Tumor-specific Activity Research Articles

Combining mathematical modeling, in vitro data and clinical target expression to support bispecific antibody binding affinity selection: a case example with FAP-4-1BBL.

The majority of bispecific costimulatory antibodies in cancer immunotherapy are capable of exerting tumor-specific T-cell activation by simultaneously engaging both tumor-associated targets and costimulatory receptors expressed by T cells. The amount of trimeric complex formed when the bispecific antibody is bound simultaneously to the T cell receptor and the tumor-associated target follows a bell-shaped curve with increasing bispecific antibody exposure/dose. The shape of the curve is determined by the binding affinities of the bispecific antibody to its two targets and target expression. Here, using the case example of FAP-4-1BBL, a fibroblast activation protein alpha (FAP)-directed 4-1BB (CD137) costimulator, the impact of FAP-binding affinity on trimeric complex formation and pharmacology was explored using mathematical modeling and simulation. We quantified (1) the minimum number of target receptors per cell required to achieve pharmacological effect, (2) the expected coverage of the patient population for 19 different solid tumor indications, and (3) the range of pharmacologically active exposures as a function of FAP-binding affinity. A 10-fold increase in FAP-binding affinity (from a dissociation constant [KD] of 0.7 nM-0.07nM) was predicted to reduce the number of FAP receptors needed to achieve 90% of the maximum pharmacological effect from 13,400 to 4,000. Also, the number of patients with colon cancer that would achieve 90% of the maximum effect would increase from 6% to 39%. In this work, a workflow to select binding affinities for bispecific antibodies that integrates preclinical in vitro data, mathematical modeling and simulation, and knowledge on target expression in the patient population, is provided. The early implementation of this approach can increase the probability of success with cancer immunotherapy in clinical development.

NIR responsive nanosystem based on upconversion nanoparticle-engineered bacteria for immune/photodynamic combined therapy with bacteria self-clearing capability

Biological engineering bacteria hold great promise in tumor therapy due to their targeted delivery, tumor penetration, and tumor-specific activation capabilities. However, the use of live bacteria raises safety concerns, as they can potentially cause infections or adverse immune responses in patients. Additionally, most biological engineering bacteria are only responsive to blue light, which has limited penetration depth within biological tissues. To address these limitations, we have developed a nanoplatform that combines dual-emission upconversion nanoparticles (referred to as DDUCNPs), which can realize dual-wavelength emission under dual-wavelength excitation, with biological engineering bacteria for tumor treatment and the self-clearance of biological engineering bacteria after therapy in the near-infrared (NIR) window. This design allows us to utilize 980 nm light, which is converted to blue light by the DDUCNPs, to activate the bacteria and promote the controlled release of tumor necrosis factor-alpha (TNF-α) for precise tumor ablation. Subsequently, we employ 808 nm excitation to achieve light conversion into the red light, thereby activating photosensitizer molecules and generating singlet oxygen (ROS) for in vivo clearance of the bacteria involved in the treatment. Simultaneously, the generated ROS also undergoes photodynamic therapy (PDT) on the tumor to enhance the therapeutic effect. By combining these elements on a single platform, our system achieves the activation and self-clearance of biological engineering bacteria in the NIR window, effectively enabling tumor treatment. This approach overcomes the limitations of blue light penetration and addresses safety concerns associated with live bacteria, offering a promising strategy for precise and controlled tumor therapy.

Chemoradiotherapy and nanomedicine: Drug mechanisms and delivery systems.

Radiotherapy is an invaluable tool in the treatment of cancer. However, when used as a monotherapy, it fails to provide curative outcomes. Chemotherapy drugs are often included to boost the effects of radiation. Key classes of radiosensitizing drugs include platinum compounds, anthracyclines, antimetabolites, taxanes, topoisomerase inhibitors, alkylating agents, and DNA damage repair inhibitors. Chemoradiotherapy suffers from not only systemic toxicities from chemotherapy drugs but also synergistic radiation toxicity as well. It is critical to deliver radiosensitizing molecules to tumor cells while avoiding adjacent healthy tissues. Currently, nanomedicine provides an avenue for tumor specific delivery of radiosensitizers. Nanoscale delivery vehicles can be synthesized from lipids, polymers, or inorganic materials. Additionally, nanomedicine encompasses stimuli responsive particles including prodrug formulation for tumor specific activation. Clinically, nanomedicine and radiotherapy are intertwined with approved formulation including DOXIL and Abraxane. Though many challenges remain, the ongoing progress evidences a promising future for both nanomedicine and chemoradiotherapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice

The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.

