Abstract: Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to 〈 6% likely due to the immune response. For one of these, no bleeds have been reported through 12 weeks of follow up; the other has had 4 bleeds through 37 weeks of observation. Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

Abstract: Sterile inflammation inducing venous thrombosis is coordinated by the damage-associated molecular pattern HMGB1 delivered by platelets. The effect of HMGB1 depends on the redox form, and disulfide HMGB1 induces NET formation, platelet aggregation, and monocyte activation.

Abstract: Background Approximately 50% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). Rare patients who naturally develop a minor antigen-specific graft-versus-leukemia T cell response show substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747; Spierings et al. (2013) Biol. Blood Marrow Transplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C/C or T/C) is an HLA-A*02:01- and haematopoietically-restricted minor histocompatibility antigen derived from the class I myosin protein, MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). Patients receiving donor lymphocyte infusion from HA-2-mismatched donors who develop HA-2-specific T cells show a graft vs leukemia response and often experience long-term remission (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747), making HA-2 an ideal candidate for TCR-engineered T cell immunotherapy of liquid tumors. Methods Using TScan's proprietary ReceptorScan platform, we discovered 1,302 HA-2-specific TCRs by screening 237 million naïve CD8 + T cells from 5 healthy HA-2-negative donors. We evaluated these TCRs using our proprietary DexScan platform to select the 15 TCRs with the highest surface expression and greatest affinity for the HA-2 peptide when transferred into primary human T cells. We further tested each TCR individually in our clinical vector backbone for surface expression, selective cytotoxicity, cytokine production, and proliferation using a panel of cell lines that express varying levels of HLA-A*02:01 and MYO1G. Finally, the top 5 TCRs were evaluated for alloreactivity using an array-based screen assessing 108 MHC-I molecules individually, and for off-target cross-reactivity using our proprietary genome-wide TargetScan platform. A lead TCR with limited alloreactivity and a narrow off-target profile was selected as our lead TSC-101 TCR. The avidity of TSC-101 for its putative off-targets was further measured in peptide-pulsed experiments to better appreciate the toxicity risks associated with our lead clinical candidate. Results and Conclusion Of the 1,302 HA-2-specific TCRs identified by our ReceptorScan platform, we identified TSC-101 as the most active TCR. TSC-101 displayed no alloreactivity to 107/108 HLAs tested and limited off-target risks in a genome-wide screens. Potential off-target peptides identified for TSC-101 displayed extremely weak avidities, predicting an absence of toxicity risks for our clinical candidate. Based on these results, TSC-101 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2022. To our knowledge, this is the first clinical grade HA-2-specifc TCR being developed for immunotherapy for liquid tumors. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Abstract: Introduction Immune-mediated hematologic disorders are relatively rare but potentially life-threatening immune-related adverse events (irAE) with immune checkpoint inhibitor (ICI) therapy (Shiuan, Beckermann et al 2017). The management strategy often relies on systemic corticosteroids and other immunosuppressant agents in steroid-refractory cases (Brahmer, Lacchetti et al 2018). We hereby describe our institution's experience in diagnosing and managing immune-related hematologic adverse events (ir-h-AE). Methods We retrospectively searched the electronic health record for all ir-h-AE diagnosed by treating providers and confirmed by study authors between 2015 and 2020. The search included patients on ICI and the following conditions: immune-related neutropenia (IRN), autoimmune hemolytic anemia (AIHA), cold agglutinin disease (CAD), immune thrombocytopenia (ITP), immune-related pancytopenia, aplastic anemia (AA), and pure red cell aplasia (PRCA). For AIHA, PRCA, and CAD, partial response (PR) was defined as hemoglobin greater than 10 g/dL but & lt; 12 g/dL and 2 g/dL above the nadir without blood product transfusion. Complete response (CR) was defined as hemoglobin of ≥ 12 g/dL and 2 g/dL above the nadir without blood product transfusion (Peñalver, Alvarez-Larrán et al. 2010). For ITP, CR was defined as platelet count ≥ 100 × 109/L and absence of bleeding. PR was defined as platelet count ≥ 30 × 109/L and ≥ two-fold increase from the baseline count and absence of bleeding (Gómez-Almaguer, Tarín-Arzaga et al. 2013). For IRN, CR was defined as absolute neutrophil count (ANC) ≥ 1500/ mm3 on 2 consecutive measurements separated by 7 days; PR was defined as ANC ≥ 500/ mm3 sustained over 7 days. JMP® 14.1 was used for data analysis. Results Seventeen patients were identified, 9 (52.9%) of whom were female (table 1). Median age was 68 years (range 26-78). The most common pre-existing malignancies were melanoma (6 patients, 35.3%), non-small cell lung cancer (4 patients, 23.5%), and gastrointestinal cancer (3 patients, 17.6%). The ir-h-AE included 7 patients (41.2%) with warm AIHA, 4 (23.5%) with ITP (1 had immune-related pancytopenia that resolved with ITP persisting), 3 (17.6%) with IRN, 1 with AA, 1 with CAD, and 1 with PRCA and mild ITP. All patients had received an ICI dose within 60 days of diagnosis (range 3-52 days, median 18 days). Only 1 patient had a concomitant chronic autoimmune disorder (antinuclear antibody-positive arthritis). Four patients (23.5%) were receiving chemotherapy. Sixteen patients (94.1%) required treatment and received corticosteroids. Eight out of 15 evaluable patients (53.3%) responded to corticosteroids only. Another 4 responders (26.7%) required additional therapies including intravenous immunoglobulins (IVIG) (4, 23.5%) and/or rituximab (4, 23.5%). Overall response rate was 71.4% for AIHA and 100% for IRN. ITP was likely underrepresented in our cohort, and the response rate of 66.7% is likely lower than the response rate in clinical practice. This could be related to the fact that many mild thrombocytopenia cases are not further evaluated in clinical practice. None of our patients died from the sequelae of an ir-h-AE. Two patients (11.8%) died within 30 days of ir-h-AE diagnosis (disseminated intravascular coagulation likely due to the underlying malignancy and sepsis from bacterial pneumonia). At the time of ir-h-AE diagnosis, the malignancy was showing evidence of response in 9 patients (52.9%), progression in 6 patients (35.3%), and stability in 2 patients (11.8%). ICI therapy was permanently discontinued in 9 patients (52.9%) and temporarily held in 6 patients (35.3%). Three of the 5 patients (60%), who were rechallenged with ICI therapy, had recurrence of the same ir-h-AE (AIHA) with adequate response to therapy. Non-hematologic irAE (7 patients, 41.2%) included 3 patients with hepatitis, 2 with thyroiditis, 2 with pneumonitis, 1 with colitis, and 1 with dermatitis. Conclusion Our clinical experience with ir-h-AE highlights the importance of exercising vigilance in assessing the hematologic parameters of patients receiving ICI therapy. While relatively rare, ir-h-AE may impact the clinical course of patients with cancer and their eligibility for therapies that have the potential of offering durable control of the underlying malignancy. ir-h-AE respond to classical therapies including corticosteroids, IVIG, and rituximab. Disclosures Block: Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Marker Therapeutics: Research Funding; Transgene: Research Funding; Immune Design: Research Funding. Kottschade:Bristol-Myers Squibb: Consultancy, Research Funding.

