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BackgroundADCs have emerged as a promising class of anticancer therapy. The choice of linker and payload in an ADC critically impacts its physiochemical properties, stability, PK/PD profile, and ultimately therapeutic index (TI) in the clinic. Monomethyl auristatin E (MMAE) is a clinically validated payload with multiple favorable attributes including broad and potent antitumor activity. On the other hand, high lipophilicity of MMAE drives the generally poor hydrophilicity and physiochemical properties of an ADC with a conventional linker such as vc-PAB. As a result, vc-PAB-MMAE (i.e. vedotin)-based ADCs are typically limited with low DAR (up to 4) and narrow TI. A novel, proprietary, protease-cleavable, and highly hydrophilic linker that can fully offset the high hydrophobicity of MMAE was developed (with the linker and MMAE collectively dubbed as LD343), and characterized in head-to-head comparisons with vedotin.MethodsPhysiochemical properties and additional characteristics of LD343-based ADCs were evaluated in a range of in vitro and in vivo studies with proprietary antibodies utilized as tool mAbs. Key in vitro analyses included HIC- and SEC-HPLC, bystander effect, and plasma stability. PK, efficacy, and tolerability were evaluated in mouse or rat models.ResultsLD343-based ADC (at DAR8) displayed high homogeneity and markedly improved hydrophilicity compared to the vedotin-based ADC (at DAR4) on HIC-HPLC, as well as excellent stability in plasma in vitro. LD343-based ADC produced robust bystander effect that was comparable to the vedotin-based ADC in a co-culture study with antigen-positive and negative tumor cells, suggesting no compromise on payload release from the hydrophilic linker. In the rat PK study, LD343 conferred stable PK of the ADC that is indistinguishable from the unconjugated parent mAb and superior to the vedotin-conjugate. In multiple cell-derived xenograft models in mice, LD343-based ADCs elicited sustained tumor regression that exceeded that of vedotin-based ADCs at the same or increased drug load. In the rat tolerability study, LD343 enabled an approximately 4-fold increase in the tolerated drug load, compared to vedotin, suggesting a simultaneous improvement in efficacy and tolerability.ConclusionsComprehensive preclinical studies demonstrated that the highly hydrophilic LD343 conferred markedly improved physiochemical properties and PK/PD characteristics associated with significantly expanded TI, compared to vedotin. LD343 is a novel and highly promising MMAE-based linker-drug platform that can broadly serve many attractive targets and antibody technologies; it represents a key contribution toward developing next-generation ADCs that may further fulfill high unmet medical need.

BackgroundSpatial transcriptomics and proteomics measurements enable high-dimensional characterization of tissues at subcellular levels, but understanding the larger-scale spatial organization of cells and extracting tissue structures of interest remain challenging tasks that require extensive human annotations. This challenge is particularly difficult in the context of cancers, where tissues structures are heterogeneous and may be defined by molecular subtypes, as well as morphological features.MethodsTo address this need for consistent identification of tissue structures, in this work, we present a novel annotation method Spatial Cellular Graph Partitioning (SCGP) that allows unsupervised identification of tissue structures that reflect the anatomical and functional units of human tissues. SCGP performs partitioning of tissue sections by conducting community detection on the graphical representations of tissue regions. Input graphs are constructed based on nodes that represent spatial units (e.g., cells) and edges connecting nodes that are spatially-close or share similar features. We further present a reference-query extension pipeline based on SCGP that enables the generalization of existing tissue structures to previously unseen samples by inserting pseudo-nodes representing reference partitions into the input graphs.ResultsOur experiments demonstrate reliable and robust partitioning on multiple datasets encompassing different tissue types including kidney, brain and skin sections. SCGP outperforms existing unsupervised annotation tools in recognizing compartments in kidney tissues with various degrees of diabetic kidney disease, and it achieves comparable accuracy in segmenting human brain sections as the state-of-the-art spatial transciptomics analysis tools. The extension pipeline is evaluated under a variety of conditions and is capable of addressing common challenges such as noise, artifact, batch effects, and phenotypic differences. Downstream analysis on SCGP-identified tissue structures reveals disease-relevant biological insights, underscoring its potential in facilitating biomedical research and driving new discoveries.ConclusionsWe demonstrate a rapid, unsupervised method to identify molecularly and spatially distinct tissue structures in spatial biology datasets. This method could aid in characterizing complex samples, such as tumor microenvironments, on multiple scales.