Sucrose-Powered Liposome Nanosensors for Urinary Glucometer-Based Monitoring of Cancer.

Timely detection of early-stage cancer holds immense potential in enhancing prognostic outcomes. There is an increasing desire for versatile tools to enable simple, sensitive, and cost-effective cancer detection. By exploiting the extraintestinal metabolic inertness and efficiency renal clearance of sucrose, we designed a liposome nanosensor using sucrose as a messenger to convert tumor-specific esterase activity into glucose meter readout, enabling economical and sensitive urinalysis for cancer detection in point-of-care testing (POCT). Our results demonstrate that the nanosensors exhibited significant signal differences between tumor-bearing and healthy mice in both orthotopic and metastatic tumor models. Additionally, efficient elimination of the nanosensors through the hepatobiliary pathway was observed with no significant toxicity. Such a non-invasive diagnostic modality significantly assists in personalized pharmacological treatment and follow-up efficacy assessment. We envision that this modular liposome nanosensor platform might be applied for economically detecting diverse diseases via a simple urinary test.

Phase III randomized trial evaluating tilsotolimod in combination with ipilimumab versus ipilimumab alone in patients with advanced refractory melanoma (ILLUMINATE 301).

LBA9516 Background: There are limited treatment options for advanced melanoma that has progressed during or after immune checkpoint inhibitor therapy. Intratumoral (IT) immunotherapy may improve tumor-specific immune activation by promoting local tumor antigen presentation while avoiding systemic toxicities. The Phase 3 ILLUMINATE-301 study (NCT03445533) evaluated tilsotolimod, a toll-like receptor 9 agonist, with or without ipilimumab in patients with anti-programmed death-1 (PD-1)-advanced refractory melanoma. Methods: Patients with unresectable Stage III–IV melanoma that progressed during or after anti-PD-1 therapy were randomized 1:1 to receive 24 weeks of tilsotolimod plus ipilimumab or 10 weeks of ipilimumab alone. Nine IT injections of tilsotolimod were administered to a single designated lesion over 24 weeks. Intravenous ipilimumab 3 mg/kg was administered every 3 weeks from Week 2 in the tilsotolimod arm and Week 1 in the ipilimumab arm. The primary endpoint was efficacy measured using objective response rate (ORR; independent review) and overall survival (OS). Results: A total of481 patients received tilsotolimod plus ipilimumab (n = 238) or ipilimumab alone (n = 243). ORRs were 8.8% in the tilsotolimod arm and 8.6% in the ipilimumab arm, with disease control rates of 34.5% and 27.2%, respectively. Median OS was 11.6 months in the tilsotolimod arm and 10.0 months in the ipilimumab arm (hazard ratio (HR) 0.96 [95% confidence interval (CI) 0.77–1.19]; P = 0.7). Grade ≥3 treatment-emergent adverse events occurred in 61.1% and 55.5% of patients in the tilsotolimod and ipilimumab arms, respectively. Conclusion: Adding tilsotolimod to ipilimumab did not improve response or OS in patients with PD-1 refractory advanced melanoma. However, the results represent the largest prospective dataset reported to date on using ipilimumab in this setting and are a valuable addition to the knowledge base. Clinical trial information: NCT03445533 .

MB2033, an anti-PD-L1 × IL-2 variant fusion protein, demonstrates robust anti-tumor efficacy with minimal peripheral toxicity

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rβγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rβγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.

Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD)2) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD)2 simultaneously engages tumor cells and PD-1+ Tcells via dual targeting, thus mediating tumor-specific Tcell activation and proliferation with mild effects on non-specific Tcells. In multiple syngeneic mouse models, αPD-1-(iRGD)2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD)2 remodels the tumor microenvironment and expands a population of "better effector" CD8+ tumor infiltrating Tcells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD)2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy.

Investigation of a potential protein biomarker signature that may predict clinical benefit of NT-I7 and pembrolizumab in patients with cold gastrointestinal tumors.

