Current Strategies for the Immunotherapy of Multiple Myeloma

The introduction of cellular immunotherapies using genetically modified T cells has revolutionized the treatment of patients with B-cell lymphomas. However, despite the progress made in this field, similarly effective immunotherapeutic approaches have not yet been identified for patients with solid tumors or other hematologic malignancies such as multiple myeloma. Here we outline the most promising novel cellular immune strategies for patients with multiple myeloma. In addition, we highlight combinatorial approaches that, it is hoped, will further optimize cellular immunotherapies for myeloma and lead to deep and durable responses and, possibly, even cures.

Introduction

Allogeneic stem cell transplantation (alloSCT) with or without donor lymphocyte infusion (DLI) was one of the earliest forms of adoptive cell transfer (ACT) in multiple myeloma and remains one of the very few therapeutic options with curative potential in patients with this malignancy. Importantly, patients in whom graft-vs-host disease (GvHD) develops are less likely to experience relapses, which supports the hypothesis of a T-cell–mediated graft-vs-myeloma (GvM) effect.[1] At the same time, GvHD-associated morbidity and mortality, which is mediated by alloreactive T cells, is a serious concern that prevents alloSCT and DLI from becoming a standard therapy for patients with multiple myeloma.[2] Therefore, it would be highly desirable to optimize ACT for multiple myeloma by separating GvHD from GvM through either enrichment or ex vivo engineering of tumor-specific T cells.

One of the first ACT approaches was the administration of autologous tumor-infiltrating lymphocytes (TILs) in patients with refractory melanoma. Solid tumors such as melanoma are highly enriched in tumor-specific T cells, but because of the immunosuppressive environment created by the tumor, these immune cells are not sufficiently able to destroy the malignant cells. By removing TILs from this immunosuppressive environment and expanding and activating them ex vivo, their effector functions can be restored. Lymphodepletion prior to infusion of TILs and simultaneous administration of high-dose interleukin (IL)-2 yielded impressive clinical results in patients with metastatic melanoma.[3,4] Two factors that prevent more widespread use of TILs for the treatment of melanoma and other solid tumors are the limited lifespan of these heavily expanded cells, and the expensive, time-consuming, and technically demanding production process.[5]

In the case of multiple myeloma, the bone marrow is enriched in tumor-reactive T cells, or marrow-infiltrating lymphocytes (MILs),[6] which theoretically can be used analogously to TILs. A clinical trial that included 25 patients with multiple myeloma who received CD3/CD28-expanded and IL-2–activated autologous MILs after myeloablative therapy showed a 90% reduction of disease burden and increased overall survival.[7] At this point, it remains to be seen whether the same limitations identified in the production of TILs will also prohibit more widespread use of MILs in multiple myeloma.

Chimeric Antigen Receptors for the Treatment of Multiple Myeloma

Chimeric antigen receptors (CARs) are artificial proteins that result from fusing antigen-binding domains to signaling domains derived from receptors and coreceptors involved in T-cell activation.[8] When expressed in immune effector cells, CARs mediate strong cytotoxic activity against cells that express the target antigen (Figure). In their most common format, CARs comprise single-chain variable fragments derived from the variable regions of antibodies or known ligands or receptors; scaffolding domains derived from CD8a or immunoglobulin heavy chains; and the signaling subunits of CD3z alone or in combination with single or multiple costimulatory proteins, such as 4-1BB, CD28, or OX40.[9]

The initial clinical use of CARs has generated remarkable results. Targeting different B-cell malignancies, such as therapy-refractory chronic lymphocytic leukemia (CLL), using CAR T cells that recognize CD19 has resulted not only in an overwhelming response rate of 57% to 75% but in lasting remissions in a substantial proportion of patients.[10,11] Currently, multiple CD19 CAR products are in late-stage clinical trials, and a large number of constructs that target other antigens have moved into the clinic.