Abstract: Both Hodgkin lymphoma (HL) and asthma are associated with relative isolation from early childhood infectious exposures (hygiene hypothesis) and a T-helper-2 (Th2)-skewed immune response. To test the hypothesis that there is some component of shared genetic etiology between HL and asthma, we examined the genetic overlap in data from genome-wide association study (GWAS) meta-analyses of the two diseases conducted in populations of European ancestry. The HL GWAS meta-analysis consisted of 1,816 HL cases and 7,877 controls, and the asthma GWAS meta-analysis consisted of 2,088 asthma cases and 2,743 controls. The combined HL-asthma GWAS data resulted in 904,634 common single nucleotide polymorphisms (SNPs) genotyped with both arrays. We observed a total of 9 common SNPs associated with both HL and asthma at p 〈 0.0001, compared to 3 SNPs expected in both data sets at that significance level. Out of 66 genetic risk variants associated with HL at a genome-wide significance level of p 〈 10-8, 5 variants replicated in the asthma GWAS dataset at p 〈 0.05. In a meta-analysis combining the HL and asthma GWAS data, we found genome-wide significant associations with two correlated SNPs (r2 =0.91) in the Th2 transcription factor gene GATA3 (rs422628, ORHL-asthma meta =1.25, p=3.36 x 10-9 and rs444929, ORHL-asthma meta = 1.26, p=2.09 x 10-8) that were not genome-wide significant in either disease alone (Table 1). The association with one SNP remained genome-wide significant in the meta-analysis combined with the nodular sclerosis subset (rs422628, ORHL-asthma meta =1.32, p=3.75 x 10-9), but not with other HL subtypes. An association with a SNP in IKZF3, a gene involved in B lymphocyte differentiation and proliferation, reached genome-wide significance only in the meta-analysis with the HL subset positive for Epstein-Barr virus (EBV) in the tumor (rs9909593, ORHL-asthma meta = 1.19, p=3.80 x 10-8). In a genetic diseasome analysis based on an ontological analysis of published GWAS data, HL, especially nodular sclerosis HL, was more closely related to asthma than to solid cancers. Recognition of overlap in genetic predisposition to HL and other immune diseases sheds light on the complex etiology of HL and may enable novel diagnostic and therapeutic approaches. Abstract 135. Table 1 Genome-wide significant results of a meta-analysis combining Hodgkin lymphoma (HL) and asthma GWAS1data. HL GWAS1 Asthma GWAS1 Combined GWAS1 SNP2 Chr3 BP4 Gene OR5 P-value6 OR5 P-value6 OR5 P-value6 rs422628a 10 8151415 GATA3 1.24 1.13 x 10-5 1.31 6.39 x10-5 1.25 3.26 x 10-9 rs444929b 10 8150030 GATA3 1.26 3.24x 10-6 1.28 8.90 x 10-4 1.26 2.09 x 10-8 rs422628c 10 8151415 GATA3 1.33 7.77 x 10-6 1.31 6.39 x10-5 1.32 3.75 x 10-9 rs9909593d 17 35223675 IKZF3 1.11 2.60 x 10-1 1.25 5.06 x 10-8 1.19 3.80 x 10-8 1Genome-wide association scan 2Single nucleotide polymorphism 3Chromosome 4Base pair position 5Odds ratio 6P-values are derived from a meta-analysis using a fixed effects model with weights proportional to the square root of the number of cases. aAll Hodgkin lymphoma cases bAll Hodgkin lymphoma cases, SNP association reported in Cozen et al., Nat Comm, 2014 cNodular sclerosis cases only dEBV-positive cases only Disclosures No relevant conflicts of interest to declare.