Auto-SCT and Immunotherapy for Plasma Cell Myeloma

REVIEW Beyond consolidation: auto-SCT and immunotherapy for plasma cell myeloma

N Lendvai1,2,5, AD Cohen3,5 and HJ Cho4

Autologous hematopoietic cell transplantation (auto-HCT) is the standard consolidation therapy for plasma cell myeloma patients following induction therapy. Auto-HCT improves disease-free survival (DFS), but is generally not curative. The allogeneic HCT experience demonstrated that T-cell immunotherapy can confer long-term DFS. Preclinical and clinical data indicate that myeloma- associated Ags elicit humoral and cellular immune responses (IRs) in myeloma patients. These ﬁndings strongly suggest that the immunotherapeutic strategies, including immune checkpoint inhibitors, therapeutic cancer vaccines and adoptive cellular therapies, are promising avenues of clinical research that may be most applicable in the minimal residual disease state following auto-HCT. These strategies are designed to prime or augment antimyeloma IRs and promote a ‘host-vs-myeloma’ effect that may result in durable DFS. Innovative clinical trials investigating immune checkpoint inhibitors and cancer vaccines have demonstrated that robust immunity against myeloma-associated Ags can be elicited in the setting of auto-HCT. A diverse array of immunotherapeutic strategies have entered clinical trials in myeloma, including PD-1/PD-L1 inhibitors, DC/myeloma cell fusion vaccines and adoptive chimeric Ag receptor T-cell therapy, and further investigation of combinations of immunologic and pharmaceutical agents are expected in the near future. In this review, we will discuss the preclinical data supporting immunotherapy in auto-HCT for myeloma, clinical investigation of these strategies and the future prospects of immunotherapy in pursuit of the goal of curative therapy.

Bone Marrow Transplantation (2015) 50, 770–780; doi:10.1038/bmt.2015.5; published online 9 March 2015

INTRODUCTION against autologous myeloma cells were detected in patients with 10,11 Autologous hematopoietic cell transplantation (auto-HCT) is a MGUS or asymptomatic PCM. The progression of disease was standard consolidation therapy for plasma cell myeloma (PCM). associated with loss of both adaptive and innate immune effector Auto-HCT was associated with superior PFS and OS in two large functions, suggesting that disabling of immune surveillance may randomized studies while a meta-analysis showed superior PFS.1–3 contribute to progression. However, these myeloma-associated Incorporation of auto-HCT and the introduction of the immuno- immune responses (IRs) could be restored ex vivo with appropriate modulatory drugs (ImiDs; thalidomide, lenalidomide and pomali- stimulation.12,13 Humoral and cellular IRs against myeloma- domide) and proteasome inhibitors (bortezomib and carfilzomib) associated Ags such as Wilm’s tumor 1 (WT1) and the type I have led to significant improvement in clinical outcomes, but cure melanoma Ag gene (MAGE) proteins MAGE-A3 and CT7 (MAGE- has remained elusive. Allogeneic HCT (allo-HCT) studies demon- C1) were detected in untreated PCM patients and after auto- or strated a plateau in OS with long-term follow-up, indicating a allo-HCT. In the allo-HCT setting, these IRs were associated with potential cure fraction.4–6 Higher relapse rates were reported in improved outcomes, and myeloma Ag-specific T cells from T-cell-depleted allo-HCT,7,8 and objective responses were patients could kill both human myeloma cell lines and autologous – observed with DLI in patients who relapsed after allo-HCT,7,9 PCM cells.14 16 MoAbs targeting immune modulating molecules strongly suggesting that immunologic therapy in the form of on T and natural killer (NK) cells, such as CD137 (4-1BB), T-cell-mediated ‘graft-vs-myeloma’ effect could control the programmed death (PD)-1 or glucocorticoid-induced TNF receptor disease. However, allo-HCT continues to have high procedure- (TNFR)-related protein, promoted cytotoxic activity against mye- related morbidity and mortality and can only be applied to a loma cells in preclinical models17,18 (AD Cohen, unpublished). minority of PCM patients owing to age and health restrictions. Auto-HCT was historically viewed as an inopportune time for Therefore, alternative strategies to confer ‘host-vs-myeloma’ active immunotherapy due to delayed immune reconstitution and immunity is an active area of research, fueled by significant poor responses to infectious disease vaccines (Table 1).19,20 June advances in understanding immune regulation and progress in and colleagues demonstrated that delayed recovery of immune immunologic therapy for other cancers. competence to vaccines could be ameliorated through the Preclinical data support the concept of harnessing the immune reconstitution of the mature autologous T-cell compartment after system to eliminate malignant plasma cells. Immune responses auto-HCT.21,22 In two studies, PBL were harvested by leukopheresis

1Myeloma Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2Department of Medicine, Weill Medical College of Cornell University, New York, NY, USA; 3Division of Hematology/Oncology, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA and 4Multiple Myeloma Service, Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Correspondence: Dr HJ Cho, Multiple Myeloma Service, Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Box 1130, One Gustave L Levy Place, New York, NY 10029, USA. E-mail: [email protected] 5These authors contributed equally to this work. Received 29 December 2014; accepted 31 December 2014; published online 9 March 2015 Transplant and immunotherapy for myeloma N Lendvai et al 771

Table 1. Biologic factors that impact immunologic therapy in the setting of auto-HCT

Favorable immunologic factors Unfavorable immunologic factors

Minimal residual disease Lymphopenia Tumor Ag release by high-dose chemotherapy Delayed recovery of immune responsiveness to vaccines Enhanced DC activation/T-cell cross-priming Delayed recovery of Ag-speciﬁc immune repertoire Increased availability of T-cell survival factors (IL-7, IL-15) Reduced suppressor populations (MDSC and Treg) Abbreviations: HCT = hematopoietic cell transplantation; MDSC = myeloid-derived suppressor cell; Treg = regulatory T cell. Many of the unfavorable factors can be mitigated by reconstitution of autologous PBL in the early post-HCT period.

abCentral Peripheral

CTL CTL

CTLA-4 PD-1 TCR

Antigen anti-PD-1 anti-CTLA-4 MHC class I anti-PD-L1 PD-L1 B7-1/2

Dendritic cell Myeloma cell Figure 1. Mechanisms of immune checkpoint inhibitors. (a) CTL recognize Ags in the context of MHC class I on APCs such as DCs in lymphoid tissue. CTLA-4 is a negative regulatory receptor that binds to B7-1/-2 on DC and downregulates CTL activity as a mechanism of central tolerance. Anti-CTLA-4 checkpoint inhibitor MoAbs such as ipilimumab and tremelimumab block CTLA-4 interactions and promote expansion of CTL. (b) PD-1 on CTL binds to its cognate receptor PD-L1 on normal and tumor cells, downregulating T-cell activity even in the context of Ag recognition through the TCR. Checkpoint inhibitors targeting PD-1 (pembrolizumab, nivolumab and pidilizumab) or PD-L1 (MPDL3280A and MEDI4736) block this mechanism of peripheral tolerance and allow CTL to kill targets such as myeloma cells. from PCM patients after vaccination with an infectious disease studies of adoptive cellular therapy for melanoma support these or tumor vaccine. Vaccine-primed T cells were expanded and findings, with the greatest response rates seen with protocols activated ex vivo by co-incubation with MoAb-conjugated beads incorporating myeloablative pretransfer conditioning regimens that ligated and activated CD3 and CD28 receptors on T cells. and autologous stem cell support.27 These activated T cells were reinfused into patients after auto-HCT, These findings support the investigation of immunologic and the patient received subsequent booster vaccinations. This therapy in conjunction with auto-HCT for consolidation of PCM. strategy effectively restored humoral and cellular immunity to the The transplant renders a minimal residual disease state that may vaccine compared with controls. In addition, several preclinical be most amenable to eradication by immunologic mechanisms. studies suggest that the early lymphopenic period after myeloa- Current investigations fall into three general categories: immune blative chemotherapy in auto-HCT is a favorable environment for checkpoint inhibitors; myeloma vaccines; and cellular therapies, the recovering immune system (or adoptively transferred lym- and trials of combinations of immunologic therapies are entering phocytes) to be stimulated through vaccination, thus enhancing the field. recognition of tumor Ags. Several mechanisms that have been demonstrated include the following: (1) tumor Ag release due to cell death; (2) enhanced DC activation and cross-priming due to CTLA-4 IMMUNE CHECKPOINT INHIBITORS greater availability of TLR agonists (for example, LPS from inflamed Cancer immunotherapy gained an added dimension with the gut); (3) increased availability of T-cell survival factors such as IL-7 introduction of immune checkpoint inhibitors, MoAbs, that are and IL-15 due to elimination of competing populations; and directed at the inhibitory cell surface molecules that normally (4) elimination of suppressor populations (for example, myeloid- function to attenuate Ag-specificT-cellIRs(Figure1).Theseagents derived suppressor cells and regulatory T cells), which impair do not attack the tumor directly; instead, they block inhibitory antitumor immunity.23–25 In addition, hematopoietic stem cells interactions and ‘take the brakes off’ of pre-existing tumor-specific enhanced the efficacy of adoptively transferred, Ag-specific CD8+ T cells, which can then lyse tumor cells. The prototype checkpoint T cells in a murine model.26 The National Cancer Institute clinical inhibitor ipilimumab is directed against CTL Ag (CTLA)-4 on T cells,

