Citations

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation. To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A). Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C). Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E). In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite pre-clinical successes, therapeutic interventions have failed to extend patient survival by more than a few months. Anti-PD-1 checkpoint inhibition monotherapy has had efficacy against some tumor types but not GBM. The aim of this study was to determine whether supplementing anti-PD-1 checkpoint blockade with an engineered extended half-life IL-2 could improve outcomes in a preclinical model of disease. Our enhanced checkpoint blockade (ECB) strategy reliably cures approximately50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extended median survival even in the mice that eventually succumbed. This therapy generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. Further, many of the characteristics of brain-tumor mediated peripheral immunosuppression, including MHC class II downregulation on APCs, are prevented by ECB combination therapy. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Notably, ECB’s efficacy is independent of host MHC class I restricted antigen presentation, being equally efficacious in MHC class I and CD8 T cell deficient mice. Conversely, ECB combination therapy is reliant on CD4 T cells and their depletion abrogates the therapy’s survival benefit. Our data shows ECB combination immunotherapy to be efficacious against the GL261 glioma model through an MHC class I independent mechanism, enhancing its off-the-shelf translational appeal relative to strategies requiring extensive knowledge of tumor-specific antigens.

Abstract PURPOSE Vertebral compression fracture (VCF) is a potential adverse effect following stereotactic body radiation therapy (SBRT) for spinal metastases. In this analysis, we developed and internally validated a risk stratification model for VCF. METHODS From an initial set of 680 treatments, we excluded those with proton therapy, prior surgical intervention, or missing data. The final dataset had 464 treatments in 313 patients. Delineations of VCF and all radiographic components of the spinal instability neoplastic score (SINS) were determined by a radiologist. Recursive partitioning analysis (RPA) was conducted using separate training (70%), internal validation (15%), and test (15%) sets. The log-rank test was used as the criterion for node splitting. RESULTS With a median follow-up of 21 months, we identified 84 VCF (18%), including 65 (77%) de novo and 19 (23%) progressive fractures. There was a median 9 months (IQR: 3 – 21) to VCF. From an initial set of 15 candidate variables, six were identified using the backwards selection method, feature importance testing, and a correlation heatmap. Four were then selected in the highest-fidelity RPA models: epidural tumor extension, lumbar location, gross tumor volume > 10 cc, and SINS > 6. One point was assigned to each variable, and the resulting multivariate Cox model had a concordance of 0.760. Each one point increase in score was associated with increasing rates of VCF. Low-risk lesions (score: 0-1, n=273) had 2-year freedom from VCF of 92%, compared to 80% for intermediate-risk (score: 2, n=99) and 56% (score: 3-4, n=92) for high-risk lesions (p < 0.0001). Cumulative incidence curves with death as a competing risk showed increased VCF with higher scores via Gray’s test (p < 0.001). CONCLUSIONS Our internally-validated model identifies a subgroup of patients with high risk for VCF who may benefit from prophylactic surgical stabilization or vertebroplasty.

Abstract Glioblastoma is associated with severe and multifaceted immunosuppression affecting all immune organs. Immunosuppression in GBM is a critical barrier to the success of immunotherapies and patient survival. We demonstrated that immunosuppression in the GL261-model of experimental GBM presents with significant thymic and spleen atrophy, MHCII downregulation, presence of potent immunosuppressive factors in serum, and sequestration of T-cells in the bone marrow. Parabiosis studies determined that soluble factors mediate immunosuppression by inhibiting T-cell proliferation, thymic involution, and loss of peripheral T-cells. In contrast, bone marrow T-cell sequestration was not mediated through soluble factors. While the immunosuppression in GBM is severe, a causative link between each facet of immunosuppression and overall survival is lacking. We used two strategies to block T-cell sequestration into the bone marrow and evaluated the extent survival was impacted in experimental GBM. First, we evaluated the extent a novel and off-the-shelf combination immunotherapy that uses extended 1/2-life IL-2 and anti-PD-1 reverses bone marrow T-cell sequestration. Sham treatment or anti-PD1 monotherapy did not alter T-cell sequestration in the bone marrow and animals had no enhanced survival. Extended 1/2-life IL-2 monotherapy and combination strategy both prevented T-cell sequestration into the bone marrow. However, only combined therapy, which also prevented MHC class II downregulation, improved survival. Second, we determined that glioma-bearing adrenalectomized mice do not present with bone marrow T-cell sequestration. However, sera of glioma-bearing adrenalectomized mice is as immunosuppressive as glioma-bearing controls. Blocking bone marrow T-cell sequestration in the presences of serum immunosuppression led to no survival benefit in glioma-bearing adrenalectomized mice compared to controls. In short, bone marrow T-cell sequestration alone does not correspond with overall survival in experimental glioma. Importantly, a concerted effort to reverse MHC class II downregulation and define inhibitory circulating factors may have the highest impact in immunotherapeutic efficacy and improving patient survival.

