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Myeloma patients may develop oligoclonal immunoglobulins, so-called abnormal protein bands (APB), after stem cell transplantation. APB do not correspond to the patient's paraprotein and confer a good prognosis. We set out to investigate whether such APB represent a humoral anti-myeloma immune response by screening immunoglobulins of 15 myeloma patients after allogeneic stem cell transplantation and a control group of healthy donors for reactivity with myeloma protein extracts. While the immunoglobulins of healthy donors did not react with myeloma protein extracts, patient-derived immunoglobulins showed variable levels of interaction, depending on the presence of APB on immunofixation. Most commonly, we detected interactions with heat-shock proteins, followed by neutral alpha-glucosidase, alpha-enolase and vimentin, as well as proliferating cell nuclear antigen and MAGEA4. More than 80% of targets were upregulated in myeloma. Heat-shock protein 60 (HSP60) was subsequently evaluated as an exemplary antigen. We found that HSP60 was aberrantly displayed on the surface of primary myeloma cells. Indeed, patient-derived APB-containing immunoglobulins recognized surface HSP60 suggesting that this antigen becomes accessible to the immune system after aberrant membrane exposition. We conclude that immunoglobulin fractions with APB recognize recurrent myeloma antigens and that this humoral response may contribute to the more favorable prognosis in patients with APB. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a hematological cancer for which immune-based treatments are currently in development. Many of these rely on the identification of highly disease-specific, strongly and stably expressed antigens. Here, we profiled the myeloma B-cell immunome both to explore its predictive role in the context of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and to identify novel immunotherapeutic targets. We used random peptide phage display, reverse immunization, and next-generation sequencing-assisted antibody phage display to establish a highly myeloma-specific epitope fingerprint targeted by B-cell responses of 18 patients in clinical remission. We found that allogeneic HSCT more efficiently allowed production of myeloma-specific antibodies compared with autologous HSCT and that a highly reactive epitope recognition signature correlated with superior response to treatment. Next, we performed myeloma cell surface screenings of phage-displayed patient transplant immunomes. Although some of the screenings yielded clear-cut surface binders, the majority of screenings did not, suggesting that many of the targeted antigens may in fact not be accessible to the B-cell immune system in untreated myeloma cells. This fit well with the identification of heat-shock proteins as a class of antigens that showed overall the broadest reactivity with myeloma patient sera after allogeneic HSCT and that may be significantly translocated to the cell surface upon treatment as a result of immunogenic cell death. Our data reveal a disease-specific epitope signature of MM that is predictive for response to treatment. Mining of transplant immunomes for strong myeloma surface binders may open up avenues for myeloma immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Multiple myeloma (MM) is the malignancy with the most frequent expression of the highly immunogenic cancer-testis antigens (CTA), and we have performed the first analysis of longitudinal expression, immunological properties, and fine specificity of CTA-specific antibody responses in MM. Methods: Frequency and characteristics of antibody responses against cancer-testis antigens MAGE-A3, NY-ESO-1, PRAME, and SSX-2 were analyzed using peripheral blood ( N = 1094) and bone marrow ( N = 200) plasma samples from 194 MM patients. Results: We found that antibody responses against CTA were surprisingly rare, only 2.6 and 3.1 % of patients evidenced NY-ESO-1- and SSX-2-specific antibodies, respectively. NY-ESO-1-specific responses were observed during disease progression, while anti-SSX-2 antibodies appeared after allogeneic stem cell transplantation and persisted during clinical remission. We found that NY-ESO-1- and SSX-2-specific antibodies were both capable of activating complement and increasing CTA uptake by antigen-presenting cells. SSX-2-specific antibodies were restricted to IgG3, NY-ESO-1 responses to IgG1 and IgG3. Remarkably, NY-ESO-1-positive sera recognized various non-contiguous regions, while SSX-2-specific responses were directed against a single 6mer epitope, SSX-2. Conclusions: We conclude that primary autoantibodies against intracellular MM-specific tumor antigens SSX-2 and NY-ESO-1 are rare but functional. While their contribution to disease control still remains unclear, our data demonstrate their theoretic ability to affect cellular anti-tumor immunity by formation and uptake of mono- and polyvalent immune complexes. [ABSTRACT FROM AUTHOR]

