In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.

Cancer stem cell (CSC) biology and tumor immunology have shaped our understanding of tumorigenesis. However, we still do not fully understand why tumors can be contained but not eliminated by the immune system and whether rare CSCs are required for tumor propagation.Long latency or recurrence periods have been described for most tumors. Conceptually, this requires a subset of malignant cells which is capable of initiating tumors, but is neither eliminated by immune cells nor able to grow straight into overt tumors. These criteria would be fulfilled by CSCs. Stem cells are pluripotent, immune-privileged, and long-living, but depend on specialized niches. Thus, latent tumors may be maintained by a niche-constrained reservoir of long-living CSCs that are exempt from immunosurveillance while niche-independent and more immunogenic daughter cells are constantly eliminated.The small subpopulation of CSCs is often held responsible for tumor initiation, metastasis, and recurrence. Experimentally, this hypothesis was supported by the observation that only this subset can propagate tumors in non-obese diabetic/scid mice, which lack T and B cells. Yet, the concept was challenged when an unexpectedly large proportion of melanoma cells were found to be capable of seeding complex tumors in mice which further lack NK cells. Moreover, the link between stem cell-like properties and tumorigenicity was not sustained in these highly immunodeficient animals. In humans, however, tumor-propagating cells must also escape from immune-mediated destruction. The ability to persist and to initiate neoplastic growth in the presence of immunosurveillance – which would be lost in a maximally immunodeficient animal model – could hence be a decisive criterion for CSCs.Consequently, integrating scientific insight from stem cell biology and tumor immunology to build a new concept of “CSC immunology” may help to reconcile the outlined contradictions and to improve our understanding of tumorigenesis.

HLA-G is a non-classical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune-regulatory functions. We previously have reported that HLA-G is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1 and soluble HLA-G5) into TE671 rendered these cells resistant to alloreactive lysis by direct inhibition of natural killer (NK) cells, and CD4 and CD8 T cells. Further, HLA-G reduced alloproliferation, interfered with effective priming of antigen-specific cytotoxic T cells and reduced antigen-specific alloreactive lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, a few HLA-G-positive cells within a population of HLA-G-negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.

Co-regulation of genes has been extensively analyzed, however, rather limited knowledge is available on co-regulations within the miRNome. We investigated differential co-expression of microRNAs (miRNAs) based on miRNome profiles of whole blood from 540 individuals. These include patients suffering from different cancer and non-cancer diseases, and unaffected controls. Using hierarchical clustering, we found 9 significant clusters of co-expressed miRNAs containing 2–36 individual miRNAs. Through analyzing multiple sequencing alignments in the clusters, we found that co-expression of miRNAs is associated with both sequence similarity and genomic co-localization. We calculated correlations for all 371,953 pairs of miRNAs for all 540 individuals and identified 184 pairs of miRNAs with high correlation values. Out of these 184 pairs of miRNAs, 16 pairs (8.7%) were differentially co-expressed in unaffected controls, cancer patients and patients with non-cancer diseases. By computing correlated and anti-correlated miRNA pairs, we constructed a network with 184 putative co-regulations as edges and 100 miRNAs as nodes. Thereby, we detected specific clusters of miRNAs with high and low correlation values. Our approach represents the most comprehensive co-regulation analysis based on whole miRNome-wide expression profiling. Our findings further decrypt the interactions of miRNAs in normal and human pathological processes.

TPS2658 Background: Growth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. A recent study [Haake et al. AACR2020; Abstract #5597] elucidated a mechanism by which GDF-15 inhibits LFA-1 activation on CD8+ T cells, thus interfering with effector T cell recruitment to tissues. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [reviewed in Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15. Methods: This is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed post or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments. Further key eligibility criteria include having received at least one prior anti-PD1/-PD-L1 treatment and having relapsed on or after it or having been refractory to it, and presenting with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for “GDF-15 Antibody-mediaTed Effector cell Relocation”. Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g. degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect) In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a „mono-followed-by-combination“-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination. The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 1 has been completed without DLT and enrollment for cohort 2 began in February 2021. Clinical trial information: NCT04725474.

e14532 Background: Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily with low to absent expression in healthy tissue. GDF-15 has been linked to feto-maternal immune tolerance, to prevention of excessive immune cell infiltration during tissue damage, and to anorexia. Various major tumor types secrete high levels of GDF-15. In cancer patients, elevated GDF-15 serum levels correlate with poor prognosis and reduced overall survival (OS). Methods: Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking was analyzed by whole blood adhesion assays, a HV18-MK melanoma-bearing humanized mouse model and a GDF-15-transgenic MC38 model. Additionally, patient GDF-15 serum levels were correlated with clinical response and overall survival in oropharyngeal squamous cell carcinoma (OPSCC) and melanoma brain metastases. Results: In whole blood cell adhesion assays GDF-15 impairs adhesion of T and NK cells to activated endothelial cells. Neutralization of GDF-15 by CTL-002 rescued T cell adhesion. In HV18-MK-bearing humanized mice CTL-002 induced a strong increase in TIL numbers. Subset analysis revealed an overproportional enrichment of T cells, in particular CD8+ T cells. As immune cell exclusion is detrimental for checkpoint inhibitor (CPI) therapy, a GDF-15-transgenic MC38 model was tested for anti-PD-1 therapy efficacy. In GDF-15 overexpressing MC38 tumors response to anti PD-1 therapy was reduced by 90% compared to wtMC38 tumors. Combining aPD-1 with CTL-002 resulted in 50% of the mice rejecting their GDF-15 overexpressing tumors. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for HPV+ OPSCC and for melanoma brain metastases. GDF-15 serum levels were significantly higher in HPV- than in HPV+ OPSCC patient (p < 0.0001). Low GDF-15 levels corresponded to longer OS in both HPV- and HPV+ OPSCC. In two independent melanoma patient cohorts treated with nivolumab or pembrolizumab low baseline serum GDF-15 levels were predictive for clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 independently predicts poor survival in aPD-1 treated melanoma patients. Conclusions: Taken together our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of T cells to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. In melanoma, patients with higher GDF-15 levels have significantly shorter survival and are less likely to respond to anti-PD1 therapy. GDF-15 may thus serve as a new predictive biomarker for anti-PD1 response, but most importantly also represents a novel target for cancer immunotherapy to improve tumor immune cell infiltration and response to anti-PD1 therapy.

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