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SummaryDespite pre-clinical murine data supporting T regulatory (Treg) cell depletion as a major mechanism by which anti-CTLA-4 antibodies function in vivo, the two main antibodies tested in patients (ipilimumab and tremelimumab) have failed to demonstrate similar effects. We report analogous findings in an immunocompetent murine model humanized for CTLA-4 and Fcy receptors (hCTLA-4/hFcyR mice), where both ipilimumab and tremelimumab fail to show appreciable Treg depletion. Immune profiling of the tumor microenvironment (TME) in both mice and human samples revealed upregulation of the inhibitory Fcy receptor, FcyRIIB, which limits the ability of the antibody Fc fragment of human anti-CTLA-4 antibodies to induce effective antibody dependent cellular cytotoxicty/phagocytosis (ADCC/ADCP). Blocking FcyRIIB in humanized mice rescues Treg depleting capacity and anti-tumor activity of ipilimumab. For another target, CC motif chemokine receptor 8 (CCR8), which is selectively expressed on tumor infiltrating Tregs, we show that Fc engineering to enhance binding to activating Fc receptors, while limiting binding to the inhibitory Fc receptor, leads to consistent Treg depletion and single-agent activity across multiple tumor models, including B16, MC38 and MB49. These data reveal the importance of reducing engagement to the inhibitory Fc receptor to optimize Treg depletion by TME targeting antibodies. Our results define the inhibitory FcyRIIB receptor as a novel immune checkpoint limiting antibody-mediated Treg depletion in tumors, and demonstrate Fc variant engineering as a means to overcome this limitation and augment efficacy for a repertoire of antibodies currently in use or under clinical evaluation in oncology.Highlights-Fully human anti-CTLA-4 antibodies are limited in their capacity to deplete T regulatory cells and drive durable anti-tumor immunity in humanized FcyR/hCTLA-4 mice-The inhibitory Fcy receptor, FcyRIIB, is upregulated in the tumor microenvironment in patients and in humanized FcyR/hCTLA-4 mice-Blocking FcyRIIB leads to rescue of Treg depletion in humanized murine models-Fc engineering can improve the depleting capacity and in vivo anti-tumor activity of anti-CTLA and anti-CCR8 antibodies targeting tumor infiltrating Tregs

Central diabetes insipidus (CDI) is an uncommon complication of acute myeloid leukemia (AML). Patients present with polyuria either preceding or at the time of diagnosis of AML. We describe the case of a 36-year-old male who presented with a subacute onset of polyuria, polydipsia, nocturia, and fatigue. After an extensive workup, he was diagnosed with AML/CMML (chronic myelomonocytic leukemia) with a normal karyotype with DNMT3A, CBFB, and PTPN11 mutations. Further workup of the polyuria with a water deprivation test helped confirm the diagnosis of CDI along with MRI findings suggestive of hypophysitis. In this report, we analyze the clinical workup for AML and CDI and report the possibility of extramedullary leukemic infiltration associated with DNMT3A mutation, which has been reported as one of the mechanisms in the existing literature. We also discuss other theories hypothesized to cause CDI in AML patients with abnormal karyotypes. Our patient progressed to AML from CMML-2 after a cycle of decitabine, with confirmed gingival and presumed central nervous system (CNS) involvement. He is in minimal residual disease (MRD)-negative complete remission after induction with a CNS-active acute lymphoblastic leukemia-2 regimen. He also received double umbilical cord blood transplantation, conditioned with cyclophosphamide, fludarabine, thiotepa, and total body irradiation (TBI) of 4 Gy. This was complicated by engraftment syndrome for which he is currently being managed. CDI of the patient was corrected by desmopressin administration.

