Introduction: SKY59 is an anti-C5 antibody applying SMART* [Fukuzawa et al., SciRep 2017] to allow for infrequent SC dosing. A three-part adaptive clinical trial was conducted in healthy volunteers (part1 -previously reported, Röth et al., Blood 2017 130:4750) and PNH patients who are treatment naive (part 2) or previously treated with eculizumab (ecu) (part 3) to establish dose, safety and efficacy of SKY59. Methods: Part 2 was an intra-patient dose escalation study. Patients received IV doses of 375mg, 500mg, and 1000mg of SKY59 on days 1, 8 and 22, respectively, followed by weekly doses of SKY59 of 170mg SC starting on day 36. In part 3 PNH patients who had been on ecu for at least 3 months received an IV loading dose of 1000mg SKY59 on day 1, 2 weeks after their last ecu dose, and were randomized to receive 170mg SC QW, 340mg SC Q2W or 680mg SC Q4W of SKY59 starting on day 8. After 5 months all patients entered an open-label extension remaining on their previous dose regimen with SKY59. Terminal complement activity was quantified using an ex vivo liposome immunoassay (LIA). The study was approved by Ethics Committees and Health Authorities and conducted according to the principles of the declaration of Helsinki. Results: 17 PNH patients were included (Table 1), 10 in part 2 and 7 in part 3 at the time of writing this abstract. PK: After SC administration, bioavailability is estimated at 100%. For a patient of 75 kg, the terminal half-life was estimated around 25 days. PD: Following IV dosing, complete complement inhibition (defined as LIA values < 10 U/mL, the LLOQ of the assay), was achieved at end of infusion in all patients and maintained for all SC dose regimens. Median baseline (BL) total C5 concentration was 107 μg/mL (range: 66.9 - 130 μg/mL) in part 1, 140 μg/mL (73.6 - 184 μg/mL) in part 2, 295 μg/mL (205 - 354 μg/mL) in part 3. Following treatment with SKY59, patients in Part 2 showed a slight increase in total C5 at week 6 to 215 μg/mL (109 - 331 μg/mL). Patients in part 3 had a significant decrease in total C5 at week 6 to 228 μg/mL (184 - 305 μg/mL), likely reflecting the antigen disposing activity of SKY59. Treatment-naive patients (including one Arg885His C5 SNP patient [Nishimura et al.; N Engl J Med. 2014]) with PNH had a rapid median reduction in LDH (-79% from BL; median 1.20 x ULN, range 0.8 to 1.7 x ULN) at 6 weeks. LDH of Patients in part 3 did not show a significant change at 6 weeks or later compared to BL except for one C5 SNP patient who normalized LDH. 6/6 of naive (non-transfusion dependent) and 1/5 switch (non-transfusion dependent) patients had an increase in hemoglobin of at least 10g/L from BL to week 20, one patient in Part 2 and one patient in Part 3 normalized their hemoglobin. Overall transfusion requirements were not changed. QoL: From BL to week 10 treatment naive patients experienced a median change of +11 points (range -1 to 28) in the FACIT fatigue score. Switch patients showed no significant short-term change from BL to week 12 on the same questionnaire. Safety: Treatment with SKY59 was well tolerated, particularly, no injection site AEs and a low incidence of headache was observed. There were 2 SAEs (break-through hemolysis due to infection and atrial fibrillation) not related to study drug. No AEs resulted in withdrawal from the study or death. In 2/7 switch patients mild-moderate non-serious, likely drug-target-drug complex (DTDC) mediated reactions with clinical manifestations similar to serum sickness were observed in the initial post-switch period (day 9 and 10 respectively). These manifestations were treated with topical steroids and resolved by day 21 with no interruption in study treatment. In patients switching from ecu to SKY59, the formation of DTDC composed of SKY59, C5, and ecu are expected due to the different binding epitopes of the two antibodies. Conclusion: SKY59 administered SC in a low volume is very well tolerated, has a good benefit/risk ratio and is efficacious in treatment naive and ecu-treated patients with PNH. Transient induction of DTDC was not associated with undue toxicity. Furthermore, SKY59 has the potential to provide treatment for patients unresponsive to ecu due to SNP and significantly reduces the treatment burden associated with chronic IV administration. Updated data will be presented at the meeting. * Sequential Monoclonal Antibody Recycling Technology Disclosures Röth: Bioverativ: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Peffault De Latour:Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Usuki:Takeda Pharmaceutical: Speakers Bureau; Boehringer-Ingelheim Japan: Research Funding; Novartis: Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical: Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Winter:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Hsu:Roche: Employment. Dieckmann:Roche: Employment. Anzures-Cabrera:Roche: Employment. Jordan:Roche: Employment. Shinomiya:Chugai: Employment. Klughammer:F. Hoffmann-La Roche Ag: Employment. Bucher:Roche: Employment. Jahreis:Genentech: Employment, Equity Ownership. Nishimura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Chugai Pharmaceuticals: Consultancy, Research Funding.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTK C481S , sensitive (10 À4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTK C481S was detected in 48Á2% (40/83) of the patients, with 80Á0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72Á7% (32/44) of all patients experiencing relapse, emergence of the BTK C481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTK C481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTK C481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10−4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax–rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

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Background and Objectives CARD9 is a CARMA-like intracellular protein that is highly expressed in innate immune cells. In contrast to its well-defined role in fungal recognition pathways in dendritic cells, its function in non-infectious autoimmune inflammation has not been described yet, despite the fact that a genome-wide association screen raised a possible association between CARD9 gene polymorphisms and a human autoimmune arthritis. Here, we investigated the role of CARD9 in autoantibody-mediated diseases by a transgenic method. Materials and Methods We used the neutrophil-, macrophage-, Fc receptor and Syk-mediated K/BxN serum transfer arthritis and the anti-collagen type VII antibody-mediated epidermolysis bullosa acquisita (EBA) models in the presence or absence of CARD9. Bone marrow neutrophils (or cultured bone marrow-derived macrophages) from wild type, Syk- or CARD9-deficient mice were stimulated through their Fcγ receptors, followed by the analysis of the superoxide release (as a short-term response) and chemokine release (as a long-term response). Results In contrast to the previously published total resistance of Syk-deficient bone marrow chimeras to experimental arthritis, the absence of CARD9 resulted in a partial, but significant decrease in arthritis severity and CARD9−/− mice showed a moderate skin inflammation in the EBA model compared to the wild type animals. Neutrophil (and monocyte) accumulation at the site of inflammation was strongly affected, while CARD9-deficient neutrophils (and monocytes) had normal migratory capacities to the joints in wild type/CARD9−/− mixed bone marrow chimeras. Surprisingly, the synovial levels of the chemokines CXCL1 and CXCL2 were dramatically reduced in the absence of CARD9 upon arthritis induction. While Syk−/− neutrophils (and macrophages) failed to produce superoxide or release chemokines compared to wild type cells when stimulated through their Fcγ receptors among in vitro conditions, CARD9−/− neutrophils showed normal short-term, but strongly reduced long-term responses. Furthermore, Fcγ receptor-mediated phosphorylation of Syk was intact in CARD9−/−neutrophils. Conclusions CARD9 plays an important role in the development and progression of autoimmune arthritis and autoimmune blistering skin disease in mice, likely by linking the Syk tyrosine kinase to chemokine production in innate immune cells.