Combination of two monoclonal antibodies with SOCS1 N- and C-terminal binding sites to address SOCS1 status in B cells and B-cell lymphoma.

INTRODUCTION: Accumulating studies show that the tumour suppressor SOCS1 is one of the most frequently mutated genes in lymphomas, often affecting the coding sequence of SOCS1 protein. Depending on the type of mutation and lymphoma concerned, SOCS1 mutations have different impacts on progression-free and overall survival. Two antibodies binding the N and C terminals of SOCS1 would be a suitable 'test pair' to identify truncated versions of SOCS1. We, therefore, compared the C-terminal antibody 424C with the N-terminal antibody 4H1. MATERIALS AND METHODS: As 424C has already been characterised, we performed a comparative analysis of anti-SOCS1 antibody 4H1 using immunohistochemistry on human tonsil tissue and chamber slides, immunoblots on SOCS1 wildtype and mutated transfected HEK293T cells and lymphoma cell lines and cross-reactivity analysis and epitope mapping with protein microarrays. RESULTS: Compared with 424C, anti-SOCS1 antibody 4H1 showed various cross-reactions with other proteins resulting in a 'pancellular' immunohistochemical staining pattern in FFPE lymphoid tissue. Like 424C, 4H1 identified SOCS1 wildtype and SOCS1 mutations in immunoblot experiments but also bound an unknown protein with high intensity. CONCLUSION: Anti-SOCS1 antibody 4H1 may be useful in a molecular setting but is disqualified as an immunohistochemical diagnostic tool due to its very broad non-specific binding.

T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

BACKGROUND & AIMS: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. METHODS: We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. RESULTS: The 2 T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. CONCLUSION: This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. LAY SUMMARY: T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.

A new reliable and highly specific monoclonal antibody to detect the C-terminal region of silencer of cytokine signaling 1.

INTRODUCTION: SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. METHODS: BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. RESULTS: Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. CONCLUSION: This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection.

High-density peptide arrays are an excellent means to profile anti-plasmodial antibody responses. Different protein intrinsic epitopes can be distinguished, and additional insights are gained, when compared with assays involving the full-length protein. Distinct reactivities to specific epitopes within one protein may explain differences in published results, regarding immunity or susceptibility to malaria. We pursued three approaches to find specific epitopes within important plasmodial proteins, (1) twelve leading vaccine candidates were mapped as overlapping 15-mer peptides, (2) a bioinformatical approach served to predict immunogenic malaria epitopes which were subsequently validated in the assay, and (3) randomly selected peptides from the malaria proteome were screened as a control. Several peptide array replicas were prepared, employing particle-based laser printing, and were used to screen 27 serum samples from a malaria-endemic area in Burkina Faso, West Africa. The immunological status of the individuals was classified as "protected" or "unprotected" based on clinical symptoms, parasite density, and age. The vaccine candidate screening approach resulted in significant hits in all twelve proteins and allowed us (1) to verify many known immunogenic structures, (2) to map B-cell epitopes across the entire sequence of each antigen and (3) to uncover novel immunogenic epitopes. Predicting immunogenic regions in the proteome of the human malaria parasite Plasmodium falciparum, via the bioinformatics approach and subsequent array screening, confirmed known immunogenic sequences, such as in the leading malaria vaccine candidate CSP and discovered immunogenic epitopes derived from hypothetical or unknown proteins.

Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT.

L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of (125)I-labeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy.

Downregulation of the activating NKp30 ligand B7-H6 by HDAC inhibitors impairs tumor cell recognition by NK cells.

