Target For Immunotherapy Of Multiple Myeloma Research Articles

Structural Basis for the Recognition of GPRC5D by Talquetamab, a Bispecific Antibody for Multiple Myeloma

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.

Role of Natural Killer T (NKT) Cells in Myeloma Biology and Therapy.

Natural Killer T (NKT) cells are distinct innate lymphocytes that recognize lipid antigens in the context of nonpolymorphic molecule CD1d. Multiple myeloma (MM) is a hematologic malignancy wherein malignant plasma cells express CD1d and are sensitive to lysis by NKT cells. Progressive malignancy in MM is characterized by NKT cell dysfunction. Several studies have tried to harness the anti-tumor properties of NKT cells in MM to mediate tumor regression. NKT cells are also attractive targets for approaches at immune redirection in MM with chimeric-antigen receptor NKT (CAR-NKT) and bispecific antibodies. In addition to the commonly studied invariant-NKT (iNKT) cells, MM patients often also exhibit alterations in type-II NKT cells and their ligands. In patients and mouse models with Gaucher disease (GD), an inherited lipid-storage disorder with markedly increased risk for MM, distinct type-II NKT cells exhibit a T-follicular helper (NKT-TFH) phenotype and provide help to lipid-specific B cells. Chronic immune activation in this setting eventually sets the stage for malignancy, which can be targeted in both mouse models and GD patients by reducing the underlying antigen. NKT cells are thus integrally linked to MM pathogenesis and an attractive target for MM immunotherapy.

Secondary Genetic Events Impact the Expression of Key Immunotargets on the Surface of Multiple Myeloma Cells

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p <0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.

Programmed Cell Death-1 and Its Ligands as Targets for Therapy of Multiple Myeloma Patients.

PurposeAmong hematological malignancies, the expression profile of programmed cell death-1 (PD-1) and its ligands in multiple myeloma (MM) is still debated by numerous research groups. In current study, we characterized the expression of PD-1 and its ligands both on RNA and protein levels in MM patients. We have also attempted to analyze whether daratumumab therapy might overcome CD38-mediated immunosuppression that inhibits in particular CD8+ T-cell function.Patients and MethodsThis study included 149 newly diagnosed MM patients and 15 relapsed/refractory MM patients before and after daratumumab treatment. The mRNA levels of PDCD1, PDCD1LG1, PDCD1LG2 and their splicing variants was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Flow cytometry was used to characterize the surface expression of PD-1 and its ligands on plasma cells, B and T cells. The surface expression of PD-1 on T cells was assessed by flow cytometry before and after daratumumab treatment.ResultsThe mRNA expression of PDCD1LG1, PDCD1LG2 and their splicing variants were higher in plasma cells as compared to bone marrow mononuclear cells (BMMCs). Our results show that the percentage of plasma cells expressing PD-L1 was significantly higher than plasma cells expressing PD-L2 (p<0.0001) in bone marrow (BM) of MM patients. There was no significant difference between the percentage of plasma cells expressing PD-1 and B cells expressing PD-1 in BM of MM patients (11.19% vs 8.91%). We also found that the percentage of CD8+PD-1+ T cells was significantly higher than CD4+PD-1+T cells in BM (p<0.0001) of MM patients. Here, we observed no change in PD-1 expression on CD4+ and CD8+ T cells after the daratumumab treatment.ConclusionThe PD-1 and its ligands might represent an interesting target for MM immunotherapy, as one would target both malignant plasma cells as well as the immune cells that play a key role in tumor escape mechanisms.

NY-ESO-1 Protein Vaccine Combining Alum, CpG ODN, and HH2 Complex Adjuvant Induces Protective and Therapeutic Anti-Tumor Responses in Murine Multiple Myeloma.

BackgroundNY-ESO-1 is an ideal target for multiple myeloma immunotherapy. Alum, CpG ODN and HH2 complex is a safe and effective adjuvant for cancer vaccine.MethodsWe constructed NY-ESO-1 protein vaccine combined with alum, CpG ODN, and HH2 complex adjuvant to immunize the BALB/c mice inoculated with NS-1 murine multiple myeloma cells. Then, we determined the immunogenicity and anti-tumor effects in prophylactic and therapeutic models by analyzing the NY-ESO-1 antibody titer, evaluating IL4/INF-γ expression, and assessing cytotoxic T lymphocytes activities. The side-effects of vaccines were also evaluated.ResultsThe group of NY-ESO-1 protein vaccine combining alum, CpG ODN, and HH2 complex adjuvant is more capable of stimulating both humoral and cellular tumor-specific immune responses to prolong the survival of the mice and inhibit tumor growth in prophylactic and therapeutic immunotherapy. The marked side-effects were not detected in immunized mice.DiscussionThe results suggest that alum, CpG ODN, and HH2 complex as a novel immune adjuvant combined cancer vaccine could improve the immunity efficiency in a murine multiple myeloma model.

