CD3 mAb Research Articles

Outcomes of Second Line Therapy for Light Chain Amyloidosis after Initial Therapy with a Bortezomib Based Regimen

Introduction: There are a lack of randomized trials to guide therapy for relapsed AL amyloidosis with treatment regimens generally extrapolated from experience in multiple myeloma. Methods: We conducted a retrospective review of bortezomib exposed patients that received second line therapy for AL amyloidosis at three Amyloidosis centres in Australia (Brisbane, Perth and Sydney). Of 190 patients treated with bortezomib based therapy upfront, 98 (52%) had transitioned to second line therapy during the study period and comprised the study cohort. Results: Baseline characteristics for the study cohort were typical for a cohort with AL amyloidosis. Median age was 66 years, with 38% of patients being male. Cardiac and renal involvement was present in 70% of patients respectively. Seven patients (7%) had frontline therapy that included daratumumab. Median duration of frontline therapy was 5 months (range 2-18 months) and median time from diagnosis to second line therapy was 9 months (range 2-65 months). We categorized treatment regimens according to commonly used combinations and drug classes to further analyse outcomes. Six categories of therapy regimens were identified; autologous stem cell transplant (ASCT) (n=17), thalidomide based (n=12), lenalidomide based (n=13), proteasome inhibitor (PI) based (n=9), Anti-CD38 monoclonal antibody (mAB) based (n=38) and melphalan based (n=4). Five patients were excluded from this analysis as they received an atypical therapy/regimen not commonly used for AL amyloidosis. Overall response rate (ORR) for the whole cohort was 84%. The ORR was lowest for thalidomide and melphalan based therapy (67% and 50% respectively) and highest for patients receiving an ASCT or anti-CD38 mAB therapy (94% and 92% respectively), Figure 1a. Median event free survival (EFS) for the cohort from second line therapy was 17 months. Median duration of follow up from second line therapy for the cohort was 26 months . Median EFS was shortest for patients treated with thalidomide and melphalan based regimens (6 and 12 months respectively) and longest for those who received an ASCT (28 months) or anti- CD38 mAB therapy (not reached), Figure 1b. When comparing immunomodulatory drugs lenalidomide treated patients had a longer EFS (median 6 months for thalidomide vs 16 months for lenalidomide, p=0.09). The median overall survival (OS) for the cohort was 81 months. OS was longest amongst patients receiving an ASCT or anti-CD38 mAB therapy (median of 91 months and not reached, respectively). Survival was similar amongst patients treated with a PI or Immunomodulatory drug (median 41 months for thalidomide, 44 months for lenalidomide and 36 months for PI based therapy). Conclusion: In bortezomib exposed AL amyloidosis patients, second line therapy with anti CD38 mAB's induced deep responses and prolonged survival. Amongst immunomodulatory agents, the second generation agents appear to be more effective. In our cohort the small group of patients receiving repeat therapy with a PI also appeared to respond to treatment. Although an intensive treatment with significant risks, our data show that in select individuals an ASCT can induce deep and long lasting remissions. Our data highlights the variability in second line therapies for AL amyloidosis given the paucity of evidence to guide treatment. The recent addition of daratumumab as a standard of care for upfront therapy further limits options in the relapsed setting and highlights the need for trials using next generation novel anti plasma cell agents in this patient population.

Rationale for and Design of Papilio-1: A Phase 1/2, Multicenter, Open-Label Study to Evaluate the Feasibility, Safety, and Efficacy of Point-of-Care-Manufactured Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells (GLPG5301) in Relapsed/Refractory Multiple Myeloma

Background and Significance Despite improvements in treatment, patients with multiple myeloma (MM) ultimately relapse or become refractory to available regimens. Triple-refractory (refractory to CD38 monoclonal antibodies [mAbs], 1 proteasome inhibitor [PI] and 1 immunomodulatory drug [IMiD]) or penta-refractory (refractory to CD38 mAbs, 2 PIs and 2 IMiDs) patients have a poor prognosis and are in urgent need of novel treatment options. Chimeric antigen receptor (CAR) T-cell therapies have been used to target B-cell maturation antigen (BCMA) and have shown good clinical activity in patients with relapsed/refractory MM (RRMM). Faster manufacturing procedures may improve clinical outcomes by lowering discontinuation rates and reducing the need for bridging therapy. GLPG5301, a BCMA-directed CAR T-cell therapy, is produced using a novel decentralized and automated point-of-care manufacturing model. This allows administration of a fresh product within 7 days of apheresis. The objective of Papilio-1 is to evaluate the feasibility, safety and efficacy of GLPG5301, manufactured at the point-of-care, in adult patients with RRMM. Study Design and Methods Papilio-1 (CTIS: 2022-500782-27-00) is a Phase 1/2, multicenter, open-label study of GLPG5301 in patients with RRMM after ≥2 prior lines of therapy. Key eligibility criteria are presented ( Table). The study will be conducted in centers across Europe. The Phase 1 primary objective is to evaluate the safety of GLPG5301 and determine the recommended Phase 2 dose (RP2D). The Phase 1 primary endpoint is incidence of serious adverse events, including dose-limiting toxicities (DLTs), until 14 days post-infusion. The Phase 2 primary objective is to evaluate GLPG5301 efficacy. The Phase 2 primary endpoint is best objective response until 2 years post-infusion. Secondary endpoints include: type, frequency and severity of adverse events; duration of response; progression-free survival (PFS); overall survival (OS); minimal residual disease; soluble BCMA levels; time to response; time to progression; GLPG5301 levels in blood and bone marrow; serum levels of chemokines and cytokines; and number of successfully manufactured GLPG5301 products. Exploratory assessments include additional GLPG5301 pharmacokinetics (PK) and pharmacodynamics (PD). Patients will undergo leukapheresis of mononuclear cells for point-of-care manufacturing of GLPG5301 after screening ( Figure). Prior to infusion of GLPG5301, patients will receive fludarabine/cyclophosphamide lymphodepleting chemotherapy, followed by a resting period of at least 2 days. GLPG5301 will be administered as a fresh product, followed by in-hospital monitoring for 7 days. Safety, efficacy, PK and PD will be assessed until end of treatment (100 days post-infusion), and monthly until study completion (2 years). Patients will be enrolled in a long-term follow-up for up to 15 years. In Phase 1, dose escalation will be based on the occurrence of DLTs occurring within 14 days of infusion, according to predefined criteria. Three dose levels (DLs) will be assessed (DL1, DL2 and DL3). Six patients will receive the lowest dose level (DL1) and be evaluated for DLTs and efficacy. The appropriate dose for the following cohort will be then determined by the Safety Review Committee. This will be repeated until RP2D is determined. Approximately 30 patients will be treated with the RP2D in Phase 2. Safety and efficacy data will be analyzed using descriptive statistics. Response status will be summarized as best response (number, percentage, 95% confidence interval) and graphically displayed using swimmer plots by dose. Other binary endpoints will be described using a similar method. PFS and OS will be assessed using the Kaplan‒Meier method and calculated from the median survival time. Summary In Papilio-1, the feasibility, safety and efficacy of GLPG5301, a point-of-care-manufactured BCMA-directed CAR T-cell therapy, will be evaluated in adult patients with RRMM.

