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Background The pleiotropic TNF-alpha:TNFR axis plays a central role in the immune system. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated and to gain further insight, we characterized a wide panel antibodies, generated from the n-CoDeR F.I.R.S.T™ target and antibody discovery platform. We identified parallel human and mouse TNFR2 specific, complete ligand (TNF-alpha) blocking antibodies and could show potent anti-tumor activity in several immune-competent models, both as single agent and in combination with anti-PD1 using a BI-1808 murine surrogate. The mechanism-of-action was shown to be FcgR dependent and likely mediated through a combination of intra-tumor T reg depletion, CD8+ T cell expansion and modulation of tumor-associated myeloid cells. These findings were confirmed using BI-1808 in a humanized mouse model. Methods To address safety of the human lead-candidate BI-1808 two toxicological studies were performed in cynomolgus monkeys. The first study was a dose-range-finding study and the second a GLP study where three doses (2, 20 and 200 mg/kg) were given weekly for four consecutive weeks followed by a recovery period of eight weeks. In addition, cytokine release was further studied in T cell stimulation assays and in a humanized mouse model. Moreover, the BI-1808 murine surrogate was used to study the relationship between dose, receptor occupancy (RO) and efficacy in immune competent mouse cancer experimental models. Results Four weekly administrations of BI-1808 to cynomolgus monkeys were well tolerated at all doses, with no associated clinical signs, and no histopathological changes. Non-adverse and reversible increases in neutrophil counts and decreases in T cells were observed at all dose levels. No drug-related adverse events were observed and consequently the NOAEL for BI-1808 was determined to be 200 mg/kg. Pharmacokinetic studies demonstrated an expected half-life of two weeks at receptor saturation. There were no indications of cytokine release in any of the systems tested. Finally, we could show that to achieve max therapeutic effect, sustained RO was needed for approximately two weeks, covering the time it takes to generate a full adaptive Immune response. Conclusions There is a clear association between RO and therapeutic effect and BI-1808 is well tolerated at doses associated with high and sustained RO. Collectively, these studies were used to determine the starting dose in upcoming phase I/II study in solid cancer aiming for first-patient in during December 2020. Ethics Approval The study on cynomolgous monkeys was conducted by Citox/Charles River Laboratories in compliance with animal health regulations, in particular: Council Directive No. 2010/63/EU of 22 September 2010 and French decree No. 2013-118 of 01 February 2013 on the protection of animals used for scientific purposes. Studies in mice were approved by the Swedish Animal Experiment Ethics Board, ethical permit/ethical license numbers 5.2.18-17196/2018 and 5.8.18-03333/2020

Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly (e.g. anti-HER2 mAb in HER2+ breast cancer, anti-CD20 mAb in B cell cancer) or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies; ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB imbues resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206) which is currently being explored in clinical trials (NCT03571568, NCT04219254, and NCT02933320). BI-1206 was found to have cytolytic activity against malignant B cells and the ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo. Emerging data demonstrate that FcγRs modulate the therapeutic activity of many therapeutic antibodies among them HER2 targeting antibodies such as Trastuzumab. Trastuzumab alone or in combination with chemotherapy significantly improves overall survival of HER2+ breast cancer patients. However, many patients remain uncured and develop Trastuzumab resistance resulting in relapse of the disease. Means of improving anti-HER2 therapy and overcoming resistance are therefore highly desirable. We report the generation of a fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy, both in tumor growth and overall survival of anti-HER2 in the syngeneic immune competent TUBO mouse model. This enhanced therapeutic efficacy is associated with increased tumor influx of myeloid and NK-cells. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-HER2 in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ulla-Carin Tornberg, Ingrid Karlsson, Steve Beers, Mark Cragg, Ingrid Teige, Björn Frendeus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity with anti-HER2 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3423.

