SOHO Supplement 2014

Humoral and Cellular Immunotherapy in ALL in Children, Adolescents, and Young Adults Jessica Hochberg,1 Nader Kim El-Mallawany,1 Mitchell S. Cairo1,2,3,4,5 Abstract Although the event-free survival for children and adolescents with acute lymphoblastic leukemia (ALL) has dramatically improved over the past half century, it has plateaued over the past decade. Children and adolescents with refractory/ relapsed ALL continue to have a dismal prognosis with hematopoietic stem cell transplant being their most viable option for cure. There is an obvious need for the development of novel agents to further enhance overall outcomes. In this review we focus on the development of humoral and cellular immunotherapeutic agents in the treatment of childhood, adolescent, and young adult ALL. Immunotherapy in various forms has shown immense promise. To date we have seen numerous safety studies using monoclonal antibody therapy, antibody conjugates, bispecific T cell and bispecific natural killer (NK) cell antibodies and genetically reengineered T and NK cells expressing targeted chimeric antigen receptors. Initial success has been found with the anti-CD20 monoclonal antibodies followed by promising results using anti-CD22 and anti-CD19 therapies alone or in combination. Genetic modification of T and NK cells to express targeted chimeric antigen receptors offers a novel immunotherapy option that demonstrates enhanced cytotoxicity in otherwise resistant tumor cells. There is great potential to combine immunotherapies to further improve overall cure rates in children, adolescents, and young adults with poor-risk ALL. A number of humoral and cellular immunotherapy strategies have been investigated and found to be effective, safe, and well tolerated. Ideally, the targeted approach of immunotherapy will result in an overall decrease in toxicities experienced by patients. Future studies are required to determine when in the course of treatment with humoral and cellular therapy will have the safest and optimal effect in ALL. Clinical Lymphoma, Myeloma & Leukemia, Vol. 14, No. S3, S6-13 ª 2014 Elsevier Inc. All rights reserved. Keywords: Antibody, CAR, Leukemia, Lymphoblastic, Monoclonal

Introduction Although the long-term event-free survival (EFS) for children and adolescents with acute lymphoblastic leukemia (ALL) has dramatically improved over the past half century, it has plateaued at approximately 85% over the past decade. Children and adolescents with poor-risk refractory or relapsed disease have a dismal prognosis. Their only curative option includes allogeneic hematopoietic stem cell transplant (alloHSCT). As the technology for molecular and Jessica Hochberg and Nader Kim El-Mallawany are equal co-primary and first authors. 1

Department of Pediatrics Department of Medicine Department of Pathology 4 Department of Microbiology and Immunology 5 Department of Cell Biology and Anatomy New York Medical College, Valhalla, NY 2 3

Submitted: Feb 21, 2014; Accepted: Apr 24, 2014 Address for correspondence: Mitchell S. Cairo, MD, Maria Fareri Children’s Hospital at Westchester Medical Center, New York Medical College, 40 Sunshine Cottage Road, Skyline #IN-D12, Valhalla, NY 10595 Fax: 914-594-2151; e-mail contact: [email protected]

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genetic identification of ALL subtypes associated with especially poor prognoses continues to progress, oncologists are aware now, more than ever, of patients who carry an extremely high risk of relapse. Considering improvements in the diagnostic capacity to recognize certain ultra high-risk patients, coupled with the lack of improvement in long-term curative rates for the unfortunate 15% of children and adolescents who experience relapsed/refractory disease, there is a great need for the development of novel agents to enhance the overall outcomes for children, adolescents, and young adults with ALL. Immunotherapy is one of the targeted anticancer strategies that has shown immense promise. Extrapolating from the success of alloHSCT in curing extremely refractory hematologic malignancies, scientists have examined the great potential of the immune system to actively eradicate cancer cells. Although the graft versus leukemia effect has been well established for years,1,2 the efficacy of donor lymphocyte infusions to treat post-alloHSCT relapse further highlights the potential of immunotherapy.3,4 More recently, cellular immunotherapy has been successfully used for hematologic malignancies and Epstein Barr viruseassociated malignancies.5 Additionally,