BACKGROUND D2C7-IT, a novel immunotoxin-based cytotoxic therapy, targets epidermal growth factor receptor (EGFR) and mutant EGFR variant III. In preclinical studies, D2C7-IT kills tumor cells and prolongs survival, but is unable to generate cures in all animals. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. Eliminating glioblastoma immunosuppression via CD40 co-stimulation is anticipated to enhance D2C7-IT-induced antitumor responses. In murine glioma models, CED of D2C7-IT+αCD40 generated cures and long-term tumor-specific adaptive immunity. Hence, we are conducting a phase 1 trial of D2C7-IT+2141-V11 (αhuman-CD40) administered via CED in rMG patients. METHODS Eligibility includes adult patients with solitary supratentorial rMG (WHO grade 3/4); ≥ 4 weeks after chemotherapy, bevacizumab, or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing doses of 2141-V11 to determine the maximum tolerated dose when administered sequentially following D2C7-IT (166,075 ng) via CED at 0.5 mL/hr. Five dose levels (DLs) are evaluated (2141-V11 at: #1: 0.70 mg; #2: 2.0 mg; #2**: 3.0 mg #2*: 4.0 mg; #3: 7.0 mg). RESULTS As of May 29, 2022, 13 patients were treated (3 patients on DL1, DL2, and DL2**; 2 patients on DL3 and DL2*). No dose-limiting toxicities were observed; however, lower DLs were added due to higher frequency of adverse events (AEs) expected with D2C7-IT+2141-V11 with DL3 and DL2* (fever, neurologic symptoms). All patients remain alive 0.7-10.6 months after therapy. Grade ≥ 2 AEs due to D2C7-IT+2141-V11 include: headache (grade 3, n = 1; grade 2, n = 4); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1); dysphasia (grade 3, n = 1); seizures (grade 2; n = 2); fever (grade 2; n = 2); and one each of grade 2 depressed level of consciousness, fatigue, and concentration impairment. Enrollment is ongoing. CONCLUSIONS Intratumoral administration of D2C7-IT+2141-V11 via CED is safe, encouraging efficacy results are observed.

e14015 Background: D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin comprising an EGFR wild-type and mutant-specific (EGFRvIII) monoclonal antibody (Ab) fragment and a genetically engineered form of the Pseudomonas exotoxin. When injected directly into the tumor by convection enhanced delivery (CED), immunotoxins induce both direct tumor killing and secondary immune responses by activation of CD4+ and CD8+ T-cells. Tumor-associated macrophages (TAMs) are the most prominent glioma-infiltrating immune cells and constitute up to 40% of the tumor mass. Upon binding of D2C7-IT to EGFR and cellular internalization, the Pseudomonas exotoxin moiety of the D2C7-IT kills residual GBM cells, upregulates proinflammatory CD40, and induces pattern recognition receptor pathway transcriptome expression. This potentially creates a proinflammatory glioma microenvironment where TAM activation may be further stimulated by sequential CED of 2141-V11, an Fc engineered anti-human CD40 agonist antibody developed at Rockefeller University. We are conducting a first in human trial of the combination of D2C7-IT + 2141-V11 administered via CED in recurrent MG patients. Methods: Eligibility includes adult patients with recurrence of a solitary supratentorial WHO grade 3 or 4 MG; ≥ 4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing levels of 2141-V11 to determine the maximum tolerated dose (MTD) of the compound administered intratumorally in conjunction with D2C7-IT. Dose escalation and de-escalation are managed using a modified Bayesian optimal interval (BOIN) design to identify the MTD. Intratumoral administration of D2C7-IT via CED (4612 ng/mL over 72 hours) is followed by a 7-hour infusion of 2141-V11, both infused at 0.5 mL/hr. 2141-V11 is dose-escalated to determine the MTD when combined with D2C7-IT. Four dose levels (DL) are planned: #1: 0.70 mg; #2: 2.0 mg; #3: 7.0 mg; #4: 21.0 mg. Results: As of February 7, 2022, three patients were treated at DL1 and DL2, and two patients at DL3. No DLTs have been observed, and all eight patients remain alive and in observation on study after 7.0, 6.5, 6.0, 4.4, 2.8, 2.4, 0.9 and 0.5 months. Early signs of tumor response have been observed, with one patient at DL1 and 2 patients at DL2 without radiographic evidence of active tumor. Grade 2 or higher AEs due to D2C7-IT and/or 2141-V11 include: headache (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1; grade 2, n = 1); dysphasia (grade 3, n = 1); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 1); and depressed level of consciousness (grade 2, n = 1). Enrollment is ongoing. Conclusions: Intratumoral administration of D2C7-IT + 2141-V11 via CED is safe, and encouraging efficacy results have been observed. Clinical trial information: NCT04547777.