The pleiotropic TNF-α:TNFR axis plays a central role in immune regulation. While the cellular expression of TNFR1 is broad, TNFR2 expression is mainly restricted to immune cells. The therapeutic potential of targeting TNFR2 for cancer treatment has been previously indicated and to gain further insight, we generated a wide panel of TNFR2-specific antibodies using the n-CoDeR F.I.R.S.T™ target and antibody discovery platforms. We identified antibodies against mouse or human TNFR2 that could block TNF-α binding and showed potent anti-tumor activity in several immune-competent models, both as single agent and in combination with anti-PD1. The mechanism-of-action was shown to be FcγR dependent and likely mediated through a combination of intra-tumor T reg depletion, CD8+ T cell expansion and modulation of innate immune cells e.g. neutrophils and myeloid cells. Toxicological studies of the human lead-candidate BI-1808 in non-human primates (NHP) demonstrated very good tolerability at doses up to 200 mg/kg, and in vitro studies with human cells showed no signs of harmful cytokine release. Furthermore, in vivo studies using immune competent mouse cancer experimental models showed a clear relationship between dose, receptor occupancy (RO) and efficacy. In both murine models and in the NHPs, soluble TNFR2 was clearly modulated by the treatment and closely correlated with RO. In addition, several changes in the immune cells (e.g. T cells and neutrophils) in the blood compartment were observed. Since January 2021, BI-1808 is evaluated in an ongoing Phase I/IIa trial. Similar to the preclinical studies, correlations between dose, RO and soluble TNFR2 has been clearly observed in the patients. In addition, and consistent with TNF-α:TNFR biology being conserved across the species, modulations of T cell and neutrophil numbers are also paralleled between the patients, NHPs and mice, increasing the likelihood of successful clinical translation of our findings. Citation Format: Linda Mårtensson, Kirsty Cleary, Petra Holmkvist, Mathilda Kovacek, Carolin Svensson, Monika Semmrich, Therese Blidberg, Mimoza Demiri, Osman Dadas, Marie Borggren, Vici Pitic, Sean H. Lim, Stephen A. Beers, Susanne Gertsson, Kristoffer S. Rohrberg, Ingrid Karlsson, Andres McAllister, Mark S. Cragg, Björn Frendeus, Ingrid Teige. BI-1808 - a first in class ligand-blocking αTNFR2 antibody for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4156.

Background: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as crossreactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dosedependent manner. Conclusion: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination. info:eu-repo/semantics/publishedVersion

