Drug-to-antibody Ratio Research Articles

Abstract 4700: Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity

Abstract Antibody-drug conjugates (ADCs) have ushered in a new era of precision cancer therapy, and the caspase-3-cleavable linker has demonstrated its potential as a game-changing addition to the ADC toolkit. The DEVD peptide, specifically cleaved by caspase and lysosomal enzymes, in combination with the KG peptide, provides the necessary flexibility to achieve targeted cancer cell apoptosis. This research explores the key attributes and actions of the linker, aiming to highlight its potential for revolutionizing ADC therapy. The caspase-3-cleavable linker operates through caspase-3 activation during apoptosis. After targeting cancer cells with specific antigens, the ADC is internalized into the cells, and the linker is cleaved by caspase-3, releasing the drug and initiating apoptosis. The specificity of caspase-3 extends the drug’s effect to neighboring cells, even those lacking the target antigen. This cascade effect ensures the continuous activation of ADCs until all cancer cells are eradicated. Firstly, it overcomes tumor heterogeneity by activating the drug outside of the cell. Traditional bystander killing effects rely on internalization followed by drug release and penetration of neighbor cancer cells. However, this linker's unique mechanism ensures that the drug is activated by caspase-3 outside the cell without the need for internalization, effectively targeting all cancer cells within the tumor tissue. This is achieved through the induced phenotype targeting effect (IPTE), which involves caspase-3 cleavage of the linker, inducing a phenotype in the tumor tissue that targets all cancer cells, regardless of their specific markers. Secondly, it maintains therapeutic efficacy even as the target antigen decreases. Current targeted therapies face a challenge as target antigens may diminish during treatment, leading to reduced drug effectiveness. However, the unique mechanism of the linker ensures that the drug continues to be activated as long as there is caspase-3 secretion, resulting in continuous activation of ADCs in the tumor, with a feedback amplification loop that ensures therapeutic efficacy until all cancer cells are eliminated. Lastly, the linker's superior physicochemical properties allow for a maximum drug-to-antibody ratio (DAR). Its hydrophilic nature enables independent drug conjugation, increasing therapeutic potential and mitigating aggregation issues. This innovation addresses the current limitations in ADC conjugation technologies. In summary, the caspase-3-cleavable linker represents a significant leap forward in the field of ADCs. Its unique attributes offer promising prospects for more effective and targeted cancer treatments. This linker is poised to revolutionize the landscape of targeted cancer therapy, bringing us one step closer to the dream of eradicating cancer cells with precision and efficiency. Citation Format: Ha Kyeong Lee, Byoungmo Kim, Hyo Won Chang, Sang Yoon Kim, Youngro Byun. Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity [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 4700.

Abstract 5804: Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload

Abstract LCB84 is an antibody-drug conjugate (ADC) that consists of an antibody targeting human trophoblast cell-surface antigen-2 (TROP2) linked to monomethyl auristatin E (MMAE) via a beta-glucuronidase cleavable linker with a drug-to-antibody ratio (DAR) of 4. LCB84 demonstrates tumor selective killing by multiple mechanisms, increasing both safety and efficacy in parallel. Firstly, the over-expression of TROP2 in many solid tumors allows for targeted delivery of MMAE by LCB84 to Trop2-positive tumor tissues. Secondly, the overexpression of beta-glucuronidase in the lysosome of most tumors facilitates the selective release of active payload within tumor cells compared to normal healthy tissues. Thirdly, the intrinsic selectivity of tubulin inhibitor payloads, such as MMAE, for proliferating cells provides additional protection for non-proliferating normal cells. MMAE is also cell-permeable in its free drug form and can lead to increased efficacy of LCB84 via a bystander effect, enabling activity in heterogenous tumors. LCB84 demonstrates superior efficacy in mouse tumor xenograft studies when compared to benchmark TROP2-targeting ADCs, including sacituzumab govitecan and datopotamab deruxtecan. LCB84 also shows an improved safety profile compared to these clinical benchmarks, as well as compared to traditional MMAE-bearing ADCs, in both in vitro and in vivo toxicity assessments. Additionally, LCB84 shows a stable preclinical pharmacokinetic profile with minimal loss of payload over time. The utilization of an ADC for target-mediated delivery, in combination with a stable beta-glucuronide trigger for selective payload release in tumors, allows for a superior preclinical therapeutic index which is expected to translate to a best-in-class ADC in the clinic. Citation Format: Stephen Slocum, Hyejung Kim, Chul-Woong Chung, Changsik Park. Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload [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 5804.

Abstract 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts [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 2616.

