Chimeric Antigen Receptor-T Cells for Leukemias in Adults: Methods, Data and Challenges

5.1 CAR T-Cell Therapeutics in Adult Leukemias: A General Introduction

Within the past several years, renewed interest in immunotherapy has been observed in multiple fields of oncology, including antibody-based therapeutics (e.g., checkpoint blockade) and adoptive cellular therapies. In the field of adult leukemia, such interest has been driven by the limitations of presently available therapies to induce durable remissions reliably in patients with relapsed or refractory leukemia. For instance, while an increasing proportion of children and young adults with B-cell acute lymphoblastic leukemia (B-ALL) have achieved long-term overall survival in recent decades, most adults diagnosed with B-ALL continue to experience relapse, and adults with relapsed or refractory B-ALL continue to have a poor prognosis when treated with standard salvage chemotherapy (Kantarjian et al. 2010; Gokbuget et al. 2012). The natural history of chronic lymphocytic leukemia (CLL) is considerably more heterogeneous, and a subset of patients will never require therapy. However, patients with unfavorable cytogenetic or molecular features, or with persistent or recurrent disease following initial purine analog-based therapy, continue to have a guarded prognosis when treated with standard therapy regimens (Tam et al. 2008; Kay et al. 2007). The development of oral molecularly targeted therapies such as ibrutinib has now brought a highly efficacious line of therapy to patients with newly diagnosed or relapsed CLL (Byrd et al. 2013, 2015). However, such therapies require an indefinite duration of treatment, are associated with few complete (vs. partial) responses, and are limited by toxicity or development of resistance in a subset of patients.

The adoptive transfer of genetically modified autologous T-cells aims to rapidly establish specific antitumor activity. This strategy requires targeting of autologous T-cells by means of a transgene-encoded antigen receptor, consisting of a chimeric antigen receptor (CAR), as will be discussed herein, or T-cell receptor (TCR) chains. The rationale for targeting CD19 specifically in B-cell malignancies is discussed in a previous chapter. To summarize, CD19 is a surface-exposed 95 kDa glycoprotein present on B-cells from early development until differentiation into plasma cells and represents an integral component of a cell surface signal transduction complex positively regulating signal transduction through the B-cell receptor (Stamenkovic and Seed 1988; Bradbury et al. 1993; Matsumoto et al. 1993; Fearon and Carter 1995). CD19 is nearly universally expressed by B-ALL, CLL, and hairy cell leukemia, while not expressed on normal tissues other than B-cells, including multipotent hematopoietic progenitor cells (De Rossi et al. 1993; Uckun et al. 1988; Schwonzen et al. 1993; Robbins et al. 1993). A CAR is a recombinant receptor construct composed of an extracellular antibody-derived single-chain variable fragment (scFv), linked to intracellular T-cell signaling domains of the T-cell receptor, thereby redirecting T-cell specificity in an HLA-independent manner (Park and Brentjens 2010). As discussed in a previous chapter, multiple generations of CARs have been developed and investigated in clinical studies. First-generation CARs consist of a target-specific scFv fused to the CD3ζ endodomain of the T-cell receptor/CD3 complex. As first-generation CAR T-cells exhibited limited persistence, expansion, and antitumor efficacy in preclinical and clinical studies, second-generation CARs incorporated cytoplasmic signaling domains of T-cell costimulatory receptors (e.g., CD28, 4-1BB) to provide a costimulatory “signal 2” to the T-cell. Third-generation CARs place multiple costimulatory domains in tandem (Fig. 5.1). Several groups have presented early data demonstrating that CAR-modified T-cells targeting CD19 can induce meaningful responses in patients with relapsed or chemotherapy-refractory B-cell leukemias (Park et al. 2016). In 2017, tisagenlecleucel, a CD19-targeted CAR T-cell product bearing a 4-1BB costimulatory domain, was approved by the US Food and Drug Administration for children and young adults <26 years old with refractory B-ALL or in second or greater relapse and became the first commercially available CAR T-cell product (U.S. Food and Drug Administration 2017). The largest clinical series described herein, reflecting treatment of adults with B-ALL and CLL, have employed second-generation CD19-targeted CAR T-cells. In this chapter, we review clinical outcomes of adults with leukemia treated with CAR T-cells, toxicities associated with CAR T-cell administration, present challenges limiting therapeutic efficacy, and future directions, including novel targets and enhancements to improve antileukemic activity.

../images/394140_1_En_5_Chapter/394140_1_En_5_Fig1_HTML.png — Fig. 5.1
Schematic depicting structure of chimeric antigen receptors in largest published series to date. *scFv* single-chain variable region fragment, TM transmembrane

