Melanoma-associated Chondroitin Sulfate Proteoglycan Research Articles

Abstract 5530: Metastasis founder cells activate immunsuppression early in human metastatic colonization

Abstract Metastatic dissemination occurs very early in melanoma, often before diagnosis, rendering prevention of metastasis formation an important therapeutic goal. However, the earliest steps of lethal metastasis in patients are incompletely understood. Therefore, in this study we searched for the earliest detectable disseminated cancer cells (DCC) in sentinel lymph node biopsies of 492 stage I-III melanoma patients. We used micromanipulator-assisted isolation and single-cell transcriptome analysis of these extremely rare DCC and identified melanoma -associated chondroitin sulfate proteoglycan (MCSP) expressing melanoma cells as strong candidates for metastasis founder cells in lymph nodes. Based on a median follow-up time of 6 years, their detection was the strongest predictor of systemic metastasis and death upon multivariable analysis. Single-cell transcriptome analysis revealed that melanoma DCC were exposed to CD8 T cell attack during the transition from single cells to metastasis-initiating clusters, activated the extracellular vesicular exosomal pathway, and expressed the immunomodulatory proteins CD155 and CD276, but rarely PD-L1. CD155- and CD276-positive extracellular vesicles from patient-derived DCC models showed an immunosuppressive activity on CD8 T cells. In conclusion, our data suggest that either direct targeting of metastasis-founder cell with MCSP or their immune escape mechanisms may be key to curing early-stage melanoma. Citation Format: Severin Guetter, Courtney Koenig, Huiqin Koerkel-Qu, Aleksandra Markiewicz, Sebastian Scheitler, Marie Katzer, Mark Berneburg, Philipp Renner, Beatrix Cucuruz, Leonhard Guttenberger, Veronika Naimer, Kathrin Weidele, Steffi Treitschke, Christian Werno, Hanna Jaser, Tonia Bargmann, Armin Braun, Florian Weber, Katharina Limm, Reinhard Rachel, Felix Baumann, Lisa Schmidleithner, Kathrin Schambeck, Anja Ulmer, Sebastian Haferkamp, Christoph Klein, Melanie Werner-Klein. Metastasis founder cells activate immunsuppression early in human metastatic colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5530.

Abstract 5979: Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model

Abstract Activity of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, is reduced in several malignant cell lines and human tissues, including melanomas, mammary, prostate, and colon. Following tail vein injection of B16F10 melanoma cells in 12-week-old female C57BL/6J mice (n=11) and subsequent treatment by tail vein injection of bioactive, recombinant human ARSB (0.2 mg/kg), on days 2 and 7, development of metastatic lung lesions was markedly reduced. The average number of lesions declined from mean value of 114 (range: 10-278; n=5) to mean value of 2.5 (range: 0-9; n=6). These findings are consistent with our previous report of significant increase in survival and decrease in size of B16F10 subcutaneous melanomas in C57BL/6J mice treated with exogenous ARSB. The underlying mechanisms leading to inhibition of melanoma proliferation have profound impact on vital signaling pathways and transcriptional events. These pathways involve more or less binding with C4S when ARSB activity is modified. Notably, galectin-3 binding with C4S increases following exogenous ARSB, leading to decline in free nuclear galectin-3. This leads to reduced expression of the melanoma biomarker chondroitin sulfate proteoglycan CSPG)-4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP). Expression of Programmed Death-Ligand 1 (PD-L1) is also reduced, declining from 1.34 ± 0.23 ng/mg protein (n=5) to 0.50 ± 0.05 ng/mg protein (n=6) following ARSB treatment. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects contribute to decline in matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Following treatment with exogenous ARSB, the serum MMP2 declined from 267 ± 38 (ng/ml; n=5) to 121 ± 16 ng/ml (n=6), consistent with the observed decline in melanoma lesions. The impact of ARSB on signaling pathways and transcriptional events demonstrates how modification of a critical sulfation and the resulting impact on binding with C4S influences vital cell processes and can suppress tumor development. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Exogenous ARSB may become an effective treatment for acquired deficiency of ARSB in melanoma. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Insug O-Sullivan. Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5979.