A variety of CAR products have been developed for the treatment of multiple myeloma, and recent data from early-phase clinical trials allow an initial assessment of their efficacy in this malignancy. A case report was published of a patient with refractory multiple myeloma treated with CAR T cells using the same construct recognizing CD19 as in earlier trials that targeted lymphoma. Importantly, while CD19 is usually absent from the majority of conventional multiple myeloma cells, targeting B cells-possibly including multiple myeloma precursor cells-represents an interesting strategy to eradicate disease. The treated patient showed a dramatic response by achieving a complete remission that lasted over 1 year.[12] Because of this therapy’s uncommon approach, however, it remains to be seen whether similar responses can be achieved in a larger cohort of patients.

Recently, two reports were published on the use of CAR T cells that target more traditional markers of multiple myeloma plasma cells: CD138[13] and the immunoglobulin kappa light chain.[14] Both trials reported few toxicities, but unfortunately objective responses were not observed for the 7 and 5 treated multiple myeloma patients, respectively.

Finally, the most promising results have been seen in trials using CAR T cells that target B-cell maturation antigen (BCMA). BCMA is specifically expressed in the later stages of development of B cells, including plasma cells and multiple myeloma cells.[15] In a dose-escalation trial, the 2 patients treated with the highest CAR T-cell dose initially had a very good partial response and a stringent complete response, respectively.[16] However, the latter patient relapsed after 17 weeks. Overall, some efficacy of CAR T cells for the treatment of multiple myeloma has been shown; however, so far no obvious ideal candidate has emerged from the initial trials.

Strategies for Improving CAR Specificity and Reactivity

A major roadblock preventing more widespread application of CARs is their lack of tissue specificity, which usually results in the unintended targeting of one or more types of healthy tissues.[11,17] This not only causes immediate potentially life-threatening toxicities but limits the group of targetable antigens to those that lack expression on all vital healthy tissues. Efforts have been made to identify novel targets exclusively expressed on tumor cells,[18-20] but extensive research in this area suggests that few truly cancer-specific surface antigens exist.

Alternative strategies have been developed that require binding of two or more antigens to confer T-cell activation. Specifically, the CD3z signaling domain and the costimulatory domains can be split into two CAR constructs with different specificities.[21] In this setting, only CAR T cells that bind to both antigens will receive a sustained activating signal. A potential drawback of this approach may be the substantial cytotoxicity exerted by CAR T cells that receive activation through the CD3z-containing CAR alone, which has been demonstrated previously.[22,23] Alternatively, activating CAR constructs could be combined with inhibitory CARs using signaling domains derived from coinhibitory T-cell receptors (TCRs).[24] Recently, researchers developed an elegant approach that requires binding of two or more CARs with a synthetic Notch construct, which induces expression of the second CAR as a consequence of binding of the first CAR.[25]

Another cause for concern in the clinical use of CAR T cells is the expansion of cells within the patient, which leads to difficult-to-predict kinetics and cumulative cytotoxic activity. To address this problem, different strategies have been developed in the preclinical setting. While most of the earliest CAR T-cell approaches rely on the generation of cells that stably express CAR constructs following retroviral transduction, an alternative method has been proposed that uses transient transfection of CAR mRNA, resulting in the clearance of CARs within days.[26] Alternatively, suicide switches[27,28] and truncated surface molecules targetable by existing US Food and Drug Administration–approved monoclonal antibodies[29] have been developed, which allow conditional deletion of CAR T cells (in case severe adverse events occur) or the removal of CAR T cells after successful treatment. However, there are potential drawbacks to this approach because of the potential induction of construct-specific immune responses that complicate later application of the same product.[30,31] In addition, one of the major parameters that determines the activity of CAR T cells is their persistence, which these approaches abrogate.[32]

A molecular switch that allows conditional activation of CAR T cells has been developed using a small molecule to trigger recruitment of separately expressed signaling domains to a truncated CAR.[33] This system addresses the problem of CAR-directed immunogenicity arising from deletion of CAR T cells via suicide genes while allowing tunable control of T-cell activation after their injection. However, to date, none of these more advanced molecular tools that aim to control CAR T-cell activity in vivo have been studied in clinical trials.