5.2 CD19-Targeted CAR T-Cells in B-ALL

The largest series to date treating adults with relapsed or refractory B-ALL with CD19-targeted CAR T-cells are summarized in Table 5.1. Important differences between these studies include the use of autologous vs. allogeneic T-cells for genetic modification, as well as different transduction methods, costimulatory domains, regimens of lymphodepleting chemotherapy, CAR T-cell doses, and CAR T-cell product composition. Investigators from Memorial Sloan Kettering Cancer Center (MSKCC) were the first to report the efficacy of CD19-targeted CAR T-cells incorporating a CD28 costimulatory domain (19-28z) in generating durable molecular remissions in five adults with relapsed ALL (Brentjens et al. 2013; Davila et al. 2014) and have since reported the largest series of adults with ALL treated with CD19-targeted CAR T-cells (Park et al. 2018). Patients’ high-risk characteristics include ≥3 prior lines of treatment (n = 32), prior allogeneic hematopoietic cell transplantation (allo-HCT, n = 19), and Philadelphia (Ph) chromosome positivity (n = 16). Subsequent to salvage therapy or allo-HCT but prior to CAR T-cell infusion, 27 patients had morphologic disease (≥5% blasts in the bone marrow [BM] or measurable extramedullary disease) and 26 patients had minimal disease (<5% blasts in BM). Patients received cyclophosphamide (Cy) alone or Cy in combination with fludarabine (Flu) as lymphodepleting chemotherapy 2 days prior to 19-28z CAR T-cell infusion. Initially, all patients received 3 × 10⁶ 19-28z CAR T-cells/kg regardless of pretreatment disease burden. However, after observing a higher incidence of treatment-related toxicities at this dose in patients with morphologic disease (see section on “Toxicities”), the CAR T-cell dose was adjusted based on the disease burden, such that patients with morphologic disease received 1 × 10⁶ 19-28z CAR T-cells/kg and patients with minimal disease continued to receive 3 × 10⁶ 19-28z CAR T-cells/kg. In the entire cohort (i.e., at all CAR T-cell doses), 44 of 53 evaluable patients achieved or remained in complete response (CR) following 19-28z CAR T-cell infusion; 32 of 44 patients in CR evaluated for minimal residual disease (MRD) by multiparameter flow cytometry or deep sequencing achieved MRD-negative CR. Similar rates of CR were observed regardless of age, disease burden prior to CAR T-cell infusion, number of prior therapies, and prior allo-HCT. Seventeen of 44 patients in CR following CAR T-cell infusion underwent allo-HCT. However, as of most updated analysis, 6-month overall survival (OS) appeared similar between those who did and did not undergo post-CAR T-cell allo-HCT. Twenty-five patients achieving CR experienced morphologic relapse during follow-up; four of these patients relapsed with CD19-negative blasts by flow cytometry. Median event-free survival among all patients and among those who achieved MRD-negative CR was 6.1 months and 12.5 months, respectively (Brentjens et al. 2013; Davila et al. 2014; Park et al. 2015, 2018). 19-28z CAR T-cells were generally detectable by flow cytometry and quantitative PCR (qPCR) for 1–6 months post-infusion (Park et al. 2018).

Table 5.1

CAR T-cell design and production and clinical aspects of largest reported clinical series to date investigating CD19-targeted CAR T-cells in the treatment of B-ALL

Institution/Reference	# of patients reported	Gene transfer method	scFv	Costimulatory domain	Lymphodepleting chemotherapy	CAR T-cell doses	Disease-related outcomes
Memorial Sloan Kettering Cancer Center (Brentjens et al. 2013; Davila et al. 2014; Park et al. 2018)	53	Gammaretrovirus	SJ25C1	CD28	Cy or Cy/Flu	1 × 10⁶ vs. 3 × 10⁶ CAR⁺ T-cells/kg	CR: 83% (MRD-negative in 67%); 17 of 44 in CR underwent allo-HSCT Median EFS: 6.1 months (all) and 12.5 months (pts in MRD-negative CR)
Fred Hutchinson Cancer Research Center (Turtle et al. 2016)	30	Lentivirus	FMC63	4-1BB	Cy 2–4 g/m² (± etoposide 100 mg/m² × 3 days) or Cy 30–60 mg/kg + Flu 25 mg/m² × 3–5 days	2 × 10⁵, 2 × 10⁶, and 2 × 10⁷ CAR⁺ T-cells/kg	CR: 10/12 (MRD-negative by flow cytometry) among pts receiving Cy or Cy/etoposide; 16/17 (MRD-negative by flow cytometry and FISH/karyotype) among pts receiving Flu/Cy Median DFS: not yet reached in Flu/Cy arm
University of Pennsylvania (Frey et al. 2014)	12	Lentivirus	FMC63	4-1BB	Investigator’s choice	6.5–8.45 × 10⁶ CAR⁺ T-cells/kg	CR: 89% (8/9) of evaluable pts, all MRD-negative; 3 non-evaluable patients died in the setting of refractory CRS
National Cancer Institute (Brudno et al. 2016)	5	Gammaretrovirus	FMC63	CD28	None (administered following allo-HCT)	4.2–7.1 × 10⁶ CAR⁺ T-cells/kg	CR: 80% (4/5, all MRD-negative)

Cy cyclophosphamide, Flu fludarabine, EFS event-free survival, DFS disease-free survival, CR complete response, MRD minimal residual disease, Allo-HCT allogeneic hematopoietic cell transplantation

