Bone marrow expands the repertoire of functional T cells targeting tumor-associated antigens in patients with resectable non-small-cell lung cancer.

The efficacy of cancer immunotherapy may be improved by increasing the number of circulating tumor-reactive T cells. The bone marrow is a priming site and reservoir for such T cells. The characteristics of bone marrow-derived tumor-reactive T cells are poorly understood in patients with non-small-cell lung cancer (NSCLC). To compare the responsiveness of tumor antigen-reactive T cells from the bone marrow with matched peripheral blood samples in patients with resectable NSCLC, we used flow cytometry, cytokine capture assays and enzyme-linked immunospot assays to examine the responsiveness of T cells to 14 tumor antigens in matched bone marrow and peripheral blood samples from patients with resectable NSCLC or benign tumors and tumor-free patients. T cells with reactivity to tumor antigens were detected in the bone marrow of 20 of 39 (51%) NSCLC patients. The panel of tumor antigens recognized by bone marrow-derived T cells was distinct from that recognized by peripheral blood-derived T cells in NSCLC patients. Unlike for peripheral blood T cells, the presence of tumor-reactive T cells in the bone marrow did not correlate with recurrence-free survival after curative intent resection of NSCLC. T cells with reactivity to tumor antigens are common in the bone marrow of patients with NSCLC. Tumor-reactive T cells of the bone marrow have the potential to significantly broaden the total repertoire of tumor-reactive T cells in the body. To clarify the role of tumor-reactive T cells of the bone marrow in T cell-based immunotherapy approaches, clinical studies are needed (ClinicalTrials.gov: NCT02515760).

A phase 1 trial extension to assess immunologic efficacy and safety of prime-boost vaccination with VXM01, an oral T cell vaccine against VEGFR2, in patients with advanced pancreatic cancer.

VXM01 is a first-in-kind orally applied tumor vaccine based on live attenuated Salmonella typhi carrying an expression plasmid encoding VEGFR2, an antigen expressed on tumor vasculature and a stable and accessible target for anti-angiogenic intervention. A recent randomized, placebo-controlled, phase I dose-escalation trial in advanced pancreatic cancer patients demonstrated safety, immunogenicity and transient, T-cell response-related anti-angiogenic activity of four priming vaccinations applied within one week. We here evaluated whether monthly boost vaccinations are safe and can sustain increased frequencies of vaccine-specific T cells. Patients with advanced pancreatic cancer were randomly assigned at a ratio of 2:1 to priming with VXM01 followed by up to six monthly boost vaccinations, or placebo treatment. Vaccinations were applied orally at two alternative doses of either 106 colony-forming units (CFU) or 107 CFU, and concomitant treatment with standard-of-care gemcitabine during the priming phase, and any treatment thereafter, was allowed in the study. Immunomonitoring involved interferon-gamma (IFNγ) ELIspot analysis with long overlapping peptides spanning the entire VEGFR2 sequence. A total of 26 patients were treated. Treatment-related adverse events preferentially associated with VXM01 were decreases in lymphocyte numbers in the blood, increased frequencies of neutrophils and diarrhea. Eight out of 16 patients who received at least one boosting vaccination responded with pronounced, i.e. at least 3-fold, increase in VEGFR2-specific T cell response over baseline levels. In the VXM01 vaccination group, VEGFR2-specific T cells peaked preferentially during the boosting phase with an average 4-fold increase over baseline levels. In conclusion, prime/boost vaccination with VXM01 was safe and immunogenic and increased vaccine specific T cell responses compared with placebo treatment.

Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens.

Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial.

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

Long-term survival after adoptive bone marrow T cell therapy of advanced metastasized breast cancer: follow-up analysis of a clinical pilot trial.

BACKGROUND: The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study. METHODS: Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays. RESULTS: Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009). CONCLUSION: In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.

Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Heparanase: a new metastasis-associated antigen recognized in breast cancer patients by spontaneously induced memory T lymphocytes.