Introduction BI-1206 is a fully human anti-FcγRIIB antagonistic monoclonal antibody. BI-1206 enhances the activity of anti-CD20 antibodies such as rituximab by blocking the interaction of the anti-CD20 with this inhibitory receptor. This combination may overcome resistance to rituximab and enhance its activity. Methods The safety and tolerability profile of BI-1206 in combination with rituximab is currently investigated in the Phase 1/2a clinical trial 17-BI-1206-02. The study population includes subjects with follicular lymphoma (FL), marginal zone lymphoma (MZL), and mantle cell lymphoma (MCL) who have relapsed or are refractory to rituximab. Induction therapy consists of four weekly administrations of rituximab and three administrations of BI-1206, both given as i.v. infusions. In the first week of treatment rituximab is administered as single agent. In the following three weeks patients receive BI-1206 followed by rituximab. In Phase 1, a 3+3 study design is used, with escalating doses of BI-1206 and a fixed dose of rituximab (375 mg/m 2), with the aim of selecting the RP2D of BI-1206 for the expansion cohort (Phase 2a). Patients showing clinical benefit are eligible for continued maintenance therapy with dosing of BI-1206 and rituximab every 8 weeks for up to 7 cycles. The assessment of the pharmacokinetics (PK) of BI-1206 includes non-compartmental analysis (NCA), and the assessment of the pharmacodynamics (PD) includes B cell depletion, FcγRIIB expression and BI-1206 receptor occupancy (RO). Results With a cut-off date of July 20 th, 2021, 16 subjects have received doses of up to 100 mg BI-1206 in combination with rituximab (375 mg/m 2). Nine out of these received all four doses and were evaluated for therapeutic benefit. Three subjects were diagnosed with MCL, 1 with MZL and 12 with FL. The most frequent adverse advents (AEs) related to BI-1206 have been infusion related reactions (IRRs). These IRRs led to dose limiting toxicities (DLTs) in 4 subjects, 2 experienced G4 platelet drops, and 2 experienced G3 and G4 liver enzyme elevations. No signs of liver damage were observed, and all patients recovered within days of the event, leaving no sequelae. These events have been milder and less frequent after implementation of a novel pre-medication regimen. No DLTs have been observed after implementation of this regimen. Clinical responses were observed already at the starting dose (30 mg). To date three complete responses (CR) have been observed, one at the 30mg and two at the 70mg dose levels. Two complete responses completed maintenance and one is still on maintenance treatment. The two subjects who developed a CR and finished the study have remained in CR over 1,5 and 2,0 years. Three subjects achieved partial responses (PR), one at 30mg and two at 70mg. One patient on 100 mg is experiencing ongoing stable disease at the cut-off date and is on maintenance treatment. One patient with a blastic form of MCL achieved complete depletion of peripheral tumor cells and achieved a PR. Increasing doses (30 mg to 70 or 100 mg) of BI-1206 gave rise to a supra-proportional increase in C max as well as an increase in the half-life of BI-1206. A trend of accumulation after consecutive doses was also seen. When comparing the serum-concentrations against the associated RO% of FcγRIIB there was a trend that higher dose levels were close to fully saturating FcgRIIB receptors for up to 72 hours. It is therefore likely that further increases in dose will give rise to complete receptor saturation, which should be maintained for extended periods of time. Conclusions Preliminary data of the clinical trial 17-BI-1206-02, where BI-1206 is combined with rituximab is promising. BI-1206 has a good safety profile that induced IRRs that are adequately managed with a novel steroid regimen. At a cut-off date of July 20 th, 2021, 3 CR, 3 PR and one SD have been observed among the 12 evaluable patients who completed the induction treatment. Importantly, the CRs have been very long lasting. This at doses associated only with transient receptor saturation. It may be speculated that doses which enable full RO% for a longer period (e.g., the entire dosing interval), may show additional clinical benefit. Since patients are currently undergoing treatment at higher doses, these data will be updated. Disclosures Karlsson: BioInvent International AB: Current Employment. Gertsson: BioInvent International AB: Current Employment. Kilany: BioInvent International AB: Current Employment. Borggren: BioInvent International AB: Current Employment. Abrisqueta: BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Carneiro: Novartis: Membership on an entity's Board of Directors or advisory committees; Pierre-Fabre: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees. Cordoba: Kyowa-Kirin: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; ADCTherapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Jerkeman: Jansen-Cilag, AbbVie, Gilead, Celgene: Other: part of a research collaboration between Karolinska Institutet and Janssen Pharmaceutica NV for which Karolinska Institutet has received grant support, Research Funding. Mercadal: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences, Inc.: Honoraria, Speakers Bureau. Cohen: Janssen, Adicet, Astra Zeneca, Genentech, Aptitude Health, Cellectar, Kite/Gilead, Loxo, BeiGene, Adaptive: Consultancy; Genentech, BMS/Celgene, LAM, BioINvent, LOXO, Astra Zeneca, Novartis, M2Gen, Takeda: Research Funding. Sancho: Takeda: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Teige: BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Mårtensson: BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. Frendéus: BioInvent International AB: Current Employment, Current equity holder in publicly-traded company. McAllister: BioInvent International AB: Current Employment, Current equity holder in publicly-traded company.