TPS4616 Background: CD40 is an immune-stimulatory receptor centrally involved in activating antigen-presenting cells and downstream anti-tumor immunity. Multiple agonistic anti-CD40 antibodies have been investigated clinically for cancer therapy, but have thus far shown limited efficacy and notable systemic toxicity. Our group has previously demonstrated that the ability of the Fc domain of anti-CD40 agonist antibodies to engage the FcγRIIB receptor, needed for optimal CD40 crosslinking, is critical for CD40 agonist activity. We engineered a fully-human anti-CD40 agonist antibody (2141-V11) with optimized Fc binding to FcγRIIB, which demonstrates significantly enhanced CD40 agonist activity. Non-muscle invasive bladder cancer (NMIBC), which remains an area of significant unmet need, represents an attractive clinical context to pursue CD40 agonist therapy given strong enrichment of CD40 in the bladder tumor microenvironment and routine use of local intravesical delivery as a potential strategy to mitigate systemic toxicity. Our preclinical studies, including in immunocompetent orthotopic murine models of bladder cancer humanized for CD40 and Fcγ receptors, revealed that intravesical delivery of 2141-V11 induced potent and durable anti-tumor immunity in both front-line and BCG-unresponsive settings without associated systemic toxicity (Garris and Wong, et al., Sci Transl Med, 2021;13:eabd1346). These data provide rationale to investigate intravesical 2141-V11 for the treatment of BCG-unresponsive NMIBC. Methods: This is a phase 1, open-label, dose-escalation study (NCT05126472) to evaluate the safety and tolerability of intravesically-delivered 2141-V11 in patients with BCG-unresponsive NMIBC who are considered ineligible for or have elected not to undergo radical cystectomy. Key inclusion criteria include adults with high-grade NMIBC (Ta, CIS, and/or T1) of urothelial histology that is unresponsive to adequate BCG therapy per consensus 2018 FDA guidelines. The primary objectives are to evaluate the safety and tolerability of 2141-V11 and to define the maximum tolerated dose and/or recommended phase 2 dose. The secondary objectives are to characterize the pharmacokinetics and evaluate the preliminary clinical activity of 2141-V11. Exploratory objectives include assessment of biomarkers of biological activity and disease resistance, their potential associations with clinical outcome measures, and patient-reported outcomes/quality of life measures. The 2141-V11 antibody is administered intravesically once weekly for 3 doses. Depending on disease status at week 13 and week 25 evaluations, patients may be eligible for re-treatment (once weekly for 3 doses) at these time points. Dose exploration utilizes a modified continual reassessment method design. Enrollment began in November 2021 and is ongoing. Clinical trial information: NCT05126472.

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase-2) is critical for tumor necrosis factor (TNF) α-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory post-synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase-2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA-evoked calcium flux and excitatory post-synaptic currents.

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that play a role in the inflammatory response. These enzymes have been well studied in the context of cancer biology and inflammation. Recent studies, however, suggest that these enzymes also play roles in brain development and neurodegenerative disease. Select MMPs can target proteins critical to synaptic structure and neuronal survival, including integrins and cadherins. Here, we show that one member of the MMP family, MMP-7, which may be released from cells, including microglia, can target a protein critical to synaptic function. Through analysis of extracts from murine cortical slice preparations, we show that MMP-7 cleaves the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor to generate an N-terminal fragment of ∼65 kDa. Moreover, studies with recombinant protein show that MMP-7-mediated cleavage of NR1 occurs at amino acid 517, which is extracellular and just distal to the first transmembrane domain. Data suggest that NR2A, which shares sequence homology with NR1, is also cleaved following treatment of slices with MMP-7, while select AMPA receptor subunits are not. Consistent with a potential effect of MMP-7 on ligand binding, additional experiments demonstrate that NMDA-mediated calcium flux is significantly diminished by MMP-7 pretreatment of cultures. In addition, the AMPA/NMDA ratio is increased by MMP-7 pretreatment. These data suggest that synaptic function may be altered in neurological conditions associated with increased levels of MMP-7.—Szklarczyk, A., Ewaleifoh, O., Beique, J.-C., Wang, Y., Knorr, D., Haughey, N., Malpica, T., Mattson, M. P., Huganir, R., Conant, K. MMP-7 cleaves the NR1 NMDA receptor subunit and modifies NMDA receptor function.

We report on efforts to develop a new airport arrival efficiency metric that takes into account capacity constraints at the arrival airport. One of the new Air Traffic Organization’s (ATO) system-wide metrics is the Airport Arrival Efficiency Rate (AAER). While the current AAER is a good system measure, a gauge for performance at and near the airport is needed. In our proposed methodology, aircraft are assigned to available slots at an airport based on their unimpeded ETAs, which has the effect of delaying the actual ETA of a flight if the demand is greater than the airport capacity. An efficiency score is computed for an airport based on the fraction of assigned slots that are not filled in a time period, and we show that this score is sensitive to the actual airport performance.