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Purpose Zolbetuximab (IMAB362) is engineered to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity. We evaluated ADCC activity and the impact of the immune-modulating drugs zoledronic acid (ZA) and interleukin-2 (IL-2) as co-treatment with zolbetuximab on relevant immune cell populations and ADCC lysis activity. Methods This phase 1, multicenter, open-label study investigated the immunological effects and activity, safety, tolerability, and antitumor activity of multiple doses of zolbetuximab alone (n = 5) or in combination with ZA (n = 7) or with ZA plus two different dose levels of IL-2 (low dose: 1 million international units [mIU] [n = 9]; intermediate dose: 3 mIU [n = 7]) in pretreated patients with advanced gastric and gastroesophageal junction (G/GEJ) adenocarcinoma. Results Twenty-eight patients with previously treated advanced G/GEJ adenocarcinoma that was CLDN18.2-expressing were enrolled into four treatment arms. Treatment with zolbetuximab + ZA + IL-2 induced short-lived expansion and activation of ADCC-mediating cell populations, namely γ9δ2 T cells and natural killer cells, within 2 days after administration; this effect was more pronounced with intermediate-dose IL-2. Expansion and activation of regulatory T cells treated with either IL2 dose was moderate and short-lived. Strong ADCC activity was observed with zolbetuximab alone. Short-lived ADCC activity was observed in several patients treated with ZA + intermediate-dose IL-2, but not lower-dose IL-2. In the clinical efficacy population, the best confirmed response was stable disease (n = 11/19; 58%). Conclusions Zolbetuximab mediates proficient ADCC in patients with pretreated advanced G/GEJ cancers. Co-treatment with ZA + IL-2 did not further improve this effect. Trial registration: NCT01671774

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) that encodes the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy in preventing coronavirus disease-19 (COVID-19)1. Here we extend our previous phase 1/2 trial report2 and present BNT162b2 prime/boost induced immune response data from a second phase 1/2 trial in healthy adults (18-55 years of age). BNT162b2 elicited strong antibody responses, with SARS-CoV-2 serum 50% neutralizing geometric mean titers up to 3.3-fold above those observed in COVID-19 human convalescent samples (HCS) one week post-boost. BNT162b2-elicited sera neutralized 22 pseudoviruses bearing SARS-CoV-2 S variants. Most participants had a strong IFNγ- or IL-2-positive CD8+ and CD4+ T helper type 1 (TH1) T cell response, detectable throughout the full observation period of nine weeks following the boost. pMHC multimer technology identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week post-boost, epitope-specific CD8+ T cells of the early differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes conserved in a broad range of variants at well tolerated doses.

An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18–55 years of age. Two doses of 1–50 μg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-μg dose) to 3.5-fold (50-μg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms. In a phase I/II dose-escalation clinical trial, the mRNA COVID-19 vaccine BNT162b1 elicits specific T cell and antibody responses that suggest it has protective potential.

Treating patients who have cancer with vaccines that stimulate a targeted immune response is conceptually appealing, but cancer vaccine trials have not been successful in late-stage patients with treatment-refractory tumours1,2. We are testing melanoma FixVac (BNT111)—an intravenously administered liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated, tumour-associated antigens that are prevalent in melanoma—in an ongoing, first-in-human, dose-escalation phase I trial in patients with advanced melanoma (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733). We report here data from an exploratory interim analysis that show that melanoma FixVac, alone or in combination with blockade of the checkpoint inhibitor PD1, mediates durable objective responses in checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. Clinical responses are accompanied by the induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens. The antigen-specific cytotoxic T-cell responses in some responders reach magnitudes typically reported for adoptive T-cell therapy, and are durable. Our findings indicate that RNA-LPX vaccination is a potent immunotherapy in patients with CPI-experienced melanoma, and suggest the general utility of non-mutant shared tumour antigens as targets for cancer vaccination. Results of an exploratory interim analysis from a phase I trial show that an RNA vaccine targeted towards four melanoma-associated antigens produces durable objective responses in patients with melanoma that are accompanied by strong CD4+ and CD8+ T-cell immunity.

