Characterization of a Novel Third-Generation Anti-CD24-CAR against Ovarian Cancer.

Novel therapeutic approaches against ovarian cancer (OC) are urgently needed because of its high rate of recurrence even after extensive surgery and multi-agent chemotherapy. We aimed to develop a novel anti-CD24 chimeric antigen receptor (CAR) as an immunotherapeutic approach against OC cells and cancer stem cells (CSC). CSC represents a subpopulation of the tumor characterized by enhanced chemoresistance as well as the increased capability of self-renewal and metastasis. We designed a codon-optimized third-generation CAR containing the highly active single chain variable fragment (scFv) "SWA11" against CD24. We equipped the human NK-cell line NK-92 with the anti-CD24 CAR and an anti-CD19 control CAR using lentiviral transduction. Engineered NK-92 cells showed high cytotoxic activity against CD24-positive OC cell lines (SKOV3, OVCAR3). This effect was restricted to CD24-expressing cells as shown after lentiviral transduction of CD24-negative cell lines (A2780, HEK-293T) with CD24 transmembrane proteins. Additionally, NK-92 cells equipped with our novel anti-CD24 CAR were highly effective against patient-derived primary ovarian cancer cells. The activation of NK cells was shown by specific IFNγ secretion upon antigen stimulation. To further reduce possible off-target effects in vivo, we applied a dual-CAR approach using an anti-CD24-CD28-41BB fusion protein linked via a 2A sequence to an anti-mesothelin-CD3ζ-CAR. The dual-CAR was simultaneously active against CD24 and mesothelin expressing cells. Our novel anti-CD24-CAR showed a highly cytotoxic effect against OC cell lines and primary OC cells and will be evaluated in future in vivo trials as a promising immunotherapeutic approach against OC.

Randomised phase II trial to investigate catumaxomab (anti-EpCAM × anti-CD3) for treatment of peritoneal carcinomatosis in patients with gastric cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) represents an unfavourable prognostic factor for patients with gastric cancer (GC). Intraperitoneal treatment with the bispecific and trifunctional antibody catumaxomab (EpCAM, CD3), in addition to systemic chemotherapy, could improve elimination of PC. METHODS: This prospective, randomised, phase II study investigated the efficacy of catumaxomab followed by chemotherapy (arm A, 5-fluorouracil, leucovorin, oxaliplatin, docetaxel, FLOT) or FLOT alone (arm B) in patients with GC and PC. Primary endpoint was the rate of macroscopic complete remission (mCR) of PC at the time of second diagnostic laparoscopy/laparotomy prior to optional surgery. RESULTS: Median follow-up was 52 months. Out of 35 patients screened, 15 were allocated to arm A and 16 to arm B. mCR rate was 27% in arm A and 19% in arm B (p = 0.69). Severe side effects associated with catumaxomab were nausea, infection, abdominal pain, and elevated liver enzymes. Median progression-free (6.7 vs. 5.4 months, p = 0.71) and overall survival (13.2 vs. 13.0 months, p = 0.97) were not significantly different in both treatment arms. CONCLUSIONS: Addition of catumaxomab to systemic chemotherapy was feasible and tolerable in advanced GC. Although the primary endpoint could not be demonstrated, results are promising for future investigations integrating intraperitoneal immunotherapy into a multimodal treatment strategy.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates. CONCLUSION: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).

An unusual hypoechoic solid mass in the pancreatic head.

Solid pancreatic lesions found on imaging procedures are suspicious for malignancy and, therefore, demand immediate diagnostic evaluation and therapy. In the case of indeterminate histology, a primary resection should be considered in order to preserve the possibility of curative surgery, although rare entities may be initially disregarded. We present here the case of a 48-year-old female patient with a hypoechoic lesion of the pancreatic head, which was clearly delineated from the surrounding pancreatic tissue. The challenging diagnosis of metastatic leiomyosarcoma could only be established by considering the long-term clinical history and former histology specimens.

Evaluation of Angiopoietin-2 as a biomarker in gastric cancer: results from the randomised phase III AVAGAST trial.