Investigators from the Fred Hutchinson Cancer Research Center (FHCRC) also reported mature results of their phase I trial of CD19-targeted CAR T-cells in 30 adults with relapsed/refractory B-ALL (Turtle et al. 2016). In contrast to MSKCC’s approach, the FHCRC’s treatment protocol uses lentiviral transduction and a CD19-targeted CAR bearing a 4-1BB costimulatory domain (rather than the CD28 costimulatory domain). In addition, CD4⁺ and CD8⁺ T-cells are expanded separately in vitro prior to infusion at a defined ratio of 1:1 CD4⁺:CD8⁺ CAR T-cells, at total infused doses of 2 × 10⁵, 2 × 10⁶, and 2 × 10⁷ CAR T-cells/kg. In support of this methodology, investigators from FHCRC have reported preclinical data demonstrating that transduced CAR T-cell subsets exhibit different effector functions, noting weak lytic activity from isolated CD4⁺ CAR T-cells, but greater IFN-γ, TNF-α, and IL-2 cytokine production from naïve CD4⁺ CAR T-cells after stimulation with CD19⁺ tumor cells, and greatest direct antitumor potency from CD8⁺ CAR T-cells with a central memory phenotype. In NOD/SCID/γc^−/− (NSG) mice engrafted with the Raji/ffluc (CD19⁺) tumor cell lines, treatment with patient-derived CAR T-cells products with defined subset composition resulted in enhanced survival (Sommermeyer et al. 2016). As such, this strategy may allow for a lower requisite overall CAR T-cell dose and more uniform product composition between patients. In the FHCRC trial, the 30 treated patients had received a median of 3 prior intensive chemotherapy regimens (range, 1–11), and 11 had experienced relapse following prior allo-HCT. Most had morphologic B-ALL at the time of infusion (median BM blast burden 21%, range, 0.014–97%), and seven had extramedullary disease. Thirteen patients received lymphodepleting chemotherapy consisting of Cy-based regimens without Flu; 10 of 12 evaluable patients achieved BM CR without evidence of disease by flow cytometry, though 7 of 10 experienced relapse at a median of 66 days following CAR T-cell infusion (Turtle et al. 2015, 2016). While five of these patients were retreated, no response was observed; an endogenous CD8⁺ T-cell response directed against the murine scFv component of the transgene was observed and hypothesized to contribute to CAR T-cell rejection and in vivo expansion failure. Of note, investigators from the National Cancer Institute (NCI) reported T-cell mediated anti-CAR responses in several pediatric patients with relapsed or refractory B-ALL treated with CD19-targeted CAR T-cells (Lee et al. 2015). As the FHCRC investigators hypothesized that greater lymphodepletion would reduce the risk of CAR T-cell rejection, they added Flu 25 mg/m²/day for 3–5 days to Cy 60 mg/kg prior to CAR T-cell infusion in 17 subsequent patients, observed BM CR by flow cytometry and cytogenetic studies in 16 of 17 patients, and noted significantly improved disease-free survival compared with patients in the prior cohort who had not received Flu (Turtle et al. 2016). Additionally, greater CD4⁺ and CD8⁺ CAR T-cell levels were observed 28 days following infusion of 2 × 10⁶ CAR T-cells in patients receiving Flu/Cy vs. Cy-based lymphodepleting chemotherapy (Turtle et al. 2016). Of note, due to greater toxicity observed in patients with >20% BM blasts treated with ≥2 × 10⁶ CAR T-cells, the investigators ultimately adopted a risk-adapted strategy in which these patients received a lower dose of 2 × 10⁵ CAR T-cells, similar to MSKCC’s approach as above (Turtle et al. 2016).

As discussed in a separate chapter, investigators from the University of Pennsylvania (UPenn) and Children’s Hospital of Philadelphia (CHOP) have reported on the use of CTL019, a CD19-targeted CAR T-cell product containing a 4-1BB costimulatory domain, in children and adolescents with relapsed or refractory B-ALL (Maude et al. 2014, 2018). This group has additionally reported on 12 adults with relapsed or refractory B-ALL treated with CTL019 following investigator’s choice of lymphodepleting chemotherapy. Eight of nine evaluable patients achieved MRD-negative CR at 1 month. However, substantial toxicity was observed as noted in a subsequent section (Frey et al. 2014). While mature data reflecting CTL019 persistence in adults with B-ALL has not yet been reported, this group has noted CTL019 persistence by qPCR and B-cell aplasia for ≥2 years following infusion in pediatric patients with B-ALL who achieved MRD-negative CR (Grupp et al. 2015).

While most studies of CD19-targeted CAR T-cell therapeutics to date have employed autologous T-cells, investigators from the NCI have additionally investigated the use of donor-derived (i.e., allogeneic) CAR T-cell products in patients with progressive B-ALL or other B-cell malignancies post-allo-HCT. Eligible patients had ≤grade I acute graft-versus-host disease (GvHD) and ≤mild chronic GvHD; DLI was not required for recipients with B-ALL. The investigators modified T-cells derived from the related or unrelated donor used for the patient’s allo-HCT to express a CD19-targeted CAR incorporating a CD28 costimulatory domain. The five reported patients with progressive or relapsed B-ALL at infusion received a single dose of 4.2–7.1 × 10⁶ CAR T-cells/kg (median 5.6 × 10⁶/kg) without antecedent lymphodepleting chemotherapy. Four of these patients achieved MRD-negative CR, with recovery of normal hematopoiesis, including normal polyclonal B-cells; remission was durable (16+ months) in one patient with B-ALL treated with this approach, and another patient underwent second allo-HCT while in MRD-negative CR (Brudno et al. 2016).

