Sutro Biopharma Research Articles

Preliminary Results of a Phase 1 Dose Escalation Study of the First-in-Class Anti-CD74 Antibody Drug Conjugate (ADC), STRO-001, in Patients with Advanced B-Cell Malignancies

Preliminary Results of a Phase 1 Dose Escalation Study of the First-in-Class Anti-CD74 Antibody Drug Conjugate (ADC), STRO-001, in Patients with Advanced B-Cell Malignancies

Continuing Medical Education and Patient Education on Follicular Lymphoma: Changes in Clinician Practice, Patient Behaviors, and Knowledge across Clinicians and Patients

BACKGROUND: Recent advances on follicular lymphoma (FL) have led to significant changes in disease management. Healthcare providers (HCPs) and their patients face multiple challenges, from treatment initiation, to navigating, selecting and sequencing treatment options while balancing the efficacy and toxicities of available options in the first‐line setting and beyond. To address these needs, in partnership with the Leukemia & Lymphoma Society, we created and measured the impact of continuing medical education (CME) to HCPs and healthcare education directed to patients. METHODS: A 1-hour online video-based CME activity for HCPs and 1-hour healthcare education activity for patients were created in 2018 to address identified practice and knowledge needs among HCPs, and knowledge, communication and self-efficacy behaviors among patients. Each activity consisted of slides, polling and live questions, and were broadcast live and on-demand for 6 months at www.OMedLive.com for HCPs, and CancerCoachLive.com, www.LLS.org and Facebook Video for patients. Test questions were administered at 3 time points (pre-, immediate post-, and 2 mos. post-activity). Data from these test questions, responses to live polling questions, and learner-submitted questions during live Q&A were analyzed to determine engagement, lessons learned and continuing education gaps. RESULTS: As of July 2019, 655 HCPs and 12,198 patients took part in the activities. HCPs reported the activity had a positive impact on patient clinical outcomes (58%) and clinical experience (60%). Significant gains in knowledge/competence were observed in 4 of 8 comparisons (avg. pre-post gain 40.75%, avg. Cohen's d effect size .789) on: immunotherapy, clinical trial data, treatment initiation, and disease progression with immunochemotherapy. With patient learners, 47% reported improved communication with their healthcare providers, 49% increased feeling in control of health care decisions, 40% improved their health-related behaviors, and 17% improved engagement in shared decision making. Significant gains in knowledge were observed in 3 of 8 comparisons (avg. pre-post gain 19.25%, avg. Cohen's d effect size .298) on: treatment for untreated advanced-stage FL, and steps to improve patient care. CONCLUSIONS: CME and patient education can yield gains in clinical practice behaviors, patient healthcare communication and self-efficacy, and treatment knowledge of FL. Together, aligned education directed to HCPs and patients can improve the ability to provide individualized treatment for patients. Continuing education for HCPs is advised on prognostic factors and initial treatment choice for untreated advanced stage FL, post-induction and management strategies for early relapse, emerging agents for FL, and balancing patient desire for therapy where observation is still viable option. For patients, further education is recommended on elements of disease monitoring, pharmacologic and non-pharmacologic treatment options for FL, side effects of therapy, strategies to address relapse, benefits of shared decision-making, and access to specialists and support groups. Disclosures Leonard: Bayer Corporation: Consultancy; Sutro Biopharma: Consultancy; Akcea Therapeutics: Consultancy; Miltenyi: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Karyopharm Therapeutics: Consultancy; Merck: Consultancy; MorphoSys: Consultancy; Gilead: Consultancy; BeiGene: Consultancy; ADC Therapeutics: Consultancy; Sandoz: Consultancy; Nordic Nanovector: Consultancy.

Subgroup Analyses of Elderly Patients Aged ≥ 70 Years in AUGMENT: A Phase III Randomized Study of Lenalidomide Plus Rituximab (R2) vs Rituximab Plus Placebo (R-Placebo) in Patients with Relapsed/Refractory (R/R) Indolent Non-Hodgkin Lymphoma (iNHL)

Subgroup Analyses of Elderly Patients Aged ≥ 70 Years in AUGMENT: A Phase III Randomized Study of Lenalidomide Plus Rituximab (R2) vs Rituximab Plus Placebo (R-Placebo) in Patients with Relapsed/Refractory (R/R) Indolent Non-Hodgkin Lymphoma (iNHL)

Preliminary Results from a Phase 1b Study of TAK-079, an Investigational Anti-CD38 Monoclonal Antibody (mAb) in Patients with Relapsed/ Refractory Multiple Myeloma (RRMM)

Preliminary Results from a Phase 1b Study of TAK-079, an Investigational Anti-CD38 Monoclonal Antibody (mAb) in Patients with Relapsed/ Refractory Multiple Myeloma (RRMM)

First-in-Human Imaging of Multiple Myeloma Using Copper-64-Labeled Daratumumab: Preliminary Results

