Amanda Przespolewski, DO

Chimeric antigen receptor (CAR) T-cell therapy is a novel form of cellular immunotherapy in which autologous T cells are genetically modified to express CARs found on tumor cells, as well as on costimulatory molecules required for T-cell activation. Although now an established treatment modality for CD19+ lymphoid malignancies, the development of CAR T-cell strategies for myeloid malignancies, specifically acute myeloid leukemia (AML), is still being explored preclinically and in phase I trials. Barriers to successful adaptation of this approach in AML include heterogeneous target antigen expression on diverse myeloid cell populations, co-expression on normal hematopoietic stem cells leading to off-target toxicity, and lack of clear anti-leukemia responses. This review will discuss the current status of CAR T-cell therapy in AML, highlighting the challenges and potential for this approach to change the treatment landscape for this disease.

Adoptive immunotherapy holds much promise for the therapy of hematologic malignancies, with CD19+ lymphoid malignancies being the showpiece for this therapy. In chimeric antigen receptor (CAR) T-cell therapy, autologous T cells are genetically engineered to express CARs found on tumor cells as well as on costimulatory molecules.

Multiple early-phase trials investigating CAR T-cell therapy in treatment-refractory lymphoid malignancies, including chronic lymphocytic leukemia and acute lymphoblastic leukemia (ALL), have demonstrated significant antitumor effects, with high complete remission rates and clear reduction in tumor burden.

Such studies have sparked interest in the development of CAR T-cell therapy for myeloid malignancies, specifically acute myeloid leukemia (AML). However, major challenges in developing targeted immunotherapeutic approaches for AML, as compared with its lymphoid counterparts, are the underlying biological heterogeneity of myeloid malignancies and the lack of truly tumor-specific surface antigens that are also not expressed on normal myeloid and hematopoietic progenitor cells. To date, investigators have explored targeting CD33, CD123, and folate receptor (FR) beta as well as numerous other antigens as CAR T-cell constructs in preclinical models and limited early phase testing.

Here, we review the current status of CAR T-cell therapy for treatment of AML, highlighting the early

stages of development of this approach, clinical challenges, and its promise to change the treatment paradigm for this disease.

Significant preclinical data supporting the efficacy of CAR T-cell therapy for AML are summarized in Table 1.

CD33 is a sialic acidbinding transmembrane receptor expressed on AML blasts, early multilineage hematopoietic progenitors, and myelomonocytic precursors, as well as, to a lesser degree, pluripotent stem cells and nonhematologic tissues.

The impetus for selection of the CD33 moiety as a target for CAR T-cell therapy arose from the known clinical activity of gemtuzumab ozogamicin, an anti-CD33 monoclonal antibodydrug conjugate, for treatment of CD33+ relapsed/refractory (r/r) patients with AML.

Kenderian et al designed CD33-targeted CAR T cells (CART-33), expressing a humanized version of the same single-chain variable fragment (scFv) of the original murine clone my96 along with either human CD8 or immunoglobulin G (igG)4 hinges.

Coculture of CART-33 with AML cells in vitro induced extensive CART-33 proliferation, degranulation, and cytokine production, as well as robust cytotoxicity at low effector-to-target ratios. Significant in vitro activity was observed against primary AML cells, with superior cytotoxicity seen with the CD4 hinge construct. Infusion of 1 x 105 CART-33 cells eradicated in vivo disease and prolonged overall survival (OS) of multiple primary CD33+ AML xenograft models (Table 1). Rafiq and colleagues generated CART-33 expressing variable light and heavy chains of the humanized M-195 antibody together with the costimulatory CD28 and CD3-zeta signaling domains with the interleukin (IL)-12 gene.

Co-culture of these T cells with AML cells resulted in enhanced interferon-&gamma; (IFN-&gamma;), IL-2, and IL-12 production. Significant cytotoxicity was observed in vitro in a range of effector-to-target ratios using AML cell lines and patient-derived CD33+ AML xenografts. O&rsquo;Hear et al engineered alternative CART- 33 cells consisting of a CD33 single-chain variable fragment linked to a CD8 leader sequence, and the cytoplasmic domains of 4-1BB and CD3&zeta;. These cells exhibited cytotoxicity against a panel of 6 human CD33+ AML cell lines as well as patient samples. Infusion of these CART-33 cells in a murine model reduced leukemia burden to <1% of control animals.

CD123, a transmembrane &alpha; subunit of the IL-3 receptor, represents an attractive immunotherapeutic target for AML given its preferential overexpression in leukemic stem cells relative to normal CD34+ cells.

Gill et al generated CD123-directed CAR T cells (CART-123) using a construct comprised of cloned light-to-heavy or heavy-to-light orientations of the mouse anti-human CD123 scFv (clone 32716 or clone 26292) together with 41BB and CD3-zeta.

These CART-123 cells demonstrated significant cytotoxicity against AML cells and primary patient samples, and they induced IFN-gamma, macrophage inflammatory protein (MIP) 1&alpha;, MIP1&beta;, IL-2, and granulocyte macrophage colony-stimulating factor (G-CSF). In vivo, this construct led to decreased disease burden in CD123+ AML cell lines and primary patient xenografts. Of note, animals rechallenged with MOLM14 AML cells following previous CART-123 therapy exhibited rapid proliferation of CART-123 cells consistent with establishment of T-cell memory. Mardios et al generated a similar CART-123 construct comprised of human igG4 Fc region, CD28, and CD3-zeta signaling domains.

These CART-123 cells produced high levels of tumor necrosis factor (TNF)-alpha and IFN-gamma, and they demonstrated both cytotoxic activity and proliferation in the presence of CD123+ AML cell lines and patient samples but not in the presence of CD123cells. Decreased disease burden and improved OS were observed in xenograft models. Cytokine-induced killer (CIK) cells possessing spontaneous antitumor activity have also been transduced to express an anti-CD123 CAR via an SFG retroviral vector, also known as a Moloney murine leukemia virus-based plasmid, utilized to transfect the cells of interest.