5.3 CD19-Targeted CAR T-Cells in CLL

Following several early clinical reports of CD20- and CD19-targeted CAR T-cells in patients with relapsed or refractory B-cell non-Hodgkin lymphoma, several groups reported their early experience treating patients with relapsed or refractory CLL with CD19-targeted CAR T-cells (see Table 5.2 for largest/most mature series). MSKCC initially reported on eight patients with purine analog-refractory CLL and bulky lymphadenopathy treated with 19-28z CAR T-cells. Seven of eight patients had additional adverse cytogenetic or molecular features (del17p, del11q, and/or unmutated IgHV). No objective responses were observed in three patients who received 1.2–3.0 × 10⁷ 19-28z CAR T-cells/kg without any prior lymphodepleting chemotherapy. A fourth patient developed fevers, hypotension, and renal failure following 19-28z CAR T-cell therapy and died within 48 h of a suspected sepsis syndrome, possibly related to subacute infection prior to CAR T-cell therapy (Brentjens et al. 2010). Four subsequent patients received Cy 1.5–3.0 g/m² followed by 0.4–1.0 × 10⁷ CAR T-cells/kg, infused in split doses over 2 days. One patient demonstrated marked reduction of peripheral adenopathy after an initial period of stable to progressed disease, two others demonstrated stable disease, and another demonstrated progression (Brentjens et al. 2011). Updated clinical results have noted objective responses in a subset of patients with CLL receiving conditioning chemotherapy prior to 19-28z CAR T-cell therapy, however (Geyer et al. 2016a). In a subsequent phase I trial at MSKCC, we enrolled patients with untreated CLL and administered 19-28z CAR T-cells to patients with residual disease following initial chemoimmunotherapy consisting of pentostatin, Cy, and rituximab (PCR). Eight such patients attained PR and then subsequently received Cy 600 mg/m² followed 2 days later by escalating doses of 19-28z CAR T-cells (3 × 10⁶, n = 3; 1 × 10⁷, n = 3; 3 × 10⁷, n = 2, in 19-28z CAR T-cells/kg), administered outpatient. Most treated patients had unfavorable cytogenetic or molecular features (unmutated IgHV, n = 7; del11q, n = 1). Two patients achieved CR as best response, three achieved stable disease (with BM PR in one), and three had progression of disease, one of whom had a rising ALC by the time of 19-28z CAR T-cell infusion and two of whom achieved BM response (MRD-positive CR in one, PR in the other) with progression noted in lymph node; median PFS was 13.6 months (Geyer et al. 2016b; Park et al. 2014). CAR T-cells were detectable for a maximum of 48 days post-infusion by flow cytometry and/or qPCR.

Table 5.2

CAR T-cell design and production and clinical aspects of selected clinical series to date investigating CD19-targeted CAR T-cells in the treatment of CLL

Institution	# of patients reported	Gene transfer method	scFv	Costimulatory domain	Lymphodepleting chemotherapy	Infused cell doses	Responses observed
National Cancer Institute (Kochenderfer et al. 2012)	4	Gammaretrovirus	FMC63	CD28	Cy 60 mg/kg × 2 days + Flu 25 mg/m² × 5 days	0.3–2.8 × 10⁷ CAR⁺ T-cells/kg	ORR: 3/4 (CR, n = 1; PR, n = 2)
National Cancer Institute (Kochenderfer et al. 2015)	4	Gammaretrovirus	FMC63	CD28	Cy 60 mg/kg × 1–2 days + Flu 25 mg/m² × 5 days	1–4 × 10⁶ CAR⁺ T-cells/kg	ORR: 4/4 (CR, n = 3; PR, n = 1)
National Cancer Institute (Brudno et al. 2016)	5	Gammaretrovirus	FMC63	CD28	None (administered following Allo-HCT)	0.4–3.1 × 10⁶ CAR⁺ T-cells/kg	ORR: 2/5 (CR, n = 1; PR, n = 1; SD, n = 1)
Fred Hutchinson Cancer Research Center (Turtle et al. 2017)	19	Lentivirus	FMC63	4-1BB	Cy 30–60 mg/kg × 1 + Flu 25 mg/m² × 3 days	2 × 10⁵, 2 × 10⁶, or 2 × 10⁷ CAR⁺ T-cells/kg; 1:1 CD4⁺:CD8⁺	ORR: 14/19 (CR, n = 4; PR, n = 10)
University of Pennsylvania (Porter et al. 2015)	14	Lentivirus	FMC63	4-1BB	Investigator’s choice	0.14–11 × 10⁸ CAR⁺ T-cells (median, 1.6 × 10⁸ cells)	ORR: 8/14 (MRD-negative CR, n = 4; PR, n = 4) Median PFS: 7 months Median OS: 29 months
University of Pennsylvania (Porter et al. 2016)	35	Lentivirus	FMC63	4-1BB	Investigator’s choice	5 × 10⁷ vs. 5 × 10⁸ CAR⁺ T-cells	ORR: 9/17 (CR, n = 6; PR, n = 3) among pts receiving 5 × 10⁸ CAR⁺ T-cells

Cy cyclophosphamide, Flu fludarabine, OS overall survival, PFS progression-free survival, CR complete response, PR partial response, MRD minimal residual disease, Allo-HCT allogeneic hematopoietic cell transplantation