BackgroundGlioblastoma multiforme (GBM) is an incurable glial tumor affecting the central nervous system. The reported median survival and the percentage of patient surviving up to 2-years in the unfavorable patients’ subgroup with unmethylated (u)MGMT promoter is 12.7 months and less than then 15% (Stupp et al., 2009), respectively. Despite immunotherapies being able to slow or eradicate numerous tumors, even those metastasized to the brain, none so far have extended survival in GBM.MethodsWe have developed a personalized hematopoietic stem cell-based immunotherapy platform delivering immunotherapeutic payloads into the TME through a subset of tumor infiltrating macrophages. Specifically, Temferon has been designed to deliver IFN-α2 within the TME by Tie-2 expressing macrophages (TEMs). Temferon is currently under testing in an open-label, Phase 1/2a dose-escalation study (NCT03866109) evaluating its safety, and biological activity in up to 27 newly diagnosed GBM patients with uMGMT. Temferon is administered by ASCT after the RTx treatment without the concurrent administration of Temozolomide, whose survival benefit is known to be marginal in the uMGMT population.ResultsAs of June 2023, 4 incremental doses of Temferon (0.5–3.0 x106 cells/kg) have been tested across 21 patients (median age at enrolment 57) assigned to seven cohorts. Median Overall Survival (OS)) after 1st surgery is 15 months (5–40 months). The haematological recovery occurred in all the patients irrespective of dose administered. The percentage of transduced cells found in the BM, reached for the highest dose tested up to the 50% at 1 month and persisted at detectable level in the long-term. Very low median concentrations of IFNα were detected in the plasma, indicating tight regulation of vector expression. Notably, as predicted by TEMs biological behaviour, in the CSF the concentration of IFNa increased concomitantly to evidence of disease progression suggesting increase tumor recruitment of TEMs and subsequent release of IFNa . The 57% of the treated patients underwent a 2nd-line treatment (either pharmacological or surgical) with an interim survival rate at 2-years of 28% (5 of 18 patients; 3 patients excluded as follow-up is below 12 months), which is higher than that reported in literature (15%). One out of the surviving patients was enrolled in a long-term follow-up study and survived up to 3 years after surgery without any 2nd-line therapy added for 2 years.ConclusionsThese data provide initial evidence on Temferon potential to counteract disease progression and improve the survival of uMGMT GBM patients.

BackgroundThe novel T-MASK (Targeted Metallo/protease Activated SuperKine) platform involves fusion of a dual tumor-targeting/masking domain to potent immune modulator(s) via a metallo-protease (MMP) sensitive linker (PSL) to achieve the following objectives: (1) reduce/fine-tune the potency of the immune modulator by steric hindrance to increase systemic tolerability and (2) promote retention in the tumor microenvironment (TME) to maximize MMP cleavage and restore full potency at the intended target site. As proof of concept, we selected as the tumor-targeting/masking domain an IL-13 superkine (MDNA213) with high selectivity and affinity for the IL-13 decoy receptor IL-13Ra2, a tumor associated antigen expressed in many aggressive solid tumors. MDNA213 is fused via a PSL to MDNA11 and MDNA223, both containing a not-alpha, beta-enhanced IL-2 fused with albumin or anti-PD1 antibody respectively. We present preliminary results on characterization of both T-MASK constructs demonstrating conditional fine-tuning of IL-2R agonism.MethodsT-MASK optimization included assessment of PSL linker and orientation of the tumor-targeting/masking domain. In vitro IL-2 and PD-1/PDL-1 reporter assays were performed to evaluate IL-2R stimulation and anti-PD1 blockade respectively. In vitro MMP assay was used to validate cleavage of T-MASK constructs and restoration of full potency.ResultsT-MASK constructs showed approximately 10–40 fold reduction in potency compared to respective non-masked versions in IL-2R induced p-STAT5 reporter cell assay. Extent of fine-tuning can be adjusted by length and composition of PSL and orientation of the domains, providing versatility to the T-MASK platform and potential to engage the complete repertoire of peripherally circulating immune cells. In the case of MDNA223 (anti-PD1-IL-2Superkine), fusion of MDNA213 to generate the T-MASK construct MDNA113 resulted in reduced IL-2R agonism but had no effect on potency of PD1/PDL-1 blockade as expected. Unmasking of MDNA113 by MMP-mediated cleavage fully restored its IL-2R signaling activity to the same level as the non-masked MDNA223 construct. Similar data were obtained with the masked version of MDNA11, demonstrating robustness of the T-MASK platform. In vivo studies are ongoing to evaluate the effect of T-MASK constructs on peripheral immune cell expansion (systemic response), tumor retention (maximize activation) and tumor growth inhibition (targeted response).ConclusionsMDNA113 is a novel T-MASK construct designed to increase tolerability while leveraging the synergy between PD1/PDL-1 blockade and IL-2R agonism for immunotherapy. Ongoing studies investigate alternative tumor-targeting/masking domains and immune modulators, including other cytokines and potent therapeutic agents, to potentially broaden the utility of the T-MASK platform.Ethics ApprovalAnimal studies are conducted in concordance with institutional guideline and approval.

