CD20 Ab Research Articles

Nanofluidic delivery implant sustains localization and maximizes efficacy of intratumoral immunotherapy

Intratumoral immunotherapy holds promise for improving cancer treatment efficacy. However, rapid clearance from the tumor upon bolus volume injections limits efficacy and impedes assessment of dose-dependent effects on the tumor immune microenvironment (TIME). To this end, we developed a drug-agnostic nanofluidic implant to deliver immunotherapy within the tumor, providing a mechanism for sustained and controlled intratumoral dosing. Diffusive drug elution from the implant reservoir is controlled by a nanofluidic membrane, which abrogates rapid drug elimination from the tumor and maximizes immunotherapy localization. Here we first achieve in vitro sustained release of agonist CD40 monoclonal antibody (mAb), anti-programmed death ligand-1 (PDL1) mAb, and immune-cell targeted polymeric prodrugs of Resiquimod (toll-like receptor 7/8,TLR 7/8) and a small molecule STING agonist. We then demonstrated in vivo sustained intratumoral drug localization of agonist CD40 Ab and anti-PDL1 Ab in the 4T1 murine model of triple-negative breast cancer (TNBC). Further, we show highly effective anti-tumor efficacy with radiation therapy and sustained intratumoral co-delivery of agonist CD40 Ab and anti-PDL1 Ab in the EMT6 TNBC murine model. Finally, we demonstrate the versatility of this implant by showing that sustained intratumoral delivery of the STING or Resiquimod polymeric prodrugs strongly inhibits 4T1 tumor growth. Collectively, these results support our nanofluidic system as a therapeutic platform technology to investigate sustained intratumoral dosing of immunotherapy.

Multifocal Acquired Demyelinating Sensory and Motor Neuropathy (MADSAM) with Cranial Nerve Involvement. Case Report