Claudin 18.2 (CLDN18.2) is contained within normal gastric mucosa epithelial tight junctions; upon malignant transformation, CLDN18.2 epitopes become exposed. Zolbetuximab, a chimeric monoclonal antibody, mediates specific killing of CLDN18.2-positive cells through immune effector mechanisms.The FAST study enrolled advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients (aged ≥18 years) with moderate-to-strong CLDN18.2 expression in ≥40% tumour cells. Patients received first-line epirubicin + oxaliplatin + capecitabine (EOX, arm 1, n = 84) every 3 weeks (Q3W), or zolbetuximab + EOX (loading dose, 800 mg/mIn the overall population, both PFS [hazard ratio (HR) = 0.44; 95% confidence interval (CI), 0.29-0.67; P0.0005] and OS (HR = 0.55; 95% CI, 0.39-0.77; P0.0005) were significantly improved with zolbetuximab + EOX (arm 2) compared with EOX alone (arm 1). This significant PFS benefit was retained in patients with moderate-to-strong CLDN18.2 expression in ≥70% of tumour cells (HR = 0.38; 95% CI, 0.23-0.62; P0.0005). Significant improvement in PFS was also reported in the overall population of arm 3 versus arm 1 (HR = 0.58; 95% CI, 0.39-0.85; P = 0.0114) but not in high CLDN18.2-expressing patients; no significant improvement in OS was observed in either population. Most adverse events (AEs) related to zolbetuximab + EOX (nausea, vomiting, neutropenia, anaemia) were grade 1-2. Grade ≥3 AEs showed no substantial increases overall (zolbetuximab + EOX versus EOX alone).In advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients expressing CLDN18.2, adding zolbetuximab to first-line EOX provided longer PFS and OS versus EOX alone. Zolbetuximab + EOX was generally tolerated and AEs were manageable. Zolbetuximab 800/600 mg/m

An effective vaccine is needed to halt the spread of the SARS-CoV-2 pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 COVID-19 vaccine trial with BNT162b1, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Here we present antibody and T cell responses after BNT162b1 vaccination from a second, non-randomized open-label phase 1/2 trial in healthy adults, 18-55 years of age. Two doses of 1 to 50 µg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those in a COVID-19 convalescent human serum panel (HCS). Day 43 SARS-CoV-2 serum neutralising geometric mean titers were 0.7-fold (1 µg) to 3.5-fold (50 µg) those of HCS. Immune sera broadly neutralised pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had TH1 skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon (IFN)γ was produced by a high fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T-cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest multiple beneficial mechanisms with potential to protect against COVID-19.

