Off-target Toxicity Research Articles

CAPN2-responsive mesoporous silica nanoparticles: A promising nanocarrier for targeted therapy of pancreatic cancer

Pancreatic adenocarcinoma (PDAC) is highly resistant to conventional chemotherapeutic interventions, resulting in exceptionally low survival rates. The limited efficacy can in part be attributed to dose limitations and treatment cessation urged by toxicity of currently used chemotherapy. The advent of targeted delivery strategies has kindled hope for circumventing off-target toxicity. We have previously reported a PDAC-specific mesoporous silica nanoparticle (MSN) containing a protease linker responsive to ADAM9, a PDAC-enriched extracellularly deposited protease. Upon loading with paclitaxel these ADAM9-MSNs reduced side effects both in vitro and in vivo, however, disappointing antitumor efficacy was observed in vivo. Here, we propose that an efficient uptake of MSNs by tumor cells might underlie the lack of antitumor efficacy of MSNs functionalized with linker responsive to extracellular proteases. Harnessing this premise to improve antitumor efficacy, we performed an in silico analysis to identify PDAC-enriched intracellular proteases. We report the identification of BACE2, CAPN2 and DPP3 as PDAC enriched intracellular proteases, and report the synthesis of BACE2-, CAPN2- and DPP3-responsive MSNs. Extensive preclinical assessments revealed that paclitaxel-loaded CAPN2- and DPP3-MSNs exhibit high PDAC specificity in vitro as opposed to free paclitaxel. The administration of paclitaxel-loaded CAPN2- and DPP3-MSNs in vivo confirmed the reduction of leukopenia and induced no organ damage. Promisingly, in two mouse models CAPN2-MSNs reduced tumor growth at least as efficiently as free paclitaxel. Taken together, our results pose CAPN2-MSNs as a promising nanocarrier for the targeted delivery of chemotherapeutics in PDAC.

Abstract LB265: IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC)

Abstract Cyclin-dependent kinases (CDK) 4/6 are key cell cycle regulators that form complexes with cyclin D1 and the master regulator p27Kip1. This ternary complex acts downstream of the ER- and HER2-pathways, which drive cell proliferation. CDK4/6 phosphorylates the Retinoblastoma protein and other substrates, and keeps p27 from inhibiting G1-associated CDK2. Targeted inhibition of CDK4/6 (CDK4/6i) blocks the growth of ER+ and HER2+ BC by dissolving the CDK4/6-cyclin D1 complex and permitting the shuttling of p27 to inhibit CDK2 complex. Approval of CDK 4/6i such as palbociclib, though a game changer for ER+ mBC, resulted in tumor resistance, leaving patients without effective treatments and a 5-yr survival of only 30%. Although CDK2 inhibitors (CDK2i) address compensatory CDK2 activity bypassing CDK4/6i, a key resistance mechanism, they lack selectivity due to CDK homology, resulting in off-target toxicity. To inhibit CDK2/4/6 simultaneously, combinations of CDK4/6i and CDK2i monotherapies were attempted, but the lack of coordination between CDK4/6 and CDK2 inhibition resulted in suboptimal efficacy. Concarlo presents a unique therapeutic rationale for directly targeting the CDK master regulator p27 to simultaneously inhibit CDK2/4/6 in a concerted manner, one that is guided by the role of p27 to turn all of the G1 CDKs ON and OFF during periods of proliferation and quiescence, respectively. This transition is facilitated by BRK (Breast tumor kinase)-mediated phosphorylation of p27. When p27 is Y phosphorylated, it converts CDK4/6 and CDK2 into open, active complexes. An endogenous BRK-spliced variant ALT can competitively bind to p27, block Y phosphorylation and lock CDK2/4/6 in the OFF conformation. Concarlo engineered a shorter variant of ALT (CCL20) and packaged in cationic liposomes for intracellular delivery, creating a novel IpY.20 drug product. As a single agent, IpY.20 inhibits cell proliferation in treatment-naïve and CDK4i-resistant HR+ cells with reduced non-tumor cell activity. In combination, IpY.20 enhances CDK4/6i responses in treatment-naïve HR+ cells, making it a potential front-line mBC therapy. While other CDKi activity is limited to cytostasis, IpY.20 shows dual cytostasis-cytotoxicity, potentially a more durable option. IpY.20 does not cause neutropenia commonly seen with other CDKis, and rescues the CDK4/6i-induced neutropenia in mice, suggesting better clinical tolerability. These findings underscore the significance of coordinated targeting of CDK2/4/6, making IpY.20 a first-in-class product uniquely exploiting p27-mediated CDK2/4/6 regulation. Direct targeting of p27 presents a new treatment paradigm bypassing the challenges of ATP-competitive CDKis, with potential to expand into tumor types with aberrant p27, CDK2/4/6 activity, or RAS/MAPK pathways that increase CDK4/6 activity. Citation Format: Grace Chen, Natalie Dudas, Kate Coleman, Carolina Guido, Natalia Zisman, Krishna Allamneni, Stacy Wister Blain. IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB265.