2152-2650/$ - see frontmatter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2014.04.015

humoral immunotherapy has established itself as an indispensable component of the arsenal of anti- agents for treating hematologic malignancies, with the greatest success being pioneered by the use of the anti-CD20 monoclonal antibody (moAb) rituximab in the treatment of mature B-cell lymphomas. Undoubtedly, there is great potential to combine effective immunotherapies with the backbone multiagent chemotherapy combinations in hopes of further improving the overall cure rates for children with ALL. Ideally, the targeted approach of immunotherapy will empower the deintensification of chemotherapy regimens and result in an overall decrease in toxicities and late effects experienced by patients. In this review we focus on the development of immunotherapeutic agents in the treatment of childhood, adolescent, and young adult ALL. The vast majority of immunotherapy strategies for ALL are specific to pre-Becell disease, which accounts for approximately 85% of all ALL cases. Immunotherapy for T-cell ALL has unfortunately not progressed on the same trajectory as its more common pre-Becell counterpart. Humoral and cellular immunotherapies are discussed with particular emphasis on novel developments.

Humoral Immunotherapy for ALL Just before the turn of the 21st century, the moAb rituximab stepped onto the scene of anticancer therapies with resounding success. Exhibiting unprecedented activity as a single-agent treatment in adult mature B-cell lymphomas, its efficacy has also been established for a variety of B-cell malignancies including mature B-cell lymphomas in children.6,7 Rituximab was initially incorporated into a pediatric clinical trial as part of the ICE (ifosfamide, carboplatin, and etoposide) relapse regimen for B-cell lymphomas.8 Its early success led to its integration into upfront therapy for mature B-cell lymphomas in children in combination with standard chemotherapy in the Berlin-Frankfurt-Munster and the Children’s

Oncology Group (COG) clinical trials.9,10 It is notable that 40 children with bone marrow and/or central nervous system (CNS) involvement treated with rituximab with standard FrenchAmerican-Britishebased chemotherapy achieved a 3-year EFS of 90%, which is distinctly greater compared with historical controls, which ranges from 75% to 80%.11 The early excitement over the success of rituximab has inspired further development of moAbs in the treatment of pediatric malignancies. Humoral immunotherapy using moAbs is an attractive therapeutic strategy for hematologic malignancies. Leukemia cells fortunately express multiple human differentiation antigens that are uncommonly found in other normal human tissues. They thereby serve as ideal targets for the development of anticancer agents with a limited side effect profile. Although rituximab and several of its early successors are unconjugated antibodies, the development of moAbs conjugated with chemotherapy, radiotherapy, cytokines, or even toxins to enhance tumor cell killing properties has provided an added boost to the potential of humoral immunotherapy. Additionally, the novel development of bispecific moAbs has successfully linked tumor cells with immune effector cells such as T or natural killer (NK) cells (Table 1). Monoclonal antibodies exert cytotoxic effects on the target cell through multiple mechanisms: direct effect (ie, induction of apoptosis or inhibition of cellular proliferation signals), antibodydependent cellular cytotoxicity (ADCC), or via complementdependent cytotoxicity. In the latter 2 examples, moAbs activate either effector cells or the complement cascade to exert cytotoxic effects on the tumor cells. The efficacy of moAbs has increased as the technology to develop them has become more sophisticated. Although the early generations of moAbs were murine or chimeric in origin, recent moAbs are often humanized or fully human, decreasing their antigenicity, thereby enhancing their efficacy in the body’s circulation. Numerous targets for moAb therapy exist in

Table 1 Unconjugated and Conjugated MoAbs in Pediatric ALL Target Antigen

Unconjugated MoAb

Origin

Conjugated MoAb

Origin

SAR3419

Humanized

Blinatumomab (BiTE)

Mouse

CD19 CD20

Rituximab

Chimeric

Ofatumumab

Human

90Y-Ibritumomab tiuxetan

Mouse

Obinutuzumab

Humanized

1311-Tositumomab

Mouse

Veltuzumab

Humanized

AME-133

Humanized Inotuzumab ozogamicin

Humanized

CD22 Epratuzumab

Humanized

CAT-3888 (BL22)

Mouse

Moxetumomab pasudotox

Mouse

90

Y-epratuzumab tetraxetan

CD40 CD52 HLA-DR

Dacetuzumab

Humanized

Humanized

Lucatumumab

Human

Alemtuzumab

Humanized

Apolizumab

Humanized

Milatuzumab

Humanized

CD7

scFvCD7:sTRAIL

Murine

Abbreviations: ALL ¼ acute lymphoblastic leukemia; BiTE ¼ bi-specific T-cell engager; MoAb ¼ monoclonal antibody.