These cells have the advantage of displaying nonhuman leukocyte antigen-restricted cytotoxicity and minimal alloreactivity. Anti-CD123 CIK CART-cells exhibited high (60%) in vitro cell kill against CD123+ THP-1 cells and primary AML blasts. An alternative CART engineered with ScFv-CD123 generated from the mAb 7G3 and CD28/OX40-zeta constructs also demonstrated decreased tumor burden in primary AML patient xenografts.

Magnani and colleagues showed the feasibility and efficacy of a nonviral methodology (the Sleeping Beauty transposon system) to produce CAR CIKs.13 CD123 CIK cells generated with this approach, with CD28/OX40/TCR&zeta; costimulatory domains, demonstrated competent in vitro T-cell responses and decreased in vivo disease burden in mice following infusion of 1 x 107 CAR CIK cells every 10 days.

Folate Receptor &beta; Targeting FR&beta; is a cell membranebound folate receptor with homology with folate receptor alpha, a well established target for CAR T cells in epithelial tumors. FR-beta is expressed in 70% of primary AML cases, with relatively limited expression in normal tissue. Using m909 scFv, FR-beta-specific CAR constructs were generated, which were shown to have significant

IFN-gamma release response to FR-beta+ AML targets compared with CD19-directed CAR T-cell controls.

The m909 CAR T cells exhibited proliferation and lysis when co-cultured with AML cells with high to moderate FR-beta expression. Further, m909 CAR T cells demonstrated in vivo antitumor activity.

Human C-Type Lectin-Like Molecule-1 Targeting

Human C-type lectin-like molecule-1 (CLL1 or CLEC12A) is another cell surface marker overexpressed in AML blasts and leukemic stem cells. Because AML blasts express CLL1 heterogeneously, CAR T cells engineered with this antigen show only modest effector

functions against primary AML cells. However, because CLL1 is preferentially overexpressed in AML cells in the marrow of patients who fail to achieve complete remission after induction chemotherapy, these CAR T-CLL1 cells may represent an effective means of eradicating minimal residual disease (MRD). Mice engrafted with primary human AML who received cytarabine followed by a single dose of CAR T-CLL1 lived significantly longer relative to untransduced mice receiving only chemotherapy (100% survival at 200 days compared with 20% in controls).

FMS-like tyrosine kinase-3 (FLT3) is expressed on the surface of normal hematopoietic progenitors as well as on most AML blasts, independent of FLT3 mutation status, leading to active investigation of FLT3 as a target for CART-directed AML therapy. T cells

have been transduced with a FLT3 CAR construct consisting of an scFv derived from a well-characterized antihuman FLT3 antibody paired with 41BB and CD3&zeta; costimulatory domains. These CART-FLT3 cells demonstrated in vitro proliferation when exposed to FLT3+ AML cell lines, and they inhibited leukemia progression in nonobese diabetic scid gamma mice engrafted with the same cells. Of note, comparison of CART-FLT3 with CART-33 cells revealed that CARTFLT3transduced cells appear to have equivalent or potentially less toxicity to normal hematopoietic progenitors than do their CD33 counterparts, suggesting a potential advantage to this targeted approach.

B7H6, a member of the B7 family, is readily detectable on AML blasts but not on healthy tissue, and it is recognized by NKp30, an activating receptor on NK cells. CAR T cells expressing NKp30 elicited B7H6-dependent potent cytolytic activity and IFN-gamma release when cocultured with various leukemia cell lines and primary blasts, but not with B7H6 myeloma lines. AML engrafted mice infused with NKp30+ CAR T cells exhibited appreciable reduction in tumor burden. Treatment was particular effective in vivo in eradicating minimal residual AML disease (defined as 5% blasts).

Lewis Y (LeY) antigen is also overexpressed on AML cells, with limited expression on normal tissues. Preclinical studies have demonstrated that LeYspecific CAR T cells produced varying amounts of IFN-gamma on exposure to AML cell lines and primary AML cells. In addition, co-culture of the Lewis Y CAR T-cell construct with these cell lines resulted in cytolytic activity.

Summarized below and in Table 2 are the available limited published clinical data on CAR T-cell therapy for AML.

In the only published case report, Wang and colleagues reported the efficacy and tolerability of autologous CART-33 for a single patient with refractory AML.

A total of 1.12 x 109 CART-33 cells were administered in escalating doses over 4 consecutive days

without conditioning therapy. Following infusion, the patient experienced a marked decrease in blast ratio from >50% pre-treatment to <6% at 2 weeks following administration. Despite persistently high levels of CAR T cells as induction of multiple immunomodulatory

cytokines (IL-6, IL-8, TNF-alpha, and IFN-gamma) in the peripheral blood, the blast percentage gradually increased to nearly 70% at 9 weeks post infusion, suggesting tumor escape mechanism. The patient ultimately succumbed to his disease at 13 weeks post CAR T-cell infusion. Toxicities included grade 4 chills, fevers with T-max 42&deg;C treated with etanercept (antiTNF-alpha) and transient hyperbilirubinemia. Two ongoing clinical trials in China are currently underway involving CART-33 cells for patients with AML.

NCT01864902 is a continuation of the phase I study by Wang and colleagues; its goal is to evaluate the safety and in vivo survival of autologous or donor-derived CART-33 cells in patients with relapsed/refractgory (r/r) AML. The second trial (NCT02799680) is a phase

I study evaluating the safety and efficacy of allogeneic CART-33 cells in patients with r/r AML.

Multiple trials for CART-123 T cells in AML are underway. A phase I trial for UCAR123 is currently enrolling patients with r/r AML (NCT03190278).

Another phase I dose escalation study is investigating the safety, tolerability, and optimal dosing of anti CD123/CD28 CAR T cells that also express truncated EGFR. Patients enrolled in this study will undergo lymphodepletion with cyclophosphamide, fludarabine, or etoposide, followed by T-cell infusion (NCT02159495). Lastly, a phase I trial evaluating donor-derived CART-123 cells in relapsed AML following allogeneic transplant is currently recruiting participants (NCT03114670).