Abstract LB_C21: Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1

Abstract In experiments in melanoma, prostate, colon, and mammary cells and tissues, the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S), acts as a tumor suppressor. Following treatment with recombinant human (rh) ARSB in previous experiments and in recent experiments in the B16F10 mouse model of malignant melanoma, specific molecular pathways have been identified that retard tumor progression. These pathways involve more or less binding with chondroitin 4-sulfate when ARSB activity is modified. Galectin-3 binding with C4S increases following exogenous ARSB, leading to lower nuclear galectin-3 and reduced expression of chondroitin sulfate proteoglycan (CSPG)4 and of carbohydrate sulfotransferase (CHST)15 in melanoma cells and tissues. Nuclear Sp1 also declines, inhibiting transcription of both CSPG4, also known as the melanoma-associated chondroitin sulfate proteoglycan (MCSP), and of CHST15, which is required for 6-sulfation of N-acetylgalactosamine 4-sulfate. These proteins are associated with more aggressive tumors, and decline in their expression is consistent with the tumor suppressive function of ARSB. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects lead to decline in expression of the matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Treatment of cultured melanoma cells with siRNA for ARSB leads to opposite effects to those of exogenous ARSB, including epigenetic effects modifying Programmed Death-Ligand 1 (PD-L1) expression. Treatment with rhARSB significantly reduces PD-L1 protein and gene expression, in contrast to increases when ARSB is silenced. The effects of rhARSB on PD-L1 expression are attributable to declines in phospho-JNK and nuclear c-Jun when free galectin-3 is less and to associated increases in histone deacetylase (HDAC) 3 expression and activity. Consistent with these effects, the ARSB-silencing induced increase in PD-L1 is inhibited by peptide inhibitors of the JNK-c-Jun interaction and by HDAC3 inhibitor. ARSB silencing reduces PD-L1 promoter acetylation, as shown by chromatin immunoprecipitation with anti-acetylhistone H3 antibody. The impact of rhARSB on signaling pathways, transcriptional events, and epigenetic mechanisms demonstrates how a critical sulfation and the resulting modification of chondroitin 4-sulfate can influence vital cell processes and can suppress tumor development. Effects on subcutaneous and pulmonary melanomas demonstrate the impact of exogenous ARSB in melanoma and suggest future prospects as a therapeutic agent. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Citation Format: Joanne K. Tobacman, Insug O-Sullivan, Sumit Bhattacharyya. Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C21.

Bispecific antibodies redirect synthetic agonistic receptor modified T cells against melanoma

BackgroundMelanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating...

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

Abstract 3511: Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche

Abstract Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. Here, we test the hypothesis that lymph-borne TDFs are sufficient to initiate the formation of the pre-metastatic LN niche. Using a murine melanoma model (YUMM1.7), we demonstrate primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, we have demonstrated that TDFs isolated from YUMM1.7-conditioned media uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however our data further demonstrates TSPs are also sufficient to activate this niche. To identify candidate TDFs in vivo that have the potential to initiate LN transformation, we adopted a novel chemical biology strategy to ubiquitously tag TDFs in vivo using non-canonical amino acids and engineered expression of mutant tRNA synthetases in YUMM1.7 melanoma cells. Using this approach, we identified several TDFs by mass spectrometry present in pre-metastatic LNs, and identified one candidate (CSPG4; Melanoma-Associated Chondroitin Sulfate Proteoglycan) as a potential mediator of pre-metastatic niche development. Shed CSPG4 is found within TSPs isolated from media conditioned by YUMM1.7, but not benign dermal, cells. We generated stable shRNA-expressing YUMM1.7 cell lines for efficient knockdown of CSPG4, and found that loss of CSPG4 confers loss of TSP-dependent pre-metastatic niche development in the draining LN. Furthermore, using an enzyme specific to chondroitins (the main glycan decorating CSPG4), we demonstrate that loss of chondroitin glycosylation of CSPG4 is sufficient to attenuate metastasis, even with the core protein present. We find that fibroblastic reticular cells in the draining LN respond to TSPs by becoming more myofibroblast like, resembling cancer-associated fibroblasts that provide direct trophic support to tumor cells. This phenotypic switch is partially rescued in the absence of CSPG4. Our results collectively indicate TSPs prime the LN for metastasis and we have identified melanoma-shed CSPG4 as a driver of pre-metastatic niche development, potentially through its influence on the LN-resident fibroblasts. Citation Format: Haley du Bois, Annie L. Chen, Katherine S. Ventre, Maria S. Steele, Triantafyllia Karakousi, Amanda W. Lund. Melanoma-shed, lymph-borne CSPG4 conditions the pre-metastatic lymph node niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3511.