Resistance to CD38 Antigen Targeted Therapy in Multiple Myeloma May Result from Underrecognized Chromosome 4 Gene Deletions

Anti-CD38 monoclonal antibodies (mAbs), daratumumab (dara) and isatuximab (isa), are approved to treat multiple myeloma (MM) in the first line and relapsed/refractory settings. Resistance portends a poor prognosis, yet underlying mechanisms have not been well characterized. Despite generally high CD38 expression on most malignant plasma cells (PCs), antigen density may impact the clinical efficacy of targeted therapy. Unfortunately, therapeutic anti-CD38 mAbs interfere with detection and quantification of CD38 surface level by most conventional clinical flow cytometry assays, limiting characterization of CD38 antigen escape. Tumor cell evasion of antigen specific therapeutics can occur with target loss or downmodulation, a phenomenon conventionally referred to as antigen escape. To elucidate mechanisms of CD38 antigen escape, we evaluated MM patients treated with anti-CD38 mAbs who underwent a marrow evaluation between 1/2017 and 10/2022 at a single institution. Eighty-one of 161 (50%) patients had become clinically refractory over their course. Among 82 (51%) patients with ≥1 chromosomal genomic array test (CGAT) performed, 10 (12%) were found to harbor a CD38 deletion ( Table). With a CGAT coverage of 31%, the estimated prevalence of CD38 deletions was 39%. The median interval from diagnosis to CD38 deletion was calculated as 6.7 years (IQR 2.8 to 11.6) for CD38-deleted patients and estimated as 12.9 years (95% CI 12 to NA; Figure) for all patients. The presence of a CD38 deletion was associated with an increasing number of chromosomal abnormalities on concurrent FISH (OR 2.00, 95% CI 1.33 to 3.29, p=0.002). Specific FISH and flow cytometry findings occurred in conjunction with a CD38 deletion. In 50% of patients, flow cytometry detected a bimodal distribution of CD38 fluorescence intensity, reflecting two populations of malignant plasma cells with distinct CD38 surface levels. No patient showed a uniform malignant plasma cell population with a normal surface CD38 level. When the CD38 (4p15.32) deletion was identified in the context of a large 4p deletion spanning the FGFR3 locus, the FGFR3 (4p16) probe could detect the relevant loss of 4p. This was seen in 6 of 8 patients with a concurrent FGFR3 deletion by CGAT. The proportion of clones harboring a CD38 deletion could be quantified by FISH over time when testing was performed on a CD138-enriched sample. Low-level CD38 deletions (≤25%) did not confer resistance to dara-containing multi-drug regimens. In two cases where such deletions were present at diagnosis, disease control is ongoing in response to dara-Pd and dara-Rd. We suggest that acquired CD38 gene deletions appear to be common, the proportion of patients harboring them increases over time irrespective of prior exposure to CD38 mAbs, and their presence facilitates tumor antigen escape. We demonstrate that CGAT can identify CD38 deletions. Conventional clinical flow cytometry assays are unable to quantify the CD38 surface level due to interference from anti-CD38 mAbs. However, we propose that the finding of a bimodal distribution of CD38 fluorescence by flow cytometry could serve as an initial screen to identify at-risk patients warranting confirmatory CD38 mutational analysis. Following characterization of a deletion, FISH could be used to monitor the clonal level longitudinally. Our suggestion that the presence of a CD38 deletion in >25% of malignant PC clones predicts for reduced CD38 mAb efficacy requires prospective validation.

Dual Co-Stimulation with CD28 and ICOS Enhances T Cell Activation and Optimizes CAR-T Cell Antitumor Activity

