Sabrina Meyer

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells

Background Multiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood. Design and Methods We performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interference-based gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing. Results We show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies. Conclusions Cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.

Cancer-testis antigen expression and its epigenetic modulation in acute myeloid leukemia†

Cancer‐testis antigens (CTA) represent attractive targets for tumor immunotherapy. However, a broad picture of CTA expression in acute myeloid leukemia (AML) is missing. CTA expression was analyzed in normal bone marrow (BM) as well as in AML cell lines before and after treatment with demethylating agents and/or histone acetylase inhibitors. Presence of selected CTA with a strictly tumor‐restricted expression was then determined in samples of patients with AML before and after demethylating therapy. Screening AML cell lines for the expression of 20 CTA, we identified six genes (MAGE‐A3, PRAME, ROPN1, SCP‐1, SLLP1, and SPO11) with an AML‐restricted expression. Analyzing the expression of these CTA in blast‐containing samples from AML patients (N = 64), we found all samples to be negative for MAGE‐A3 and SPO11 while a minority of patients expressed ROPN1 (1.6%), SCP‐1 (3.1%), or SLLP1 (9.4%). The only CTA expressed in substantial proportion of patients (53.1%) was PRAME. Following demethylating treatment with 5′‐aza‐2′‐deoxycytidine, we observed an increased or de novo expression of CTA, in particular of SSX‐2, in AML cell lines. In AML patients, we detected increased expression of PRAME and induction of SSX‐2 after demethylating therapy with 5‐azacytidine. With the exception of PRAME, CTA are mostly absent from AML blasts. However, demethylating treatment induces strong expression of CTA, particularly of SSX‐2, in vitro and in vivo. Therefore, we propose that CTA‐specific immunotherapy for AML should preferentially target PRAME and/or should be combined with the application of demethylating agents opening the perspective for alternative targets like CTA SSX‐2. Am. J. Hematol., 2011.

The cytokine/chemokine pattern in the bone marrow environment of multiple myeloma patients.

OBJECTIVE The interaction of multiple myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing pattern, survival, and proliferation of malignant plasma cells. We aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT). MATERIALS AND METHODS We determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines, as well as in the plasma derived from BM and peripheral blood samples of 10 newly diagnosed MM patients, 20 MM patients who had received allogeneic stem cell transplantation (alloSCT), and 20 healthy donors. RESULTS Besides cytokines/chemokines known to be secreted by myeloma cell lines, such as interleukin-1 receptor antagonist (IL-1RA), IL-8, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, and MIP-3α, we also detected significant levels of epidermal growth factor (EGF), hepatocyte growth factor (HGF), IL2R, IL-12p40/p70, IL-22, interferon-γ (IFN-γ)-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), and regulated on activation normally T-cell expressed and secreted (RANTES) in culture supernatants. The BM environment in MM patients evidenced elevated concentrations of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and monokine induced by IFN-γ. Additionally, in the BM of MM patients post alloSCT, we found selectively elevated concentration of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin. Eotaxin levels were particularly high in patients with chronic graft-vs-host disease. CONCLUSIONS Our study demonstrates characteristic cytokine/chemokine patterns in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin, might not only be developed into diagnostic instruments and/or predictive biomarkers, but are also potential targets for future myeloma- or graft-vs-host disease-specific therapies.

An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body

OBJECT In the framework of our current study we set out to develop an optimized assay for the quantification of antigen-specific B cells in different compartments of the human body. METHODS Mononuclear cells (MNC) derived from the peripheral blood, bone marrow (BM), or human tonsils were incubated with different combinations of stimuli. The stimulated cells and culture supernatants were then applied to IgG-ELISPOT and ELISA read-out assays and tetanus toxoid (TT)-specific B cell responses were quantified. RESULTS We found that a combination of CD40L, CpG, and IL21 was optimal for the induction of TT-specific IgG-producing cells from memory B cell (mBc) precursors. This cocktail of stimuli led to a proliferation-dependent induction of CD19(intermediate)CD38(high)CD138(high)IgD(negative) terminally differentiated plasma cells. Applying our optimized methodology we were also able to quantify mBc specific for cytomegalovirus and influenza virus A. Most importantly, the same method proved useful for the comparison of mBc frequencies between different compartments of the body and, accordingly, we were able to demonstrate that TT-specific mBc preferably reside within tonsillar tissue. CONCLUSION Here, we optimized an assay for the quantification of antigen-specific B cells in different human tissues demonstrating, for example, that TT-specific mBc preferably reside in human tonsils but not in the BM or the peripheral blood. We suggest that our approach can be used for the enumeration of mBc specific for a wide variety of Ag (microbial, tumor-related, auto-antigens), which will lead to significant improvements regarding our knowledge about the biology of humoral immunity.