B-cell maturation antigen-specific chimeric antigen receptor T cells for multiple myeloma: Clinical experience and future perspectives.

Despite major advances in the treatment of multiple myeloma (MM), it remains a largely incurable disease with long-term control often dependent on continuous therapy. More effective, better tolerated treatments are therefore required to achieve durable remissions and to improve the quality of life of MM patients. Adoptive immunotherapy employing T cells expressing chimeric antigen receptors (CAR) is currently among the most promising treatment approaches in cancer. Within the target portfolio for MM immunotherapy, B-cell maturation antigen (BCMA) is among the most widely studied target antigens. BCMA is consistently expressed on MM cells and, importantly, is not expressed in critical healthy tissue. For this reason, it is an ideal target for MM immunotherapy. Several clinical trials evaluating different BCMA-targeting CAR constructs have been initiated and early results are very promising. However, in this rapidly developing clinical landscape, the ultimate role of BCMA-specific CAR-T cell therapy remains unclear. In this review, we will summarize currently available clinical data on BCMA-directed CAR-T cells and discuss potential future perspective for this promising treatment approach in MM.

Aberrant RHAMM Splicing in Multiple Myeloma (MM) and Its Implications for Immunotherapy

Background: Receptor for hyaluronan-mediated motility (RHAMM) or CD168 has been a promising target for MM immunotherapy because it is overexpressed in MM cells. RHAMM has been tested as a target for an anti-RHAMM peptide vaccination approach in MM and other hematological malignancies. Although RHAMM peptide-induced immune response in patients, clinical outcomes were mixed, that can be a result of equal expression levels of RHAMM in different subpopulation of bone marrow (BM) cells in MM patients and healthy donors (HD). To enhance current RHAMM-peptide and future immunotherapeutic approaches, we investigated the cause of altered RHAMM mRNA splicing in MM patients. mRNA splicing has the potential to produce numerous mis-spliced genes, creating novel disease markers; some resulting proteins are likely to contain neoantigens selectively expressed on MM tumor cells. Methods/results: Splicing alterations can be caused by single nucleotide variations (SNVs) that affect splicing regulatory elements (SREs), or by deregulated splicing factors (SFs). We evaluated the incidence of SNVs located in the vicinity of the RHAMM. We identified a total of 57 SNVs: 72% SNVs are in the intronic region, and 28% are in the RHAMM coding region. We used the "HEXplorer" tool and predicted that four SNVs have the potential to contribute to aberrant RHAMM splicing in MM either by altering SF binding to SREs or by impacting splice site selection. Predicted SNVs were evaluated using an in vivo splicing assay to identify SNV-clusters causing aberrant RHAMM splicing. We have observed progressive overexpression of core SF PTBP1/2 (polypyrimidine track-binding protein) in MM patients and associated with disease progression. Since SNVs on the RHAMM modulate canonical SF binding sites, we tested the effects of PTBP1/2 deregulation on RHAMM splicing. We expressed PTBP1/2 in H929 cells, and then evaluated the RHAMM splicing pattern in transfected cells at a single cell (SC) level. SC analyses showed that overexpression of PTBP1/2 increased (2.5-fold) the RHAMM-V3:FL ratio in MM cells. SC analyses also identified overexpression of the RHAMM-V3 splice variant in 18% of H929 SCs expressing PTBP1, and in 37% of cells expressing PTBP2, confirmed at the single cell (SC) level. In BM-infiltrating myeloid cells, analyses showed 50% of myeloid cells express the RHAMM-V3 variant alone, and 79% of plasma cells (PCs) express this variant in combination with RHAMM-FL. Moreover, the RHAMM-V3/FL ratio in PCs is elevated (2.6-fold), further confirming a correlation between the RHAMM variant ratio and the clinical outcome. Next, we determined RHAMM-V3/FL ratios in BM stromal cells from 16 MM patients: MM-BMSC samples exclusively express the RHAMM-V3 in combination with RHAMM-FL and the RHAMM-V3:FL is 1.8 fold. In BMSC samples derived from healthy donors (HD), we detected relatively low-level expression of RHAMM-FL as compared to expression levels of RHAMM-FL in MM patients, while RHAMM-V3 transcripts were undetectable. SC analysis of RHAMM FL and splice variant transcripts in MM BMSC and HD-BMSC agreed with the analyses done on the MM HD-BMSC bulk population. We did not detect any MM BMSC cells expressing RHAMM-V3 alone and the RHAMM V3/FL ratio was 1.6-fold, which is lower than that in MM-PCs. MM-BMSC screening also identified a new splice variant of RHAMM, that was absent in MM PCs or in MM myeloid cells. Conclusions: Our study suggests that aberrant RHAMM splicing in MM can result from SNPs/SNVs affecting SRE due to the upregulation of PTBP1/2. Our study is the first to show that the RHAMM-V3 variant is associated with PTBP2 overexpression. The identification of cell type-specific RHAMM splicing events identifies novel targets for improved immunotherapy in MM. Disclosures Chu: Celgene: Honoraria; AstraZeneca: Honoraria; Gilead: Honoraria; Teva: Consultancy; Amgen Inc.: Honoraria. Anderson:C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board.