Introduction BI-1206 is a fully-human IgG1 monoclonal antibody that exquisitely recognizes and blocks FcγRIIB. BI-1206 enhances the activity of anti-CD20 antibodies such as rituximab by preventing interaction with FcγRIIB and may thus overcome resistance to those treatments. BI-1206 is currently in clinical investigation in combination with rituximab for the treatment of indolent NHL. This report presents a preliminary pharmacokinetic evaluation of the data generated in the trial. Methods The safety and tolerability profile of BI-1206 in combination with rituximab is currently investigated in the Phase 1/2a clinical trial 17-BI-1206-02. The study population includes patients with follicular lymphoma (FL), marginal zone lymphoma (MZL), and mantle cell lymphoma (MCL) who have relapsed or are refractory to rituximab. BI-1206 and rituximab are administered as i.v. infusions once per week for four weeks. In Phase 1a, a 3+3 study design is used, with escalating doses of BI-1206 and a fixed dose of rituximab (375 mg/m2), with the aim of selecting the RP2D of BI-1206 for an expansion cohort in Phase 2a. Patients showing clinical benefit, are eligible for continued maintenance therapy with dosing of BI-1206 and rituximab every 8 weeks. The assessment of the pharmacokinetics (PK) of BI-1206 included non-compartmental analysis (NCA), and the assessment of the pharmacodynamics (PD) included receptor occupancy (RO%). PK modelling was conducted to further characterize the PK behavior and to provide predictions of upcoming dose levels. In addition, the effect of BI-1206 on the PK of rituximab was investigated by comparing PK parameters of rituximab to literature values of rituximab monotherapy. Results Up to 100 mg BI-1206 has been administered in combination with rituximab (375 mg/m2). Increasing doses of BI-1206 from 30 mg to 70 or 100 mg gave rise to a supra-proportional increase in Cmax as well as an increase in the half-life of BI-1206. A trend of accumulation after consecutive doses was also seen. When comparing the serum-concentrations against the associated RO% of FcγRIIB there is a trend that higher doses almost fully saturate the receptors immediately after dosing and for up to 72 hours. It is likely that increasing the dose further, will give rise to full receptor saturation, which should be maintained for an extended period. Clinical response, assessed by reduction of tumour size, has been observed at the 70 mg cohort. This at a dose which typically does not saturate receptors for the entire dose interval. It may therefor be speculated that doses which enable full RO% over the entire dosing interval, may show additional clinical benefit for patients. PK modelling showed that there was a significant contribution of a non-linear component on the elimination of BI-1206, which may be attributed to receptor binding. A two-compartment model with linear (non-saturating) and non-linear (saturating) elimination best describes the data. Using the model for predictions of upcoming doses revealed that doses close to the ones already administered may be sufficient for full receptor saturation during the entire dosing interval. Finally, the Cmax after one dose of rituximab was in the same range as previously reported values, indicating no substantial effect on the PK of rituximab. Consequently, at the current dose levels, there is no apparent need for dose-adjustments for rituximab. Conclusions This report presents preliminary data of the clinical trial 17-BI-1206-02, where BI-1206 is combined with rituximab. The presented data is encouraging, both in terms of first clinical response against tumors, as well as showing signs of overcoming target-mediated drug disposition, which may allow weekly or even less frequent dosing at clinically relevant dose levels. Disclosures Jerkeman: Roche:Research Funding;Celgene:Research Funding;Gilead:Research Funding;Abbvie:Research Funding;Janssen:Research Funding.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Lindell Andersson:BioInvent International AB:Current Employment.Karlsson:BioInvent International AB:Current Employment.Borggren:BioInvent International AB:Current Employment.Abrisqueta:Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria, Speakers Bureau;AbbVie:Consultancy, Honoraria, Speakers Bureau;Roche:Consultancy, Honoraria, Speakers Bureau.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