© 2015 Macmillan Publishers Limited Bone Marrow Transplantation (2015) 770 – 780 Transplant and immunotherapy for myeloma N Lendvai et al 772 which has an important role in downregulating inflammatory MYELOMA VACCINES responses and inhibiting activation and expansion of auto-reactive Therapeutic tumor vaccines cover a broad range of delivery 28 T cells in the spleen and lymph nodes. T-cell receptors (TCRs) bind platforms, including cell-based protein or peptide, plasmid DNA to Ags displayed by Ag-presenting cells, primarily DCs, in the context and engineered viruses, and target different cancer-associated of MHC class I and II, resulting in the expansion of Ag-specific T cells. Ags (Table 3). Short synthetic peptide vaccines, composed of CTLA-4 is expressed on T cells late in the IR and binds to its cognate defined antigenic epitopes, were among the first therapeutic receptors B7-1 and -2 on DCs, resulting in the attenuation of T-cell tumor vaccine formulations. Their design was fueled by research activity. Ipilimumab blocks CTLA-4 binding to B7-1 and -2, that defined the rules for peptide binding to common MHC class I antagonizing this critical mechanism of ‘central’ tolerance at the and II molecules and presentation to T cells. However, recent work 29 sites where Ag-specific immunity is primed and expanded. suggests that short peptides may be tolerogenic,49 and these have Ipilimumab was approved for metastatic melanoma on the basis given way to investigation of synthetic long peptides (SLPs) and of phase 3 results demonstrating superior OS compared with heteroclitic peptides. Heteroclitic peptides, selected on the basis 30,31 chemotherapy. Autoimmune adverse events reflecting the of in vitro or in silico testing, contain amino-acid substitutions that critical role of CTLA-4 in central tolerance were observed in these increase the stability of the peptide/MHC complex and often trials, including gastrointestinal inflammation, rashes and uveitis. generate robust cytotoxic T-cell responses that cross-react with These adverse events were effectively managed by close monitoring the native peptide epitope.50–55 WT1 has emerged as a potentially and prompt institution of steroids. Ipilimumab therapy was important target in PCM, after it was shown that the patients who correlated with the emergence of humoral and cellular IRs against received DLI following T-cell-depleted allo-HCT developed tumor-associated Ags, suggesting that CTLA-4 blockade acts in part increased WT1-specific T-cell responses post DLI, which were by breaking tolerance to tumor cells, thus promoting protective associated with reduction in paraprotein.56 An ongoing study at – antitumor immunity.32 34 Tremelimumab, a second-generation anti- the Memorial Sloan-Kettering Cancer Center targets residual CTLA-4 MoAb, is in clinical trials for solid tumors and hematologic myeloma post auto-HCT with a heteroclitic WT1 peptide vaccine malignancies (Table 2). combined with three WT1 SLPs and delivered with montanide and GM-CSF adjuvant. Similarly, a SLP encompassing the signal domain of MUC1 with GM-CSF adjuvant was investigated in a PD-1/PD-L1 CHECKPOINT INHIBITORS phase I/II study of 15 post-auto-HCT PCM patients with stable or The PD-1/PD-L1 (PD-ligand 1) axis is a second set of checkpoint progressive residual disease and MUC1+ plasma cells.57 MUC1 35,36 inhibitor targets in clinical investigation. PD-1 is also expressed signal peptide domain contains several MHC class I/II and B-cell on T cells and its cognate ligand PD-L1 is expressed on normal epitopes, and in this study MUC1-specific cellular and humoral IRs tissues (Figure 1). This is an important mechanism of ‘peripheral’ were noted. Response assessment revealed disease stabilization in tolerance, preventing autoimmune activity. PD-L1 is also the majority of the patients, lasting for up to 26 months after expressed on many tumor cells, and clinical trials of MoAbs study completion (ongoing follow-up), including in patients directed against PD-1/PD-L1 are underway based on the hypoth- entering the study with PD. esis that blocking this inhibitory signal will antagonize peripheral Idiotype (Id) has long been recognized as a potential target for tolerance and promote T-cell activity against tumor cells. myeloma immunotherapy. Id-KLH (keyhole limpet hemocyanin) Pembrolizumab, a MoAb against PD-1, was recently granted the administered with GM-CSF to PCM patients at least 100 days post- FDA approval for relapsed melanoma for patients who progressed auto-HCT resulted in anti-Id T-cell responses. Subsequent studies through ipilimumab and BRAF inhibitor therapy. Pembrolizumab have sought to use DC-based therapy to mobilize protective IRs. demonstrated OS benefit in phase 1 and 2 trials in this population Reichardt et al.58 treated 12 post-auto-HCT PCM patients with Id- and advanced phase trials are underway for melanoma and lung pulsed autologous DC followed by five s.c. boosts of Id-KLH. Two cancer.37,38 Similarly, MoAbs against PD-L1 have shown activity in patients developed an Id-specific, cellular proliferative IR and one solid tumors.39 Autoimmune adverse effects were also observed in developed a transient Id-specific cytotoxic T-cell (CTL) response. clinical trials for anti-PD-1/PD-L1 MoAbs, although in general, The two patients that developed cellular IR also were in CR after they were less severe. Clinical trials are also underway combining auto-HCT (before vaccination) and they remained in CR at the time anti-CTLA-4 and anti-PD-1/PD-L1 MoAbs.40 In addition, the anti- of the publication of the report; therefore, the clinical impact of PD-1 MoAb pidilizumab (CT-011) could be safely combined with this approach remains to be determined. A larger series of a total auto-HCT in an early-phase trial for lymphoma.41 of 26 patients had similar results,59 and other Id-based strategies PD-L1 is expressed on primary myeloma cells from PCM are under investigation.60 patients, and several studies have demonstrated that blockade Rosenblatt et al.61 used DC/PCM cell fusions in the early post- of the PD-1/PD-L1 axis augments T and NK cell-mediated auto-HCT setting as additional consolidation. Vaccination resulted antimyeloma activity in vitro as well as in murine myeloma in the expansion of myeloma-reactive lymphocytes and expansion – models.17,42 45 These antimyeloma effects can be further of T cells with specificity for myeloma-associated Ags such as enhanced by combining PD-1/PD-L1 blockade with irradiation, MUC1. Encouragingly, a number of late responses (several months cell-based vaccines, hematopoietic stem cell transplant or post-auto-HCT) were observed in the vaccinated patients, raising lenalidomide,17,46,47 providing a rationale for combination therapy the possibility that the vaccine may have impacted residual trials. A phase I trial of the anti-PD-1 Ab nivolumab in various disease in these patients. This promising strategy is moving hematologic malignancies included 27 relapsed/refractory PCM forward in a randomized phase 2 BMT-CTN study. Importantly, this patients and demonstrated prolonged stable disease in two- strategy is now being combined with immune checkpoint thirds.48 Current trials in myeloma include a phase 1 study of inhibition after in vitro studies showed that PD-1 blockade pembrolizumab, lenalidomide and dexamethasone for relapsed prevented the DC/PCM fusion vaccine-mediated increase in T-cell PCM (NCT02036502), a phase 1/2 study of pidilizumab and expression of PD-1, promoted vaccine-induced polarization of lenalidomide for relapsed PCM (NCT02077959) and a phase 2 T cells toward an activated phenotype expressing Th1 cytokines, study of pidilizumab and auto-HCT with or without a myeloma cell and decreased regulatory T cells.62 In the ongoing study, PCM fusion vaccine (NCT01067287) that will be discussed in greater patients receive serial infusions of CT-011 alone or in conjunction detail (Table 2). Protocols for diverse checkpoint inhibitors and with DC/PCM fusion vaccine following auto-HCT. In the CT-011 combinations in the context of auto-HCT are planned and will alone cohort, a dramatic and persistent expansion of myeloma- open in the next year. specific T cells was noted.63