Background: The Chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has revolutionized anticancer therapy. CAR-T cell expansion and persistence are strikingly associated with the intrinsic programmes of T cells. However, the current process for CAR-T cells manufacturing resulted in high frequencies of exhausted and senescent CAR-T cells with reduced mitochondrial mass. T cell receptor (TCR) signaling cooperates with co-stimulatory signaling to regulate T cells activation and differentiation. Considering that ICOS has a well-defined role in T cell differentiation, especially with regard to the incorporation of ICOS into CAR to boost CAR-T cells antitumor activity, we therefore hypothesize that TCR activation in synergy with dual CD28 and ICOS co-stimulation rapidly triggers T cell activation and may be a potential approach to optimize CAR-T cells antitumor activity. Methods: In this study, we established a culture system using plate-coated CD3 in combination with CD28 and ICOS mAbs to simultaneously stimulate T cells, and further studied whether dual CD28 and ICOS co-stimulation could reprogram the transcriptional, epigenetic and proteomic states of T cells and optimize CAR-T cells antitumor activity. In vitro CAR-T cell phenotype and cytotoxicity were detected via flow cytometry, co-culture killing assay and degranulation assay. NSG mice were inoculated with Raji cells to establish xenograft mouse model, and the tumor load and survival time were dynamically monitored after treatment with CAR-T cells. Results: As expected, dual co-stimulation potently enhanced T cells activation and expansion via reprogramming their transcriptome, epigenome and proteome. Mechanistically, dual CD28 and ICOS co-stimulation rapidly upregulated the pathways such as ribosome, citrate cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) to drive T cells exiting from quiescence. Moreover, metabolic pathways are rewired in CD28 and ICOS-stimulated T cells to guarantee sufficient energy for cell proliferation. Meaningfully, dual co-stimulation confers CAR-T cells with high lentiviral transfection efficiency, possibly correlating with highly open chromatin accessibility in CD28 and ICOS-stimulated T cells. CAR-T cells manufactured with dual CD28 and ICOS co-stimulation demonstrated significantly enhanced OXPHOS and mitochondrial biogenesis, resulting in a superior expansion and being more effective at killing tumor cells in immunodeficient animals. Conclusions: Collectively, our data indicated that activating CD28 and ICOS signaling during CAR-T cells preparation is a potential strategy for optimizing CAR-T cells antitumor activity.

Low Dose Rituximab Efficiently Clears Circulating CLL Cells and Maintains Sensitivity to Antibody Dependent Cellular Phagocytosis (ADCP)

Introduction: We have recently shown that high frequency (2 x week) low dose (50 mg)(HFLD) rituximab (RTX) and acalabrutinib is highly effective initial therapy for patients (n=38) with progressive CLL (ClinicalTrials.gov NCT03788291, PMID:36689726). The first dose of 50 mg of IV rituximab administered at 25 mg/h decreased the median circulating CLL cell count by 84% from pre-treatment baseline at 1h with no further decrease in the count during the remainder of the infusion (PMID:37003030). During the first hour of infusion, median CLL cell membrane CD20 levels decreased to 65% of baseline and were 41% of baseline at the end of the infusion. Median serum rituximab concentration was 2 mcg/ml at 1h and 10 mcg/ml at the end of the infusion. Median complement (CH50) concentration decreased to 91% of baseline at 1 h and to 81% of baseline at the end of the infusion. The cessation of clearance of CLL cells from the circulation within 1h of starting rituximab therapy is thus unlikely to be caused by low rituximab levels, decreased CLL cell CD20 expression or low serum complement levels. We and others have previously shown that ADCP is the primary mechanism of clearance of anti-CD20 mAb opsonized B cells in both humans and mice. We hypothesized that clearance of circulating CLL cells decreases because residual circulating CLL cells are resistant to mAb induced ADCP. To test this hypothesis, we measured in vitro ADCP sensitivity of CLL cells sampled from patients before and after rituximab treatment. Methods: Blood specimens were collected from 13 patients prior to starting therapy (C1D1_pre), 1h after start of IV rituximab infusion (C1D1_1h), at end of rituximab infusion (C1D1_post), and 48 h after initiation of therapy (C1D3_pre). Sensitivity of circulating CLL cells to in vitro ADCP was measured using live cell time-lapse high-content microscopy imaging of phagocytosis of CLL cells by human monocyte derived macrophages (hMDM) as previously described (PMID:32005699). Phagocytosis was quantified using a void index and sensitivity of samples was compared using the area under the curve of the 2h void index plot (AUC). Experiments were done in triplicate with co-cultures of hMDM and CLL cells without mAb as controls. Rituximab (10 mcg/ml) was used to test CD20 mAb mediated ADCP and alemtuzumab (10 mcg/ml) to test non-CD20 mediated ADCP. Results: Figure (median and 95% confidence intervals) shows that neither rituximab nor alemtuzumab induced ex-vivo ADCP as measured by median void index AUC was significantly altered by prior in vivo exposure of CLL cells to rituximab (Wilcoxon Match-Paired Sign test; all p>0.05). Discussion: The rapid decrease in circulating CLL cells induced by IV rituximab occurred only within the first hour after initiation of mAb infusion. Our measurements show that serum rituximab and complement concentrations and CLL cell membrane CD20 levels should be adequate to support additional CLL cell clearance after 1h. We now demonstrate that residual circulating CLL cells remain sensitive to in vitro ADCP by both rituximab and alemtuzumab. This suggests that despite reduced levels of surface antigen, circulating cells remain sensitive to mAb mediated clearance. Alternative explanations for the limited clearance of circulating CLL cells include: 1. Saturation of the limited cytotoxic capacity of the innate immune effector cells (Kupffer cells and splenic macrophages) compatible with our previously reported data on finite macrophage ADCP capacity (PMID:32556153); 2. Mobilization of CLL cells from lymphoid tissue; and 3. Temporary sequestration of rituximab opsonized CLL cells in tissue (e.g. spleen or lungs), which is unlikely given the observed decrease in lymph node size following initiation of therapy. Ongoing research is testing these hypotheses.

Prophylactic Tocilizumab to Prevent Cytokine Release Syndrome (CRS) with Teclistamab Administration