Increased expression and secretion of heparanase (Hpa) by tumor cells promotes tumor invasion through extracellular matrices, tissue destruction, angiogenesis, and metastasis. Here, we show the existence in breast cancer patients of Hpa-specific T lymphocytes by fluorescence-activated cell sorting flow cytometry using Hpa peptide-MHC class I tetramers. We furthermore show memory T-cell responses in a high proportion of breast cancer patients to Hpa-derived HLA-A2-restricted peptides, leading to production of IFN-gamma and to generation of antitumor CTLs lysing breast cancer cells. Such CTLs recognized endogenously processed respective Hpa peptides on Hpa-transfected and Hpa-expressing untransfected breast carcinoma cells. According to these results and to the fact that such cells were not found in healthy people, Hpa seems to be an attractive new tumor-associated antigen and its HLA-A2-restricted peptides ought to be good candidates for peptide vaccination to reactivate memory immune responses to invasive and metastatic cancer cells.

Melanoma-reactive T cells in the bone marrow of melanoma patients: association with disease stage and disease duration.

Using IFN-gamma enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients.

Tumor selective replication of Newcastle disease virus: association with defects of tumor cells in antiviral defence.

To investigate tumor-selective viral replication, we compared several tumorigenic human cell lines to nontumorigenic human cells from the blood for the sensitivity to become infected by a recombinant lentogenic strain of Newcastle Disease Virus (NDV) with incorporated transgene EGFP (NDFL-EGFP). Although fluorescence signals in nontumorigenic cells were only weak or missing completely, a massive and long-lasting transgene-expression was observed in all tumor cell lines. The majority of tumor cells (50-95%) could be infected, and viral replication was associated with an increase in the cell surface density of viral antigens. To clarify the underlying mechanism of the observed difference in virus susceptibility we examined the kinetics of interferon-induced antiviral enzymes because NDV is a strong type-I interferon inducer. This analysis revealed several defects of tumor cells in their antiviral defence responses: They showed no response to UV-inactivated NDV, whereas nontumorigenic cells reacted with induction of high-levels of the antiviral enzymes PKR and MxA. Upon coincubation with live NDV, tumor cells showed a delayed response in the increased expression of the antiviral enzymes in comparison with PBMC. In nontumorigenic cells the replication cycle of NDV stopped after the production of positive-strand RNA, while tumor cells continued in the replication cycle and copied viral genomes 10-50 hr after infection. These results can explain the tumor selective replication behavior of this interesting antineoplastic virus.

Heparanase expression at the invasion front of human head and neck cancers and correlation with poor prognosis.

PURPOSE: Head and neck squamous cell carcinomas (HNSCC) are characterized by a poor prognosis due to aggressive, recurrent tumor growth. Expression of the extracellular matrix-degrading enzyme heparanase was associated with poorer prognosis in several cancers. We analyzed the presence of heparanase in HNSCC tissues and tumor cells and its potential prognostic significance. EXPERIMENTAL DESIGN: We analyzed the expression of the active form of heparanase in HNSCC tissues in corresponding tumor cell cultures and after xenotransplantation of tumor cell cultures into NOD/Scid mice by immunohistochemistry, Western blot analysis, and reverse transcription-PCR in altogether 25 patients and did a comparison with clinicopathologic data of the patients. RESULTS: Heparanase expression in situ was detected in all tumor biopsies in the tumor stroma and in tumor cells from 13 of 19 primary tumors and 9 of 12 lymph node metastases. Heparanase was localized in disseminated tumor cells, in tumor cell clusters invading adjacent stromal tissues, and in tumor cells at the tumor invasion front. Lymph node metastases expressed higher levels of heparanase compared with corresponding primary tumors. In contrast to a heterogeneous expression pattern in tumor tissues, all corresponding HNSCC tumor cell cultures showed a rather homogeneous heparanase expression on the mRNA and protein levels. Comparison of heparanase expression in situ and in corresponding tumor cell cultures in vitro or after xenotransplantation into NOD/Scid mice revealed that heparanase expression was regulated in vivo. Lack of heparanase in tumor cells from primary tumors or lymph node metastases was correlated with prolonged disease-free survival and overall survival. CONCLUSION: Heparanase expression seems to be involved in the invasiveness and aggressiveness of HNSCC.