BACKGROUND: The multifocal acquired demyelinating sensory and motor neuropathy (MADSAM) is a rare adult onset subvariant of the chronic inflammatory demyelinating polyneuropathy (CIDP). The latter affects 1-9 cases per million adults and may pose a diagnostic challenge (antibody and electrophysiological overlap). The hallmark of the disease spectrum is an immune mediated structural myelin breakdown. Urgent differentiation of MADSAM from the other variants of CIDP is indicated due to its distinct pathogenesis, management, and long-term therapy response to immunomodulators. The disease presents with progressive asymmetrical motor and sensory deficits initially located typically in one limb. Rarely the disease manifests with cranial nerve involvement. The prognosis of patients suffering from MADSAM is reliant on rapid diagnosis and therapeutic response, but neurological deficits reside. Due to its rarity and diagnostic challenge, misdiagnosis is common. THE CASE: A 84 years old Caucasian male diagnosed with multifocal acquired demyelinating sensory and motor neuropathy (MADSAM) presented to our department with progressive neurological deficits. His neurological examination and history revealed paresthesia, hypesthesia, diminished vibration sense (pallhypesthesia), distally pronounced paresis in the upper extremities with a wrist drop on the left hand. Gait examination exposed coordination deficits. The history of the patient and the initial presentation of the disease in 1998 exemplifies the diagnostic challenge of MADSAM, due its mimicry of other diseases, like multifocal motor neuropathy (MMN). The initial symptoms were asymmetrical motor and sensory deficit starting at the upper extremities. Before the patient presented to our facility, he was diagnosed with neuroborreliosis and treated accordingly. The correct diagnosis was set at our department according to clinical presentation, nerve conduction velocity parameters and conduction block during electroneurography, typical changes in nerve ultrasound suggesting an inflammatory neuropathy and fasciculations of the gastrocnemius muscle. After an ineffective steroid therapy and long standing intravenous immunoglobulins the patient is now on a therapeutic scheme of 1000mg Rituximab (CD20 Ab) every 6 months and 1000mg Mycophenolatmofetil. The patient subjectively reports improvement of his status and slower progression of the disease since the Rituximab paradigm. Furthermore, an evident atrophy of the orbicularis oculi muscle was now noted during follow up examination, which highlights facial nerve involvement. CONCLUSION: This case exemplifies the challenge of diagnosing the multifocal acquired sensory and motor neuropathy, but prompt diagnosis and therapeutic intervention is associated with a better prognosis and slower progression of the disease in patients suffering from MADSAM.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Richter's transformation (RT) is characterized by transformation of chronic lymphocytic leukemia to an aggressive lymphoma, most commonly CD20+ large B-cell lymphoma (LBCL). Prognosis for patients (pts) with RT is poor and no standard of care exists; there remains a major unmet medical need. Glofitamab is a T-cell engaging bispecific antibody (Ab) with a novel 2:1 (CD20:CD3) format. In a Phase I/II study (NCT03075696), fixed-duration glofitamab monotherapy demonstrated durable responses and a manageable safety profile in pts with relapsed/refractory LBCL (Dickinson et al. 2022). We report the efficacy and safety of glofitamab monotherapy in pts with RT after a median follow-up of 40.6 months (range: 0.3–53.9). Methods: All pts had RT and had received ≥1 prior regimen including ≥1 anti-(a) CD20 Ab. Pts received obinutuzumab pretreatment (1000 or 2000mg) 7 days before the first glofitamab dose and intravenous glofitamab at a fixed dose (0.6, 16, or 25mg) or with step-up dosing (SUD) in Cycle 1 (target dose 16 or 30mg) every 3 weeks for up to 12 cycles. Responses were assessed using Lugano 2014 criteria. Cytokine release syndrome (CRS) events were graded by ASTCT criteria. Results: As of 10 October 2022, 11 pts had received glofitamab at a fixed dose (0.6–25 mg, n = 5) or with SUD (2.5/10/16 mg, n = 3; 2.5/10/30 mg, n = 3). Median age was 71 years (range: 48–76); six pts were aged >70 years. Overall, 91.0% of pts had Ann Arbor stage III–IV disease and 45.5% had an IPI score ≥3. Median number of prior therapies was 3 (range: 1–4) and 54.6% of pts had ≥3 prior therapies. Most pts were refractory to a prior aCD20 Ab-containing regimen (90.9%) and all to their most recent regimen (100%); 54.5% of pts were refractory to their initial therapy. Investigator-assessed overall response rate and complete response (CR) rate were 63.6% and 45.5%, respectively. Median time to CR was 3 months (95% CI: 2.5–not estimable [NE]). Complete responses were durable and most (4/5; 80%) had been ongoing for ≥24.9 months at data cut (Figure). CRS occurred in 72.7% of pts, was primarily associated with the initial doses, and was mostly Grade (Gr) 1 (27.3%) or Gr 2 (27.3%); Gr 3 (9.1%, n = 1) or Gr 4 (9.1%, n = 1) events were uncommon. Glofitamab-related neurologic adverse events (AEs) potentially consistent with immune effector cell-associated neurotoxicity syndrome occurred in 5 pts (Gr 3, n = 1 [syncope]; Gr 1, n = 4). No glofitamab-related Gr 5 (fatal) AEs or glofitamab-related AEs leading to discontinuation were reported. The research was funded by: NCT03075696 is sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing assistance, under the direction of all authors, was provided by Dikeledi Matlebjane, MSc of Ashfield MedComms, an Inizio company, and was funded by F. Hoffmann-La Roche Ltd. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, ongoing trials Conflicts of interests pertinent to the abstract C. Carlo-Stella Employment or leadership position: Humanitas University Consultant or advisory role: Sanofi, ADC Therapeutics, Karyopharm Tx, Celgene/BMS, Roche, Merck Sharp & Dohme, Scenic Biotech, Novartis Honoraria: Celgene/BMS, Incyte, Roche, Janssen Oncology, Merck Sharp & Dohme, Astra-Zeneca, Gilead Research funding: Sanofi, ADC Therapeutics, Roche Other remuneration: Travel, accommodation, expenses - Takeda, Janssen Oncology M. Hutchings Consultant or advisory role: Takeda, Roche, Genmab, Janssen, Abbvie Research funding: Celgene, Genmab, Roche, Takeda, Novartis, Janssen, Merck, Abbvie, AstraZeneca E. Mulvihill Employment or leadership position: F. Hoffmann-La Roche Stock ownership: F. Hoffmann-La Roche Other remuneration: Travel, accommodation, expenses - F. Hoffmann-La Roche J. Relf Employment or leadership position: Roche Products Limited Stock ownership: Roche, F-Star Therapeutics and Harpoon Therapeutics B. Byrne Employment or leadership position: Roche Products Ltd, Welwyn Garden City, United Kingdom Stock ownership: Roche Products Ltd, Welwyn Garden City, United Kingdom L. Lundberg Employment or leadership position: F. Hoffmann-La Roche Stock ownership: F. Hoffmann-La Roche Other remuneration: Patent with F. Hoffmann-La Roche M. Dickinson Consultant or advisory role: Novartis, BMS, Gilead, Roche, Janssen, Abbvie, Genmab Honoraria: Roche, Amgen, MSD, Janssen, BMS, Novartis, Gilead, Abbvie Research funding: Novartis, Roche, Takeda, Celgene, MSD, Abbvie, Lilly Other remuneration: Travel, accommodation, expenses - Roche