Abstract 547: Recombinant immunotoxins targeting B-cell maturation antigen induce complete responses in bone marrow myeloma mouse model

Abstract Background: Multiple myeloma (MM) is a B-cell malignancy for which new treatments are urgently needed. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed on MM and an ideal target for immunotherapy of MM. Recombinant immunotoxins (RITs) are chimeric proteins composed of the Fv or Fab portion of an antibody fused to a fragment of Pseudomonas Exotoxin A (PE). One of these, Lumoxiti, is FDA approved for the treatment of drug-resistant hairy cell leukemia, another B-cell malignancy. LMB-75 is an immunotoxin with an anti-BCMA dsFv of mAb BM306 fused to domain III of PE, and LMB-70 is an anti-BCMA Fab fused to domain III of PE. We previously treated H929 myeloma cells grown as subcutaneous tumors with anti-BCMA immunotoxins that are very effective in killing cultured cells and cells from patients. We observed tumor growth inhibition but not complete tumor responses. Goal: Here we used H929 cells to develop a bone marrow mouse model that is more relevant to human disease and to determine if these immunotoxins are more active against H929 cells growing in bone marrow. Methods: The NCI-H929 cell line was transfected with luciferase and GFP, recycled through the bone marrow of a mouse and enriched by flow to isolate cells with high GFP expression, which were used to inoculate tumors IV into NSG mice. Mice were treated IV with immunotoxins and tumor burden was assessed using bioluminescence imaging. Results: H929-luc-GFP cells grow uniformly in the bone marrow of mice, cause hind limb paralysis around day 40, and provide a relevant in vivo model for testing RITs. We achieved complete responses when treating mice with 1.5 mg/kg LMB-75 or LMB-70 given QODX5 beginning on days 4 or day 8. Mice in all treatment groups were disease free at 3 months. An immunotoxin targeting CD22, which is not expressed on H929 cells, or an anti-BCMA immunotoxin with a mutation that inactivates toxin activity did not have any anti-tumor activity. Conclusions: LMB-75 and LMB-70 induce complete, durable remissions of H929-luc-GFP cells growing in mouse bone marrow without toxicity to mice. These promising results support further pre-clinical development of these agents for MM. Citation Format: Zoe Shancer, Xiu-fen Liu, Emily King, Tapan Bera, Ira Pastan. Recombinant immunotoxins targeting B-cell maturation antigen induce complete responses in bone marrow myeloma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 547.

Bone marrow IRF4 level in multiple myeloma: an indicator of peripheral blood Th17 and disease.

Interferon regulator factor 4 (IRF4) is characterized to be a member of interferon regulatory family, which is predominantly expressed in bone marrow plasma cells of patients with multiple myeloma (MM). Recent studies indicated IRF4 is critical for T-help cells (Th17) differentiation and interleukin-17 (IL-17) secretion. Here, a total of 58 MM patients were enrolled in this study, the proportions of Th17 cells and T regulatory (Treg) cells in peripheral blood mononuclear cells (PBMCs) were determined by flow cytometric analysis. Immunohistochemistry was employed to detect the IRF4 expression in bone marrow. Herein, we observed a significant increase of IRF4 in bone marrow accompanied with a notable up-regulation of Th17 cells in PBMC within MM patients compared with healthy donors. Furthermore, the proportions of Th17 cells and serum IL-17 levels were higher in patients with stage III than stage I & II MM patients, and those parameters were positively correlated with the expression of IRF4 in these cases. These results for the first time indicate that a crosstalk between IRF4 and Th17 cells is associated with MM prognosis, and IRF4 may be served an important target for MM immunotherapy.

Novel anti-myeloma immunotherapies targeting the SLAM family of receptors

ABSTRACTTreatment for multiple myeloma (MM) has significantly advanced in the last decade with the introduction of proteasome inhibitors and immunomodulatory therapies. Unfortunately, MM continues to cause significant morbidity and most patients eventually succumb to the disease. As in other areas of cancer, immunotherapy in MM has also evolved and holds promise to deliver long-lasting remissions or even cure. The signaling lymphocyte activation molecules (SLAM) family of surface proteins represents a group of potential targets for immunotherapy in MM as some of the family members are expressed consistently on plasma cells and also on myeloma propagating pre-plasma cells. Here, we review the SLAM family members in detail, describe their tissue distribution, biologic pathways, as well as relevant pre-clinical studies and clinical trials in MM. Our review demonstrates the value of SLAM family receptors as potential targets for anti-myeloma immunotherapies and outlines how immunotherapeutic approaches can be developed.