Table 2. Immune checkpoint inhibitors entering clinical trials for PCM

Target Checkpoint inhibitor Dose/route Common adverse Notable immune-mediated Clinical trials reactions adverse reactions for PCM

CTLA-4 Ipilimumaba 3 mg/kg i.v. every 3 weeks (four Fatigue Enterocolitis NCT01592370 doses) Diarrhea Hepatitis NCT00060372 Rash Dermatitis NCT01822509 Pruritis Neuropathy Colitis Endocrinopathy Tremelimumab 15 mg/kg i.v. every 90 days Diarrhea/colitis Enterocolitis Other dosing schedules under Nausea Hepatitis investigation Fatigue Endocrinopathy Rash Uveitis/iritis Pruritis Pancreatitis Vomiting Anorexia Abdominal pain PD-1 Pembrolizumaba 2 mg/kg i.v. every 3 weeks Fatigue Enterocolitis NCT02036502 Cough Hepatitis NCT01953692 Nausea Nephritis NCT01592370 Pruritis Endocrinopathy Hypophysitis NCT02077959 Rash Anorexia Constipation Arthralgia Diarrhea Nivolumab Multiple dosing schedules under Fatigue Enterocolitis investigation Rash Vitiligo Diarrhea Endocrinopathy Pneumonitis Pidilizumab Multiple dosing schedules under Diarrhea Not reported at this time investigation Rash PD-L1 MPDL3280Ab Multiple dosing schedules under Pyrexia Not reported at this time NCT01375842 investigation Anemia Anorexia Fatigue Nausea MEDI4736 Multiple dosing schedules under Diarrhea Not reported at this time investigation Fatigue Rash Vomiting Pyrexia Abbreviations: FDA = Food and Drug Administration; PCM = plasma cell myeloma; PD-1 = programmed death 1; PD-L1 = programmed death ligand 1. aFDA approved. bFDA breakthrough therapy designation. Adverse events (AEs) are reported primarily from solid tumor trials including melanoma and lung cancer except for pidilizumab (heme malignancy and auto-HCT for lymphoma; AEs reported but conﬁrmation of immune-mediated AEs not reported). Early-phase clinical trials for these agents are underway primarily in ‘basket’ trials for relapsed hematologic malignancies and protocols for relapsed myeloma. Trials in the context of auto-HCT are combinations with vaccine therapy (see Table 3) are also open. Further trials investigating checkpoint inhibitors in auto-HCT for myeloma are expected in 2015.

As noted earlier, post-auto-HCT immune deficiency can be Patients received lenalidomide maintenance starting on day corrected by a combination of vaccine and adoptive reconstitu- 100.64 Dextramer staining demonstrated MAGE-A3-specific tion of activated autologous T cells.21,22 This strategy was applied CD8 T cells in 7 of 8 evaluable HLA-A2+ patients, whereas in a phase 1 trial where patients received T cells on day 2 after vaccine-specific cytokine-producing T cells were generated in auto-HCT. HLA-A2+ patients received a multipeptide tumor Ag 19 of 25 patients. Further studies are needed to determine the vaccine derived from human telomerase reverse transcriptase clinical impact of this approach. In a new ‘twist’ on an old theme, (hTERT) and survivin; ‘universal’ tumor Ags that are often Id-KLH vaccination is being tested on this platform in an ongoing overexpressed in myeloma. Tumor Ag vaccine administration study (Table 3). was restricted to HLA-A2+ patients because the vaccine is a The authors recently completed a study integrating several of cocktail of peptides that are known to be presented to CD8 these important strategies, in which recombinant MAGE-A3 T cells in the context of MHC I molecules expressed in HLA-A2+ protein was administered with AS15 adjuvant (containing mono- patients. Patients also received the 7-valent pneumococcal phosphoryl lipid A, QS21 and CpG7909) pre- and post auto-HCT, conjugate vaccine regardless of HLA type.21 Ten of 28 patients coupled with infusion of vaccine-primed, autologous PBL early had IRs to the hTERT/survivin vaccine, but these were weaker after auto-HCT. All patients had MAGE-A+ myeloma cells at than the pneumococcal conjugate vaccine responses. There baseline. In contrast to the previously discussed studies, the was no difference in OS between the patients who received the vaccine-primed PBL were not manipulated ex vivo. Robust hTERT/survivin vaccine and those who did not. In a subsequent humoral IRs to MAGE-A3 were detected in all 12 patients tested study of the same design, 27 PCM patients received poly-ICLC/GM- that persisted at least 1 year.65 MAGE-A3-specific CD4 T cell CSF-primed MAGE-A3 vaccine ± montanide, before auto-HCT responses were detected in the 3 patients tested so far, but CD8 and activated T cells and booster vaccinations after auto-HCT. T-cell activity has not been shown yet.

Table 3. Vaccine trials in auto-HCT for PCM

Vaccine Vaccine formulation Adjuvant/concomitant therapy Clinical trial Comments References identiﬁer rnpatadimnteayfrmyeloma for immunotherapy and Transplant Recombinant MAGE-A3+AS15 Recombinant MAGE-A3/H. ﬂu protein D/His-tag AS15 adjuvant (monophosphoryl NCT01380145 Robust humoral and CD4 T-cell responses 106 adjuvant fusion protein lipid A, QS21, CpG7909) observed autologous, unmanipulated PBL infusion (day +3 post-auto-HCT)

– Anti-PD-1 ± DC/myeloma Autologous monocyte-derived DC/ autologous Pidilizumab (CT-011) NCT01067287 Robust expansion of myeloma-speciﬁc T cells in 61,63 8 05McilnPbihr Limited Publishers Macmillan 2015 © 780 fusion myeloma cell fusion NCT00458653 response to CT-011. Clinical data not yet available. Cohort receiving vaccine+CT-011 accruing

Autologous tumor vaccine Irradiated autologous tumor cells GM-CSF-secreting K562 cells NCT00024466 Tumor-speciﬁc humoral and cellular responses 107 +GM-CSF observed. Clinical impact uncertain

Autologous Id-KLH conjugate Id-KLH conjugate GM-CSF NCT00019097 Anti-Id proliferative T-cell response in 2 of 11 and 58,108 Lendvai N +GM-CSF Id-speciﬁc DTH in 8 of 10. Long-term follow-up showed recovery of TCR diversity. PFS and OS were comparable to case-matched controls α receiving IFN or steroid maintenance al et

CT7, MAGE-A3 and WT1 CT7, MAGE-A3 and WT1 mRNA-electroporated DC NA NCT01995708 Currently accruing mRNA-electroporated DC

WT1 analog peptide vaccine WT1 peptides Montanide ISA 51 VG NCT01827137 Currently accruing +GM-CSF GM-CSF

Activated T cells+hTERT hTERT/survivin peptides+PCV for Montanide ISA 51, GM-CSF NCT00834665 Ag-speciﬁc CD8+ T-cell response seen in 10/28 21 multipeptide vaccine HLA-A2+ patients CD3/CD28-activated autologous patients. No improvement in OS compared with PCV alone for HLA-A2 − patients. T-cell infusion (day +2 the PCV only arm post-auto-HCT)

MAGE-A3+poly-ICLC vaccine/ GL-0817 protein composed of linked class I and Toll-like receptor 3 agonist NCT01245673 MAGE-A3–speciﬁc CD8+ T cells seen in 7 of 8 64 vaccine-primed, activated class II epitopes of MAGE-A3 and the HIV-1-TAT poly-ICLC (Hiltonol), Montanide, patients, vaccine-speciﬁc cytokine-producing T cells membrane translocation sequence (Trojan GM-CSF T cells were generated in 19 of 25 patients peptide), which delivers the peptides into the ER1 CD3/CD28-activated autologous and facilitates the formation of MHC class I T-cell infusion (day +2 complexes post-auto-HCT)

Id-pulsed autologous DC+IL-2 Id (isolated from autologous serum) loaded NA NCT00616720 Phase II study of 27 patients who were compared 60 or IFNg autologous DC (APC8020) with historical control (n = 124). Two-year OS beneﬁt, in spite of no difference in PFS, TTP. Immune responses were not reported