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Immunotherapy in Pediatric ALL Table 2 List of Antigenic Targets for Humoral Immunotherapy in Pediatric B-Cell Precursor ALL CD5 CD19 CD20 CD22 CD23

CD37 CD40 CD52 CD74 CD80

Death receptors HLA-DR Surface Immunoglobulin

Abbreviation: ALL ¼ acute lymphoblastic leukemia.

B-cell ALL (Table 2), however, current strategies focus on the following antigens: CD22, CD19, and CD20. In this section we expand more on the development of humoral immunotherapy targeting these specific antigens.

CD22

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Expressed in > 95% of children with B-cell ALL, the presence of CD22 shifts from the cytoplasmic domain in developing B cells to the cell surface in later stages of B-cell development.12 The humanized moAb epratuzumab targets the extracellular domain of CD22 and was one of the original moAbs used in a clinical trial for pediatric ALL. Its mechanisms of tumor cell killing include ADCC, phosphorylation of the CD22 antigen, and the inhibition of cell proliferation.13 Epratuzumab was initially studied in COG trials for pediatric ALL patients experiencing early relapse. In the phase I trial, it was given twice weekly for 4 doses, and then subsequently given weekly for an additional 4 doses in combination with standard reinduction chemotherapy; the results were promising. Of 15 patients, 9 achieved complete remission (CR), with 7 having no evidence of minimal residual disease (MRD).14 Unfortunately, the phase II portion of the trial did not yield equally convincing results. Epratuzumab was administered twice weekly for 8 total doses in combination with the same standard reinduction chemotherapy regimen. Compared with historical controls, there was no distinct increase in the rate of achieving a second CR. Of patients who did achieve CR, 42% were MRD-negative, which is favorable compared with only 25% of historical controls who achieved MRD-negative disease status.15 Subsequently, conjugated forms of anti-CD22 moAbs have been developed. Inotuzumab ozogamicin is also a humanized moAb directed at CD22 that conjugates the moAb with calicheamicin, an antitumor antibiotic. It was recently studied in a clinical trial that included adults and children. However, of 49 total patients, there were only 3 children. The overall response rate was 57%; CR was achieved in 18% of patients.16 Other conjugated anti-CD22 moAbs have recently been developed. CAT-3888 (BL22) and its second-generation successor moxetumomab pasudotox (formerly known as CAT-8015 or HA22) are moAbs conjugated with a truncated form of the pseudomonas exotoxin A. Moxetumomab pasudotox exhibits a greater affinity for CD22, and promising results have surfaced from preclinical studies and early reports of a clinical trial.17,18 When internalized after binding of the moAb, these immunotoxin conjugates precipitate cellular toxicity resulting in cell death. Moxetumomab pasudotox is currently being evaluated in a clinical trial in children, adolescents, and young adults with relapsed or refractory

Clinical Lymphoma, Myeloma & Leukemia September 2014

CD22-positive (CD22þ) ALL or non-Hodgkin lymphoma (NHL) (NCT00659425). Epratuzumab has also been conjugated to the radioisotope yttrium 90 (90Y-epratuzumab tetraxetan) and studied in clinical trials for adults with relapsed or refractory ALL. The experience with this agent in children is limited, and currently there are no clinical trials using this radioisotope conjugated moAb in children. However, potential use in the future might center on its incorporation into pre-alloHSCT conditioning regimens.