2563 Background: Microsatellite stable colorectal (MSS-CRC) and pancreatic cancer (PDAC) are immunologically cold tumors due to low immunogenicity and lack of genomic diversity. NT-I7, a long-acting IL-7, and pembrolizumab (pembro) show efficacy in these hard-to-treat indications. While a limited set of patients (pts) achieve objective response, frequency and duration of the disease control rate point to a larger subset obtaining clinical benefit. To identify novel predictive biomarkers, we analyzed baseline peripheral and biopsy samples from pts based on treatment duration. Methods: Open-label Phase 2a study in pts with relapsed/refractory checkpoint inhibitor-naïve MSS-CRC and PDAC; NT-I7 1200 µg/kg IM every 6 weeks (Q6W), pembro 200 mg IV Q3W. Subjects were grouped by treatment duration, measured in NT-I7 doses administered before treatment discontinuation for any cause: 1 dose was short (ST), 2-3 doses medium (MT) and ≥4 doses long (LT). Correlative studies included peripheral (proteomics, T cell receptor sequencing [TCRseq], single cell RNA sequencing [scRNAseq]), and biopsy (genomics, transcriptomics, TCRseq). Results: As of 02OCT2023, 53 evaluable pts completed or discontinued treatment; 5 are still on follow up. ST group included 21 pts, MT 22 and LT 10 (including all 5 partial responders). Tumor biopsies were confirmed MSS with low tumor mutational burden (TMB). LT pts had similar age (59.0 [53.0-71.5] vs 66.0 [47.3-73.5]; ST vs LT) and lower baseline tumor burden (81.0 mm vs 58.0 mm; ST vs LT, p = 0.022). Biopsy analysis showed LT pts had, at baseline, upregulated pathways related to immune activity despite confirmed cold tumor status. Baseline scRNAseq in peripheral blood showed that stem-like CD8 T cells (precursors of exhausted [TPEX] and stem-cell memory [TSCM]) had a differential activation pattern in LT pts; those pathways were enriched in memory effector subsets in ST pts. Preserved antigen-specific stemness may be needed for NT-I7 + pembro efficacy. Baseline concentrations of 3 proteins that can be produced by growing tumors were significantly increased in ST pts. Pts were classified based on elevated levels of these potential biomarkers: POSITIVE (≥2 biomarkers; 20 pts) and NEGATIVE (≤1 biomarker; 33 pts). Pts with NEGATIVE signature at baseline had significantly higher overall survival, regardless of ST, MT or LT status (13.2 vs 8.9 months, p = 0.030). Conclusions: Preserved tumor-specific TPEX activity may be required for NT-I7 + pembro activity based on its presence in LT pts, who remained on treatment the longest. According to this analysis, there are 3 potentially predictive protein biomarkers that may help identify a pt subset more likely to experience clinical benefits from the combination treatment of NT-I7 + pembro. Further verification of the predictive nature of this signature in independent cohorts is ongoing. Clinical trial information: NCT04332653 .