Natural killer (NK) cells are central effector cells during innate immune responses against cancer. Natural cytotoxicity receptors expressed by NK cells such as NKp30 are involved in the recognition of transformed cells. Recently, the novel B7 family member B7-H6, which is expressed on the cell surface of various tumor cells including hematological malignancies, was identified as an activating ligand for NKp30. To investigate expression and regulation of B7-H6, we generated monoclonal antibodies. Our study reveals that B7-H6 surface protein and messenger RNA (mRNA) expression in various tumor cell lines was downregulated upon treatment with pan- or class I histone deacetylase inhibitors (HDACi) as well as after small interfering RNA-mediated knockdown of the class I histone deacetylases (HDAC) 2 or 3. B7-H6 downregulation was associated with decreased B7-H6 reporter activity and reduced histone acetylation at the B7-H6 promoter. In certain primary lymphoma and hepatocellular carcinoma samples, B7-H6 mRNA levels were elevated and correlated with HDAC3 expression. Finally, downregulation of B7-H6 on tumor cells by HDACi reduced NKp30-dependent effector functions of NK cells. Thus, we identified a novel mechanism that governs B7-H6 expression in tumor cells that has implications for potential cancer treatments combining immunotherapy with HDACi.

Dickkopf-3, an immune modulator in peripheral CD8 T-cell tolerance.

In healthy individuals, T cells react against incoming pathogens, but remain tolerant to self-antigens, thereby preventing autoimmune reactions. CD4 regulatory T cells are major contributors in induction and maintenance of peripheral tolerance, but a regulatory role has been also reported for several subsets of CD8 T cells. To determine the molecular basis of peripheral CD8 T-cell tolerance, we exploited a double transgenic mouse model in which CD8 T cells are neonatally tolerized following interaction with a parenchymal self-antigen. These tolerant CD8 T cells have regulatory capacity and can suppress T cells in an antigen-specific manner during adulthood. Dickkopf-3 (DKK3) was found to be expressed in the tolerant CD8 T cells and to be essential for the observed CD8 T-cell tolerance. In vitro, genetic deletion of DKK3 or blocking with antibodies restored CD8 T-cell proliferation and IL-2 production in response to the tolerizing self-antigen. Moreover, exogenous DKK3 reduced CD8 T-cell reactivity. In vivo, abrogation of DKK3 function reversed tolerance, leading to eradication of tumors expressing the target antigen and to rejection of autologous skin grafts. Thus, our findings define DKK3 as a immune modulator with a crucial role for CD8 T-cell tolerance.

Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma-based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T-cell antigen CD3 that efficiently prevents tumor growth in a mouse B-cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B-cell non-Hodgkin and lymphocyte-predominant Hodgkin lymphoma, as well as on a subset of CD4(+) T cells known as follicular T-helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co-stimulation-independent activation of CD8(+) and CD4(+) T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN-γ, TNF-α, IL-6 and IL-10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B-cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody-based therapies in the treatment of B-cell malignancies.

Neurofibromin specific antibody differentiates malignant peripheral nerve sheath tumors (MPNST) from other spindle cell neoplasms.

Malignant peripheral nerve sheath tumors (MPNST) derive from the Schwann cell or perineurial cell lineage and occur either sporadically or in association with the tumor syndrome neurofibromatosis type 1 (NF1). MPNST often pose a diagnostic challenge due to their frequent lack of pathognomonic morphological or immunohistochemical features. Mutations in the NF1 tumor suppressor gene are found in all NF1-associated and many sporadic MPNST. The presence of NF1 mutation may have the potential to differentiate MPNST from several morphologically similar neoplasms; however, mutation detection is hampered by the size of the gene and the lack of mutational hot spots. Here we describe a newly developed monoclonal antibody binding to the C-terminus of neurofibromin (clone NFC) which was selected for optimal performance in routinely processed formalin-fixed and paraffin-embedded tissue. NFC immunohistochemistry revealed loss of neurofibromin in 22/25 (88 %) of NF1-associated and 26/61 (43 %) of sporadic MPNST. There was a strong association of neurofibromin loss with deletions affecting the NF1 gene (P < 0.01). In a series of 256 soft tissue tumors of different histotypes NFC staining showed loss of neurofibromin in 2/8 myxofibrosarcomas, 2/12 (16 %) pleomorphic liposarcomas, 1/16 (6 %) leiomyosarcomas, and 4/28 (14 %) unclassified undifferentiated pleomorphic sarcomas. However, loss of neurofibromin was not observed in 22 synovial sarcomas, 27 schwannomas, 23 solitary fibrous tumors, 14 low-grade fibromyxoid sarcomas, 50 dedifferentiated liposarcomas, 27 myxoid liposarcomas, 13 angiosarcomas, 9 extraskeletal myxoid chondrosarcomas, and 7 epitheloid sarcomas. Immunohistochemistry using antibody NFC may substantially facilitate sarcoma research and diagnostics.