Investigators from UPenn have treated >40 patients with relapsed or refractory CLL with CTL019 (Frey et al. 2014). This group published an initial report describing durable CR in a patient with refractory, p53-deficient CLL following pentostatin + Cy followed by 1.5 × 10⁵ CTL019/kg (Porter et al. 2011). The investigators subsequently published detailed follow-up on 14 patients with relapsed or refractory CLL treated on their pilot study with CTL019. Treated patients had several high-risk features, including a median of five prior therapies, loss of TP53 or chromosome 17p (n = 6), and unmutated IgHV (n = 9). Patients received one of several lymphodepleting regimens, including bendamustine (n = 6), Cy/Flu (n = 3), or Cy/pentostatin, followed by infusion of CTL019 (median dose, 1.6 × 10⁸ CTL019) over 1–3 days. Eight of 14 patients completed the full 3-day regimen, with others receiving only one (n = 3) or two fractions (n = 3) due to fevers within 24 h of infusion. Eight of 14 patients attained objective response at median follow-up of 19 months, including 4 patients who achieved MRD-negative CR, of whom 3 have experienced durable responses (28–53 months), while 1 died of unrelated causes 21 months post-CTL019 infusion with no evidence of disease. Four patients achieved PR within the first month of infusion, lasting 5–13 months, and two patients with PR completely cleared detectable CLL from the blood and BM. Six patients had no objective response and experienced progressive disease within 9 months of CTL019 infusion. Median OS for all patients was 29 months, 18-month OS was 71%, and 18-month PFS was 28.6%. Though subgroup analysis is limited considerably by the small number of patients, number of previous therapies, abnormalities of chromosome 17p, IgHV mutation status, and CTL019 dose did not appear to be correlated with response (Porter et al. 2015). CTL019 expansion peaked within 1 month of infusion, and patients achieving CR exhibited greater peak expansion than those who did not achieve CR. Persistence of CTL019 by qPCR or flow cytometry and B-cell aplasia were noted through last follow-up (as long as ≥4 years) in patients achieving MRD-negative CR (Porter et al. 2015). UPenn additionally presented a phase II dose optimization study in which 28 patients with relapsed or refractory CLL were randomly assigned to receive either 5 × 10⁷ or 5 × 10⁸ CTL019 (stage 1) and subsequent patients received 5 × 10⁸ CTL019 (stage 2) following conditioning chemotherapy. Among 17 patients who received the higher CTL019 dose, 9 achieved objective response (CR, n = 6, and PR, n = 3) (Porter et al. 2014, 2016).

Several other groups have reported results in patients with CLL treated with CD19-targeted CAR T-cells on prospective studies enrolling patients with different B-cell malignancies. Investigators from the NCI have included several patients with CLL in their prospective trials of CD19-targeted CAR T-cell therapies in B-cell NHL. In their initial series, they treated eight patients with indolent B-NHL, including CLL, with Flu/Cy followed by CD19-targeted CAR T-cell, and IL-2 post-infusion. Three of four patients with CLL experienced objective response, including one patient who achieved a durable CR (Kochenderfer et al. 2012). In a subsequent study that omitted IL-2 following CAR T-cell infusion, they again observed responses in all four enrolled patients with relapsed CLL; three of these patients achieved CR, ongoing as of the time of publication (Kochenderfer et al. 2015). Investigators from the FHCHC also reported on 19 patients with relapsed and refractory CLL treated with CD19-targeted CAR T-cells, with 4 patients achieving CR (Turtle et al. 2017). Finally, as above, investigators from the NCI have additionally investigated the use of CD19-targeted allogeneic CAR T-cell infusion post-allo-HCT, including in patients with progression of CLL post-allo-HCT who had ≤grade I acute GvHD (aGvHD), including following at least ≥1 donor lymphocyte infusion (DLI). Patients received no antecedent lymphodepleting chemotherapy. Best responses were CR (n = 1), PR (n = 1), and stable disease (n = 1) among the five enrolled patients with CLL (Brudno et al. 2016).

5.4 Toxicities Associated with CD19-Targeted CAR T-Cells

The principal toxicities of CD19-targeted CAR T-cells observed to date include cytokine release syndrome (CRS), a spectrum of neurologic toxicities, and the on-target/off-tumor effect of B-cell aplasia. CRS may be defined broadly as a systemic inflammatory response in the hours to days following CAR T-cell infusion characterized by fevers, myalgias, malaise, and, in more severe cases, capillary leak syndrome with hypotension, hypoxia, and, more rarely, acute kidney injury and coagulopathy. CRS appears to arise in the setting of brisk CAR T-cell activation and expansion and marked elevations in pro-inflammatory cytokines (Davila et al. 2014). Severe CRS may be treated initially using the IL-6 receptor inhibitor tocilizumab, with the addition of lymphotoxic corticosteroids if symptoms persist. Intravenous immune globulin (IVIG) may be used to manage hypogammaglobulinemia in the setting of B-cell aplasia.

