Potent Cell Killing Research Articles

Abstract C115: Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate

Abstract STRO-002 is a novel folate receptor alpha (FolRα)-targeting antibody-drug conjugate (ADC) currently undergoing evaluation in clinical trials at various phases of development in ovarian and endometrial cancer and pediatric AML. Here, we describe the favorable properties of the STRO-002 ADC and summarize its activity in preclinical models. STRO-002 is an ADC composed of an anti-FolRα antibody conjugated to four tubulin-targeting hemiasterlin warheads. Incorporation of non-natural amino acids, using Sutro’s XpressCF® and XpressCF+® cell-free expression and conjugation systems, allows for site-specific conjugation of the hemiasterlin payload, yielding a homogenous and high-fidelity ADC. As an ADC, STRO-002 internalizes rapidly into FolRα+ tumor cells and exhibits potent target-dependent cell killing on xenograft tumor cell lines. Further characterization of STRO-002 and the hemiasterlin payload reveals significant ADC bystander activity and a lower sensitivity to drug efflux via the P-gp drug pump compared to other microtubule-targeting warheads. In addition to robust direct and bystander cell killing, STRO-002 is also capable of triggering immunogenic cell death (ICD). In in vitro cell killing assays, STRO-002 induces all three hallmarks of ICD—HMGB1 and ATP release and surface exposure of calreticulin—and in in vivo vaccination studies, injection of STRO-002-killed tumor cells was effective at establishing a protective immune response against subsequent tumor challenge, identifying STRO-002 as a bonafide ICD inducer.In vivo, STRO-002 treatment demonstrates robust efficacy in ovarian xenograft models, even when treatment is initiated in large, established tumors. In addition, combination therapy with STRO-002 and carboplatin, anti-VEGF, or anti-PD-L1 results in greater efficacy than treatment with single agents alone, demonstrating combination benefit with current standard-of-care therapies. To better understand the activity of STRO-002 in more-translatable preclinical models, we turned to patient-derived xenografts (PDX) and found that STRO-002 exhibits good anti-tumor activity in endometrial cancer and non-small cell lung cancer PDX studies, suggesting potential clinical benefit in these indications. In conclusion, STRO-002 exhibits multiple advantageous properties as an ADC and shows good anti-tumor activity, either in combination or as a single agent, in both cell-derived xenograft and PDX models. Citation Format: Robert Yuan, Andrew McGeehan, Sihong Zhou, Jennifer Smith, Dayson Moreira, Krishna Bajjuri, Cuong Tran, Gang Yin, Alice Yam, Helena Kiefel, Xiaofan Li. Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C115.

Abstract A044: Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies

Abstract Background: CD38 is a transmembrane protein that is often overexpressed in multiple myeloma (MM) and other hematologic cancers. The success of anti-CD38 monoclonal antibodies, like daratumumab and isatuximab, in the treatment of MM supports the potential of CD38-targeting therapy candidates in development, including chimeric antigen receptor (CAR) engineered natural killer (NK) cells. Because CD38 is expressed on normal hematopoietic cells and other tissues, it is important to assess the affinity and epitope-specificity of the CD38-targeting moiety to limit potential on-target/off-tumor toxicities. To determine how different biophysical characteristics impact CAR activity, we identified a diverse panel of CD38-specific antibodies, which were shown to display a wide range of affinities and epitopes towards CD38 and converted these clones into CARs to evaluate their expression and target-specific and non-specific activation in a high-throughput (HTP) CD38-knockout (CD38 KO) Jurkat screen. Methods: Antibodies against CD38 were identified through hybridoma screening. Humanized mice were immunized with the extracellular domain of CD38, and lymphocytes from lymph nodes were fused with SP2/0 cells to generate hybridoma cells. Supernatants from single cell sorted hybridoma clones were screened against CD38+ and CD38 KO Jurkat cells and were assessed for affinity and epitope via surface plasmon resonance detection with the Carterra LSA. 96 unique clones were converted into CARs and transduced into CD38 KO Jurkat cells. CAR-expressing Jurkat cells were assayed for CAR expression and activation (via CD69 expression) when cultured overnight in the presence or absence of MM.1R target cells. Seven CARs were selected for additional characterization in primary NK cells. NK cells from healthy donors were expanded on our proprietary NKSTIM cell line, knocked out for CD38 using a CRISPR-Cas system, and transduced with CD38 CAR constructs. CD38 CAR NK-mediated cytotoxicity against MM.1S target cells was assessed by IncuCyte® S3 live cell analysis system. Results: Hybridoma screening identified many unique CD38-specific antibodies, displaying a wide range of affinities and epitopes towards CD38. Despite these biophysical differences, many of these antibodies, when converted to CARs, confer target-specific activity. Multiple epitopes on CD38 may be targeted and even low affinity antibodies were sufficient for target-specific CAR activation. Furthermore, the seven selected CAR candidates that were expressed in CD38 KO donor NK cells displayed potent in vitro target cell killing against MM.1S target cells. Conclusions: In summary, we have identified and characterized a range of CD38-specific antibodies. When converted into CARs, these targeting moieties mediate target-specific activation, despite differences in CD38 epitope and in affinity, demonstrating the utility of HTP CAR screening. Moreover, expression of the top seven CAR candidates in CD38 KO NK cells resulted in potent target cell killing, supporting further pre-clinical evaluation in relevant models. Citation Format: Emily N Kang, Jacinda T Chen, Bing Li, Kate Jamboretz, Nitin Patel, Daniel Bedinger, Kyle T Pratt, Jessica Hsieh, Luxuan TE Buren, Chao Guo, Ivan Chan, James B Trager, Sasha Lazetic. Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A044.