BACKGROUND: In the phase III AVAGAST trial, the addition of bevacizumab to chemotherapy improved progression-free survival (PFS) but not overall survival (OS) in patients with advanced gastric cancer. We studied the role of Angiopoietin-2 (Ang-2), a key driver of tumour angiogenesis, metastasis and resistance to antiangiogenic treatment, as a biomarker. METHODS: Previously untreated, advanced gastric cancer patients were randomly assigned to receive bevacizumab (n=387) or placebo (n=387) in combination with chemotherapy. Plasma collected at baseline and at progression was analysed by ELISA. The role of Ang-2 as a prognostic and a predictive biomarker of bevacizumab efficacy was studied using a Cox proportional hazards model. Logistic regression analysis was applied for correlations with metastasis. RESULTS: Median baseline plasma Ang-2 levels were lower in Asian (2143 pg ml(-1)) vs non-Asian patients (3193 pg ml(-1)), P<0.0001. Baseline plasma Ang-2 was identified as an independent prognostic marker for OS but did not predict bevacizumab efficacy alone or in combination with baseline VEGF. Baseline plasma Ang-2 correlated with the frequency of liver metastasis (LM) at any time: Odds ratio per 1000 pg ml(-1) increase: 1.19; 95% CI 1.10-1.29; P<0.0001 (non-Asians) and 1.37; 95% CI 1.13-1.64; P=0.0010 (Asians). CONCLUSIONS: Baseline plasma Ang-2 is a novel prognostic biomarker for OS in advanced gastric cancer strongly associated with LM. Differences in Ang-2 mediated vascular response may, in part, account for outcome differences between Asian and non-Asian patients; however, data have to be further validated. Ang-2 is a promising drug target in gastric cancer.

Tropism-modified AAV vectors overcome barriers to successful cutaneous therapy.

Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.

[Immunotherapy for gastrointestinal cancer]. / Immuntherapie bei gastrointestinalen Tumoren.

Cancers of gastrointestinal tract make up the largest group of solid tumour diseases in Germany. The prognosis at diagnosis is often critical. Drug therapies reduce the risk of relapse after resection and can halt the progression of metastatic disease. Immunotherapies contribute increasingly to the treatment of gastrointestinal tumours. Monoclonal antibodies (mAB) against surface receptors from the epidermal growth factor receptor family (EGFR, Her2) are well established. The effect is partly based on the interruption of the oncogenic downstream signalling cascades and partly on immune effector mechanisms such as antibody-dependent cellular cytotoxicity. In clinical practice mAB directed against programmed cell death protein 1 (PD-1), its ligand (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) - so-called immune checkpoint inhibitors - play an increasing role and change the natural history of some subgroups of gastrointestinal cancers, especially those with deficient DNA mismatch repair which leads to genomic microsatellite instability.

Adeno-Associated Virus Vectors-a Target of Cellular and Humoral Immunity-are Expanding Their Reach Toward Hematopoietic Stem Cell Modification and Immunotherapies.

All current market-approved gene therapy medical products for in vivo gene therapy of monogenic diseases rely on adeno-associated virus (AAV) vectors. Advances in gene editing technologies and vector engineering have expanded the spectrum of target cells and, thus, diseases that can be addressed. Consequently, AAV vectors are now being explored to modify cells of the hematopoietic system, including hematopoietic stem and progenitor cells (HSPCs), to develop novel strategies to treat monogenic diseases, but also to generate cell- and vaccine-based immunotherapies. However, the cell types that represent important new targets for the AAV vector system are centrally involved in immune responses against the vector and its transgene product as discussed briefly in the first part of this review. In the second part, studies exploring AAV vectors for genetic engineering of HSPCs, T and B lymphocytes, and beyond are presented.

Exceptional Response of BRAFV600E-Mutated Acinar Cell CUP to BRAF/MEK Inhibition.

Complete remission of BRAF V600E-driven ACC CUP by BRAF/MEK inhibition underscores importance of precision oncology.

Comparative bioinformatic analysis of KRAS, STK11 and KEAP1 (co-)mutations in non-small cell lung cancer with a special focus on KRAS G12C.