Abstract 1885: Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment

Abstract ROR1 is a type I transmembrane protein that belongs to the ROR family. It is a receptor for Wnt family signaling molecules Wnt5a, and plays a critical role in various cellular processes, such as cell proliferation, survival, and migration. Being an oncofetal protein, ROR1 exhibits limited expression in most normal tissues. However, it is abnormally expressed in various hematological and solid cancers, contributing to the development and progression of many types of cancer. Due to its overexpression in cancer, ROR1 presents as a highly attractive target for antibody-drug conjugate (ADC) therapy. The current clinical results of ROR1 ADCs have been promising in treating patients with relapsed and/or refractory (R/R) hematologic malignancies. BR111 is anti-ROR1 ADC that consists of a biparatopic antibody, stably conjugated to an antimitotic agent. The biparatopic antibodies demonstrate superior binding affinity to ROR1-expressing cancer cells compared to single epitope antibodies. Notably, our unique and innovative conjugation platform CysX™, not only prevents payload detachment during circulation and reducing the off-target toxicity, but also enables a uniformed drug-to-antibody ratio (DAR) of 4. Both in vitro and In vivo studies have highlighted the remarkable antitumor activity of BR111. Preclinical data have shown great safety profile in cynomolgus monkeys. BR111 demonstrated a wider therapeutic window for cancer treatment, outperforming the lead anti-ROR1 ADC currently in phase II/IIl trial. The promising outcome holds great potential as a therapeutic treatment for hematological and solid cancers, offering a better safety profile. BR111 has great potential in further clinical evaluations. Citation Format: Xiaobei Zhao, Jie Zhu, zhenhua Wu, Yiqun Li, Jing Li, Yaqiong Zhou, Lei Nie, Haibin Wang, Gang Chen. Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment [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 1885.

Abstract 1862: ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications

Abstract Background: Folate Receptor alpha (FRα) is a validated cancer target that is prevalently expressed in multiple cancers with high unmet need, including ovarian cancer and other gynecological cancers, NSCLC, endometrial cancer and TNBC. Due to FRα’s favorable expression profile, multiple antibody-drug conjugates (ADCs) are being explored in this setting. Here we present the preclinical characterization of a new anti-FRα ADC, ZW191. ZW191 is a bystander active antibody drug conjugate (ADC) comprised of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor (TOPO1i) payload, ZD06519, via a maleimidocaproyl (MC) anchor and a glycyl glycyl phenylalanyl glycyl (GGFG)-aminomethyl (AM) cleavable linker at a drug-to-antibody ratio (DAR) of 8. ZW191 is projected to undergo IND submission in 2024. Materials and Methods: The novel antibody and drug-linker components of ZW191 were generated, characterized, and optimally integrated. The apparent binding affinity and cellular internalization of the ZW191 antibody were determined in FRα-expressing cells. Tumor spheroid cancer cell cultures were utilized to determine ZW191’s degree of tissue penetration and its cytotoxicity. The bystander activity of ZW191 was assessed using antigen positive and negative co-culture experiments. The anti-tumor activity of ZW191 was evaluated in a panel of ovarian cancer, NSCLC, endometrial cancer and TNBC patient-derived xenografts (PDX) spanning a range of FRα expression. ZW191 was evaluated in toxicology and pharmacokinetic (PK) studies performed in non-human primates (NHP). Results: The antibody component of ZW191 features a favorable binding profile with strong binding to FRα, and drives superior tumor spheroid penetration, cellular internalization, and payload delivery compared to FRα targeted antibodies used in multiple other ADCs. ZW191 demonstrates potent activity in FRα expressing cell cultures and effective bystander activity. In a panel of PDX models representing a range of FRα expression, ZW191 demonstrates compelling anti-tumor activity across ovarian and endometrial cancers, NSCLC and TNBC. ZW191 demonstrates an encouraging tolerability profile in NHP, with favorable PK. The promising efficacy, tolerability, and PK support the potential of ZW191 as a novel therapeutic agent that may help address unmet need in patients with high and low FRα expressing cancers. Citation Format: Sam Lawn, Andrea Hernandez Rojas, Raffaele Colombo, Jodi Wong, Kaylee Wu, Vincent Fung, Manuel Lasalle, Mark E. Petersen, Lemlem Degefie, Araba Sagoe-Wagner, Dunja Urosev, Luying Yang, Ambroise Wu, Catrina Kim, Amos Chua, Kurt Stahl, Geoffrey C. Winters, John C. Fann, Jamie Rich, Stuart Barnscher. ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications [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 1862.