CD19 Another antigen that is present on most pre-B ALL cells, CD19 is another target for moAb immunotherapy in pediatric ALL with great potential. SAR3419 is a humanized moAb conjugated with maytansinoid, a tubulin polymerization inhibitor. Other conjugated anti-CD19 moAbs have been developed, but they have not been well incorporated into the clinical setting because of the limitations of toxicity and availability.19 Preclinical testing for SAR3419 was initially demonstrated with B-cell NHL20 and in CD19þ ALL xenograft models.21 Based on these favorable preclinical results in lymphoma and leukemia, its utility in the clinical setting has also been explored. A recent trial in adults with refractory/relapsed B-cell NHL achieved a reduction in tumor size in 74% of patients, including 7 of 15 patients (47%) who were refractory to rituximab.22 There is currently a phase II trial evaluating SAR3419 in relapsed/refractory CD19þ ALL (NCT01440179). Blinatumomab is a bispecific moAb that represents one of the most novel advances in humoral immunotherapy. A bispecific T-cell engager, this antibody construct binds CD19 and CD3. In doing so, it directs an effector CD3þ cytotoxic T cell in close proximity to the CD19þ tumor cell. Based on favorable results from clinical trials in adults with relapsed/refractory NHL,23 blinatumomab also demonstrated safety and remarkable efficacy in adults with MRDþ B-cell ALL. Of 21 patients with persistence or relapse of MRD, 16 became negative after treatment with a 4-week continuous infusion of blinatumomab. The probability of relapse-free survival in this cohort was 78% with a median follow-up of 405 days.24 A long-term follow-up this study was performed demonstrating a 61% relapse-free survival at a median follow-up of 33 months. For the 9 patients in this cohort who went on to receive alloHSCT, 6 are in sustained CR. Of the 11 who did not receive alloHSCT, 6 are also in CR. This included 4 of 6 Philadelphia chromosomeenegative patients who received no further therapy after blinatumomab.25 Based on this immense promise from clinical trials in adults, blinatumomab is currently under investigation in children with refractory ALL, with second or later bone marrow relapse, or with marrow relapse after alloHSCT. Initial reports established the maximum tolerated dose at 15 mg/m2/d given as a continuous intravenous infusion over 28 days, followed by a 14-day treatmentfree interval. The dose-limiting toxicity in the pediatric trial was cytokine release syndrome (CRS); in adults CNS-related toxicity was also notable. To reduce the risk of CRS, a dose-escalating approach was used, with 5 mg/m2/d for the first 7 days, followed by an escalation to the maximum tolerated dose of 15 mg/m2/d. With the dose escalation protocol, none of the 11 patients

Jessica Hochberg et al experienced CRS. The overall response rate was 41%, with 32% of patients achieving CR.26 Considering that this group of patients was characterized by notoriously refractory leukemia, these early results are very promising. A phase II clinical trial investigating the efficacy, safety, and tolerability of blinatumomab in pediatric and adolescent patients with relapsed/refractory B-cell ALL is under way (NCT01471782).

CD20 Typically considered an antigen associated with mature B-cells, CD20 is expressed in just < 50% of childhood B-cell precursor ALL patients. Jeha et al investigated a cohort of 353 patients, of whom 169 had CD20 expression > 20%. The 5-year EFS was similar for patients with and without CD20 expression.27 Although moAbs targeting CD20 have mostly been used in mature B-cell NHL in children, multiple clinical trials have investigated their efficacy in adults with ALL.28,29 Perhaps its greatest utility in the setting of pediatric ALL would be in combination with other moAbs with multiagent chemotherapy in children with the CD20þ immunophenotype. Several newer generation humanized and fully human anti-CD20 moAbs have recently been developed, demonstrating greater biological efficacy than their stalwart predecessor, rituximab. Ofatumumab (human), obinutuzumab (humanized), veltuzumab (humanized), and AME-133 (humanized) are all anti-CD20 moAbs that demonstrate improvements in a variety of mechanisms of action. Either exhibiting a stronger affinity for the CD20 antigen,30,31 more stable binding,32 or eliciting an enhanced cytotoxic response to the tumor cell,33 these novel moAbs have expanded the horizon of possibilities in treating CD20þ hematologic malignancies, even in patients with rituximab-resistant disease.34 Obinutuzumab is a type II moAb with the capacity to promote direct cell death of the target cell without cross-linking with other antibodies. It has a glycoengineered fragment crystallizable region that has been shown to cause significantly enhanced ADCC.35 Its efficacy has been established in clinical trials for chronic lymphocytic leukemia36 and B-cell NHL.37,38 More recently, it has been investigated in xenograft models for B-cell ALL. At a dose of 30 mg/kg/dose given weekly, obinutuzumab successfully achieved a significantly improved decrease in tumor luminescence and a statistically significant survival advantage compared with rituximab (at equal doses) and with lower doses of obinutuzumab.39 These preliminary studies in 2 different B-cell precursor ALL mouse models might serve as a foundation to further investigate these novel antiCD20 moAbs in the treatment of the subset of CD20þ B-cell precursor ALL patients.