Evaluation of the Potential of Umbilical Cord Mesenchymal Stromal Cell–Derived Small Extracellular Vesicles to Improve Rotator Cuff Healing: A Pilot Ovine Study

Background: Despite significant advancements in surgical techniques to repair rotator cuff (RC) injuries, failure rates remain high and novel approaches to adequately overcome the natural biological limits of tendon and enthesis regeneration of the RC are required. Small extracellular vesicles (sEVs) derived from the secretome of human multipotent mesenchymal stromal cells (MSCs) have been demonstrated to modulate inflammation and reduce fibrotic adhesions, and therefore their local application could improve outcomes after RC repair. Purpose: In this pilot study, we evaluated the efficacy of clinical-grade human umbilical cord (hUC) MSC-derived sEVs (hUC-MSC-sEVs) loaded onto a type 1 collagen scaffold in an ovine model of acute infraspinatus tendon injury to improve RC healing. Study Design: Controlled laboratory study. Methods: sEVs were enriched from hUC-MSC culture media and were characterized by surface marker profiling. The immunomodulatory capacity was evaluated in vitro by T-cell proliferation assays, and particle count was determined by nanoparticle tracking analysis. Twelve skeletally mature sheep were subjected to partial infraspinatus tenotomy and enthesis debridement. The defects of 6 animals were treated with 2 × 1010 hUC-MSC-sEVs loaded onto a type 1 collagen sponge, whereas 6 animals received only a collagen sponge, serving as controls. Six weeks postoperatively, the healing of the infraspinatus tendon and the enthesis was evaluated by magnetic resonance imaging (MRI) and hard tissue histology. Results: CD3/CD28-stimulated T-cell proliferation was significantly inhibited by hUC-MSC-sEVs (P = .015) that displayed the typical surface marker profile, including the presence of the MSC marker proteins CD44 and melanoma-associated chondroitin sulfate proteoglycan. The local application of hUC-MSC-sEVs did not result in any marked systemic adverse events. Histologically, significantly improved Watkins scores (P = .031) indicated improved tendon and tendon-to-bone insertion repair after sEV treatment and lower postcontrast signal of the tendon and adjacent structures on MRI suggested less residual inflammation at the defect area. Furthermore, the formation of osteophytes at the injury site was significantly attenuated (P = .037). Conclusion: A local, single-dose application of hUC-MSC-sEVs promoted tendon and enthesis healing in an ovine model of acute RC injury. Clinical Relevance: Surgical repair of RC tears generally results in a clinical benefit for the patient; however, considerable rerupture rates have been reported. sEVs have potential as a cell-free biotherapeutic to improve healing outcomes after RC injury.

Extracellular Vesicle Surface Markers as a Diagnostic Tool in Transient Ischemic Attacks.