Abstract 479: Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots

Abstract Acute Myeloid Leukemia (AML) treatment faces considerable challenges, in particular the delivery of therapeutics with sufficient potency and specificity. Traditional Antibody-Drug Conjugates (ADCs) offer a strategic approach to cancer therapy by combining the targeting capabilities of monoclonal antibodies with the cell-killing effect of cytotoxic drugs. However, the limitations in drug-to-antibody ratio (DAR) and the challenges in controlling the release kinetics have often hindered their therapeutic window suggesting novel approaches are necessary. Our approach integrates DNA nanobot technology to facilitate targeted delivery of anthracycline agents directly to CD37-expressing leukemic cells. CD37, a tetraspanin superfamily antigen, has shown promise as a target for AML due to its selective expression on immune cells and favorable internalization properties. By harnessing DNA origami assembly techniques, we developed a customizable platform capable of accommodating a significantly higher payload of chemotherapeutic agents than traditional ADCs. This increased payload potential, paired with the precision targeting of CD37, presents a potent therapeutic strategy with the possibility of reduced systemic toxicity. Here we present preclinical evidence demonstrating targeted delivery of our DNA nanobot drug delivery device and anthracycline payload to CD37+ AML target cells in vitro. In addition, we show targeted efficacy in vitro and in vivo of our anthracycline-loaded DNA nanobot-enabled delivery system, which significantly outperformed free anthracycline in AML target cells. Collectively, our findings suggest an improvement in the therapeutic index of anthracycline and antimitotic agents as well as the ability to combine multiple drug pathways in a single delivery device. The implications of this work extend beyond AML, offering a versatile platform that could revolutionize ADC chemistry and targeted drug delivery. We propose that our DNA nanobot delivery system signifies a pivotal advancement in the field of targeted cancer therapy, with the potential to overcome longstanding barriers in the treatment of AML and potentially other malignancies. Citation Format: Nicholas Vantangoli, Patrick D. Halley, Jeffrey R. Spitzner, John C. Byrd, Karilyn T. Larkin, Carlos E. Castro, Christopher R. Lucas. Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots [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 479.

Abstract 6582: Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors

Abstract Background: Nectin-4 is a cell membrane adhesion protein overexpressed in several solid tumors, including urothelial, breast, lung, ovarian, and pancreatic cancers, with limited expression in normal tissues, making it an ideal target for antibody-drug conjugates (ADC). Enfortumab Vedotin (EV, PADCEV®) is an ADC targeting Nectin-4 with a monomethyl auristatin E (MMAE) payload approved for the treatment of patients with urothelial carcinoma (UC), but with limited clinical activity reported beyond UC. In addition, EV causes peripheral neuropathy and skin toxicity, leading to treatment discontinuation. To overcome these limitations and the mechanism of resistance to EV, IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 with an improved therapeutic index, was developed. Methods: In vitro studies were performed to investigate internalization, tumor cell killing and bystander effect, antibody-dependent cellular cytotoxicity (ADCC) and complement activation. In vivo efficacy studies were performed on cell line-derived xenografts (CDX) of triple negative breast cancer, patient-derived xenografts (PDX) of urothelial carcinoma and syngeneic tumor models expressing human Nectin-4. IPH45 efficacy was compared to EV. Toxicology studies to determine the dose range were conducted in rat and non-human primates (NHP). Results: IPH45 consists of a humanized IgG1 targeting a unique epitope in human Nectin-4, non-overlapping with EV, a cleavable linker, and a topoisomerase I inhibitor with a high drug-to-antibody ratio (DAR). In vitro, IPH45 is internalized upon binding to its target and induces direct killing of Nectin-4 expressing tumor cells as well as bystander killing of Nectin-4 non-expressing tumor cells in mixed culture. IPH45 mediates ADCC and complement-mediated killing. IPH45 demonstrates activity in CDX and PDX models with similar effective dose as EV. Moreover, IPH45 shows anti-tumor efficacy in vitro and in vivo in primary and secondary EV resistant models. In dose-range finding toxicology studies in rats and NHP, IPH45, unlike EV, does not elicit skin toxicity. IPH45 remains well tolerated up to the highest dose tested, suggesting a potentially larger therapeutic index than MMAE-based ADCs. Pharmacokinetic analyses in mice, rats, and NHP show minimal release of free toxin in the serum and half-life compatible with Q2W or less frequent dosing schedules in clinic. Conclusion: IPH45 is a topoisomerase I ADC targeting Nectin-4 with the potential for larger therapeutic index than EV, improved safety and dosing regimen, and ability to overcome primary and secondary resistance to EV or other Nectin-4 ADC in development. IPH45 is progressing to the clinic where the translatability of this unique profile into improved patient outcomes will be assessed across indications. Citation Format: Romain Remark, Cécile Bonnafous, Laura Chiossone, Caroline Soulas, Cyril Perrier, Guillaume Habif, Sivan Bokobza, Aurélie Maguer, Rachel Courtois, Julie Lopez, Grégory Fenaux, Olivier Benac, Barbara Carrette, Robin Letay-Drouet, Raja Bonifay, Séverine Augier, Léa Simon, Benjamin Rossi, Ariane Morel, Agnès Represa, Nicola Beltraminelli, Yannis Morel, Carine Paturel, Eric Vivier. Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing 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 6582.