OBJECTIVES: Mutations in STK11 (STK11MUT) and KEAP1 (KEAP1MUT) occur frequently in non-small cell lung cancer (NSCLC) and are often co-mutated with KRAS. Several studies linked the co-occurrence of KRASMUT + STK11MUT, as well as KRASMUT + KEAP1MUT to reduced response to immune checkpoint inhibitors (ICI) and even a negative impact on survival. Data focusing STK11 + KEAP1 co-mutations or the triple mutation (KRAS + STK11 + KEAP1) are scarce. The recent availability of KRAS-G12C inhibitors increases the clinical relevance of those co-mutations in KRAS-mutated NSCLC. MATERIALS AND METHODS: We present a comprehensive bioinformatic analysis encompassing six datasets retrieved from cBioPortal. RESULTS: Independent of the treatment, triple mutations and STK11MUT + KEAP1MUT were significantly associated with a reduced overall survival (OS). Across treatments, OS of patients with a KRAS G12C triple mutation was significantly reduced compared to patients with KRAS G12C-only. Under ICI-therapy, there was no significant difference in OS between patients harboring the KRAS G12C-only and patients with the KRAS G12C triple mutation, but a significant difference between patients harboring KRAS non-G12C and KRAS non-G12C triple mutations. Triple mutated primary tumors showed a significantly increased frequency of distant metastases to bone and adrenal glands compared to KRAS-only mutated tumors. Additionally, our drug response analysis in cancer cell lines harboring the triple mutations revealed the WNT pathway inhibitor XAV-939 as a potential future drug candidate for this mutational situation. CONCLUSION: The triple mutation status may serve as a negative prognostic and predictive factor across treatments compared to KRASMUT-only. KRAS G12C generally seems to be a negative predictive marker for ICI-therapy.

Modifying immune responses to adeno-associated virus vectors by capsid engineering.

De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.

Plasma EBV DNA as a prognostic factor in EBV associated gastric cancer: a multicenter, prospective study (EBV PRESAGE study).

Purpose: The Cancer Genome Atlas Research Network identified Epstein-Barr-Virus (EBV)-positive gastric cancer as a distinct molecular subtype. The prevalence is 8-9% and the histological examination shows pronounced lymphocytic infiltration, elevated levels of IFN-γ and consequently overexpression of PD-L1. The role of plasma EBV DNA load as a prognostic factor in patients with this cancer subtype is still to be defined. Methods and analysis: The present multicenter prospective observational study "EBV PRESAGE", involving German and Italian cancer centers, aims to evaluate the prognostic role of plasma EBV DNA in EBV-related gastric cancer (GC). The objective is to study the association between plasma EBV DNA load at different consecutive time points and the patient's prognosis. Every patient with a new diagnosis of gastric cancer (including gastroesophageal junction adenocarcinoma) will be screened for Epstein-Barr encoded small Region (EBER) on tissue biopsies using in situ hybridization (ISH). If EBER ISH is positive, blood analysis for plasma EBV DNA will be conducted. The plasma EBV quantitative analysis will be centralized, and extraction, detection, and quantification of EBV DNA in plasma samples will be performed using real-time PCR. Discussion: We hypothesized that plasma EBV DNA represents a non-invasive tool for monitoring EBV-related GC and might be valuable as a prognostic marker.

Capsid-modified adeno-associated virus vectors as novel vaccine platform for cancer immunotherapy.

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for in vivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular application. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in conditions of a late tumor challenge, i.e., 80 days post-vaccination, between 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector particles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.

Nivolumab and ipilimumab in recurrent or refractory cancer of unknown primary: a phase II trial.

Cancer of unknown primary has a dismal prognosis, especially following failure of platinum-based chemotherapy. 10-20% of patients have a high tumor mutational burden (TMB), which predicts response to immunotherapy in many cancer types. In this prospective, non-randomized, open-label, multicenter Phase II trial (EudraCT 2018-004562-33; NCT04131621), patients relapsed or refractory after platinum-based chemotherapy received nivolumab and ipilimumab following TMBhigh vs. TMBlow stratification. Progression-free survival (PFS) represented the primary endpoint; overall survival (OS), response rates, duration of clinical benefit and safety were the secondary endpoints. The trial was prematurely terminated in March 2021 before reaching the preplanned sample size (n = 194). Among 31 evaluable patients, 16% had a high TMB ( > 12 mutations/Mb). Overall response rate was 16% (95% CI 6-34%), with 7.7% (95% CI 1-25%) vs. 60% (95% CI 15-95%) in TMBlow and TMBhigh, respectively. Although the primary endpoint was not met, high TMB was associated with better median PFS (18.3 vs. 2.4 months) and OS (18.3 vs. 3.6 months). Severe immune-related adverse events were reported in 29% of cases. Assessing on-treatment dynamics of circulating tumor DNA using combined targeted hotspot mutation and shallow whole genome sequencing as part of a predefined exploratory analysis identified patients benefiting from immunotherapy irrespective of initial radiologic response.

Expression of platelet-derived growth factor-C and insulin-like growth factor I in hepatic stellate cells is inhibited by miR-29.