NKG2D ligands are upregulated on the cell surface of malignant hematopoietic cells, and thus they may serve as another CAR T cell targeting candidate. Nikiforow and colleagues recently conducted a feasibility and safety trial of CAR T cells targeting NKG2D in 6 patients with AML/myelodysplastic syndromes. Single doses of CAR T cells were well tolerated with no dose-limiting toxicities or cytokine release. Following single dosing, no patients had appreciable tumor response 28 days after infusion. Future studies investigating the effects of multiple CAR T-cell infusions are currently underway.

Anti-LeY CAR T cells were evaluated in a phase I trial of relapsed AML at Peter MacCallum Cancer Centre in Australia (NCT01716364). In this study, patients received fludarabine and cytarabine followed by CAR T-cell infusion. Out of 4 evaluable patients, 1 achieved a transient cytogenetic remission (5 months), 1 experienced a transient reduction in peripheral blood blasts, and 1 had stable disease for 23 months. No grade 3 or 4 toxicities or cytokine release syndrome were observed. Although all patients eventually relapsed, anti-LeY CAR T cells persisted for up to 10 months.

Despite the dramatic effects of CAR T-cell therapy on preclinical models, numerous clinical challenges remain as barriers to the successful implementation of this novel immunotherapeutic approach for treatment of myeloid malignancies in patients. These include: a) heterogeneous antigen expression on diverse AML cell populations; b) potential for off-target toxicities to normal myeloid progenitor and hematopoietic stem cells in patients; and c) potential for life-threatening complications of potent T-cell activation in vivo, specifically cytokine release syndrome and fatal neurological events. To date, much emphasis has been placed on finding optimal antigenic targets, minimizing off-tumor effects including cytokine release syndrome, and abrogation of unchecked cytotoxic T-cell (CTL) activity

resulting in myeloablative hematological toxicity.

Given the heterogeneous expression of CD33, CD123, and FR&beta; across AML cell profiles, there is vested interest in broadening the targeting milieu of CAR T-cell therapies. Ongoing studies by Perna and colleagues are aimed at identifying novel CAR antigenic targets for AML therapy.

Probing of the AML surfaceome for candidate molecules, followed by flow cytometric validation analyses, recently resulted in 4 novel antigen candidates, including 2 G-protein coupled receptors not previously reported. In pairing candidate antigens, validation studies have shown 3 distinct antigen combinations that are ~100% expressed on AML cells but are absent on normal cells.

Multiple safety mechanisms are also currently being incorporated into CAR T-cell technology to address concerns regarding potential off-target toxicity and the CTL activity on normal myeloid and hematopoietic progenitors. For instance, CART-33 therapy reduces CD34+ CD38 hematopoietic stem cells as well as CD34+ CD38&ndash; myeloid progenitors and peripheral myeloid cells in mice engrafted with human fetal CD34+ cells.

In addition, of concern, significant ablation of normal hematopoiesis has also been observed following CART-123 infusions.

Suicide gene systems as a means of mitigating damage to normal cells during adoptive cell therapies

have previously been shown to be efficacious in controlling graft versus host disease (GVHD) in allogeneic hematopoietic stem cell transplant recipients. This technology is currently being adapted to engineer CAR T cells.

CART-33 cells with available activating elimination gene components to truncate T-cell activity have also been designed.

Genetically engineered systems using inducible caspase-9mediated apoptosis pathways have also been explored to abrogate CAR T-cell toxicity against normal hematopoietic progenitors.

The GoCAR-T platform, combining a CD123-specific CAR construct with a costimulatory switch, has recently developed.

To improve efficacy in targeting heterogeneous myeloid malignancies, dual targeting of CD123 and CD33 utilizing the universal CAR (UniCAR) model has recently emerged as an innovative approach.

This modality provides added flexibility to antigen selection strategies by allowing for the introduction of multiple targeting modules (TMs) either simultaneously or in sequence during treatment. During in vitro evaluation utilizing this model, dual-specific anti-CD123-CD33 TMs showed enhanced anti-leukemic effects, as evidenced by the low concentrations of bispecific TMs needed to elicit target-cell lysis. In subsequent studies, UniCAR cells interacting with dual-specific TMs effectively eradicated >80% blast cells in primary AML samples. T-cell specific cytokines (IFN-gamma, G-CSF, IL-13) were also detected following treatment, indicating viable UniCAR activation in response to bispecific TMs. By targeting multiple antigenic variants either simultaneously or sequentially, it is also feasible to reduce the emergence of antigen-loss tumor escape mechanisms. The same investigators recently demonstrated that UniCAR cells exhibit superior antitumor reactivity to conventional CAR T cells at low effector-to-target ratios.

Infusion of cells engineered to express CD123-specific target modules resulted in a) inhibition of AML engraftment in murine hosts, and b) long-term persistence. UniCAR cells obtained from said mice retained cytotoxic potential against AML cell lines for at least 4 months after transplantation, supporting the potential for long-term immunoprotection.

The use of UniCAR/UniTARG (UniTARG is an antibody-based platform with universal effector arm with individual targeting molecules), a flexible, modular CAR platform, also represents an alternative means of modulating off-tumor, on-target effects with CART therapy.

Conventional, second-generation CAR signaling is contingent on tumor surface antigen recognition by scFv expressed on retargeted T cells. Within the UniCAR paradigm, there is a separation of the antigen-binding and antigen-signaling aspects of the system. Engineered cells express a UniCAR that has specificity for a short peptide motif derived from human nuclear protein rather than a specific tumor antigen. Such cells remain physiologically inert after reinfusion until specific TMs carrying both tumor specific antigen and the nuclear peptide motif are introduced. Guzman and colleagues have taken this approach 1 step further by engineering CD123-directed T cells that no longer express T-cell receptor (TCR); this was done as a means to avoid the potentially life-threatening clinical effects of potent T-cell activation (ie, cytokine release syndrome, fatal cerebral edema) that have been reported in CART-19 T-cell therapy in patients with ALL.

These specifically modified UCAR123 cells have minimal to no risk of inducing GVHD in recipients, but they retain anti-leukemic activity as demonstrated by effective in vitro elimination of AML cells and complete AML eradication in the marrow of patient-derived xenografts. Mice treated with these modified UCAR123 cells also showed significant increases in OS as compared with control mice.