Current Challenges in CAR Immunotherapy for Hematologic Malignancies

While the advent of CAR T cells has reinvigorated the field of adoptive T-cell therapy and led to encouraging initial clinical results, many obstacles remain. In particular, a major issue is the frequent occurrence of cytokine release syndrome (CRS), an often life-threatening complication that arises from the potent activation of, and cytokine secretion by, CAR T cells. CRS, which results from efficient recognition and targeting of tumor cells, appears to be closely linked to CAR T-cell efficacy.[34-36] It is therefore unclear whether CRS can be addressed without compromising antitumor activity.

To date, the vast majority of cases can be controlled in the intensive care setting by neutralization of IL-6, a cytokine centrally involved in T-cell activation and proliferation.[37] However, it should be noted that, until recently, patients treated with CAR T cells have been relatively young and otherwise healthy. Older patients, such as those with multiple myeloma, who have additional comorbidities may show more severe symptoms and respond less favorably to IL-6 neutralization. Additional measures to control the activation state of CAR T cells in vivo, such as those described previously, may be necessary to provide safe treatments for these patients.

Escape variants are another problem commonly observed in patients treated with modalities that target single antigens. For example, patients treated with CD19-specific CAR T cells have been shown to relapse with CD19-negative blasts.[38] Different parameters govern the likelihood of target antigen downregulation, such as general transcriptional plasticity, functional relevance of the targeted antigen, and intratumoral heterogeneity. To address these issues, it will be essential to identify ideal antigens, which would be shared among most if not all tumor cells and patients and which are functionally essential for the malignant phenotype.[39]

An initially unexpected problem observed in patients treated with CD19 CAR T cells, which recently resulted in a temporary halt of one of the largest clinical CAR T-cell trials, is the occurrence of neurologic toxicities; these have been life-threatening in some cases.[35,36] While the mechanism of this complication remains incompletely understood, it may be linked to the targeting of CD19, since similar symptoms have been observed in patients treated with the bispecific anti-CD19/CD3 antibody blinatumomab.[40,41] However, no conclusive evidence about the probable causes has been provided, and the recent halt serves as an important reminder that the biologic implications of CAR T-cell treatment remain incompletely understood. Treatment will require careful monitoring, especially when novel antigens are targeted.

TCR-Engineered T Cells as an Adoptive Immunotherapy for Multiple Myeloma

TCR engineering is a promising alternative approach, which solves the majority of the technical and logistical problems associated with the generation of sufficient numbers of TILs. The TCR is a heterodimer that consists of the TCR α and β chain. Peptide antigen recognition is usually mediated by the hypervariable CDR3 region located in the variable parts of both receptor chains, whereas human leukocyte antigen (HLA)-binding residues are located mainly in the CDR1 and CDR2 loops. Major histocompatibility complex (MHC) class I molecules preferentially present 8 to 10 short amino acid peptide fragments to CD8 T cells, whereas CD4 T cells recognize longer peptides of 15 to 20 amino acids presented by MHC class II molecules. By genetically transferring the antigen-specific receptor chains to patient-derived primary T cells, they acquire the ability to recognize and destroy tumor cells that express the respective antigens. In contrast to CAR T cells, which recognize surface molecules through direct recognition by their binding domains, TCR T cells are able to detect intracellular peptide antigens presented by HLA molecules (see Figure).

Potential TCR Target Antigens in Multiple Myeloma

The ability of TCR T cells to recognize intracellular antigens considerably broadens the repertoire of potential target molecules compared with that of CAR T cells. This includes mutated neo-epitopes, which can differ from their wild-type variants by just one amino acid. As long as the peptide fragment that contains the mutated residue is presented by HLA molecules, TCRs are much more likely to recognize these antigens than are CAR T cells, which frequently bind to conformational epitopes. Point mutations are able to elicit robust T-cell responses that can be exploited for therapeutic use.[42,43] Mutations in multiple myeloma occur more frequently than in other hematologic malignancies, with an average of 3,000 genomic mutations compared with 1,500 to 2,000 mutations in CLL, acute myeloid leukemia, and acute lymphocytic leukemia.[44] However, the frequency of mutational epitopes that are immunogenic is very low, given that only a fraction of peptides that contain the mutation will be presented by HLA molecules.