Among adults with B-ALL treated with CAR T-cells, the incidence and severity of severe CRS appears to be associated with underlying disease burden and with CAR T-cell dose (Davila et al. 2014; Park et al. 2018; Turtle et al. 2016; Frey et al. 2014). Classification systems for CRS severity vary between reported studies to date, complicating cross-trial comparisons (Lee et al. 2015; Porter et al. 2015). At MSKCC, severe CRS (i.e., requiring vasopressors or mechanical ventilation) has been observed almost exclusively among adults with morphologic B-ALL (vs. MRD alone) at the time of CAR T-cell infusion. After 3 of 11 patients at MSKCC with morphologic B-ALL experienced fatal treatment-related toxicity following infusion of 3 × 10⁶ 19-28z CAR T-cells/kg, CAR T-cell dose was lowered to 1 × 10⁶ 19-28z CAR T-cells/kg for patients with morphologic B-ALL and maintained at 3 × 10⁶/kg for those patients with MRD only. Subsequently, no grade 5 toxicity was observed in patients treated according to this risk-adapted approach (Park et al. 2015, 2018). The FHCRC reported 25 of 30 adults with B-ALL treated with CD19-targeted CAR T-cells developed any CRS between 6 h and 9 days following CAR T-cell infusion, most commonly characterized by fevers and/or hypotension and elevated levels of IL-6 and IFN-γ; 7 patients had severe CRS requiring ICU care, and 2 patients died of treatment-related toxicity (severe CRS in one, irreversible neurologic toxicity in the other). Pretreatment disease burden and CAR T-cell dose were correlated with the incidence of CRS. After the first two patients were treated with 2 × 10⁷ CAR T-cells/kg developed severe treatment-related toxicities, this dose level was determined to be too toxic. Moreover, as all six patients with >20% BM blasts treated with ≥2 × 10⁶ CAR T-cells/kg required ICU care for CRS and developed severe neurologic toxicity, they implemented a risk-adapted approach in which patients with >20% BM blasts received 2 × 10⁵ CAR T-cells/kg and those with ≤20% BM blasts received 2 × 10⁶ CAR T-cells/kg. Thereafter, only one of ten patients with >20% BM blasts required ICU care (Turtle et al. 2016). Investigators from UPenn noted severe CRS in 11 of 12 adults with B-ALL treated with CTL019 on a phase I trial, including 3 patients with CRS refractory to tocilizumab and corticosteroids who died within 3–15 days of infusion; all treated patients had high pretreatment disease burden, and patients with fatal CRS had received a higher dose of CTL019 compared to the others (Frey et al. 2014). While data describing CRS in patients with CLL treated with CD19-targeted CAR T-cells are more limited, a preliminary report describing MSKCC’s initial phase I trial utilizing 19-28z CAR T-cells in patients with CLL noted that all patients became febrile following 19-28z CAR T-cell infusion and two developed hypotension (Brentjens et al. 2011). In their subsequent trial of CAR T-cell therapy as consolidation following PCR chemoimmunotherapy in patients with CLL, four of five patients receiving ≥1 × 10⁷ 19-28z CAR T-cells/kg were readmitted with fevers and mild, self-limited CRS not requiring ICU transfer, anti-cytokine therapy, or corticosteroids (Geyer et al. 2016b; Park et al. 2014). UPenn reported 9 of 14 CLL patients treated with CTL019 on their phase I trial developed ≥grade 1 CRS, 5 of whom required tocilizumab and/or corticosteroids and 4 of whom required ICU admission; in abstract form, they reported 19 of 35 patients with CLL treated with CTL019 on their phase II study developed any CRS, of whom 4 required tocilizumab ± corticosteroids (Porter et al. 2014, 2015, 2016). A similar proportion of patients with CLL or other B-NHL treated by the NCI with CD19-targeted CAR T-cells experienced fever (12 of 15) or hypotension (4 of 15) consistent with CRS (Kochenderfer et al. 2015).

A range of generally reversible neurologic toxicity has been observed following CAR T-cell infusion in children and adults, including delirium, seizure-like activity, confusion, word-finding difficulty, aphasia, and frank obtundation (Davila et al. 2014; Turtle et al. 2016; Porter et al. 2015; Kochenderfer et al. 2015). These neurologic toxicities may occur independently of CRS. Twenty-two of 53 adults with B-ALL treated with 19-28z CAR T-cells at MSKCC have experienced ≥grade 3 neurologic toxicity, including 14% of patients with MRD only at the time of CAR T-cell infusion, suggesting a less intimate correlation between the development of such toxicity and tumor burden (Park et al. 2015, 2018). FHCRC reported ≥grade 3 neurologic toxicity in 15 of 30 adults with B-ALL treated with CD19-targeted CAR T-cells, either concurrent with or after resolution of CRS, including generalized seizures in 3 patients (Turtle et al. 2016). Investigators from UPenn reported neurologic toxicity including hallucinations, confusion, and delirium in 6 of 14 adults (≤grade 2, n = 5) with relapsed/refractory CLL treated with CTL019 (Porter et al. 2015). The NCI has reported a similar incidence and spectrum of neurologic toxicity in patients with CLL and other B-NHL treated with CD19-targeted CAR T-cells (Kochenderfer et al. 2015). A correlation between CAR T-cell concentrations in the CSF has been postulated, but not consistently observed, and the pathogenesis of such toxicity remains uncertain (Davila et al. 2014; Lee et al. 2015).