Abstract CT096: Phase I first-in-human MUC1* targeted autologous CAR T cells for the treatment of metastatic breast cancers

Abstract Purpose: First-in-human Phase I study for advanced MUC1* positive breast cancer with autologous T cells engineered to express either a chimeric antigen receptor, huMNC2-CAR44 or huMNC2-CAR22, which specifically bind to a cleaved form of MUC1 (MUC1*); evaluate the safety and preliminary anti-tumor activity. Methods: MUC1* (muk 1 star) is the growth factor receptor form of MUC1, created by cleavage and release of the N-terminal portion of MUC1. The targeting antibody, huMNC2, only recognizes the conformational epitope created when MUC1 is cleaved by specific tumor-associated enzymes that are correlated with poor prognosis. huMNC2 does not bind to full-length MUC1, which is expressed on all normal epithelial cells. huMNC2-scFv was incorporated into huMNC2-CAR44, comprising a CD8 hinge and transmembrane region, 4-1BB costimulatory domain and wild-type CD3z. huMNC2-scFv was also incorporated into huMNC2-CAR22 wherein the hinge, transmembrane and co-stimulatory portions were derived from CD28 and CD3z bears the 1XX mutations to increase in vivo persistence. Inclusion criteria require that the patient’s tumor is at least 30% MUC1* positive and that patient has progressed through 2 or 3 prior therapies, while in the metastatic setting. Patients receive standard Cy/Flu lymphodepletion approximately 3-days before CAR T treatment, administered at 1 of 4 dose levels ranging from 3.3X10^5 up to 1.0X10^7 CAR+ T cells. Results: To date, 8 patients have been treated with huMNC2-CAR44. No patients experienced neuro toxicities. No off-target toxicities were observed. 3 patients experienced CRS Grade 1-3. In 6 of the 8 patients, side effects were non-existent or minimal. However, one patient experienced a Grade 5 SAE that was deemed to be possibly related to the treatment. Best responses include Partial Responses and Stable Disease at a low CAR-T dose. Greatest efficacy was observed for patients whose biopsy showed H Scores >120. Patients are currently being enrolled for treatment with huMNC2-CAR22, where the 1XX mutations are expected to increase in vivo persistence, durability of response and reduced incidence of CRS. Conclusions: These data support a conclusion that the MUC1* antibody, huMNC2, is safe and could have high therapeutic value as a CAR T treatment for solid tumors with moderate to high antigen density. As the huMNC2-CAR22 (1XX) trial proceeds, we will assess if patient responses mirror our animal results that show that the 1XX mutations confer increased persistence, reduced exhaustion and the ability to kill tumors with low antigen density. Citation Format: Cynthia Carol Bamdad, Joanne E. Mortimer, Yuan Yuan, Jennifer M. Specht, Andrew K. Stewart, Benoit J. Smagghe, Stephen C. Lin, Mark G. Carter, Tim W. Synold, Mark D. Fleming, Stanley R. Hamilton, Vishwas Parekh, Danica M. Walkley, Qing Liu-Michael, Kevin R. Yi, Jac-Leen S. Nash, Michael J. Nash, Stephen J. Forman. Phase I first-in-human MUC1* targeted autologous CAR T cells for the treatment of metastatic breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT096.

Abstract LB043: VBC101-F11: An innovative EGFR/cMet bispecific antibody drug conjugate (ADC) targeting key oncogenic drivers in solid tumors

Abstract EGFR and cMet are two classic growth factor receptors frequently co-expressed in solid tumors at significantly high levels, while only limited to moderate expression in normal tissues. Beyond their co-expression patten, the aberrant or over expression in their pathway are implicated in oncogenesis. Notably, MET pathway activation either through increased cMet expression or its ligand, is often associated with resistance to EGFR tyrosine kinase inhibitors (TKI). Therefore, the interplay between EGFR and cMet, coupled with their differential expression in tumors versus normal tissue substantiates the rationale for a bispecific approach that targets both receptors to enhance tumor specificity. VBC101-F11 is a bispecific anti-EGFR/cMet ADC engineered to deliver clinically validated cytotoxic agents to tumor cells with a drug-to antibody ratio (DAR) of 4. This innovative design distinguishes VBC101-F11 from other EGFR/cMet-targeting agents on the market or in clinical trials (ABBV399 or AZD9592), optimizing both efficacy and safety. 1. VBC101-F11 features a low-affinity EGFR arm coupled with a high-affinity cMet arm. Incorporating the low-affinity EGFR arm enhances binding avidity and internalization, resulting in a superior cytotoxicity with a 5-fold improvement in IC50 compared to ABBV-399 (targeting cMet only). This nuanced EGFR arm design ensures a reduction in EGFR-related off-target toxicity in normal tissues. 2. The biparatopic design in cMet arm further underscores our strategic rationale by demonstrating a synergistic effect in terms of binding, internalization, and functional blocking with a 10-fold improvement in EC50 compared to the monospecific parental antibodies that target a single epitope. This ensures VBC101-F11’s maximized efficacy in tumor cells. 3. VBC101-F11’s innovative nanobody-based format sets our molecule apart. Living imaging data in mouse model reveals that VBC101-F11 (90kD) achieves superior tumor penetration and accumulation from 1 hour to over 90 hours surpassing the mAb ABBV399 and bispecific antibody AZD9592, both at 150kD. 4. VBC101-F11 has demonstrated outstanding molecular developability in stress tests, particularly in human and non-human primate (NHP) plasma, where both the antibody component and the entire ADC molecule exhibit remarkable stability, positioning VBC101-F11 as a viable candidate for future Chemistry, Manufacturing, and Controls (CMC) development. 5. In in vivo CDX models using various lung cancer cell lines, VBC101-F11 equipped with MMAE warhead displayed a marked superiority in tumor growth inhibition (TGI of 60% vs. 17%) at a single dose of 3mg/kg compared to MMAE conjugating ADC ABBV-399. Further payload exploration, such as DNA replication inhibitor, has also yielded promising results, with VBC101-F11-new payload showing comparable efficacy to the same class ADC AZD9592 in models with low EGFR and high cMet expression (100% TGI) and in those with moderate EGFR/cMet expression (~ 60% TGI). In summary, VBC101-F11, with unique design and distinguished attributes, has demonstrated its remarkable potential in the landscape of anti-EGFR/cMet therapies. The encouraging efficacy observed in preclinical models supports the molecule’s advancement into clinical development, positioning it as a best-in-class bispecific ADC for treating tumors in patients. Citation Format: Wei (Vivian) Wang, Jing Li, Lin Ma, Man Xu, Qun Yin. VBC101-F11: An innovative EGFR/cMet bispecific antibody drug conjugate (ADC) targeting key oncogenic drivers in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB043.