BackgroundThe results of adaptive immune receptor (AIR) repertoire diversity assays can be affected by various biases from differences in conditions in the RT-PCR and NGS sequencing steps. Spike-in synthetic controls can be used as calibration standards to address these biasesMethodsIn this study, we synthesized near full-length BCR and TCR constructs that mimic seven different IGH, IGK, IGL, TRB, TRA, TRG and TRD genes. To test our spike-in controls, three sets of variants at different concentrations were added to the RNA samples before the reverse transcription reaction with Cellecta’s DriverMap™ Adaptive Immune Receptor (AIR) Profiling Assay. The DriverMap™ protocol uses reverse gene-specific primers with unique molecular identifiers (UMI), allowing UMI-based correction of amplification biases during data analysisResultsCalculating UMI-based correction and comparing that with spike-in controls enabled us to differentiate between real and background sequences and estimate the average sequencing error rate at 0.4%-0.8% per base, which is within the reported range of Illumina sequencing.ConclusionsThis suggests that our spike-in controls are reliable and may be used as a universal tool to correct AIR protocol biases and calculate error and mutation rates in different AIR profiling assays.

BackgroundBispecific T Cell Engagers (BiTEs), consisting of an anti-CD3 scFv fused to an anti-tumor antigen scFv, are highly effective in the treatment of relapsed/refractory Acute Lymphoblastic Leukemia (ALL). However, the short half-life of BiTEs necessitates continuous intravenous administration at high doses for four-week increments. To overcome these pharmacokinetic shortcomings, we developed a method to engineer plasma cell precursors to continuously secrete transgenic biologics. Plasma cells were chosen for their high antibody production capacity (thousands of Ig molecules/cell/sec) and long-term survival (persisting for decades), making them a highly attractive cell-based platform for continuous biologic delivery.1 MethodsTo demonstrate proof-of-concept, we integrated a transgene coding for a bispecific non-Ig anti-CD3:CD19 scFv into the CCR5 safe-harbor locus of primary human B cells with CRISPR/Cas9, then initiated differentiation into plasma cells using a modified feeder-free culture system. Cells were characterized by flow cytometry, indel frequency, and droplet digital PCR. Edited cell supernatant was tested for therapeutic protein production with a timed ELISA and in vitro function with a cytolytic activity assay incorporating co-cultured T effector and CD19 expressing tumor cells. We also assessed in vivo anti-tumor activity of anti-CD3:CD19 scFv engineered B cell medicines (BeCMs) in NSG mice harboring a patient-derived xenograft (PDx). To avoid targeting of the BeCM by CD3+ cells, CD19 was knocked-out via a multiplexed CRISPR/Cas9 editing protocol. Mice were then inoculated with a luciferized B-ALL PDx line. Autologous T cells were delivered 24 hours and 72 hours following tumor transfer, and IVIS imaging was performed over the course of 17 days. A control GFP-engineered BeCM arm and PBS-dosed cohort were monitored in parallel, with n=6 mice per group.ResultsFlow cytometric analysis confirmed robust differentiation of BeCMs along the plasma cell lineage. We observed >85% cutting efficiency and 40–50% targeted integration, which translated to secretion rates (0.6–0.8 μg BiTE/106 cells/day) that were sufficient for in vitro