One of the most promising groups of tumor-associated antigens currently pursued as targets for immunotherapeutic approaches in multiple myeloma are cancer-testis antigens (CTAs). Multiple myeloma is considered the malignancy with the highest expression of CTAs. Because of their mainly intracellular expression, CTAs are not easily accessible to conventional CAR T cells but can be recognized by TCR-engineered T cells. Several features make them very promising as target molecules for ACT[45]:

• Their expression is highly restricted to malignantly transformed cells. In healthy tissues, their expression is low to absent. Only germline tissues express considerable amounts of CTAs, but because of the absence of MHC molecules on those cells, they are practically “invisible” to T cells.

• By promoting cell proliferation and suppression of apoptosis, CTAs are often of functional relevance for tumor cells.

• CTAs are immunogenic, and for most members of this group, spontaneous humoral and cellular immune responses in patients are detectable.

NY-ESO-1

NY-ESO-1 is expressed in many different malignancies, including multiple myeloma. We previously reported that the malignant plasma cells of 7% of patients with multiple myeloma express NY-ESO-1.[46] Multiple myeloma patients with a poor prognosis-as determined by metaphase cytogenetic abnormalities-and patients with relapsing multiple myeloma show elevated expression of NY-ESO-1, which indicates the tumor-promoting features of the antigen.[47] NY-ESO-1 is considered a highly immunogenic CTA that elicits spontaneous humoral and cellular immune responses as well as vaccine-induced T-cell responses.[47,48]

Several clinical trials have targeted NY-ESO-1 with TCR-engineered T cells. High objective response rates were achieved in synovial cell sarcoma and melanoma using an affinity-enhanced NY-ESO-1–specific TCR.[49,50] In a recent clinical trial, this affinity-enhanced TCR was used in patients with multiple myeloma. Reprogrammed T cells were applied 2 days after autologous stem cell transplantation and showed high persistence and bone marrow homing. Eighty percent of patients experienced objective clinical responses, which highlights the potential of NY-ESO-1 as a target for receptor-engineered T cells.[51]

MAGE-C2

MAGE-C2 is one of the most frequently expressed CTAs in multiple myeloma, with expression rates of 55% to 60%,[46,52] and it is detectable in osteolytic lesions in the majority of patients.[53] MAGE-C2 exerts tumor-promoting effects for several malignancies. Its expression can serve as a predictive marker for prostate cancer recurrence, and it can be indicative of lymph node metastases in melanoma.[54,55] We and others have shown that MAGE-C2 can directly promote tumor growth[56] and that it suppresses apoptosis in multiple myeloma cells.[57] It has a high immunogenic potential and elicits spontaneous humoral[58] and cellular immune[59] responses. Our group characterized several CD4 and CD8 T-cell epitopes that are recognized by T cells from patients with multiple myeloma.[60] Because of its frequent expression, multiple myeloma–promoting features, and high T-cell immunogenicity, MAGE-C2 is one of the most interesting candidates as a target for ACT.

MAGE-C1

Another CTA, MAGE-C1, is expressed in the majority of patients with multiple myeloma, and its expression increases with progressive disease stages.[61,62] We have shown that MAGE-C1 expression in multiple myeloma strongly predicts concomitant expression of other CTAs, such as MAGE-A3 and MAGE-C2.[63] Similar findings were reported for the concomitant expression of NY-ESO-1, which was also identified as an interaction partner of MAGE-C1, indicating involvement in the regulation of expression of other CTAs.[64]

A number of studies have shown that MAGE-C1 can serve as a target for myeloma-specific T cells.[65-68] The occurrence of MAGE-C1–specific T cells after alloSCT in patients with multiple myeloma correlated with the development of complete remission, whereas patients who lacked these T cells exhibited a progressive disease course, which indicates antitumor activity in vivo.[69] The functional relevance of MAGE-C1 for multiple myeloma cells is also supported by our data that show its knockdown leads to the induction of apoptosis, including multiple myeloma precursor cells, which are considered highly resistant to chemotherapy.[70]