IL-21-armored B7H3 CAR-iNKT cells exert potent antitumor effects

CD1d-restricted invariant NKT (iNKT) cells play a critical role in tumor immunity. However, the scarcity and limited persistence restricts their development and clinical application. Here, we demonstrated that iNKT cells could be efficiently expanded using modified cytokines combination from peripheral blood mononuclear cells. Introduction of IL-21 significantly increased the frequency of CD62L-positive memory-like iNKT cells. iNKT cells armoring with B7H3-targeting second generation CAR and IL-21 showed potent tumor cell killing activity. Moreover, co-expression of IL-21 promoted the activation of Stat3 signaling and reduced the expression of exhaustion markers in CAR-iNKT cells invitro. Most importantly, IL-21-arming significantly prolonged B7H3 CAR-iNKT cell proliferation and survival invivo, thus improving their therapeutic efficacy in mouse renal cancer xerograph models without observed cytokine-related adverse events. In summary, these results suggest that B7H3 CAR-iNKT armored with IL-21 is a promising therapeutic strategy for cancer treatment.

And-Gate CAR T-Cells to Improve Tumour Specificity and Targeting of Low-Expression Antigens in Multiple Myeloma

Whilst chimeric antigen receptor (CAR) T cells have shown remarkable success, further novel targets are required. However, our previous cell surface proteomics work in multiple myeloma (MM) showed that target choice is highly limited by an absence of truly tumour-specific antigens, and often very low on-tumour expression of target proteins in some primary samples. One approach to improve tumour specificity is to use combinatorial antigen recognition, or “AND-gates”, whereby a CAR T cell must recognise two different antigens for full activation and target cell elimination. It has also been reported that this approach might overcome low target expression. We therefore decided to explore the potential for AND-gate targeting in MM and identify a suitable target pairing. To determine a comprehensive list of viable AND-gate combinations for MM, we first integrated our MM proteomics data with a normal tissue proteomics dataset. From 777 extracellular proteins expressed in primary MM, we identified a total of 287,890 possible pairwise combinations. We next excluded any pairs with overlapping expression in vital, healthy tissues, reducing potential combinations to 664. Additional filtering steps to remove difficult-to-target proteins and combinations where either target was expressed on T cells - thus avoiding T cell fratricide - resulted in a final list of 92 AND-gate targets. From our prioritised candidates, we selected TNFRSF8 and TMPRSS11E as an exemplar pairing. By mass spectrometry, both TNFRSF8 and TMPRSS11E were expressed across all 8 profiled primary MM samples at comparable levels to existing CAR T cell targets, such as GPRC5D, and had no predicted overlapping expression in normal tissue. To test our AND-gate combination, we designed a ‘split-CAR’ system in which TNFRSF8 was targeted by a 1 st generation CAR with an intracellular signalling domain, but without a co-stimulatory receptor (CAR-only construct), and TMPRSS11E was targeted by a construct with a chimeric co-stimulatory receptor (CCR), but lacking an intracellular signalling domain (CCR-only construct). Constructs were initially validated in a Jurkat cell line model of T cell activation. Jurkats were transformed with CCR-only, CAR-only, or both constructs, as well as with full 2 nd generation CARs targeting TNFRSF8 or TMPRSS11E. These cells were co-cultured with the TNFRSF8+TMPRSS11E+ human MM cell line XG1 or the TNFRSF8+TMPRSS11E- erythroleukemic cell line K562. As anticipated, neither the CCR-only nor the CAR-only Jurkats were activated, with no production of IFNγ or IL2 ( Fig. 1A). Whilst Jurkats expressing the full 2 nd generation TMPRSS11E CAR were activated in co-culture with XG1, Jurkats expressing the TNFRSF8 2 nd generation CAR were not, despite expression of TNFRSF8 on XG1. Jurkats co-expressing both components of the split-CAR were markedly activated when co-cultured with TNFRSF8+TMPRSS11E+ XG1 cells (but not with TNFRSF8+TMPRSS11E- K562 cells), as demonstrated by a large increase in expression of CD69, IFNγ, and IL2 ( Fig. 1A). Thus, the split-CAR system showed a very high degree of specificity, requiring both antigens to be co-expressed on the target cell. Moreover, it was activated even when full 2 nd generation CARs against TNFRSF8 were not. Next, we sought to investigate the cytolytic activity of our split-CAR constructs in donor T cells. In a cytotoxicity assay, T cells expressing the CAR-only or CCR-only construct did not exhibit significant killing of XG1 cells ( Fig. 1B). In parallel with the Jurkat reporter assays, only 2 nd generation TMPRSS11E CAR T cells, and not 2 nd generation TNFRSF8 CAR T cells, were cytotoxic to XG1. Finally, donor T cells expressing both components of the split-CAR demonstrated potent killing of dual-positive XG1 cells ( Fig. 1B), but not single-positive K562 cells. We have thus developed an analytical pipeline for the discovery of AND-gate CAR T cell targets for MM, also applicable to other tumours. This approach led to the development of an extremely potent, but highly selective, split-CAR targeting TNFRSF8 and TMPRSS11E. Importantly, our split-CAR targeted very low levels of TNFRSF8 antigen that were insufficient to trigger activation of a 2 nd generation TNFRSF8 CAR-T. In summary, our work suggests that AND-gate targeting can increase not just specificity, but potency, even against very low expression tumour targets in MM. Link to preprint https://doi.org/10.1101/2023.04.04.535580