Abstract PO4-18-07: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors

Abstract Background: MYC deregulation is a hallmark of triple negative breast cancer (TNBC) and is associated with aggressive tumors and poor clinical outcomes. Although MYC remains undrugged, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9) is a critical regulator of oncogenic MYC expression and an important MYC cofactor. KB-0742 is an oral CDK9 inhibitor that demonstrates promising preclinical activity against TNBC. In a real-world cohort, TNBCs have higher MYC expression and higher rates of MYC genomic amplification than other breast cancer subtypes. In primary patient-derived cell lines, KB-0742 treatment results in stronger cytotoxic effects in TNBC as compared to other subtypes. In patient-derived organoids and patient-derived xenografts, CDK9 inhibition by KB-0742 drives antiproliferative and anti-tumor growth effects, including in models that are resistant to standard of care. KB-0742 strongly downregulates MYC protein levels at doses that only partially inhibit CDK9 activity, suggesting a therapeutic window for tumor-specific activity (Saffran, D.C. et al, 2021). KB-0742 is currently being evaluated in a phase 1/2 dose escalation and cohort expansion study in patients with TNBC and other transcriptionally addicted tumors (NCT04718675). Trial design: This phase 1/2 study includes two parts: dose escalation (part 1) and cohort expansion (part 2). Part 2 includes cohort A (solid tumors with high prevalence of MYC overexpression including TNBC, non-small cell lung cancer and ovarian) and cohort B (other transcriptionally addicted tumor types including sarcomas, adenoid cystic carcinoma, nut midline carcinoma and small cell lung cancer). KB-0742 is dosed orally once daily for 3 consecutive days, followed by 4 days, off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria: Part 1 dose escalation is open to patients with relapsed or refractory solid tumors. Part 2 is defined by tumor indications in cohorts A and B. Eligibly criteria include age > 18 years (≥ 12 years old and with a body weight ≥ 40 kg part 2 for cohort B), acceptable organ function, and ECOG PS < 2. Specific aims: Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, preliminary anti-tumor activity, and identifying a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. The Modified Continuous Reassessment Method (mCRM) (Goodman et al., Stat Med 1995) will guide dose escalation and MTD. RP2D nomination is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response to KB-0742 in patients is assessed every other cycle starting from cycle two after treatment using RECIST 1.1 criteria. Target accrual: Targeted total enrollment is 170 patients. For additional information, contact clinicaltrials@kronosbio.com Citation Format: Monica Mita, Alain Mita, Miguel Villalona-Calero, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Gregory Cote, Richard Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-07.

Abstract LB124: Targeting NaPi2b with a novel dual TOP1i ADC that shows excellent biophysical properties and high efficacy in vivo

Abstract The Araris site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-NaPi2b ADC with two different Topoisomerase-1 inhibitors (TOP1i) as payloads that shows excellent efficacy vs anti-NaPi2b ADCs that were unsuccessful in clinical trials due to low therapeutic indices (TI), preventing administration at therapeutically active doses. The Araris ADC was designed to combine two features in one ADC to maximize tumor-specific activity by using two different TOP1i payloads: one potent TOP1i that is able to exert bystander activity to address tumor heterogeneity and low-target expression and one exatecan derivative (DAR2) that accumulates in cells (low bystander activity) to achieve greater potency. This novel ADC is characterized by a well-defined drug-to-antibody ratio (DAR) of 4 (DAR2+2), high homogeneity and purity and shows high stability under stressed conditions. In in vitro assays on various target-positive cell lines with different NaPi2b expression levels, the Araris ADC demonstrated nM-potency in cell cytotoxicity assays, similar to the clinical NaPi2b ADCs lifastuzumab vedotin and upifitamab rilsodotin but being much less toxic to target-negative cells. Moreover, this ADC showed excellent stability in mouse and human sera, exemplified by the absence of payload deconjugation or linker cleavage. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile comparable to the unmodified parent antibody. Strikingly, in a high-expressing OVCAR-3 xenograft model, the Araris ADC administered as a single dose at 9 mg/kg on day 0 led to very high anti-tumor activity, essentially leading to tumor eradication and a long lasting anti-tumor response in all treated animals. We here show first encouraging results on a novel concept of combining TOP1i payloads that have two different features in one ADC to maximize efficacy and tolerability. We believe that this concept in combination with a stable payload attachment at low DAR and an excellent exposure may help to develop ADCs with an improved therapeutic index for various solid tumor indications. The initial results indicate that this ADC may overcome the limitations of current clinical programs against NaPi2b and has the potential to be a first-in-class ADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Lia Kallenberger, Emma Renard, Romain Bertrand, Rachael Fay, Roger Santimaria, Patrick Maurhofer, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Targeting NaPi2b with a novel dual TOP1i ADC that shows excellent biophysical properties and high efficacy in vivo [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 LB124.

Abstract 7558: Novel liquid biopsy based determination of lung cancer histology using a comprehensive epigenomic platform reveals enhancer activity as a key determinant for accurate classification