Activated T cells ± Id-KLH Id-KLH conjugate CD3/CD28-activated autologous NCT01426828 Randomization to Id-KLH vaccine or KLH alone. conjugate vaccine T-cell infusion (day +2 post-auto- Currently accruing HCT) Abbreviations: DTH = delayed-type hypersensitivity; ER = endoplasmic reticulum; HCT = hematopoietic cell transplantation; His = histidine; hTERT = human telomerase reverse transcriptase; Id = idiotype; KLH = keyhole limpet hemocyanin; NA = not applicable; PCM = plasma cell myeloma; PCV = pneumococcal conjugate vaccine; PD-1 = programmed death 1; PD-L1 = programmed death ligand 1; TTP = time to progression; WT1 = Wilm’s tumor 1. A broad range of vaccine platforms and antigenic targets are under investigation in auto-HCT for plasma cell myeloma. Notably, investigation into combination therapy with anti-PD-1 checkpoint inhibitor is underway (NCT01067287, NCT00458653). 05McilnPbihr iie oeMro rnpatto 21)770 (2015) Transplantation Marrow Bone Limited Publishers Macmillan 2015 ©

Table 4. Cellular therapies in auto-HCT for PCM

Cellular therapy Cellular therapy product Concomitant/ activating therapy Comments Clinical trial identiﬁer/ references

CART19 for multiple myeloma Autologous T cells transduced with CD19- Salvage MEL auto-HCT (CART19 cells Attempts to target CD19+ clonogenic PCM NCT02135406 speciﬁc CAR incorporating CD3ζ/4-1BB infused day +12) precursor cells. Currently accruing signaling domains

Activated MILs CD3/CD28-activated autologous BM Upfront MEL auto-HCT (MIL infused day Feasible and safe. Six CR/10PR in 22 patients. Ex NCT0056609866 lymphocytes +3) vivo MIL proliferation to PCM lines correlated with response

Activated MILs ± allogeneic CD3/CD28-activated autologous BM Upfront MEL auto-HCT (MIL infused day Randomization to MIL infusion ± vaccine. Accrual NCT01045460 GM-CSF/myeloma cell vaccine lymphocytes +3) GM-CSF-secreting allogeneic completed myeloma cell line

Activated MILs ± tadalafil CD3/CD28-activated autologous BM Upfront MEL auto-HCT (MIL infused day Randomized trial. Tadalafil (PDE-5 inhibitor) used NCT01858558 lymphocytes +3) Tadalafil to inhibit suppressive MDSCs.Currently accruing

Co-stimulated autologous T cells CD3/CD28-activated autologous PBL Upfront or salvage MEL auto-HCT Seminal study showing proof of principle that NCT0004685222 (T cells infused day +12 or day +100) early (day +12) infusion of primed (?) activated T cells could restore immune competence post- auto-HCT

Activated, NY-ESO1/LAGE- Autologous, CD25-depleted, CD3/CD28- Upfront or salvage auto-HCT (T cells Restricted to HLA-A2+ patients. Transduction NCT0135228678,79 Lendvai myeloma N for immunotherapy and Transplant peptide-speciﬁcTCR activated T cells transduced with transgenic, infused day +2) feasible. Transgenic T cells expanded, homed to transgenic T cells afﬁnity-enhanced TCR recognizing shared BM, and persisted 46 months. Progression NY-ESO1/LAGE epitope associated with loss of T cells or Ag loss by

myeloma cells al et

Expanded NK-enriched cells Haplo-identical, CD3-depleted, IL-2-activated Salvage Flu/Dex/MEL with delayed Takes advantage of KIR mismatch to enhance NK NCT0008945373 PBL (containing at least 1 × 106/kg (day +14) auto-HCT (NK cells infused cell recognition/lysis of PCM cells. Demonstrated CD56+ CD3- NK cells) days 0 and +2) IL2 days +1 to +11 proof of principle. Limited ex vivo expansion and in vivo persistence of donor NK cells. Signiﬁcant IL2-associated toxicity

Expanded NK cells Haplo-identical, peripheral blood-derived, MEL auto-HCT (up to 10 NK cell Better puriﬁcation and expansion steps allow for NCT01040026 bead-puriﬁed, IL-2 and IL-15 expanded infusions days +3 to +30) repeated infusions. No IL2 given to patients. NK cells Accrual ongoing.

Cord blood NK cells Allogeneic, cord blood-derived, CD3-depleted, MEL (d − 7) with delayed auto-HCT Large-scale expansion of NK cells achieved from NCT01729091 K562-based aAPC+IL2-expanded NK cells (NK cells infused day − 5) Lenalidomide frozen, widely available source. Accrual ongoing days − 8to− 2 Low dose IL2 days − 5to− 1 Abbreviations: aAPC = artificial Ag-presenting cell; auto-HCT = autologous hematopoietic cell transplantation; CAR = chimeric Ag receptor; Dex = dexamethasone; Flu = fludarabine; MDSC = myeloid-derived suppressor cell; MEL = high-dose melphalan; MIL = marrow-infiltrating lymphocyte; NK = natural killer; PCM = plasma cell myeloma; PDE-5 = phosphodiesterase-5. Notably, combination therapy with autologous myeloma cell vaccine is under investigation (NCT01045460). – 780 775 Transplant and immunotherapy for myeloma N Lendvai et al 776

Table 5. Comparison of TCR transgenic vs CAR T cells

TCR transgenic T cells CAR T cells

Potential targets Intracellular or surface Ags Surface Ags only Potential for afﬁnity enhancement of receptor Yes Yes to improve target binding Restricted to particular HLA type Yes No Potential for mispairing of receptor with Yes No endogenous TCR Potential for on-target, off-tumor toxicity Yes Yes (for example, via recognition of normal cells expressing Ag) Demonstration of serious off-target toxicity Yes (recognition of similar epitope expressed by a Yes (cytokine-release syndrome, different protein in normal tissue (for example, anaphylaxis to murine sequences in heart muscle)) CAR construct) Demonstration of durable clinical efﬁcacy in Not to date Yes hematologic malignancies (ongoing trials in myeloma, AML) (ALL, CLL, non-Hodgkin’s lymphoma) Abbreviation: CAR = chimeric Ag receptor.

CELLULAR THERAPIES the inhibitory KIR signal is not provided and NK cell activation and As described above, several studies have now demonstrated the cytotoxicity occurs. This KIR ‘mismatch’ has been associated with safety and feasibility of adoptively transferring autologous PBL in improved outcomes in patients with myeloma and other conjunction with vaccination and auto-HCT. These studies, which hematologic malignancies undergoing allo-HCT, presumably due 69–71 utilized polyclonal PBL, relied upon a concomitantly administered to increased NK cell activity against the tumor cells. Van Rhee tumor vaccine to drive the IR toward a particular Ag or Ags. In and colleagues attempted to harness this NK cell vs myeloma addition to these vaccine-based approaches, a number of other effect in conjunction with an auto-HCT. Following lymphodeplet- cellular therapy strategies (Table 4) is being explored in ing conditioning with fludarabine, dexamethasone and melpha- conjunction with auto-HCT to target myeloma, including transfer lan, 10 patients with relapsed myeloma received haplo-identical, of marrow-infiltrating lymphocytes (MILs), killer Ig-like receptor KIR-mismatched, IL2-activated NK cell-enriched infusions on days (KIR)-mismatched NK cells, and genetically-modified T cells 0 and +2, followed by IL2 administration days +1 to +11, followed expressing transgenic TCRs (Tg TCRs) or chimeric Ag receptors by infusion of autologous stem cells on day +14. Toxicities were (CARs). primarily related to IL2 (fevers/rigors and hypotension) or neutropenic infections. Fifty percent of patients achieved a CR fi or near CR. Donor NK cells were able to lyse KIR-mismatched PCM Marrow-in ltrating lymphocytes cell lines or primary PCM cells ex vivo, and transiently expanded Borrello and colleagues at Johns Hopkins demonstrated that MILs in vivo, but were eliminated by day 14, likely by residual recipient were phenotypically and functionally different from PBL, with T cells.72 This study demonstrated the proof of principle that this greater proportion of central memory T cells, greater recognition approach could be feasible, but was limited by the inability to and killing of myeloma cells upon re-activation, and greater generate large numbers of activated NK cells ex vivo, and by 13 capacity for trafficking to the marrow/tumor microenvironment. limited persistence of these cells in vivo. To overcome these This led to a phase I trial of adoptive transfer of autologous, limitations, this group is currently exploring ex vivo NK cell ex vivo-activated MILs on day +3 following high-dose melphalan activation with 4-1BB ligand and IL-15, as well as alternative and auto-HCT in myeloma patients. Initial results from 22 patients conditioning regimens, to enhance the expansion and persistence demonstrated that the generation and infusion of large numbers of transferred NK cells,73 with ongoing clinical studies in relapsed/ of activated MILs was feasible and safe, with mild autologous refractory myeloma (NCT01313897). Ongoing pilot trials at other GVHD of the skin (not requiring therapy) seen in 32%. Clinical centers exploring alternative expansion techniques before infu- responses included 6 CR (27%) and 10 PR (45%). MILs recovered sion of haplo-identical NK cells derived from peripheral blood74 post-HCT had greater recognition of PCM cells ex vivo, and ex vivo (NCT01040026) or umbilical cord blood75 (NCT01729091) follow- proliferation and IFN-gamma production by marrow T cells post ing auto-HCT will provide further insight into the safety and HCT correlated with depth of response.66 On the basis of this potential efficacy of this approach in myeloma patients. initial experience, a randomized phase II trial is currently underway (NCT01858558) comparing auto-HCT ± activated MILs in patients TCR transgenic T cells with high-risk myeloma. All patients in this study also receive Another adoptive therapy strategy involves transducing auto- tadalafil, a phosphodiesterase type 5 inhibitor, post HCT, based on logous T cells to express a Tg TCR specific for a particular tumor Ag the data suggesting that this agent may inhibit the function of peptide-MHC complex. This approach has been successfully myeloid-derived suppressor cells, a population of granulocytic and employed in several pilot studies in solid tumors,76,77 with monocytic cells that accumulate in the tumor microenvironment objective tumor regressions noted. In an interim analysis of a in multiple cancers, including myeloma, and suppress the activity phase II study (NCT01352286) in myeloma, 12 HLA-A0201+ high- of infiltrating immune cells via upregulation of inducible nitric risk or relapsed patients undergoing auto-HCT were treated on oxide synthase and arginase-1.67,68 day +2 with autologous T cells engineered to express an affinity- enhanced, Tg TCR recognizing a peptide epitope shared by the NK cell transfer NY-ESO-1 and LAGE-1 Ags. Transgenic T cells were well tolerated, NK cell activation is controlled by a balance of activating and expanded, and homed to marrow, and persisted as far out as inhibitory signals transduced through various surface receptors, 1-year postinfusion in some patients.78 Clinical activity was including KIRs that bind to MHC class I molecules. When a target encouraging in this population, with 7 of 11 evaluable patients cell (for example, a tumor cell) lacks the appropriate MHC I ligand, achieving VGPR or better.79 Although this strategy merits further