Introduction: Teclistamab is a T-cell redirecting bispecific antibody that targets both CD3 on the T-cell surface and B-cell maturation antigen (BCMA) expressed on myeloma cells. Teclistamab gained accelerated approval for RRMM patients that had previously been treated with 4 prior lines of therapy including a PI, IMID and a CD38 monoclonal antibody. This approval was based on the results of the MajesTEC-1 study demonstrating an ORR of 63%. Cytokine release syndrome (CRS), a known complication of T-cell engaging therapies, occurred in 72% patients which required a REMS program to monitor the patients in hospital for 48 hours post both priming doses and the target dosing. Several approaches are in place to minimize the risk of CRS to allow for the administration of teclistamab safely as an outpatient. We have evaluated the role of administering tocilizumab prophylactically to mitigate the incidence and the severity of CRS. Methods: A total of 48 patients were admitted to the Emory University hospital from December 2022 until July 2023 as mandated by REMS program for teclistamab step-up dosing followed by the first full dose at least 48 hours apart (0.06, 0.3, and 1.5mg/kg) with premeds per institutional guidelines (Benadryl 50 PO, Tylenol 650 PO, and Dexamethasone 16mg PO 30 minutes prior to each dose). Upon evaluation of our first 15 patients, the median time to CRS from the administration of the first-priming dose was 48 hours. We subsequently administered tocilizumab 8mg/kg IV over an hour (max dose of 800 mg) prophylactically at 44 hours (4 hours prior to the second step-up dose level) for the next 33 patients. CRS was graded per the American Society for Transplantation and Cellular Therapy criteria and managed according to institutional guidelines. Results: The median age of the patients was 66 years (range, 44-82). All patients were IMID, PI and CD38 mab refractory. The rate of all grade CRS amongst the entire cohort was 21 of 48 patients (43.8%). CRS occurred in 10 of the 33 patients (30.3%) who received prophylactic tocilizumab, compared to 11 of 15 (73.3%) of patients that did not. The majority the events in the prophylactic tocilizumab group are grade 1 [8 (24.2%)]. 1 patient had grade 2 and another had grade 3 CRS (patient with an aggressive relapse at time of teclistamab administration, requiring 3 doses of tocilizumab every 8 hours). The median number of tocilizumab doses was 1 (range, 1-3). The median duration of CRS was 1 day (range, 1-3). 1 dose of steroids (outside of premeds) was administered in the 1 patient who experienced grade 3 CRS but in no additional patients treated with prophylactic tocilizumab. Concurrent immune effector cell neurotoxicity syndrome (ICANS) was also decreased with the incorporation of prophylactic tocilizumab (20% vs 6.1%, respectively). All ICANS in the prophylactic cohort were grade 1 and managed symptomatically. The median duration of ICANS was 1 day. The non-prophylactic cohort experienced 3 of 15 (20%) patients with readmissions, with 1 additional patient going to hospice. None of the patients in the prophylactic cohort were readmitted to the hospital within 14 days of discharge. Conclusion: Prophylactic tocilizumab prior to the second priming dose has decreased the incidence and severity of CRS in heavily refractory RRMM patients receiving teclistamab. These results are lower in absolute incidence and severity when compared to the MajesTEC-1 trial where CRS was seen in 72% of patients. Prophylactic tocilizumab prevented usage of steroids, prevented dose delays, prevented readmission to the hospital, and possibly reduced the occurrence of ICANS. These data make a case for early incorporation of prophylactic tocilizumab and support outpatient administration of teclistamab in most patients.

A Phase 1, Open-Label, Dose Escalation and Dose Expansion Study of CLN-978 (CD19XCD3XHSA) in Patients with Relapsed/Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (B-NHL)

Background: Patients with B-cell non-Hodgkin lymphoma (B-NHL) who are refractory to/or have relapsed (R/R) after treatment with anti-CD20 monoclonal antibodies, either alone or in combination with standard-of-care chemotherapy, have a poor prognosis. Response rates tend to decrease and duration of response shortens with each successive line. CD19 represents a significant target for the development of novel immunotherapies for the treatment of patients with R/R B-NHL, many of whom develop disease that is refractory to CD20-containing regimens. CD19-directed therapies approved for B-NHL include an Fc-enhanced anti-CD19 monoclonal antibody, a CD19-directed antibody-drug conjugate, and several CD19-directed CAR-T cell products. While CAR-T approaches are an important alternative for some B-NHL subtypes, many patients still relapse, and challenges remain with toxicity and accessibility in the community setting. Consequently, substantial unmet needs remain for effective treatments with broad applicability across B-NHL subtypes and practice settings. CLN-978 is a fully human, half-life extended, single-chain T cell engaging construct targeting CD19 and CD3, with a human serum albumin (HSA) binding moiety for half-life extension. Preclinically, CLN-978 effectively redirects T cells to kill CD19-expressing malignant cells and has demonstrated potent in vitro and in vivo efficacy against cancer cells expressing very low levels of human CD19. Study Design and Methods: This is a phase 1, open-label, multi-center, first-in-human, dose escalation, and dose expansion study of CLN-978 in patients with R/R B-NHL. Adult patients (≥ 18 years of age) with one of the following CD19+ B-cell histologies: diffuse large B-cell lymphoma - de novo or transformed, high-grade B-cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or marginal zone lymphoma (nodal, extranodal, or mucosa-associated) for whom there is no available approved standard therapy will be recruited. Other key eligibility criteria include: ≥ 2 lines of prior chemotherapy, Eastern Cooperative Oncology Group Performance Status 0-2. Key exclusion criteria include prior treatment with allogeneic hematopoietic stem cell transplantation or any other investigational CD19 x CD3 T cell engager. Prior CAR-T therapy, unconjugated or radio-conjugated CD19 mAb, and CD19 antibody-drug conjugate are allowed. The study consists of 2 parts: 1) a dose escalation phase commencing with accelerated titration in single-patient cohorts followed by a conventional 3+3 dose escalation design to identify the recommended phase 2 dose (RP2D) and 2) a dose expansion phase to further characterize the safety and preliminary efficacy of CLN-978 in disease-specific cohorts at the RP2D. CLN-978 will be subcutaneously (SC)-administered once weekly (QW) in 28-day cycles until progressive disease, intolerable toxicity, or a maximum of 24 cycles of treatment. Initial administration of CLN-978, inclusive of potential priming doses and the first target dose, will be administered on an inpatient basis. Subsequent doses may be given in the outpatient setting. Based on emerging data, less frequent administration schedules may be implemented in Cycle 4 and beyond as approved by the Safety Review Committee. Safety, pharmacokinetic (PK), pharmacodynamic, and preliminary efficacy assessments will guide the selection of the dose and schedule for further evaluation. The primary endpoint is safety. Key secondary endpoints include PK, anti-drug antibodies, overall response rate, duration of response, time to response, time to subsequent anti-lymphoma therapy, progression-free survival, and overall survival. Approximately 90 patients (30 in dose escalation and 60 in dose expansion) will be enrolled at approximately 6 sites in the U.S., reflecting both community and academic centers. The study is currently enrolling (NCT05879744).