Cancer Testis Antigen, Ropporin, Is a Potential Target for Multiple Myeloma Immunotherapy

Despite recent improvements in standard pharmacologic treatments of multiple myeloma (MM), immunotherapy may prove to be more effective due to its higher specificity and lower toxicity. A novel cancer/testis antigen, ropporin, is a testis-specific protein localized in the sperm flagella. Comparing ropporin expression in healthy and MM samples, we did not detect ropporin expression in the normal tissues, but positive signals were found in 44% of the MM primary samples. The immunogenicity of ropporin was confirmed by the presence of specific antibodies detected by enzyme-linked immunosorbent assay in patients' serum. Our results show that ropporin is a novel cancer/testis antigen for MM. Except for in the testis, an immune privileged site, ropporin was not expressed in normal tissues, but was present in MM cell lines and patients' samples. Noteworthy, we show for the first time that ropporin was present at the cell surface of MM plasma cells. We suggest that ropporin is a promising target for MM immunotherapy, as we were able to generate human leukocyte antigen class I-restricted cytotoxic lymphocytes able to kill autologous MM cells.

Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103

Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL). Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by (51)Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004). MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.

Serological identification and bioinformatics analysis of immunogenic antigens in multiple myeloma.

Identifying appropriate tumor antigens is critical to the development of successful specific cancer immunotherapy. Serological analysis of tumor antigens by a recombinant cDNA expression library (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. We applied SEREX to the cDNA expression library of cell line HMy2, which led to the isolation of six known characterized genes and 12 novel genes. Known genes, including ring finger protein 167, KLF10, TPT1, p02 protein, cDNA FLJ46859 fis, and DNMT1, were related to the development of different tumors. Bioinformatics was performed to predict 12 novel MMSA (multiple myeloma special antigen) genes. The prediction of tumor antigens provides potential targets for the immunotherapy of patients with multiple myeloma (MM) and help in the understanding of carcinogenesis. Crude lysate ELISA methodology indicated that the optical density value of MMSA-3 and MMSA-7 were significantly higher in MM patients than in healthy donors. Furthermore, SYBR Green real-time PCR showed that MMSA-1 presented with a high number of copy messages in MM. In summary, the antigens identified in this study may be potential candidates for diagnosis and targets for immunotherapy in MM.

SPAN-Xb Expression in Myeloma Cells Can Be Upregulated by Pharmacologics.

SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood 2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines.Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb.We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression.Table 1Cellspre-5-azacytidinepost-5-azacytidineARK-B0456ARP-12601376H9290116IM90532KMS-1100MM1-R0428RPMI 82262721348U2660984SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposureTable 2PharmacologicsSPAN-Xb mRNA5-azacytidine532IL-74GM-CSF9IL-7 + GM-CSF365-azacytidine + IL-710885-azacytidine + GM-CSF9525-azacytidine + IL-7 + GM-CSF1632medium only0SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics

Sperm protein 17 (Sp17) is a suitable target for immunotherapy of multiple myeloma

AbstractSperm protein 17 (Sp17) is a protein recently identified as a novel cancer-testis (CT) antigen in multiple myeloma (MM). Because this tumor antigen demonstrates a very restricted normal tissue expression, Sp17 may be an excellent target for tumor vaccine of MM. In this study, we determined the ability to generate Sp17-specific HLA class I–restricted cytotoxic T lymphocytes (CTLs) from the peripheral blood of 4 patients with MM, 3 consecutive Sp17+patients, and 1 Sp17− patient. Dendritic cells were generated from monocytes of 4 patients with MM and used to present a recombinant Sp17 protein to autologous T cells. Following 4 rounds of antigen stimulation, the CTLs were tested for their ability to kill autologous targets in an Sp17-dependent and HLA-class I– restricted manner in standard cytotoxicity assays. Despite previous chemotherapy and the immunosuppression so often associated with MM, CTL generation was successful in all 4 patients, irrespective of the Sp17 status of their tumors. Most importantly, the CTLs were able to lyse autologous tumor cells that expressed Sp17. Tumor cell lysis in all cases appeared to be mainly mediated by perforin and could be blocked by concanamycin A. We conclude that Sp17 is a suitable target for immunotherapy of MM. Our findings provide the basis for a clinical study aimed at inducing a cellular immune response directed at Sp17+ MM.