Bone Marrow Transplantation (2015) 770 – 780 © 2015 Macmillan Publishers Limited Transplant and immunotherapy for myeloma N Lendvai et al 777 study, it is limited by applicability only to particular HLA types, recently opened at the University of Pennsylvania (NCT02135406). the potential for recombination with the endogenous TCR and the This is the first study of CART19 cells in myeloma, as well as the potential for off-target toxicity due to T-cell recognition of first study combining CAR T cells with an auto-HCT. The rationale alternative peptides with unexpected homology to the tumor for giving CART19 cells in myeloma, which typically is CD19- Ag peptide. This latter toxicity was demonstrated in two patients negative, is to target clonogenic PCM precursor cells that are who received affinity-enhanced TCR transgenic T cells specific for thought to reside within the CD19+ B-cell compartment,99,100 and a MAGE-A3 peptide and who developed fatal cardiac toxicities may contribute to resistance to high-dose melphalan and other – within a few days after infusion. Subsequent studies demonstrated therapies.101 103 The primary endpoint is safety and tolerability, that the Tg TCR also recognized an epitope from titin, a cardiac and an important secondary endpoint is an intrapatient compar- myocyte protein, leading to massive cardiac T-cell infiltration and ison of time to progression (TTP) after salvage auto-HCT+CART19 cardiomyocyte necrosis.80 Thus, very careful selection of potential cells to TTP after first auto-HCT. As TTP after salvage auto-HCT is peptide targets, with extensive in silico and in vitro testing for typically significantly shorter than after first auto-HCT,104,105 any cross-reactivity to normal tissue epitopes, is necessary for this improvement in this expected TTP would provide a preliminary approach. signal of efficacy that would support additional studies in larger numbers of patients. CAR T cells The CAR approach combines the effector function, trafficking and CONCLUSIONS long-lived persistence of T cells with the ability of Abs to recognize Novel drugs and auto-HCT have revolutionized the care of PCM, surface Ags in a non-MHC restricted manner. This is one of several dramatically improving response rates and OS. However, broadly key differences (Table 5) of CAR T cells compared with TCR applicable curative therapy remains an elusive goal. Immunother- transgenic T cells, which recognize peptides only in the context of apy in the peri-auto-HCT setting is a promising path to achieving fi a speci c MHC molecule. The tumor-binding function of a CAR is this goal by augmenting consolidation therapy to eradicate fi accomplished by engineering T cells to express an Ag-speci c minimal residual disease. The next generation of clinical single-chain variable fragment (scFv), containing the VH and VL investigation faces the challenge of combining these approaches chains joined by a peptide linker, which is then linked to an into safe, effective methods to efficiently induce or enhance host- intracellular signaling domain that mediates T-cell activation. First vs-myeloma immunity. As noted, a few forward thinking trials are generation CARs contain a minimal TCR signaling domain already combining immune checkpoint inhibitors with vaccines or ζ consisting of TCR . Second generation CARs contain double co- immunomodulatory drugs, and it is likely that combination ζ stimulatory signaling domains: either CD28 and TCR or 4-1BB and immunotherapy will be necessary to elicit and sustain protective TCRζ, which augment the expansion and persistence of the CAR- 81 antimyeloma immunity. Correlative analysis is an indispensible transduced T cells. Adoptive transfer of second-generation CAR component of these investigations, as understanding of the T cells targeting the B-cell marker CD19 (called CART19 cells) has mechanisms of IR to myeloma will be necessary to rationally demonstrated remarkable clinical activity in relapsed/refractory combine and sequence cellular therapy, vaccines and immune B-cell malignancies such as pre-B-cell ALL, CLL and B-cell non- modulation with auto-HCT. Hodgkin’s lymphomas, in some cases inducing remissions lasting 43 years after a single infusion.82–85 CART19 therapy can also cause serious toxicities, including prolonged B-cell aplasia that CONFLICT OF INTEREST may require intravenous immunoglobulin (IVIG) replacement, and ADC has received research funding from Bristol-Myers Squibb; has been on the a cytokine-release syndrome that may require intensive care advisory board of Bristol-Myers Squibb and Janssen. The remaining authors declare support and cytokine-specific therapies such as the IL6-receptor no conflict of interest. antagonist tocilizumab. This CAR approach is now being explored in myeloma, with a REFERENCES number of potential targets identified, including CD138, CD38, CD44v, kappa light chain, Lewis Y, CS1/SLAMF7 and BCMA. 1 Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF et al. A pro- Preclinical studies have shown that T cells or in some cases, NK spective, randomized trial of autologous bone marrow transplantation and cells expressing CARs specific for these targets can kill myeloma chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J 86–91 Med 1996; 335:91–97. cells in vitro and in xenograft models, and several have now 2 Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, Hawkins K et al. High-dose entered early-phase clinical trials. One target of interest is B-cell chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. maturation Ag (BCMA). Several studies have demonstrated near N Engl J Med 2003; 348: 1875–1883. universal BCMA expression in PCM cell lines and primary patient 3 Koreth J, Cutler CS, Djulbegovic B, Behl R, Schlossman RL, Munshi NC et al. High- PCM cells, and signaling through BCMA provides a survival signal dose therapy with single autologous transplantation versus chemotherapy for to myeloma cells and promotes resistance to antimyeloma newly diagnosed multiple myeloma: A systematic review and meta-analysis of therapies.92–96 BCMA surface expression appears higher in PCM randomized controlled trials. Biol Blood Marrow Transplant 2007; 13:183–196. 4 Bensinger WI, Maloney D, Storb R. Allogeneic hematopoietic cell transplantation cells compared with normal plasma cells, and protein expression 38 – has not been reported in other normal cells or tissues, other than for multiple myeloma. Semin Hematol 2001; : 243 249. 97,98 5 Gahrton G, Tura S, Ljungman P, Belanger C, Brandt L, Cavo M et al. Allogeneic some mature B-cell subsets and plasmacytoid DC. Carpenter bone marrow transplantation in multiple myeloma. European Group for Bone 97 et al. demonstrated that BCMA-redirected CAR T cells could Marrow Transplantation. N Engl J Med 1991; 325:1267–1273. proliferate, degranulate and produce cytokines when exposed to 6 Bensinger WI, Buckner CD, Anasetti C, Clift R, Storb R, Barnett T et al. Allogeneic BCMA-expressing targets and could specifically kill PCM cell lines marrow transplantation for multiple myeloma: an analysis of risk factors on and primary patient PCM cells. Adoptive transfer of BCMA CAR outcome. Blood 1996; 88: 2787–2793. T cells into immunodeficient mice harboring palpable RPMI-8226 7 Crawley C, Lalancette M, Szydlo R, Gilleece M, Peggs K, Mackinnon S et al. myeloma xenografts eradicated the tumor in 100% of mice. These Outcomes for reduced-intensity allogeneic transplantation for multiple data have led to a pilot clinical trial that opened in August 2014 at myeloma: an analysis of prognostic factors from the Chronic Leukaemia Working Party of the EBMT. Blood 2005; 105:4532–4539. the National Cancer Institute, using CD3/CD28 BCMA CAR T cells 8 Huff CA, Fuchs EJ, Noga SJ, O'Donnell PV, Ambinder RF, Diehl L et al. Long-term for relapsed/refractory myeloma (NCT02215967). follow-up of T cell-depleted allogeneic bone marrow transplantation in Finally, a pilot study investigating CART19 infusion in conjunc- refractory multiple myeloma: importance of allogeneic T cells. Biol Blood Marrow tion with salvage autologous stem cell transplant for myeloma has Transplant 2003; 9: 312–319.

