VT68.2: An Antibody to Chondroitin Sulfate Proteoglycan 4 (CSPG4) Displays Reactivity against a Tumor-Associated Carbohydrate Antigen.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

Carbohydrate (Chondroitin 4) Sulfotransferase-11-Mediated Induction of Epithelial-Mesenchymal Transition and Generation of Cancer Stem Cells.

INTRODUCTION: We have previously shown that the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) is elevated in human breast cancer tissues, and that its expression in human breast cancer cell lines is associated with aggressive behavior of cells. The clinical significance of CHST11 expression is unknown, and its function in breast cancer cells is not fully understood. OBJECTIVE: The current study was performed to define the clinical significance of this gene and address its biological function in promoting the aggressive behavior of breast cancer cells. METHODS: Publicly available datasets were analyzed to determine the correlation of CHST11 expression with breast cancer survival. MCF-7 cells were transfected with the human CHST11 gene, and MCF-7-CHST11 cells with stable expression of the gene were established. Morphology and metastatic capacity of transfected cells were monitored in vitro. E-cadherin and ß-catenin expression was compared by immunofluorescence. The expression of genes involved in epithelial-mesenchymal transition (EMT) and pluripotency was determined using real-time PCR. The Wnt inhibitor, Wnt-C59, was used to examine the involvement of Wnt in CHST11-mediated morphology. RESULTS: The elevated expression of CHST11 in breast tumor specimens was significantly associated with poor survival among patients. MCF-7-CHST11 cells displayed morphological characteristics consistent with EMT, together with a significantly higher proliferation rate, enhanced migratory potential, and more robust anchorage-independent growth. MCF-7-CHST11 cells showed decreased expression of E-cadherin and increased accumulation of ß-catenin, as assessed by immunofluorescence. Consistently, increased expression of CHST11 resulted in upregulation of key EMT and stem cell markers. Morphological transition in MCF-7-CHST11 cells was partially reversed by co-incubation with an inhibitor of the Wnt pathway. CONCLUSIONS: Our findings support a role for CHST11 in induction of EMT and stem cell-like properties. Our data also associate the expression levels of CHST11 in breast tumor specimens with patients' survival. The results have a significant implication for CHST11 expression level as a novel molecular signature for predictive and prognostic purposes in breast cancer. Moreover, with a possible role in driving tumor cell aggressiveness, CHST11 expression might be further considered as a potential therapeutic target for breast cancer.

Tumor-associated Glycans and Tregs in Immunogenicity of an Autologous Cell-based Vaccine.

Development of cancer vaccines targeting tumor-associated antigens (TAAs) is an alternative approach to chemotherapy with sustained anti-tumor effects. The success of active immunotherapy has been hampered by tumor-induced immune suppressors. Regulatory T cells (Tregs) are a population of immune suppressors with a proven role in regulating anti-tumor immune responses. Removing or subduing Tregs activity leads to more robust anti-tumor immune responses. Here, we used a cell-based vaccination strategy in the 4T1 murine mammary model to examine whether bulk removal of certain TAAs, using their glycan profile, can affect the immunogenicity of the vaccine. We employed affinity columns of several lectins that are reactive with breast cancer cell lines to deplete lectin-reactive TAAs, while enriching for other antigens. Wheat germ agglutinin (WGA), concanavalin A (Con A), Vicia villosa (VVA), and Griffonia simplicifolia lectin-I (GS-I) were used to fraction crude tumor secreted antigens (TSA). Fractions were tested for their ability to stimulate Tregs and their anti-tumor efficacy. We observed that crude TSA activated Tregs and activation of CD4+CD25+ cells led to an inhibitory function on CD4+CD25- effector cells. Immunization of mice with GS-I- and VVA-depleted fractions significantly delayed tumor establishment and inhibited lung metastases. Depletion of WGA-reactive glycoconjugates led to activation of Tregs, larger tumors and more distant metastases. The data indicate that TAAs can be enriched using their glycan expression pattern to weaken immune suppression and improve anti-tumor response. Therefore, the efficacy of autologous cancer cell vaccination can be improved through enrichment for certain TAAs using carbohydrate specificity.

Antibodies: At The Nexus of Antigens and Cancer Vaccines.

This review describes the development of monoclonal antibodies and the inception of their use in cancer therapy, their impact on defining cancer biomarkers, and their structural utility in new cancer vaccine development.

Harnessing Antibody Polyspecificity for Cancer Immunotherapy.

