify the native TCR and MHC or to completely remove it. Genome editing technologies provide several methods to modify a target gene, such as zinc-finger nuclease (ZFN) technology,⁸⁹ transcription activatorlike effector nucleases (TALEN)s⁹⁰ and clustered regularly-interspaced short palindromic repeats (CRISPR).⁹¹

TALEN-mediated editing approach was recently employed and reported.^{90, 92} Here, both αβ TCR and CD52 molecules were edited generating CART cells that lack both molecules. An 11 month old infant with refractory ALL (relapsed after a mismatched allogeneic transplantation) was recently treated with this approach. This patient achieved a MRD positive remission after using third party allogeneic CART19 with no evidence of epithelial GVHD (there was a concern for transfusion associated GVHD). Chimerism analysis at one month showed that 7% of cells were of the third party source, indicating the persistence of CART cells and no rejection.⁹²

Finally, CRIPSR/Cas9 multiplex system is being investigated to generate CART cells deficient in the expression of endogenous TCR, human leukocyte antigen (HLA) class I and programmed cell death protein 1 (PD1), as a way to develop allogeneic universal CART cells. Efficiency was over 70% percent gene editing at the protein level by multiplex gene disruption. Preclinical evaluation of CART19 generated with this approach showed potent anti-tumor activity and no evidence of GVHD in animal models. ⁹³

An alternative approach to obviate the issue of GVHD from allogeneic donors would be to use cell types other than T cells. Natural killer cells do not cause GVHD and several groups have published preclinical data on CAR-modified NK cells.^{94, 95} Clinical trials using CAR-NK cells are in progress (NCT02742727, NCT02839954). Notably, using NK cells would still mandate some HLA matching since NK cells express HLA class I molecules and could therefore be rejected in the setting of a host-versus-graft direction mismatch.

Lymphodepleting chemotherapy prior to CART cell infusion

Preclinical studies have shown that lymphodepleting chemotherapy prior to adoptive cell transfer results in depletion of regulatory T cells and other immune cells that can act as a competing sink for cytokines. This in turn augmented T cell function and dramatically improved the antitumor efficacy of transferred T cells.⁹⁶ The role and optimal agents and combinations of lymphodepleting chemotherapy are unknown. A few of the first pediatric patients treated with CART19 received no lymphodepleting chemotherapy and continue to be in remission more than 3 years later,¹³ suggesting that the function and activity of CART19 cells are not entirely dependent on the lymphodepleting chemotherapy. Most of the current clinical trials, however, incorporate some form of chemotherapy regimen. This commonly involves cyclophosphamide or the combination of FluCy in patients with CLL or ALL. In patients with non-Hodgkin lymphoma, several lymphodepleting regimens have been used by different groups, such as fludarabine, bendamustine, cyclophosphamide or lymphoma specific chemotherapeutic combinations. Recently, correlative studies from the FHRCR clinical trial showed that the addition of fludarabine to cyclophosphamide as a lymphodepleting regimen improved persistence, expansion and the CR rates in patients with lymphoma. ⁴¹ When FluCy combination was used, the CR rates were 42%, compared to 8% when cyclophosphamide was used alone, however cases of severe cerebral edema were reported.^{41, 42}

No lymphodepleting chemotherapy was administered in the allogeneic CART19 trial conducted at the NCI. This included 20 adults with progressive B-cell malignancies following allo-HCT (B-NHL, n=10, B-ALL, n=5, CLL, n=5). Despite that, objective responses were observed in 8 patients. In patients with ALL, 4 of 5 achieved MRD negative CR. ⁶³ The responses observed following CART cell infusion without antecedent lymphodepletion also suggest that lymphodepletion is not necessary for allogeneic CART-cell function.