Abstract 739: AMT-253, a first-in-class MUC18-targeting antibody-drug conjugate, for the treatment of MUC18-positive solid tumors

Abstract MUC18, also known as MCAM (melanoma cell adhesion molecule) or CD146, is a type I transmembrane glycoprotein originally identified as a cell adhesion molecule of melanoma that plays important roles in tumor growth and progression including tumor angiogenesis, epithelial-mesenchymal transition (EMT), and metastasis. MUC18 is overexpressed in a variety of solid tumors including (but not limited to) melanoma, osteosarcoma, head and neck, lung, esophagus, breast, ovarian, and cervical cancer and hepatocellular carcinoma, with restricted presence on normal healthy tissues, mainly on blood vessels and a minor subset of T helper cells. Limited expression in normal tissues and high expression in tumors makes MUC18 a promising target for the development of antibody drug conjugate (ADC). AMT-253 is a novel ADC targeting MUC18. It is composed of a humanized MUC18-targeting IgG1 antibody conjugated with Exatecan, a topoisomerase I inhibitor, attached to a proprietary self immolative T moiety linker through reduced interchain disulfide bonds with a drug-to-antibody ratio (DAR) of 8. AMT-253 was stable in vitro with less than 0.5% of payload releasing when incubated with human, monkey, mouse or rat plasma for 21 days at 37°C. AMT-253 selectively bound to cell surface MUC18 and was efficiently internalized into MUC18-positive cancer cells. AMT-253 exhibited antiproliferative activity against several MUC18-positive cancer cell lines and demonstrated potent bystander killing effect in vitro. Treatment with AMT-253 resulted in tumor growth inhibition or regression in a panel of MUC18 positive cell line derived xenograft (CDX) or patient derived xenograft (PDX) models. In addition, AMT-253 was well tolerated in a GLP compliant toxicity study in cynomolgus monkeys at dose levels ≤45 mg/kg. Taken together, our preclinical in vivo efficacy and toxicity studies demonstrated that AMT-253 has a wide therapeutic window and could potentially provide benefits for cancer patients with highly unmet medical needs. AMT-253 is currently being evaluated in a Phase I first-in-human clinical trial (NCT05906862). Citation Format: Jing Shi, Jiaqing Yan, Yaling Huang, Jun Guo, Yan Kong, Xun Meng, Shu-Hui Liu. AMT-253, a first-in-class MUC18-targeting antibody-drug conjugate, for the treatment of MUC18-positive 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 739.

Abstract 1909: Advancing a novel tubulin-inhibitor ADC technology: The Adcentrx auristatin platform offers enhanced efficacy and safety profiles compared to vedotin technology

Abstract Auristatins represent an important class of anti-mitotic antibody-drug conjugate (ADC) payloads with potent cytotoxic effects on rapidly dividing cancer cells. Most notably, monomethyl auristatin E (MMAE) stands as the most extensively validated compound, laying the foundation for the vcMMAE (vedotin) linker-payload technology that has contributed to the approval of several ADCs, including brentuximab vedotin, polatuzumab vedotin-piiq, tisotumab vedotin-tftv, and enfortumab vedotin-ejfv. However, while these first-generation ADCs have paved the way for a paradigm shift in cancer treatment away from traditional chemotherapy, there remains a discernible opportunity for improvement in both efficacy and safety. In this context, we introduce the Adcentrx auristatin platform, which substantially expands the therapeutic window of auristatin-ADCs beyond the vedotin technology. Through systematic exploration, we have synthesized >100 proprietary auristatin analogues with a range of distinctive properties. We identified optimized linker-payloads by strategically pairing lead payload candidates with our proprietary i-ConjugationTM, an irreversible, stable cysteine conjugation technology, and fine-tuned linkers. The resulting ADCs with a drug-to-antibody ratio (DAR) of 8 were extremely hydrophilic with excellent in vivo pharmacokinetics, and sustained DAR retention for a period of 21-days. Comparative assessments against vedotin-ADCs revealed that Adcentrx-ADCs demonstrated superior efficacy across multiple targets and indications in various mouse xenograft models. In Sprague-Dawley rats, Adcentrx-ADCs exhibited remarkable tolerability, albeit their high drug loading of 8. In summary, our preclinical studies underscore the significant enhancement in the therapeutic index achieved by the Adcentrx auristatin platform. Furthermore, this technology presents a promising alternative mode of action to camptothecins, exhibiting tolerance to targets expressed in non-proliferative normal tissues but without the burden of severe dose-limiting toxicities such as interstitial lung disease. Citation Format: Dong Jun Lee, Sabine Rottmann, Anna Wang, Peter P. Challita, Andrew M. Hau, Wes Sisson, Mario M. Kuo, Kris Zhang, Monica Leung, Alexis Mahloch, Erin Nye, Paul Datta, Sam Janssen, Felipe Acosta, Oscar Betancourt, Maria Shahmoradgoli, Alexander Chu-Kung, MaoJun Guo, Pia M. Challita-Eid, Hui Li. Advancing a novel tubulin-inhibitor ADC technology: The Adcentrx auristatin platform offers enhanced efficacy and safety profiles compared to vedotin technology [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 1909.