Surface molecule CD229 as a novel target for the diagnosis and treatment of multiple myeloma

Background To date, multiple myeloma remains an incurable malignancy due to the persistence of minimal residual disease in the bone marrow. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cell populations. We, therefore, investigated functionally relevant immunoreceptors specifically associated with myeloma cells as well as their clonogenic precursors. Design and Methods Potential target proteins were identified using antibody arrays against phosphorylated immunoreceptors with lysates from myeloma cell lines. CD229 expression was confirmed in primary myeloma cells by reverse transcriptase polymerase chain reaction, western blot, fluorescence-activated cell sorting, and immunohistochemistry. Apoptosis, clonogenic growth, and sensitivity to chemotherapy were determined following short-interfering RNA-mediated downregulation of CD229. Antibody-dependent cellular and complement-dependent cytotoxicity were analyzed using a monoclonal antibody against CD229 to demonstrate the antigen’s immunotherapeutic potential. Results Our screening assay identified CD229 as the most strongly over-expressed/phosphorylated immunoreceptor in myeloma cell lines. Over-expression was further demonstrated in the CD138-negative population, which has been suggested to represent myeloma precursors, as well as on primary tumor cells from myeloma patients. Accordingly, CD229 staining of patients’ bone marrow samples enabled the identification of myeloma cells by flow cytometry and immunohistochemistry. Down-regulation of CD229 led to a decreased number of viable myeloma cells and clonal myeloma colonies, and enhanced the anti-tumor activity of conventional chemotherapeutics. Targeting CD229 with a monoclonal antibody resulted in complement- and cell-mediated lysis of myeloma cells. Conclusions Our results demonstrate that the immunoreceptor CD229 is specifically over-expressed on myeloma cells including their clonogenic precursors and contributes to their malignant phenotype. Monoclonal antibodies against this protein may represent a promising diagnostic and immunotherapeutic instrument in this disease.

Role of Interleukin 16 in Multiple Myeloma

BACKGROUND Multiple myeloma is a malignancy characterized by the expansion of a plasma cell clone that localizes to the human bone marrow. Myeloma cells and bone marrow stromal cells produce soluble factors that promote the survival and progression of multiple myeloma. Interleukin 16 (IL-16) is involved in regulating the migration and proliferation of normal leukocytes. However, the role of IL-16 in human cancers, including multiple myeloma, is unclear. METHODS We investigated IL-16 expression in cell lines (n = 10) and in the bone marrow of myeloma patients (n = 62) and healthy bone marrow donors (n = 12) by quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. Transfection of two human multiple myeloma cell lines with small interfering RNAs was used to examine the effect of IL-16 gene silencing on apoptosis by flow cytometry, on proliferation by bromodeoxyuridine incorporation, and on colony formation. Protein neutralization assays were performed by treating multiple myeloma cells with a monoclonal antibody against the carboxyl-terminal fragment of IL-16. All statistical tests were two-sided. RESULTS IL-16 was strongly overexpressed in the bone marrow of myeloma patients compared with healthy donors. Myeloma cell lines as well as primary tumor cells from myeloma patients constitutively expressed IL-16 and its receptors CD4 and/or CD9 and spontaneously secreted soluble IL-16. Silencing of IL-16 reduced the proliferative activity of myeloma cells by approximately 80% compared with untreated cells (mean relative proliferative activity IL-16 siRNA vs untransfected cells, EJM cells: 20.1%, 95% confidence interval [CI] = 14.3% to 26.0%, P = .03; KMS-12-BM cells: 22.8%, 95% CI = 5.5% to 40.0%, P = .04), and addition of a recombinant carboxyl-terminal IL-16 peptide reversed that effect. A monoclonal antibody directed against IL-16 or its receptors had a comparably strong growth-inhibiting effect on the tumor cells. CONCLUSIONS IL-16 is an important growth-promoting factor in multiple myeloma and a candidate for novel diagnostic, prognostic, and therapeutic applications for this incurable human malignancy.

The trifunctional antibody catumaxomab amplifies and shapes tumor-specific immunity when applied to gastric cancer patients in the adjuvant setting

Background: Patients with gastric cancer benefit from perioperative chemotherapy, however, treatment is toxic and many patients will relapse. The trifunctional antibody catumaxomab targets EpCAM on tumor cells, CD3 on T cells, and the Fcγ-receptor of antigen-presenting cells. While in Europe catumaxomab is approved for treating malignant ascites, it has not been investigated in the perioperative setting and its exact immunological mode of action is unclear. Methods: In our study, gastric cancer patients received neoadjuvant platinum-based chemotherapy, one intraoperative application of catumaxomab, and 4 postoperative doses of intraperitoneal catumaxomab. Immunomonitoring was performed in 6 patients before surgery, after completion of catumaxomab treatment, and one month later. Results: Intraperitoneal application of catumaxomab caused an increased expression of activation markers on the patients’ T cells. This was accompanied by a transient decrease in numbers of CXCR3+ effector T cells with a T-helper (Th)-1 phenotype in the peripheral blood. All patients evidenced pre-existing EpCAM-specific CD4+ and/or CD8+ T cells. While these cells transiently disappeared from the blood stream after intraperitoneal application of catumaxomab, we detected increased numbers of peripheral EpCAM-specific cells and a modified EpCAM-specific T-cell repertoire 4 weeks after completion of treatment. Finally, catumaxomab also amplified humoral immunity to tumor antigens other than EpCAM. Conclusions: Our findings suggest that catumaxomab exerts its clinical effects by (1) activating peripheral T cells, (2) redistributing effector T cells from the blood into peripheral tissues, (3) expanding and shaping of the pre-existing EpCAM-specific T-cell repertoire, and (4) spreading of anti-tumor immunity to different tumor antigens.