Extracellular vesicles (EVs) are promising biomarkers for cerebral ischemic diseases, but not systematically tested in patients with transient ischemic attacks (TIAs). We aimed at (1) investigating the profile of EV-surface antigens in patients with symptoms suspicious for TIA; (2) developing and validating a predictive model for TIA diagnosis based on a specific EV-surface antigen profile. We analyzed 40 subjects with symptoms suspicious for TIA and 20 healthy controls from a training cohort. An independent cohort of 28 subjects served as external validation. Patients were stratified according to likelihood of having a real ischemic event using the Precise Diagnostic Score, defined as: unlikely (score 0–1), possible-probable (score 2–3), or very likely (score 4–8). Serum vesicles were quantified by nanoparticle tracking analysis and EV-surface antigen profile characterized by multiplex flow cytometry. EV concentration increased in patients with very likely or possible-probable TIA (P<0.05) compared with controls. Nanoparticle concentration was directly correlated with the Precise Diagnostic score (R=0.712; P<0.001). After EV immuno-capturing, CD8, CD2, CD62P, melanoma-associated chondroitin sulfate proteoglycan, CD42a, CD44, CD326, CD142, CD31, and CD14 were identified as discriminants between groups. Receiver operating characteristic curve analysis confirmed a reliable diagnostic performance for each of these markers taken individually and for a compound marker derived from their linear combinations (area under the curve, 0.851). Finally, a random forest model combining the expression levels of selected markers achieved an accuracy of 96% and 78.9% for discriminating patients with a very likely TIA, in the training and external validation cohort, respectively. The EV-surface antigen profile appears to be different in patients with transient symptoms adjudicated to be very likely caused by brain ischemia compared with patients whose symptoms were less likely to due to brain ischemia. We propose an algorithm based on an EV-surface-antigen specific signature that might aid in the recognition of TIA.

Ralph A. Reisfeld, PhD: In Memoriam (1926–2020)

![][1] The sad news that Ralph A. Reisfeld, PhD, passed away on December 6, 2020 reached us early this year, and we would like to reflect on Ralph's long career and distinctive contribution to biomedical science and in particular to the field of cancer research. Ralph was well known

Flow Cytometry for Detection and Quantification of Micrometastases in Sentinel Lymph Nodes from Patients with Primary Melanoma

BackgroundDetection of micrometastases in the regional lymph nodes is one of the most important prognostic factors for melanoma patients. Our aim was to evaluate the suitability of flow cytometry for rapid quantification of disseminated melanoma cells in sentinel lymph nodes (SLN). Methods132 SLNs from 104 patients diagnosed with melanoma were analyzed by flow cytometry, utilizing the extracellular marker melanoma-associated chondroitin sulfate proteoglycan, in addition to quantitative immunocytology and conventional histopathology, including immunohistochemistry. For quantification, the number of melanoma-positive cells per million lymph node cells (disseminated cancer cell density, DCCD) detected by flow cytometry was compared to the DCCD obtained by immunocytology. ResultsCompared to histopathology and immunocytology, flow cytometry exhibited a sensitivity of 50% and a specificity of 85%. DCCDs of immunocytology and flow cytometry of the 37 immunocytologically positive SLNs showed a positive correlation (Spearman's ρ = 0.7, P < 0.0001). In 10 SLNs from 9 patients with high tumor load, the flow cytometric DCCD was 8-fold higher on average than the immunocytologic DCCD. ConclusionsAlthough flow cytometry is not yet suitable for early detection of metastatic melanoma, it promises to become a valuable tool for rapidly quantifying tumor load in high-risk patients.

Mathematical-model-guided development of full-thickness epidermal equivalent

Epidermal equivalents prepared with passaged keratinocytes are typically 10–20 μm thick, whereas intact human epidermis is up to 100 μm thick. Our established mathematical model of epidermal homeostasis predicted that the undulatory pattern of the papillary layer beneath the epidermis is a key determinant of epidermal thickness. Here, we tested this prediction by seeding human keratinocytes on polyester textiles with various fiber-structural patterns in culture dishes exposed to air, aiming to develop a more physiologically realistic epidermal model using passaged keratinocytes. Textile substrate with fiber thickness and inter-fiber distance matching the computer predictions afforded a three-dimensional epidermal-equivalent model with thick stratum corneum and intercellular lamellar lipid structure. The basal layer structure was similar to that of human papillary layer. Cells located around the textile fibers were proliferating, as indicated by BrdU and YAP (Yes-associated protein) staining and expression of melanoma-associated chondroitin sulfate proteoglycan. Filaggrin, loricrin, claudin 1 and ZO-1 were all appropriately expressed. Silencing of transcriptional coactivator YAP with siRNA disturbed construction of the three-dimensional structure. Measurement of trans-epidermal water loss (TEWL) indicated that the model has excellent barrier function. Our results support the idea that mathematical modeling of complex biological processes can have predictive ability and practical value.