Introduction: 18Fluoro-deoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and non-malignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with copper-64 via the chelator DOTA (64Cu-DOTA-Dara), led to improved sensitivity and specificity over that of FDG (Caserta et al, Blood 2018). Herein, we report the results of a Phase 1 trial (NCT#03311828) designed to 1) assess the safety and feasibility of 64Cu-DOTA-Dara PET/CT and 2) to evaluate and characterize the ability of 64Cu-DOTA-Dara to accurately detect or exclude MM lesions compared with FDG PET/CT. Methods: Patients with biopsy-proven MM and/or a plasmacytoma received an FDG PET/CT scan within 60 days of enrollment. On Day 0, patients were infused with unlabeled ("cold") Dara at one of four dose levels (0 mg, 10 mg, 45 mg, 95 mg) to optimize biodistribution of radioconjugated 64Cu-DOTA-Dara, especially in the liver and the spleen. Within 6 hours of unlabeled Dara administration, patients received 64Cu-DOTA-Dara at a dose of 13.63-16.68 mCi (~5 mg). Whole-body PET scans were obtained at 24 hours and at 48 hours (the latter scan encompassing known tumors). 64Cu-DOTA-Dara standardized uptake values (SUV) were evaluated in MM lesions and normal organs, which were then compared with values from standard FDG PET/CT. Biopsies were performed on accessible discordant lesions. Results: A total of 10 Dara-naïve patients were imaged. Patients were treated with 0 (n=3), 10 (n=3), 45 (n=3), or 95 mg (n=1) of unlabeled Dara. Four patients had newly diagnosed disease, one had biochemical relapse, one had a recurrent plasmacytoma by MRI, and four had possible recurrence by standard PET/CT. No significant adverse events were observed from either cold or 64Cu-DOTA-Dara. With the exception of the one patient with a recurrent plasmacytoma, radiolabeled antibody was eliminated from systemic circulation in subjects analyzed at the first three dose levels within 30 min post injection. In the patient with a recurrent plasmacytoma, the radiolabeled antibody was elevated in the blood for over two days. One newly diagnosed patient had extensive disease by FDG PET and had a biopsy-proven target lesion in the sternum, which had an SUVmax of 14.7 on 64Cu-DOTA-Dara PET/CT vs. 3.3 onFDG PET/CT. A second biopsy from the same patient was taken from a discordant iliac crest lesion (positive for 64Cu-DOTA-Dara but negative for FDG PET/CT) that showed 20-30% MM cell infiltration. In another patient, an iliac crest lesion was 64Cu-DOTA-Dara positive and FDG-negative; biopsy revealed 6% plasmacytosis in the bone. A third patient had an FDG PET/CT positive pleural lesion with an SUVmax of 8.98 and negative on 64Cu-DOTA-Dara (Figure 1A). The lesion did not show recurrence upon biopsy. Furthermore, 64Cu-DOTA-Dara PET/CT yielded superior imaging of bone lesions in the calvarium (Figure 1B). Escalating doses of unlabeled Dara decreased liver and spleen uptake by 64Cu-DOTA-Dara. Conclusions: In this ongoing study, 64Cu-DOTA-Dara PET/CT imaging is to date safe and provides whole body imaging of MM. Further dose escalation of cold Dara (145 mg, 195 mg) is planned to optimize background interference. This modality has the potential to improve sensitivity and specificity over FDG PET/CT scanning in early-stage MM as well as in recurrent disease. Disclosures Krishnan: Celgene, Janssen, Sanofi, BMS: Consultancy; Sutro BioPharma, zPredicta: Consultancy; Amgen, Takeda: Speakers Bureau; Celgene, Z Predicta: Other: Stock Ownership; Takeda: Research Funding. Palmer:Gilead Sciences: Consultancy. Rosenzweig:Celgene, Takeda: Speakers Bureau. Wu:ImaginAb, Inc.: Consultancy, Other: Board Member.

Tandem Autologous-Autologous Vs. Autologous-Allogeneic Transplantation for Newly Diagnosed Multiple Myeloma: Pooled Analysis of 1,338 Patients from Four Trials with Long-Term Follow up

Introduction: Tandem autologous transplant (auto- auto) has been studied as a method to increase remission rates and reduce relapse in the upfront therapy of MM. The use of autologous followed by reduced intensity conditioning allogenic transplantation (auto-allo) offers the potential of long-term graft-versus-myeloma (GVM) effect, but with the risk of graft versus host disease and potentially higher non-relapse mortality (NRM). Trials comparing these two strategies relied on availability of HLA-matched sibling donors for arm allocation (biological "randomization") and have yielded conflicting results, in part due to trial size or limited follow up. A pooled analysis of multiple trials with extended follow up provides the best opportunity to compare these two transplant strategies. Methods: We obtained individual patient data from participants of 4 trials comparing auto-auto vs. auto-allo after brief induction therapy, namely BMT CTN 0102 (N=709), NMAM2000 (N=357), PETHEMA/GEM2000 (N=110), and the Torino consortium trial (N=162). In all 4 trials arm allocation was by biological "randomization". Patients were designated high risk if beta-2 microglobulin ≥ 4.0 mg/L at diagnosis or presence of deletion of chromosome 13 by metaphase karyotyping. Time to event outcomes were analyzed by intention to treat, from the time of first autologous transplant. Main outcomes analyzed were overall survival (OS) and progression free survival (PFS). Secondary outcomes analyzed were NRM and risk of relapse, treated as competing risks, and post relapse survival. Results: There were 1,338 patients included in the analysis, 899 in auto-auto and 439 in auto-allo. Median follow up of survivors is 118.5 months. Characteristics of the two arms are displayed in Table 1. Median OS was 78.0 months in auto-auto and 98.3 months in auto-allo (HR= 0.85, P=0.003, Figure 1). OS was 59.8 % vs. 62.3% at 5-years (P=0.37) and 36.4% vs. 44.1% at 10 years (P=0.01) for auto-auto and auto-allo respectively. PFS was also improved in auto-allo (HR= 0.84, P=0.004) with 5-year PFS of 23.4 vs. 30.1% (P=0.01) and 10-year PFS of 14.4% vs. 18.7% (P=0.06). For the 214 high risk patients (125 auto-auto, 89 auto-allo) there was superior 5-year and 10-year PFS with auto-allo, but no difference in OS. Risk of NRM was higher in auto-allo (10 year 8.3% vs. 19.7%, P<0.001), while risk of disease progression was higher in auto-auto (10 year 77.2% vs. 61.6%, P<0.001). There were 685 progressions in auto-auto and 266 in auto-allo. Median post relapse overall survival was 41.5 months in auto-auto and 62.3 months in auto-allo (HR= 0.71, P<0.001, Figure 2). Five years post relapse, 37.0% of patients were alive in auto-auto vs. 51.1% in auto-allo (P<0.001). Conclusion: Long-term follow up using a large pooled dataset of 4 trials indicates durable, long-term disease control with an auto-allo strategy. Despite higher NRM, there was a reduction in the risk of relapse and superior post relapse survival in auto-allo. This supports the hypothesis of a durable GVM effect enhancing myeloma control with subsequent therapies. Disclosures Costa: Janssen: Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy; Karyopharm: Consultancy; Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; Sanofi: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding. Pasquini:Pfizer: Consultancy; Medigene: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Kite Pharmaceuticals: Research Funding; BMS: Research Funding. Bladé:Janssen, Celgene, Amgen, Takeda: Membership on an entity's Board of Directors or advisory committees; Irctures: Honoraria. Schonland:Sanofi: Research Funding; Takeda: Honoraria, Research Funding; Prothena: Honoraria; Medac: Other: Travel grant; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria. Giralt:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Kite: Consultancy; Johnson & Johnson: Consultancy, Research Funding; Actinium: Consultancy, Research Funding; Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy; Amgen: Consultancy, Research Funding. Patriarca:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Stadtmauer:Abbvie: Research Funding; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Celgene: Consultancy; Takeda: Consultancy. Krishnan:Celgene, Janssen, Sanofi, BMS: Consultancy; Sutro BioPharma, zPredicta: Consultancy; Amgen, Takeda: Speakers Bureau; Celgene, Z Predicta: Other: Stock Ownership; Takeda: Research Funding.