CD24 controls Src/STAT3 activity in human tumors.

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.

Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells is L1CAM dependent.

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.

Humoral response to catumaxomab correlates with clinical outcome: results of the pivotal phase II/III study in patients with malignant ascites.

The trifunctional antibody catumaxomab is a targeted immunotherapy for the intraperitoneal treatment of malignant ascites. In a Phase II/III trial in cancer patients (n = 258) with malignant ascites, catumaxomab showed a clear clinical benefit vs. paracentesis and had an acceptable safety profile. Human antimouse antibodies (HAMAs), which could be associated with beneficial humoral effects and prolonged survival, may develop against catumaxomab as it is a mouse/rat antibody. This post hoc analysis investigated whether there was a correlation between the detection of HAMAs 8 days after the fourth catumaxomab infusion and clinical outcome. HAMA-positive and HAMA-negative patients in the catumaxomab group and patients in the control group were analyzed separately for all three clinical outcome measures (puncture-free survival, time to next puncture and overall survival) and compared to each other. There was a strong correlation between humoral response and clinical outcome: patients who developed HAMAs after catumaxomab showed significant improvement in all three clinical outcome measures vs. HAMA-negative patients. In the overall population in HAMA-positive vs. HAMA-negative patients, median puncture-free survival was 64 vs. 27 days (p < 0.0001; HR 0.330), median time to next therapeutic puncture was 104 vs. 46 days (p = 0.0002; HR 0.307) and median overall survival was 129 vs. 64 days (p = 0.0003; HR 0.433). Similar differences between HAMA-positive and HAMA-negative patients were seen in the ovarian, nonovarian and gastric cancer subgroups. In conclusion, HAMA development may be a biomarker for catumaxomab response and patients who developed HAMAs sooner derived greater benefit from catumaxomab treatment.

CD24 Ala57Val polymorphism predicts pathologic complete response to sequential anthracycline- and taxane-based neoadjuvant chemotherapy for primary breast cancer.

Overexpression of CD24 is an independent prognostic factor for breast cancer. Recently, two polymorphisms in the CD24 gene were linked to disease risk and progression in autoimmune diseases. Here, we evaluated the clinical relevance of these polymorphisms with respect to their potential to predict a pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) for primary breast cancer (PBC), one of the strongest prognostic factors in this setting. A total of 257 patients were randomized to either doxorubicin/cyclophosphamide (AC) or doxorubicin/pemetrexed (AP), both followed by docetaxel (Doc) as NCT for T2-4 N0-2 M0 PBC as part of an international, multicenter, randomized phase II trial. CD24 polymorphisms were analyzed on germ line DNA and correlated with clinicopathologic variables and pCR. No significant associations were found between either of the polymorphisms and any of the clinicopathologic variables. In a multivariate analysis, CD24 Val/Val genotype was the only significant predictor of pCR (OR: 4.97; P = 0.003). The predictive potential was significant in both treatment arms and in the hormone receptor-positive subgroup. There was no correlation between CD24 3'UTR (TG/Del) genotype and pCR. We did not observe any association between CD24 genotype and CD24 protein expression or in vitro chemosensitivity, but there was a significant correlation between CD24 Val/Val and intratumoral lymphocyte aggregates. In conclusion, CD24 Ala/Val SNP is a strong and independent predictor of pCR after NCT for PBC and may affect immune functions rather than tumor characteristics. Further evaluation of the CD24 function and validation of its predictive potential are clearly warranted.

Sulforaphane increases drug-mediated cytotoxicity toward cancer stem-like cells of pancreas and prostate.