Furthermore, there has been interest in developing multiple targeted CAR T cells against both tumor and nontumor surface antigens. Given the high mortality and morbidity associated with viral infections following intensive chemotherapy and allogeneic stem cell transplant for acute leukemias, Zhou et al recently engineered CD123-directed CAR T cells that also recognize virus-specific epitopes of Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus.

Both EBV-specific and multi-virusspecific CAR T cells demonstrate virus-specific cytotoxicity against EBV and mediate AML cell lysis. This approach may ultimately yield a 1-stop multidimensional treatment option for AML and unresolved common viral infections in this patient population.

Recent studies have investigated the possibility of combining CAR T cells with checkpoint inhibitors to further enhance antileukemic activity. Emerging data in many disease types have shown that exposure of malignant cells to directed CAR T cells induces expression of the programmed death (PD) ligands 1 and 2 on tumor cells. Subsequent upregulation of the receptor for PD-1 on CAR T cells constitutes an exhausted T-cell phenotype. Kenderian et al demonstrated that in vitro co-culture of human AML cells (primary AML patient samples and the AML cell line MOLM14) with AML-directed CAR T cells enhanced expression of multiple coregulatory molecules (PD-1 and TIM-3) on the T cells.

Exposure of CAR T cells harvested from AML-bearing murine hosts to PD-1 or TIM-3 blockade effectively prevented immune exhaustion and resulted in increased CAR T-cell proliferation and cytokine production. In vivo administration of PD-1 or TIM-3 blockade with CAR T-cell therapy translated into improved disease control and OS in leukemia mouse models. This combination warrants further clinical investigation in future clinical trials.

Lastly, another promising cellular immunotherapeutic approach for AML is NK cell therapy. Patients with AML exhibit impaired NK cell function. Allogeneic NK cell infusions in nontransplanted and transplanted patients with AML have been shown to mediate anti-leukemic effects without inducing graftversus-host activity. Sources of NK cell infusions include alloreactive or haploidentical cells, as well as NK cells derived from normal hematopoietic progenitor cells in umbilical cord blood or bone marrow, and G-CSF-mobilized peripheral stem cells. The ability to generate high numbers of NK cells ex vivo from CD34+ hematopoietic precursors may allow this technology to serve as a universal &ldquo;off the shelf&rdquo; cellular therapy for most patients with AML. To date, adoptive NK cell therapy has shown promising activity preclinically and in early-phase clinical trials in limited numbers of patients. NK cell therapy may be particularly promising for the treatment of patients with chemoresistant disease as a bridge to transplant or for treating patients in the MRD state. Ongoing clinical investigations are addressing optimal cell dosage, toxicities, duration of NK cell persistence, and how best to maintain the in vivo activation status of infused NK cells, ie through concomitant cytokine support (IL-2, IL-15).

Emerging rigorous preclinical data have paved the way for the further clinical development of CAR T-cell therapy for AML. However, many clinical challenges remain. These include appropriate target selection, lack of potent anti-leukemic effects, and risks of myeloablative and cytolytic T-cell toxicities. At present, both a) the identification of new targetable antigens, and b) the development of a flexible modular CAR platform capable of dual or sequential targeting of antigens, hold promise to address these issues. The results of ongoing clinical trials testing the safety of numerous CAR T-cell constructs in patients with AML are eagerly awaited. Once completed, further studies of CAR T cells for treatment of overt AML disease, as well as for MRD following chemotherapy and/or transplantation, should be explored.

Articles

Although preclinical data are promising, there are many clinical challenges in developing CAR T-cell therapy in acute myeloid leukemia.

Chimeric Antigen Receptor T-Cell Therapy in Acute Myeloid Leukemia

Department of Pharmaceutical Services, Adjunct Professor of University,&nbsp;Buffalo School of Pharmacy and Pharmaceutical Sciences,&nbsp;Erie County Medical Center,&nbsp;Buffalo, NY

Associate Professor of Medicine, Department of Medicine,&nbsp;Assistant Professor of Immunology,&nbsp;Department of Immunology,&nbsp;Roswell Park Cancer Institute

Bruton&rsquo;s tyrosine kinase (BTK) is a crucial component of the B-cell receptor-signaling pathway that has been associated with the pathogenesis of B-cell malignancies. Ibrutinib is an oral BTK inhibitor that showed promising antitumor activity in preclinical lymphoma models. Subsequently, ibrutinib was tested in prospective clinical studies and was found to be active in various B-cell malignancies, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenstr&ouml;m&rsquo;s macroglobulinemia (WM).

In addition, ibrutinib appears to be well tolerated in patients with lymphoma/leukemia. Ongoing clinical studies are incorporating this novel agent in the management of other subtypes of B-cell lymphoid malignancies and testing the drug&rsquo;s antitumor activity as a single agent or in combination with other agents in patients with CLL/MCL or WM.

Non-Hodgkin&rsquo;s lymphoma (NHL) is a diverse group of malignancies that exhibit distinct biology, clinical behavior, and outcomes. It is the sixth-most diagnosed cancer in the United States. NHL accounts for 4% to 5% of the cancer cases and 3% to 4% of all cancer-related deaths. B-cell lymphoma constitutes a large majority of NHL seen in the United States and includes subtypes such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and Waldenstr&ouml;m&rsquo;s macroglobulinemia (WM). These subtypes of NHL have been treated with traditional intravenous systemic chemotherapy with or without monoclonal antibodies (mAbs) targeting CD20 with varying degrees of efficacy. The early use of rituximab as a single agent or in combination with various systemic chemotherapy regimens (ie, CVP [cyclophosphamide, vincristine, prednisone], CHOP [cyclophosphamide, doxorubicin, vincristine, and prednisone], fludarabine) has resulted in improved response rates, duration of remission, and improved overall survival (OS) in patients with follicular lymphoma (FL) and with DLBCL.

Two recently published retrospective analyses suggest that the use of mAbs targeting CD20 (rituximab) in combination with systemic polychemotherapy improves the OS in patients with FL when compared with chemotherapy alone.

On the other hand, a significant number of patients with certain subtypes of B-cell lymphoma relapsed after initial response or failed to achieve a durable remission, indicating the need for drug discovery. Moreover, elderly patients or those with significant comorbid conditions may require alternative effective and less toxic regimens (ie, nonchemotherapy regimens).