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.

Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

Abstract 740: CNTN4 as a novel target for solid cancer with antibody-drug conjugate

Abstract As a novel immune checkpoint, we previously confirmed that contactin 4 (CNTN4) regulates T cell activity negatively by binding to amyloid precursor protein (APP) on T cells. CNTN4 is highly expressed in various types of tumors, including gallbladder, pancreas, stomach, endometrium, liver, prostate, bladder cancer, melanoma, and other tumors, with positive rates over 70% through immunohistochemistry analysis in contrast to low-level expression in normal tissues. Based on the tumor-specific expression profile of CNTN4, we evaluated the potential of CNTN4 as a novel target for antibody-drug conjugate (ADC). First, the internalization of the anti-CNTN4 antibody in the IND submission stage, GENA-104A16, into CNTN4-positive cancer cells was confirmed. For investigation of the potential of targeting CNTN4 using ADC, clinically validated various linker-payloads (MMAE, MMAF, SN-38, and exatecan derivate) were conjugated to GENA-104A16 using thiol maleimide conjugation with average drug-to-antibody ratio (DAR) in the range of 4.0 to 4.8. After preparing ADCs, we confirmed the binding affinity for CNTN4 was maintained as high even after the payload conjugation through an ELISA. In vitro studies have demonstrated the highly cytotoxic effect of MMAF conjugate among the conjugates of GENA-104A16 on CNTN4-positive cancer cells compared to negative cancer cells. And there was no cytotoxic effect on normal cells. Furthermore, treating MMAF conjugate of GENA-104A16 showed promising efficacy in the Pan02 pancreatic orthotopic mouse model and the patient-derived xenograft model. These results suggest that CNTN4 could be a highly potential ADC target that could expand the options for cancer treatment strategies. Citation Format: Mi Young Cha, Hyunuk Kim, Seungmin Byun, Youngeun Ha, Kitae Park, Hyunkyung Yu, Bu-Nam Jeon, Mira Kim, Soojung Moon, Kyung Mi Park, Hansoo Park. CNTN4 as a novel target for solid cancer with antibody-drug conjugate [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 740.

Abstract 5810: The impact of scaffold, linker, homogeneity and payload selection on the efficacy and tolerability of anti-tubulin ADCs

Abstract Antibody-drug conjugates (ADCs) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The properties of an ADC, including its activity and safety profile, can be highly influenced by its characteristics and individual components, commonly viewed as the antibody, payload, linker, and drug-to-antibody ratio (DAR). However, multiple other characteristics can also significantly influence an ADC’s overall drug-like properties, including DAR homogeneity, method of bioconjugation, hydrophilicity, and charge balance. Due to the multiparametric nature of ADC platform optimization, there are few examples in preclinical research, and even fewer in the clinical realm, where comparisons across platforms have been investigated for the same target, both preclinically and clinically, to elucidate how deliberate modifications in molecular design can improve drug-like properties and clinical outcomes. To that end, here we describe the improvements observed in preclinical characteristics, as well as their translational relevance to corresponding clinical characteristics. Two anti-NaPi2b ADCs were produced using two different platforms, XMT-1536 (Dolaflexin platform) and XMT-1592 (Dolasynthen platform), conjugated to the same antibody and employing the same payload. The Dolasynthen platform was deliberately designed, taking into account preclinical and clinical learnings from earlier ADC platforms, to improve both safety and efficacy. In addition, comparisons to clinically approved anti-tubulin ADC platforms are made to provide further context. Citation Format: Scott D. Collins, Natalie Keirstead, Marc Damelin, Dorin Toader, Katsu Ishida, Niyanta N. Kumar, Kelly L. Lancaster, Kalli C. Catcott, Annika Yau, Mohan Bala, Timothy B. Lowinger. The impact of scaffold, linker, homogeneity and payload selection on the efficacy and tolerability of anti-tubulin 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 5810.