MAGE-A3

Because of its expression in a wide variety of cancers and its absence from any healthy tissues accessible to the immune system, MAGE-A3 became an attractive target for novel cancer immunotherapies.[71] However, the first clinical trials that used TCR-engineered T cells to target MAGE-A3 reported fatal neurotoxicity and cardiotoxicity in some patients.[72,73] Toxicities in both trials were caused by the cross-reactivity of the engineered receptors to the MAGE-A12[74] and titin[75] proteins expressed in the respective healthy tissues. In both cases, affinity-enhanced TCRs were used, which had passed all preclinical testing without indicating any off-target toxicities. Because of the nature of target recognition by TCRs-including processed peptide antigens and different HLA contexts, studying the cross-reactivity of TCRs is considerably more complex than for other compounds such as monoclonal antibodies; however, strategies that assess the safety of novel therapeutic TCRs will need to be improved to minimize patient risk.

SSX proteins

The family of synovial sarcoma X chromosome breakpoint (SSX) proteins includes 10 highly homologous members that are specifically expressed in tumor and testis tissue.[76,77] The SSX antigens were first described as being involved in the common t(X;18)(p11.2;q11.2) chromosomal translocation in synovial sarcoma.[78,79] The SSX family members share 73% to 92% amino acid sequence homology.[76] SSX-1, SSX-2, SSX-4, and SSX-5 are expressed in different tumor entities, including multiple myeloma.[46,77,80] Sixty-one percent of patients with multiple myeloma show expression of at least one of the four SSX antigens. Their expression correlates with an adverse prognosis, and SSX-2 has the strongest association with reduced survival.[80] SSX family members, especially SSX-2, are highly immunogenic and elicit humoral and cellular immune responses.[81-83] Their heterogeneous intratumoral expression pattern, however, may pose a problem for future immunotherapeutic approaches because it may result in the outgrowth of antigen-negative tumor cells. Methyltransferase inhibitors can induce and enhance the expression of SSX and other CTAs on the mRNA and the protein level, which indicates the potential for future combination treatments.[84,85]

Checkpoint Inhibitors as a Component of Multimodal Immunotherapy for Multiple Myeloma

Traditionally, cancer research has focused almost exclusively on the tumor cell itself as the therapeutic target in human malignancies. However, recent evidence points to an important role of the tumor microenvironment in promoting carcinogenesis, tumor progression, and tumor escape from detection and elimination by the immune system. For multiple myeloma in particular, an immunologic dysregulation in the local microenvironment is a hallmark of the disease.[86] Thus, it has been shown that in patients with myeloma, bone marrow stromal cells significantly inhibit the T-cell–mediated lysis of multiple myeloma cells in a cell-cell contact-dependent manner. In addition, it has been demonstrated that different types of immune cells in the tumor microenvironment are dysregulated and functionally impaired; they create an immunosuppressive tumor milieu in patients with myeloma and seem to play a critical role in multiple myeloma progression.[87-92]

Programmed death 1 (PD-1), a member of the CD28 family of receptors, is a transmembrane protein that is expressed on the surface of antigen-activated and -exhausted T and B cells and has two ligands, PD-L1 and PD-L2.[93-95] Our group and others have demonstrated that PD-L1 is downregulated in normal plasma cells, while it is overexpressed by myeloma cell lines and primary myeloma tumor cells from patients with multiple myeloma.[96-99] In addition, PD-1 is overexpressed on T cells from patients with myeloma, and PD-1/PD-L1 interactions are likely to play an important role in the local immunosuppression in multiple myeloma.[98-100] Recently, our group showed that PD-L1 is expressed not only on malignant plasma cells in the bone marrow of patients with myeloma but also on chemotherapy-resistant and myeloma-propagating precursor cells.[101] Thus, immunotherapies that use anti–PD-1/PD-L1 strategies (see Figure) can potentially lead to prolonged remission and perhaps even cure in patients with multiple myeloma.