The occurrence of SOX2-specific autoantibodies seems to be associated with an improved prognosis in patients with monoclonal gammopathy of undetermined significance (MGUS). However, it is unclear if SOX2-specific antibodies also develop in established multiple myeloma (MM). Screening 1094 peripheral blood (PB) sera from 196MMpatients and 100 PB sera from healthy donors, we detected SOX2-specific autoantibodies in 7.7% and 2.0% of patients and donors, respectively. We identified SOX2211-230 as an immunodominant antibody-epitope within the full protein sequence. SOX2 antigen was expressed in most healthy tissues and its expression did not correlate with the number of BM-resident plasma cells. Accordingly, anti-SOX2 immunity was not related to SOX2 expression levels or tumor burden in the patients' BM. The only clinical factor predicting the development of anti-SOX2 immunity was application of allogeneic stem cell transplantation (alloSCT). Anti-SOX2 antibodies occurred more frequently in patients who had received alloSCT (n = 74). Moreover, most SOX2-seropositive patients had only developed antibodies after alloSCT. This finding indicates that alloSCT is able to break tolerance towards this commonly expressed antigen. The questions whether SOX2-specific autoantibodies merely represent an epiphenomenon, are related to graft-versus-host effects or participate in the immune control of myeloma needs to be answered in prospective studies. [ABSTRACT FROM AUTHOR]

Simple Summary: Multiple myeloma is a hematological malignancy of antibody-producing plasma cells in the bone marrow. Nucleotides released from cells in the tumor microenvironment act as inflammatory danger signals. CD38 and other enzymes on the surface of cancer cells hydrolyze these nucleotides to immunosuppressive mediators, thereby hampering anti-tumor immune responses. Daratumumab and other CD38-specific antibodies mediate killing of tumor cells by natural killer cells, macrophages, and the complement system. Here, we investigated whether CD38-specific antibodies also inhibit the enzyme activity of CD38-expressing tumor cells, thereby providing a potential second mode of action. Our results showed that daratumumab and nanobody-based heavy chain antibodies inhibit the ADPR cyclase but not the NAD+ hydrolase activity of CD38. Thus, there remains a need for better CD38-inhibitory antibodies. The nucleotides ATP and NAD+ are released from stressed cells as endogenous danger signals. Ecto-enzymes in the tumor microenvironment hydrolyze these inflammatory nucleotides to immunosuppressive adenosine, thereby, hampering anti-tumor immune responses. The NAD+ hydrolase CD38 is expressed at high levels on the cell surface of multiple myeloma (MM) cells. Daratumumab, a CD38-specific monoclonal antibody promotes cytotoxicity against MM cells. With long CDR3 loops, nanobodies and nanobody-based heavy chain antibodies (hcAbs) might bind to cavities on CD38 and thereby inhibit its enzyme activity more potently than conventional antibodies. The goal of our study was to establish assays for monitoring the enzymatic activities of CD38 on the cell surface of tumor cells and to assess the effects of CD38-specific antibodies on these activities. We monitored the enzymatic activity of CD38-expressing MM and other tumor cell lines, using fluorometric and HPLC assays. Our results showed that daratumumab and hcAb MU1067 inhibit the ADPR cyclase but not the NAD+ hydrolase activity of CD38-expressing MM cells. We conclude that neither clinically approved daratumumab nor recently developed nanobody-derived hcAbs provide a second mode of action against MM cells. Thus, there remains a quest for "double action" CD38-inhibitory antibodies. [ABSTRACT FROM AUTHOR]

The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated. [ABSTRACT FROM AUTHOR]