Targeting the diverse glycan repertoire expressed on tumor cells is considered a viable therapeutic strategy to deal with tumor cell heterogeneity. Inherently polyspecific, natural, glycan-reactive antibodies are purported to be protective in thwarting infections and in cancer immunotherapy. Tumor-associated carbohydrate antigens (TACAs) are related to pathogen glycans, to which nascent or natural antibodies exist and IgM responses are elicited. To capture the polyspecific nature of anticarbohydrate responses, we have focused on the rational design of carbohydrate mimetic peptides (CMPs) cross-reactive with TACA reactive antibodies. In particular, we have focused on the development of CMPs that display reactivity to GD2 and Lewis Y (LeY) reactive monoclonal antibodies. They would serve as templates for pan-immunogens inducing biosimilar polyreactive antibodies. In the design, we relied on structural analyses of CMP's enhanced binding to the templates using molecular modeling. Glycan reactivity patterns of affinity CMP-purified human antibodies further refined specificity profiles in comparison with the immune response to the CMP in clinical trials. In this study, we further define the molecular characteristics for this mimicry by considering the polyspecificity of LeY and GD2 reactive antibodies binding to the lacto-ceramide core Galß(1,4)Glcß(1-1')Cer. Binding to this minimum building block can be capitalized on for cancer therapy and diagnostics and illustrates a new approach in designing cancer vaccines taking advantage of the latent polyspecificity of antibodies and the relevance of natural antibodies in antigen discovery and design.

Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells.

INTRODUCTION: We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. METHODS: Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group). RESULTS: The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002). CONCLUSIONS: Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.

Glycan mediated immune responses to tumor cells.

Preclinical animal studies convincingly demonstrate that tumor immunity to self-antigens can be actively induced and can translate into effective anti-tumor responses. Among the most challenging of clinical targets for cancer immunotherapy is Tumor Associated Carbohydrate Antigens (TACA). The molecular characterization of TACA suggest that these glycans are both altered and self-antigens. A new appreciation of the interaction of glycans with immune effector cells that will benefit immunotherapy strategies is emerging as more information on the nature of molecular interactions of glycan recognition molecules is obtained. Carbohydrate recognition affects more or less every aspect of the innate and adaptive immune response and their role in immunotherapy of cancer should be considered beyond the existing paradigm of traditional TACA based-vaccines.

P10s-PADRE vaccine combined with neoadjuvant chemotherapy in ER-positive breast cancer patients induces humoral and cellular immune responses.

Breast cancer patients diagnosed with HR+/HER2- tumors face a persistent risk of distant recurrence long after completion of their treatment. Strategies to induce anti-tumor immune responses could complement standard-of-care therapies for these patients. The current study was performed to examine the feasibility, safety and immunogenicity of adding P10s-PADRE to standard-of-care chemotherapy in HR+/HER2- early-stage breast cancer patients. Twenty-five subjects were treated in a single-arm Phase Ib clinical trial. Five different immunization schedules were considered to evaluate the feasibility of eliciting an immune response. The primary immunogenicity endpoint was antibody titer. The expression of several activation markers on natural killer (NK) cells and serum concentrations of Th1/Th2 cytokines were also examined. The percentage of tumor-infiltrating lymphocytes (TILs) was determined. Antibody response was superior in schedule C where 3 weekly immunizations preceded the first dose of chemotherapy. A significant change in CD16, NKp46 and CD94 expression levels on NK cells and a rise in serum content of IFN-γ was observed after treatment. Schedule C showed an increase in TILs in residual lesions. The combination therapy is safe and immunogenic with treatment schedule C being immunologically promising. Randomized trials focused on long-term survival outcomes are needed to evaluate clinical benefits.

Bridging innate and adaptive antitumor immunity targeting glycans.

Effective immunotherapy for cancer depends on cellular responses to tumor antigens. The role of major histocompatibility complex (MHC) in T-cell recognition and T-cell receptor repertoire selection has become a central tenet in immunology. Structurally, this does not contradict earlier findings that T-cells can differentiate between small hapten structures like simple glycans. Understanding T-cell recognition of antigens as defined genetically by MHC and combinatorially by T cell receptors led to the "altered self" hypothesis. This notion reflects a more fundamental principle underlying immune surveillance and integrating evolutionarily and mechanistically diverse elements of the immune system. Danger associated molecular patterns, including those generated by glycan remodeling, represent an instance of altered self. A prominent example is the modification of the tumor-associated antigen MUC1. Similar examples emphasize glycan reactivity patterns of antigen receptors as a phenomenon bridging innate and adaptive but also humoral and cellular immunity and providing templates for immunotherapies.

Immunomodulatory effects of radiofrequency ablation in a breast cancer model.