Ibrutinib (PCI-32765) is a novel, oral, targeted agent that inhibits Bruton&rsquo;s tyrosine kinase (BTK), suppressing the B-cell receptor (BCR) signaling pathway with proven activity in certain subtypes of NHL. It is emerging as a therapeutic option for patients with a variety of B-cell lymphoma because of its clinical activity, tolerability, and easy administration. The US Food and Drug Administration (FDA) has approved ibrutinib for the treatment of patients with relapsed/refractory CLL or MCL and for patients with previously untreated CLL (p53 deletion) and WM.

Ibrutinib forms a covalent bond with cysteine 481 on the BTK enzyme and inhibits BTK activity at Tyr-223.

Ibrutinib&rsquo;s empiric formula is C25H24N6O2 and its molecular weight is 440.50.

Bruton&rsquo;s tyrosine kinase is a Tec family kinase that is involved in the BCR signaling pathway.

The BCR pathway and BTK are implicated in NHL pathogenesis as a main driver for proliferation, differentiation, and survival. In normal physiologic conditions, BTK is activated upstream by Src family kinases Blk, Lyn, and Fyn.

BTK when activated will then phosphorylate phospholipase-C&gamma; leading to calcium mobilization and activation of nuclear factor-&kappa;B (NF-&kappa;B) and mitogen-activated protein (MAP) kinase pathways.

BTK is overexpressed in a variety of lymphoid malignancies, including CLL, MCL, DLBCL, WM, and multiple myeloma (MM).

Inhibition of BTK therefore shuts down further activation of the BCR signaling pathway and can technically arrest the proliferation of lymphoid malignancies.

Ibrutinib is rapidly absorbed, with peak plasma concentrations occurring after 1 to 2 hours.

A variety of ibrutinib doses had been studied in multiple phase I clinical trials in doses ranging from 280 mg to 840 mg.

In a phase I study seeking to determine the maximum tolerated dose of ibrutinib in combination with standard doses of rituximab combined with CHOP (R-CHOP), the steady state was achieved after 1 cycle of therapy.

Doses in the ibrutinib + R-CHOP study ranged from 280 mg to 560 mg daily. The amount of time that a drug is present at the maximum concentration in serum (Tmax) values were not dose dependent, and the maximum plasma concentration of the drug (Cmax) for the 420-mg and 560-mg dose levels were 190 ng/mL and 187 ng/mL, respectively, on day 1 of cycle 2. The area under the curve (AUC) increased the dose proportionally.

AUC in the first 24 hours of therapy was 502 ng.h/mL from a single dose of 560 mg.23 AUC increased across the dose range in cycle 2 with a maximum of AUC24 with 560-mg dose at 1014 ng.h/mL and AUC 882 ng.h/mL. Plasma concentrations decreased biphasically with an initial mean half-life of 2 to 3 hours.

Mean apparent terminal half-life ranged from 4 to 8 hours. Most (>80%) of ibrutinib after single-dose administration was excreted in the feces within 48 hours and 7.8% was excreted in the urine.

Total excretion (>90%) occurred within 168 hours.

Ibrutinib binds reversibly to plasma proteins at 97% in vitro and has a volume of distribution at steady state (Vdss) of 683 L.16 Ibrutinib is metabolized primarily by CYP3A4 and, to a minor extent, by CYP2D6.

There was no difference between poor and extensive CYP2D6 metabolizers and unchanged ibrutinib exposure.

Ibrutinib&rsquo;s half maximal inhibitory concentration is 0.5 nmol/L.

Ibrutinib when combined with R-CHOP showed that BTK was >90% occupied at the 560-mg dose level, 4 hours after receiving the first dose.

These data follow another pharmacodynamics study, which showed that at 2.5 mg/kg, >95% of BTK occupancy occurred 4 hours post dose.

Nearly complete occupancy was observed in patients with AUCs exceeding 160 ng.h/mL. No decrease was observed in immunoglobulin levels over the course of 12 cycles. There was an observed decrease in anti-immunoglobulin E (IgE)-stimulated basophil degranulation.

Ibrutinib&rsquo;s activity as a single agent was evaluated in patients with relapsed or refractory CLL or with small lymphocytic lymphoma (SLL) through a phase Ib/II clinical trial.

In this study, 85 patients were enrolled, 65% of whom had advanced disease. Two-thirds of the patients were also considered to have high-risk disease (ie, with 17p13.1 or 11q22.3 deletions). The median number of prior treatments was 4 (range 1-12). The patients were each assigned to 1 of 3 cohorts: the first 2 groups (n = 51) received ibrutinib at 420 mg daily, and the third cohort of patients received ibrutinib at 840 mg daily (n = 34). Treatment was continued until disease progression or unacceptable toxicities.

The overall response rate (ORR) was 71% for patients treated with ibrutinib at 420 mg (2 complete responses [CRs] and 34 partial responses [PR]).

Similar activity was found in patients treated at the higher dose level (71%, 24 PR). The clinical activity observed appeared to be durable. The 26-month estimated progression-free survival (PFS) was 75% and the OS rate was 83%.

This study demonstrated that ibrutinib was active irrespective of high-risk prognostic features.

For example, patients possessing a 17p13.1 deletion exhibited a 68% response rate, including 1 CR, whereas patients without the deletion showed a response rate of 71%. However, mutations in the immunoglobulin variable-region heavy-chain gene (

) appeared to affect responses. Four of the 12 patients (33%) with this mutation had a PR or CR, while 53 of 69 patients (77%) without this mutation were found to have a PR or CR that was statistically significant (

= .005). In addition, ibrutinib was found to result in sustained improvement in cytopenias, which was defined as an increase of more than 50% or a hemoglobin concentration of greater than 11 g/dL, an absolute neutrophil count higher than 1500 cells/mm,

or a platelet count greater than 100,000 cells/mm.

Of note, a transient lymphocytosis was observed in these patients, which correlated with a decrease in size of lymph nodes, spleen, or both. The lymphocytosis did not elicit symptoms from patients, and resolved with continued treatment.