Proteasome Inhibitors Impair the Innate Antiviral Immune Response and Potentiate Pelareorep-Based Viral Therapy in Multiple Myeloma

INTRODUCTION: Pelareorep is the infusible form of human reovirus (RV). Our single-agent phase 1 RV trial in relapsed multiple myeloma (MM) showed that pelareorep treatment selectively infected MM cells, as viral RNA was found in myeloma cells and not the bone marrow (BM) stroma. However, we did not observe apoptosis. Our ongoing phase 1 trial, which combines the proteasome inhibitor carfilzomib with RV, has demonstrated RV infection, apoptosis, and clinical responses. We investigated the molecular mechanisms behind the role of a PI (carfilzomib) in this setting. RESULTS: In all MM cell lines we tested (n=4), independently from their sensitivity to RV infection (Stiff et al., Mol Cancer Ther, 2016), viral replication and apoptosis was impaired when MM cells were directly exposed to PIs (carfilzomib and bortezomib). When this experiment was repeated in the setting of the bone marrow cellular fraction or peripheral blood mononuclear cells (PBMCs), it had the opposite effect, as the addition of PI to RV increased RV replication and apoptosis in MM cells. When we washed PBMCs after overnight exposure to RV+PI or to either single agent, then added MM cells, we observed higher infection and apoptosis in cancer cells co-cultured with RV+PI compared to levels from PBMCs treated with each of the single agents, suggesting that PIs increase the ability of PBMCs to serve as a reservoir for infectious reovirus. Monocytes (CD14+) can engage in phagocytosis of reovirus (Berkeley et al., Cancer Immunol Res, 2018), and accordingly we found that RV genome and capsid protein production were detected in CD14+ cells, but not in CD14-depleted PBMCs, and increased upon PI treatment compared to that in RV-treated CD14+ cells. Given that the NF-κB complex is a key proinflammatory signaling pathway associated with the early innate-antiviral immune response, and because PIs can block the degradation of the NF-κB inhibitor IκBα upon phosphorylation, we investigated the effect of the specific IκBα inhibitor Bay-11 in RV viral replication. Our data show that either PI or Bay-11 can inhibit RV-induced IκBα phosphorylation and its subsequent degradation upon RV infection in CD14+ cells, an effect associated with higher capsid formation in RV-treated CD14+ cells in combination with PI or Bay-11, compared to levels from RV alone. Cytokine profiling in PBMCs and CD14+ cells treated with RV in combination with either PI or Bay-11 showed a significant decrease in IFN-α and IFN-β (IFNs) levels and a concomitant increase in RV replication, in contrast to levels from RV alone (p<0.001). We then decided to investigate whether CD14 depletion could affect RV delivery to the cancer cells invivo. Upon intra-femoral injection of 5TGM1 MM cells into syngeneic C57BL/KaLwRij mice, the mice in which the monocytes were depleted by clodronate-liposome treatment before intravenous RV injection showed lower capsid protein formation in the BM MM cells compared to that in mice where the monocytic population was intact. Because monocytes respond to infection by dividing into macrophages to eliminate pathogens, we wanted to investigate whether PI could impair this effect. Intriguingly, although higher levels of viral genome were detected in PI+RV-treated CD14+ cells compared to RV-treated cells (p<0.01), PI abolished ex-vivo RV-induced monocyte differentiation (n=3, p<0.001), without affecting the ability of RV-infected CD14+ cells to induce higher MM cell killing as compared to that from the CD14+ fraction treated with either single agent. Immune killing assays showed that RV-infected MM cells are more susceptible than non-infected cells to T cell effectors (p<0.001), a mechanism that is further potentiated by the addition of PI. In fact, in contrast to the PI-induced blocking of monocyte expansion and activation in response to the virus (n=3, p<0.001), our data suggest that the PI potentiate the expansion of CD8+ T cells, both ex vivo and in two RV+PI treated patients we analyzed so far. CONCLUSIONS: Here we report for the first time that PIs enhance pelareorep entry, infection, and killing of myeloma cells through its effect on the CD14+ fraction. Reovirus infection and replication within CD14+ cells are augmented by PI-induced NF-κB inhibition of the early innate pro-inflammatory immune response. We also report for the first time that carfilzomib induces direct T cell activation and potentiates T cell killing activity against RV-infected MM cells. Disclosures Krishnan: Takeda: Research Funding; Celgene, Z Predicta: Other: Stock Ownership; Amgen, Takeda: Speakers Bureau; Sutro BioPharma, zPredicta: Consultancy; Celgene, Janssen, Sanofi, BMS: Consultancy. Sborov:Celgene: Honoraria; Janssen: Consultancy. Hofmeister:Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees.