Other MoAbs Alemtuzumab is a humanized moAb targeting CD52, an antigen that is highly expressed on B and T lymphocytes. Its major role in pediatric oncology is to provide in vivo T-cell depletion in conditioning regimens before alloHSCT. However, because CD52 is expressed on most T- and B-ALL cells, it does serve as a potential target for humoral immunotherapy. Its efficacy appeared equivocal in a small phase II COG clinical trial evaluating alemtuzumab in relapsed ALL. Only 13 patients were enrolled, with 1 patient achieving CR.40 Nonetheless, there remains a potential role for

alemtuzumab to be incorporated into conditioning regimens for ALL patients who undergo alloHSCT. Although cyclophosphamide and total body irradiation have been the mainstays in conditioning regimens for alloHSCT in pediatric ALL for many years, perhaps there is room for alemtuzumab to exert a dual benefit in eradicating ALL cells while simultaneously performing in vivo T-cell depletion in preparation for alloHSCT. Favorable outcomes have been established in adult and pediatric ALL, with the added benefit of lower rates of graft versus host disease.41,42 Additionally, there would be a role for alemtuzumab in T-cell and B-cell precursor ALL. Although most humoral immunotherapeutic strategies in pediatric ALL target B-cell antigens, a preclinical trial in a T-cellespecific moAb has demonstrated some potential for T-cell ALL. An antiCD7 moAb linked to the tumor necrosis factorerelated apoptosisinducing ligand caused potent CD7-restricted apoptosis in a variety of malignant T-cell lines, including blood cell samples from patients with T-cell ALL.43 This has yet to be tested in animal models, however it does serve as 1 of the few examples of humoral immunotherapy for T-cell ALL.

Cellular Immunotherapy Chimeric Antigen Receptors A promising approach to immunotherapy is the adoptive transfer of immune effector cells that have been genetically modified to express chimeric antigen receptors (CARs) to enhance antigen-specific binding and overcome inhibitory signals, thereby enhancing cytotoxicity against otherwise resistant tumor cells. CARs combine an extracellular antigen binding transmembrane domain with intracellular signal transduction domains to direct specificity to enhance immune effector cell cytotoxicity (Figure 1).44 The specificity of a CAR is achieved by linking the variable heavy and variable light antibody domains to construct a single-chain fragment variable region. Newer generation CARs have also included additional ligands or cytokines to redirect and enhance specificity to receptors (eg, cytokine receptors, costimulatory molecules).45,46 The surface receptor is then fused to 1 or more cytoplasmic signaling domains to enhance cytotoxicity. There has been a progressive evolution from a single signaling molecule (CD3z) to the addition of co-stimulatory molecules with 41BB and CD28 being most commonly used (Figure 2).47,48 In this way, we have been able to see not only dramatic advances in direct targeting of multiple tumor antigens but improved effector cell functioning as well. T lymphocytes and NK cells transduced with CARs have been shown to recognize surface molecules of target tumor cells and specifically enhance cytotoxicity against tumor targets in vivo and in vitro.45,49 CAR-transduced T-cells have been developed and tested in multiple clinical trials for lymphoid malignancies and various solid tumors. Recent trials have included CAR T-cell therapy against CD19, CD20 (lymphoid malignancies), GD2 (neuroblastoma), CEA (adenocarcinoma), PSMA (prostate cancer), CEA (breast, colorectal cancer), IL13R (glioblastoma), and HER2 (lung, osteosarcoma), to name a few. In general, infusions have been well tolerated with few long-term toxicities. Efficacy against specific targets has varied.49 Most success has been seen in trials targeting lymphoid malignancies.