© 2015 Macmillan Publishers Limited Bone Marrow Transplantation (2015) 770 – 780 Transplant and immunotherapy for myeloma N Lendvai et al 778 9 Kroger N, Shimoni A, Zagrivnaja M, Ayuk F, Lioznov M, Schieder H et al. Low-dose 31 Robert C, Thomas L, Bondarenko I, O'Day S M DJ, Garbe C et al. Ipilimumab plus thalidomide and donor lymphocyte infusion as adoptive immunotherapy after dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011; allogeneic stem cell transplantation in patients with multiple myeloma. Blood 364: 2517–2526. 2004; 104: 3361–3363. 32 Kitano S, Tsuji T, Liu C, Hirschhorn-Cymerman D, Kyi C, Mu Z et al. Enhancement 10 Dhodapkar MV, Geller MD, Chang DH, Shimizu K, Fujii S, Dhodapkar KM et al. of tumor-reactive cytotoxic CD4+ T cell responses after ipilimumab A reversible defect in natural killer T cell function characterizes the progression treatment in four advanced melanoma patients. Cancer Immunol Res 2013; 1: of premalignant to malignant multiple myeloma. J Exp Med 2003; 197: 235–244. 1667–1676. 33 Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S et al. 11 Dhodapkar MV, Krasovsky J, Osman K, Geller MD. Vigorous premalignancy- Immunologic correlates of the abscopal effect in a patient with melanoma. N specific effector T cell response in the bone marrow of patients with monoclonal Engl J Med 2012; 366: 925–931. gammopathy. J Exp Med 2003; 198: 1753–1757. 34 Weber JS, Hamid O, Chasalow SD, Wu DY, Parker SM, Galbraith S et al. 12 Dhodapkar MV, Krasovsky J, Olson K. T cells from the tumor microenvironment Ipilimumab increases activated T cells and enhances humoral immunity in of patients with progressive myeloma can generate strong, tumor-specific patients with advanced melanoma. J Immunother 2012; 35:89–97. cytolytic responses to autologous, tumor-loaded dendritic cells. Proc Natl Acad 35 Naidoo J, Page DB, Wolchok JD. Immune checkpoint blockade. Hematol Oncol Sci USA 2002; 99: 13009–13013. Clin North Am 2014; 28:585–600. 13 Noonan K, Matsui W, Serafini P, Carbley R, Tan G, Khalili J et al. Activated marrow- 36 Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to infiltrating lymphocytes effectively target plasma cells and their clonogenic activate anti-tumor immunity. Curr Opin Immunol 2012; 24:207–212. precursors. Cancer Res 2005; 65: 2026–2034. 37 Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R et al. Safety and tumor 14 Atanackovic D, Arfsten J, Cao Y, Gnjatic S, Schnieders F, Bartels K et al. responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 2013; 369: Cancer-testis antigens are commonly expressed in multiple myeloma and 134–144. induce systemic immunity following allogeneic stem cell transplantation. Blood 38 Robert C, Ribas A, Wolchok JD, Hodi FS, Hamid O, Kefford R et al. 2007; 109: 1103–1112. Anti-programmed-death-receptor-1 treatment with pembrolizumab in 15 Goodyear O, Piper K, Khan N, Starczynski J, Mahendra P, Pratt G et al. CD8+ ipilimumab-refractory advanced melanoma: a randomised dose-comparison T cells specific for cancer germline gene antigens are found in many patients cohort of a phase 1 trial. Lancet 2014; 384:1109–1117. with multiple myeloma, and their frequency correlates with disease burden. 39 Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P et al. Safety and Blood 2005; 106: 4217–4224. activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 16 van Rhee F, Szmania SM, Zhan F, Gupta SK, Pomtree M, Lin P et al. NY-ESO-1 is 2012; 366: 2455–2465. highly expressed in poor-prognosis multiple myeloma and induces spontaneous 40 Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM et al. humoral and cellular immune responses. Blood 2005; 105: 3939–3944. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 2013; 369: 17 Benson DM Jr, Bakan CE, Mishra A, Hofmeister CC, Efebera Y, Becknell B et al. The 122–133. PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: 41 Armand P, Nagler A, Weller EA, Devine SM, Avigan DE, Chen YB et al. Disabling a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody. Blood immune tolerance by programmed death-1 blockade with pidilizumab after 2010; 116: 2286–2294. autologous hematopoietic stem-cell transplantation for diffuse large B-cell 18 Murillo O, Arina A, Hervas-Stubbs S, Gupta A, McCluskey B, Dubrot J et al. lymphoma: results of an international phase II trial. J Clin Oncol 2013; 31: Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in 4199–4206. mouse models of myeloma. Clin Cancer Res 2008; 14: 6895–6906. 42 Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement 19 Anderson KC, Soiffer R, DeLage R, Takvorian T, Freedman AS, Rabinowe SL et al. of PD-L1 on tumor cells in the escape from host immune system and tumor T-cell-depleted autologous bone marrow transplantation therapy: analysis of immunotherapy by PD-L1 blockade. Proc Natl Acad Sci USA 2002; 99: immune deficiency and late complications. Blood 1990; 76: 235–244. 12293–12297. 20 Guillaume T, Rubinstein DB, Symann M. Immune reconstitution and 43 Liu J, Hamrouni A, Wolowiec D, Coiteux V, Kuliczkowski K, Hetuin D et al. immunotherapy after autologous hematopoietic stem cell transplantation. Blood Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and 1998; 92: 1471–1490. increase expression after stimulation with IFN-{gamma} and TLR ligands via a 21 Rapoport AP, Aqui NA, Stadtmauer EA, Vogl DT, Fang HB, Cai L et al. MyD88-, TRAF6-, and MEK-dependent pathway. Blood 2007; 110:296–304. Combination immunotherapy using adoptive T-cell transfer and tumor antigen 44 Rosenblatt J, Glotzbecker B, Mills H, Vasir B, Tzachanis D, Levine JD et al. PD-1 vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to 2011; 117: 788–797. autologous dendritic cell/myeloma fusion vaccine. J Immunother 2011; 34: 22 Rapoport AP, Stadtmauer EA, Aqui N, Badros A, Cotte J, Chrisley L et al. 409–418. Restoration of immunity in lymphopenic individuals with cancer by vaccination 45 Tamura H, Ishibashi M, Yamashita T, Tanosaki S, Okuyama N, Kondo A et al. and adoptive T-cell transfer. Nat Med 2005; 11: 1230–1237. Marrow stromal cells induce B7-H1 expression on myeloma cells, 23 Paulos CM, Wrzesinski C, Kaiser A, Hinrichs CS, Chieppa M, Cassard L et al. generating aggressive characteristics in multiple myeloma. Leukemia 2013; 27: Microbial translocation augments the function of adoptively transferred 464–472. self/tumor-specific CD8+ T cells via TLR4 signaling. J Clin Invest 2007; 117: 46 Hallett WH, Jing W, Drobyski WR, Johnson BD. Immunosuppressive effects 2197–2204. of multiple myeloma are overcome by PD-L1 blockade. Biol Blood Marrow 24 Zhang B, Bowerman NA, Salama JK, Schmidt H, Spiotto MT, Schietinger A et al. Transplant 2011; 17: 1133–1145. Induced sensitization of tumor stroma leads to eradication of established cancer 47 Kearl TJ, Jing W, Gershan JA, Johnson BD. Programmed death receptor-1/ by T cells. J Exp Med 2007; 204:49–55. programmed death receptor ligand-1 blockade after transient lymphodepletion 25 Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ et al. to treat myeloma. J Immunol 2013; 190: 5620–5628. Removal of homeostatic cytokine sinks by lymphodepletion enhances the 48 Lesokhin AM, Ansell SM, Armand P, Scott EC, Halwani A, Gutierrez M et al. efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med 2005; Preliminary Results of a Phase I Study of Nivolumab (BMS-936558) in Patients 202: 907–912. with Relapsed or Refractory Lymphoid Malignancies. American Society of 26 Wrzesinski C, Paulos CM, Gattinoni L, Palmer DC, Kaiser A, Yu Z et al. Hematology Annual Meeting 2014, San Francisco, CA, USA. Hematopoietic stem cells promote the expansion and function of adoptively 49 Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, van der Burg SH, Offringa R. transferred antitumor CD8 T cells. J Clin Invest 2007; 117: 492–501. Superior induction of anti-tumor CTL immunity by extended peptide vaccines 27 Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U et al. Adoptive involves prolonged, DC-focused antigen presentation. Eur J Immunol 2008; 38: cell therapy for patients with metastatic melanoma: evaluation of intensive 1033–1042. myeloablative chemoradiation preparative regimens. J Clin Oncol 2008; 26: 50 Dyall R, Bowne WB, Weber LW, LeMaoult J, Szabo P, Moroi Y et al. Heteroclitic 5233–5239. immunization induces tumor immunity. J Exp Med 1998; 188: 1553–1561. 28 Egen JG, Kuhns MS, Allison JP. CTLA-4: new insights into its biological function 51 Keogh E, Fikes J, Southwood S, Celis E, Chesnut R, Sette A. Identification of new and use in tumor immunotherapy. Nat Immunol 2002; 3: 611–618. epitopes from four different tumor-associated antigens: recognition of naturally 29 Wolchok JD, Hodi FS, Weber JS, Allison JP, Urba WJ, Robert C et al. Development processed epitopes correlates with HLA-A*0201-binding affinity. J Immunol of ipilimumab: a novel immunotherapeutic approach for the treatment of 2001; 167: 787–796. advanced melanoma. Ann NY Acad Sci 2013; 1291:1–13. 52 Parkhurst MR, Salgaller ML, Southwood S, Robbins PF, Sette A, Rosenberg SA 30 Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB et al. et al. Improved induction of melanoma-reactive CTL with peptides from the Improved survival with ipilimumab in patients with metastatic melanoma. N Engl melanoma antigen gp100 modified at HLA-A*0201-binding residues. J Immunol JMed2010; 363:711–723. 1996; 157: 2539–2548.