MicroRNAs are short noncoding, endogenous RNA species that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of mRNAs. Recently, it was shown that miR-29 inhibits expression of extracellular matrix proteins such as collagens, suggesting an antifibrotic function of miR-29. In the present study, we now investigated the role of miR-29 in profibrogenic growth factor expression as a further central mechanism of fibrosis. Screening of databases revealed putative miR-29 target sequences in the mRNA of platelet-derived growth factor (PDGF)-B, PDGF-B receptor, PDGF-C, vascular endothelial growth factor-A, and insulin-like growth factor (IGF)-I. To analyze miR-29 interaction with the predicted binding sites, we cloned the 3'-UTR sequences of the putative targets in fusion to the luciferase-reporter coding sequence. Functional miR-29 binding to PDGF-C and IGF-I mRNA sequences, but not to the corresponding mutants, was then proven by reporter assays. Hepatic stellate cells (HSC) that transdifferentiate into myofibroblasts, producing extracellular matrix proteins and profibrogenic growth factors, for example, the members of the PDGF family, are crucial for liver fibrosis. Myofibroblastic transition of primary HSC resulted in the loss of miR-29, but in a significant increase of PDGF-C and IGF-I. Compensation of reduced miR-29 levels by miR-29 overexpression in myofibroblastic HSC was followed by a definitive repression of IGF-I and PDGF-C synthesis. After experimental fibrosis, induced by bile-duct occlusion, miR-29 expression was shown to be reduced, but IGF-I and PDGF-C expression was upregulated, correlating inversely to the miR-29 pattern. Thus, we conclude that miR-29, downregulated during fibrosis, acts as an antifibrogenic mediator not only by targeting collagen biosynthesis, but also by interfering with profibrogenic cell communication via PDGF-C and IGF-I.

Analysis of Tie2-expressing monocytes (TEM) in patients with colorectal cancer.

Tie2-expressing monocytes (TEM) promote tumor angiogenesis and growth in experimental cancer models. The role of TEM in cancer patients is unknown. We studied TEM in healthy volunteers and colorectal cancer (CRC) patients. Although TEM were detectable in the blood and tumor lesions of CRC patients, their frequency and functional phenotype showed no correlation with levels of angiopoietin-2 or vascular endothelial growth factor, microvessel density, tumor markers, tumor stage, or outcome of antiangiogenic therapy. These unexpected findings are at odds with murine tumor models and question the diagnostic or therapeutic value of TEM in human cancer.

[What is confirmed in the treatment of colon cancer?] / Was ist gesichert in der Therapie des Kolonkarzinoms.

Colorectal cancer is the second most common cancer diagnosed in Germany and is the third most frequent cause of cancer-related death in both males and females. The majority of colorectal cancers occur via the adenoma-carcinoma sequence of origin. This means that colorectal cancers can be endoscopically detected in premalignant stages and can be curatively treated within the framework of early detection. Screening colonoscopy and, to a lesser extent, fecal occult blood testing, have led to a reduction in the colon cancer-related incidence and mortality. The acceptance and the use of screening colonoscopy should therefore be developed further. Treatment strategies for colorectal cancer are based on TNM staging, supplemented by anatomical and histopathological risk features as well as individual patient characteristics and treatment preferences. The molecular tumor profile is increasingly used to complement decision-making in the surgical, adjuvant and palliative treatment of colorectal cancer. Colon and rectal cancer have many similarities; however, they differ in the preoperative, surgical and adjuvant treatment strategies. This article focuses on colon cancer.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

[Gastric Cancer: diagnosis and current treatment strategies]. / Diagnostik und Therapie des Magenkarzinoms.

Gastric and esophago-gastric junction (EGJ) adenocarcinoma are leading causes of cancer mortality and morbidity worldwide. In this short update article, we outline novel key developments in this field. Molecular defined subtypes can guide treatment decisions and create a roadmap for development of future agents in distinct subgroups of gastric cancer. In locally advanced EGJ cancer chemo-radiotherapy or perioperative chemotherapy are recommended treatment strategies. The FLOT regimen has evolved as a new standard for perioperative chemotherapy due to improved survival outcomes. For gastric cancer with limited metastasis, multimodality treatment approaches are under investigation. Systemic treatment over several lines along with best supportive care represents a standard in the management of metastatic disease. First line treatment should include trastuzmab in case of Her2 positivity. Immune checkpoint inhibitors are very effective in microsatellite instable tumors even in late treatment lines. In perspective, checkpoint inhibitors hold promise to become part of the standard treatment in the near future and novel Her2 targeted treatment approaches currently under development show promising results.