Abstract 1902: ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio (DAR) of 8. ADRX-0706 utilizes a novel tubulin inhibitor payload AP052 with a proprietary cleavable linker utilizing Adcentrx’ proprietary i-ConjugationTM technology, which results in excellent biophysical features. Most noteworthy, ADRX-0706 displays a very stable PK profile with overlapping curves for total antibody and ADC as well as full DAR retention in circulation as determined by mass spectrometry. This translates to superior efficacy of ADRX-0706 over EV in multiple in vivo cancer cell line- and patient-derived xenograft mouse models across indications. Detailed PK/PD characterization of ADRX-0706 validates the mechanism of action as an anti-mitotic agent inducing cell cycle arrest and apoptosis. In addition, ADRX-0706 treatment leads to increased PD-L1 expression in mouse pharmacology models, which is a consequence of immunogenic cell death as described for this payload class. These findings are in line with emerging data on EV and provide rationale for combination regimens with checkpoint inhibitors. Besides the improvement in efficacy, ADRX-0706 was also designed for superior safety. A payload distribution study in tumor-bearing mice reveals a preferential and increased delivery of payload to tumors and lower payload delivery to normal tissues as well as lower free payload levels in plasma for ADRX-0706 compared to EV. Taken together, these data provide association of the improved drug-linker stability of ADRX-0706 to an improved Nectin-4 directed delivery of AP052 payload to the tumor and lower exposure in normal tissues, which may translate to an improved therapeutic window. In summary, the preclinical efficacy and safety properties of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Maria Shahmoradgoli, Andrew Hau, Dong Jun Lee, Anna Wang, Peter P. Challita, Oscar Betancourt, Wes Sisson, Mario M. Kuo, Kris Zhang, Albert Goldson, Sam Janssen, Paul Datta, Felipe Acosta, Alexander Chu-Kung, Hui Li, Pia M. Challita-Eid, Sabine Rottmann. ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window [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 1902.

Abstract 3125: TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors

Abstract Tumor-associated carbohydrate antigens (TACAs) are short oligosaccharides expressed on the surface of cancer cells and play key roles in cellular communication, immune evasion, apoptosis and metastasis. TACAs are therefore interesting targets for antibody-based therapies in cancer. We have generated an antibody (TCX-101) against a TACA structure with improved binding affinity compared to other monoclonal antibodies (mAbs) against the same target as shown by surface plasmon resonance (~ 10x superior KD), as well as high level of specificity against glycan species related to the target, as accessed by glycan array, flow cytometry, and immunocytochemistry or immunofluorescence. Immunohistochemistry studies on fresh-frozen tissue microarrays showed high expression of the target in various solid tumors (e.g. gastrointestinal, lung, and breast) and only low expression in normal tissues. In vitro, TCX-101 has ~ 4x higher maximum binding, ~ 2x faster binding kinetics, ~ 4x faster cellular internalization and induces ~ 2x higher antibody-dependent cellular cytotoxicity than a reference antibody. Monomethyl auristatin E (MMAE) was used in a drug-antibody ratio (DAR) of 4 to generate an antibody-drug conjugate (ADC). This ADC was effective in in vitro toxicity assays against murine B16 melanoma cells induced to express the target (measurable effect on WT cells), as well as human breast cancer MCF7 WT cells (~ 25x less activity on target-knockout MCF7 cells, Table 1). When tested in vivo, TCX-101 was highly effective in two different xenograft mouse models (B16, MCF7) with more than 95% tumor growth inhibition compared to vehicle, and no signs of toxicity (no weight loss, no histophatological findings). A fully humanized version of TCX-101 with improved binding properties (EC50 6.27 nM vs. 8.13 nM) was generated and will be evaluated in further in vivo experiments. In conclusion, TCX-101 is a promising ADC for the treatment of various solid tumors. IC50 [nM] TCX-101-MMAE 4 DAR B16 KI 7.64 B16 WT no killing observed MCF7 WT 1.97 MCF7 KO 45.97 Citation Format: Fausto G. Gomes, Francesco Muraca, Gustavo M. Schmidt Garcia Moreira, Kathrin Rösch, Maria Bräutigam, Peter Sondermann, Matthias Ocker. TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the 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 3125.

Abstract 2601: Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker

Abstract Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2. Citation Format: Yulia Liubomirski, Galia Tiram, Anna Scomparin, Samer Gnaim, Sayantan Das, Sachin Gholap, Liang Ge, Eilam Yeini, Omri Shelef, Arie Zauberman, Nir Berger, Doron Kalimi, Mira Toister-Achituv, Christian Schröter, Stephan Dickgiesser, Jason Tonillo, Min Shan, Carl Deutsch, Stanley Sweeney-Lasch, Doron Shabat, Ronit Satchi-Fainaro. Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker [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 2601.