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Immunotherapy in Pediatric ALL Figure 1 Antitumor Effects Mediated by CAR-Engrafted T Cells. CAR-Modified T Cells Can Recognize Tumor Cells via Binding of the CAR to Its TAA Independent of TCR-MHC/Peptide Interactions. As a Result, T Cells Are Activated and Can Efficiently Eliminate Tumor Cells by the Secretion of Perforin and Granzymes and the Expression of FASL and TRAIL. In Addition, Other Tumor-Infiltrating Immune Cells Can Be Activated by the Secretion of Various Cytokines

Abbreviations: CAR ¼ Chimeric Antigen Receptor; FASL ¼ FAS ligand; IFN ¼ Interferon; IL ¼ Interleukin; MHC ¼ Major histocompatibility complex; TAA ¼ Tumor associated antigen; TCR ¼ T cell receptor; TNF ¼ Tumor necrosis factor; TRAIL ¼ Tumor Necrosis FactoreRelated Apoptosis-Inducing Ligand. Reproduced from Cartellieri M et al. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer. J Biomed Biotechnol 2010; 2010:956304.

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In clinical trials, CAR-modified T cells that target CD19 and CD20 have been reported to be effective in adults with CLL50 and B-cell lymphomas.51 Recently, there has also been success in children with relapsed and refractory pre-BeALL who received autologous infusions of T cells transduced with an anti-CD19 CAR.52 Grupp et al reported on their use of autologous CD19-CAR T cells in 10 adults with CLL and 2 children with high-risk refractory/relapsed pre-BeALL. Autologous T cells were collected, expanded, and then genetically modified using a lentivirus vector to express an antiCD19 CAR along with CD3z and CD137 (41BB) as a costimulatory molecule. Cells were then infused back into the patient where they noted robust expansion in vivo of approximately 1000fold greater than their original engraftment levels, with modified cells detected in peripheral blood, bone marrow, and cerebral spinal fluid. The infusions were found to be safe and well tolerated, however, a significant macrophage activation type syndrome occurred in each patient, prompting investigation into simultaneous immunomodulatory drugs to prevent massive cytokine release.52 CR was achieved in each patient, demonstrating promising antileukemic efficacy in patients with high-risk disease who had previously been refractory to traditional chemotherapy and targeted antibody therapy.52 Similar results have also been demonstrated in adults with relapsed ALL using CD19 CAR with CD28 as a co-stimulatory molecule with CD3z. All patients with persistent disease with T-cell infusion demonstrated rapid tumor eradication and achieved MRD-CRs assessed using deep sequencing polymerase chain

Clinical Lymphoma, Myeloma & Leukemia September 2014

reaction.53 The ability to obtain complete sustained responses allows for the possibility of repeated maintenance transfusions or as a bridge to bone marrow transplant in patients previously unable to benefit. Although T cells have been the main focus to date of CAR therapy, there has been increasing data to support a role for modified NK cell immunotherapy, particularly in the setting after bone marrow transplant. NK cells are particularly attractive after alloHSCT because of the concern for developing graft versus host disease secondary to T-cell infusion. However, their effectiveness is limited by inhibitory human leukocyte antigen signals. Imai et al were able to expand a population of NK cells in vitro using coculture of peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecule 41BB ligand and membrane-bound interleukin-15. They went on to show that genetic modification of these expanded NK cells with an antiCD19 receptor linked to CD3z and 41BB co-stimulatory molecules was able to overcome typical NK resistance. These activated NK cells had increased interferon and other cytotoxic cytokine production, showing markedly enhanced NK-cellemediated killing of leukemic cells.45 Our group also has demonstrated an antitumor effect of anti-CD20 CAR-modified expanded peripheral blood NK (PBNK) cells against disseminated CD20þ Burkitt lymphoma (BL) in vivo using human CD20þ BL xenografted nonobese diabetic severe combined immune deficiency mice.54,55 Using a similar engineered K562 coculture expansion approach, we were able to show that multiple injections of expanded anti-