Radiofrequency ablation (RFA), a minimally invasive surgical procedure has an increasing application in the surgical treatment of tumors. Data indicate that RFA might stimulate anti-cancer immunity possibly through the induction of necrosis and heat shock proteins (HSP) expression. This study tests a hypothesis that RFA leads to bidirectional immunoregulation. Experimental rat breast tumors were treated with RFA, surgical excision or sham operation. RFA resulted in the highest NK cells infiltration, increased HSP70 expression and activation of caspase-3 enzyme in the tumor margins. A significant reduction of the circulatory regulatory T (Treg) cells was found in both RFA- and excision-treated rats, although less pronounced in the RFA-group. The splenocyte proliferation to tumor cell lysate was stronger in the RFA-treated rats in comparison with untreated tumor-bearing rats. The potential role of self-HSP for immunomodulation was examined using in vitro proliferation assay to tetanus toxoid using human peripheral leukocytes. The response to the tetanus toxoid was significantly suppressed by HSP90 plus auto-antibodies versus HSP90 or auto-antibodies alone. In conclusion, RFA reduced the circulatory Tregs although not as efficient as tumor excision. HSPs plus natural antibodies suppress the anti-tumor response probably by stimulating Tregs. Therefore, RFA may play a role in anti-cancer therapy if combined with Tregs suppression.

Association of DNA-Methylation Profiles With Immune Responses Elicited in Breast Cancer Patients Immunized With a Carbohydrate-Mimicking Peptide: A Pilot Study.

Immune response to a given antigen, particularly in cancer patients, is complex and is controlled by various genetic and environmental factors. Identifying biomarkers that can predict robust response to immunization is an urgent need in clinical cancer vaccine development. Given the involvement of DNA methylation in the development of lymphocytes, tumorigenicity and tumor progression, we aimed to analyze pre-vaccination DNA methylation profiles of peripheral blood mononuclear cells (PBMCs) from breast cancer subjects vaccinated with a novel peptide-based vaccine referred to as P10s-PADRE. This pilot study was performed to evaluate whether signatures of differentially methylated (DM) loci can be developed as potential predictive biomarkers for prescreening subjects with cancer who will most likely generate an immune response to the vaccine. Genomic DNA was isolated from PBMCs of eight vaccinated subjects, and their DNA methylation profiles were determined using Infinium® MethylationEPIC BeadChip array from Illumina. A linear regression model was applied to identify loci that were differentially methylated with respect to anti-peptide antibody titers and with IFN-γ production. The data were summarized using unsupervised-learning methods: hierarchical clustering and principal-component analysis. Pathways and networks involved were predicted by Ingenuity Pathway Analysis. We observed that the profile of DM loci separated subjects in regards to the levels of immune responses. Canonical pathways and networks related to metabolic and immunological functions were found to be involved. The data suggest that it is feasible to correlate methylation signatures in pre-treatment PBMCs with immune responses post-treatment in cancer patients going through standard-of-care chemotherapy. Larger and prospective studies that focus on DM loci in PBMCs is warranted to develop pre-screening biomarkers before BC vaccination. Clinical Trial Registration: www.ClinicalTrials.gov, Identifier: NCT02229084.

The Promise of Anti-idiotype Revisited.

The promise of idiotype-based therapeutics has been disappointing forcing a new look at the concept and its potential to generate an effective approach for immunotherapy. Here, the idiotype network theory is revisited with regard to the development of efficacious anti-idiotype vaccines. The experience of polyclonal anti-Idiotype reagents in animal models as well as an understanding of the immune response in humans lends to the proposition that polyclonal anti-Idiotype vaccines will be more effective compared to monoclonal-based anti-Idiotype vaccines. This novel strategy can be adapted in Biotech-standard production of therapeutic antibodies.

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries.

Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients' sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

Expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry.

BACKGROUND: The number of breast cancer patients diagnosed with ductal carcinoma in situ (DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences. METHODS: For detection of TACA expression, specimens were stained with Griffonia simplicifolia lectin-I (GS-I) and Vicia vilosa agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors. RESULTS: We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; P = 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; P = 0.0004). CONCLUSION: The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk.

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens for cancer vaccination.

Mechanisms of broad cross-protection, as seen in viral infection and also applied to vaccines, emphasize preexisting antibodies, CD8+ memory T cells, and accelerated B-cell responses reactive with conserved regions in antigens. Another practical application to induce broad-spectrum responses is making use of multispecific antigen recognition by antibodies and T cells. Antibody polyreactivity can be related to the capacity of the antigen-combining site to accommodate a number of different small epitopes or to attain different conformations. A better understanding of the functionality of molecular interactions with graded specificity might help the design of polyreactive immunogens inducing antibody responses to a predefined set of target antigens. We have found this approach useful in targeting tumor-associated carbohydrate antigens in cancer vaccine development. Using combinatorial libraries and pharmacophore design principles, carbohydrate mimetic peptides were created that not only induce antibodies with multiple specificities, but also cellular responses that inhibit tumor growth in vivo.