Potential Factors That Impact Lenalidomide/R-CHOP Efficacy in Previously Untreated Diffuse Large B-Cell Lymphoma in the ROBUST and ECOG-ACRIN 1412 Studies

Potential Factors That Impact Lenalidomide/R-CHOP Efficacy in Previously Untreated Diffuse Large B-Cell Lymphoma in the ROBUST and ECOG-ACRIN 1412 Studies

A Mixed Methods Approach to Generate Meaningful Change Estimates for the EORTC QLQ-MY20

IntroductionIn addition to efficacy and safety, novel regimens for the treatment of multiple myeloma (MM) should be assessed in terms of disease-related symptoms, the adverse events patient's experience, and their effect on health-related quality of life (HRQOL). Patient-reported outcome (PRO) measures capture these experiences from the patient's perspective. However, an interpretative challenge of PROs is understanding the magnitude of change that can be considered clinically meaningful.The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30, supplemented with the multiple myeloma-specific module (QLQ-MY20), are commonly used PRO measures in MM trials. However, the minimally important difference (MID) in QLQ-MY20 scores, between groups or for an individual who may be categorized as a responder, has not been established. Previously, estimates based on the statistical variability of scores, or a universal 10-point threshold, have been applied to approximate an MID or responder definition (RD).This study aims to provide empirical estimates of the MID and RD for the QLQ-MY20 in an adult newly diagnosed (ND) and relapsed/refractory (RR) MM population using clinical trial data and qualitative patient interviews.MethodsData were pooled from three completed Phase III clinical trials assessing the efficacy and safety of carfilzomib-containing regimens in patients with RRMM (ASPIRE [NCT01080391], ENDEAVOR [NCT01568866]) or NDMM (CLARION [NCT01818752]). A recommended MID or RD should come from triangulating estimates from several different techniques. MIDs were estimated quantitatively by evaluating the mean PRO score changes in patients grouped as ‘improved’, ‘stable’ or ‘deteriorated’ according to external anchors. The anchors were selected based on variables common to the source datasets expected to reflect changes in PRO scores, and assessed for suitability through correlations. Those included were based on a PRO measuring chemotherapy-induced neuropathy (FACT/GOG-Ntx), ECOG performance status, and matched CTCAE toxicity events. RDs for deteriorations and improvements in health were estimated using the same anchor measures through receiver operating characteristic (ROC) curves, supported by ‘distribution-based’ estimates incorporating the statistical variability of scores. Qualitative telephone interviews were conducted to obtain patient insights from patients with MM regarding what constitutes meaningful change, to further support RD estimates and triangulate quantitative findings. This directly utilizes patient input to define an important treatment benefit and translate HRQOL change scores into tangible and clinically relevant impacts of the treatment on a patient. Interview participants were independent to the clinical trial samples and recruited via patient associations and clinical sites in the US and UK. Ethical approval was obtained for the study.ResultsThe pooled sample from the clinical trials comprised 2,147 patients for analysis; 14 additional patients (to date) were interviewed. MID and RD estimates varied as a result of the different methods, anchors and time points used to derive them. Based on the clinical trial sample, the range of MID and RD estimates for the QLQ-MY20 subscales were calculated and are presented in Table 1. The qualitative interview findings to date suggest that patients consistently understood the subscale concepts, relevance and response options for each domain of the QLQ-MY20. Patients discussed the concept of meaningful change at the domain level and provided reliable meaningful change estimates. The quantitative and qualitative findings will be triangulated to derive weighted estimates recommended for use in future studies. A single global estimate may not be appropriate for all subscales, in which case a range may be recommended.ConclusionsUpdated data based on full triangulation of the qualitative and quantitative evidence will be presented at the annual meeting. Findings from the current study will inform the development of new recommended interpretation guidelines for QLQ-MY20. These values will facilitate power analysis when designing studies and enable consistent interpretation of treatment effect on patients' HRQOL by regulators, payers and clinicians. DisclosuresYucel:Amgen: Employment, Equity Ownership. Trigg:Adelphi Values: Employment; Amgen: Consultancy. Sully:Adelphi Values Ltd: Employment; Amgen: Consultancy. Harper:Adelphi Values: Employment; Amgen: Consultancy. Bonner:Amgen: Consultancy; Adelphi Values: Employment. Shah:Nkarta: Consultancy; Amgen: Consultancy; Sutro Biopharma: Research Funding; University of California San Francisco: Employment; Indapta Therapeutics: Consultancy; Bristol Myers Squibb: Consultancy; Bluebird: Research Funding; Janssen: Research Funding; Kite: Consultancy; Nekktar: Consultancy; Teneobio: Consultancy; Celgene: Research Funding; Indapta Therapeutics: Equity Ownership; Sanofi: Consultancy; Takeda: Consultancy. Panjabi:Amgen: Employment, Equity Ownership. Cocks:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Endomag Ltd.: Consultancy; Adelphi Values: Employment.

Novel Allosteric Inhibitors of Heat Shock Protein 70 As Agents to Probe Protein Homeostasis and Overcome Proteasome Inhibitor Resistance in Multiple Myeloma