An additional phase II clinical trial evaluated ibrutinib in previously untreated and relapsed or refractory CLL with TP53 mutations.

The study enrolled patients over 18 years of age with CLL who had deletion 17p13.1 or a

mutation. Ibrutinib was administered at a dose of 420 mg orally once daily until disease progression or unacceptable toxicity.

The study enrolled 51 patients with CLL who had the

deletion or mutation, 35 with previously untreated CLL, and 16 with relapsed or refractory CLL.

Most patients enrolled had unmutated IgVH and/or advanced Rai-stage disease. In addition, 47 patients were identified as having deletion 17p13.1, and 4 had a

mutation. At the time of follow-up (24 weeks), 48 of the patients (15 with relapsed/refractory and 33 with untreated CLL) were evaluable for response. An objective response was noted in 44 patients, with 24 patients having a PR and 20 patients with a PR and lymphocytosis. Eighteen of the 33 previously untreated patients had a PR. Of the 15 patients with relapsed or refractory CLL, 12 demonstrated an objective response, 6 had a PR, and 6 had a PR and lymphocytosis. The authors also noted that the depth and rate of responses increased with time. For example, PR was recorded in 6 patients at 8 weeks and in 24 patients at 24 weeks.

In addition, all tissue compartments demonstrated rapid disease control with at least a 50% mean reduction of tumor burden in both the lymph nodes and spleen at 8 weeks.

Disease improvement was noted in the bone marrow when compared with the lymph nodes and spleen. For example, at 8 weeks, a 50% decrease in tumor burden of the bone marrow, lymph node, and spleen was documented in 16 of 36 patients, 31 of 44 patients, and 30 of 38 patients, respectively. At the completion of treatment, a 50% reduction in disease was found in the bone marrow, lymph node, and spleen in 34 of 41 patients, 42 of 45 patients, and 38 of 40 patients, respectively. At 24 months, the approximated PFS for all patients was 82%, and OS was 80% at 24 months. For patients who were previously untreated, the OS at 24 months was estimated at 84% versus 74% for patients who were previously treated.

The combination of rituximab with ibrutinib was evaluated in patients with high-risk CLL through a single-arm phase II study.

Eligible patients included those with relapsed/refractory high-risk CLL or those with previously untreated CLL with deletion 17p13.1,

mutation, or 11q deletion. High-risk disease was defined as having a remission less than 36 months after chemotherapy treatment. Ibrutinib was administered as 420 mg daily continuously (each cycle comprised of 28 days). Rituximab was dosed at 375 mg/m

. During the first cycle, rituximab was administered weekly.

For cycles 2 through 6, rituximab was administered once every 28 days. After completing 6 cycles, rituximab was discontinued and patients were maintained on single-agent ibrutinib until disease progression or unacceptable toxicities.

The study enrolled 40 patients, 36 of whom had previously treated high-risk CLL and 4 who had deletion 17p13.1 or a

mutation and had not received previous therapy.

At the time of follow-up, 39 patients were evaluable for response assessment. A PR was found in 34 (87%) of patients, and CR was found in 3 patients. Two of the patients achieving CR had deletion 17p13.1 or

mutation and were previously untreated. The time to achieve response was 5.72 months, and the median duration of response was 15.44 months. In addition, the 18-month PFS for all patients was 78%, and was 72.4% in patients with deletion 17p13.1 or

mutation. Furthermore, a sustained improvement in cytopenias was noted in 15 of 24 patients with baseline thrombocytopenia and 15 of 17 patients with baseline anemia.

Ibrutinib was compared head to head with ofatumumab in patients with CLL/SLL who had relapsed or refractory disease after at least 1 therapy.

The study enrolled 391 patients at 67 sites across the United States and Europe. Patients were stratified based on resistance to purine analogue or whether they had chromosome deletion 17p13.1. Ibrutinib was given at 420 mg daily until progression or toxicity; ofatumumab was given intravenously at 300 mg on week 1, then 2000 mg weekly for 7 weeks and then every 4 weeks for 16 weeks. As a result of other phase II data, those who progressed on ofatumumab were allowed to cross over to receive ibrutinib.

The primary endpoint of this trial was PFS assessed by an independent review board. Treatment-related leukocytosis was not considered to be progressive disease.

In the ibrutinib group, patients had received a median of 3 prior therapies, while the ofatumumab group had received 2 prior therapies. Otherwise, baseline demographic characteristics were balanced between treatment groups. Ibrutinib therapy resulted in a longer PFS (not reached at a median follow-up of 9.4 months). In contrast, the median PFS in the ofatumumab group was 8.1 months; 57 patients in the ofatumumab group crossed over and received ibrutinib. Similar results were observed in the analysis of patients with deletion 17p13.1 treated with ibrutinib versus ofatumumab. The median PFS was not reached in the ibrutinib group and was only 5.8 months in patients treated with ofatumumab. Ibrutinib also significantly improved OS.

After 1 year, 90% of patients receiving ibrutinib were still alive compared with 81% of patients receiving ofatumumab. When data were assessed on various subgroups according to deletion 17p13.1, previous purine analogue therapy, Rai stage at baseline, and bulky disease, ibrutinib significantly improved OS in all group categories compared with ofatumumab.

A phase II study assessed the efficacy of ibrutinib in relapsed or refractory MCL.

Patients were eligible for the study after a confirmed diagnosis of MCL with cyclin D1 overexpression or translocation breakpoints t(11;14) and measurable disease (lymph nodes &ge;2 cm). Exposure to previous therapy was required, but patients were ineligible if they had received more than 5 therapies. Ibrutinib was administered orally at a dose of 560 mg daily.

A total of 111 patients received ibrutinib single agent, with an approximated median follow-up of 15.3 months.

A PR was observed in 47% of patients, and a CR in 21% of patients, with an ORR of 68%. These response rates were also found to improve over time. In patients with lymph nodes &ge;5 cm in diameter, 63% had a response to treatment (27 of 43 patients).

The median duration of response was estimated to be 17.5 months, and the median time to response was found to be 1.9 months.