Despite intense efforts to develop treatments against pancreatic cancer, agents that cure this highly resistant and metastasizing disease are not available. Considerable attention has focused on broccoli compound sulforaphane (SF), which is suggested as combination therapy for targeting of pancreatic cancer stem cells (CSCs). However, there are concerns that antioxidative properties of SF may interfere with cytotoxic drugs-as suggested, e.g., for vitamins. Therefore we investigated a combination therapy using established pancreatic CSCs. Although cisplatin (CIS), gemcitabine (GEM), doxorubicin, 5-flurouracil, or SF effectively induced apoptosis and prevented viability, combination of a drug with SF increased toxicity. Similarly, SF potentiated the drug effect in established prostate CSCs revealing that SF enhances drug cytotoxicity also in other tumor entities. Most importantly, combined treatment intensified inhibition of clonogenicity and spheroid formation and aldehyde dehydrogenase 1 (ALDH1) activity along with Notch-1 and c-Rel expression indicating that CSC characteristics are targeted. In vivo, combination treatment was most effective and totally abolished growth of CSC xenografts and tumor-initiating potential. No pronounced side effects were observed in normal cells or mice. Our data suggest that SF increases the effectiveness of various cytotoxic drugs against CSCs without inducing additional toxicity in mice.

Functional analysis of bispecific antibody (EpCAMxCD3)-mediated T-lymphocyte and cancer cell interaction by single-cell force spectroscopy.

The atomic force microscopy (AFM) is a powerful tool to analyze forces generated on cellular interactions on the single-cell level. This highly sensitive device can record changes in force in the pico-Newton range, which equals single molecule bonds. Here, we have used single-cell force spectroscopy by AFM to investigate the interaction between T cells and tumor cells that is induced by the bispecific antibody HEA125xOKT3 (specificity anti-EpCAMxCD3). We show that HEA125xOKT3 induces a specific increase in adhesion force between T cells and cancer cells. The adhesive force that is generated on cell-cell contact is dependent on the applied force on initial contact and the duration of this initial contact. In summary, HEA125xOKT3 has been found to mediate contact formation by distinct processes. It induces direct cell-cell interaction, which results in the activation of T-cell signaling, facilitates the formation of supramolecular activation clusters and ultimately of an immune synapse.

Loss of EpCAM expression in breast cancer derived serum exosomes: role of proteolytic cleavage.

OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40-100 nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. METHODS: We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. RESULTS: Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). CONCLUSION: Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood.

Co-Stimulatory Bispecific Antibodies Induce Enhanced T Cell Activation and Tumor Cell Killing in Breast Cancer Models.

Although T cell-recruiting CD3-binding bispecific antibodies (BiMAb) have been proven to be clinically effective for hematologic malignancies, the success of BiMAb targeting solid tumor-associated antigens (TAA) in carcinomas so far remains poor. We reasoned that provision of co-stimulatory BiMAb in combination with αTAA-αCD3 BiMAb would boost T cell activation and proliferative capacity, and thereby facilitate the targeting of weakly or heterogeneously expressed tumor antigens. Various αTAA-αCD3 and αTAA-αCD28 BiMAb in a tetravalent IgG1-Fc based format have been analyzed, targeting multiple breast cancer antigens including HER2, EGFR, CEA, and EpCAM. Moreover, bifunctional fusion proteins of αTAA-tumor necrosis factor ligand (TNFL) superfamily members including 4-1BBL, OX40L, CD70 and TL1A have been tested. The functional activity of BiMAb was assessed using co-cultures of tumor cell lines and purified T cells in monolayer and tumor spheroid models. Only in the presence of tumor cells, αTAA-αCD3 BiMAb activated T cells and induced cytotoxicity in vitro, indicating a strict dependence on cross-linking. Combination treatment of αTAA-αCD3 BiMAb and co-stimulatory αTAA-αCD28 or αTAA-TNFL fusion proteins drastically enhanced T cell activation in terms of proliferation, activation marker expression, cytokine secretion and tumor cytotoxicity. Furthermore, BiMAb providing co-stimulation were shown to reduce the minimally required dose to achieve T cell activation by at least tenfold. Immuno-suppressive effects of TGF-ß and IL-10 on T cell activation and memory cell formation could be overcome by co-stimulation. BiMAb-mediated co-stimulation was further augmented by immune checkpoint-inhibiting antibodies. Effective co-stimulation could be achieved by targeting a second breast cancer antigen, or by targeting fibroblast activation protein (FAP) expressed on another target cell. In tumor spheroids derived from pleural effusions of breast cancer patients, co-stimulatory BiMAb were essential for the activation tumor-infiltrating lymphocytes and cytotoxic anti-tumor responses against breast cancer cells. Taken together we showed that co-stimulation significantly potentiated the tumoricidal activity of T cell-activating BiMAb while preserving the dependence on TAA recognition. This approach could provide for a more localized activation of the immune system with higher efficacy and reduced peripheral toxicities.

