No abstract

Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapymediated delivery. We have previously described R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region. To evaluate the potential of R1a-B6 for immunoprophylaxis, we have reformatted it as an Fc fusion for adeno-associated viral (AAV) vector delivery. Our findings demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6 fused to Fc fragments of different isotypes equipped either, with or without antibody dependent cellular cytotoxicity (ADCC) activity, was able to drive sustained high-level expression (0.5-1.1 mg/mL) in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and avian influenza A/Vietnam/1194/2004 (H5N1). This data suggests that R1a-B6 is capable of cross-subtype protection and ADCC was not essential for R1a-B6 efficacy. Our findings demonstrate AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response.

Background: All antibodies approved for the treatment of cancer are monoclonal IgGs, and no IgE therapy has yet been tested in humans. The biology of IgE, compared with IgG, offers potential for enhanced immune surveillance and superior effector cell potency against tumor cells. IgE antibodies in preclinical cancer models are not associated with allergic toxicity even in immunocompetent animals, and in vivo efficacy compares favorably with equivalent IgGs. Methods: We conducted a first-in-human first-in-class trial of MOv18, a chimeric monoclonal IgE, in patients with solid tumors expressing folate receptor-alpha, the antigen recognized by this antibody. Antigen expression was deemed positive in the presence of >5% membrane staining of any intensity using the mouse clone BN3.2. Intravenous treatment was administered weekly for 6 weeks, then two-weekly. The trial incorporated pre-treatment skin prick testing with MOv18 IgE, and an ex vivo basophil activation test (BAT) using patient whole blood, with the aim of predicting systemic allergic toxicity and excluding patients at potential risk. Safety, efficacy, markers of immune response, and pharmacokinetics have been evaluated in 24 patients to date, at total doses ranging from 0.07 to 3.0mg. Results: Treatment was well tolerated in almost all patients. The most common toxicity was readily manageable urticaria, without systemic symptoms, signs or tryptase elevation. One patient treated at the 0.5mg dose experienced anaphylaxis, with tryptase elevation, despite a negative pre-dose skin prick test. This was the only patient in the trial with baseline circulating basophils that could be activated by ex vivo exposure to MOv18 IgE. This BAT assay was subsequently used to ensure no further patient with reactive basophils was exposed. Maximum tolerated dose has not yet been reached. Dose-dependent increases in Cmax were observed, and plasma concentrations of 70-100ng/mL achieved at the 1.5mg dose are comparable to typical levels of endogenous IgE. No consistent anti-drug antibody response has been detected. Preliminary evidence of anti-tumor activity was seen in a patient with ovarian cancer at a total MOv18 IgE dose of 0.7mg. Shrinkage of peritoneal metastases was accompanied by a tumor marker reduction meeting Gynecologic Cancer InterGroup criteria for response. Conclusions: These results support for the first time the safety of IgE as a treatment for cancer, and provide preliminary evidence for anti-tumor efficacy of this new therapeutic class. The mechanism of cutaneous toxicity is being investigated. Clinical testing of class-switched IgE versions of approved IgG-based therapeutic antibodies is warranted. Citation Format: James Spicer, Bristi Basu, Ana Montes, Udai Banerji, Rebecca Kristeleit, Gareth J. Veal, Christopher Corrigan, Stephen Till, George Nintos, Timothy Brier, Ionut G. Funingana, Joo Ern Ang, Kam Zaki, Annie Griffin, Claire Barton, Paul Jones, Sarah Mellor, Susan Brook, Katie Stoddart, Christopher Selkirk, Simon Carroll, Heike Lentfer, Natalie Woodman, Amy Pope, Giulia Pellizzari, Mano Nakamura, Kristina M. Ilieva, Atousa Khiabany, Chara Stavraka, Hannah Gould, Jitesh Chauhan, Heather Bax, Sarah Pinder, Debra Josephs, Sophia Karagiannis. Phase 1 trial of MOv18, a first-in-class IgE antibody therapy for cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT141.

Background: The development and rapid uptake of immune checkpoint inhibitors (CPI) has changed the outlook for patients with cancer. However, CPIs have different adverse event (AE) profiles to other systemic therapies, and prompt AE management is essential to assure optimal outcomes. In order to understand what and when adverse events are experienced, reported and managed during CPI treatment, a mixed methods study was conducted, including a case note review of patients who were receiving immunotherapy and semi-structured interviews with patients to understand their experience, management and reporting of AEs after receiving immune CPI treatment. Methods: This mixed methods study was conducted at a large cancer hospital in the United Kingdom. A case note review identified how and where patients reported AEs. Data relating to patients with lung, bladder, prostate and head & neck cancers who received CPI treatment between 01/04/2015 and 31/07/2018 were extracted from eprescribing databases and clinical data were included for analysis at a single time point (31 July 2018). Semistructured interviews were conducted with patients receiving CPI treatment, exploring experience of AEs and reasons for delays in AE reporting and management. Results: Sixty-two patients were included in the case note review, with 78 AEs being experienced by 36 patients (58%), including one patient experiencing 10 AEs. Serious AEs were experienced by 12 patients (19%) and ten AEs (17%) required oral steroids as treatment. The majority of AEs were reported to clinicians prior to further dosing, although milder AEs were often not addressed until subsequent clinic appointments. Interviews with 13 patients yielded major themes: variability, causality, decision making and impact. Conclusion: Most CPI-associated AEs are manageable if reported and treated promptly. Both the case note review and interviews found that reporting of non-serious AEs is often left until routine clinic visits, despite impacting patient experience, leaving the opportunity for AEs to be left unreported and implying a potential benefit for real time monitoring. Our study highlights a need to provide patients with reminders around AEs and their timely reporting even when apparently innocuous; patients must understand that AEs can occur at any cycle and even following treatment completion.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.