Cannabinoids and the endocannabinoid system in immunotherapy: helpful or harmful?

Numerous studies in various cancer models have demonstrated that ingredients of cannabis can influence tumor growth through the endocannabinoid system (ECS), a network of molecules (mediators, receptors, transporters, enzymes) that maintains homeostasis and protection in many tissues. The main constituents of the ECS are the classical cannabinoid (CB) receptors, such as CB1 and CB2, their endogenous ligands (endocannabinoids), and the endocannabinoids’ synthesizing and degrading enzymes. The role of the ECS in cancer is still unclear and its effects often depend on the tumor entity and the expression levels of CB receptors. Many studies have highlighted the tumor cell-killing potential of CB1 agonists. However, cannabis is also known as an immunosuppressant and some data suggest that the use of cannabis during immunotherapy worsens treatment outcomes in cancer patients. CB receptors are widely present in immune cells, and together with monoacylglycerol lipase, the 2-arachidonoylglycerol degrading enzyme, they could be critically involved in the regulation of the immune cell profile of the tumor microenvironment (TME), and hence in tumor progression. So far, data on the impact of the ECS in the immune-TME are still vague. In this review, we discuss the current understanding of the ECS on immunoregulation during tumor growth, and how it might affect the outcome of cancer immunotherapy.

Conformation-selective rather than avidity-based binding to tumor associated antigen derived peptide-MHC enables targeting of WT1-pMHC low expressing cancer cells by anti-WT1-pMHC/CD3 T cell engagers.

T cell engagers, a category of T cell-retargeting immunotherapy, are rapidly transforming clinical cancer care. However, the lack of tumor-specific targets poses a significant roadblock for broad adaptation of this therapeutic modality in many indications, often resulting in systemic on-target off-tumor toxicity. Though various tumor-derived intracellular mutations provide a massive pool of potential tumor-specific antigens, targeting them is extremely challenging, partly due to the low copy number of tumor associated antigen (TAA)-derived pMHC on tumor cell surface. Further, the interplay of binding geometry and format valency in relation to the capacity of a T cell engager to efficiently target low density cell-surface pMHC is not well understood. Using the Wilms' tumor 1 (WT1) oncoprotein as a proof-of-principle TAA, combined with an array of IgG-like T cell engager modalities that differ in their anti-TAA valency and binding geometry, we show that the ability to induce an immunological synapse formation, resulting in potent killing of WT1 positive cancer cell lines is primarily dependent on the distinct geometrical conformations between the Fab arms of anti-WT1-HLA-A*02:01 and anti-CD3. The augmented avidity conferred by the binding of two anti-WT1-HLA-A*02:01 Fab arms has only minimal influence on cell killing potency. These findings demonstrate the need for careful examination of key design parameters for the development of next-generation T cell engagers targeting low density TAA-pMHCs on tumor cells.

Targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Evaluation of Two Chemoenzymatic Glycan Remodeling Approaches to Generate Site-Specific Antibody-Drug Conjugates.