5.4.1 Expert Point of View

CD19-targeted CAR-modified T-cells have emerged as one of the most effective available therapies in treating adults with relapsed or refractory B-ALL, including those with relapse following allo-HSCT, with high rates of MRD-negative CR now observed in multiple reported series, despite differences in therapeutic strategies, including differences in CAR design (e.g., scFv, costimulatory domain), lymphodepleting chemotherapy, and CAR T-cell dose, source (e.g., autologous vs. allogeneic), and product composition. A more modest proportion of patients with refractory CLL treated with second-generation CAR T-cells achieve durable clinical benefit, though long-term CRs have been observed in several reports. Potential strategies for extending this technology to non-B-cell leukemias are noted in the following section.

CAR T-cell therapies remain a new and evolving approach in the treatment of refractory adult leukemia, and further review of mature data will be required to draw firm conclusions regarding optimal therapeutic strategy. The broad, clinically relevant themes of studies to date have suggested the importance of costimulation and adequate lymphodepletion in promoting CAR T-cell persistence and expansion. Limited data suggests a trend toward longer persistence of CAR T-cells containing a 4-1BB (vs. CD28) costimulatory domain, such as CTL019. However, treatment with this 4-1BB-containing CAR T-cell product additionally appears associated with a greater incidence of CD19⁻ B-ALL escape variants when relapse occurs post-CR following CAR T-cell therapy. Additionally, differences in clinical trial design and patient selection make it extremely difficult to conclude whether costimulatory domain selection affects the incidence of relapse after achieving CR or long-term EFS/OS (Park et al. 2018; Turtle et al. 2016; Maude et al. 2014; Grupp et al. 2015).

Several lines of evidence support the need for adequate lymphodepletion prior to CAR T-cell therapy; while the exact mechanisms remain unclear, lymphodepleting chemotherapy may enhance antigen-presenting cell activation and eradicate immune-suppressive regulatory T-cells and homeostatic cytokine “sinks.” The absence of significant rise in pro-inflammatory cytokine levels, poor CAR T-cell expansion, and limited clinical efficacy have been observed in adults with B-cell malignancies treated with CAR T-cell products without antecedent lymphodepleting chemotherapy (Brentjens et al. 2011; Cruz et al. 2013). Additionally, investigators from the FHCRC noted that the addition of Flu to Cy appeared to enhance CAR T-cell persistence and expansion, decrease the incidence of transgene-directed immune responses, and possibly improve EFS in adults with relapsed B-ALL treated with CAR T-cells (Turtle et al. 2016). Potential strategies for enhancing the persistence, expansion, and clinical efficacy of CAR T-cells in patients with CLL are discussed in a subsequent section.

Optimizing CAR T-cell therapeutics in adult leukemias additionally warrants consideration of management following CAR T-cell therapy. Challenges proximal to therapy include acquiring greater understanding of the pathogenesis and optimal management of treatment-related neurologic toxicity and in prevention and management of refractory CRS. Challenges more distal to therapy include defining an optimal consolidation strategy for patients with B-ALL achieving MRD-negative CR and management of escape variants (i.e., loss of target tumor-associated antigen) at relapse. In MSKCC’s experience to date treating refractory B-ALL with 19-28z CAR T-cells, we have identified no significant difference in OS among patients achieving MRD-negative CR, regardless of whether the patient underwent subsequent allo-HCT (Park et al. 2015, 2018). However, in other series utilizing this approach, including the NCI’s experience in treating pediatric patients with relapsed B-ALL, most patients achieving MRD-negative CR have undergone allo-HCT (Lee et al. 2015); whether consolidative allo-HCT or >1 cycle of CAR T-cell therapy will improve long-term EFS in this setting remains uncertain. We and others have additionally described loss of detectable CD19 by flow cytometry in relapsing B-ALL blasts in a subset of patients with B-ALL treated with CD19-targeted CAR T-cell therapy, a finding that parallels reports of CD19-negative relapse following therapy with the bispecific T-cell engager blinatumomab (Park et al. 2018; Turtle et al. 2016; Lee et al. 2015; Maude et al. 2014; Frey et al. 2014; Grupp et al. 2015; Topp et al. 2014). One of several potential mechanisms underlying this phenomenon appears to be alternative splicing of CD19 mRNA compromising the target epitope, and in turn, CAR T-cell efficacy, while preserving cytoplasmic domains required for kinase recruitment and signaling to permit leukemic maintenance (Sotillo et al. 2015). Antigen escape might be treated by the use of CAR T-cells targeting other immature B-cell antigens (e.g., CD22), as is being investigated in several ongoing studies (NCT02650414, NCT02315612), and might be prevented by use of CAR T-cells targeting multiple tumor-associated antigens at once or by therapies designed to enhance early T-cell expansion and overcome inhibitory effects of the tumor microenvironment as described in the next section.