BackgroundLipo-MERIT is an ongoing, first-in-human, open-label, dose-escalation Phase I trial investigating safety, tolerability and immunogenicity of BNT111 in patients with advanced melanoma. BNT111 is an RNA-LPX vaccine targeting the melanoma tumor-associated antigens (TAAs) New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma-associated antigen 3 (MAGE-A3), and transmembrane phosphatase with tensin homology (TPTE). A previous exploratory interim analysis showed that BNT111, alone or combined with immune checkpoint inhibition (CPI), has a favorable adverse event (AE) profile, gives rise to antigen-specific T-cell responses and induces durable objective responses in CPI-experienced patients with unresectable melanoma.1 Here, we present preliminary data in patients with no evidence of disease (NED) at trial inclusion in the BNT111 monotherapy subgroup.MethodsPatients with stage IIIB/C and IV pre-treated cutaneous melanoma were intravenously administered with BNT111 using a prime/repeat boost protocol. Patients were treated in seven dose escalation cohorts (7.2 to 400 µg total RNA) and three expansion cohorts to further explore dose levels of 14.4, 50 and 100 μg. In this analysis, patients receiving BNT111 monotherapy were grouped as having evidence of disease (ED) or NED, and immunogenicity, efficacy and safety were evaluated. Vaccine-induced immune responses were analyzed using an interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay directly ex vivo.ResultsAs of May 24, 2021, 115 patients have received BNT111 within the Lipo MERIT trial. Of 71 patients treated with BNT111 monotherapy, 38 patients had ED and 33 patients had NED after prior therapies. Baseline characteristics were similar between the two groups. ELISpot data revealed comparable BNT111-induced T-cell responses against at least one TAA in ED vs. NED patients (14/22 [64%] and 19/28 [68%] patients with available ELISpot-evaluable samples, respectively), suggesting that BNT111 has the ability to induce T-cell immunity irrespective of the presence of a detectable tumor. As previously reported for ED patients, vaccine-induced CD4+ as well as CD8+ T-cell responses were also observed in NED patients, with a substantial fraction of de novo induced responses undetectable prior to vaccination. In NED patients, clinical efficacy was promising; median disease-free survival was 34.8 months (95% confidence interval: 7.0–not reached). The safety profile was similar in ED vs. NED patients; 38/38 (100%) and 32/33 (97%) patients experienced related treatment-emergent AEs, respectively, of which the majority were mild-to-moderate flu-like symptoms.ConclusionsImmunogenicity and safety profiles of BNT111 monotherapy were comparable in ED and NED patients. Promising signs of clinical activity were observed in NED patients.AcknowledgementsThe authors would like to acknowledge Camilla West (BioNTech SE) for medical writing support.Trial RegistrationClinicaltrials.gov: NCT02410733; EudraCT No. 2013-001646-33.ReferencesSahin U, Oehm P, Derhovanessian E, et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 2020;585(7823):107–112.Ethics ApprovalEthics & Institutional Review Board approval was obtained prior to initiation of the trial (2018-13393_21-AMG federführend).

Vaccine protects against B1.1.7 variant The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B1.1.7 (VOC 202012/01) variant that emerged in late 2020 in the United Kingdom has many changes in the spike protein gene. Three of these are associated with enhanced infectivity and transmissibility, and there are concerns that B.1.1.7 might compromise the effectiveness of the vaccine. Muik et al. compared the neutralization efficacy of sera from 40 subjects immunized with the BioNTech-Pfizer mRNA vaccine BNT162b2 against a pseudovirus bearing the Wuhan reference strain or the lineage B.1.1.7 spike protein (see the Perspective by Altmann et al.). Serum was derived from 40 subjects in two age groups 21 days after the booster shot. The vaccine remained effective against B.1.1.7 with a slight but significant decrease in neutralization that was more apparent in participants under 55 years of age. Thus, the vaccine provides a significant “cushion” of protection against this variant. Science, this issue p. 1152; see also p. 1103, Despite the many genetic changes in the B.1.1.7 (VOC 202012/01) 2020 UK variant of SARS-CoV-2, the BioNTech-Pfizer mRNA vaccine remains protective., Recently, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage called B.1.1.7 (variant of concern: VOC 202012/01), which is reported to spread more efficiently and faster than other strains, emerged in the United Kingdom. This variant has an unusually large number of mutations, with 10 amino acid changes in the spike (S) protein, raising concerns that its recognition by neutralizing antibodies may be affected. In this study, we tested SARS-CoV-2-S pseudoviruses bearing either the Wuhan reference strain or the B.1.1.7 lineage spike protein with sera of 40 participants who were vaccinated in a previously reported trial with the messenger RNA–based COVID-19 vaccine BNT162b2. The immune sera had slightly reduced but overall largely preserved neutralizing titers against the B.1.1.7 lineage pseudovirus. These data indicate that the B.1.1.7 lineage will not escape BNT162b2-mediated protection.