Fc-glycosite-specific antibody-drug conjugation represents a promising direction for the preparation of site-specific antibody-drug conjugates (ADCs). In the present research, we conducted a systemic evaluation of two endoglycosidase-catalyzed chemoenzymatic glycoengineering technologies to prepare glycosite-specific ADCs. In the first two-step approach, the antibody was deglycosylated and then reglycosylated with a modified intact N-glycan oxazoline. In the second one-pot approach, antibodies were deglycosylated and simultaneously glycosylated with a functionalized disaccharide oxazoline. For the comprehensive evaluation, we first optimized and scaled-up the preparation of azido glycan oxazolines. Afterwards, we proved that the one-pot glycan-remodeling approach was efficient for all IgG subclasses. Subsequently, we assembled respective ADCS using two technology routes, with two different linker-payloads combinations, and performed systemic in vitro and in vivo evaluations. All the prepared ADCs achieved high homogeneity and illustrated excellent stability in buffers with minimum aggregates, and exceptional stability in rat serum. All ADCs displayed a potent killing of BT-474 breast cancer cells. Moving to the mouse study, the ADCs prepared from two technology routes displayed potent and similar efficacy in a BT-474 xenograft model, which was comparable to an FDA-approved ADC generated from random conjugation. These ADCs also demonstrated excellent safety and did not cause body weight loss at the tested dosages.

Dose Escalation of ISB 1342, a Novel CD38xCD3 Bispecific Antibody, in Patients with Relapsed / Refractory Multiple Myeloma (RRMM)

Introduction: ISB 1342 is a humanized CD38xCD3 bispecific T-cell engager using Ichnos proprietary Bispecific Engagement by Antibodies based on the T cell receptor (BEAT ®) platform, designed to simultaneously bind CD38 on multiple myeloma (MM) cells and CD3ε on T cells, to induce tumor killing. This mechanism of action is different from existing monospecific CD38 targeting therapies and was designed to overcome resistance to daratumumab in MM. ISB 1342 exhibits potent killing of the primary MM cells and MM cell lines with low sensitivity to daratumumab ( Pouleau B. et al. Blood 2023; 142 (3): 260-273). We report here, the updated findings from the weekly dose-escalations of intravenous (IV) and subcutaneous (SC) injections of an ongoing, single-agent phase 1 study (NCT03309111) of ISB 1342 in patients with RRMM. Methods: Adult patients with RRMM that relapsed after or were refractory to prior therapies, including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody; received ISB 1342 on a weekly (QW) schedule by IV or SC injection with a priming dose on the first day in 2 parallel dose-escalation schemas. Patients had measurable disease per the International Myeloma Working Group (IMWG) criteria (2016). Dose escalation in the QW schedule began at 0.2/0.3 mg/kg priming/treatment dose and followed a “3 + 3” design. The primary study objectives are to determine the maximum tolerated dose (MTD) and select a recommended phase 2 dose (RP2D) of ISB 1342. Secondary objectives includ evaluation of anti-myeloma activity, pharmacokinetics (PK), pharmacodynamics, and immunogenicity of ISB 1342. Results: As of July 18, 2023, based on ongoing clinical database, 39 subjects had received weekly IV infusions of ISB 1342 in 8 dose-escalation groups from 0.2/0.3 mg/kg to 4.0/16.0 mg/kg as priming/treatment dose; and 7 additional subjects had received weekly SC injections at 2.0/8.0 mg/kg. Two different formulations have been tested in SC group. Median number of cycles administered was 2. The majority were male (57%) and white (74%); 13% were Black or African American. The median age was 69 years (range, 54-76) and the median time since diagnosis was 6.6 years (range, 2.8-22.2). The median number of prior anti-myeloma lines of therapy was 6 (range 1-12). Twenty-one (67%) subjects were considered to be triple-class refractory and 18 (48%) penta-drug refractory. Forty-one (89%) subjects experienced treatment-related adverse events (TRAEs) of any grade. Most TRAEs were grade 1-2, including infusion related reactions (37%), cytokine release syndrome (CRS, 34%, all grade 1or 2), anemia (24%), neutropenia (24%), and thrombocytopenia (17%). Five subjects in SC group (71% of SC) had injection site reaction, all grade 1 or 2. Grade 3 or higher TRAEs occurring in more than 5% of subjects were infusion related reaction (no grade 4), anemia (no grade 4), neutropenia, leukopenia. No Grade 5 TRAE was observed. After QW IV infusion, ISB 1342 serum concentration peaked at the end of infusion and then showed bi-exponential decline. ISB 1342 serum exposures increased in a dose linear fashion across the tested IV infusion dose range. The limited PK data after SC injection suggest slow presentation of ISB 1342 into the systemic circulation leading to lower C max and delayed T max relative to IV. Transient increases in serum cytokines were observed within 24 hours after ISB 1342 dosing, including IFNg, TNFa, IL-2, IL-6 and IL-10. Transient increases in T-cell activation were observed by flow cytometry at 24-48 hours after ISB 1342 dosing at 1.0 / 4.0 μg/kg and above, based on increased expression of CD69 on peripheral blood CD4+ and CD8+ T-cell populations (Figure 1). Comparison of the available data from subjects treated at 2 / 8 μg/kg (Cohort 109) indicated reduced T-cell activation following SC compared to IV administration of ISB 1342. The efficacy signals observed in Cohorts 108 and 109 are consistent with the estimates of the lower boundary of the predicted efficacious dose range using a quantitative systems pharmacology (QSP) model based on preclinical data. Updated clinical data, including response rates and safety data, will be present at the meeting. Conclusions: Treatment with ISB 1342 was well tolerated at higher dose levels evaluated. Observed CRS events were moderate. No increased risk of infection has been observed . Updated safety, efficacy, biomarker and PK data will be presented.