functional assay tumor cytolytic activity. Significant reduction in tumor burden (bioluminescent flux, area under the curve) was observed in vivo in the anti-CD3:CD19 scFv cohort compared to the controls, which was in concordance with heightened in vivo T cell activation. The ~1000 pg/mL BiTE detected in mouse plasma demonstrates that BeCM-derived biologics can meet or even exceed the steady-state plasma concentrations achieved with clinically relevant doses.2 ConclusionsThese findings underscore the clinical potential of BeCMs as an emerging platform for sustained delivery of anti-tumor biologics.ReferencesHung KL, Meitlis I, Hale M, Chen C, Singh S, Jackson SW, Miao CH, Khan IF, Rawlings DJ, James RG. Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. Mol. Ther. 2018; 26:456–467.Franquiz MJ and Short NJ. Blinatumomab for the Treatment of Adult B-Cell Acute Lymphoblastic Leukemia: Toward a New Era of Targeted Immunotherapy. Biologics. 2020; 14: 23–34.Ethics ApprovalDeidentified human PBMCs were acquired under informed consent from the Fred Hutch Specimen Processing and Research Cell Bank (protocol #3942).

BackgroundICT01 is a first-in-class anti-BTN3A mAb that selectively activates γ9δ2 T cells, leading to remodeling of the tumor microenvironment by activated γ9δ2 T, CD8 T, and NK cells (EVICTION-NCT04243499). However, many cancer patients have very low circulating γ9δ2 T cells that limit their response to ICT01. In the EVICTION-2 trial, a novel regimen of ICT01 plus low dose subcutaneous (LDSC) IL-2 is being investigated in patients with advanced-stage solid tumors to increase the number of γ9δ2 T cells that generate a more efficacious anti-tumor immune response.MethodsICT01 (1, 5, 20 or 75 mg, IV Q3W) is given in combination with IL-2 (Proleukin®, 1 or 2 MIU/m2, SC) on days 1–5 of the first 3 cycles and will be continued alone thereafter. Per dose combination two patients are enrolled for dose escalation based on the BOIN simulations to identify a dose regimen that safely expands γ9δ2 T cells, which will be expanded to 6 pts for recommended phase 2 dose regimens. The study received ethics approval for all sites involved.ResultsNineteen patients have completed at least one cycle of ICT01 plus IL-2. Treatment-related adverse events were mainly mild to moderate infusion-related reactions, comparable to those observed with ICT01 or IL-2 monotherapy. No dose-limiting toxicities were reported. A 2–11x increase of γ9δ2 T cell counts above baseline was observed for all 3 cycles across all cohorts peaking around day 8 to 15, which appeared greater at low doses where ICT01 was rapidly cleared, while prolonged target occupancy/activation by high doses prevented γ9δ2 T cells from remaining in the circulation. Activation, mobilization, and proliferation of CD8 T cells (2–3x) and NK cells (2–9x) cycles was similarly observed. Elevated levels of IFNγ, TNFα, IL-6 and IL-8 peaked at ~4 hours post ICT01/IL-2 dose that returned to baseline despite expansion of γ9δ2 T cells. Increased Tregs by flow cytometry appear to be greater at lower doses of ICT01, which may be similar to effects observed in NHPs. Response data by RECIST1.1 every 8 weeks and IHC of tumor biopsies collected at baseline and on Day 28 will be presented.ConclusionICT01 plus LDSC IL-2 produces a broad anti-tumor immune response that is durable across multiple treatment cycles, which appears different to prior attempts to expand g9d2 T cells with bisphosphonates or phosphoantigens.