Background Claudin 18.2 (CLDN18.2) is physiologically confined to gastric mucosa tight junctions; however, upon malignant transformation, perturbations in cell polarity lead to CLDN18.2 epitopes being exposed on the cancer cell surface. The first-in-class monoclonal antibody, zolbetuximab (formerly known as IMAB362), binds to CLDN18.2 and can induce immune-mediated lysis of CLDN18.2-positive cells. Patients and methods Patients with advanced gastric, gastro-oesophageal junction (GEJ) or oesophageal adenocarcinomas with moderate-to-strong CLDN18.2 expression in ≥50% of tumour cells received zolbetuximab intravenously every 2 weeks for five planned infusions. At least three patients were enrolled in two sequential cohorts (cohort 1300 mg/m2; cohort 2600 mg/m2); additional patients were enrolled into a dose-expansion cohort (cohort 3600 mg/m2). The primary end point was the objective response rate [ORR: complete and partial response (PR)]; secondary end points included clinical benefit [ORR+stable disease (SD)], progression-free survival, safety/tolerability, and zolbetuximab pharmacokinetic profile. Results From September 2010 to September 2012, 54 patients were enrolled (cohort 1, n = 4; cohort 2, n = 6; cohort 3, n = 44). Three patients in cohort 1 and 25 patients in cohorts 2/3 received at least 5 infusions. Antitumour activity data were available for 43 patients, of whom 4 achieved PR (ORR 9%) and 6 (14%) had SD for a clinical benefit rate of 23%. In a subgroup of patients with moderate-to-high CLDN18.2 expression in ≥70% of tumour cells, ORR was 14% (n = 4/29). Treatment-related adverse events occurred in 81.5% (n = 44/54) patients; nausea (61%), vomiting (50%), and fatigue (22%) were the most frequent. Conclusions Zolbetuximab monotherapy was well tolerated and exhibited antitumour activity in patients with CLDN18.2-positive advanced gastric or GEJ adenocarcinomas, with response rates similar to those reported for single-agent targeted agents in gastric/GEJ cancer trials. ClinicalTrials.gov number NCT01197885.

16 Background: Physiologically, the tight junction protein CLDN18.2 is present only in the gastric mucosa. Upon malignant transformation, CLDN18.2 epitopes are exposed on the cell surface and accessible to targeted therapy. Zolbetuximab (formerly IMAB362) is a chimeric mAb that mediates specific killing of CLDN18.2+ cancer cells through immune effector mechanisms; single-agent activity has been reported in G/GEJ cancer. Methods: Patients (pts) with advanced HER2-negative (HER–) G/GEJ cancer with CLDN18.2 expression of ≥ 2+ staining intensity with the anti-CLDN18 43-14A mAb in ≥ 40% tumor cells were eligible (NCT01630083). Patients were randomized 1:1 to receive first-line EOX ± zolbetuximab (loading dose 800 mg/m2, then 600 mg/m2) Q3W. The study was extended with an exploratory arm of 1000 mg/m2 zolbetuximab + EOX Q3W (data not presented). Primary endpoint was PFS; secondary endpoints included OS, ORR, and safety/tolerability. Results: A total of 161 pts (G, n = 128; GEJ, n = 27; esophagus n = 6) were randomized to receive zolbetuximab (800/600 mg) + EOX (n = 77) or EOX alone (n = 84). In all, 45% of pts had diffuse type histology per Lauren’s classification. Median PFS was longer with zolbetuximab + EOX (7.5 mo) versus EOX alone (5.3 mo; P < 0.0005; HR 0.44; 95% CI 0.29, 0.67). Median OS (13 vs 8.4 mo; P = 0.0008; HR 0.56; 95% CI 0.40, 0.79) and ORR (39 vs 25%; P = 0.022) were also significantly higher with zolbetuximab + EOX versus EOX alone. Increased efficacy was more pronounced in pts with high CLDN18.2 expression (≥ 2+ staining intensity in ≥ 70% tumor cells). Zolbetuximab + EOX was generally well tolerated. Most AEs considered related to zolbetuximab and EOX (ie, nausea, vomiting, neutropenia, anemia) were of grade ≤ 2 severity; there was no significant increase in grade ≥ 3 events with the addition of zolbetuximab to EOX. Conclusions: Addition of zolbetuximab to first-line chemotherapy resulted in significantly longer PFS and OS and higher ORR with manageable toxicity. A phase III trial is evaluating the combination of zolbetuximab plus mFOLFOX6 as first-line treatment of CLDN18.2+/HER2-negative, advanced/metastatic G or GEJ adenocarcinoma (NCT03504397). Clinical trial information: NCT01630083.