5.4.2 Future Directions

The dramatic responses observed in many patients with refractory B-ALL treated with CD19-targeted CAR T-cells have led several groups of investigators to consider potential strategies for extending CAR T-cell therapy to acute myeloid leukemia (AML) and T-cell leukemias. While CD19 is in many ways an ideal target surface antigen for B-cell malignancies, as previously discussed, selection of a target for AML is far more challenging given the lack of known surface antigens unique to malignant myeloid blasts and not expressed on normal hematopoietic cells or myeloid precursors. Using a lentiviral anti-CD123 vector costimulated via 4-1BB, investigators from UPenn noted that CD123-targeted CAR T-cells (CART123) exhibited potent antileukemic activity in NSG mice bearing human AML cell lines (e.g., MOLM14) as well as NSG mice transgenic for IL-3, stem cell factor, and GM-CSF (NSGS mice) bearing patient-derived AML samples. However, CART123 administration appeared to ablate normal hematopoiesis in NSG mice engrafted with human CD34⁺ hematopoietic progenitor cells, even at 1 month post-infusion (Gill et al. 2014). Administration of T-cells transduced with an anti-CD33 lentiviral vector (encoding an scFv derived from the anti-CD33 monoclonal antibody-drug conjugate gemtuzumab ozogamicin and a 4-1BB costimulatory domain) leads to severe hematopoietic toxicity in similar humanized xenograft models (Kenderian et al. 2015). As such, this group subsequently investigated a transiently expressed mRNA CD33-targeted CAR as a potential means to circumvent the myeloablation observed with CART123 and CART33 and thereby expand the therapeutic index of CAR T-cell therapy for AML. The mRNA construct is electroporated into human T-cells to generate the RNA-CART33 product, resulting in high-level CAR expression that diminished over 7 days, with cytotoxicity decreasing over time post-electroporation. MOLM14-engrafted NSG mice exhibited enhanced survival following Cy + RNA-CART33 vs. Cy + untransduced T-cells (Kenderian et al. 2015). Further development of “biodegradable” CAR T-cells and other strategies to modulate the therapeutic effect of CAR T-cells with potent antileukemic activity may permit safer application of CAR T-cell therapeutics in AML. Alternatively, CAR T-cells with greater hematologic toxicity might be used in patients with AML as a bridge to allo-HCT, as is being investigated in ongoing clinical trials utilizing CD33-targeted and CD123-targeted CAR T-cells (NCT02623582, NCT01864902). There also remains an unmet need for effective therapies for patients with relapsed or refractory T-cell malignancies, including T-ALL. However, the potential use of CAR T-cells targeted to T-cell antigens raises immediate concerns of on-target/off-tumor toxicity against endogenous T-cells and infused CAR T-cells (i.e., fratricide). Investigators from the Baylor College of Medicine/Texas Children’s Hospital have conducted preclinical studies of CD5-targeted CAR T-cells, as CD5 is expressed on the surface of most T-ALL and T-NHL. Specifically, human T-cells transduced with a retrovirus encoding a CD5-targeted CAR costimulated via CD28 eliminate malignant T-cell lines in vitro and in xenograft NSG mouse models in vivo while exhibiting only limited fratricide, with sparing of native central and effector memory T-cells as well as virus-specific T-cells (Mamonkin et al. 2015).

The more modest observed clinical efficacy of CD19-targeted CAR T-cells in CLL, compared with B-ALL, may be related in part to a hostile tumor microenvironment. CLL cells exploit a variety of mechanisms to escape elimination from the endogenous immune system, including upregulation of inhibitory ligands inducing impairment of T-cell immunologic synapses (e.g., CD200, PD-L1), production of soluble plasma factors leading to suppression of NK cytotoxicity (e.g., BAG6), release of exosomes promoting a cancer-associated fibroblast phenotype in stromal cells and supporting leukemic maintenance, and induction of CD8⁺ T-cell exhaustion (as marked by decreased proliferative and cytotoxic capacity and increased expression of exhaustion markers PD-1, CD160, and CD244) (Reiners et al. 2013; Ramsay et al. 2012; Gorgun et al. 2005; McClanahan et al. 2015; Riches et al. 2013; Paggetti et al. 2015). Several strategies to overcome this inhibitory microenvironment have been described and are being investigated in forthcoming clinical studies, including further genetic modification of CD19-targeted CAR T-cells with an additional costimulatory ligand, such as 4-1BB ligand (4-1BBL) or CD40 ligand (CD40L), or incorporation of the pro-inflammatory cytokine IL-12 (Pegram et al. 2012, 2015; Stephan et al. 2007). Several investigators have hypothesized that coadministration of checkpoint inhibitors targeting the programmed death-1 (PD-1)/PD-L1 pathway may abrogate CAR T-cell exhaustion, deplete myeloid-derived suppressor cells at disease sites, and thereby enhance antitumor response (John et al. 2013). Additionally, ibrutinib appears to have immunomodulatory effects independent of inhibition of Bruton’s tyrosine kinase (BTK), including inhibition of interleukin-2-inducible T-cell kinase (ITK), which may in turn enhance Th1 responses (Sagiv-Barfi et al. 2015). Investigators from the University of Pennsylvania have observed decreased levels of PD-1 expression on endogenous CD8⁺ T-cells in patients with CLL treated with ibrutinib, as well as superior CTL019 expansion ex vivo in patients treated with ibrutinib for ≥5 months, and enhanced proliferation and engraftment in vivo in patients with CLL treated with ibrutinib prior to CTL019 infusion (Fraietta et al. 2016). Early clinical data from MSKCC supports the potential efficacy of ibrutinib in promoting ex vivo T-cell expansion and enhancing clinical response (Geyer et al. 2016a). These findings suggest a potential role for combination therapy with ibrutinib and CD19-targeted CAR T-cells in future clinical studies in CLL.

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