Introduction Administration of therapeutic monoclonal antibodies (mAb) is often accompanied by infusion related reactions (IRR), of ill-determined mechanism. During the clinical development of BI-1206 (NCT02933320 and NCT03571568), a fully human mAb that binds specifically to hFcγRIIB (hCD32B), frequent IRR were observed, independent of sustained FcγRIIB blockade. At 100mg/patient, BI-1206 showed transient receptor saturation for up to 72h, whereas IRR most often resolved within 24h. Administration of BI-1206 was aslo associated with transient thrombocytopenia but not increased bleeding, and most episodes resolved within a week. Elevated transaminases (i.e. alanine transaminase (ALT) and aspartate transaminase (AST)) and a transient cytokine release was also observed alongside thrombocytopenia. Methods Six to eight-week-old C57BL/6 mice were injected either through intravenous (i.v.), intra-peritoneal (i.p.) or subcutaneous (s.c.) routes with a surrogate mouse anti-FcγRIIB (AT130-2 IgG2a) in doses ranging from 1-400μg/mouse. Mice were monitored post injection for changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Serum concentration of AT130-2 were quantified using an in-house ELISA. Platelet counts were analysed in fresh blood using a Vetscan (Triolab). Transaminases were analysed by IDEXX BioResearch. Cytokines were analysed with V-plex Proinflammatory Panel 1 Mouse kit (MesoScale Discovery), including interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL, 4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO and tumor necrosis factor (TNF)-α. Results Following administration of 200μg AT130-2 i.v., rapid onset of IRR was seen within 5-7 minutes. Blood sampling of these mice indicated reduced blood pressure. The mice recovered 10-15 minutes post IRR onset and no macroscopic symptoms were observed by 1h. Tolerability was dose-dependent and showed a clear pattern of increasing IRR with regards route of administration in the order s.c. A decrease in platelet count was seen at the same time as IRRs after both i.v. and i.p. administration. The decrease was transient and normalised within 8h post injection. AST, ALT, IL-5, IL-6, IL-10 KC/GRO, TNF-α showed a transient increase, all peaking 1-3 post injection, except for IL-5. IL-5 showed a delayed peak 3-8h post injection. To investigate whether premedication with corticosteroids could inhibit the IRR and associated toxicities, mice were premedicated with betamethasone or dexamethasone 16-24h s.c. and 1h i.v. pre injection of AT130-2. Development of IRR and platelet reduction was completely inhibited with premedication. Also, the increase in liver transaminases and cytokine release was significantly reduced. A single dose of premedication at 1h pre injection did not inhibit the IRR nor prevent the decrease in platelet count, Whilst a single dose premedication 16-24h pre injection reduced the IRR and platelet decrease, it not completely block the changes, indicating that two doses of corticosteroids are optimal. Conclusions Here we demonstrate anin vivomodel that recapitulates the tolerability profile seen with BI-1206 in human cancer subjects, including IRR, decreased platelet count, elevated transaminases and transient cytokine release. In the mouse model, there was a correlation between IRR with high dose and rapid increase in serum concentration, rather than FcγRIIB saturation following administration of anti-FcγRIIB without IRR. This pre-medication regimen has been implemented in clinical trials and shown to improve tolerability to BI-1206. Disclosures Karlsson: BioInvent International AB:Current Employment.Mårtensson:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Borggren:BioInvent International AB:Current Employment.Kovacek:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Blidberg:BioInvent International AB:Current Employment.Oldham:BioInvent International AB:Research Funding.Jerkeman:Roche:Research Funding;Abbvie:Research Funding;Janssen:Research Funding;Gilead:Research Funding;Celgene:Research Funding.Dyer:BioInvent International AB:Research Funding.Davies:Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte:Consultancy;Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead:Research Funding;Celegene, Roche, Kite Pharma, Celegene:Honoraria;Roche:Other: TRAVEL, ACCOMMODATIONS, EXPENSES.Córdoba:Roche:Honoraria, Other: travel and accommodation;Abbvie:Honoraria, Other: travel and accommodation;Janssen:Honoraria, Other: travel and accommodation;Takeda Farmacéutica España S.A.:Speakers Bureau;Gilead:Honoraria, Other: travel and accommodation.Abrisqueta:AbbVie:Consultancy, Honoraria, Speakers Bureau;Janssen:Consultancy, Honoraria, Speakers Bureau;Celgene:Consultancy, Honoraria;Roche:Consultancy, Honoraria, Speakers Bureau.Beers:BioInvent International AB:Research Funding.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Lim:BioInvent International AB:Patents & Royalties, Research Funding.Cragg:BioInvent International AB:Patents & Royalties, Research Funding.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly e.g. anti-CD20 mAb in B cell cancer, or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies;ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB may precipitate resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206). Based on its cytolytic activity against B cell cancer cells and ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo, BI-1206 is being explored in clinical trials (NCT03571568; NCT04219254). Emerging data demonstrate that FcγRs can modulate the therapeutic activity of ICB antibodies (anti-PD-1/PD-L1 and anti-CTLA-4). While both anti-CTLA-4 and anti-PD-1/L1 mAb are effective and approved for cancer therapy, and combined checkpoint blockade improves therapeutic responses, anti-CTLA-4 therapies are associated with tolerability issues. Means of enhancing the anti-CTLA-4 therapeutic window are therefore desirable in order to deliver this therapy to more patients. Recent preclinical and retrospective clinical data indicate that anti-CTLA-4 antibodies may promote antitumor immunity by dual mechanisms; “releasing the brakes” on T effector cells and depleting immune suppressive intratumoral T regulatory (Treg) cells. Anti-CTLA-4 induced Treg depletion was shown to be FcγR-dependent. Here, we report the generation of a novel, fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy of submaximally efficacious doses of anti-CTLA-4 in responsive (MC38) or resistant (CT26) syngeneic immune competent mouse models. Of relevance to the clinical setting where low dose anti-CTLA-4/anti-PD-1 combination therapies have been approved for different indications, we further demonstrate that triplet therapy significantly enhanced survival in the checkpoint blockade-poorly responsive B16 tumor model. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-CTLA-4 ICB in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ali Roghanian, Ulla-Carin Thornberg, Ingrid Karlsson, Stephen A. Beers, Mark S. Cragg, Ingrid Teige, Bjorn L. Frendéus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1642.

