Sarah-Katharina Kays

Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

Janus kinase (JAK) inhibitors provide limited depth and durability of response in myelofibrosis. We evaluated pelabresib-a bromodomain and extraterminal domain (BET) inhibitor-plus ruxolitinib (a JAK inhibitor) compared with placebo plus ruxolitinib as first-line therapy. In this phase 3 study (MANIFEST-2), JAK inhibitor-naive patients with myelofibrosis were randomized 1:1 to pelabresib 125 mg once daily (QD; 50-175 mg QD permitted) for 14 days followed by a 7-day break (21-day cycle), or to placebo in combination with ruxolitinib 10 or 15 mg twice daily (BID; 5 mg QD-25 mg BID permitted). Primary endpoint was reduction in spleen volume of ≥35% from baseline at week 24. Key secondary endpoints were absolute change in total symptom score (TSS) and TSS50 response (≥50% reduction in TSS from baseline at week 24). The primary endpoint was met in 65.9% of patients randomized to pelabresib-ruxolitinib (n = 214) versus 35.2% to placebo-ruxolitinib (n = 216) (difference, 30.4%; 95% confidence interval (CI), 21.6, 39.3; P < 0.001). Absolute change in TSS was -15.99 versus -14.05 (difference, -1.94; 95% CI, -3.92, 0.04; P = 0.0545) and TSS50 was achieved in 52.3% versus 46.3% (difference, 6.0%; 95 CI, -3.5, 15.5) with pelabresib-ruxolitinib versus placebo-ruxolitinib. Exploratory analyses of proinflammatory cytokine amounts and bone marrow morphology showed greater improvement with the combination. Thrombocytopenia and anemia were the most common treatment-emergent adverse events, occurring in 52.8% (13.2% grade ≥3) versus 37.4% (6.1% grade ≥3) and 44.8% (23.1% grade ≥3) versus 55.1% (36.5% grade ≥3), respectively. Pelabresib in combination with ruxolitinib is well tolerated, improves signs of underlying myelofibrosis pathobiology and provides substantial clinical benefit over standard-of-care JAK inhibitor monotherapy. ClinicalTrials.gov identifier: NCT04603495 .

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the anti-tumoral activity of MV-CD133, we here pursued arming technologies, receptor extension and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super-cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data suggest new concepts and approaches towards enhancing the oncolytic activity of CD133-targeted oncolytic viruses, but also raise awareness about careful toxicity testing of novel virus types.