Thermosensitive polymer prodrug nanoparticles prepared by an all-aqueous nanoprecipitation process and application to combination therapy

Despite their great versatility and ease of functionalization, most polymer-based nanocarriers intended for use in drug delivery often face serious limitations that can prevent their clinical translation, such as uncontrolled drug release and off-target toxicity, which mainly originate from the burst release phenomenon. In addition, residual solvents from the formulation process can induce toxicity, alter the physico-chemical and biological properties and can strongly impair further pharmaceutical development. To address these issues, we report polymer prodrug nanoparticles, which are prepared without organic solvents via an all-aqueous formulation process, and provide sustained drug release. This was achieved by the “drug-initiated” synthesis of well-defined copolymer prodrugs exhibiting a lower critical solution temperature (LCST) and based on the anticancer drug gemcitabine (Gem). After screening for different structural parameters, prodrugs based on amphiphilic diblock copolymers were formulated into stable nanoparticles by all-aqueous nanoprecipitation, with rather narrow particle size distribution and average diameters in the 50–80 nm range. They exhibited sustained Gem release in human serum and acetate buffer, rapid cellular uptake and significant cytotoxicity on A549 and Mia PaCa-2 cancer cells. We also demonstrated the versatility of this approach by formulating Gem-based polymer prodrug nanoparticles loaded with doxorubicin (Dox) for combination therapy. The dual-drug nanoparticles exhibited sustained release of Gem in human serum and acidic release of Dox under accelerated pathophysiological conditions. Importantly, they also induced a synergistic effect on triple-negative breast cancer line MDA-MB-231, which is a relevant cell line to this combination.

In Situ Nanofiber Formation Blocks AXL and GAS6 Binding to Suppress Ovarian Cancer Development.

Anexelekto (AXL) is an attractive molecular target for ovarian cancer therapy because of its important role in ovarian cancer initiation and progression. To date, several AXL inhibitors have entered clinical trials for the treatment of ovarian cancer. However, the disadvantages of low AXL affinity and severe off-target toxicity of these inhibitors limit their further clinical applications. Herein, by rational design of a nonapeptide derivative Nap-Phe-Phe-Glu-Ile-Arg-Leu-Arg-Phe-Lys (Nap-IR), a strategy of in situ nanofiber formation is proposed to suppress ovarian cancer growth. After administration, Nap-IR specifically targets overexpressed AXL on ovarian cancer cell membranes and undergoes a receptor-instructed nanoparticle-to-nanofiber transition. In vivo and in vitro experiments demonstrate that in situ formed Nap-IR nanofibers efficiently induce apoptosis of ovarian cancer cells by blocking AXL activation and disrupting subsequent downstream signaling events. Remarkably, Nap-IR can synergistically enhance the anticancer effect of cisplatin against HO8910 ovarian tumors. It is anticipated that the Nap-IR can be applied in clinical ovarian cancer therapy in the near future.

Tumor-targeted and stimulus-responsive polymeric prodrug nanoparticles to enhance the anticancer therapeutic efficacy of doxorubicin

Polymeric prodrug nanoparticles have gained increasing attention in the field of anticancer drug delivery because of their dual functions as a drug carrier and a therapeutic agent. Doxorubicin (DOX) is a highly effective chemotherapeutic agent for various cancers but causes cardiotoxicity. In this work, we developed polymeric prodrug (pHU) nanoparticles that serve as both a drug carrier of DOX and a therapeutic agent. The composition of pHU includes antiangiogenic hydroxybenzyl alcohol (HBA) and ursodeoxycholic acid (UDCA), covalently incorporated through hydrogen peroxide (H2O2)-responsive peroxalate. To enhance cancer cell specificity, pHU nanoparticles were surface decorated with taurodeoxycholic acid (TUDCA) to facilitate p-selectin-mediated cancer targeting. TUDCA-coated and DOX-loaded pHU nanoparticles (t-pHUDs) exhibited controlled release of DOX triggered by H2O2, characteristic of the tumor microenvironment. t-pHUDs also effectively suppressed cancer cell migration and vascular endothelial growth factor (VEGF) expression in response to H2O2. In animal studies, t-pHUDs exhibited highly potent anticancer activity. Notably, t-pHUDs, with their ability to accumulate preferentially in tumors due to the p-selectin targeting, surpassed the therapeutic efficacy of equivalent DOX and pHU nanoparticles alone. What is more, t-pHUDs significantly suppressed VEGF expression in tumors and mitigated hepato- and cardiotoxicity of DOX. Given their cancer targeting ability, enhanced therapeutic efficacy and minimized off-target toxicity, t-pHUDs present an innovative and targeted approach with great translational potential as an anticancer therapeutic agent.