Introduction: Clinical efficacy of proteasome inhibitors (PIs) has established multiple myeloma (MM) as the paradigm cancer type in which to therapeutically target protein homeostasis. However, PI resistance is one of the key clinical challenges in this disease. While mechanisms of PI resistance appear complex, proteomic analysis showed multiple heat shock proteins to be highly upregulated in PI-resistant cell line models (Soriano et al, Leukemia (2016) 30:2198). Furthermore, HSPA1B, which encodes a cytosolic Hsp70, is one of the genes which best-stratifies PI-sensitive versus resistant MM cell lines (Mitra et al, Blood Cancer J (2017) 7:e581). These findings support the hypothesis that Hsp70 family chaperones may be critical in maintaining plasma cell protein homeostasis by reducing unfolded protein burden in the PI-refractory setting, indicating potential as a therapeutic target. Targeting Hsp70 may also therefore be effective in combination with PIs or other agents targeting MM protein homeostasis. Here, we profiled a series of novel, allosteric Hsp70 inhibitors of the “JG” class (Shao et al, J Med Chem (2018) 61:6163). We aimed to evaluate their role as anti-myeloma therapeutics as well as their utility as tool compounds to better define targetable protein homeostasis networks in MM.Methods: “JG” compounds were screened in quadruplicate against a panel of 13 cell lines: 9 MM, one AML, 3 immortalized normal bone marrow stromal, in 384-well plates with viability measured by CellTiter-Glo. The parental compound JG98 was tested in combination with the p97 inhibitor CB-5083, the PI bortezomib, and Hsp90 inhibitor 17-DMAG in RPMI-8226, MM1.S, KMS34, HS5, and CMK cell lines for 72 hours, with synergy and antagonism quantified using the ZIP score method. Murine studies were performed using luciferase-labeled RPMI-8226 implanted in NOD scid gamma (NSG) mice (n = 5 mice per arm). For cell surface mass spectrometry experiments, RPMI-8226 and AMO-1 cells were treated with JG342 at the LD20 for 48 hours prior to biotinylation of cell surface glycoproteins (n=3), followed by enrichment for biotinylated proteins with Neutravidin beads and LC-MS sample processing and analysis. For pulsed-SILAC experiments (n = 2), MM1.S cells were grown in light medium for six doublings, followed by exchange to heavy medium (Arg10 and Lys8) with JG342 treatment at the LD10 or DMSO, with timepoint collections performed at 16, 21, and 26 hours, followed by sample processing and LC-MS analysis. All mass spectrometry data was analyzed in MaxQuant and statistical analysis was performed in Perseus and R.Results: Supporting our hypothesis, drug screening of 16 JG compounds revealed consistently greater potency versus a PI-resistant AMO-1 cell line versus the parental line (Fig. A). We focused on the lead compound JG342, which exhibits sub-µM LC50s (140-500 nM) against a panel of myeloma cell lines and a favorable therapeutic index against bone marrow stromal cells. However, all tested JG molecules, including JG342, showed unfavorable pharmacokinetics in NSG mouse models and could not demonstrate in vivo anti-MM efficacy at achievable doses. Therefore, we focused on JG molecules as tool compounds to probe protein homeostasis in MM and further validate Hsp70 as a therapeutic target in this disease. Combination assays using JG98, bortezomib, CB-5083, and 17-DMAG revealed surprisingly diverse, cell line-specific protein homeostasis wiring networks leading to differential synergism and antagonism in dual therapies targeting the proteostasis machinery. To evaluate potential combination effects of Hsp70 inhibition with immunotherapies, we employed cell surface proteomics and found that JG342 leads to broad changes in the MM surface proteome including decrease in expression of BCMA (Fig. B). Lastly, we employed pSILAC mass spectrometry to profile JG342 mediated changes in protein synthesis and degradation. Calculated protein half-lives revealed a distinct subset of differentially regulated proteins after Hsp70 inhibition including aminotransferases and DNA chaperones.Conclusions: Inhibition of Hsp70 is a promising therapeutic strategy to overcome PI resistance in myeloma, but new molecules with more favorable in vivo properties are needed to benefit patients. The “JG” molecules also provide a novel set of tool compounds for exploring protein homeostasis biology not only in MM but across biological systems. [Display omitted] DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Integrating Phosphoproteomics and Transcriptional Classifiers Reveals “Hidden Signaling” in Multiple Myeloma Including Differential KRAS and NRAS Mutant Effects

Introduction: Multiple myeloma (MM) is a complex disease that requires a sophisticated treatment strategy. Currently, no kinase inhibitors have been approved for MM despite their potential for supplementing current combination therapies. Previous functional studies have explored kinase dependency in MM by either a small molecule inhibitor library (Dhimolea et al. 2014 ASH) or RNA interference (Tiedemann et al. 2010, Blood 115:1594). However, owing to their off-target effects, these approaches are imprecise at dissecting signaling networks driving MM growth and survival. Here, we aim to improve diagnostic and prognostic measures and recommend small molecule-based treatments for MM patients by identifying vulnerable signaling patterns in disease using integrated transcriptome- and phosphoproteome-based predictive models.Methods: We employed two methods for measuring cellular signaling activity within a tumor sample. The first involves an unbiased phosphoproteome profiling of eight human myeloma cell lines (HMCL) in biological triplicates. Cells were lysed and digested with trypsin prior to enrichment for phosphorylated peptides by Fe3+-IMAC. Samples were analyzed on a Thermo Q-Exactive Plus mass spectrometer and data processed in MaxQuant to identify and quantify phosphorylation sites. To infer relative kinase activities, we applied kinase set enrichment analysis (KSEA). Drug screening was performed in 384-well plates with CellTiter-Glo as viability readout as previously described (Lam et al. 2018, Haematologica 103:1218). For transcriptome analysis, we implemented the gene expression-based signaling pathway prediction model called PROGENy (Schubert et al. 2018, Nat Comm. 9:20). We applied PROGENy to RNAseq data on 64 MM cell lines (www.keatslab.org) as well as plasma cells from >1000 patients in the MMRF CoMMpass study (research.themmrf.org). Furthermore, using our recently described approach (Way et al. 2018, Cell Rep. 23:172), we built a machine learning classifier to predict RAS genotype from the transcriptomic profiles of MM patients. A tenth of the data set was withheld for testing while the rest was used to train the multiclass logistic regression classifier with sparse penalty.Results: We measured the KSEA-predicted activities of 297 kinases across eight tested MM cell lines. Initially, we were surprised to find high predicted activity in the Ras signaling pathway for KRAS-codon 12 mutant cell lines but low predicted activity in NRAS-mutant cell lines (Fig. A: AMO1 harbors a non-canonical KRAS mutation at codon 146). We further explored this finding with our machine learning-based Ras classifier built on transcriptional data in the CoMMpass study. We identified 311, 405, and 390 genes whose expressions are characteristic of the WT RAS, KRAS mutant, and NRAS mutant genotype, respectively, with surprisingly limited overlap between KRAS and NRAS transcriptional signatures. Building on our KSEA analysis, we next performed a kinase inhibitor screen to evaluate the predictive value of the inferred kinase activities for drug sensitivity. Of 12 screened compounds, mTOR inhibitor INK128 displayed the strongest correlation between drug response and predicted kinase activity. Furthermore, we probed the potential of using pathway activity signatures as prognostic and therapeutic markers. To this end, we applied PROGENy to RNAseq data derived from CoMMpass patients and found that the MAPK signature stratifies patient survival with statistical significance, while the presence and absence of RAS mutations carry no prognostic value (Fig. B). Finally, by integrating RNAseq and drug screen data from the Cancer Dependency Map, we identified three compounds whose inhibitory effects strongly correlate with MAPK activity scores while no significant difference in drug sensitivity was detected between RAS WT and mutants.Conclusion: Both phosphoproteomics and a machine learning-based transcriptional classifier highlight a striking difference in the pattern of signaling between NRAS and KRAS mutants. In addition, we have demonstrated that PROGENy scores possess clinical value for prognostic and therapeutic use based on patient transcriptome data. Taken together, uncovering the cellular signaling networks dysregulated in MM may lead to improved precision medicine, particularly in stratifying patients who may benefit most from kinase inhibitor therapy. DisclosuresWiita:TeneoBio: Research Funding; Sutro Biopharma: Research Funding.