In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Glofitamab in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and ≥ 2 prior therapies: Pivotal phase II expansion results.

7500 Background: Glofitamab is a T-cell engaging bispecific antibody (Ab) with a novel 2:1 configuration that confers bivalency for CD20 (B cells) and monovalency for CD3 (T cells). In a Phase I/II study (NCT03075696), escalating glofitamab doses were highly active and well tolerated in pts with R/R B-cell lymphomas, with obinutuzumab pretreatment (Gpt) and Cycle (C) 1 step-up dosing providing effective CRS mitigation. For the first time, we present pivotal Phase II expansion results in pts with R/R DLBCL and ≥2 prior therapies. Methods: All pts had DLBCL (DLBCL NOS, HGBCL, PMBCL, or trFL) and had received ≥2 prior regimens including ≥1 anti-(a) CD20 Ab and ≥1 anthracycline. IV Gpt (1000mg) was given 7 days before the first glofitamab dose. IV glofitamab was then given as step-up doses on Day (D) 1 (2.5mg) and D8 (10mg) of C1 and at the target dose (30mg) on D1 of C2–12 (21-day cycles). The primary endpoint was CR rate (best response during initial treatment) assessed by Independent Review Committee (IRC) using Lugano 2014 criteria. CRS was assessed using ASTCT criteria. Results: As of Sep 14, 2021, 107 pts had received ≥1 dose of study treatment (median age: 66 yrs [21–90]; Ann Arbor stage III–IV disease: 74%; IPI score ≥3: 54%; DLBCL NOS: 74%). Median prior therapies was 3 (2–7); 59% had ≥3 prior therapies and 35% had received prior CAR T-cells (CAR-Ts). Most pts were refractory to a prior aCD20 Ab-containing regimen (85%) and to their most recent regimen (85%). Many were refractory to their initial therapy (59%) and to prior CAR-Ts (32%). After a median follow-up of 9 months (0.1–16), ORR and CR rates by IRC were 50.0% and 35.2%, respectively. CR rates were consistent in pts with and without prior CAR-Ts (32% vs 37%). Median time to CR was 42 days (95% CI: 41–48). The majority of CRs (33/38; 87%) were ongoing at data cut. An estimated 84% of complete responders and 61% of responders remained in response at 9 months. At data cut, the projected 12-month OS rate was 48%, and 92% of complete responders were alive. These results are consistent with earlier Phase I data in 100 pts treated with target glofitamab doses ≥10mg (CR rate: 34%; estimated 20-month CR rate in complete responders: 72%). CRS occurred in 68% of pts, was primarily associated with the initial doses, and was mostly Gr 1 (51%) or Gr 2 (12%); Gr 3 (3%) and Gr 4 (2%) events were uncommon. All but 2 CRS events were resolved at data cut. Glofitamab-related neurologic AEs potentially consistent with ICANS occurred in 3 pts (all Gr 1–2). No glofitamab-related Gr 5 (fatal) AEs occurred. Glofitamab-related AEs leading to discontinuation were uncommon (3 pts, 3%). Conclusions: Fixed-duration glofitamab induces durable complete remissions and has favorable safety in pts with R/R DLBCL and ≥2 prior therapies, including those with prior exposure to CAR-Ts. Glofitamab is a promising new therapy for pts with heavily pretreated and/or highly refractory DLBCL. Clinical trial information: NCT03075696.

Identification of polo-like kinase 1 as a therapeutic target in murine lupus.