BackgroundImmune checkpoint blockade (ICB) therapies including anti-CTLA4 and anti-PD1/L1 antibodies have revolutionized cancer treatment landscape by eliciting durable remission.1 However, despite early response, most cancer patients eventually become resistant to the ICB therapies. The DNAM1 axis, an additional immunomodulatory pathway, is a potent regulator of innate and adaptive immunity.2 The immunoglobulin receptors TIGIT and PVRIG, which interact with their ligands PVR and PVRL2 in DNAM1 axis respectively, are reported to contribute to primary or acquired resistance to PD-1/L1 blockade. Here, we report the internally discovered D3L-002, an anti-TIGIT×PVRIG bispecific antibody (bsAb), bound to TIGIT and PVRIG simultaneously and effectively blocked the interaction between TIGIT/PVR and PVRIG/PVRL2. D3L-002 could restore the function of exhausted T/NK cells and result in potent anti-tumor activity.MethodsThe affinity of D3L-002 was examined by surface plasmon resonance (SPR) method. Cellular binding and in vitro blocking assays were measured by FACS or ELISA. T cell activation was detected via TCR/NFAT signaling and IFN-γ cytokine secretion. NK cell function was examined by degranulation and target cell lysis. Antibodies’ efficacy was studied in syngeneic mouse tumor models.ResultsD3L-002, a IgG1 tetravalent bsAb with symmetric IgG-single-chain variable fragment (scFv) structure, showed high binding affinity to the extracellular domain of human TIGIT (KD < 1 nM) and PVRIG (KD < 10 nM) respectively. D3L-002 bound to human TIGIT and PVRIG expressing cells with sub-nanomolar EC50 and was cross-reactive to respective cynomolgus homologs. Moreover, it demonstrated strong TIGIT/PVR and PVRIG/PVRL2 receptor-ligand interaction blocking activities in both cellular and protein level assays. This potent blocking translated well into enhanced functions in vitro. D3L-002 was able to activate TCR/NFAT signaling in Jurkat cells and showed better activity than anti-TIGIT and anti-PVRIG parental mAb alone, or their combination. D3L-002 could also enhance degranulation and cytotoxicity of primary NK cells and reinvigorate exhausted CD8+ T cells. In MC38 model, D3L-002 showed a trend of better anti-tumor efficacy than parental anti-TIGIT (P>0.05) and anti-PVRIG mAb (P<0.01). In addition, the combination of D3L-002 with anti-PDL1 mAb showed more potent efficacy than Atezolizumab monotherapy (P<0.01). MoA study indicated that D3L-002 monotherapy treatment depleted Treg, while combination with Atezolizumab induced CD8+ T cell proliferation in tumor microenvironment.ConclusionsD3L-002 is a novel TIGIT×PVRIG bsAb which demonstrated potent anti-tumor effect via TIGIT and PVRIG co-blocking in both in vitro and in vivo models. D3L-002 might provide a novel treatment approach for solid tumors and overcome resistance to current ICB therapies.ReferencesSharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348(6230):56–61.Alteber Z, Kotturi MF, Whelan S, Ganguly S, Weyl E, Pardoll DM, Hunter J, Ophir E. Therapeutic targeting of checkpoint receptors within the DNAM1 axis. Cancer Discov. 2021;11:1–13.

BackgroundLymphocyte-activated gene 3 (LAG-3) is an immune checkpoint receptor protein that negatively regulates T-cell activation. The objective of this study (ClinicalTrials.gov identifier: NCT05909436) was to evaluate the safety, tolerability, and preliminary efficacy of GLS-012, a LAG-3-blocking antibody, monotherapy and in combination with zimberelimab (anti-PD-1 mAb) in patients with advanced melanoma after progression on standard treatment.MethodsThe dose escalation stage of this study has a traditional 3 + 3 dose escalation design with planned cohorts of fixed-dose GLS-012 with 40, 80, 240, 480, 800 mg Q3W and followed by a dose escalation of combination therapy with less than and equal to the recommended phase 2 dose (RP2D) of GLS-012 and a fixed-dose zimberelimab with 360 mg Q3W. DLT observation period was 21 days after the first dose. The primary endpoints were the number of patients with a dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in the dose escalation stage. In dose expansion stage, patients received intravenous 240mg GLS-012 Q3W as monotherapy, and were assigned to receive RP2D GLS-012 combined with a fixed-dose 360 mg zimberelimab Q3W as combination therapy. The primary endpoint was safety in the dose expansion stage.ResultsAs of May 18th, 2023, 17 patients with advanced melanoma after progression on standard treatment (median age: 55 yr [range: 33–75]; ECOG PS: 0 [n = 12], 1 [n = 5]) were enrolled for GLS-012 monotherapy dose escalation and dose expansion. The median number of medications was 4 (range: 1~9). All the 13 patients in the dose escalation cohorts have completed the first cycle of treatment (DLT observation period). No DLT was observed, and MTD was not reached. The most common treatment-related adverse event (TRAE) was decreased white blood cell count which happened in 2 patients. No Grade ≥3 TRAE was observed. By the investigator-assessment per RECIST 1.1, in 14 evaluable patients, stable disease (SD) was observed with 8 patients.ConclusionsGLS-012 has acceptable toxicity and shows preliminary antitumor activity in patients with advanced melanoma after progression on standard treatment.Trial RegistrationClinicaltrials.gov identifier: NCT05909436Ethics ApprovalThe study (Triumph-01) was approved by the ethics committee of Beijing Cancer Hospital (approval number: 2022YW114). All participants provided signed informed consent before any study procedure.