Bone Marrow Transplantation (2015) 770 – 780 © 2015 Macmillan Publishers Limited Transplant and immunotherapy for myeloma N Lendvai et al 779 53 Pinilla-Ibarz J, Korontsvit T, Zakhaleva V, Roberts W, Scheinberg DA. Synthetic for relapsed myeloma in the setting of autologous stem cell transplantation. Br J peptide analogs derived from bcr/abl fusion proteins and the induction of Haematol 2008; 143: 641–653. heteroclitic human T-cell responses. Haematologica 2005; 90: 1324–1332. 73 Garg TK, Szmania SM, Khan JA, Hoering A, Malbrough PA, Moreno-Bost A et al. 54 Tangri S, Ishioka GY, Huang X, Sidney J, Southwood S, Fikes J et al. Structural Highly activated and expanded natural killer cells for multiple myeloma features of peptide analogs of human histocompatibility leukocyte antigen class immunotherapy. Haematologica 2012; 97: 1348–1356. I epitopes that are more potent and immunogenic than wild-type peptide. J Exp 74 Siegler U, Meyer-Monard S, Jorger S, Stern M, Tichelli A, Gratwohl A et al. Good Med 2001; 194:833–846. manufacturing practice-compliant cell sorting and large-scale expansion of 55 Chen JL, Dunbar PR, Gileadi U, Jager E, Gnjatic S, Nagata Y et al. Identification of single KIR-positive alloreactive human natural killer cells for multiple infusions to NY-ESO-1 peptide analogues capable of improved stimulation of tumor- leukemia patients. Cytotherapy 2010; 12: 750–763. reactive CTL. J Immunol 2000; 165:948–955. 75 Shah N, Martin-Antonio B, Yang H, Ku S, Lee DA, Cooper LJ et al. Antigen 56 Tyler EM, Jungbluth AA, O'Reilly RJ, Koehne G. WT1-specific T-cell responses in presenting cell-mediated expansion of human umbilical cord blood yields log- high-risk multiple myeloma patients undergoing allogeneic T cell-depleted scale expansion of natural killer cells with anti-myeloma activity. PLoS One 2013; hematopoietic stem cell transplantation and donor lymphocyte infusions. Blood 8: e76781. 2013; 121:308–317. 76 Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. 57 Carmon L, Avivi I, Kovjazin R, Zuckerman T, Dary L, Gatt ME et al. Phase I/II study Cancer regression in patients after transfer of genetically engineered lympho- exploring ImMucin, a pan-major histocompatibility complex, anti-MUC1 signal cytes. Science 2006; 314:126–129. peptide vaccine, in multiple myeloma patients. Br J Haematol (epub ahead of 77 Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME et al. Tumor print 11 December 2014; doi:10.1111/bjh.13245). regression in patients with metastatic synovial cell sarcoma and melanoma 58 Reichardt VL, Okada CY, Liso A, Benike CJ, Stockerl-Goldstein KE, Engleman EG using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol et al. Idiotype vaccination using dendritic cells after autologous peripheral blood 2011; 29:917–924. stem cell transplantation for multiple myeloma--a feasibility study. Blood 1999; 78 Kalos M, Rapoport AP, Stadtmauer EA, Vogl DT, Weiss BM, Binder-Scholl GK et al. 93 – : 2411 2419. Prolonged T cell persistence, homing to marrow and selective targeting of 59 Liso A, Stockerl-Goldstein KE, Auffermann-Gretzinger S, Benike CJ, Reichardt V, antigen positive tumor in multiple myeloma patients following adoptive transfer van Beckhoven A et al. Idiotype vaccination using dendritic cells after auto- of T cells genetically engineered to express an affinity-enhanced T cell receptor logous peripheral blood progenitor cell transplantation for multiple myeloma. against the cancer testis antigens NY-ESO-1 and Lage-1. Blood 2012; 120: 6 – Biol Blood Marrow Transplant 2000; :621 627. 755 (ASH Annual Meeting Abstracts). 60 Lacy MQ, Mandrekar S, Dispenzieri A, Hayman S, Kumar S, Buadi F et al. Idiotype- 79 Rapoport AP, Stadtmauer EA, Vogl DT, Weiss BM, Binder-Scholl GK, Brewer JE pulsed antigen-presenting cells following autologous transplantation for multi- et al. Adoptive transfer of gene-modified T-cells engineered to express high- ple myeloma may be associated with prolonged survival. Am J Hematol 2009; 84: affinity TCRs for cancer-testis antigens (CTAs) NY-ESO-1 or Lage-1, in MM 799–802. patients post auto-SCT. Blood 2012; 120: 472 (ASH Annual Meeting Abstracts). 61 Rosenblatt J, Avivi I, Vasir B, Uhl L, Munshi NC, Katz T et al. Vaccination with 80 Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L et al. dendritic cell/tumor fusions following autologous stem cell transplant induces Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in immunologic and clinical responses in multiple myeloma patients. Clin Cancer myeloma and melanoma. Blood 2013; 122:863–871. Res 2013; 19: 3640–3648. 81 Barrett DM, Singh N, Porter DL, Grupp SA, June CH. Chimeric antigen receptor 62 Rosenblatt J, Glotzbecker B, Mills H, Vasir B, Tzachanis D, Levine JD et al. PD-1 therapy for cancer. Annu Rev Med 2014; 65:333–347. blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to 82 Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K et al. Efficacy and toxicity autologous dendritic cell/myeloma fusion vaccine. J Immunother 2011; 34: management of 19-28z CAR T cell therapy in B cell acute lymphoblastic 409–418. leukemia. Sci Transl Med 2014; 6: 224ra25. 63 Rosenblatt J, Avivi I, Vasir D, Uhl L, Katz T, Somaiya P et al. Blockade of PD-1 in 83 Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor- combination with dendritic cell/myeloma fusion cell vaccination following modified T cells in chronic lymphoid leukemia. N Engl J Med 2011; 365:725–733. autologous stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 84 Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, S109–S109. Stetler-Stevenson M et al. Chemotherapy-refractory diffuse large B-cell 64 Rapoport AP, Aqui NA, Stadtmauer EA, Vogl DT, Xu YY, Kalos M et al. lymphoma and indolent B-cell malignancies can be effectively treated with Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/ autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and Oncol. (e-pub ahead of print 25 August 2014; doi: JCO.2014.56.2025). costimulated autologous T cells. Clin Cancer Res 2014; 20: 1355–1365. 65 Cohen AD, Lendvai N, Gnjatic S, Jungbluth AA, Bertolini S, Pan L et al. MAGE-A3 85 Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ et al. Chimeric recombinant protein (recMAGE-A3) immunotherapy and autologous peripheral antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371 – blood lymphocyte (PBL) infusion is safe and induces robust humoral immune : 1507 1517. responses in multiple myeloma (MM) patients undergoing autologous stem cell 86 Jiang H, Zhang W, Shang P, Zhang H, Fu W, Ye F et al. Transfection of chimeric transplantation (autoSCT). J Clin Oncol 2013; 122:154–154. anti-CD138 gene enhances natural killer cell activation and killing of multiple 8 – 66 Noonan K, Huff CA, Davis Sproul JM, Lemas MVM, Rudraraju L, Luznik L et al. myeloma cells. Mol Oncol 2014; : 297 310. Phase I/II study of marrow infiltrating lymphocytes (MILs) generates measurable 87 Mihara K, Bhattacharyya J, Kitanaka A, Yanagihara K, Kubo T, Takei Y et al. T-cell myeloma-specific immunity in the autologous stem cell transplant (SCT) setting. immunotherapy with a chimeric receptor against CD38 is effective in eliminating 26 – 53rd ASH, Annual Meeting and Exposition 10–13 December 2011, San Diego, CA, myeloma cells. Leukemia 2012; : 365 367. USA, Abstract 997. 88 Vera J, Savoldo B, Vigouroux S, Biagi E, Pule M, Rossig C et al. T lymphocytes fi 67 Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W et al. Phospho- redirected against the kappa light chain of human immunoglobulin ef ciently 108 – diesterase-5 inhibition augments endogenous antitumor immunity by reducing kill mature B lymphocyte-derived malignant cells. Blood 2006; :3890 3897. myeloid-derived suppressor cell function. J Exp Med 2006; 203: 2691–2702. 89 Casucci M, Nicolis di Robilant B, Falcone L, Camisa B, Norelli M, Genovese P et al. 68 Ramachandran IR, Martner A, Pisklakova A, Condamine T, Chase T, Vogl T et al. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid Myeloid-derived suppressor cells regulate growth of multiple myeloma by leukemia and multiple myeloma. Blood 2013; 122:3461–3472. inhibiting T cells in bone marrow. J Immunol 2013; 190: 3815–3823. 90 Peinert S, Prince HM, Guru PM, Kershaw MH, Smyth MJ, Trapani JA et al. 69 Kroger N, Zabelina T, Berger J, Duske H, Klyuchnikov E, Binder T et al. Donor KIR Gene-modified T cells as immunotherapy for multiple myeloma and haplotype B improves progression-free and overall survival after allogeneic acute myeloid leukemia expressing the Lewis Y antigen. Gene Ther 2010; 17: hematopoietic stem cell transplantation for multiple myeloma. Leukemia 2011; 678–686. 25: 1657–1661. 91 Chu J, Deng Y, Benson DM, He S, Hughes T, Zhang J et al. CS1-specific chimeric 70 Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Marsh SG et al. Donor antigen receptor (CAR)-engineered natural killer cells enhance in vitro and killer cell Ig-like receptor B haplotypes, recipient HLA-C1, and HLA-C mismatch in vivo antitumor activity against human multiple myeloma. Leukemia 2014; 28: enhance the clinical benefit of unrelated transplantation for acute myelogenous 917–927. leukemia. J Immunol 2014; 192: 4592–4600. 92 Claudio JO, Masih-Khan E, Tang H, Goncalves J, Voralia M, Li ZH et al. A molecular 71 Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A et al. compendium of genes expressed in multiple myeloma. Blood 2002; 100: Effectiveness of donor natural killer cell alloreactivity in mismatched hemato- 2175–2186. poietic transplants. Science 2002; 295: 2097–2100. 93 Novak AJ, Darce JR, Arendt BK, Harder B, Henderson K, Kindsvogel W et al. 72 Shi J, Tricot G, Szmania S, Rosen N, Garg TK, Malaviarachchi PA et al. Infusion of Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for haplo-identical killer immunoglobulin-like receptor ligand mismatched NK cells growth and survival. Blood 2004; 103:689–694.