4038 Background: Claudin 18.2 (CLDN18.2), a gastric mucosa tight junction protein, is aberrantly expressed in various cancers. In the FAST Phase 2 trial (NCT01630083), IMAB362, an anti-CLDN18.2 monoclonal antibody, administered in combination with EOX chemotherapy, prolonged survival compared to EOX alone in patients with advanced/recurrent gastric, gastroesophageal junction (GEJ), and esophageal cancers ineligible for trastuzumab. The aim of the present analysis was to assess tumor CLDN18.2 expression and co-expression with HER2 in the FAST population. Methods: Tumor tissue samples from patients screened for inclusion into the FAST trial were analyzed for CLDN18.2 expression using a CE-marked, validated immunohistochemistry (IHC) assay. CLDN18.2 expression was centrally scored based on staining intensity and percentage of stained tumor cells. In a subset of tissue samples with known HER2 status, overall HER2 expression and co-expression with CLDN18.2 were determined. Results: Tissue samples from 730 patients with gastric, GEJ, and esophageal cancer were screened for the FAST study. Of these 730 samples, 685 (94%) were assessed by central IHC; 49% (n=333/685) met the FAST CLDN18.2 expression criterion (≥2+ intensity in ≥40% of tumor cells) for inclusion. Across the 154 tissue samples with known HER2 status, the majority (84%; n=129/154) were HER2–. Furthermore, 94 of these 154 samples (61%) met the FAST CLDN18.2 expression criterion, of which 14% (n=13/94) co-expressed HER2 (Table). Conclusions: In tissue samples from patients screened for the FAST trial, nearly half met CLDN18.2 expression inclusion criterion. In samples with known HER2 status, co-expression of CLDN18.2 and HER2 occurred in 14% of the samples that met study eligibility. These data suggest CLDN18.2 may serve as a non-HER2 overlapping targetable alteration in a distinct subpopulation of patients with gastric, GEJ, or esophageal cancers. Clinical trial information: NCT01630083. [Table: see text]

Therapeutic vaccination with tumor antigen-encoding RNAs is being investigated in various clinical trials. Typically, the RNA vaccine is administered intradermally, subcutaneously or intranodally with the intention to get expression of the encoded antigens in local antigen-presenting cells (APCs). We have developed a novel class of RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application, which allow systemic targeting of APCs. RNA(LIP) is a novel nanoparticulate formulation of lipid-complexed mRNA which selectively delivers the functional mRNA to APCs in lymphoid compartments body-wide for efficient mRNA uptake and expression of the encoded antigen by APCs. Moreover, this formulation has intrinsically strong adjuvant activity, mimics a systemic viral infection and induces synchronized activation of potent adaptive as well as type-I-IFN-mediated innate immune responses (Kranz et al., Nature 2016). The first-in-human phase I/II dose escalation Lipo-MERIT trial (NCT02410733) conducted in four German study centers assesses the safety, tolerability, and biological efficacy of RNA(LIP) immunotherapy in patients with stage IIIB/C and IV melanoma. This trial is the first to investigate intravenous administration of a RNA-based cancer vaccine. Following antigen expression stratification on routinely collected tumor samples, eligible patients are treated with repeated dosing of the tetravalent Lipo-MERIT vaccine composed of RNA(LIP) products encoding the shared melanoma-associated antigens NY-ESO-1, tyrosinase, MAGE-A3, and TPTE. Pharmacodynamic activity and immunogenicity of the vaccine is investigated by concerted immune monitoring and correlative biomarker studies. Clinical activity is assessed following imaging according to irRECIST1.1. As of January 2018, >50 patients have been treated with escalating or constant dosing under the guidance of an independent data safety and monitoring board. The Lipo-MERIT vaccine was generally well-tolerated and no dose-limiting toxicities (DLTs) were observed so far. Further patient enrollment is continuing. Preliminary data on the assessment of vaccine-induced immune responses and clinical responses from the first patients treated will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Heidrun Mitzel-Rink, Jochen Utikal, Christoffer Gebhardt, Jessica C. Hassel, Roland Kaufmann, Andreas Pinter, Evelyna Derhovanessian, Christine Anft, Sebastian Attig, Alexandra Deubel, Mustafa Diken, Maike Gold, Claudia Guertler, Heinrich Haas, Ludwig Heesen, Alexandra Kemmer-Brück, Lena M. Kranz, Klaus Kuehlcke, Andreas Kuhn, Peter Langguth, Ulrich Luxemburger, Daniel Maurus, Martin Meng, Felicitas Müller, Richard Rae, Fatih Sari, Katharina Schreeb, Doreen Schwarck-Kokarakis, Malte Stein, Dirk Jäger, Stephan Grabbe, Sebastian Kreiter, Christoph Huber, Özlem Türeci, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT156.