Novel Clinically Translatable Iron Oxide Nanoparticle for Monitoring Anti-CD47 Cancer Immunotherapy.

A novel clinically translatable iron oxide nanoparticle (IOP) is currently being tested in phase 2 clinical trials as a magnetic resonance imaging (MRI) contrast agent for hepatocellular carcinoma diagnosis. The purpose of our study is to evaluate if this IOP can detect activation of tumor-associated macrophages (TAMs) due to CD47 mAb-targeted immunotherapy in 2 mouse models of osteosarcoma. The toxicity, biodistribution, and pharmacokinetics of IOP were evaluated in 77 female and 77 male rats. Then, 24 female BALB/c mice with intratibial murine K7M2 tumors and 24 female NOD scid gamma mice with intratibial human 143B osteosarcoma xenografts were treated with either CD47 mAb (n = 12) or control antibody (n = 12). In each treatment group, 6 mice underwent MRI scans before and after intravenous infusion of either IOP or ferumoxytol (30 mg Fe/kg). Tumor T2* values and TAM markers F4/80, CD80, CD206, and Prussian blue staining were compared between different experimental groups using exact 2-sided Wilcoxon rank sum tests. Biodistribution and safety evaluations of IOP were favorable for doses of less than 50 mg Fe/kg body weight in female and male rats. Both IOP and ferumoxytol caused negative enhancement (darkening) of the tumor tissue. Both murine and human osteosarcoma tumors treated with CD47 mAb demonstrated significantly shortened T2* relaxation times after infusion of IOP or ferumoxytol compared with controls (all P 's < 0.05). Higher levels of F4/80 + CD80 + were found in murine and human osteosarcomas treated with CD47 mAb compared with sham-treated controls (all P 's < 0.05). In addition, murine CD47 mAb-treated tumors after infusion of either IOP or ferumoxytol showed significantly higher numbers of Prussian blue-positive cells compared with controls ( P < 0.05). There was no significant difference of F4/80 + CD206 + cells among any of the groups (all P 's > 0.05). Iron oxide nanoparticle-enhanced MRI can be used to diagnose CD47 mAb-mediated TAM-activation in osteosarcomas.

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

Antibody response to SARS-CoV-2 vaccination or infection in a prospective cohort of children with neuroinflammatory diseases.

Immune medications affect antibody responses to SARS-CoV-2 vaccination in adults with neuroinflammatory disorders, but little is known about antibody responses in children with neuroinflammation and on immune treatments. Here we measure antibody levels in response to SARS-CoV-2 vaccination in children receiving anti-CD20 monoclonal antibodies, or fingolimod. Children under 18 years of age with pediatric-onset neuroinflammatory disorders who received at least two mRNA vaccines were included. Plasma samples were assayed for SARS-CoV-2 antibodies (spike, spike receptor binding domain-RBD, nucleocapsid) and neutralization antibodies. Seventeen participants with pediatric onset neuroinflammatory diseases were included: 12 multiple sclerosis, one neuromyelitis optica spectrum disorder, two MOG-associated disease, and two autoimmune encephalitis. Fourteen were on medications (11 on CD20 monoclonal antibodies-mAbs, one on fingolimod, one on steroids, one on intravenous immunoglobulin) and three were untreated. Nine patients also had pre-vaccination samples available. All participants had seropositivity to spike or spike RBD antibodies except for those receiving CD20 mAbs. However, this proportion was higher in children than in an adult MS patient cohort. The most significant contributor to antibody levels was duration of DMT. SARS-CoV-2 antibodies are decreased in children on CD20 monoclonal antibodies than on other treatments. Treatment duration associated with vaccination responses.

299-OR: A Combinatorial Approach with Monoclonal Antibodies to SerpinB13 and CD3 Is Therapeutically Superior to Single Antibody Use in Autoimmune Diabetes

A successful approach to autoimmune diabetes therapy may require simultaneous insulin-producing cell regeneration and T-cell mediated autoimmunity resolution in pancreatic islets. While CD3 antibody has been proved to reduce islet inflammation, the monoclonal antibody (mAb) to serpinB13 protease inhibitor, developed in our laboratory, was shown to cause regenerative changes in beta cells. Whether a combination of both antibodies is superior to single antibody use in the treatment of autoimmune diabetes is unclear. To address this non-obese diabetic (NOD) female mice at late pre-diabetic stage were injected with serpinB13 mAb (clone B29, 10, 40 or 200 micrograms/animal) and CD3 mAb (clone 145-2C11; at a suboptimal dose of 10 micrograms). All experimental injections were controlled with nonspecific hamster IgG and mouse IgG (clone HB-158). After the last antibody injection, random blood glucose levels were assessed for 20 weeks, and mice were declared diabetic after two measurements of glucose exceeding 300 mg/dL. We found that the combination of 40 micrograms of serpinB13 mAb with CD3 mAb offered a significant level of protection from diabetes (p=0.0027, Kaplan-Meyer with log rank test), while the combination with 10 micrograms of serpinB13 mAb had no effect. The higher dose of 200 micrograms of serpinB13 mAb showed a trend in favor of diabetes inhibition. Interestingly, at 6 to 7 weeks after completing the treatment with 40 micrograms of serpinB13 mAb alone, NOD mice showed a strong tendency to remain healthy, and separated from the double-negative control, which continued to progress to diabetes. Our results indicate that a combinatorial approach with mAbs to serpinB13 and CD3 is beneficial in autoimmune diabetes. The observations also suggest that an intermediate dose of serpinB13 mAb, and a sufficient time following administration of this mAb, is critical for its positive impact on diabetes prevention. Disclosure B.Szepietowska: None. S.V.Avdulov: None. Y.Kryvalap: None. J.Czyzyk: None. Funding JDRF (2-SRA-2022-1215-S-B)