Abstract 6352: Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types

Abstract Traditional antibody-drug conjugates leverage the specificity of antibodies to deliver cytotoxic payloads to tumor microenvironments (TMEs), thereby reducing systemic toxicities associated with non-targeted chemotherapeutics. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to selectively deliver synthetic immune agonists to the TME without systemic immune activation. The first generation of ISACs and the majority of approved and investigational ADCs contain payloads linked to native lysine or cysteine residues, which can lead to heterogenous drug-antibody ratios (DARs), reduced antibody stability, and labile conjugation, all of which limit therapeutic index. Here we describe the generation and preclinical characterization of ARX622, a site-specific TLR7-selective agonist ISAC targeting HER2. To avoid the instability issues of previous generation ADCs and ISACs, ARX622 has a homogenous and stable DAR via oxime bond-mediated payload conjugation to a synthetic amino acid, para-acetyl phenylalanine (pAF), which is recombinantly incorporated at defined heavy chain positions. Similar to cytotoxic ADCs built on this site-specific conjugation platform, ARX622 ISAC and total mAb pharmacokinetic (PK) profiles in non-human primates (NHPs) are indistinguishable, demonstrating in vivo stability and minimal payload deconjugation. Accordingly, ARX622 displays an extended PK profile in mice compared to a DAR-matched random cysteine ISAC containing the same antibody and payload. In the presence of HER2-expressing cells, ARX622 selectively induces proinflammatory cytokine production, type I and III IFN secretion, and APC maturation. In a syngeneic tumor model, ARX622 promotes complete tumor regressions, immunologic memory, and epitope spreading. In xenograft systems, single doses of ARX622 induce complete regression of large, established tumors (>1000mm3), and robustly inhibit tumor growth in mice that have progressed through prior treatment with HER2-targeted mAbs or ADCs. In HER2-low xenografts, ARX622 monotherapy inhibits tumor growth, and promotes complete regression of late-stage (800mm3) tumors when sequentially combined with a HER2-ADC. Based on PK data at efficacious doses in mice and tolerated exposures in NHP toxicity studies, ARX622 has an estimated therapeutic index of ~60x. Taken together, ARX622’s mAb-like properties, high stability, broad immune mechanism induction, strong anti-tumor activity alone or in combination with ADCs, and wide therapeutic index make it a strong candidate for further development toward treating patients with a variety of HER2-expressing solid tumors. Citation Format: David Mills, Ji Young Kim, Erica Wood, Keith Tatsukawa, Nick Knudsen, Jay Nelson, Lillian Skidmore, Kedar GC, Hon Tran, Manoj Pal, Hamidreza Hashemi, Ying Buechler, Shawn Zhang, Dan O'Connor. Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types [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 6352.

Abstract 5797: Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing

Abstract Introduction: ADCs have had tremendous impact on patient outcomes in breast cancer and are now second line therapy for stage IV HER2 positive metastatic breast cancer. However, many patients fail to respond or relapse after treatment with ADC therapies due to tumor heterogeneity and resistance to ADC payloads. We are developing next generation ADCs that deliver targeted combination chemotherapies with a single molecule. Method: CatenaBio has developed a novel system capable of attaching distinct payloads at different sites to the same antibody, enabling the production of highly stable single-molecule targeted combination therapies, Multi-Payload-Conjugates (MPCs), with a well-defined drug-antibody ratio (DAR). Results: We screened combinations of different payloads targeting several different mechanisms of cell division attached to trastuzumab at different DARs to optimize tumor cell killing. These targeted combination ADCs demonstrated robust killing in both HER2 high and low tumor cell lines. Additionally, these novel MPCs show potent inhibition of tumor growth in a HER2+ xenograft model of cancer. Conclusion: Antibody Drug Conjugates have revolutionized the treatment of high HER2 positive breast cancer. More recently advances have been made in the design of ADCs to expand indications to include HER2 low as well as HER2 negative patients. Multi-payload Conjugates® offer the next step in ADC design and allow for the combination of multiple mechanisms of action in a single MPC that are highly effective across multiple breast cancer cell lines and HER2 expression levels. These molecules offer the potential to circumvent tumor resistance pathways and deliver deeper and more durable responses. Citation Format: Richard L. Kendall, Marco Lobba, Saurabh Johri, Chanez Symister, Devin Trinter, Maxwell Nguyen, Daniel Gutierrez, Samantha Brady, Andrew Lau, Matthew B. Francis. Next generation antibody drug conjugates: Multi-payload conjugates targeting multiple mechanisms of cell killing [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 5797.