Holoprosencephaly (HPE) is the most frequently observed human embryonic forebrain defect. Recent evidence indicates that the two major forms of HPE, classic HPE and midline interhemispheric (MIH) HPE, are elicited by two different mechanisms. The only gene known to be associated with both forms of HPE is Zic2. We used the zebrafish Danio rerio as a model system to study Zic knockdown during midline formation by looking at the close homolog Zic1, which is expressed in an overlapping fashion with Zic2. Zic1 knockdown in zebrafish leads to a strong midline defect including partial cyclopia due to attenuated Nodal and Hedgehog signaling in the anterior ventral diencephalon. Strikingly, we were able to show that Zic1 is also required for maintaining early forebrain expression of the retinoic acid (RA)-degrading enzyme cyp26a1. Zic1 LOF leads to increased RA levels in the forebrain, subsequent ventralization of the optic vesicle and down-regulation of genes involved in dorsal BMP signaling. Repression of BMP signaling in dorsal forebrain has been implicated in causing MIH HPE. This work provides a mechanistical explanation at the molecular level of why Zic factors are associated with both major forms of HPE.

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)-anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co-receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue-specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss-of-function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams-Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.

Formation of a multicellular organism is a complex process involving differentiation and morphogenesis. During early vertebrate development, the radial symmetric organization of the egg is transferred into a bilateral symmetric organism with three distinct body axes: anteroposterior (AP), dorsoventral, and left-right. Due to cellular movements and proliferation, the body elongates along the AP axis. How are these processes coupled? Two recent publications now indicate that cell migration as well as orientated cell divisions contribute to axis elongation. The processes are coupled through the planar cell polarity pathway.1 At the same time, the AP axis is patterned independently of convergent extension. This process, however, is required for cell migration and represents a cue for polarized cell motility during gastrulation. Thus, it is AP polarity that instructs individual cells how to orientate with respect to the embryonic axis and provides positional information for the process of convergent extension.(2)

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/β-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca2+-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.2 BioEssays 24:881–884, 2002. © 2002 Wiley Periodicals, Inc.

The RNA-binding protein Hermes [RNA-binding protein with multiple splicing (RBPMS)] is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss of function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localization, causes similar defects in arborization. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behavior and an increase in the density of presynaptic puncta, suggesting that reduced arborization is accompanied by increased synaptogenesis to maintain synapse number.