Background and objectives Kidney biopsy is the current gold standard to diagnose membranous nephropathy. Approximately 70%–80% of patients with primary membranous nephropathy have circulating anti-phospholipase A2 receptor antibodies. We previously demonstrated that in proteinuric patients with preserved eGFR and absence of associated conditions ( e.g., autoimmunity, malignancy, infection, drugs, and paraproteinemia), a positive anti-phospholipase A2 receptor antibody test by ELISA and immunofluorescence assay confirms the diagnosis of membranous nephropathy noninvasively. These data have not been externally validated. Design, setting, participants, & measurements The clinical and pathologic characteristics of patients with a positive anti-phospholipase A2 receptor antibody test at the Mayo Clinic, the University Hospital Vall D’Hebron (Barcelona), and the Columbia University Medical Center (New York) were retrospectively reviewed. Biopsy findings and presence or absence of a potential associated condition were assessed. Results From a total of 276 patients with positive anti-phospholipase A2 receptor serology, previously reported patients ( n =33), kidney transplant recipients ( n =9), pediatric patients ( n =2), and patients without kidney biopsy ( n =69) were excluded. Among the 163 remaining patients, associated conditions were identified in 47 patients, and 15 patients had diabetes mellitus. All 101 patients of the final cohort had a primary diagnosis of membranous nephropathy on kidney biopsy. In the 79 patients with eGFR≥60 ml/min per 1.73 m 2 , none of the biopsy findings altered diagnosis or management. Among the 22 patients with decreased eGFR, additional findings included superimposed acute interstitial nephritis ( n =1). Conclusions In patients with preserved eGFR and absence of associated conditions or diabetes, a positive anti-phospholipase A2 receptor test by either ELISA >20 RU/ml or a positive immunofluorescence assay confirms the diagnosis of membranous nephropathy, precluding the requirement for a kidney biopsy.

Abstract IntroductionRadiotherapy is considered standard of care for adjuvant peri-operative treatment of many spinal tumors, including those with instrumented fusion. Unfortunately, radiation treatment has been linked to increased risk of pseudoarthrosis. Newerfocused radiotherapy strategies with enhanced conformalitycould offer improved fusion rates for these patients, but this has not been confirmed.MethodsWe performed a retrospective analysis of patients at three tertiary care academic institutions withprimary and secondary spinal malignancies that underwent resection, instrumented fusion, and peri-operative radiotherapy. Two board certified neuro-radiologists used theLenke fusion score to grade fusion status at6 and 12-months after surgery. Secondary outcomes includedclinical pseudoarthrosis, wound complications, and the effect of radiation timing, radiobiological dose delivered, the use of photons versus protons, tumor type, tumor location,and use of autograft on fusion outcomes.ResultsAfter reviewof 1252 spinal tumor patients, there were 60 patients with at least 6 months follow-up that were included in our analyses. Twenty-five of these patients received focused radiotherapy,20 patients received conventional radiotherapy, and 15 patients were treated with protons. There was no significant difference between the groups for covariates such assmoking status,obesity, diabetes, intraoperative use of autograft, and use of peri-operative chemotherapy. There was a significantly higher rate of fusion for patients treated with focused radiotherapy compared to those treated with conventional radiotherapy at 6-months (64.0% versus 30.0%, Odds ratio: 4.15, p=0.036) and 12-months (80.0% versus 42.1%, OR: 5.50, p=0.022). There was a significantly higher rate of clinical pseudoarthrosis in the conventional radiotherapy cohortcompared to patients in the focused radiotherapy cohort (19.1% versus 0%,p=0.037). There was no difference in fusion outcomes for any of the secondary outcomes except for use of autograft. The use ofintra-operative autograft was associated with an improved fusion at 12-months (66.7% versus 37.5%, OR: 3.33, p=0.043). ConclusionFocused radiotherapy may be associated withan improved rate of fusion and clinical pseudoarthrosis when compared to conventional radiation delivery strategiesin patients with spinal tumors.Use of autograft at the time of surgery may be associated with improved 12-month fusion rates.Further large-scale prospective and randomized controlled studies are needed to better stratify the effects of radiation delivery modality in these patients.