Renal-clearable luminescent gold nanoparticles incorporating active and bio-orthogonal tumor-targeting for drug delivery and controlled release

The use of ultrasmall gold nanoparticles (AuNPs) as drug delivery systems (DDSs) represents certain challenges, including limited targeting efficiency, uncontrolled drug release, and off-target drug toxicity. Herein, we synthesize a series of second near-infrared (NIR-II) region emitting AuNPs by incorporating both c(RGDfC) peptide and cucurbit[7]uril (CB[7]) for active and bio-orthogonal tumor-targeting, respectively. The CB[7]/c(RGDfC)-AuNPs exhibited rapid tumor accumulation and prolonged retention, which were utilized as nanocarriers to load oxaliplatin (OX) to afford OX@CB[7]/c(RGDfC)-AuNPs for targeted drug delivery and tumor chemotherapy. The mice treated with OX@CB[7]/c(RGDfC)-AuNPs achieved superior therapeutic outcomes compared to three times the dose of OX treatment (H-OX group). Meanwhile, the AuNPs showed excellent biosafety as opposed to obvious hepatotoxicity and severe hepatorenal toxicity observed in the mice treated with low dose of OX and H-OX, respectively. This renal-clearable DDS holds promise for precise drug delivery and controlled release, offering new perspectives and methods for clinical cancer treatment.

Synthetic biology approaches for enhancing safety and specificity of CAR-T cell therapies for solid cancers

CAR-T cell therapies have been successful in treating numerous hematologic malignancies as the T cell can be engineered to target a specific antigen associated with the disease. However, translating CAR-T cell therapies for solid cancers is proving more challenging due to the lack of truly tumor-associated antigens and the high risk of off-target toxicities. To combat this, numerous synthetic biology mechanisms are being incorporated to create safer and more specific CAR-T cells that can be spatiotemporally controlled with increased precision. Here, we seek to summarize and analyze the advancements for CAR-T cell therapies with respect to clinical implementation, from the perspective of synthetic biology and immunology. This review should serve as a resource for further investigation and growth within the field of personalized cellular therapies.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. Invitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Affinity targeting of therapeutic proteins to the bone surface-local delivery of sclerostin-neutralizing antibody enhances efficacy.

Currently available biotherapeutics for the treatment of osteoporosis lack explicit mechanisms for bone localization, potentially limiting efficacy and inducing off-target toxicities. While various strategies have been explored for targeting the bone surface, critical aspects remain poorly understood, including the optimal affinity ligand, the role of binding avidity and circulation time, and, most importantly, whether or not this strategy can enhance the functional activity of clinically relevant protein therapeutics. To investigate, we generated fluorescent proteins (eg, mCherry) with site-specifically attached small molecule (bisphosphonate) or peptide (deca-aspartate, D10) affinity ligands. While both affinity ligands successfully anchored fluorescent protein to the bone surface, quantitative radiotracing revealed only modest femoral and vertebral accumulation and suggested a need for enhanced circulation time. To achieve this, we fused mCherry to the Fc fragment of human IgG1 and attached D10 peptides to each C-terminus. The mCherry-Fc-D10 demonstrated an ~80-fold increase in plasma exposure and marked increases in femoral and vertebral accumulation (13.6% ± 1.4% and 11.4% ± 1.3% of the injected dose/g [%ID/g] at 24h, respectively). To determine if bone surface targeting could enhance the efficacy of a clinically relevant therapeutic, we generated a bone-targeted sclerostin-neutralizing antibody, anti-sclerostin-D10. The targeted antibody demonstrated marked increases in bone accumulation and retention (20.9 ± 2.5% and 19.5 ± 2.5% ID/g in femur and vertebrae at 7days) and enhanced effects in a murine model of ovariectomy-induced bone loss (bone volume/total volume, connectivity density, and structure model index all increased [P< .001] vs untargeted anti-sclerostin). Collectively, our results indicate the importance of both bone affinity and circulation time in achieving robust targeting of therapeutic proteins to the bone surface and suggest that this approach may enable lower doses and/or longer dosing intervals without reduction in biotherapeutic efficacy. Future studies will be needed to determine the translational potential of this strategy and its potential impact on off-site toxicities.

Abstract 2260: Transcriptomic immuno-oncology investigation of B-cell lymphomas using a novel comparative workflow