Human antibody VH domains targeting uPAR as candidate therapeutics for cancers.

The high expression of uPAR has been linked to tumor progression, invasion, and metastasis in several types of cancer. Such overexpression of uPAR makes it a potential target for immunotherapies across common cancers such as breast, colorectal, lung, ovarian cancer, and melanoma. In our study, two high-affinity and specific human VH domain antibody candidates, designed as clones 3 and 115, were isolated from a phage-displayed human VH antibody library. Domain-based bispecific T- cell engagers (DbTE) based on these two antibodies exhibited potent killing of uPAR-positive cancer cells. Thus, these two anti-uPAR domain antibodies are promising candidates for treating uPAR positive cancers.

Potential of moderate-Z superparamagnetic nanoparticles in improving the efficacy of cancer theranostics

The radiosensitization studies of superparamagnetic nanoparticles (SPMNPs) are fewer than those of high-Z NPs due to the notion that only high-Z NPs can enhance the radiation damage through the physical effects. Recent studies have shown that, in addition to physical effects, chemical and biological effects of NPs contribute equally to the observed radiation damage enhancement. Therefore, in this study, the cytotoxicity, anticancer, and radiosensitization potential of the hydrothermally synthesized moderate-Z Zn0.4M0.6Fe2O4 SPMNPs (M = Mn, Ni, Cu, and Zn) against HT-29 Colon cancer cells were studied. The SPMNPs were characterized by XRD, XPS, VSM, FTIR, FE-SEM, and DLS techniques. The observed values of saturation magnetization of the SPMNPs were explained by cationic distribution obtained via Rietveld refinement and deconvolution method from XRD and XPS results, respectively. The cytotoxicity, anticancer, and radiosensitization potential of the SPMNPs against astrocytes and HT-29 colon cancer cells were evaluated by MTT assay and clonogenic assay. The SPMNPs exhibited differential cell killing, anticancer, and radiosensitization potential in colon cancer cells. Zn0.4Mn0.6Fe2O4 SPMNPs exhibited a significant damage enhancement of 3.67 fold compared to gamma radiation alone in cancer cells. However, in vivo studies are required before considering these NPs for clinical studies.

Penetration of Nanobody-Dextran Polymer Conjugates through Tumor Spheroids.

Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.

Co-Expression of an IL-15 Superagonist Facilitates Self-Enrichment of GD2-Targeted CAR-NK Cells and Mediates Potent Cell Killing in the Absence of IL-2.

In contrast to T lymphocytes, natural killer (NK) cells do not require prior sensitization but are rapidly activated upon encountering virally infected or neoplastic cells. In addition, NK cells can be safely applied in an allogeneic setting, making them important effector cells for the development of off-the-shelf therapeutics for adoptive cancer immunotherapy. To further enhance their therapeutic potential, here, we engineered continuously expanding NK-92 cells as a clinically relevant model to express a humanized second-generation chimeric antigen receptor (CAR) with a composite CD28-CD3ζ signaling domain (hu14.18.28.z) that targets the disialoganglioside GD2, which is expressed at high levels by neuroblastoma cells and other tumors of neuroectodermal origin. In a separate approach, we fused an IL-15 superagonist (RD-IL15) to the GD2-CAR via a P2A processing site. Lentivirally transduced NK-92/hu14.18.28.z and NK-92/hu14.18.28.z_RD-IL15 cells both displayed high and stable CAR surface expression and specific cytotoxicity toward GD2-positive tumor cells. GD2-CAR NK cells carrying the RD-IL15 construct in addition expressed the IL-15 superagonist, resulting in self-enrichment and targeted cell killing in the absence of exogenous IL-2. Furthermore, co-culture with RD-IL15-secreting GD2-CAR NK cells markedly enhanced proliferation and cytotoxicity of bystander immune cells in a paracrine manner. Our results demonstrate that GD2-CAR NK cells co-expressing the IL-15 superagonist mediate potent direct and indirect antitumor effects, suggesting this strategy as a promising approach for the further development of functionally enhanced cellular therapeutics.