Acute psychological stress increases peripheral blood CD3+CD56+ natural killer T cells in healthy men: possible implications for the development and treatment of allergic and autoimmune disorders.

Abstract Acute psychological stress has primarily been investigated regarding its effects on conventional lymphocytes such as natural killer (NK) cells and CD4+ and CD8+ T cells. However, it might be important to focus on more “specialized” lymphocyte subsets, playing a role, for instance, in allergic conditions and autoimmunity, to identify links between stress, the immune system and somatic diseases. Using flow cytometry we determined frequencies of circulating T helper (Th)1-type (CD226+) and Th2-type (CRTH2+) T cells, γδ T cells, conventional CD56+ natural killer T (NKT) cells and invariant NKT cells (iNKT) in healthy young males (N = 31; median age 26 years) undergoing a laboratory computer-based stressor lasting 12 min. We found that acute psychological stress induced a prolonged increase in CD4+ and CD8+ T cells expressing a Th2 phenotype. We also detected an acute increase in CD4− and CD8− double negative γδ T cells. Finally, we found that the well-known increase in NK cells under stressful conditions was paralleled by a significant increase in numbers of conventional CD56+ NKT cells. In contrast, numbers of iNKT was not altered by stress. This study adds further evidence to a psychoneuroimmunological model proposing that under stressful conditions certain lymphocyte subsets, including iNKT and less mature T cells, are retained in lymphoid tissues while antigen-experienced effector-type T cells with a Th2 phenotype, γδ T cells and conventional CD56+ NKT cells are mobilized into the peripheral blood. We suggest that in the case of frequent stress exposure, this might result in the promotion of, for example, allergic conditions.

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy.

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies.

Abstract 515: Generation of MAGE-C2/CT10-specific T cell clones for immunotherapy of multiple myeloma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective Multiple myeloma (MM) is an incurable neoplasm derived from bone marrow-residing malignant plasma cells. The median life expectancy of MM patients ranges between 3 and 5 years despite the application of conventional chemotherapy including novel agents as well as stem cell transplantation (SCT). Cancer-testis (CT) antigens such as MAGE-C2/CT10 are commonly and specifically expressed in myeloma and, therefore, represent attractive target structures for T cell-based immunotherapies. Methods We screened sera of MM-patients for MAGE-C2/CT10-specific IgG antibodies in an ELISA using recombinant full-length protein. In sera-positive patients frequencies of MAGE-C2/CT10-specific CD4+ and CD8+ T cells were determined following a single cycle of in vitro presensitization of peripheral T cells using a panel of overlapping 20mer peptides spanning the complete sequence of MAGE-C2/CT10. In an IFN-gamma-ELISPOT, which served as a read-out assay, presensitized T cells were re-exposed to MAGE-C2/CT10 peptides pulsed on autologous APC (PHA-stimulated CD4+ T cells [T-APC] or EBV-transformed B-lymphocytes). Stimulated T cells responding to MAGE-C2/CT10 underwent single-cell FACS-sorting of live IFN-gamma secreting T cells using a cytokine secretion assay. After generation of MAGE-C2/CT10-specific CD4+ and CD8+ T cell-clones we identified the composition of the specific T-cell-receptors (TCR) and amplified the receptor chains via PCR. Jurkat-cells were transduced with the specific TCR and specifity and affinity of the resulting genetically modified T cells was evaluated. Results In all 5 of 7 MM-patients with an antibody response against MAGE-C2/CT10 we also detected significant T cell responses against the same antigen. All of these patients evidenced MAGE-C2/CT10 CD4+ T cells and 3/5 patients also showed concomitant CD8+ T cell responses against MAGE-C2/CT10. Overall, CD8+ T cell responses were directed against a total number of 5 different epitopes of MAGE-C2/CT10 whereas CD4+ T cells recognized a total of 13 single peptide sequences. Interestingly, in one patient CD4+ and CD8+ T cells recognized the same MAGE-C2/CT10 20mer peptide. From those myeloma patients evidencing T cell responses against MAGE-C2/CT10 we were able to generate a total number of 44 T cell-clones specific for 8 different epitopes of the CT antigen. Until now, we have identified and cloned the specific TCR of 7 T cell clones. Preliminary experiments suggest that Jurkat cells transduced with the specific TCR recognize naturally processed MAGE-C2/CT10 and are of high avidity. Conclusions Our results demonstrate that the CT antigen MAGE-C2/CT10 induces spontaneous antibody as well as CD4+ and CD8+ T cell responses in myeloma patients. Cloning of these spontaneously occurring T cells seems to be an efficient way to generate TCR-transduced T cells for the MAGE-C2/CT10-specific immunotherapy of MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 515. doi:1538-7445.AM2012-515