Abstract BACKGROUND: Accurate molecular characterization of lung cancer including histology classification and therapeutic target profiling is critical for treatment planning and resistance monitoring. For example, detecting lineage transformation from lung adenocarcinoma (LUAD) to small cell lung carcinoma (SCLC) following treatment, an established resistance mechanism, is challenging due to deteriorating patient health and complicated invasive procedures. Here, we describe an accurate lung cancer histological classification using an innovative, multimodal epigenomic liquid biopsy method that comprehensively profiles tumor-specific transcription activation from 1 mL of patient plasma. METHODS: 37 LUAD, 34 SCLC patient plasma samples collected from commercial biobanks, and reference cell lines were profiled for enhancer, promoter and DNA methylation activity to identify epigenomic loci characteristic of LUAD and SCLC. The resulting map of select quantified loci was used as input to a machine learning algorithm that distinguishes SCLC from LUAD, in a cross validated training and testing approach. RESULTS: The epigenomic maps derived from lung cancer patient plasma were highly informative of the regulatory programming of the tumor cells. The enhancers and promoters most strongly linked to lung cancer profiles across the patient population include key, histology-specific driver genes such as NKX2-1, FOXA1, FOXA2, ONECUT2, DLL3, and ASCL1. The resulting classifier was able to accurately predict histology with a cross validated AUC greater than 0.94. In silico simulation results suggest that accurate classification can be achieved with ctDNA content below 1%. Further correlative examination of the SCLC enhancer landscape revealed a partitioning of SCLC patients into molecular subtypes characterized by active enhancer marks at subtype-specific transcripts, including ASCL1, POU2F3, NEUROD1, and YAP1. The transcriptional regulation of ADC targets is apparent through comprehensive epigenomic profiling of plasma in both SCLC and LUAD, with the expression of Claudin 18.2, SEZ6, and others showing a strong dynamic range independent of ctDNA levels. This represents a novel liquid biopsy approach to distinguish LUAD from SCLC and provide simultaneous insight into expression of key targets at clinically relevant ctDNA levels. CONCLUSION: These data demonstrate the feasibility of epigenomic mapping from 1mL of plasma in lung cancer patients to distinguish lung cancer histology. The resulting data is biologically interpretable through the lens of transcriptional activation of key histology specific genes. This liquid-biopsy based approach may provide a non-invasive alternative to invasive and challenging tissue biopsies for biomarker investigation and therapeutic decision making. Citation Format: Jamey Guess, Aparna Gorthi, Anthony D'ippolity, Jonathan Beagan, Travis Clark, Michael Coyne, Tyrone Tamakloe, Baovy Tran, Hyun-Hwan Jeong, Kristian Cibulskis, Hathairat Sawaengsri, Juliann Chmielecki, Corrie A. Painter, J Carl Barrett, Matthew Eaton. Novel liquid biopsy based determination of lung cancer histology using a comprehensive epigenomic platform reveals enhancer activity as a key determinant for accurate classification [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 7558.

Abstract 277: Cancer-associated fibroblasts, educated by tumor cells, differentially facilitate cancer cell invasion in cell-line based human gastric tumoroids

Abstract Purposes: Gastric cancer can be categorized into two subtypes: intestinal and diffuse. The latter is notorious for frequent peritoneal metastasis, resulting in a poorer prognosis. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play crucial roles in tumor invasion and metastasis. However, little is known about whether CAFs are associated with the differential progression between the two subtypes of gastric tumors. This study investigated whether differential cancer invasion may be attributed to tumor-specific CAF activation, using a cell-line-based human gastric tumoroid model. Methods: Heterotypic tumor spheroids (TS) were prepared with human gastric cancer lines of intestinal (MKN28, MKN74) and diffuse type (MKN45, KATO III) mixed with human gastric fibroblasts (HGFs) using a minipillar-array chip previously developed. Invasion phenotype and expression of epithelial-mesenchymal transition (EMT)-related proteins were evaluated in heterotypic TSs. mRNA expression in HGFs isolated from heterotypic TSs was analyzed and compared between the two subtypes. Results: While both heterotypic TSs displayed increased invasion by HGFs, the diffuse type TS showed a higher level of invasion with individual or amoeboid migration and loose binding of cancer cells with HGF. In contrast, the intestinal type TS showed collective migration and tight binding between cancer cells and HGFs. HGFs in each heterotypic TS exhibited differential morphology, matrix remodeling patterns, and gene expression in a tumor subtype-specific manner. Compared to normal HGFs, HGFs isolated from diffuse heterotypic TS showed enriched expression of cytoskeleton-associated genes and several integrins, whereas those of intestinal heterotypic TS exhibited increased expression levels of inflammatory cytokines. Conclusions: This study established tumoroid models for human gastric cancer subtypes, intestinal and diffuse, using heterotypic TS culture. Our results suggest that the subtype-specific invasion ability of tumor cells is driven by CAFs that are differentially educated by tumor cells in the tumor microenvironment. Our model not only advances our understanding of CAFs' role in tumor progression but also offers an in vitro model for assessing potential therapeutic targets and drug candidates for human gastric cancers. Citation Format: Hyun Ju Hwang, Saet-Byeol Lee, Hyo-Jeong Kuh. Cancer-associated fibroblasts, educated by tumor cells, differentially facilitate cancer cell invasion in cell-line based human gastric tumoroids [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 277.