Retrospective Analysis of Modified Hyper-CVAD Therapy Combined with Proteasome Inhibition for Patients with Multiple Myeloma and Renal Insufficiency or Renal Failure

Background: While novel agents have allowed for successful outpatient treatment of patients with multiple myeloma (MM), multi-agent infusional chemotherapy regimens continue to have a role, particularly in high burden disease states. However, the use of platinum-based regimens is limited in patients with renal insufficiency and renal failure. We have been using a modified hyperCVAD regimen, termed HyperCD (Table 1), with omission of vincristine, and inclusion of a proteasome inhibitor (PI, either bortezomib or carfilzomib) in these aggressive clinical presentations for rapid tumor reduction. Here we present our experience with this treatment modality, with analysis of treatment outcomes and toxicities in the renal insufficiency subset.Methods:We performed a single-center chart review of all MM patients at our institution with available electronic medical records who received at least one HyperCD cycle in the setting of concurrent renal insufficiency (defined by estimated eGFR <50mL/min by CKD-EPI at treatment start) between 01/2012 and 12/2016. Data on demographics and disease characteristics were collected. Response was determined by IMWG criteria. Progression free survival (PFS) and overall survival (OS) were calculated by Kaplan Meier method, with PFS including progression or death from any cause and censoring at last known follow-up for live patients.Results:Baseline characteristics: Sixty-seven patients were included in this analysis (Table 2), including 46 non-dialysis dependent and 21 dialysis dependent patients. Median age was 62 years (range 29 - 85). 19% had known prior high-risk cytogenetics defined by t(4;14), t(14;16) and/or 17p(del). Median number of prior lines of therapy was 2 (range 0-14). 34% had prior autologous transplantation. Prior therapies included alkylator therapy in 67%, immunomodulator in 64%, anti-CD38 antibody therapy in 13%, and PI in 87%. Reasons for HyperCD administration included rapidly progressive disease (63%), hypercalcemia (30%), extensive bony (16%) or soft tissue disease (9%), primary (4%) or secondary plasma cell leukemia (7%), and stem cell mobilization (22%). Disease status at time of treatment included new diagnosis (13%), insufficient prior response (18%), and relapsed/refractory or relapsed disease (69%). The median number of cycles administered per patient was 1 (range 1-4), with 55% receiving 1 cycle and 45% receiving 2 or more cycles. The median creatinine at therapy start was 2.93 (range 1.15-8.98).Responses: Of 23 patients on dialysis, 8 (35%) were able to discontinue either during or sometime after therapy. Median percent improvement in creatinine after therapy among non-dialysis patients was 24% (p=0.0001 by wilcoxon signed rank). The overall response rate (ORR) was 69% (46/67), with 1% complete responses (CR), 28% very good partial responses (VGPR), and 39% partial responses (PR). The median time to PR or better was 1.1 months, with a median PFS of 5.1 months, and median OS of 21 months. 87% of patients were able to bridge to other therapy including autologous transplantation (22%). 81% of patients have progressed or died at last follow-up, with disease being the most common cause of death (29/34, 85%).Therapy related toxicities: Median duration of severe neutropenia (ANC</=500/µl) was 4 days (range 0-17) and severe thrombocytopenia (platelets </= 50,000/µl) 7 days (range 0-29), per cycle. 24% of patients had neutropenic fever with documented infections in 37%. Other toxicities included severe bleeding (4%), deep vein thrombosis (7%), and cardiac events (25%), including tachyarrhythmia (9%), demand ischemia (3%), heart failure (2%), pulmonary hypertension (3%), and volume overload (9%). One patient (2%) had potential treatment related mortality (TRM) approximately 3 months after therapy and 1 patient (2%) died within 30 days from progressive disease.Conclusion: HyperCD-based regimens yielded relatively high response rates in MM patients with renal insufficiency, including renal failure. Several patients discontinued dialysis, with improvement in renal function also seen in non-dialysis patients. While TRM was low, hematologic, infectious, and cardiac toxicities were common. The majority of patients were able to successfully bridge to other therapy. HyperCD-based regimens may thus be a potential alternative to platinum based therapies in myeloma patients requiring intensive infusional chemotherapy. [Display omitted] DisclosuresShah:Nekktar: Consultancy; Teneobio: Consultancy; Amgen: Consultancy; Sutro Biopharma: Research Funding; Janssen: Research Funding; Nkarta: Consultancy; Indapta Therapeutics: Equity Ownership; Takeda: Consultancy; Celgene: Research Funding; Indapta Therapeutics: Consultancy; Bluebird: Research Funding; University of California San Francisco: Employment; Kite: Consultancy; Sanofi: Consultancy; Bristol Myers Squibb: Consultancy. Wong:Janssen: Research Funding; Roche: Research Funding. Martin:Sanofi: Research Funding; Amgen: Research Funding; Roche: Consultancy. Wolf:Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy.