#6402 IMPACT OF DARATUMUMAB AND BELATACEPT ON HLA ANTIBODIES IN KIDNEY TRANSPLANT CANDIDATES WITH 100% CPRA: EARLY RESULTS OF ATTAIN (ITN090ST)

Abstract Background and Aims Despite high allocation priority, &amp;lt;10% of kidney transplant candidates with cPRA 100% can find a compatible donor. Current desensitization strategies are ineffective due to antibody rebound. Adding costimulation blockade to plasma cell (PC) depletion prevents antibody rebound in nonhuman primates by countering nodal B cell and Tfh expansion. Method ATTAIN is a pilot, phase I/II trial of daratumumab, a CD38 mAb used in multiple myeloma, plus belatacept, a high affinity CTLA4-Ig, to desensitize kidney transplant candidates with cPRA ≥99.9%. Enrolled subjects receive daratumumab (6 doses: 8 mg/kg) and belatacept (4 doses: 10 mg/kg) over 10 weeks with bone marrow and blood assessments pre-and post-treatment. The primary efficacy endpoint (PE) is a composite of (1) elimination of ≥1 HLA antibody specificity, (2) ≥50% reduction in the MFI of ≥3 HLA antibody specificities, or (3) kidney transplant from a previously incompatible donor. Target accrual is 15, enrolled in 2 cohorts (5+10). Results Cohort 1 (n = 5, mean age 44, 60% with previous transplant) has been enrolled and treated, with 5-31 weeks follow-up to date. The treatment was tolerated well in all 5 patients who showed a significant reduction in most HLA antibodies after treatment without manifestation of rebound. 3 of 5 participants reached the PE and 5 of 5 had &amp;gt;50% bone marrow (BM) PC depletion. One patient received a kidney transplant from a previously incompatible deceased donor and is doing well at 7 months post-transplant without rejection or rebound of HLA antibody. Treatment was temporarily paused in 3 subjects due to AEs (acute cholecystitis and COVID, upper GI bleed, fevers); no cases of opportunistic infection or malignancy occurred. Conclusion A novel HLA desensitization regimen consisting of PC depletion with Daratumumab and costimulatory blockade with Belatacept appears safe and successful in the ongoing ATTAIN clinical trial. In the initial subject, this regimen led to transplant without HLA antibody rebound or acute rejection. Longer follow-up and additional subjects are needed to confirm these promising results. ATTAIN (NCT04827979) is a trial conducted by the Immune Tolerance Network and sponsored by NIAID (award UM1AI109565).

Pharmacogenomic Analyses Implicate B Cell Developmental Status and MKL1 as Determinants of Sensitivity toward Anti-CD20 Monoclonal Antibody Therapy.

Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG+ cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.

Degradation of CD47‐SIRPα axis by pomalidomide potentiates CD20 antibody‐dependent cellular phagocytosis against B‐cell lymphoma

Introduction: Anti-CD20 monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell lymphomas (BCL). The chemo-free combination with lenalidomide (Len) and rituximab (R2) as a first-line therapy has shown promising activity in indolent lymphomas. However, the efficacy of R2 regimen is yet unsatisfactory in aggressive settings, thereby novel synergistic strategy with next-generation CD20 mAbs and/or newer immunomodulatory drugs (IMiD) are urgently needed. Pomalidomide (Pom) is more powerful than Len in immunomodulatory properties including redirection of tumor-associated macrophages (TAM), which may confer a therapeutic opportunity to potentiate antibody-dependent cellular phagocytosis (ADCP) in elimination of lymphoma cells. This study aimed to evaluate the macrophage-based anti-lymphoma activities of Pom plus CD20 mAbs. Methods: BCL cell lines (SU-DHL-4, Raji and Daudi) were co-cultured with human monocyte-derived macrophages and treated with various combinations with IMiDs (Len or Pom) plus CD20 mAbs (rituximab or obinutuzumab). Phagocytosis and cell viability of lymphoma cells were tested by flow cytometry. Protein mass spectrometry and co-immunoprecipitation (co-IP) were performed to identify Pom-redirected neosubstrates of cereblon (CRBN). IMiD-responsive CrbnI391V mice were bred to Eμ-Myc lymphoma mice to evaluate the in vivo immunomodulatory effects of Pom on macrophages. Results: In vitro co-culture systems showed that Pom plus obinutuzumab elicited the strongest ADCP effects, resulting in robust phagocytosis of tumor cells by macrophages and markedly decreased tumor cell number, when compared to other combinations including Len plus rituximab (R2), Len plus obinutuzumab (GALEN) or Pom plus rituximab. Mechanistically, we identified CD47, a well-known negative regulator of macrophage phagocytosis, as a novel CRBN neosubstrate in BCL cells using mass spectrometry screening and co-IP validation. Interestingly, we found that only Pom rather than Len or thalidomide could efficiently induce degradation of total and surface CD47 in SU-DHL-4 cells. We further confirmed that knockdown of CRBN abrogated Pom-induced CD47 degradation, indicating the on-target effect. More importantly, based on the murine spontaneous lymphoma models (Eμ-Myc CrbnI391V mice), we found administration of Pom elicited a shift from M2 (CD206+) to M1 (CD86+) phenotype of tumor-associated macrophages and significant decrease of SIRPα+ populations in both M1 and M2 subtypes. Conclusion: Our study provides evidence for the superior effects of Pom plus obinutuzumab by harnessing the anti-lymphoma activity of TAMs, and suggests degradation of CD47-SIRPα axis as a novel mechanism underlying the drug synergy. Further preclinical and clinical investigations are needed to determine the in vivo synergistic efficacy and potential toxicity of this combined treatment. The research was funded by: This study was supported by the National Natural Science Foundation of China (No. 81470336 to KZ). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Immunotherapy No conflicts of interests pertinent to the abstract.