Rapid ultrasound-assisted synthesis, characterization, DFT, molecular docking, and anticancer activity of palladium and zinc complexes with 2,6-dimethoxybenzoic acid: A comprehensive study

In this work, we present a thorough investigation encompassing various aspects of zinc and palladium complexes synthesized with 2, 6-dimethoxybenzoic acid. The complexes were prepared through an ultrasonic reaction, offering a rapid, cost-effective, and environmentally friendly method. Characterization of the complexes, prepared in a 1:2 metal-to-ligand ratio, was carried out using elemental analysis, infrared spectra, proton nuclear magnetic resonance, molar conductivity, and melting point measurements. The results revealed that the ligand exhibited chelating bidentate behavior, forming coordination bonds through both oxygen atoms of the carbonyl group. The prepared complexes were evaluated for their anticancer activity against breast cancer and colon cancer cell lines. Remarkably, the complexes exhibited significantly higher cancer cell-killing potential compared to the commonly used drug 5-fluorouracil, with cell death rates reaching 99% for both complexes. To gain insights into the underlying mechanisms of this enhanced anticancer activity, molecular docking studies were conducted, revealing a binding energy of no more than -9.14 kcal/mol between the complexes and the amino acids of the cancer cells. Furthermore, theoretical studies employing Density Functional Theory (DFT) were performed to provide additional diagnostic insights. These studies encompassed molecular structural analysis, computational vibrational properties, natural bond orbital analysis (NBO) and FMOs, electronic properties (UV–Vis spectra), average local ionization energy (ALIE), electrostatic potential (ESP), electron localization function (ELF), and reduced density gradient (RDG/NCI) analyses. The comprehensive investigation presented in this study sheds light on the synthesis, characterization, theoretical analysis, and remarkable anticancer potential of zinc and palladium complexes with 2,6-dimethoxybenzoic acid.

Iron-Gallic Acid Peptide Nanoparticles as a Versatile Platform for Cellular Delivery with Synergistic ROS Enhancement Effect.

Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron-gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H2O2 in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1-7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

CAMMA 2: A phase I/II trial evaluating the efficacy and safety of cevostamab in patients with relapsed/refractory multiple myeloma (RRMM) who have triple-class refractory disease and have received a prior anti-B-cell maturation antigen (BCMA) agent.

TPS8064 Background: Multiple myeloma (MM) remains an incurable disease. Combinations of established agents, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 antibodies, are used in all lines of therapy, but re-exposure to previously received classes of therapy is associated with decreasing response rates and duration of response (Moreau et al. 2021). BCMA-targeted agents have recently been approved for the treatment of patients with RRMM whose disease has been exposed or become refractory to PIs, IMIDs and anti-CD38 antibodies (triple-class exposed or refractory MM). Although effective, the majority of patients who respond to anti-BCMA agents eventually relapse. Proven salvage therapies for these patients are currently lacking and represent a new unmet medical need. Cevostamab is an immunoglobulin G1-based T-cell-engaging bispecific antibody that targets Fc receptor-homolog 5 (FcRH5) on myeloma cells and CD3 on T cells. Dual binding results in T-cell activation and potent killing of MM cells. In an ongoing Phase I study (GO39775; NCT03275103), cevostamab monotherapy showed promising activity in heavily pre-treated patients with RRMM, including those with prior exposure to anti-BCMA agents (Trudel et al. 2021). Methods: CAMMA 2 (CO43476; NCT05535244) is an open-label, multicenter Phase I/II trial evaluating cevostamab monotherapy in patients with RRMM who are triple-class refractory and have previously received an anti-BCMA agent. Prior anti-BCMA antibody-drug conjugates or chimeric antigen receptor (CAR) T cells are permitted in Cohorts A1 and B1, while prior anti-BCMA bispecific antibodies are allowed in Cohorts A2 and B2. In A1 and A2, cevostamab is administered by intravenous infusion and is initiated with step dosing (0.3mg on Cycle [C] 1 Day [D] 1 and 3.3mg on C1D2, D3, or D4 depending on the emergence and resolution of cytokine release syndrome [CRS] after the initial administration), with the target dose of 160mg given on C1D8 and on D1 of each subsequent 21-day cycle. Patients are hospitalized for the C1 administrations only and treatment is continued until disease progression or unacceptable toxicity occurs. CRS is managed per protocol based on the clinical presentation and may involve corticosteroid and tocilizumab treatment. Primary objectives are evaluation of efficacy (primary endpoint: objective response rate by investigator assessment using IMWG criteria) and safety. Secondary objectives include assessment of quality of life, pharmacokinetics, pharmacodynamics, and immunogenicity. Cohorts B1 and B2 will evaluate the efficacy and safety of cevostamab at the recommended Phase II dose in this setting. As of February 2023, enrolment into Cohorts A1 and A2 is ongoing. Clinical trial information: NCT05535244 .