Abstract Patients with B-cell lymphoma currently have limited treatment options, often with serious side effects; for example, B-cell lymphoma patients undergoing chemo- and radiation therapies experience poor health from off-target toxicity. Similarly, though CD19 CAR T-cell therapy is very effective, patients suffer lifelong B-cell aplasia as a side effect. Comparing a cancer to an autoimmune disease in the same tissue type may grant us additional insights on the immune dysregulation of the cancer, leading to development of more immune-aware cancer treatments. We introduce a new data-driven comparative workflow for the analysis of immuno-oncology attributes by comparing a cancer and an autoimmune disease in the same tissue type. As an example use case, we compare primary B-cell lymphomas to primary B-cells from systemic lupus erythematosus with the goal of delineating new mechanisms that can be leveraged to treat B-cell lymphomas. Our analysis will utilize bulk RNA-sequencing (RNA-seq) of B-cells for initial discovery, followed by single-cell RNA-sequencing (scRNA-seq) of Peripheral Blood Mononuclear Cells (PBMCs) for validation. Our analytical framework incorporates differential gene analysis, GO Terms, known protein-protein interactions, principal component analysis, and machine learning for biomarker detection. Here, we describe our novel tool in detail and share the results of our lymphoma use case to gain a better understanding of the targetable immune-related attributes of B-cell lymphoma. We expect that this novel tool, along with the results of our B-cell lymphoma use case, will contribute to the continued development of immune-focused treatment options for B-cell lymphoma and other cancers. Citation Format: Naomi Rapier-Sharman, Michael T. Told, Sehi Kim, Kim L. O'Neill, Brett E. Pickett. Transcriptomic immuno-oncology investigation of B-cell lymphomas using a novel comparative workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2260.

Abstract 7179: OBI-992, a novel TROP2 targeting antibody drug conjugate demonstrates superior in vivo PK/PD properties and a favorable safety profile

Abstract Background: TROP2 targeting antibody-drug conjugates (ADC) have demonstrated promising clinical responses for the treatment of various solid tumors. However, these ADCs still exhibit serious safety issues, such as hematologic toxicities and interstitial lung disease. OBI-992, a novel TROP2 targeting ADC, which is derived from the conjugation of an anti-TROP2 antibody with a topoisomerase I inhibitor, exatecan, via an enzyme-cleavable linker. OBI-992 is designed to have high serum stability and broad therapeutic index. Herein, we describe the results from preclinical studies of OBI-992, including in vitro and in vivo stability, pharmacokinetics/pharmacodynamics (PK/PD) profile, and safety evaluation. Material and Methods: To characterize OBI-992, the linker stability was evaluated by ex vivo serum stability and rat PK studies. PK/PD properties, including systemic and tumor exposures as well as receptor occupancy, were evaluated in a human lung cancer xenograft model. In vitro toxicity testing, including ADC toxicity on differentiating neutrophils and FcγR mediated toxicity, was evaluated using human monocyte THP-1 cells. A 6-week repeat dose toxicity study in cynomolgus monkeys was conducted to evaluate the safety and toxicokinetics of OBI-992. Results: Ex vivo serum stability demonstrated that OBI-992 exhibited better linker stability than the benchmark datopotamab deruxtecan (Dato-DXd). In vivo PK evaluation in rats showed that OBI-992 had lower Cmax and AUC of free payload than Dato-DXd, indicating a better PK profile of OBI-992 in rats. A PK/PD study in tumor-bearing mice revealed that OBI-992 exhibited higher tumor exposure of free payload than Dato-DXd, resulting in a better antitumor efficacy. In addition, the antitumor effect positively correlated with percentage of receptor occupancy in a dose-dependent manner. In vitro cytotoxicity testing demonstrated that OBI-992 had lower toxicity in differentiating neutrophils and THP-1 cells compared to Dato-DXd, suggesting that OBI-992 may cause less off-target toxicity. Toxicokinetics of OBI-992 in cynomolgus monkeys revealed that the systemic exposure of ADC was similar to that of total antibody. Furthermore, the systemic exposure of ADC and total antibody were found to increase in a dose-proportional manner. Major toxicities in monkeys were target-related skin lesions. The highest non-severely toxic dose (HNSTD) was determined to be 60 mg/kg. Conclusions: OBI-992 exhibits remarkable antitumor efficacy and a favorable safety profile, supporting further clinical development of OBI-992 in TROP2 positive cancers. Citation Format: Chi-Sheng Shia, Shih-Ni Wen, Ren-Yu Hsu, Jyy-Shiuan Tu, Hui-Wen Chang, Wan-Fen Li, Ming-Tain Lai. OBI-992, a novel TROP2 targeting antibody drug conjugate demonstrates superior in vivo PK/PD properties and a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7179.

Abstract 5121: Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies

Abstract The success of CD19-directed chimeric antigen receptor (CAR)-T cell therapy for ALL has yet to be matched in solid pediatric malignancies. Moreover, immune checkpoint blockade has shown limited benefit in pediatric cancer. Despite this unmet need, there is a lack of reliable preclinical models to facilitate novel target discovery, validate therapeutic benefit, anticipate on- or off-target toxicity, and understand mechanisms of resistance. Syngeneic murine model systems are a critical intermediate for preclinical translation, preserving the complex and heterogenous immune landscape and host-immunotherapy interactions. However, model interpretation and validity are limited as expression and function of murine targets and immune effector cells are not always conserved. We have developed a syngeneic immunocompetent C57BL/6 neuroblastoma system to study CAR efficacy, toxicity, and mechanisms of resistance, as well a variety of vaccination strategies. Specifically, we have built two murine CAR T constructs containing a 100% conserved antigen-directed scFv joined to orthologous murine TCR signaling machinery. In parallel, we engineered the murine neuroblastoma cell line 9464D to stably express surface antigen Gpc2 (Bosse, Cancer Cell 2017), or a novel chimeric single chain trimer of a 9mer peptide from intracellular oncoprotein PHOX2B discovered on HLA-A*24:02 (Yarmarkovich, Nature 2023) as Phox2b/A24*02/H2-kb to generate tumor-bearing B6 or B6-A24-Tg mice. We are currently using this system to demonstrate how vaccination approaches enhance the persistence and potency of CART directed to both targets and are extending our syngeneic murine constructs to additional TH-MYCN allograft models. To further study the innate and adaptive immune response to various therapeutics, we have developed peripheral blood mononuclear cell (PBMC) humanized models. Our autologous humanized PDX mouse models of neuroblastoma use immunocompromised NSG mice engrafted with a PDX and humanized with patient-matched PBMCs to evaluate personalized vaccines in vivo. We have shown the ability to stably engraft both lymphoid and myeloid populations in various human cytokine transgenic NSG variants (i.e. SGM3, Flt3L), allowing for T/B cell and APC crosstalk, thus providing a robust means to study targeted interventions. We are currently using this autologously humanized PDX model to test the ability of multivalent, personalized neoantigen vaccines developed specific to the neoantigen landscape of individual PDXs, with a focus on endogenous T cell responses of patient PBMC against their own PDX in vivo. Citation Format: Timothy T. Spear, Elisabeth Posthill, David Groff, Anna M. Giudice, Kristopher R. Bosse, Leyuan Ma, Stephen P. Schoenberger, John M. Maris. Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5121.