Abstract 3206: Tumor-targeted nanoparticles for cancer therapy

Abstract Introduction: Untargeted cancer therapy is often toxic, limiting the maximum tolerated dose (MTD) and thus potential cure. Targeted therapy with antibody-drug conjugates (ADCs) has demonstrated greater efficacy, as with those directed at HER2 for triple negative breast cancer and other HER2 expressing tumors. However, ADC drug-antibody ratio (DAR) is limited, typically 3 to 1. In contrast, antibody-targeted nanoparticles deliver thousands of drug molecules per nanoparticle, with potentially greater anti-tumor effect. Experimental Procedures: Antibody targeted nanoparticles (TNP) were created that preferentially deliver cytotoxic molecules to tumor cells. TNPs were created with various antibodies and payloads and evaluated for their efficacy against 12 different adult and pediatric malignancies (see below), using tumor xenografts in NOG-SCID mice. Survival of control, free drug, and TNP treated animals was compared. MED was also determined. Non-tumor bearing animals treated at the same dose and schedule were evaluated for evidence of toxicity. PK/PD metrics were obtained and drug delivery to normal and tumor tissue was evaluated microscopically and by mass spectroscopy. Data: Plasma TNP drug levels persist for 48 hours, compared to free drug which is undetectable by 2 hours. Microscopically, nanoparticles accumulate in tumor tumor tissue but not normal tissue. Drug levels are typically 15 times higher in tumor tissue than normal tissue by quantitative mass spectroscopy. The minimum effective dose (MED) is reduced 25-fold for Ewing sarcoma, from 50 mg/kg to 2 mg/kg. TNPs also cross the blood-brain barrier (BBB), and were used to treat orthotopic GBM PDX xenografts, prolonging animal survival 3-fold over control or free drug treated animals. Ewing sarcoma, osteosarcoma, triple negative breast cancer, GBM, hepatoma, and adult ALL, bearing animals all showed complete tumor ablation and 100% survival when treated biweekly at 10mg/kg. Pancreatic, prostate, and ovarian Ca showed 100% survival in TNP treated mice after all control animals had died but remained tumor bearing with slow but progressive tumor growth when treated identically. Lung and colorectal Ca and melanoma bearing animals treated similarly with TNPs showed 50-80% survival at the time of control animal death. No animal in any study showed evidence of off-target toxicity. Conclusions: Tumor antigen targeted, cytotoxic molecule containing nanoparticles have been created that show broad utility in the treatment of at least a dozen different adult and pediatric cancers. Six of 12 types showed complete tumor ablation when treated at 10mg/kg biweekly injections, and the remainder showed enhanced anti-tumor effect and survival compared to free drug treatment. We conclude that use of tumor antigen targeted, small molecule cytotoxic containing nanoparticles results in dramatically enhanced tumor response and in many case, complete tumor ablation where the same free drug fails to do so. Citation Format: HyungGyoo Kang, Bryon Upton, Jon Nagy, Timothy Triche. Tumor-targeted nanoparticles for cancer therapy [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 3206.

Abstract 2623: Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index

Abstract TUB-030 is a novel Antibody-Drug Conjugate (ADC) targeting the oncofetal 5T4 antigen, a glycoprotein, that is functionally involved in regulation of the Wnt signalling pathway and promoting cellular motility and adhesion. Immunohistochemical analysis identified 5T4 overexpressed in many types of cancers, including bladder, lung, breast, stomach, esophagus, head and neck, colon, and ovarian. Analysis in fresh frozen healthy human tissue with the therapeutic mAb candidate of TUB-030 revealed limited cross reactivity. Ethynylphosphonamidate conjugation chemistry was used to build TUB-030 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting 5T4 using a cleavable linker system. Overall, TUB-030 contrasts previous ADC designs targeting 5T4, implementing several layers of differentiation in terms of antibody, payload and conjugation technology. In vitro, TUB-030 is characterized by sub-nanomolar affinity to 5T4, efficient target-mediated internalization and strong cytotoxicity towards cancer cells from different tumor indications with a broad range of 5T4 expression levels. Differently to previous 5T4-targeting ADCs, TUB-030 has efficient bystander activity against co-cultured target-negative cells, which facilitates targeting of tumors with heterogenous 5T4 expression. Pharmacokinetic analysis showed that TUB-030 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation and antibody-like clearance profile contrasting maleimide-conjugated ADCs and enabling most efficient and continued delivery of Exatecan to the tumor site. Single-dose treatment with TUB-030 results in long-lasting tumor regression with high complete remission rates across a variety of both cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) cancer models of multiple tumor indications in vivo, including models with low target expression. Preliminary repeat-dose toxicological assessment of TUB-030 in a pharmacologically relevant non-human primate species demonstrated that TUB-030 is well tolerated. The preclinical results underline that TUB-030 with novel ADC technology has the potential to enable 5T4 as a therapeutic target and provide treatment options in numerous solid cancer indications with high unmet medical need supporting further development towards the clinic. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index [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 2623.