IntroductionThe signalling cascades that contribute to lupus pathogenesis are incompletely understood. We address this by using an unbiased activity‐based kinome screen of murine lupus.MethodsAn unbiased activity‐based kinome screen (ABKS) of 196 kinases was applied to two genetically different murine lupus strains. Systemic and renal lupus were evaluated following in vivo PLK1blockade. The upstream regulators and downstream targets of PLK1 were also interrogated.ResultsMultiple signalling cascades were noted to be more active in murine lupus spleens, including PLK1. In vivo administration of a PLK1‐specific inhibitor ameliorated splenomegaly, anti‐dsDNA antibody production, proteinuria, BUN and renal pathology in MRL.lpr mice (P < 0.05). Serum IL‐6, IL‐17 and kidney injury molecule 1 (KIM‐1) were significantly decreased after PLK1 inhibition. PLK1 inhibition reduced germinal centre and marginal zone B cells in the spleen, but changes in T cells were not significant. In vitro, splenocytes were treated with anti‐mouse CD40 Ab or F(ab’)2 fragment anti‐mouse IgM. After 24‐h stimulation, IL‐6 secretion was significantly reduced upon PLK1 blockade, whereas IL‐10 production was significantly increased. The phosphorylation of mTOR was assessed in splenocyte subsets, which revealed a significant change in myeloid cells. PLK1 blockade reduced phosphorylation associated with mTOR signalling, while Aurora‐A emerged as a potential upstream regulator of PLK1.ConclusionThe Aurora‐A → PLK1 → mTOR signalling axis may be central in lupus pathogenesis, and emerges as a potential therapeutic target.

Mosunetuzumab Monotherapy Is an Effective and Well-Tolerated Treatment Option for Patients with Relapsed/Refractory (R/R) Follicular Lymphoma (FL) Who Have Received ≥2 Prior Lines of Therapy: Pivotal Results from a Phase I/II Study

Mosunetuzumab Monotherapy Is an Effective and Well-Tolerated Treatment Option for Patients with Relapsed/Refractory (R/R) Follicular Lymphoma (FL) Who Have Received ≥2 Prior Lines of Therapy: Pivotal Results from a Phase I/II Study

R-CHOP14 As a Standard of Care in Primary Mediastinal B Cell Lymphoma: A 10-YEARS Experience of Lysa Centers