Molecular and clinical dissection of CD24 antibody specificity by a comprehensive comparative analysis.

CD24 is a small, highly glycosylated cell surface protein that is linked to the membrane through a glycosyl-phosphatidylinositol anchor. It is overexpressed in many human carcinomas and its expression is linked to bad prognosis. Lately, lack or low expression of CD24 was used to identify tumor stem cells resulting in conflicting data on the usefulness of this marker. In many immunohistochemical studies, the mAb SN3b was used but the epitope and specificity of this antibody have never been thoroughly investigated. In other studies based mainly on cytofluorographic analysis, the mAb ML-5 was applied. In this study, we compared the epitope of mAb SN3b to the CD24 mAbs SWA-11 and ML-5 that both bind to the core protein of CD24. Using tissue microarrays and affinity-purified CD24 glycoforms, we observed only a partial overlap of SN3b and SWA11 reactivity. The mAb SN3b recognizes sialic acid most likely on O-linked glycans that can occur independently of the CD24 protein backbone. The SN3b epitope was not related to common sialylated cancer-associated glycan structures. Both SN3b epitope positive or negative CD24 glycoforms supported the binding of P-selectin and Siglec-5. In breast cancer, the SN3b reactivity was associated with bad prognosis, whereas SWA11 was not. In renal cell cancer, the SN3b epitope was completely absent but SWA11 reactivity was a prognostic factor. Our results shed new light on the tumorbiological role of CD24 and resolve discrepancies in the literature related to the use of different CD24 mAbs.

CD133 is indicative for a resistance phenotype but does not represent a prognostic marker for survival of non-small cell lung cancer patients.

Despite advances in anticancer treatment, lung cancer still has poor prognosis. Recently, a cancer stem cell (CSC) hypothesis has emerged describing a small subset of tumor cells with stem cell properties. CSCs found in many solid tumors express CD133 antigen on the cell surface. The presence of CSC is correlated with poor survival of patients with glioblastomas, colon or prostate cancers. In this study, we evaluated whether CD133 expression in non-small cell lung cancer (NSCLC) has a prognostic value in patients' survival. We also analyzed whether CD133 positivity of NSCLC correlates with the expression of resistance-related proteins, angiogenic factors, oncogenes, proliferative activity or apoptosis. CD133 expression was retrospectively examined in a total of 88 cases of previously untreated NSCLC by immunohistochemistry. We found no correlation between CD133 positivity or the amount of CD133(+) cells with NSCLC patients' survival, expression of oncogenes c-myc, c-N-ras, c-jun, c-fos, c-erbB1, c-erbB2 or p53, angiogenic factors VEGF, VEGFR-1, FGF, FGFR-1, tissue factor and with proliferative activity or apoptosis in NSCLC tissues. However, there was a significant association between the expression of resistance-related proteins glutathione S-transferase, thymidylate synthase, catalase, O(6)-methylguanine-DNA methyltransferase and p170 and CD133. Because CD133 expression is linked to a resistant phenotype, detection of CD133(+) cells may be useful to predict efficacy of cytotoxic therapy but CD133 is not a strong prognostic parameter for survival of patients with NSCLC.

Clever-1/Stabilin-1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation.

Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature--traffic via lymphatics and inflamed blood vessels.