In addition, the median time to a CR was determined to be 5.5 months. Among all of the patients receiving ibrutinib, the estimated PFS was found to be 13.9 months. For those patients achieving a PR, the median PFS was found to be 17.5 months. Median PFS was not yet reached for patients attaining a CR.

Ibrutinib monotherapy was evaluated in patients with WM. Results of a phase II study resulted in FDA approval of ibrutinib in patients with this rare lymphoid disorder. The study evaluated the safety and efficacy of ibrutinib in patients with relapsed/refractory WM who had previously received 1 line of therapy.

Patients received ibrutinib 420 mg orally once daily for up to 2 years until progression or unacceptable toxicity. Sanger sequencing was performed to detect baseline MYD88 L265P and Whim-like

mutations in patients. Both mutations are commonly found in patients with WM and had been implicated in its pathogenesis. These mutations had been associated with an increase (

) in sensitivity to ibrutinib. The study enrolled 63 patients, of whom 17 had refractory disease. The median age was 63 years, hemoglobin was 10.5 g/dL, serum immunoglobulin M (IgM) was 3610 mg/dL, and serum M-protein was 2.14 g/dL. The median number of cycles was 6 (2-15 cycles) and the ORR to ibrutinib was 81%, including 4 very good PR (VGPR), 32 PR, and 15 patients with minimal response (no complete response was seen). When the clinical data were reported, 11 patients had stable disease and continued to receive ibrutinib. Responses correlated with

mutations. Patients with wild-type CXCR4 had a response rate of 77% versus a mutated rate of 30%. Surrogate markers, such as decreases in serum IgM, M-protein and increases in hemoglobin, improved in wild-type CXCR4. These patients also had peripheral lymphocytosis.

Subsequent studies evaluated the safety and efficacy of incorporating ibrutinib into standard chemo-immunotherapy regimens in B-cell malignancies. A phase I/Ib trial evaluated the addition of ibrutinib to rituximab and bendamustine in patients with relapsed or refractory NHL.

Patients were assessed for maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT). A total of 46 patients were evaluated, including 17 patients with MCL, 16 with DLBCL, and 12 with FL. Patients were at least 18 years of age and had received at least 1 prior therapy, with the exception of patients with MCL, who were allowed to be treatment na&iuml;ve (n = 5). Prior therapy with rituximab and bendamustine was not an exclusion criterion. Of the 43 patients who had previous therapy, the median number of therapies was 3 and 38% were refractory to previous therapy.

Patients were treated at 2 dosing levels. Dose level 1 consisted of ibrutinib 280 mg daily on days 1 through 28.14 Dose level 2 used ibrutinib at 560 mg daily on days 1 through 28. Six patients were treated at dose levels 1 and 2, with MTD defined as the highest dose level where less than or equal to 1 of 6 patients experienced DLT. After MTD was determined, 3 cohorts of 10 patients with either FL, DLBCL, or MCL were treated with ibrutinib at the MTD or at the recommended phase II dose, if MTD was not reached. In both dose levels, rituximab was given on day 1 at 375 mg/m

on days 1 and 2. Growth factor use and dose reductions were allowed for grade 3 or 4 neutropenia for cycles 2 through 6.

A total of 33 patients responded to therapy and 52% achieved a CR.

Thirteen patients with MCL, 5 with DLBCL, and 5 with FL had a CR. The median time to response was 2.8 months and median time to CR was 2.9 months. The median duration of response was not reached at time of publication (2.8-28 months). Of the patients who did respond, 6 patients have progressed and 2 have died. Median PFS and OS were not reached. Patients completed a median of 8 cycles of therapy (1-34 cycles), with 35% receiving therapy at cycles 7-34. Ten patients received pegfilgrastim, with 4 patients requiring bendamustine dose reduction and 18 patients requiring ibrutinib dose reductions.

Ibrutinib was evaluated in combination with standard R-CHOP21 in patients with previously untreated CD20-positive B-cell lymphoma in an open-label, phase I/IIb study.

Eligible patients had DLBCL, MCL, or FL. Rituximab and CHOP chemotherapy were administered at standard doses on day 1 of a 21-day cycle for 6 to 8 cycles. Ibrutinib was dosed daily beginning on day 3 of the first cycle. The study was divided into a dose-escalation phase (part 1) and an efficacy-safety phase (part 2, only for DLBCL patients). Seventeen patients were enrolled in part 1 of the study, 7 at the 280 mg-dose level, 4 at the 420 mg dose level, and 6 at the 560 mg dose level.

All the patients enrolled had newly diagnosed lymphoma and most had stage IV disease.

The study enrolled 33 patients in phase II of the study and their median age was 60.2 years. Most patients (73%) in both phases of the trial had DLBCL. In the study, 29 patients completed 6 cycles of therapy, 4 patients prematurely discontinued (3 from adverse events: gastritis, rituximab infusion reaction, and 1 death), and 1 discontinued for noncompliance. The 2 patients who did not complete part 1 of the trial included the patient with compliance issues and the patient with rituximab infusion reaction. Of those with DLBCL, 88% of patients (n = 24) completed 6 cycles of therapy. Of all the evaluable patients, 94% achieved a response to therapy, with 72% having a CR. Activity was observed in both subtypes to DLBCL: germinal center B-cell (GCB) or non-GCB DLBCL.

Ibrutinib is FDA approved for the treatment of relapsed/refractory MCL, relapsed/refractory CLL/ SLL, or CLL/SLL with 17p deletion, and for WM.

Ibrutinib is dispensed in 140-mg capsules, requiring patients to take multiple capsules per dose. The approved dosing for CLL and WM is 420 mg by mouth daily and for MCL is 560 mg daily. Patients should take ibrutinib with a glass of water and should not crush, break, or open the capsules. Ibrutinib&rsquo;s Cmax was 2 times higher and AUC was 1.65 times higher after being administered with food, likely because of first-pass metabolism.

Investigators believe, however, that the effects of food on absorption do not require any consideration to time of administration. If a dose is missed, the dose can be taken the same day, if remembered.

Extra doses should not be taken to make up for a missed dose.

Ibrutinib should be interrupted for any grade 3 or greater nonhematologic or neutropenic infection, or for grade 4 hematologic toxicities.