Introduction: Primary mediastinal B cell lymphoma (PMBL) is clinically and biologically distinct from the other subtypes of diffuse large B cell lymphoma (DLBCL), typically affecting young female patients (pts) with a bulky mediastinal mass. Standard treatment (TRT) is a combination of anti-CD20 antibody (Ab) and anthracycline-based chemotherapy. We aimed to compare patients' outcomes after CHOP delivered every 21 days (CHOP21) or 14 days (CHOP14) or ACVBP combined with anti-CD20 Ab in real life. Methods: All Pts treated in LYSA centers were eligible in this retrospective analysis. Inclusion criteria were as follow: age ≥18 years (yrs), newly diagnosed PMBL, TRT with CHOP or ACVBP plus anti-CD20 Ab between 2006 and 2017, and patient non-opposition statement. Primary endpoint was progression-free survival (PFS), secondary endpoints were: overall survival (OS), response rate (Lugano 2014) and total metabolic tumor volume (TMTV). Results: 313 pts were enrolled from 25 LYSA centers in France and Belgium. In all, median age at diagnosis was 32 (18-88) yrs. Majority of pts were female pts (60.7%) and presented at diagnosis with a good performance status (0-1: 81.8%), stage I-II (57.5%), elevated LDH (85%), Bulk&amp;gt;10cm (58.5%) and low/low-intermediate aaIPI 0-1 (56.7%). Pts in the CHOP21 (n=57) group were older (median 40 yrs, vs 33 vs 29.5, p&amp;lt;0.001), had more frequent low/low-intermediate risk aaIPI (71.4%, vs 55.6% vs 52.5%, p=0.044) and displayed a lower proportion of elevated LDH (66.7%, vs 88.2% vs 83.9%, p=0.014) as compared to CHOP14 (n=76) and ACVBP (n=180), plus anti CD20 Ab (Rituximab: n=296, obinutuzumab: n=17). 229 pts (73.2%) received intrathecal prophylaxis. Median number of chemotherapy cycles for CHOP21, CHOP14 and ACVBP groups were: 6 (1-8), 7.5 (1-8) and 4 (1-4), respectively. Consolidation ASCT was performed for 1 (1.8%), 24 (31.6%) and 46 (25.6%) pts, (p&amp;lt;0.001) and mediastinal RT was delivered to 2 (3.5%), 11 (14.5%) and 4 (2.2%) pts, respectively (p&amp;lt;0.001). ASCT + RT was done for 0, 5 (6.6%) and 3 (1.7%) pts, respectively (p=0.043). Baseline TMTV assessment was available for 233 pts (74.4%). Complete metabolic response rates at end of TRT were comparable between CHOP21, CHOP14 and ACVBP groups: 81.1%, 90.9%, and 85.5%, respectively (p=0.46). 37 (11.8%) pts progressed including 32 (10.2%) who displayed primary refractory disease and 6 (16.2%) pts who relapsed after consolidation ASCT. CNS relapse occurred in 9 (2.9%) pts. Median time between ASCT and relapse was 3 (2-58) months. Patients received the following salvage TRTs: high dose chemotherapy (HDC: R-ICE/R-DHAOX-like) (n=30) followed by second-line consolidation ASCT (n=11/30) and post-ASCT mediastinal RT (n=5/11); salvage RT without chemotherapy (n=1); other regimens (R-CHOP, R-GEMOX) (n=3); none (n=3). 2-yrs second PFS (PFS2) rates in pts who had previously received CHOP21, CHOP14 and ACVBP were: 20.5% vs 62.5% vs 18.8% (p=0.43). Only HDC + ASCT strategy granted disease control (2-yrs PFS2: 32.3%). Median follow up was 44 (1-153) months and the CHOP21, CHOP14, ACVBP 5-yrs PFS and 5-yrs OS were: 74.7% (95%CI: 64-97.1%), 89.4% (82.7-96.6%), 89.4% (84.8-94.2%) (p=0.018, Figure A); and 81% (70-94.4%), 100% (100-100%), 92.4% (88.4-96.7%) (p=0.0036, Figure B), respectively. In a multivariate model including TRT group, consolidation ASCT and/or RT, aaIPI, Bulk&amp;gt;10cm and TMTV≥360cm3, CHOP14 was not associated with better PFS as compared to ACVBP and CHOP21 (p=0.1548). Baseline higher TMTV (≥ 360 cm3) was associated with lower PFS in multivariate analysis, independently of TRT (HR=0.41 [0.2-0.85], p=0.02). All grades TRT-related adverse events were similar between the groups, except for an excess of febrile neutropenia (5.3% vs 5.3% vs 24.4%, p&amp;lt;0.001) and mucositis (1.8% vs 3.9% vs 22.8%, p&amp;lt;0.001) in the ACVBP group. Twenty-two pts died (CHOP21: n=8, ACVBP: n=14) mainly due to lymphoma progression (n=15; 68.2%). 2 toxic deaths were observed (CHOP21: n=1, ACVBP: n=1). Secondary malignancies appeared in 7 pts (CHOP21: n=2, ACVBP: n=5), including 3 cases of acute myeloid leukemia. Conclusion These results confirm the favorable outcome of PMBL pts treated with CHOP14 and ACVBP plus anti CD20 Ab. The toxicity of ACVBP was more pronounced and CHOP14 was associated with a better OS. Baseline TMTV≥360cm3 is a highly predictive factor of unfavorable outcome in PMBL pts, independently of TRT. We recommend R-CHOP14 as standard of care in PMBL. Figure Disclosures Camus: ROCHE: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); PFIZER: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AMGEN: Honoraria; JANSSEN: Honoraria. Decazes:Bayer: Other: travel, accomodations, expenses. Bernard:Janssen: Other: Travel and accommodation . Gandhi Laurent:Abbevie, Pfizer, Takeda, Roche: Other: Travel; Roche, Takeda, Iqone, Accord: Consultancy; Roche, Takeda, Accord: Honoraria. Laribi:amgen: Research Funding; novartis: Honoraria, Research Funding; takeda: Research Funding; abbvie: Honoraria, Research Funding. Houot:Gilead: Honoraria; Kite: Honoraria; Roche: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; MSD: Honoraria. Tilly:BMS: Honoraria.

DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing.

BackgroundDuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A.MethodsT-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys.FindingsDuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable.InterpretationBased on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies.FundingGenmab

Transcriptomic and Metabolomic Profiling Identifies Calcium-Dependent Signaling Mechanisms As a Novel and Exploitable Target to Overcome Anti-CD20 Resistance in Non-Hodgkin Lymphoma (NHL)

Introduction: Anti-CD20 antibody drugs such as rituximab (RTX) and obinutuzumab (OBZ) are used ubiquitously in the treatment of B cell NHL. However, therapeutic resistance to anti-CD20 antibodies occurs quite commonly, especially in indolent NHL. Known mechanisms of resistance include loss of CD20 expression, poor immunoreactivity and dysfunctional apoptotic responses. However, few therapeutic strategies have been established to overcome resistance. Identification of new, targetable anti-CD20 resistance mechanisms are needed. Methods: Anti-CD20 RTX resistant (RR) and OBZ resistant (OR) cells were developed using SUDHL4 and SUDHL10 cultured with chronic low antibody (Ab) drug concentrations. Development of anti-CD20 resistance were characterized by CD20 immunophenotyping, gene expression profiling and systems biology analysis. Natural killer (NK)-mediated ADCC activity (AcellaTox-Glo) with real-time interaction kinetics by microfluidics was utilized for immunological characterization of anti-CD20 resistance. Calcium agonists (ionomycin, thapsigargin &amp; PMA) inducible calcium release was evaluated using Fluo-4 cell based assay. Western blot analysis was used to investigate BCR, autophagy and proteosomal signaling pathways. Metabolomic profiling was performed using Mass Spectrometry. Results: Microfluidic analyses of co-encapsulated single NK and SUDHL10 cell 1:1 effector-target (E:T) ratio revealed that NK cells engaged briskly with target cells within &amp;lt;50 minutes and resulted in &amp;gt;75% cell death within 120 minutes in the presence of anti-CD20 Ab drugs in RTX &amp; OBZ-sensitive cells (P&amp;lt;0.001). These interaction kinetics indicated that CD20 Ab drugs facilitated rapid engagement &amp; sustained tumor-NK interactions to promote cell death. However, ADCC assays using anti-CD20 Abs and primary NK cells revealed a marked decrease in NK mediated ADCC activity in RR (11%, P=0.0002) and OR (17%, P=0.001) cells compared with RTX- or OBZ-sensitive NHL B cells. Transcriptomic and secretomic analyses revealed downregulation of calcium dependent inflammatory responses (i.e., IFN, NFAT, IL6, IL22 &amp; complementary pathways) with concurrent upregulation of nucleotide metabolism in both RR and OR cells. Subsequently, analysis for 'store operated' calcium demonstrated lack of thapsigargin-releasable calcium in anti-CD20 resistant cells (Fig A-B) resulting in significant depolarization of NHL cells. While depolarization is relevant to BTK activation and mitogenic responses, we also observed activation of the pro-survival pathway, JNK (by phosphorylation), corresponding to lower calcium levels in anti-CD20 resistant NHL cells. Lower calcium levels also modulated proteasomal function and promoted glucose-dependent metabolic activity. Additionally, metabolic profiling identified increased glucose uptake and significant increases in the pool sizes of AMP, GMP, CMP and UMP in both RR and OR cells compared with parental SUDHL-4 cells, which validated increases in nucleotide metabolism as predicted by transcriptomic studies. Thus, we hypothesized that calcium-dependent mechanisms may be exploited to induce cell death in anti-CD20 resistant cells. We delineated the impact of reversing depolarization with ivermectin, inhibition of BTK with acalibrunitib, and blocking autophagy and proteasomal function using chloroquine and bortezomib, respectively, in RR &amp; OR cells. Findings indicated that co-targeting with acalibrunitib &amp; ivermectin resulted in significantly decreased cell viability in SUDHL-4 RR cells compared with parental SUDHL4 cells (P&amp;lt;0.0001). Furthermore, chloroquine or bortezomib treatment resulted in increased CD20 expression both in parental and anti-CD20 resistant cells (Fig C-D). In vivo studies of these therapeutics alone &amp; combined are under evaluation in CD20 sensitive and resistant human xenograft NHL models and will be reported at the meeting. Conclusions: We identified calcium as a critical modulator of anti-CD20 resistance in B cell NHL. Furthermore, rational multi-targeting of cellular voltage potential with BTK signaling, autophagy, and proteasomal activity resulted in CD20 re-expression and prominent enhancement of cell death in resistant NHL cells. Collectively, these data suggest that small molecule compounds targeting ionic signaling, many of which are already approved for human use, may be harnessed as novel cancer therapies. Disclosures Evens: Pharmacyclics: Consultancy, Honoraria; Verastem: Consultancy, Honoraria; Tesaro: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria.