Identifying Factors in Multiple Myeloma Controlling Response to B-Cell Maturation Antigen (BCMA)-Targeted Immunotherapy Using CRISPR-Based Functional Genomics

Introduction: Multiple myeloma (MM) is the second most common blood cancer in the United States. Recent breakthroughs in immunotherapy have the potential to transform MM treatment. An immunotherapy target that shows considerable promise in myeloma is the B-cell maturation antigen (BCMA). BCMA is specifically expressed in myeloma cells and plasma cells, making it an ideal target in myeloma. Results from two early-phase clinical trials using anti-BCMA therapy, showed remarkable response in most patients. Although immunotherapy has been promising, recent findings suggest that patients can develop resistance to such therapies by lowering the levels of the target. Here we employ our innovative CRISPR-interference/activation (CRISPRi/a)-based functional genomics platform to identify mechanisms that regulate BCMA expression, which would enable us to design strategies to improve the efficacy of available BCMA immunotherapy agents.Methods: The CRISPRi/a platform utilizes a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor domain to silence genes (CRISPRi) or to activator domains to activate transcription (CRISPRa). The CRISPR machinery is targeted to specific genes using single guide-RNAs (sgRNA). We have engineered a panel of myeloma cell lines to express components of the CRISPRi system. Here we transduced CRISPRi-AMO1 cell line with sgRNAs targeting the human genome. The sgRNA expressing cells were stained with a fluorescent-tagged BCMA antibody and FACS sorted into cells expressing high and low levels of BCMA. The cells were processed for next generation sequencing to determine the frequency of sgRNA in each of these populations. To develop BCMA chimeric antigen receptor-T (CAR-T) cells, CD8+ T cells were transduced with BCMA CAR construct specifically recognizing BCMA. To examine the anti-myeloma activity of the BCMA CAR-T cells, CAR-T cells were co-cultured with MM cell lines at a ratio of 1:2 (Effector:Target) for 24hrs. The cells were analyzed by flow cytometry for expression of CD69, an activation marker on T cells and for apoptosis of cancer cells using propidium iodide.Results: Our FACS-based genome-wide CRISPRi screen identified several genes and pathways regulating BCMA cell surface expression. In addition to previously reported gamma-secretase complex and transcription factor POU2AF1 we identified genes involved in peroxisome biogenesis, subunits of the proteasome, transcription factors and a few druggable targets regulating cell surface expression of BCMA. We are currently validating the novel genes identified from our primary genome-wide screen in a panel of MM cell lines and developing MM cell lines expressing CRISPRa machinery to perform gain-of-function screens that will complement our CRISPRi screen. Furthermore, we have developed active CAR-T cells targeting BCMA and demonstrated its efficacy in MM cell lines expressing different levels of BCMA. We are currently testing the novel genes identified from our CRISPRi screen in combination with BCMA CAR-T cells to identify genes that alter sensitivity to BCMA immunotherapy.Conclusions: Our studies have identified several novel genes and pathways regulating BCMA expression including some druggable targets. Through these studies, we expect to uncover mechanisms regulating expression of BCMA and its impact on sensitivity to BCMA immunotherapy, and pinpoint potential combination therapy targets that pre-empt resistance to BCMA immunotherapy. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy.Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega).Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here.Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Initial Results from a Phase 1 Clinical Study of bb21217, a Next-Generation Anti Bcma CAR T Therapy

Initial Results from a Phase 1 Clinical Study of bb21217, a Next-Generation Anti Bcma CAR T Therapy

IFITM3-Mediated Regulation of Cell Membrane Dynamics Is Essential for Malignant B-Cell Transformation

Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts.Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to “trap”intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion.While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis.In mechanistic studies, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization.Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Subcutaneous Delivery of Daratumumab in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM): Pavo, an Open-Label, Multicenter, Dose Escalation Phase 1b Study

Subcutaneous Delivery of Daratumumab in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM): Pavo, an Open-Label, Multicenter, Dose Escalation Phase 1b Study

Targeting CD74 with Novel Antibody Drug Conjugates (ADCs) for the Treatment of B-Cell Non-Hodgkin's Lymphoma (NHL)

CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. High expression of CD74 has been confirmed in follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and other types of NHL with immunohistochemistry (IHC) using the LL1 antibody (Stein et al. Clin Cancer Res 2007). We employed site-specific conjugation technology to generate novel CD74-targeting ADCs, SP7676 and SP7675 (STRO-001) that exhibit a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The human anti-CD74 IgG1 antibody (SP7219) used for ADCs SP7676 and STRO-001 was engineered using novel Fab-based ribosome display methods enabling selection from ~1012 different antibody variants. Hundreds of unique Fabs from this selection were converted to IgGs and expressed directly in Sutro's proprietary cell-free protein synthesis platform, Xpress CFTM, for extensive screening. The top antibody lead derived from this screen is further tested to identify the best sites for conjugation of linker-warheads. Sutro's SP7219 emerged as the top performing antibody and was conjugated to noncleavable DBCO-maytansinoid linker-warheads to form the ADCs SP7676 and STRO-001. Since conjugation sites were selected based on highest stability both in vitro and in vivo, these novel ADCs lose little drug moiety in circulation and have potential for improved PK, safety and activity profiles. In vitro cell proliferation/cytotoxicity assays show potent activity in 1) DLBCL (germinal center B-cell-like [GCB] and "double-hit") lines: SU-DHL-4, IC50 - 1nM; SU-DHL-6, IC50 - 0.4 nM; WSU-NHL, IC50 - 1.6 nM; Pfeiffer, IC50 - .09 nM; NUDUL-1, IC50 - 0.4 nM; HT, IC50 - 0.7 nM; OCI-LY-19, IC50 - 0.7 nM; WSU-DLBCL2, IC50 - 0.3 nM; 2) mantle cell lymphoma (MCL) cell lines: Mino, IC50 - 0.4-0.7 nM; JVM-2, IC50 - 1.7-2.9 nM; Jeko-1, IC50 - 0.4 - 0.6 nM; 3) Ph+ acute lymphoblastic leukemia (ALL): SUP-B15, IC50 - 3.9-4.6 nM; and 4) CLL (EBV-transformed): JVM-13, IC50 - 3.0-3.4 nM. SP7676 elicited strong anti-tumor response in the OCI-LY-10 lymphoma xenograft model with 100% of animals achieving complete regression of tumors at 3mg/kg every 3 days x 5 doses and 10 mg/kg weekly x 3 doses. In the WSU-DLCL2 "double-hit" lymphoma xenograft model, administration of SP7676 (with re-dosing at time of re-growth) produced tumor regressions at 10 mg/kg every 3 days x 5 (6/8 mice tumor free, remaining 2 with small tumors) and 10 mg/kg weekly x 3 (tumor regression in most animals, 4/8 tumor free). Additionally, STRO-001 exhibits dose-dependent tumor growth inhibition in SUDHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. Exploratory testing of our lead candidate, STRO-001 in cynomologous monkeys showed dose-dependent B-cell depletion at 1 - 30 mg/kg doses on Day 1 and 15, confirming the intended pharmacodynamic effect. Our preliminary data demonstrate that SP7676 and STRO-001 generate potent cell killing activity across multiple B-cell lymphoma/leukemia cell lines in vitro, and anti-tumor activity in preclinical B-cell NHL xenografts. Evaluation of STRO-001 in other cell lines and xenograft models and in combination studies is ongoing. GLP toxicology and other IND-enabling studies are planned. DisclosuresLi:Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Brown:Celgene: Employment. Narla:Celgene: Employment. Barnes:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Hoffmann:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

Discovery and Preclinical Development of Novel CD74-Targeting Antibody-Drug Conjugates (ADCs) with Significant Activity in Multiple Myeloma (MM) Cell Lines and Xenograft Models

CD74, also known as HLA-DR-associated invariant chain, is a type II transmembrane glycoprotein highly expressed in many B-cell malignancies. The limited expression of CD74 in normal tissues suggests it may be a suitable ADC target for these tumor types. Accordingly, we engineered an anti-CD74 human IgG1 antibody (SP7219) using novel Fab-based ribosome display methods. The selected Fabs were readily reformatted and directly screened as IgGs using Sutro’s unique high-throughput, cell-free protein synthesis platform, Xpress CFTM. We then developed novel, potent ADCs, SP7676 and SP7675 (STRO-001), comprised of our lead antibody (SP7219) conjugated to non-cleavable DBCO-maytansinoid linker-warheads with an average drug-antibody ratios (DAR) of 2. We used site-specific conjugation technology which results in a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The sites for conjugation were selected based on highest cell killing activity and stablity in vitro and in vivo. Both ADCs demonstrate potent cell killing activity across multiple B-cell tumor lines in vitro, and anti-tumor activity in preclinical multiple myeloma xenograft models. In vitro cytotoxicity assays show nanomolar potency of STRO-001 in four MM cell lines: Mc/CAR (IC50 0.8 nM), MM.1S (IC50 10-11 nM), U266B1 (IC50 8.5 -9.3 nM), and ARP-1 (IC50 4.3-22 nM). CD74 cell surface expression is required for ADC anti-proliferative effect but the expression level does not seem to correlate with in vitro potency. SP7676 elicited a robust anti-tumor response in the ANBL-6 multiple myeloma xenograft model. Durable regressions were observed in all mice at ≥ 3 mg/kg, with equivalent efficacy (regression) at 3 mg/kg (every 3 days x5) and 10 mg/kg (every 3 days x5 or weekly x3). SP7676 also elicited a clear survival benefit in a disseminated multiple myeloma CAG xenograft model starting at 1mg/kg every 3 days x 5 doses. Similarly, both SP7676 and STRO-001 inhibited the formation of internal visceral tumors in the ARP-1 xenograft model after 3 weekly doses of 3 mg/kg. Evaluation of our lead candidate, STRO-001 in additional MM cell lines and primary patient samples is planned. The tolerability of STRO-001 in non-human primates is under evaluation. STRO-001 was administered to cynomolgous monkeys in an exploratory dose-escalating study up to 30 mg/kg x 2 doses on Day 1 and 15. STRO-001 reduces normal B-cell populations at ≥1 mg/kg after a single dose, providing pharmacodynamic evidence of B-cell targeting while other hematopoietic lineages are mostly affected only at the highest dose studied. Anticipated hematologic toxicities were readily reversible at 1, 3 and 10 mg/kg and target organs of interest were identified. Based on these encouraging data, STRO-001 is advancing to IND-enabling studies for the treatment of CD74 expressing multiple myeloma and other B-cell malignancies. DisclosuresAbrahams:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Narla:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Hoffmann:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

Forum discusses biosimilars, better biologicals

Although FDA has yet to license the country’s first biosimilar, there already is talk of whether the marketplace wants similar or better. With so-called biobetters, said William J. Newell, chief executive officer of eight-year-old Sutro Biopharma Inc. in South San Francisco, California, “we’re not exploring new territory; we’re not exploring new biology. We’re leveraging existing biology” to develop new pharmaceuticals. Examples include darbepoetin alfa and peginterferon alfa. The commercial success of biobetters will depend on how much better these new biologicals are than the already licensed ones, Newell said. “In the current economic environment, whether it’s in the U.S. or internationally, I think if you’re going to be just a small incremental improvement, biosimilars are going to beat you any day of the week,” Newell told an audience at Bio Investors Forum, October 25–26 in San Francisco. The reason, he explained, is cost. From a cost-verses-benefit point of view, an accepted treatment regimen will beat a costly biobetter that offers only a small improvement in outcome or toxicity.