BACKGROUND: Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable.OBJECTIVES: To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach.METHODS: The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase.RESULTS: Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains.CONCLUSION: The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.

The development of therapeutic and prophylactic HIV vaccines for African countries is urgently needed, but the question of what immunogens to use needs to be answered. One approach is to include HIV envelope immunogens derived from HIV-positive individuals from a geographically concentrated epidemic with more limited viral genetic diversity for a region-based vaccine. To address if there is a basis for a regional selected antibody vaccine, we have screened two regionally separate cohorts from Guinea-Bissau and Denmark for neutralizing antibody activity and antibody-dependent cellular cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory activity than that from Guinea-Bissau individuals against both local and nonlocal virus strains. Interestingly, an opposite pattern was observed with ADCC activity, where Guinea-Bissau individual plasma demonstrated higher activity than Danish plasma and was specifically against the local circulating subtype. Thus, on basis of samples from these two cohorts, no local-specific neutralizing activity was detected, but a local ADCC response was identified in the Guinea-Bissau samples, suggesting potential use of regional immunogens for an ADCC-inducing vaccine.

The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKRCCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines.

For a CD8 epitope-based vaccine to match different geographic locations, the targeted epitopes for cytotoxic T-lymphocytes (CTLs) must be present in the local circulating HIV-1 strains. Secondly, the vaccine epitopes should match the host population HLA types. We characterized two new HIV-1 isolates from Guinea-Bissau. Also, we have identified 15 subdominant CD8 epitopes representing common HLA super-types theoretically covering most HLA alleles in any population. Herein we demonstrate that the selected vaccine epitopes are well conserved and simultaneously present in sequences from West Africa and Denmark. Use of the selected epitopes will likely ensure ≥10 immune targets in the majority of candidates for experimental therapeutic vaccination in both geographic regions. Our results warrant testing of the selected vaccine epitopes in both geographic locations.

The phenotype of human immunodeficiency virus type 1 (HIV-1) commonly evolves between and within infected individuals, at virus transmission, and during disease progression. This evolution includes altered interactions between the virus and its coreceptors, i.e., chemokine receptors, as well as mannose C-type lectin receptors (CLRs). Transmitted/founder viruses are predominantly restricted to CCR5, whereas the subsequent intrapatient evolution of HIV-1 coreceptor use during progressive disease can be subdivided into two distinct pathways. Accordingly, the CCR5-restricted virus population is either gradually replaced by virus variants able to use CXCR4 or evolves toward an altered, more flexible use of CCR5. Despite a strong dependency on these coreceptors for host cell entry, HIV-1 also interacts with other cell surface molecules during target cell attachment, including the CLRs. The virus interaction with the CLRs may result either in the efficient transfer of virus to CD4(+) T cells or in the degradation of the virus in endosomal compartments. The determinants of the diverse outcomes depend on which CLR is engaged and also on the glycan makeup of the envelope glycoproteins, which may evolve with the strength of the immune pressure during the disease course. With the current clinical introduction of CCR5 antagonists and the development of additional entry inhibitors, knowledge on the evolution and baseline characteristics of HIV-1 interactions with coreceptor and CLR interactions may play important roles for individualized and optimized treatment strategies. This review summarizes our current understanding of the evolution of HIV-1 interactions with these receptors.

Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (

The phenotype of human immunodeficiency virus type 1 (HIV-1) commonly evolves between and within infected individuals, at virus transmission, and during disease progression. This evolution includes altered interactions between the virus and its coreceptors, i.e., chemokine receptors, as well as mannose C-type lectin receptors (CLRs). Transmitted/founder viruses are predominantly restricted to CCR5, whereas the subsequent intrapatient evolution of HIV-1 coreceptor use during progressive disease can be subdivided into two distinct pathways. Accordingly, the CCR5-restricted virus population is either gradually replaced by virus variants able to use CXCR4 or evolves toward an altered, more flexible use of CCR5. Despite a strong dependency on these coreceptors for host cell entry, HIV-1 also interacts with other cell surface molecules during target cell attachment, including the CLRs. The virus interaction with the CLRs may result either in the efficient transfer of virus to CD4(+) T cells or in the degradation of the virus in endosomal compartments. The determinants of the diverse outcomes depend on which CLR is engaged and also on the glycan makeup of the envelope glycoproteins, which may evolve with the strength of the immune pressure during the disease course. With the current clinical introduction of CCR5 antagonists and the development of additional entry inhibitors, knowledge on the evolution and baseline characteristics of HIV-1 interactions with coreceptor and CLR interactions may play important roles for individualized and optimized treatment strategies. This review summarizes our current understanding of the evolution of HIV-1 interactions with these receptors.