Background: Question prompt lists (QPLs) are structured sets of disease-specific questions intended for patient use, enhancing the patient-physician communication by encouraging patients to ask relevant questions during a consultation. Recently, a preliminary 78 question gastroesophageal reflux disease (GERD) specific QPL was created by 12 esophageal experts through a modified Delphi (RAND/University of California, Los Angeles) technique. Patients’ perspectives and opinions on each question, however, had not been accounted for in the preliminary expert’ version. Aim: The aim was to modify a preliminary experts’ QPL, specific to adults with GERD, following patient perspectives and opinions. Methods: A preliminary GERD QPL was modified through patient input and opinions. Thirty-eight patients with a clinical diagnosis of GERD followed at Stanford University Esophageal Clinic between January and November 2019 were consented to modify the preliminary 78 question expert QPL version. After receiving the QPL in Qualtrics (Provo, UT) by a direct e-mail invitation, patients independently rated questions on a 5-point Likert scale, where 1=“should not be included,” 2=“unimportant,” 3=“don’t know/depends,” 4=“important,” and 5=“essential.” Questions were accepted for inclusion in the QPL with an a priori interagreement of 80% ranking in the range of 4 to 5. At the end, patients were encouraged to propose additional questions to incorporate into the QPL by open-endedly asking “Are there questions we didn’t ask, that you think we should?” Results: Twenty-three patients with GERD (19 female, median age 64) fully participated and modified the existing QPL (60.5%). Of the 78 questions from the preliminary GERD QPL, 66 questions (84.6%) were accepted for inclusion. The question with the highest agreement among patients rating a question as essential consisted of “what habits, food, and drinks do I have to avoid?” (82.6%). Questions eliminated because of disagreement included “What is the natural history of GERD,” “Do I have a high chance to die from my Barrett’s?,” and “Why are you prescribing an antidepressant to treat my GERD?” Nine patients suggested additional questions totaling to 16 separate questions, including “What type of surgeries are there to help GERD?,” “What stage is my GERD?,” “What are the odds/percentage of getting cancer from GERD?” Incorporating the suggested questions, the final GERD QPL—created by esophageal experts and modified by patients—consisted of 82 questions. Conclusion: Esophageal experts and GERD patients have a high level of agreement on important questions, though there is some variation in perspective. Future studies can simplify this list and measure the impact of a shared GERD QPL on patients’ decisional conflict and perceived involvement in care.

Abstract Motivation Genomic data are prevalent, leading to frequent encounters with uninterpreted variants or mutations with unknown mechanisms of effect. Researchers must manually aggregate data from multiple sources and across related proteins, mentally translating effects between the genome and proteome, to attempt to understand mechanisms. Materials and methods P2T2 presents diverse data and annotation types in a unified protein-centric view, facilitating the interpretation of coding variants and hypothesis generation. Information from primary sequence, domain, motif, and structural levels are presented and also organized into the first Paralog Annotation Analysis across the human proteome. Results Our tool assists research efforts to interpret genomic variation by aggregating diverse, relevant, and proteome-wide information into a unified interactive web-based interface. Additionally, we provide a REST API enabling automated data queries, or repurposing data for other studies. Conclusion The unified protein-centric interface presented in P2T2 will help researchers interpret novel variants identified through next-generation sequencing. Code and server link available at github.com/GenomicInterpretation/p2t2.