Phase 1 study of tasquinimod, an S100A9 inhibitor, alone and in combination with IRd for relapsed and refractory multiple myeloma (RRMM).

8042 Background: S100A9, a protein produced by myeloid-derived suppressor cells (MDSCs) in the bone marrow microenvironment, promotes multiple myeloma (MM) progression and confers therapeutic resistance. Tasquinimod (tasq), an oral S100A9 inhibitor, has pre-clinical anti-myeloma effects alone and combined with proteasome inhibitor (PI) and immunomodulator (Imid) therapy (Lin C, EHA 2020: EP896). Tasq previously improved PFS in prostate cancer patients (pts) (JCO 2016;34(22):2636-43). We are conducting a phase 1 trial (NCT04405167) of tasq as a single agent and in combination with ixazomib (ixa), lenalidomide (len), and dexamethasone (dex) (IRd) in pts with RRMM. Methods: We enrolled pts with RRMM refractory to, intolerant of, or with contraindication to len, pomalidomide (pom), bortezomib, carfilzomib (cfz), and a CD38 monoclonal antibody. Tasq was given in 28-day cycles as shown below, with a 3+3 dose escalation scheme. For combination therapy, pts received ixa (4 mg days 1/8/15), len (25 mg days 1-21, with adjustments for renal dysfunction), and dex (40 mg qwk). Results: Of 15 pts, median age was 70y (range 56-84); 53% were male; 27% were African American and 73% Caucasian. Pts had received a median of 8 prior lines of therapy (range 4-17), and all were refractory to ≥1 Imid, 87% to ≥1 PI, and 100% to a CD38 mAb, with 87% triple class refractory and penta-exposed. 10 pts received single-agent tasq at dose levels 1 (3 pts), 2 (3 pts), and 3 (4 pts). The most common treatment-emergent adverse events (TEAEs) were fatigue (all grade [gr] 1/2), anemia (5 pts, gr 3 in 2), anorexia (5 pts, gr 3 in 2), pain (5 pts, all gr 1/2), nausea/vomiting (4 pts, all gr 1/2), and neuropathy (3 pts, all gr 1/2). No dose-limiting toxicities (DLTs) were observed, but at dose level 3, 2 pts stopped for intolerable toxicities, and we identified dose level 2 as the single-agent MTD. 5 pts have received tasq with IRd at dose levels 1 (3 pts) and 2 (2 pts). Gr &gt; = 3 TEAEs were gr 4 thrombocytopenia in 1 pt (improved with dose reductions of ixa and len), gr 3 insomnia in 1 pt, and gr 5 COVID pneumonia in 1 pt. No tasq-related DLTs were observed. On single-agent tasq, 3 pts with progressive disease at study entry had stabilization of serum markers while on study. Of 5 pts on the IRd combination, 1 has had a partial response that is ongoing after 10 months, despite being previously refractory to ixa/pom and cfz/pom combinations. Conclusions: Tasquinimod, an S100A9 inhibitor, is well tolerated in pts with RRMM as a single-agent and in combination with IRd, with a single-agent MTD of 1 mg daily after a 1-week dose escalation. Tasq has anti-myeloma activity in combination with IRd, as evidenced by a partial response in a patient previously refractory to Imid/PI combination therapy. Enrollment continues to tasq with IRd at dose level 2. Clinical trial information: NCT04405167 . [Table: see text]

CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia.

Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells. Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells. The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.

Successful AAV8 re-administration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

A major challenge to AAV mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. Here we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 mAb to suppress anti-AAV NAbs and enable re-administration of AAV vectors of the same capsid in mice. An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted alkaline phosphatase was used for AAV re-administration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV re-administration was determined by the secretion of hSEAP in blood. In näive mice, an eight-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV re-administration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP re-administration. Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV mediated gene therapy.

Treg cell depletion in adult mice results in activation of antigen-presenting cells prior to fatal autoimmune disease

Abstract Studies in mice expressing the diphtheria toxin receptor (DTR) exclusively on regulatory T (Treg) cells (Foxp3-DTR mice) demonstrated the critical importance of Treg cells in maintaining normal immune homeostasis. Adult Foxp3-DTR mice injected with DT begin to die from day 10 after DT treatment. Marked expansion of almost all immune cell types was observed prior to death. The purpose of the present studies was to examine in-depth changes in lymphocytes and antigen-presenting cells at early time points after Treg depletion to determine the most critical cell types controlled by Treg in the steady state. Complete depletion of Treg was observed 2 days after treatment, but profound activation of CD4 +and CD8 +T cells as measured by induction of CD44 expression and vigorous proliferation as measured by Ki-67 incorporation were not seen on day 2 but seen on day 6 of DT treatment. Curiously, most of the CD44 hiKi-67 +T cells were CD25 −and plasma levels and intracellular levels of IL-2 were not increased suggesting that early activation of CD4 +and CD8 +T cells was not the result of a burst of IL-2 production. Increased expression of CD80, CD86, CD40, and PD-L1 could be detected on CD11c +dendritic cells (DCs) on day 6. Anti-CD80/CD86 mAbs reversed the activation of T cells provoked by depletion of Treg cells, while anti-IL-2 or anti-IL-2Rb mAbs had no effect. Taken together, these studies suggest that disruption of the regulation of Treg-mediated control of DC activation represents the first step in the complex autoimmune disease seen after Treg depletion. This work was supported by the Intramural Research Program of NIAID, NIH. This work was supported by the Intramural Research Program of NIAID, NIH.