Once symptoms resolve to at least grade 1, ibrutinib can be resumed at the previous dose. If toxicity reoccurs, the dose can be reduced by 140 mg. A further reduction may be considered, if necessary. After 2 dose reductions, ibrutinib should be discontinued. Renal clearance is not a major factor in elimination, so no renal adjustment of dose is warranted.

Ibrutinib has not been studied in renal failure requiring dialysis. Because ibrutinib is metabolized through the liver, there are dosing adjustments recommended for patients with Child-Pugh Class A hepatic impairment at baseline.

The recommended starting dose is 140 mg daily. Use is not recommended for Child-Pugh Class B and C.

Compared with chemotherapy traditionally used to treat NHL, ibrutinib is a well-tolerated alternative. Concomitant chemotherapy should be carefully considered when attributing side effect profiles to ibrutinib. Mild grade 1-2 toxicities included nausea and vomiting (15%-59%), diarrhea (23%-55%), fatigue (18%-28%), peripheral edema (28%), constipation (25%), pyrexia (22%), arthralgia (22%), and respiratory symptoms, including pneumonia (13%-33%).

Most grade 1 and 2 side effects did not require any additional treatment. In a phase II trial, 57% of patients taking ibrutinib experienced grade 3 and 4 adverse events.

Most common grade 3 and 4 side effects were hematologic, with thrombocytopenia (6%-11%) and neutropenia (16%) the most common.

The most common grade 3 and 4 nonhematologic toxicities were pneumonia (6%), fatigue (5%), dyspnea (4%), and diarrhea (2%).

Basal-cell carcinoma and squamous cell carcinomas were reported in 4% of patients taking ibrutinib, and 3% of patients developed nonskin cancers developed during the course of the clinical trials.

Other more rare side effects included atrial fibrillation (3%-9%) and ocular toxicity (3%). Ibrutinib induced atrial fibrillation in about 5% of patients in a long-term observational follow-up, occurring anywhere from 2 months to 101 months after initiation of therapy.

Incidence may be higher based on the lack of routine screening. Echocardiographic studies showed mild to severe left atrial dilatation as well as moderate left ventricular failure in some patients. The etiology of atrial fibrillation is thought to be through inhibition of the cardiac PI3K-Akt signaling pathway.

In addition, 23% of patients in an observation study were found to have isolated systolic hypertension with increases of systolic blood pressure >20 mm Hg.

Ibrutinib is a novel inhibitor of Bruton&rsquo;s tyrosine kinase (BTK) that is administered orally once a day.

Ibrutinib is well tolerated and its side effect profile is manageable compared with traditional chemotherapy.

Ibrutinib has evidence of activity in a variety of B-cell, non-Hodgkin&rsquo;s lymphoma, including MCL, CLL, WM, and subtypes of diffuse large B-cell lymphoma (DLBCL; ie, activated B-cell DLBCL).

Hematologic side effects are common with ibrutinib, including cytopenias. Other side effects include petechiae (14%), bruising, and other bleeding events. There have been several cases of subdural hematomas, with and without concomitant anticoagulation use.

Ibrutinib has been shown to cause collagen-mediated inhibition of platelet aggregation ex vivo.

and has been shown to affect the platelet adhesion to the Von Willebrand factor (VWF) at arterial sheer rate because of its inhibition of BTK.

These explanations may suggest the cause of the bleeding events beyond thrombocytopenia observed in patients treated with ibrutinib. Rates of neutropenia varied from trial to trial; however, when ibrutinib was administered alone, grade 3 or higher neutropenia was about 16%.

Serum IgG, IgA, and IgM levels remained unchanged after therapy.

Ibrutinib should be avoided with major CYP3A4 inducers or inhibitors because they significantly increase or decrease its Cmax and AUC, respectively.

Concomitant administration with ketoconazole (a strong CYP3A4 inhibitor) resulted in a 24-fold and a 29-fold increase in ibrutinib&rsquo;s AUC and Cmax, respectively.

Administration with rifampin (a strong CYA4 inducer) resulted in a 10-fold and a 13-fold decrease on ibrutinib AUC and Cmax values, respectively. There was no change in half-life with either interaction. Consider making dose adjustments in patients requiring medications with moderate to strong CYP3A4-inducing or -inhibiting properties. Despite no formal publications studying dose adjustments, it is recommended that the dose of ibrutinib be decreased to 140 mg daily when given with moderate inhibitors.

While ibrutinib is a relatively new option in the treatment of B-cell NHL, it has convincing antitumor activity as a single agent in a wide variety of B-cell lymphoma subtypes and is a viable therapeutic option for patients. It has shown to have activity in WM, CLL, MCL, and DLBCL, as well as subtypes of NHL. It has the added benefit of being administered orally once a day and having a better-tolerated side effect profile compared with other agents. On the other hand, clinicians must be cautious when prescribing this agent in frail elderly patients with comorbid conditions, because drug interactions alter ibrutinib levels and may result in excessive toxicity. Additional preclinical and clinical studies will further define its role as a single agent or in combination with rituximab, chemotherapy, or other targeted agents in the management of patients with treatment-na&iuml;ve and relapsed or refractory B-cell lymphoma.

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015.

Piro LD, White CA, Grillo-Lopez AJ, et al. Extended rituximab (anti-CD20 monoclonal antibody) therapy for relapsed or refractory low-grade or follicular non-Hodgkin&rsquo;s lymphoma.

McLaughlin P, Grillo-Lopez AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.

Leget GA, Czuczman MS. Use of rituximab, the new FDA-approved antibody.

Czuczman MS, Grillo-Lopez AJ, White CA, et al. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti- CD20 monoclonal antibody and CHOP chemotherapy.

Hiddemann W, Kneba M, Dreyling M, et al. Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advanced-stage follicular lymphoma compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group.

Marcus R, Imrie K, Belch A, et al. CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced follicular lymphoma.

Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma.

While ibrutinib is a relatively new option in the treatment of B-cell NHL, it has convincing antitumor activity as a single agent in a wide variety of B-cell lymphoma subtypes and is a viable therapeutic option for patients.

Amanda Przespolewski, DO | Authors

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