BackgroundHigh-throughput screening (HTS) programs are increasingly adopting high-content technologies that can better inform the selection of drug candidates early on in the pipelines. For cancer immunotherapy, proteomics tools to investigate interactions between cancer and immune cells compromise either content or cost, limiting access to phenotypic data. The affordable gold-standard in proteomics, the ELISA, has proven difficult to scale. At fault has been the cross-reactivity between ELISA reagents when multiplexing beyond a few dozen antibody pairs. Here, we describe the nELISA: a massively-parallelized high-throughput miniaturized ELISA with a content, cost and throughput amenable to HTS, and demonstrate its applicability to characterize immune phenotypes in co-culture systems.MethodsTo overcome the long-standing cross-reactivity issue, the nELISA uses DNA oligos to pre-assemble each pair of antibodies onto a spectrally barcoded microparticle set. The resulting reagents are fully-integrated nELISA sensors that can be read-out on commercial cytometers, enabling highly-multiplexed and high-throughput analysis. Using this approach, we developed a comprehensive inflammatory panel containing 191 cytokines, chemokines, proteases, growth factors, and soluble receptors. Our results show that the nELISA can maintain single-plex specificity, sensitivity, and quantification as content is scaled to 191-plex. Furthermore, the nELISA performs at a throughput of 1536 samples/cytometer/day, yielding >300,000 data points in a single day, at a cost amenable to high-throughput screening.ResultsTo demonstrate the nELISA’s utility in HTS, we ran the largest PBMC secretome screen to date, in which >7000 PBMC samples were treated with various inflammatory stimuli, and further perturbed with a selected library of 80 recombinant protein ‘perturbagens’. 191 secreted proteins were profiled in all samples, resulting in ~1.4M datapoints (figure 1A). The nELISA profiles were able to capture phenotypes associated with specific stimulation conditions, individual donors, and potent cytokine perturbagens. By compensating for stimulation and donor differences, we clustered perturbagens according to their effects on PBMC secretomes, identifying well-established cell responses such as Th1 or Th2. Novel phenotypic effects were also identified, such as distinct responses to the near identical CXCL12 alpha and beta isoforms (figure 1B). Interestingly, we observed important similarities between PBMC responses to the cytokine drugs IFN beta and IL-1 Receptor antagonist, supporting the use of anakinra as a replacement for IFN beta in certain indications.ConclusionsThe nELISA captures broad secretome ranges and subtle differences in immune phenotypes, revealing critical insights in cell-based screens. Thus, the nELISA is a powerful new tool for cancer immunotherapy assays, including phenotypic screening, target identification/deconvolution, and discovery of markers of target engagement.Abstract 61 Figure 1High-throughput screen of PBMC responses demonstrates the use of the nELISA for drug discovery. (A) Screen design: PBMCs isolated from six donors were treated with inflammatory stimuli at indicated concentrations, and further perturbed with 80 recombinant cytokine \"perturbagens\", generating a total of 7,392 samples; after 24 hours, concentrations of 191 secreted proteins were measured in the supernatant of each sample using the nELISA. (B) UMAP dimensionality reduction of the entire nELISA dataset; datapoints are colored (from left supernatant of each sample using the nELISA. (B) UMAP dimensionality reduction of the entire nELISA dataset; datapoints are colored (from left to right by stimulation condition, by donor, by stimulation concentration, or by individual cytokind perturbagens with strong effects, as indicated.