Fe(III)-coordination-driven fabrication of self-supplying oxygen and charge-convertible nanoplatform for enhanced photodynamic therapy of tumor

The hypoxic microenvironment of tumors severely limits the effect of oxygen-dependent photodynamic therapy. Coordination-induced self-assemblies fabricate from metal ions and polymer ligands present potential advantages in catalytic reaction-mediated O2 production, high drug loading efficiency and system stability, as well as enhanced tumor targeting. In this work, self-supplying O2 and pH-induced charge-convertible nanoplatforms (termed as Sp-NPs) were fabricated through coordination-induced self-assembly of meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) modified and carboxylated pH sensitive polymeric ligand, poly (isobutylene-alt-maleic anhydride-conjugated-methoxy-poly(ethyleneglycol)-conjugated-imidazole-conjugated-ethylenediamine-grafted-TCPP) [PIMA-mPEG-API-EDA-TCPP], and ferric ions (Fe3+) for enhanced photodynamic therapy of cancer. The Sp-NPs displays uniform elliptic nanoparticles with size of around 90 nm and excellent stability in a variety of biomimetic biological media. Moreover, Sp-NPs bearing charge-convertible feature can flip the surface charge from negative to positive in the weakly acidic tumor extracellular condition, resulting in enhanced the cellular internalization. After entering more acidity tumor cells by endocytosis, Sp-NPs can be detonated to activate photosensitizers and release Fe3+ that can catalyze endogenous hydrogen peroxide (H2O2) to produce O2, leading to a prominent enhancement of fluorescence imaging and reactive oxygen species (ROS) generation in the tumor. Importantly, this nanoplatform exhibits a more potent cancer cell killing efficiency with good biosafety compared to that were treated with free TCPP. Therefore, incorporation self-oxygenation-boosted ROS strategy and pH-induced charge-convertible mechanism into this platform offered a great promise for cancer therapy.

Abstract LB217: A novel bispecific LILRB4/CD3 antibody with potent killing of monocytic acute myeloid leukemia cells

Abstract Background: Acute myeloid leukemia (AML) remains one of the highest unmet needs among human cancers. LILRB4 is specifically expressed on M4 myelomonocytic and M5 monocytic AML cells with a lack of expression on normal hematopoietic stem cells and progenitors, making LILRB4 an attractive target for a T-cell redirecting bispecific antibody to treat AML. Using a proprietary bispecific antibody platform, we engineered and optimized a LILRB4/CD3 bispecific antibody demonstrating potent and specific killing of monocytic AML cells in vitro and in vivo. The strong chemistry, manufacturing and control (CMC) attributes and cross-reactivity to non-human primate LILRB4 and CD3 favor this bispecific for rapid advancement to the clinic. Experimental Procedures: Binding EC50 values for LILRB4/CD3 bispecific antibodies were determined by flow cytometry analysis. The selectivity of the anti-LILRB4 modality to other LILRA/B proteins was assessed by ELISA. The potency of T-cell mediated killing of LILRB4-expressing AML cells was determined by co-culturing human peripheral T cells and monocytic AML THP-1 cells. A human peripheral mononuclear cell (PBMC) assay evaluated potency against LILRB4-positive primary cells (CD14+ monocytes), using CD19+ B cells as a negative control. Tumor bioluminescence kinetics was performed in NSG mice administrated intravenously with human T cells and THP-1-luciferase cells to measure T cell-directed killing of AML cells in vivo. Single intravenous dose of 5 mg/kg was used for PK evaluation in human FcRn transgenic mice. Results: Two top candidates were selected using biophysical criteria: 1) A monovalent anti-LILRB4 arm together with a monovalent anti-CD3 (1+1); 2) A bivalent anti-LILRB4 arm together with a monovalent anti-CD3 (2+1). The binding EC50 values of the 2+1 or the 1+1 variants to LILRB4 in the THP-1 cells were 0.7 nM and 1.3 nM, respectively. ELISA showed the anti-LILRB4 arm to be highly selective for LILRB4. Both 2+1 and 1+1 variants demonstrated potent killing of the THP-1 cells with EC50 values of 0.5 pM and 4.3 pM, respectively. In the PBMC assay, both variants induced killing of monocytes while sparing B cells, supporting the specificity in inducing T-cell directed killing of the LILRB4-expressing primary cells. The anti-CD3 arm was designed to have a low affinity for CD3 to mitigate the risk of cytokine release while ensuring sufficient T cell activation. Both variants showed potent tumor-growth inhibition at doses as low as 0.2 mg/kg in the NSG mice with the 2+1 molecule showing superiority in vivo. The PK profile of the 2+1 bispecific in human FcRn-transgenic mice was similar to what is expected of human IgG1 with linear clearance. Conclusions: A novel bispecific LILRB4/CD3 (2+1) has been identified for development to treat AML based on its potency and specificity in killing monocytic AML cells in both in vitro and in vivo preclinical studies. Citation Format: Tao Huang, Kyu Hong, Krista McCutcheon, Jing-Tyan Ma, Ying Zhu, Mi Deng, Cheng Cheng Zhang, Paul Woodard, Hong Xiang, Xiao Min Schebye, X. Charlene Liao. A novel bispecific LILRB4/CD3 antibody with potent killing of monocytic acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB217.