Abstract 1337: Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma

Abstract AXL is a receptor tyrosine kinase (RTK) that is commonly expressed in metastatic melanoma and several other forms of cancer. Activation of AXL signaling is linked to tumor cell growth, invasion, metastatic transformation, drug resistance, and immune response regulation. However, treatment of metastatic melanoma continues to present clinical challenges due to its capacity to rapidly develop drug resistance, which manifests under the influence of AXL signaling. As such, AXL may be a viable antigen target for immunotherapies. Despite their successes in hematological malignancies, chimeric antigen receptor (CAR)-T cell therapies remain limited by their susceptibility to causing off-target toxicity. In contrast, natural killer (NK) cells are not associated with such risks, which makes them clinically appealing as an alternative to T cells. However, studies on the application of CAR-NK cells to treat solid tumors remain acutely scarce. To explore the clinical viability of CAR-NK cells for the treatment of metastatic melanoma, AXL-specific CAR-NK cells were generated by transfecting NK cells obtained from NK cell lines. Preliminary data from a cytotoxicity assay indicated that the cytotoxicity of CAR-NK92-MI cells against AXL-positive melanoma cell lines was significantly stronger (up to a 10-fold difference) than the corresponding parental cells. Further in vitro characterization of this CAR-NK cell line and other CAR-NK cell lines, as well as in vivo evaluation of their efficacy in a mouse xenograft model of melanoma metastasis, is underway. Citation Format: Winston Hibler, Glenn Merlino, Yan Lin Yu. Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1337.

Abstract 7183: Liver toxicity of amanitin-based antibody drug conjugates (ATACs) is caused by unspecific uptake of the ATAC into liver cells

Abstract Background: Antibody drug conjugates (ADCs) were developed to increase the therapeutic window (TW) of cytotoxins by combining their efficacy with the precision of antibodies (Ab). Nevertheless, adverse events, still limit the use of ADCs. The tox profile of an ADC is determined by its on- and off-target effects. On-target tox is caused by specific binding of the ADC to healthy target-positive cells; off-target tox is caused by target-independent binding of the ADC, e.g. via the Ab backbone or by payload release in circulation. In this study, we unravel the off-target tox mechanisms of ADCs that use amatoxins (RNA polymerase II inhibitor) as payload (ATACs) and use this knowledge to improve the TW of ATACs. Amatoxins are distinct from other ADC payloads as they are hydrophilic and thus require active transport to pass the cell membrane. The primary transporter of amatoxins in humans is the OATP1B3 transporter, which is exclusively expressed on hepatocytes. Thus, unspecific ATAC tox can be caused by free amatoxin, premature released in circulation and uptake by liver cells via OATP1B3, or by uptake of the ATAC via a yet unknown mechanism. Material and Methods: ATACs are based on cysteine-reactive and site-specific amatoxin-linker constructs by HDP. In vitro: Binding of ATACs to FcγRI by BLI; Cytotoxicity of ATACs on HEKwt, HEK-OATP1B3 and THP1 cells. In vivo: Single i.v. dose of ATAC; Tolerability in mice; Efficacy in s.c. tumor models; Blood sampling for PK analysis; ADC/total Ab detection by ELISA; DAR-loss analysis and toxin measurement by (intact) LC-MS. Results: Liver tox caused by premature release of amatoxin in circulation was investigated thoroughly in vivo. DAR-loss, survival analysis, ADC and Ab measurements, indicated that the release of amatoxin in circulation cannot be the root-cause for off-target ATAC toxicity. Further, it was ruled out that the ATAC is taken up by OATP1B3 in vitro and in vivo. Thus, uptake of the intact ATAC via yet unknown mechanisms is responsible for liver tox. As liver tox is also known for other ADCs, several uptake mechanisms are described, one of which being FcγR-mediated uptake. Indeed, ATACs bind to the FcγR and trigger target-independent cytotoxicity on FcγR+ cells. Point mutations at L234 and L235 (LALA mutation), the amino acids in the Fc part of the Ab responsible for FcγR binding, were shown to reduce FcγR binding. The LALA mutation significantly increased the tolerability of ATACs in mice and monkeys, whereas the anti-tumor efficacy was not impaired, leading to an improved TW. Conclusion: Our data show that liver toxicity of ATACs is caused by unspecific uptake of the ATAC into liver cells at least partially mediated by FcγR. This knowledge was used to optimize the Ab backbone to minimize unspecific uptake into liver cells. Insertion of a LALA mutation to avoid uptake via FcγR reduced off-target toxicity of ATACs leading to better tolerability and an increased TW. Citation Format: Christian Orlik, Kristin Decker, Marija Vranic, Marisa Schmitt, Andreas Pahl, Michael Kulke, Torsten Hechler. Liver toxicity of amanitin-based antibody drug conjugates (ATACs) is caused by unspecific uptake of the ATAC into liver cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7183.