Abstract 5677: MB101, a novel PD-L1 x CD3 targeted bispecific antibody, exhibits potent tumor-killing efficacy with minimal off-tumor toxicity

Abstract Bispecific antibody (bsAb) which targets tumor-associated antigens and simultaneously induces CD3-mediated T cell activation, is one of the promising therapeutic approaches for cancer treatment. However, despite numerous CD3-based bsAb have been developed, the clinical application faces pronounced hurdles, such as increased on-target off-tumor toxicity and cytokine release syndrome (CRS) by using high-affinity anti-CD3 moieties. Accordingly, there are still needs for the development of CD3-based bsAb with more favorable properties, which are tumor-specific targeting and potent tumor killing activity without excessive cytokine release. Thus, we developed MB101, a novel PD-L1 x CD3 bsAb with low affinity to CD3, which is uncoupled potent tumor cytotoxicity from severe cytokine release. MB101 showed a strong binding affinity to PD-L1 (KD = 0.4 nM) but a weak binding affinity to CD3 (KD = 1.0 μM), indicating preferentially targeting to PD-L1+ tumor cells. MB101 showed an effective tumor cell killing activity with higher Emax compared to avelumab mono- or combination treatment with CD3 mAb in PD-L1+ MDA-MB-231 cells. T cell-mediated cytotoxicity stimulated by MB101 in co-culture with target cells was accompanied by lower secretion of pro-inflammatory cytokines than CD3 mAb and bsAb comparator with high-affinity anti-CD3 arm. Additionally, in the absence of target cells, MB101 treatment to PBMC induced a very low level of pro-inflammatory cytokines, suggesting the possibility of reduced CRS risk. Furthermore, we proved that T cell-mediated cytotoxicity by MB101 was increased in a PD-L1 expression-dependent manner of tumor cells, but not normal cells, indicating minimal on-target off-tumor toxicity. Consistently with in vitro results, MB101 effectively regressed tumor volume (TGI=83%) without induction of excessive cytokine release in human CD3 knock-in mouse model. In addition, MB101 showed superior tumor growth inhibition (TGI=70%) than that of avelumab (TGI=44%) in humanized mouse xenograft model. In summary, MB101 selectively triggers robust PD-L1-dependent tumor cytotoxicity while inducing very low cytokine release, suggesting that MB101 might be a promising therapeutic option as CD3-based bsAb for cancer treatment. Citation Format: Suna Kim, Hyojoo Bang, Yongjun Jung, Sunjung Cho, Youngsam Kim, Sang-hyun Park, Seok Chan Kang, Youngjin Park, Sungyoub Jung. MB101, a novel PD-L1 x CD3 targeted bispecific antibody, exhibits potent tumor-killing efficacy with minimal off-tumor toxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5677.

Abstract 6308: Preclinical development of ABBV-319: a CD19-targeting glucocorticoid receptor modulator (GRM) agonist antibody-drug conjugate (ADC) for the treatment of B-cell malignancies

Abstract Introduction: Glucocorticoids are key components of standard-of-care treatment regimens (e.g., R-CHOP, Hyper-CVAD) for several B-cell malignancies. However, prolonged systemic glucocorticoid treatment results in glucocorticoid-associated adverse events and acquired resistance that limit its therapeutic potential. ABBV-319 is a novel CD19-targeting ADC engineered to reduce glucocorticoid-associated toxicity observed with systemic glucocorticoids while possessing three distinct mechanisms of action (MoA) to increase efficacy: 1) antibody-mediated delivery of GRM to activate glucocorticoid receptor (GR) induced cell death in cancer cells, 2) inhibition of CD19 signaling, and 3) enhanced Fc-mediated cancer cell killing via afucosylation of the antibody backbone. Results: We identified a GRM agonist that is 15 and 150 times more potent at driving GR transcriptional activation and cell death compared to clinical glucocorticoids dexamethasone and prednisolone, respectively. The conjugation of GRM agonist as the payload on ABBV-319 enables potent GRM-driven anti-cancer activity against malignant B-cell lines in vitro as well as in cell-line and patient derived xenograft (CDX and PDX) models in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic glucocorticoids (e.g., prednisolone) at its maximum tolerated dose in mice. The CD19 monoclonal antibody (mAb) also reduced proliferation of a subset of B-cell malignant cell lines through inhibition of the PI3K/AKT pathway. Moreover, afucosylation of the CD19 mAb in ABBV-319 enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payloads. ABBV-319 bound similarly to both V158 (high-affinity) and F158 (low-affinity) FcγRIIIa allotypes and mediated potent ADCC in co-culture assays with human peripheral blood mononuclear cells (PBMCs). Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ hematopoietic stem cell-engrafted NSG-Tg(Hu-IL15) mice, demonstrating that the three MoA (GR-driven cell death, CD19 signaling inhibition, and ADCC) collectively contribute to anti-tumor activity in vivo. ABBV-319 also displayed on-target depletion of normal human B-cells but did not affect peripheral NK cell counts in mice. Furthermore, CITE-seq profiling revealed that ABBV-319 treatment of human PBMCs activated GRM-induced signature genes restricted to B-cells, demonstrating the specificity of CD19-mediated delivery of GRM. Conclusion: ABBV-319 has potent anti-tumor activity from three distinct MoA and exhibits safety improvements compared to systemic glucocorticoids, supporting its recent progression into Phase I clinical trial. Citation Format: Chewei Anderson Chang, Ethan Emberley, Aloma L. D'Souza, Weilong Zhao, Cormac Cosgrove, Axel Hernandez Jr, Anatol Oleksijew, Paul Ellis, Luis Rodriguez, Gail Bukofzer, David Peetz, Wissam Assaily, Raghuveer Singh Mali, Wei Liu, Danqing Xu, Gregory K. Potts, Shaun McLoughlin, Kimberley McCarthy, Zhaomei Zhang, Jos Campbell, Isabella Sturdevant, Gloria Zhang, Tyler Curran, Jon D. Williams, Erwin Boghaert, Milan Bruncko, Christopher C. Marvin, Adrian Hobson, Michael Mcpherson, Tamar Uziel, Marybeth Pysz, Xi Zhao, Alex Bankovich, Kevin J. Freise, Susan Morgan-Lappe, James W. Purcell. Preclinical development of ABBV-319: a CD19-targeting glucocorticoid receptor modulator (GRM) agonist antibody-drug conjugate (ADC) for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6308.