Laura Bover

A lyophilized one-step kit of Mab FC-5.01, reactive with human breast cancer and malignant melanomas, was formulated. A direct method based on the reduction of protein disulphide bridges using 2-mercaptoethanol was used with methylene diphosphonate as the transchelating ligand. The labeled species were analyzed by ITLC thin layer chromatography and high performance liquid chromatography. Labeling efficiency was greater than 90%.

IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplificati...

Immunotherapies such as checkpoint blockade therapies are known to enhance anti-melanoma CD8+ T cell immunity, but only a fraction of patients treated with these therapies achieve durable immune response and disease control. It may be that CD8+ T cells need help from other immune cells to generate effective and long-lasting anti-tumor immunity or that CD8+ T cells alone are insufficient for complete tumor regression and cure. Melanoma contains significant numbers of B cells; however, the role of B cells in anti-melanoma immunity is controversial. In this study, B16 melanoma mouse models were used to determine the role of B cells in anti-melanoma immunity. C57BL/6 mice, B cell knockout (KO) C57BL/6 mice, anti-CD19, and anti-CXCL13 antibody-treated C57BL/6 mice were used to determine treatment efficacy and generation of tumor-specific CD8+ T cells in response to PD-L1 blockade alone or combination with TLR-7/8 activation. Whole transcriptome analysis was performed on the tumors from B...

FC-2.15 is an IgM monoclonal antibody (MAb) obtained by immunizing Balb/c mice with tumor epithelial cells from a human undifferentiated primary breast carcinoma. FC-2.15 reacts with 93.9% (31/33) of human breast primary tumors, independently of their histology and hormone receptor content. Moreover, FC-2.15 reacts with 79.6 ± 13.8% (mean ± SD) of total breast malignant tumor cells and with 88.7 ± 9.9% of proliferating tumor cells. It recognizes other neoplasia such as colon cancer, squamous carinoma and melanoma. Among the normal tissues examined, strong cross-reactivity was found with kidney proximal convolute tubules, bone marrow myeloid progeny, peripheral granulocytes and large bowel epithelium. Through Western blots, FC-2.15 recognizes three major bands of Mr 160 kDa, 130 kDa and 115 kDa in membrane extracts of MCF-7 cells grown in nude mice and of human breast carcinoma and three major bands of 250 kDa, 185 kDa and 105 kDa in membrane extracts of peripheral granulocytes. This...

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte–associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a β-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain–containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy.

The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminish...

Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted

We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated “outside-in” and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class II-enriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the β-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP-...

Introduction: GSK3174998, a humanized IgG1 agonistic anti-OX40 monoclonal antibody (mAb) identified in collaboration between GSK and MDACC is currently in Phase I clinical development. Critical for the development of more effective cancer immunotherapy are agents that stimulate effector T cells (Teff) and inhibit the immunosuppressive function of regulatory T cells (Treg) that typically infiltrate tumors. OX40 is a tumor necrosis factor receptor superfamily member expressed on the surface of activated CD4+ and CD8+ T cells. OX40 agonism stimulates both immune effector and memory functions and attenuation of Tregs. Therefore, OX40 agonistic mAbs are ideal candidates to potentially increase the efficacy of immune-checkpoint blocking antibodies, like anti-PD1 (aPD1). Methods: GSK3174998 has suitable cross-reactivity to cynomolgus monkey OX40 to inform directly on toxicology, pharmacokinetic and pharmacodynamic (PD) preclinical endpoints. However, to understand the antitumor efficacy of OX40 agonism in vivo, studies were performed using a surrogate mAb to murine OX40 (OX86) alone or in combination with a surrogate aPD1 antibody in A20 lymphoma and CT26 colon carcinoma syngeneic tumor models. Intratumoral (i.e. tumor infiltrating T cells) and peripheral (blood) PD biomarkers, including T cell intracellular and surface protein expression, cytokine production and gene regulation were analyzed. Results: GSK3174998 was well tolerated in monkeys up to 100 mg/kg. In vitro T-cell activation of OX40 with GSK3174998 resulted in enhanced CD4+ and CD8+ effector T-cell proliferation, both in plate-bound as well as soluble PBMC assays. Suppression of Treg differentiation was observed with GSK3174998 as compared with an Fc-disabled mAb, which did not demonstrate these effects. In vitro GSK3174998 induced Th1 cytokine production (IFN and TNFa) and this was further enhanced by the addition of pembrolizumab. In vitro OX86 demonstrated similar characteristics to GSK3174998. In vivo OX86 induced a significant dose-dependent, durable anti-tumor response as monotherapy, which was significantly enhanced when combined with an aPD1 checkpoint inhibitor. Preclinical efficacy correlated with PD changes in several immunological markers including T-cell proliferation and activation. In silico and IHC analysis of expression of OX40 and PDL1 in human tumors was utilized to prioritize cancers most likely to respond to monotherapy and combination therapy for the first-time-in-human (FTIH) clinical study. Conclusions: GSK3174998 is a potent anti-OX40 agonist that engages the immune system via several T-cell-mediated pathways and may further enhance the antitumor activity observed with PD1 inhibition. Preclinical studies provide a strong rationale to support the ongoing FTIH Phase I study of GSK3174998 administered alone and in combination with pembrolizumab to patients with selected advanced solid tumors. Citation Format: Carlo Toniatti, Niranjan Yanamandra, Kui Voo, Amin Al-Shami, Laura Bover, Peter Morley, Sara Brett, Tim Lofton, Jennifer Greer, Ningping Feng, Ignacio Ivan Wistuba, Sabyasachi Bhattacharya, Christopher Hopson, David Kilian, Heather Jackson, Paul Bojczuk, Mili Mandal, Junping Jing, Kevin French, Roopa Srinivasan, Axel Hoos. Engaging the immune system with GSK3174998, a potent anti-OX40 agonist antibody. [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 4864.