Abstract 5733: Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs

Abstract Topoisomerase 1 (TOP1) inhibitors are a class of cytotoxic small molecules that have demonstrated effectiveness as therapeutic agents in cancer. However, the broad clinical application has been hindered by poor pharmacokinetic profiles and off-target toxicities. Antibody-drug conjugates (ADCs) enable the targeted delivery of cytotoxic payloads by specifically targeting cell-surface antigens expressed on cancers reducing systemic exposure and toxicity. TOP1-ADCs have received FDA approval for HER2- (trastuzumab-deruxtecan) and Trop2- (sacituzumab-govitecan) expressing solid tumors, demonstrating the promise for this drug class to improve the lives of patients. We have developed a novel drug-linker using a potent TOP1 inhibitor with desirable ADME properties, and a hydrophilic glycoside linker as a platform technology enabling ADCs with favorable physicochemical and biological properties. The TOP1 inhibitor payload was optimized for potency, with reduced P-glycoprotein efflux and enhanced permeability. The hydrophilicity of the linker facilitates the development of highly loaded ADCs (8 drugs/mAb) with high plasma stability, low aggregation, and pharmacokinetics similar to parental antibody. The resultant ADCs are potent and immunologically specific with activity in multidrug-resistant tumor models and bystander activity in heterogenous antigen models. Human cancer cell derived xenograft models demonstrated potent and specific ADC anti-tumor activity with several targets and indications. Improved activity was observed compared to corresponding govitecan- and deruxtecan-based ADCs. ADCs prepared from this drug-linker technology were well tolerated in rats after repeat administration of 60 mg/kg every 4 days for 4 doses. The excellent anti-tumor activity in several models and favorable safety profile demonstrates this platform TOP1 drug-linker technology has the potential for development as ADCs against many cancer targets. Citation Format: Ryan Lyski, Lauren Bou, Johann Sigurjonsson, David Meyer, Calvin Neace, David Ortiz, Katie Snead, Kaleb Smith, Steven Jin, Nicole Stevens, Julia Cochran, Kaveh Alizadeh, Narayana Yeddula, Erica McKinney, Adam Peterson, Lindsay Dotloe, Jessica Simmons, Christopher Carosino, Kim Emmerton, Nancy Everds, Scott Jeffrey, Peter Senter. Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5733.

Abstract 2615: Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of solid tumors

Abstract cMET (mesenchymal-epithelial transition factor), which belongs to the MET family, is a type of receptor tyrosine kinase that is expressed on the surfaces of various epithelial cells. The elevation of cMET can promote the development and progression of multiple cancers. cMET- ADCs have shown promising clinical activity with highest cMET levels, indicating tumor cMET levels may be limited for efficacy. YL211, a novel cMET-ADC was developed by leveraging on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform), which could release the payload both in the tumor cell and in the tumor microenvironment. YL211 is comprised of an anti-cMET humanized monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker. The novel linker-payload and site-specific conjugation of YL211 results in a homogeneous product with a DAR (Drug-to-Antibody Ratio) of 8. Furthermore, the advancement in linker-payload design has facilitated the development of more hydrophilic ADCs, which exhibit minimal impact on the antibody's characteristics while being less prone to aggregation and showcasing lower systematic clearance. Along with release of the payload in the tumor microenvironment extracellularly, upon binding to cMET on tumor cell surfaces, the antigen-ADC complexes are internalized to lysosomal vesicles and subsequently release the toxic payload. The cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. These findings translated in vivo where YL211 showed superior efficacy in causing tumor regressions and good tolerability in both cell lines and patient-derived xenograft (CDX and PDX) mouse models with both low to high cMET expression levels (H358, CR5088, ect.). The in vivo pharmacokinetics in monkeys showed that YL211 is highly stable with less than 0.1% (molar ratio) payload released in circulation. GLP safety evaluations demonstrated good safety profiles and no off-target toxicity in monkeys. These non-clinical data suggest the stability of the linker in circulation as well as efficient release of the payload in tumors with improved therapeutic window. Taken together, preclinical data suggest that YL211 could be a promising treatment strategy for cMET positive cancer patients. Citation Format: Liang Xiao, Wei Lian, Shuai Song, Qigang Liu, Qing Zong, Sasha Stann, Jiaqiang Cai, Tongtong Xue. Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2615.