Abstract 697: Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines

Abstract The proteoglycan CSPG4 [chondroitin sulfate proteoglycan 4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2)] has been associated with progression of melanoma, and intervention to reduce CSPG4 has been considered as treatment of melanoma. In recent work, we have reported that decline in the enzyme arylsulfatase B (ARSB, N-acetylgalactosamine 4-sulfatase) is associated with malignancy, including prostate, mammary, and colon. ARSB is the enzyme that removes 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate (DS), and thereby regulates the degradation of the sulfated glycosaminoglycan (GAG) chain. Hence, the absence of ARSB leads to the accumulation of more highly sulfated C4S or DS. In prior published work, decline in ARSB and the associated increase in C4S led to transcriptional events, mediated through a decline in galectin-3 binding to the more highly sulfated C4S present. Transcription factors activator protein 1 (AP-1) and Specificity protein 1 (Sp1) interacted with galectin-3 to mediate the increased transcription of versican and Wnt9A. In this study, the mechanism by which decline in ARSB increased expression of CSPG4 and. increased the invasiveness of melanoma cell lines was addressed. Techniques included: cell culture of normal melanocytes (ATCC PCS 200-013) and melanoma cell lines obtained from radial or vertical growth phase [WM1552C, WM1552C/mock (mock CSPG4 transfection), WM1552C/MCSP (CSPG4 transfected), WM1552C/MCSPÄCD (transfected with CSPG4 without the cytoplasmic domain), WM35] and from metastatic melanoma (1205 Lu); QPCR of CSPG4; ARSB activity assay using the exogenous substrate 4-methylumbelliferylsulfate; silencing of ARSB by siRNA; invasion assay; measurements of total sulfated glycosaminoglycans and C4S using 1,9-dimethylmethylene blue dye; and measurements of matrix metalloproteinase (MMP) activity. The results show that ARSB activity was reduced in the metastatic melanoma cells to 51% of the level in the normal melanocytes. Silencing of ARSB was associated with increase to 2.8 times the baseline level in the mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) in the normal melanocytes. Invasiveness of the melanoma cells increased by 70% when ARSB was silenced, with corresponding increase in MMP activity. Since CSPG4 has C4S attachments, the effects of ARSB on CSPG4 expression may affect the availability of C4S GAG chains to interact with extracellular matrix components and affect invasiveness. The findings indicate that decline in ARSB and the associated increase in C4S have profound effects on the invasive potential of melanoma and that increased attention to ARSB and C4S may yield new therapeutic interventions. Citation Format: Sumit Bhattacharyya, Leo Feferman, Kaoru Terai, Arkadiusz A. Dudek, Joanne K. Tobacman. Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 697.

Guiding the Killer and Bringing in Accomplices: Bispecific Antibody Treatment for Malignant Melanoma

Discovery of oncogene and immune checkpoint targeting has transformed melanoma therapy in the last 5 years. However, treatment of primary or secondary drug-resistant melanoma remains a challenge. Agents designed to activate the cell death machinery directly, for example by activating the death receptors expressed by melanoma cells, could break drug resistance, and they may achieve long-lasting therapeutic success. He et al. report their studies of an MCSPxDR5 bispecific, tetravalent antibody that can simultaneously target death receptor 5 (DR5, TRAIL-R2) and melanoma-associated chondroitin sulfate proteoglycan (MCSP). This antibody can exert strong and selective DR5-dependent cytotoxic activity against MCSP-expressing melanoma cells. Crosslinking of the antibody with Fcγ-receptors increased the cytotoxic potential further, without compromising its selectivity. This approach offers a novel immunotherapeutic tool via coupling of three cooperating processes: delivering the death receptor agonist to the malignant cell population, potent activation of DR5-mediated cell death signaling, and recruitment of Fcγ-receptor-carrying immune cells that can mount an immune response against the tumor cells.

Redirected Lysis of Human Melanoma Cells by a MCSP/CD3-bispecific BiTE Antibody That Engages Patient-derived T Cells

Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8 T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.

Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction

To identify adult human buccal epithelial stem cells (SCs) on the basis of two parameters (high p63 expression and greater nucleus/cytoplasmic (N/C) ratio) and to evaluate clinical efficacy of ex-vivo expanded autologous epithelium in bilateral limbal SC-deficient (LSCD) patients. The epithelial cells were isolated from buccal biopsy and cultured on human amnion in culture inserts with 3T3 feeder layer. The SCs were identified on the basis of two-parameter analysis using confocal microscopy, surface markers, and colony-forming efficiency (CFE). The cultured epithelium was transplanted in 10 LSCD patients followed by penetrating keratoplasty in 4 patients. The clinical outcome was followed up to 3 years. A distinct population (3.0±1.7%) of small cells expressing high levels of p63 with greater N/C ratio was observed in buccal epithelium. The N/C ratio was found to be more appropriate than cell diameter for two-parameter analysis. These cells located in the basal layer were negative for connexin-43 and positive for melanoma-associated chondroitin sulfate proteoglycan, containing holoclones with 0.2% CFE, thus representing the SC population. After transplantation of cultured epithelium with increased (sixfold) SC content, anatomical and visual improvement was observed at 13-34 months in 3/10 LSCD patients. The two-parameter SC marker is useful to identify and quantify buccal epithelial SCs. The transplantation of bioengineered SC-rich buccal epithelium is a strategy for corneal surface reconstruction in bilateral LSCD. However, further studies are required to optimize the culture conditions and to look for other sources of adult SCs for better visual outcome.

Expression of the Stem Cell Markers Nestin and CD133 on Circulating Melanoma Cells

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01), and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.

Eradication of melanomas by targeted elimination of a minor subset of tumor cells

Proceeding on the assumption that all cancer cells have equal malignant capacities, current regimens in cancer therapy attempt to eradicate all malignant cells of a tumor lesion. Using in vivo targeting of tumor cell subsets, we demonstrate that selective elimination of a definite, minor tumor cell subpopulation is particularly effective in eradicating established melanoma lesions irrespective of the bulk of cancer cells. Tumor cell subsets were specifically eliminated in a tumor lesion by adoptive transfer of engineered cytotoxic T cells redirected in an antigen-restricted manner via a chimeric antigen receptor. Targeted elimination of less than 2% of the tumor cells that coexpress high molecular weight melanoma-associated antigen (HMW-MAA) (melanoma-associated chondroitin sulfate proteoglycan, MCSP) and CD20 lastingly eradicated melanoma lesions, whereas targeting of any random 10% tumor cell subset was not effective. Our data challenge the biological therapy and current drug development paradigms in the treatment of cancer.

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

BackgroundAdvanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to 1. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and 2. activate apoptotic TRAIL-signaling.ResultsTreatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.ConclusionsAnti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.

Abstract 2435: Validation of Cynomolgus monkeys as relevant species for safety assessment of a novel human BiTE antibody platform for cancer therapy