Abstract 2600: Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan

Abstract The Araris’ site-specific and one-step conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using two different Topoisomerase-1 inhibitors (TOP1i) as payloads that shows superior anti-tumor efficacy compared to trastuzumab deruxtecan (T-DXd) in head-to-head in vivo studies. For proof-of concept and comparison to T-DXd, trastuzumab was used as the targeting antibody. The Araris ADC was designed to combine two features in one ADC to maximize tumor-specific activity by using two different exatecan-based payloads: one that is able to exert bystander activity to address tumor heterogeneity and low-target expression and one that accumulates in cancer cells (no bystander activity) to achieve greater potency. We found that the ADC was well-defined with a drug to antibody ratio (DAR) of 4 (2+2), as expected and no signs of aggregation under stressed conditions (40°C) during a period of 14 days. In in-vitro assays on target positive cell-lines, the ADC demonstrated taget-specific cell-cytotoxicity in the low nM-range similarly to T-DXd. Importantly, the non-bystander exatecan showed an increased intracellular concentration (up to 4x) and the bystander capable exatecan demonstrated high bystander activity in co-cultured, target-negative cell lines. In mouse pharmacokinetic studies, the ADC showed excellent stability in circulation with no signs of payload loss or linker-cleavage with a PK profile comparable to the unconjugated trastuzumab antibody which is key for a maximal and tumor-specific payload delivery. Finally, a head-to-head study vs T-DXd was done in a challenging, medium HER2-expressing breast cancer model (JIMT-1), known to be resistant against the FDA-approved ADC T-DM1 (trastuzumab emtansine) and for T-DXd only showing limited activity (Ogitani et al., 2016). Impressively, it was found that the dual-TOP1i DAR4 Araris ADC showed superior anti-tumor efficacy compared to the payload-dose-adjusted T-DXd (DAR8). Using published doses for T-DXd, the Araris ADC was administered at ADC doses of 2 × 10mg/kg on days 1 and 8, resulting in superior anti-tumor activity and tumor eradication compared to T-DXd lasting for more than 47 days. T-DXd dosed at 2 × 5mg/kg (same payload dose) on days 1 and 8, only showed a very limited tumor growth inhibition with tumor regrowth occurring in all animals already at around 21 days. We here show first encouraging results on a novel concept of combining TOP1i payloads that have two different features in one ADC to maximize efficacy. We believe that this concept in combination with a stable payload attachment at low DAR and an excellent exposure may help to develop ADCs with an improved therapeutic index for various solid tumor indications. Citation Format: Philipp Spycher, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Patrick Maurhofer, Lia Kallenberger, Emma Renard, Bernd Schlereth, Dragan Grabulovski, Isabella Attinger-Toller. Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan [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 2600.