Immunotherapy could play an important role in eradicating chemotherapy-resistant myeloma cells from the bone marrow of patients, and monoclonal antibodies that target PD-L1 or PD-1 have shown significant clinical efficacy in different solid tumors[102,103] and also in hematologic malignancies.[104] It has been demonstrated repeatedly in myeloma mouse models that the administration of anti–PD-L1 or anti–PD-1 antibodies can suppress the growth of myeloma cells.[105-107]

Unfortunately, it seems that anti–PD-1/PD-L1 approaches alone are not as effective in multiple myeloma as they are in other tumor entities. In a phase I study with the monoclonal anti–PD-1 antibody nivolumab, none of the 27 patients with myeloma who were enrolled showed an objective clinical response.[108] However, subsequent findings indicated that anti–PD-1 approaches can lead to substantial clinical results if they are combined with other immune-related therapies, such as immunomodulatory drugs (IMiDs).[98] Accordingly, two recent clinical trials support the idea that the combination of anti–PD-1 approaches with IMiDs can result in significant responses in patients with relapsed/refractory multiple myeloma.

The KEYNOTE-023 study was an open-label, phase I, nonrandomized trial that evaluated the safety and efficacy of the anti–PD-1 antibody pembrolizumab in combination with lenalidomide and low-dose dexamethasone in patients with relapsed/refractory multiple myeloma. Of the 17 patients included in the recently presented report, 53% had three or more prior therapies, 41% were refractory to IMiDs, and 18% had double-refractory disease. Remarkably, even in this heavily pretreated and highly refractory group, 76% achieved an objective clinical response, which included patients with IMiD-refractory or double-refractory disease.[109]

In a different phase II clinical trial, 24 patients with relapsed/refractory multiple myeloma were treated with pembrolizumab combined with pomalidomide and dexamethasone. These patients had also been heavily pretreated with a median of three prior lines of therapy. As many as 75% had undergone a previous autologous transplant, and 96% were refractory to their most recent therapy. All patients had received both IMiDs and proteasome inhibitors; 75% were double-refractory to both types of drugs, and an additional 21% were refractory to lenalidomide alone. Although this was a highly refractory patient population, objective responses were observed in 50% of patients.[110] Overall, data from both trials clearly indicate that combining anti–PD-1 strategies with another agent capable of modulating the bone marrow microenvironment of multiple myeloma carries significant clinical potential.

To increase the number of myeloma-specific effector T cells targetable by anti–PD-1/PD-L1 strategies, one could also combine checkpoint inhibitors with the adoptive transfer of genetically modified T cells. It was shown using a TCR-transgenic mouse model that high-avidity, tumor-specific T cells were capable of initially delaying tumor growth; however, persistence in the tumor microenvironment resulted in tolerization. Blockade of PD-1 signals and depletion of tumor-associated dendritic cells prevented or reduced T-cell tolerization and restored antitumor immunity.[111]

Instead of only blocking PD-1/PD-L1 using a monoclonal antibody, one could also target this axis on a molecular level in the context of the adoptive transfer of genetically modified T cells. Accordingly, one group recently showed that transcription activator–like effector nuclease–mediated inactivation of the PD-1 gene in melanoma-reactive CD8 T cells enhanced the persistence of PD-1 gene–modified T cells at the tumor site and increased tumor control.[112] A different group recently introduced a genetically engineered switch receptor construct, comprising the truncated extracellular domain of PD-1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T cells. Treatment of tumor-bearing mice with PD-1/CD28 CAR T cells led to significant tumor regression as a result of enhanced CAR T-cell infiltration, decreased susceptibility to tumor-induced immunosuppression, and attenuation of inhibitory receptor expression, compared with treatment with CAR T cells alone or anti–PD-1 antibodies.[113] We believe that these novel molecular approaches could easily be combined with current strategies using genetically modified T cells and lead to superior clinical efficacy.

Conclusions and Perspectives

Preliminary clinical results indicate that novel immunotherapies using the adoptive transfer of genetically modified T cells, such as CAR T cells or TCR-transduced T cells, have the potential to lead to better clinical responses in multiple myeloma than historical approaches such as tumor vaccination. Innovative techniques to reduce the toxicities associated with these new cellular immunotherapies will need to be explored in preclinical and clinical studies. Finally, it is hoped that multimodal approaches, such as the combination of genetically modified T cells with anti–PD-1/PD-L1 or even anti–cytotoxic T-lymphocyte–associated antigen 4 strategies, will further enhance antimyeloma treatment efficacy and yield durable and deep clinical responses.[114,115]

Financial Disclosure:The authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

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