Abstract Nonclinical safety assessment of biopharmaceuticals for human use relies on their cross-reactivity with the respective target in other species, the absence of which precludes testing in commonly used animal models. This is the case for a first-generation of T cell-engaging BiTE antibodies, which solely cross-react with T cells of chimpanzee. BiTE antibodies are designed to transiently connect the CD3 subunit of T cell receptors on T cells with an antigen on target cells. This can very effectively activate T cells, leading to a potent redirected lysis of target cells. Here, we have generated a series of novel human anti-CD3 antibodies that cross-react with non-human primate species, including Cynomolgus monkeys (Macaca fascicularis) and marmoset (Callithrix jacchus). The variable domains of a selected anti-CD3 antibody were used to generate next-generation BiTE antibodies specific for (1) CD33, a target antigen for treatment of acute myelogenous leukemia, (2) melanoma-associated chondroitin sulfate proteoglycan (MCSP), a potential target for treatment of melanoma, and (3) epidermal growth factor receptor 1 (EGFR), a validated target for treatment of colorectal cancer. In vitro, CD33-, MCSP- and EGFR-specific BiTE antibodies showed comparable binding to CD3 on human and macaque T cells, and to CHO cells expressing CD33, MCSP and EGFR of human or macaque origin. In human and macaque cell-based assays, bispecific binding triggered redirected target cell lysis as well as conditional T cell activation, as previously described for first-generation BiTE antibodies. When continuously infused for one week to Cynomolgus monkeys, the CD33-specific BiTE antibody caused depletion of CD33-expressing peripheral blood cells at tolerated doses, and reached a maximum tolerated dose (MTD) at 120 µg/m2/day. The EGFR-specific BiTE antibody reached an MTD at 150 µg/m2/kg. In contrast, the MCSP-specific BiTE antibody was well-tolerated up to a dose of 5,000 µg/m2/day. Our primate data suggest that the tolerability and toxicity of CD33-, MCSP- and EGFR-specific BiTE antibodies were apparently determined by target cell-dependent bispecific binding, and not by monovalent binding to CD3 on macaque T cells. Our data validate the Cynomolgus monkey as a relevant species for assessing the safety and tolerability of next-generation BiTE antibodies that use a more broadly cross-reactive anti-CD3 human single-chain antibody. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2435.

Abstract 1921: Nonadherent human melanoma cell lines from lymph node metastases coexpress B cell markers and melanoma antigens

Abstract Introduction Some atypical human melanoma cell lines are nonadherent to plastic culture flasks for unknown reasons. We have identified five nonadherent human melanoma cell lines derived from lymph node metastases that coexpress B cell markers CD20, CD19, CD21 and CD22 and melanoma antigens when grown in Iscove's-10% fetal bovine serum (FBS) growth medium. Methods The human melanoma cell lines were prepared from metastatic tumors of consenting patients by mechanical mincing into a single cell suspension. Human melanoma cell lines used in this study were grown in Iscove's medium supplemented with 10% FBS, L-glutamine and antibiotics in a humidified 37oC CO2 incubator and passaged at least 9 times, making it highly unlikely that any lymphocytes from the original tumor remained in the cultures. All human melanoma cell lines showed expression of at least one of the melanoma antigens Mart-1, gp100 (HMB45), TRP75 and melanoma-associated chondroitin sulfate proteoglycan (MCSP) as shown by flow cytometry on a FACS Calibur (BD Biosciences). Two different antibodies, anti-human CD20 FITC clone 2H7 (BD Biosciences) and anti-human MS4A1/CD20 fluorescein clone 396444 (R&amp; D Systems), substantiated the CD20 expression on the nonadherent melanoma cell lines. Isotype controls proved specific staining. Nonadherent melanoma cell lines were tested for CD19, CD20, CD21 and CD22, CD34 and CD133. Cells were stained for 15 minutes at room temperature protected from light, washed with 0.5% BSA-PBS, fixed in 1% paraformaldehyde-PBS and analyzed on the flow cytometer within 24 hours of fixation. Intracellullar staining for Mart-1, gp100 and TRP75 used Cytofix / Cytoperm and Perm / Wash reagents (BD Biosciences) according to the manufacturer's instructions, staining for 30 minutes at room temperature. Results The five nonadherent melanoma cell lines contained human melanoma cells that coexpressed CD20 and one of the four melanoma antigens Mart-1, gp100, TRP75 or MCSP. Percentages of double positive cells varied with CD20+ Mart-1+ tending to be the most frequent and expressed by the majority of the cells of the nonadherent melanoma cell lines. All five nonadherent melanoma cell lines expressed the B cell markers CD19, CD20, CD21 and CD22. However, none expressed the hematopoetic stem cell marker CD34. The nonadherent human melanoma cell lines contained over 90% CD20+ cells and a small subpopulation of about 3% or less that coexpressed CD133. The immunophenotype of these atypical nonadherent melanomas suggests that CD20 may not be a stem cell marker but rather expressed with other B cell markers as a result of cell fusion events with B cells. Conclusion Nonadherent human melanoma cell lines derived from lymph node metastases that coexpress B cell markers CD19, CD20, CD21 and CD22 lend to speculation of their origin from cell fusion events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1921.