Formation of a multicellular organism is a complex process involving differentiation and morphogenesis. During early vertebrate development, the radial symmetric organization of the egg is transferred into a bilateral symmetric organism with three distinct body axes: anteroposterior (AP), dorsoventral, and left-right. Due to cellular movements and proliferation, the body elongates along the AP axis. How are these processes coupled? Two recent publications now indicate that cell migration as well as orientated cell divisions contribute to axis elongation. The processes are coupled through the planar cell polarity pathway.1 At the same time, the AP axis is patterned independently of convergent extension. This process, however, is required for cell migration and represents a cue for polarized cell motility during gastrulation. Thus, it is AP polarity that instructs individual cells how to orientate with respect to the embryonic axis and provides positional information for the process of convergent extension.(2)

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.

Aim: IMAB362 is a monoclonal antibody specifically targeting claudin 18 isoform 2 (CLDN18.2), which is expressed on gastric cancer cells, whereas it is only present on a fraction of healthy stomach cells. This may reduce the risk of target-related side effects. Single-agent IMAB362 appears safe in patients with advanced gastro-esophageal cancer (GEC) based on data from a phase I trial. Methods: This international, multicenter, non-randomized phase IIa study (NCT01197885) investigated the efficacy and safety of repeated doses of IMAB362 (300 and 600 mg/m2) in patients with metastatic, refractory/recurrent, CLDN18.2-positive GEC (i.e. cancer of the stomach, the lower esophagus and the GE junction). Patients received bi-weekly IMAB362 at 300 or 600 mg/m2 as 2-hour IV infusion. Response rate (RECIST criteria), progression-free-survival (PFS) and safety (NCI-CTCAE v3.0) were assessed. Pharmacokinetics (PK) were determined and an extensive biomarker program was conducted. Results: 54 patients were included in this study: 4 patients received 300 mg/m2 and 50 patients 600 mg/m2. The median age was 62 (range 45–66) years in the 300 mg/m2 and 60 (range 35–77) years in the 600 mg/m2 dose group. More than 50% of patients had received chemotherapy in the 6 months preceding this study. The full analysis set comprised 40 patients (all in 600 mg/m2 group) including 9 patients who continued to receive IMAB362 following the study period. The response rate was 10% and the disease control rate was 30% (best observed response: PR, n = 4 and SD, n = 8). Median PFS was 102 days (95% CI, 70–146 days). All observed adverse events were of grade 1–3, adverse events of grade 4/5 did not occur. Nausea and vomiting were the most common study drug-related adverse events in 31 (n = 8 with grade 3 nausea) and 27 (n = 13 with grade 3 vomiting) of 54 patients, respectively. PK supports 3-weekly IV dosing. Conclusions: Clinical efficacy of IMAB362 at repeated doses was observed in several patients. IMAB362 at 600 mg/m2 appears safe and feasible in patients with advanced late-stage GEC. Hence, IMAB362 may provide a promising new treatment option for this patient population and further clinical evaluation is ongoing. Disclosure: U. Sahin: Inventor of patents on CLDN18.2; O. Tureci: Employed by Ganymed Pharmaceuticals AG and inventor of patents on CLDN18.2. All other authors have declared no conflicts of interest.

Background The neurons in the vertebrate retina arise from multipotent retinal progenitor cells (RPCs). It is not clear, however, which progenitors are multipotent or why they are multipotent. Results In this study we show that the homeodomain transcription factor Vsx2 is initially expressed throughout the retinal epithelium, but later it is downregulated in all but a minor population of bipolar cells and all Müller glia. The Vsx2-negative daughters of Vsx2-positive RPCs divide and give rise to all other cell types in the retina. Vsx2 is a repressor whose targets include transcription factors such as Vsx1, which is expressed in the progenitors of distinct non-Vsx2 bipolars, and the basic helix-loop-helix transcription factor Ath5, which restricts the fate of progenitors to retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors fates. Foxn4, expressed in the progenitors of amacrine and horizontal cells, is also negatively regulated by Vsx2. Conclusion Our data thus suggest Vsx2-positive RPCs are fully multipotent retinal progenitors and that when Vsx2 is downregulated, Vsx2-negative progenitors escape Vsx2 repression and so are able to express factors that restrict lineage potential.