Polyacrylamide hydrogel has in the last decade gained popularity as an injectable filler for facial augmentation due to its features of non-toxicity, biocompatibility, safety profile, and immediate effect. However, as all types of injections carry the risk of infection and since the polyacrylamide hydrogel is a non-degradable implant, the possibility of bacterial biofilm formation exists. Theoretically, the risk of infection and subsequent biofilm formation can be avoided by using prophylactic antibiotic treatment prior to the time of injection.This retrospective study of outcomes following polyacrylamide hydrogel injections includes 657 subjects from one centre, which had facial injections from 2001 and 2011. Until 2007 prophylactic antibiotics were not given prior to treatment, but in September 2007 a single oral dose of azithromycin (Zitromax) and moxifloxacin (Avelox) was introduced as prophylactic antibiotics. A total of 496 subjects were injected before 2007 without antibiotic prophylactic treatment, and 161 subjects received these two antibiotics prior to treatment from September 2007.The prophylactic antibiotics (azithromycin and moxifloxacin) significantly reduced the incidence of clinical signs of inflammation/infections from 7 to 2% (P=0.03).Even though the incidence of inflammation/infections following injection of polyacrylamide hydrogel is relatively low, it may be reduced further by using prophylactic antibiotic treatment. Based on our experience, we recommend prophylactic antibiotics to patients who have facial augmentation with polyacrylamide hydrogel in order to avoid infection and risk of biofilm formation due to contamination during injection with naturally occurring micro flora from skin and lips.

The binding of human immunodeficiency virus (HIV) to C-type lectin receptors may result in either enhanced trans-infection of T-cells or virus degradation. We have investigated the efficacy of HIV-1 utilization of DC-SIGN, a C-type lectin receptor, in the setting of intrauterine or intrapartum mother-to-child transmission (MTCT). Viruses isolated from HIV-1-infected mothers at delivery and from their vertically infected children both shortly after birth and later during the progression of the disease were analysed for their use of DC-SIGN, binding and ability to trans-infect. DC-SIGN use of a child’s earlier virus isolate tended to be reduced as compared with that of the corresponding maternal isolate. Furthermore, the children’s later isolate displayed enhanced DC-SIGN utilization compared with that of the corresponding earlier virus. These results were also supported in head-to-head competition assays and suggest that HIV-1 variants displaying efficient DC-SIGN use are not selected for during intrauterine or intrapartum MTCT. However, viruses with increased DC-SIGN use may evolve later in paediatric HIV-1 infections.

For a CD8 epitope-based vaccine to match different geographic locations, the targeted epitopes for cytotoxic T-lymphocytes (CTLs) must be present in the local circulating HIV-1 strains. Secondly, the vaccine epitopes should match the host population HLA types. We characterized two new HIV-1 isolates from Guinea-Bissau. Also, we have identified 15 subdominant CD8 epitopes representing common HLA super-types theoretically covering most HLA alleles in any population. Herein we demonstrate that the selected vaccine epitopes are well conserved and simultaneously present in sequences from West Africa and Denmark. Use of the selected epitopes will likely ensure ≥10 immune targets in the majority of candidates for experimental therapeutic vaccination in both geographic regions. Our results warrant testing of the selected vaccine epitopes in both geographic locations.

BACKGROUND: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. PRINCIPAL FINDINGS: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4(+) T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. CONCLUSIONS: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.

Following the publication of this article (Journal of Translational Medicine, 8:72), it was noted that a co-author had been omitted from the authors list. The submitting authors would like to apologise to Hannes Uchtenhagen for this error. The Competing interests and Authors' contributions sections have now been updated to reflect this amendment.