Abstract 6706: An anti-claudin 18.2/CD3 bispecific antibody for the treatment of claudin 18.2 positive gastric cancer

Abstract The claudin18.2 (CLDN18.2) protein shows four transmembrane domains (TMDs) with two extracellular loops (ECL), ECL-1 and ECL-2. It is a highly selective biomarker with limited expression in normal tissues and often exhibits abnormal expression during the occurrence and development of various primary malignant tumors. The specific expression pattern supports the notion that CLDN18.2 is a distinctive molecule for targeted therapy in various cancers, particularly gastric and pancreatic cancers. Various drugs targeting CLDN18.2, including monoclonal antibodies, ADCs, CAR-T, and bispecific antibodies, have also entered clinical trials. As of this writing, there are currently no commercially available products worldwide that specifically target CLDN18.2. However, Zolbetuximab (IMAB362) has successfully reached the Phase III end point, indicating positive clinical outcomes and establishing CLDN18.2 as a promising cellular and antibody therapeutic option. Here, we describe LNF2007, an anti-CLDN 18.2/CD3 bispecific antibody that can redirect CD3+ effector T cells to CLDN18.2+ tumor sites, resulting in the activation of T cells and subsequent killing of target cells. The design of LNF2007 focuses on selecting a relatively weak and reasonable affinity to CD3, accompanied by the suppression of Fc-mediated effector functions. This aim is to minimize off-target toxicity and maximize therapeutic efficacy. In vitro data demonstrate that LNF2007 binds more strongly to CLDN18.2+cells than to CD3+ cells. LNF2007 exhibited good dose-dependent activation and cytotoxicity against engineered cell lines BXPC3-hCLDN18.2-Luc and CHOK1-hCLDN18.2-Luc, which are positive for CLDN18.2 expression .In the in vivo pharmacodynamics assay, LNF2007 also demonstrated superior efficacy in the humanized NUGC4-CLDN18.2 model and BxPC3-CLDN18.2 model. Furthermore, a 4-week repeated dose (up to 1 mg/kg) toxicity study in cynomolgus monkeys showed that LNF2007 was safe and well tolerated. In vitro cytokine release was lower in the LNF2007 group than that in the AMG 910 and AZD5863 (two another anti-claudin18.2/CD3 bispecific antibody) groups. In conclusion, the novel anti-CLDN18.2 × anti-CD3 bispecific antibody LNF2007 demonstrates similar efficacy to AMG 910 and AZD5863 and is safer. LNF2007 provides a promising therapy for claudin 18.2-positive cancer patients.Taishan Industry Leading Talents Project of Shandong Province was a sponsor of this research. Citation Format: Lili Zhao, Guimin Zhang, Zhong Liu, Jinhua Xu, Lizhi Zhao, Guangyan Li, Bin Li, Zhongsong Zhu, Wei Peng, Yuqiang Zhu, Zhaowei Chen, Zhenyu Li. An anti-claudin 18.2/CD3 bispecific antibody for the treatment of claudin 18.2 positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6706.

Abstract 6656: Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus

Abstract Major challenges for CAR-T cell therapy in solid tumors include various limitations due to the issue of appropriate antigen selection, tumor heterogeneity, and the immune-suppressive tumor microenvironment (TME). To overcome these hurdles, we’ve developed a novel therapy, CLM-202 which is a combination therapy of oncolytic Newcastle disease virus (NDV) and NDV F protein-targeted CAR-T cell therapy (NDV F CAR-T). After NDV infects tumor cells, NDV envelope protein F is expressed on the tumor cell surface. NDV F CAR-T recognizes NDV F protein and kills the tumor cells. This approach can be applied to patients with various indications, regardless of the presence of the target protein. Using NDV to deliver antigens minimizes the risk of tumor recurrence due to tumor heterogeneity. Furthermore, it mitigates off-target toxicity by targeting oncolytic virus-derived proteins, not human proteins. Oncolytic NDV also triggers a potent immune response within the tumor microenvironment, thereby altering the immunosuppressive conditions of the tumor and improving CAR-T efficacy. Therefore, CLM-202 can be a multipurpose solution for solid tumors. We have developed a second-generation CAR-T cell therapy, NDV F CAR-T, which can be used in combination with an oncolytic virus known as NDV. NDV F CAR-T is designed to specifically target the F protein expressed when cells are infected by NDV. It incorporates a single-chain variable fragment (scFv) derived from an antibody that can bind specifically to the F protein of NDV-infected cells. To assess the activity of NDV F CAR-T in tumor cells against a variety of solid tumors as indications, we conducted several experiments. We initially confirmed the expression of the F protein in NDV-infected tumor cells. We also conducted cytotoxicity tests to confirm NDV F CAR-T's ability to induce death in NDV-infected tumor cell lines. Furthermore, to assess the efficacy of NDV F CAR-T in an in vivo model, we established an orthotopic mouse model. In this model, we administered NDV via intratumoral injection, followed by the single-dose administration of NDV F CAR-T to perform the efficacy test. We have confirmed that NDV infects all solid tumor cell lines representing different indications and induces the expression of the F protein in infected tumor cells. Furthermore, we have observed that NDV infection in these diverse tumor cells results in robust tumor cell-killing activity by NDV F CAR-T and the subsequent activation of CAR-T. Moreover, in an orthotopic mouse model, we have demonstrated that the administration of NDV followed by NDV F CAR-T induces potent tumor growth inhibition. In summary, our research proposes a novel therapeutic strategy, CLM-202 that utilizes NDV F CAR-T and NDV to overcome the limitations of CAR-T cell therapy in solid tumors, including tumor heterogeneity and immune-suppressive tumor microenvironments. This approach aims to effectively treat a wide range of indications. Citation Format: Soyoung Choi, Kiwan Kim, Sangeun Lee, Jeongeun Choi, Song-Jae Lee, Yong Gu Lee, Jeonghun Kim, Hoyoung Lee, Kisoon Kim, Man-Seong Park, Seong-Won Song. Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6656.