CART cell combinations

Several combinatorial approaches with CART cell therapy are being evaluated in preclinical and early phase clinical studies. Combinatorial antigen targeting by CART cell therapy has been developed and investigated. Patients with CD19 negative relapses have extremely limited options and their outcomes are poor. Therefore, targeting more than one antigen is an appealing strategy to prevent antigen loss relapses. In fact, CART cells targeting CD19/CD20, CD19/CD20, or CD19/CD123 have been developed and shown to prevent or treat antigen loss relapses after CART cell therapy in preclinical studies by different investigators. ^{87, 88, 97}

CART cell combinations with the Bruton’s tyrosine kinase inhibitor (BTK) ibrutinib have been investigated in B cell malignancies,^{98, 99} and is now being studied clinically (NCT02640209). In preclinical studies, it has been shown that this approach results in a two pronged hit of B cell malignancies and enhances CART cell activity through down-regulation of inhibitory receptors on T cells.^{98, 99} These combinations are in early stage clinical trials. Other combinations have been investigated such as the combination with lenalidomide, or indoleamine 2,3-dioxygenase (IDO) inhibitors, or checkpoint inhibitors.¹⁰⁰ The combination with checkpoint inhibitors are of importance as this approach seems to be synergistic in preclinical evaluation ¹⁰⁰ and could potentially be of particularly valuable in CLL or solid tumors where response rates are low. These combinations are in different stages of development of early phase clinical trials.

CONCLUSIONS

Decades of research in transplant immunology, molecular biology, virology and cellular immunity are coming to fruition as we enter an exciting time in CART cell development. The remarkable success of CART19 likely presages further advances in adoptive T-cell therapy. The long lasting clinical remissions and B cell aplasia demonstrate the functional persistence of these CARTs and highlight the potential for a paradigm shift in adoptive immunotherapy; the effector cells can be measured and their activity quantified, thus giving some measure of confidence in the presence of long term immune surveillance. ¹⁴ Relating to the decades-long experience with allo-HCT, it may therefore be possible to accomplish our treasured goal of augmenting cell-mediated anti-leukemia effects without inducing GVHD.

Several hundred patients have been treated with CART19 ^{13–18, 41} to date and over a dozen with other CARTs (CARTs directing against BCMA⁵², CD20⁵⁰, CD22⁵¹, CD30⁵³, CD33⁵⁴ and CD123⁵⁵). We have learned that outcomes after CART19 are variable depending on the disease, the patient, and the CAR composition and manufacturing. These variables need to be taken into consideration when trying to determine the relative roles of allo-HCT and CART cell therapy. Ideally these questions will be tested in carefully designed clinical trials in due course. Based on our and others experience, we can hazard several broad recommendations.

1. For patients with refractory CD19+ malignancies (such as ALL), CART cells may be used before allogeneic HCT to induce remission and possibly increase the opportunities for successful allogeneic HCT.

2. For some patients with refractory malignancies, CART cells may induce sustained remissions (particularly CLL and NHL, and possibly ALL) and could be considered instead of allogeneic HCT.

3. For patients (particularly with ALL) who have short (<6 months) persistence of CART19, or who initially achieve then lose B cell aplasia (whose B cells become detectable, allogeneic HCT should be considered after CART cells.

4. For patients with relapsed B cell malignancy after allogeneic HCT (especially for ALL), CART cells are likely to be more effective than other maneuvers such as DLI.

These recommendations are quite speculative, and in this context we concede that it is too early to attempt to further finesse these recommendations based on the nuances of CART cell manufacture and design.

A few important challenges currently face the field of CART cell therapy; first the optimal structure of a CAR remains an area of active investigation. Secondly, development of systems that control the fate of CART cells will be an integral part of next generation CART cell development and is of particular importance when CARTs are used for targets other than B cells.^73–75 Finally, the future CART cell development will include continued efforts to develop a universal off the shelf CAR construct in order to enhance scalability and feasibility of this approach.^{90, 92, 93} With the rapid advances in genome editing, these techniques will likely be translated to the clinic at a very rapid pace.

Figure 2.

Schema of how chimeric antigen receptor T cell therapy can be incorporated in hematopoietic cell transplantation. MRD=minimal residual disease, NHL=non-Hodgkin lymphoma, MM=multiple myeloma, ALL=acute lymphoblastic leukemia, R/R=relapsed refractory

Highlights.

• CART cell therapy is an effective and potentially curative form of immunotherapy

• CART cells can induce remissions as a bridge to allo-HCT in B cell malignancies

• In some patients, CART cells can induce sustained remission and replace allo-HCT

• For patients relapsing after allo-HCT, CART cell therapy is a very effective option