EUROPRISE is a Network of Excellence sponsored from 2007 to 2011 by the European Commission within the 6th Framework Program. The Network encompasses a wide portfolio of activities ranging from an integrated research program in the field of HIV vaccines and microbicides to training, dissemination and advocacy. The research program covers the whole pipeline of vaccine and microbicide development from discovery to early clinical trials. The Network is composed of 58 partners representing more than 65 institutions from 13 European countries; it also includes three major pharmaceutical companies (GlaxoSmithKline, Novartis and Sanofi-Pasteur) involved in HIV microbicide and vaccine research. The Network displays a dedicated and informative web page: http://www.europrise.org. Finally, a distinguishing trait of EUROPRISE is its PhD School of students from across Europe, a unique example in the world of science aimed at spreading excellence through training. EUROPRISE held its second annual conference in Budapest in November, 2009. The conference had 143 participants and their presentations covered aspects of vaccine and microbicide research, development and discovery. Since training is a major task of the Network, the students of the EUROPRISE PhD program summarized certain presentations and their view of the conference in this paper.

Background At early stages of infection CCR5 is the predominant HIV-1 coreceptor, but in approximately 50% of those infected CXCR4-using viruses emerge with disease progression. This coreceptor switch is correlated with an accelerated progression. However, those that maintain virus exclusively restricted to CCR5 (R5) also develop AIDS. We have previously reported that R5 variants in these "non-switch virus" patients evolve during disease progression towards a more replicative phenotype exhibiting altered CCR5 coreceptor interactions. DC-SIGN is a C-type lectin expressed by dendritic cells that HIV-1 may bind and utilize for enhanced infection of T cells in trans. To further explore the evolution of the R5 phenotype we analyzed sequential R5 isolates obtained before and after AIDS onset, i.e. at the chronic stage and during end-stage disease, with regard to efficiency of DC-SIGN use in trans-infections. Results Results from binding and trans-infection assays showed that R5 viruses emerging during end-stage AIDS disease displayed reduced ability to use DC-SIGN. To better understand viral determinants underlying altered DC-SIGN usage by R5 viruses, we cloned and sequenced the HIV-1 env gene. We found that end-stage R5 viruses lacked potential N-linked glycosylation sites (PNGS) in the gp120 V2 and V4 regions, which were present in the majority of the chronic stage R5 variants. One of these sites, amino acid position 160 (aa160) in the V2 region, also correlated with efficient use of DC-SIGN for binding and trans-infections. In fitness assays, where head-to-head competitions between chronic stage and AIDS R5 viruses were setup in parallel direct and DC-SIGN-mediated infections, results were further supported. Competitions revealed that R5 viruses obtained before AIDS onset, containing the V2 PNGS at aa160, were selected for in the trans-infection. Whereas, in agreement with our previous studies, the opposite was seen in direct target cell infections where end-stage viruses out-competed the chronic stage viruses. Conclusion Results of our study suggest R5 virus variants with diverse fitness for direct and DC-SIGN-mediated trans-infections evolve within infected individuals at end-stage disease. In addition, our results point to the importance of a glycosylation site within the gp120 V2 region for efficient DC-SIGN use of HIV-1 R5 viruses.

Contains fulltext : 109278.pdf (Publisher’s version ) (Open Access) Europrise is a Network of Excellence supported by the European Commission within the 6th Framework programme from 2007 to 2012. The Network has involved over 50 institutions from 13 European countries together with 3 industrial partners and 6 African countries. The Network encompasses an integrated program of research, training, dissemination and advocacy within the field of HIV vaccines and microbicides. A central and timely theme of the Network is the development of the unique concept of co-usage of vaccines and microbicides. Training of PhD students has been a major task, and some of these post-graduate students have here summarized novel ideas emanating from presentations at the last annual Europrise meeting in Prague. The latest data and ideas concerning HIV vaccine and microbicide studies are included in this review; these studies are so recent that the majority have yet to be published. Data were presented and discussed concerning novel immunisation strategies; microbicides and PrEP (alone and in combination with vaccines); mucosal transmission of HIV/SIV; mucosal vaccination; novel adjuvants; neutralizing antibodies; innate immune responses; HIV/SIV pathogenesis and disease progression; new methods and reagents. These - necessarily overlapping topics - are comprehensively summarised by the Europrise students in the context of other recent exciting data.