Abstract 3251: Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation

Abstract CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin®molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action. Citation Format: Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3251.

Abstract 4936: Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers

Abstract A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Bibhav Poudel, Tara Robinson, Brian Christmas, Kayla Cruz, Skylar Woolman, Christine Rafie, Blake Scott, Valerie Wall, Todd Armstrong, Elizabeth Jaffee. Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4936.

A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment.

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Abstract 2613: Combination agonist and antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers

Abstract Immune tolerance by multiple mechanisms is a major obstacle in the implementation of successful immunotherapy. T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs) suppress the immune response, and the presence of extensive stromal networks within the tumor microenvironment (TME) can prevent T cell infiltration in many tumor types. Furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 is expressed on antigen presenting cells, and CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the TME. Our study asks the question of whether combining a T cell inducing vaccine and PD-1 inhibition with CD40 agonistic Ab can induce T cell priming and tumor infiltrating lymphocyte (TIL) activation in non-immunogenic solid malignancies. We utilized an orthotopic transplant model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a granulocyte macrophage colony-stimulation factor secreting vaccine (GVAX) + anti-PD-1 Ab alone or in combination with CD40 agonistic Ab, and monitored for tumor clearance and survival. A separate cohort of mice were analyzed by IHC and multi-color flow cytometry to assess T cell infiltration and activation, myeloid maturation, and MDSCs after one week of treatment. We demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD-1 Ab + CD40 Ab relative to CD40 Ab alone, although endogenous T cell infiltration remained low across treatment groups. However, adoptive transfer of neu-specific TCR transgenic cells revealed 100% tumor clearance in the combination-treated mice, along with significant T cell infiltration and activation as measured by IFNγ, Granzyme B and TNFα-secreting CD8+ T cells. In contrast, only 20% of mice treated with GVAX + anti-PD-1 Ab were able to clear tumor, and none of the mice receiving vehicle or CD40 Ab alone had long-term survival. Trends were also observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice, and warrant further investigation. In conclusion, GVAX, anti-PD-1 Ab and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in breast cancer. These results support further studies more broadly in non-immunogenic solid tumors. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Brian Christmas, Blake Scott, Tara Robinson, Todd Armstrong, Elizabeth Jaffee. Combination agonist and antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2613. doi:10.1158/1538-7445.AM2017-2613

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells.

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.

Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies.

Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Abstract Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death &amp; ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (&amp;gt;1.5-fold) reveals the involvement of the BCR (PLCG2, BTK &amp; GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 &amp; RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1&amp; PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 &amp; 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

Comparative Phosphoproteomics Study Between Obinutuzumab (GA101) Vs. Rituximab (RTX) Against RTX Sensitive/Resistant Burkitt Lymphoma (BL): Differentially Phosphorylated B Cell Receptor, Fc-Gamma Receptor, Phagocytosis and Natural Killer Cell-Mediated Cytotoxicity Signaling Pathway Proteins

Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL.Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA).Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line.Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. [Display omitted] DisclosuresKlein:Roche: Employment.

Tumor immunologic heterogeneity influences response to radiation and combination immunotherapy