Abstract 719: A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models

Abstract PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, however, 70-90% of patients do not derive durable benefit. In preclinical settings, delivery of PD-1 blockade and IL-2 agonism in cis via a bispecific molecule, comprising a PD-1 antibody fused to IL-2, has demonstrated superior activity compared to delivery of individual components or their combination. Targeting of IL-2 to PD-1+ cells in cis has been shown to drive a unique differentiation path endowing CD8+ T cells with enhanced functionality. However, we show that the IL-2 component in an unmasked PD1/IL2 bispecific molecule dominates its pharmacology, driving marked peripheral activation of immune cells, rapid IL-2 receptor-mediated clearance and systemic toxicity. To address these challenges and enable tumor-specific activity, we designed a PD1/IL2 bispecific molecule that incorporates a single domain antibody-based mask to bind to the IL-2 component and maintain it in a quiescent state until activated by prevalent and dysregulated proteases in the tumor microenvironment (TME). In preclinical models, the affinity-optimized mask prevented peripheral IL-2 receptor binding, associated toxicity and receptor-mediated clearance. Improved pharmacokinetics of our tumor-activated PD1/IL2 enabled PD1 antibody-like exposures and is projected to achieve complete blockade of PD1 signaling. Tumor-activated PD1/IL2 significantly inhibited tumor growth compared to anti-PD-1 alone or vehicle and demonstrated improved tolerability compared to an unmasked PD1/IL2 bispecific in mouse models. In addition, pharmacodynamic activity of tumor-activated PD1/IL2 was restricted to the TME. Consistent with the masked design, we observed no significant change in CD8+ T cells in the periphery and significant increases in antigen specific CD8+ T cells, memory CD8+ T cells and TCF1+ stem-like CD8+ T cells in the TME. Ex vivo protease cleavage assays performed in human samples demonstrated efficient activation of tumor-activated PD1/IL2 by multiple tumor types and minimal activation in human plasma. In non-human primates, tumor-activated PD1/IL2 achieved tolerable exposures that were comparable to those of existing PD-1 blocking immunotherapies. Taken together, these preclinical data suggest that tumor-activated PD1/IL2 has the potential to improve upon existing PD-1/PD-L1 immunotherapies by inhibiting PD-1/PD-L1 axis and simultaneously directing immunostimulatory IL-2 to antigen-experienced effector cell populations in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Stephanie Hsiao, Parker Johnson, Lisa Quinn, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Bernard Lanter, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Jason Hedges, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jennifer O'Neil, Carl U. Bialucha. A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models [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 719.

Abstract 38: AB-2100, a PSMA-inducible CA9-specific CAR T cell product for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model

Abstract Common challenges of CAR-T cell therapies in solid tumors, such as clear cell renal cell carcinoma (ccRCC), include insufficient therapeutic potency and lack of tumor specificity. We have developed AB-2100, an autologous integrated circuit T (ICT) cell product, generated via CRISPR-mediated knock-in of a single transgene into a safe-harbor locus. AB-2100 encodes a transcriptionally regulated sequential AND gate that comprises a priming receptor (PrimeR) specific for PSMA and an inducible CAR targeting CA9 antigen, which is widely expressed on local and metastatic lesions. AB-2100’s sequential AND logic-gate confers tumor-specific activity by priming off of PSMA-expressing tumor vasculature to induce CA9 CAR expression. This unique feature of the logic gate is intended to increase the safety profile of AB-2100 given that PSMA and CA9 are predicted to have limited co-expression in normal tissues. Additional functionality includes short-hairpin RNAs (shRNA) against Fas and TGFBR designed to prevent tumor-mediated resistance, and a synthetic pathway activator (SPA) that drives constitutive STAT3 signaling and enhanced T cell cytotoxicity and expansion. Mechanism of action studies demonstrate that AB-2100 can prime off of PSMA-expressing endothelial cells and induce tumor-specific killing of CA9 tumor cells, leading to the eradication of ccRCC targets in vitro. AB-2100 also exhibited selective killing of dual antigen expressing tumors in vivo using a dual-flank subcutaneous xenograft model. Preclinical xenograft studies also demonstrated that TGFBR shRNA and SPA modules enhanced antitumor activity of ICTs. When AB-2100 potency was evaluated in the subcutaneous A498 xenograft model, treatment with AB-2100 resulted in complete and durable anti-tumor responses. In summary, preclinical data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC. Citation Format: Suchismita Mohanty, Jeremy Chen, Alma Gomez, Angela Boroughs, Irene Scarfo, Laura Lim, Kevin Dang, Marvin Chew, Rakesh Sudhakah, Michelle Nguyen, Thomas J. Gardner, Beatriz Millare, James Zhang, Darrian Moskowitz, Stanley Zhou, Neroli H. Xie, Nickolas Attanasio, Amanda Fearon, Ivan Chan, Vibhavari Sail, Vince Thomas, Jennesa Smith, Jennifer McDevitt, Levi Gray-Rupp, Alba Gonzalez, Christopher Murriel, W. Nicholas Haining. AB-2100, a PSMA-inducible CA9-specific CAR T cell product for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model [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 38.

Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRβ) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRβ cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRβ becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

Transferrin-inspired iron delivery across the cell membrane using [(L2Fe)2(μ-O)] (L = chlorquinaldol) to harness anticancer activity of ferroptosis.

Although iron is a bio-essential metal, dysregulated iron acquisition and metabolism result in production of reactive oxygen species (ROS) due to the Fenton catalytic reaction, which activates ferroptotic cell death pathways. The lipophilic Fe(III)-chelator chlorquinaldol (L; i.e., 5,7-dichloro-8-hydroxy-2-methylquinoline) strongly favors the formation of a highly stable binuclear Fe(III) complex [(L2Fe)2(μ-O)] (1) that can mimic the function of the Fe(III)-transferrin complex in terms of the strong binding to Fe(III) and facile release of Fe(II) when the metal center is reduced. It should be noted that the cellular uptake of 1 is not transferrin receptor-mediated but enhanced by the high lipophilicity of chlorquinaldol. Once 1 is transported across the cell membrane, Fe(III) can be reduced by ferric reductase or other cellular antioxidants to be released as Fe(II), which triggers the Fenton catalytic reaction, thus harnessing the anticancer activity of iron. As the result, this transferrin-inspired iron-delivery strategy significantly reduces the cytotoxicity of 1 in normal human embryonic kidney cells (HEK 293) and the hemolytic activity of 1 in human red blood cells (hRBCs), giving rise to the unique tumor-specific anticancer activity of this Fe(III) complex.

Tumor-Specific Activity of Precision Medicines in the NCI-MATCH Trial.

National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) is a precision medicine basket trial designed to test the effectiveness of treating cancers based on specific genetic changes in patients' tumors, regardless of cancer type. Multiple subprotocols have each tested different targeted therapies matched to specific genetic aberrations. Most subprotocols exhibited low rates of tumor shrinkage as evaluated across all tumor types enrolled. We hypothesized that these results may arise because these precision cancer therapies have tumor type-specific efficacy, as is common among other cancer therapies. To test the hypothesis that certain tumor types are more sensitive to specific therapies than other tumor types, we applied permutation testing to tumor volume change and progression-free survival data from 10 published NCI-MATCH subprotocols (together n = 435 patients). FDR was controlled by the Benjamini-Hochberg procedure. Six of ten subprotocols exhibited statistically significant evidence of tumor-specific drug sensitivity, four of which were previously considered negative based on response rate across all tumors. This signal-finding analysis highlights potential uses of FGFR tyrosine kinase inhibition in urothelial carcinomas with actionable FGFR aberrations and MEK inhibition in lung cancers with BRAF non-V600E mutations. In addition, it identifies low-grade serious ovarian carcinoma with BRAF v600E mutation as especially sensitive to BRAF and MEK co-inhibition (dabrafenib plus trametinib), a treatment that received accelerated FDA approval for advanced solid tumors with BRAF v600E mutation. These findings support the value of basket trials because even when precision medicines do not have tumor-agnostic activity, basket trials can identify tumor-specific activity for future study.

The Limitations of Current T Cell-Driven Anticancer Immunotherapies Can Be Overcome with an Original Extracellular-Vesicle-Based Vaccine Strategy.

The emergence of tumors associated with defects in immune surveillance often involve the impairment of key functions of T lymphocytes. Therefore, several anticancer immunotherapies have focused on the induction/strengthening of the tumor-specific activity of T cells. In particular, strategies based on immune checkpoint inhibitors, CAR-T cells, and mRNA vaccines share a common goal of inducing/recovering an effective antitumor cytotoxic activity, often resulting in either exhausted or absent in patients' lymphocytes. In many instances, these approaches have been met with success, becoming part of current clinic protocols. However, the most practiced strategies sometimes also pay significant tolls in terms of adverse events, a lack of target specificity, tumor escape, and unsustainable costs. Hence, new antitumor immunotherapies facing at least some of these issues need to be explored. In this perspective article, the characteristics of a novel CD8+ T cell-specific anticancer vaccine strategy based on in vivo-engineered extracellular vesicles are described. How this approach can be exploited to overcome at least some of the limitations of current antitumor immunotherapies is also discussed.