A Cell Type Selective YM155 Prodrug Targets Receptor-Interacting Protein Kinase 2 to Induce Brain Cancer Cell Death.

Glioblastoma (GBM) is the most prevalent and aggressive primary central nervous system (CNS) malignancy. YM155 is a highly potent broad-spectrum anti-cancer drug that was derived from a phenotypic screen for functional inhibitors of survivin expression, but for which the relevant biomolecular target remains unknown. Presumably as a result of its lack of cell-type selectivity, YM155 has suffered from tolerability issues in the clinic. Based on its structural similarity to the GBM-selective prodrug RIPGBM, here, we report the design, synthesis, and characterization of a prodrug form of YM155, termed aYM155. aYM155 displays potent cell killing activity against a broad panel of patient-derived GBM cancer stem-like cells (IC50 = 0.7-10 nM), as well as EGFR-amplified and EGFR variant III-expressing (EGFRvIII) cell lines (IC50 = 3.8-36 nM), and becomes activated in a cell-type-dependent manner. Mass spectrometry-based analysis indicates that enhanced cell-type selectivity results from relative rates of prodrug activation in transformed versus non-transformed cell types. The prodrug strategy also facilitates transport into the brain (brain-to-plasma ratio, aYM155 = 0.56; YM155 = BLQ). In addition, we determine that the survivin-suppressing and apoptosis-inducing activities of YM155 involve its interaction with receptor-interacting protein kinase 2 (RIPK2). In an orthotopic intracranial GBM xenograft model, aYM155 prodrug significantly inhibits brain tumor growth in vivo, which correlates with cell-type selective survivin-based pharmacodynamic effects.

Abstract 3997: Preclinical characterization of ARX517, a next-generation anti-PSMA antibody drug conjugate for the treatment of metastatic castration-resistant prostate cancer

Abstract Prostate cancer is the most common cancer, and the second leading cause of cancer death, among men in the United States. Metastatic castration-resistant prostate cancer (mCRPC) is an advanced stage of disease in which patients ultimately fail androgen-deprivation therapies and exhibit a poor survival rate. Recently, prostate-specific membrane antigen (PSMA) has been validated as a prostate cancer tumor antigen with its over-expression in prostate tumors and low level of expression in select normal tissues. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX517, an anti-PSMA targeted next-generation antibody drug conjugate (ADC), for treatment of mCRPC patients. ARX517 is composed of a humanized anti-PSMA antibody site-specifically conjugated to drug linker AS269 (a potent tubulin inhibitor), yielding a drug-to-antibody ratio of 2. After binding to PSMA expressed on the surface of tumor cells, ARX517 is internalized and delivers a cytotoxic payload which inhibits tubulin polymerization and induces cellular apoptosis. In vitro testing of ARX517 in prostate cancer cell lines with variable PSMA expression demonstrated highly specific and potent sub-nanomolar activity in cells with high PSMA expression. To minimize premature payload release, ARX517 employs a non-cleavable PEG linker and stable oxime conjugation chemistry to enhance stability in circulation. ARX517 exhibited a long terminal half-life and high serum exposure in mice. The serum stability of ARX517 should effectively deliver more payload to target tumor cells, and in multiple CDX and PDX prostate cancer models, ARX517 showed dose-dependent anti-tumor activity in both enzalutamide-sensitive and enzalutamide-resistant models. Repeat dose toxicokinetic studies in non-human primates demonstrated ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 elicited highly specific, potent cell killing in cell lines with high PSMA expression, inhibited tumor growth in enzalutamide-sensitive and enzalutamide-resistant CDX and PDX models, demonstrated a tolerable safety profile in cynomolgus monkeys, and has a clear therapeutic index based on preclinical serum exposure data. The strong preclinical data and recent clinical validation of PSMA as a mCRPC target provide rationale for evaluation of ARX517 as a potential prostate cancer treatment. ARX517 is currently in a Phase 1 dose escalation trial (ARX517-2011 [NCT04662580]) in the United States. Citation Format: Lillian Skidmore, David Mills, Ji Young Kim, Prathap Shastri, Nick A. Knudsen, Jeff Steen, Jay Nelson, Ying Buechler, Feng Tian, Shawn Zhang. Preclinical characterization of ARX517, a next-generation anti-PSMA antibody drug conjugate for the treatment of metastatic castration-resistant prostate cancer. [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 3997.