mAbs such as anti-CD20 rituximab are proven therapies in B-cell malignancies, yet many patients develop resistance. Novel therapies against alternative targets are needed to circumvent resistance mechanisms. We sought to generate mAbs against human B-cell-activating factor receptor (BAFF-R/TNFRSF13C), which has not yet been targeted successfully for cancer therapy. Novel mAbs were generated against BAFF-R, expressed as a natively folded cell surface immunogen on mouse fibroblast cells. Chimeric BAFF-R mAbs were developed and assessed for and monotherapy cytotoxicity. The chimeric mAbs were tested against human B-cell tumor lines, primary patient samples, and drug-resistant tumors. Chimeric antibodies bound with high affinity to multiple human malignant B-cell lines and induced potent antibody-dependent cellular cytotoxicity (ADCC) against multiple subtypes of human lymphoma and leukemia, including primary tumors from patients who had relapsed after anti-CD20 therapy. Chimeric antibo...

Oncolytic viruses selectively lyse tumor cells, disrupt immunosuppression within the tumor and reactivate anti-tumor immunity, but they have yet to live up to their therapeutic potential. Immune checkpoint modulation has been efficacious in a variety of cancer with an immunogenic microenvironment, but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies would likely result in both effective and specific cancer therapy. To test the hypothesis, we first constructed oncolytic adenovirus Delta-24-RGDOX expressing the immune co-stimulator OX40 ligand (OX40L). Like its predecessor Delta-24-RGD, Delta-24-RGDOX induced immunogenic cell death and recruit lymphocytes to the tumor site. Compared to Delta-24-RGD, Delta-24-RGDOX exhibited superior tumor-specific activation of lymphocytes and proliferation of CD8+ T cells specific to tumor-associated antigens, resulting in cancer-specific immunity. Delta-24-RGDOX mediated more potent anti-gli...

ABSTRACT Inflammation can promote both oncogenesis and tumor rejection. Chronic inflammation promotes cancer cell survival and metastasis, while acute inflammation triggers cancer cell destruction. Chronic inflammation is often linked with the presence of type 2-polarized macrophages (M2), which are induced by TH2 cytokines, IL-4 and IL-13. Human breast cancer tumor microenvironment displays features of Th2 inflammation that promote tumor development. The underlying molecular mechanisms are unknown. Here we show that breast cancer cells produce cytokine TSLP that induces DCs to express OX40L in vitro. OX40L+ DCs were found in primary breast cancer tumor infiltrates. These DCs drive IL- 13+TNFα+IL-10negCD4+ inflammatory Th2 cell development in vitro. TSLP-neutralizing antibodies block the upregulation of OX40L by DCs induced by breast cancer cells, thereby blocking their capacity to induce inflammatory Th2 cells in vitro. Antibodies neutralizing TSLP, OX40L, or TSLPR inhibit tumor development and IL-13 secretion in the xenograft model. Furthermore,PAR2-signaling is involved in TSLP regulation in cancer cells. Thus, TSLP-driven inflammation might contribute to breast cancer pathogenesis and development via Th2 polarization; and TSLP production by cancer cells is regulated via PAR2-signaling.

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.

In human breast cancer the proliferating cells appear to differ from those containing estrogen receptors (ER) as shown by studies on isolated cellular subpopulations. In this paper the in vitro effect of 17-beta-estradiol on cell proliferation in 30 primary breast tumors was studied. The effect of several estradiol concentrations was assayed, and the influence of diethylstilbestrol, tamoxifen, and nafoxidine was also tested. The response to these compounds was measured through the thymidine labeling index (TLI). When exposed to 10(-9) mol/l and 10(-8) mol/l estradiol, 14 of 19 ER-positive tumors and six of 11 ER-negative tumors were induced to further proliferate. The TLI increase over the control was 219% (P less than 0.05) at 10(-9) mol/l E2 and 258% (P less than 0.05) at 10(-8) mol/l E2 for ER-positive tumors, and 233% (0.1 less than P less than 0.2) at 10(-9) mol/l E2 and 321% (0.1 less than P less than 0.2) at 10(-8) mol/l E2 for ER-negative tumors. The addition of diethylstilbestrol and antiestrogens in vitro inhibited, to varying degrees, the estradiol-induced increase in the TLI irrespective of the ER-status. The response to E2 was correlated with the expression of the ras p21 protein and carcinoembryonic antigen. It was found that the ras p21 protein is preferentially expressed in ER-negative tumors, the opposite being true for carcinoembryonic antigen. The ras p21 protein is preferentially expressed in those ER-positive tumors that do not respond to estradiol with an increase in the TLI.

Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcεRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcεRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.

Previous studies from our laboratory have demonstrated that human melanoma cell lines and tumors expressed high levels of the extracellular protein SPARC. In order to demonstrate its role in human melanoma progression, IIB-MEL-LES human melanoma cells were transfected with SPARC full length c-DNA in the antisense orientation. In vivo studies demonstrated that all the control mice injected with parental cells developed tumors, while none of the mice injected with cells obtained from three different clones with diminished levels of SPARC expression, developed tumors. These studies suggest that SPARC may play a key role in human melanoma progression.