© 2015 Macmillan Publishers Limited Bone Marrow Transplantation (2015) 770 – 780 Transplant and immunotherapy for myeloma N Lendvai et al 780 94 Moreaux J, Legouffe E, Jourdan E, Quittet P, Reme T, Lugagne C et al. BAFF and 102 Matsui W, Wang Q, Barber JP, Brennan S, Smith BD, Borrello I et al. Clonogenic APRIL protect myeloma cells from apoptosis induced by interleukin 6 depriva- multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer tion and dexamethasone. Blood 2004; 103: 3148–3157. Res 2008; 68:190–197. 95 Bellucci R, Alyea EP, Chiaretti S, Wu CJ, Zorn E, Weller E et al. Graft-versus-tumor 103 Leung-Hagesteijn C, Erdmann N, Cheung G, Keats JJ, Stewart AK, Reece DE et al. response in patients with multiple myeloma is associated with antibody Xbp1s-negative tumor B cells and pre-plasmablasts mediate therapeutic response to BCMA, a plasma-cell membrane receptor. Blood 2005; 105: proteasome inhibitor resistance in multiple myeloma. Cancer Cell 2013; 24: 3945–3950. 289–304. 96 Neri P, Kumar S, Fulciniti MT, Vallet S, Chhetri S, Mukherjee S et al. 104 Olin RL, Vogl DT, Porter DL, Luger SM, Schuster SJ, Tsai DE et al. Second auto-SCT Neutralizing B-cell activating factor antibody improves survival and inhibits is safe and effective salvage therapy for relapsed multiple myeloma. Bone osteoclastogenesis in a severe combined immunodeﬁcient human multiple Marrow Transplant 2009; 43:417–422. myeloma model. Clin Cancer Res 2007; 13: 5903–5909. 105 Michaelis LC, Saad A, Zhong X, Le-Rademacher J, Freytes CO, Marks DI et al. 97 Carpenter RO, Evbuomwan MO, Pittaluga S, Rose JJ, Raffeld M, Yang S et al. B-cell Salvage second hematopoietic cell transplantation in myeloma. Biol Blood maturation antigen is a promising target for adoptive T-cell therapy of multiple Marrow Transplant 2013; 19:760–766. myeloma. Clin Cancer Res 2013; 19: 2048–2060. 106 Cohen AD, Lendvai N, Gnjatic S, Jungbluth AA, Bertolini S, Pan L et al. MAGE-A3 98 Tai YT, Mayes PA, Acharya C, Zhong MY, Cea M, Cagnetta A et al. Novel anti-B-cell recombinant protein (recMAGE-A3) immunotherapy and autologous peripheral maturation antigen antibody-drug conjugate (GSK2857916) selectively induces blood lymphocyte (PBL) infusion is safe and induces robust humoral immune killing of multiple myeloma. Blood 2014; 123: 3128–3138. responses in multiple myeloma (MM) patients undergoing autologous stem cell 99 Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y et al. Char- transplantation (autoSCT). American Society of Hematology Annual Meeting 2013, acterization of clonogenic multiple myeloma cells. Blood 2004; 103: 2332–2336. New Orleans, LA, USA. 100 Bergsagel PL, Smith AM, Szczepek A, Mant MJ, Belch AR, Pilarski LM. In multiple 107 Borrello I, Biedrzycki B, Sheets N, George B, Racke F, Loper K et al. Autologous myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral tumor combined with a GM-CSF-secreting cell line vaccine (GVAX(R)) following blood cells expressing CD38, CD56, and monotypic Ig light chain. Blood 1995; 85: autologous stem cell transplant (ASCT) in multiple myeloma. ASH Annual 436–447. Meeting Abstracts 2004; 104: 440. 101 Boucher K, Parquet N, Widen R, Shain K, Baz R, Alsina M et al. Stemness of B-cell 108 Massaia M, Borrione P, Battaglio S, Mariani S, Beggiato E, Napoli P et al. Idiotype progenitors in multiple myeloma bone marrow. Clin Cancer Res 2012; 18: vaccination in human myeloma: generation of tumor-speciﬁc immune responses 6155–6168. after high-dose chemotherapy. Blood 1999; 94:673–683.