Background Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and MF-associated symptoms. These hallmarks result from dysregulation of the JAK/STAT pathway and BET-mediated MF target gene modulation. Pelabresib (CPI-0610; pela) is an investigational oral small-molecule drug designed to inhibit BET-mediated gene transcription involved in MF pathogenesis. Preclinical data support the potential of combining pela with therapiesthat target overlapping pathways, such as JAK/STAT, to improve response by inhibiting the molecular drivers of MF. The Phase 3 MANIFEST-2 trial was initiated based on compelling data from Arm 3 of the ongoing Phase 2 MANIFEST study (NCT02158858), which is evaluating the combination of pela and ruxolitinib (rux) in JAK inhibitor (JAKi) treatment-naïve patients (pts) with MF. In Arm 3 of MANIFEST, primary endpoint analyses at 24 Weeks (Wks) for the 84 pts enrolled reported SVR35 (≥35% reduction in spleen volume from baseline) in 68% of pts and TSS50 (≥50% reduction in Total Symptom Score [TSS] from baseline) in 56% of pts (Mascarenhas, et al. 2023). Aims MANIFEST-2 (NCT04603495) is a global, Phase 3, randomized, double-blind, active-control study of pela + rux versus placebo + rux in JAKi treatment-naïve pts with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pela + rux. Here we present the study design and baseline patient stratification for MANIFEST-2. Results from the primary analysis of MANIFEST-2 will be presented at the ASH Annual Meeting 2023. Methods The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a DIPSS score of Intermediate-1 (Int-1) or higher, platelet count ≥100 × 10 9/L, spleen volume ≥450 cm 3 by CT or MRI, ≥2 symptoms with an average score ≥3 or a TSS of ≥10 using the MFSAF v4.0, peripheral blast count &amp;lt;5% and an ECOG performance status ≤2. Patient randomization was stratified by DIPSS risk category (Int-1 vs Int-2 vs High), platelet count (&amp;gt;200 × 10 9/L vs 100-200 × 10 9/L) and spleen volume (≥1800 cm 3 vs &amp;lt;1800 cm 3). Double-blind treatment of pela (125-175 mg) or placebo was administered once daily for 14 consecutive days, followed by a 7-day break, which is considered one cycle of treatment. Rux (5-25 mg) was administered twice daily based on platelet counts and spleen response for all 21 days of the cycle. The primary endpoint is SVR35 response at Wk 24. Secondary endpoints include TSS50, percentage change in TSS, safety, pharmacokinetics, changes in bone marrow fibrosis, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 wks. Results A total of 591 pts were screened at 138 sites. Following the screening process, 431 pts from North America, Europe, Asia and Australia were randomized. The number of pts assigned to each randomization stratum are presented in Table 1; the majority of pts presented with DIPSS Int-1 or -2, had a platelet count above 200 × 10 9/L and spleen volume below 1800 cm 3. The study opened for enrolment in November 2020; the first patient received their initial treatment on April 22, 2021, and the last patient received their first treatment on March 2, 2023. The last patient is expected to reach the 24-wk primary endpoint on August 31, 2023, following which a primary analysis will occur. As of June 27, 2023 there were 383 pts ongoing in the study. Conclusion The Phase 3 MANIFEST-2 study was initiated based on compelling data from the ongoing Phase 2 MANIFEST study, in which the combination of pela and rux in Arm 3 demonstrated significant clinical activity and disease-modifying potential without clinically relevant treatment-limiting toxicity. MANIFEST-2 has completed recruitment and will provide definitive efficacy results of combination therapy in JAKi treatment-naïve pts with MF. As DIPSS Int-1 pts have been excluded or underrepresented in prior randomized trials in MF, MANIFEST-2 will also provide important insights in assessing the benefits of starting treatment at an earlier stage of the disease. Primary results from the pivotal Phase 3 MANIFEST-2 trial of pela and rux versus placebo and rux in JAKi treatment-naïve pts may have the potential to influence the MF treatment paradigm and will be presented at the ASH Annual Meeting 2023.

Hematopoietic stem cells (HSCs) are an important target cell population for gene therapy since they can reconstitute the entire hematopoietic system. HSC-enriched cell populations can be recognized based on cell surface marker expression, such as CD34, which is broadly expressed on immature and partially differentiated cells. In mice, co-expression of CD34 and CD105 was previously shown to be relatively more specific for the most immature, long-term repopulating HSCs. Here, we evaluated whether CD105, which is expressed on 30%-80% of CD34(+) cells, is a marker also for human long-term repopulating HSCs. Therefore, we tracked the mature progeny of CD34(+) cells transduced with the CD105-targeted lentiviral vector CD105-LV in xenotolerant mice. Transduction was blocked with soluble CD105 protein confirming specificity. Importantly, CD105-LV transduced human CD34(+) cells engrafted in NOD-scid IL2Rγ(-/-) mice with up to 20% reporter gene-positive cells detected long term in all human hematopoietic lineages in bone marrow (BM), spleen, and blood. In addition, competitive repopulation experiments in mice showed a superior engraftment of CD105-LV transduced CD34(+) cells in BM and spleen compared with cells transduced with a conventional nontargeted lentiviral vector. Thus, human CD34(+)/CD105(+) cells are enriched for early HSCs with high repopulating capacity. Targeting this cell population with CD105-LV offers a novel gene transfer strategy to reach high engraftment rates of transduced cells and highlights the applicability of receptor-targeted vectors to trace cell subsets offering an alternative to prospective isolation by surface markers.