Clinical trials of CTLA-4 and PD-1/PD-L1 antibodies (Ab) have shown responses limited to 10-30% of patients. Increasing evidence suggests radiation (RT) can enhance responses to checkpoint therapies. However, understanding of tumor-derived factors that influence response is limited, and many preclinical models are designed to be immunogenic. We sought to develop a model of immunologic heterogeneity to study factors that influence responses to RT and immunotherapy. Two tumor clones Py117 and Py8119 derived from the PyMT mammary carcinoma in C57Bl/6 mice engraft efficiently and upon RT the Py117 but not Py8119 tumors regressed. There was no significant difference detected in radiosenstivity by clonogenic survival, hypoxia by pimonidazole staining, or angiogenesis through Meca32 staining. Tumor dissociation 10 days after 12 Gy treatment revealed a significant increase in CD45+ leukocytes, CD8+ T cells and decreased CD4+ T cells, myeloid derived suppressor cells, and macrophages in Py117 tumors but minimal changes in Py8119 tumors. Secondly, there was elevated MHCI and induced PD-L1 expression in Py117 compared to Py8119 tumors suggesting a greater response may be obtained with immunotherapy. Mice treated with 12 Gy, 12Gy + PD-1 Ab, and a single dose of CTLA-4 Ab 3 days prior to RT and PD-1 therapy revealed prolonged response with RT + PD-1 and RT + PD-1 + CTLA-4 groups, significant by repeated measures analysis. Notably 2/8 tumors treated with RT + PD-1 and 6/8 mice treated with RT + PD-1 +CTLA-4 were undetectable at day 81. Implantation of tumors with different ratios of Py117 and Py8119 cells revealed that 50% inoculation of Py117 cells leads to an immune response against both tumor clones characterized by greater tumor MHCI expression, CD8+ T cell infiltration and decreased macrophage infiltration. We hypothesized that treating the unresponsive Py8119 tumors with CD40 agonist would replicate the findings of the mixing experiment. CD40 Ab in combination with 20 Gy RT resulted in a greater antitumor response with significant decrease in tumor size not seen in either treatment alone. Evaluation of the tumor microenvironment revealed MHCI expression was enhanced, macrophage infiltration was decreased and CD8+ T cells were increased. These results support that tumor immunologic heterogeneity can influence immune responses after radiation and targeting suppressive factors in the tumor microenvironment can enhance responses. This model of heterogeneity without genetic manipulation is ideal to study tumor-derived factors that enhance or suppress the immune response after RT and could inform clinical approaches to radiation and immunotherapy combinations.

A Comparative Global Phosphoproteomics Analysis of Obinutuzumab (GA101) Versus Rituximab (RTX) Against RTX Sensitive and Resistant Burkitt Lymphoma (BL) Demonstrates Differential Phosphorylation of Signaling Pathways Proteins

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate phosphorylation of signaling pathway proteins altered differentially after obinutuzumab or RTX treatment against RTX sensitive/ resistant BL cell lines.Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs . Cell death was evaluated by staining with AnnexinV/7AAD and analyzed by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer). For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji, /Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database.Results: Obinutuzumab, compared to RTX, significantly enhanced cell death in Raji 45.1±3.3% vs. 32.7±6.8%, (p=0.005) & Raji4RH 15.8±2.2% vs. 2.1±1.5% (p=0.001), respectively. Overall survival of mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in Raji (p=0.05) & Raji4RH (p=0.024), respectively.In Raji, 329 proteins were differentially phosphorylated (>1.5-fold change) between obinutuzumab vs. RTX. Of these proteins, 171 were expressed at higher levels in obinutuzumab than in RTX. Proteins differentially phosphorylated in response to obinutuzumab and RTX treatment were involved in the B-cell receptor (BCR) signaling pathway (LYN, BTK, CD19, PLCG2, INPP5D, NFATC1 and PIK3AP1), the spliceosome (TRA2A, DDX46 and PRPF31), and the cell cycle signaling pathway (WEE1, MMC3, GSK3B and CCNH). (Fig.1A)Obinutuzumab and RTX also resulted in a differential phosphorylation of 606 proteins in Raji4RH. These proteins were involved in the spliceosome (CDC42, TRA2A and DDX42), tight junction (HCLS1, PRKCD, EPB41 and MYH2) and nucleotide excision repair (POLD3, CCNH and LIG1) pathways. (Fig.1B)Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B) was validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, reveled up regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line.Conclusions: These data suggest that obinutuzumab vs. rituximab treatment result in global changes in BL proteins involved in BCR, spliceosome, cell cycle, nucleotide excision repair & tight junction signaling pathway. Furthermore, BCR signaling pathways appear more affected by obinutuzumab compared to RTX in Raji cell lines as compared to Raji4RH. Further, these data revealed the utility of unbiased phosphoproteome interrogation of obinutuzumab vs. rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.