Jessica Hochberg et al Figure 2 Evolution of CD19-Directed CARS and Their Use in Clinical Trials Targeting Lymphoid Malignancies. All Generations of CARs Contain a Transmembrane Structural Domain, and an Extracellular scFv, Which Is Derived From a Human-CD19-Specific Mouse Monoclonal Antibody—Either FMC63 (IgG2a) or SJ25C1 (IgG1). First-Generation CARs Contain a Single Cytoplasmic Signaling Domain (CD3-z), Which Links Antigen Recognition to Intracellular Signal Transduction Pathways. SecondGeneration CARs Contain CD3-z and a Co-Stimulatory Signaling Domain, Either CD28 or 41BB (Also Known as CD137, a Member of the Tumor Necrosis Factor Receptor Superfamily). Compared With First-Generation CARs, Second-Generation CARs Induce Superior Antitumor Responses in Preclinical Studies and in Patients With B-Cell Malignancies. ThirdGeneration CARs Contain 2 Co-Stimulatory Domains, CD28 and 41BB, in Addition to Signaling Domain CD3-z. Baylor (Baylor College of Medicine): Clinical Trials NCT01853631, NCT00586391; NCI (National Cancer Institute): Clinical Trials NCT01087294, NCT00924326, NCT01593696; MSKCC (Memorial Sloan-Kettering Cancer Center): Clinical Trials NCT01840566, NCT01860937, NCT01044069, NCT00466531, NCT01416974; Penn (Abramson Cancer Center of the University of Pennsylvania): Clinical Trials NCT01747486, NCT01029366, NCT01551043

Abbreviations: CAR ¼ Chimeric Antigen Receptor; scFv ¼ Single-Chain Variable Fragment. Reproduced from Tothova and Steensma. Treatment of B-ALL With CD-19-Specific Chimeric Antigen Receptor T Cells. Hematologist 2013; 10:1-16.

CD20 CAR PBNK cells can not only mediate tumor regression in a BL xenograft mouse model, but significantly increased the survival of mice compared with controls (Figure 3).

These results indicate the therapeutic potential of multiple injections of anti-CD19 or CD20 CAR-expanded T or NK cells and provide a future direction for examining the antitumor activity

Figure 3 Cartoon Illustrating Mechanism of Peripheral Blood NK Expansion by K562-mbIL15-41BBL

Abbreviation: NK ¼ Natural Killer.

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Immunotherapy in Pediatric ALL against antibody- or chemotherapy-resistant lymphoma and leukemia before and after bone marrow transplantation.

3.

Conclusion

4.

Humoral and cellular immunotherapy for childhood, adolescent, and young adult ALL continues to be a rapidly growing area of research with much promise based on early results. The identification of ideal tumor antigen targets remains paramount to this success. Ideally these targets should have all of the following characteristics: (1) differentially expressed on tumor versus normal cells; (2) high-density expression; (3) antigenic; (4) required for critical cell biological function and/or survival; (5) not shed or secreted; and (6) no modulation after antibody (ligand) binding.56 CD19, CD20, and CD22 are found in abundance on leukemic cells and meet all of these criteria. Although these remain the main targets of immunotherapy for ALL at this time, there is increasing awareness of the array of additional targets on leukemic cells that either alone or in combination (eg, blinatumomab) can offer greater efficacy. Whether cell death occurs through direct lysis, apoptosis, antibody- or complement-dependent cellular cytotoxicity depends on the targeted antigen and the effector cell involved. Ultimately, combined approaches will prove to be of greatest benefit, particularly in relapsed or refractory disease. To date we have seen that moAbs, antibody conjugates, bispecific T-cell and bispecific NK-cell antibodies all show significant activity against ALL. Additionally, genetically reengineered T and NK cells expressing targeted CAR have shown impressive activity in vitro and in vivo against ALL. Optimizing dosing schedules and managing toxicities are of obvious importance moving forward but there is nothing to suggest that this cannot be accomplished readily. In summary, a number of humoral and cellular immunotherapy strategies have been investigated in adult and pediatric acute leukemias. To date, these approaches appear to be safe and well tolerated. The optimal dose and schedule of moAbs remain to be determined, particularly when administered in conjunction with combination chemotherapy. Maximal benefit is likely to occur in the setting of MRD, after bone marrow transplant or as a replacement for certain cytotoxic agents in multiagent chemotherapy upfront regimens. Future studies are required to determine when in the treatment protocol that humoral and cellular therapy will have the safest and optimal effect in ALL.

Acknowledgment The authors thank Erin Morris, RN, BSN, for her expert editorial assistance in the preparation of this report.

5. 6. 7.

8.

9. 10.

11.

12. 13. 14. 15.

16. 17. 18. 19. 20. 21.

22.

23.

Disclosure Mitchell S. Cairo is a Scientific Advisor to Roche Pharmaceuticals. The remaining authors have stated that they have no conflicts of interest.

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