Potent Cytotoxic Payload Research Articles

Use of a Cyclic α-Alkylidene-β-Diketone as a Cleavable Linker Strategy for Antibody-Drug Conjugates.

In the fast-evolving landscape of targeted cancer therapies, the revolutionary class of biotherapeutics known as antibody-drug conjugates (ADCs) are taking center stage. Most clinically approved ADCs utilize cleavable linkers to temporarily attach potent cytotoxic payloads to antibodies, allowing selective payload release under tumor-specific conditions. In this study, we explored the utilization of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), a cyclic β-diketone featuring an active alkylidene group, to develop a novel chemically labile linker. This linker was designed to exploit the difference in reduction potential between the intracellular compartment and plasma. Upon reduction of an azido trigger strategically installed neighboring the cyclic β-diketone, the resulting nucleophilic primary amine reacts with the alkylidene group facilitated by a favorable ring closure reaction in accordance with Baldwin's rules. Consequently, this reaction enables the simultaneous release of the attached cytotoxic payload. The therapeutic utility of this novel linker strategy was demonstrated by separate conjugation of the linker to two epidermal growth factor receptor (EGFR)-targeting ligands to afford a peptide-drug conjugate and an ADC. This work comprises a significant contribution to the bioconjugation field by introducing the alkylidene cyclic β-diketone as a tunable scaffold used for the temporary conjugation of therapeutic agents to peptides and proteins.

Late-Stage Desulfurization Enables Rapid and Efficient Solid-Phase Synthesis of Cathepsin-Cleavable Linkers for Antibody-Drug Conjugates.

The synthesis of linker-payloads is a critical step in developing antibody-drug conjugates (ADCs), a rapidly advancing therapeutic approach in oncology. The conventional method for synthesizing cathepsin B-labile dipeptide linkers, which are commonly used in ADC development, involves the solution-phase assembly of cathepsin B-sensitive dipeptides, followed by the installation of self-immolative para-aminobenzyl carbonate to facilitate the attachment of potent cytotoxic payloads. However, this approach is often low yield and laborious, especially when extending the peptide chain with components like glutamic acid to improve mouse serum stability or charged amino acids or poly(ethylene glycol) moieties to enhance linker hydrophilicity. Here, we introduce a novel approach utilizing late-stage desulfurization chemistry, enabling safe, facile, and cost-effective access to the cathepsin B-cleavable linker, Val-Ala-PABC-MMAE, on resin for the first time.

Efficacy of Antibody Drug Conjugates Alone and in Combination with other Agents in Metastatic Urothelial Carcinoma: A Scoping Review.

Antibody drug conjugates represent a promising class of antineoplastic agents comprised of a monoclonal antibody linked to a potent cytotoxic payload for targeted delivery of chemotherapy to tumors. Various antibody drug conjugates have demonstrated impressive efficacy in patients with metastatic urothelial carcinoma in clinical trials, leading to two FDA approved therapies and several other agents and combinations in clinical development. A comprehensive systematic review was undertaken utilizing the principles of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Queried databases included Ovid MEDLINE, Ovid Embase, Web of Science Core Collection and Cochrane CENTRAL Trials. The search sought to identify prospective therapeutic clinical trials in humans with metastatic urothelial carcinoma with a single-arm or randomized controlled trial design investigating antibody drug conjugate-containing regimens. The literature search yielded 4,929 non-duplicated articles, of which 30 manuscripts and conference abstracts were included, which derived from 15 clinical trials including 19 separate cohorts with efficacy outcome results. Eleven trials investigated ADC monotherapy, while two investigated combination regimens, and the remaining two studies were mixed. Five unique ADC targets were represented including Nectin-4, Trop-2, HER2, Tissue Factor, and SLITRK6. Twelve clinical trial cohorts required prior treatment (63%). Objective response rate was reported for all studies and ranged from 27-52% for ADC monotherapies and 34-75% for ADC plus anti-PD-1 agents. Time to event outcome reporting was highly variable. In addition to enfortumab vedotin and sacituzumab govitecan, various HER2-targeted antibody drug conjugates and ADC-anti-PD-1 combination regimens have demonstrated efficacy in clinical trials and are poised for clinical advancement.

A phase I study of EO-3021 in adult patients with solid tumors likely to express CLDN18.2.

TPS429 Background: Claudin 18 isoform 2 (CLDN18.2) belongs to a family of tight junction proteins with broad expression in gastric and gastroesophageal junction (GEJ), pancreatic, esophageal, and other solid tumors. Expression of CLDN18.2 in normal tissue is limited to the gastric mucosa, making it a promising antibody-drug conjugate (ADC) therapeutic target. Currently, there are no approved therapies targeting CLDN18.2. EO-3021 (also known as SYSA1801) is an ADC comprised of a fully human CLDN18.2 monoclonal antibody (mAb) specifically conjugated at glutamine 295 with a cleavable linker and MMAE with a homogenous drug-to-antibody ratio (DAR) of 2. In preclinical models, EO-3021 selectively delivers a potent cytotoxic MMAE payload directly to cancer cells expressing CLDN18.2, retains antibody-dependent cellular toxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, and exhibits a bystander effect.1 In an ongoing Phase 1 study in China (NCT05009966), EO-3021 exhibited signs of anti-tumor activity in patients with gastric cancer and had an acceptable safety profile.2 Outside of Greater China, EO-3021 is being evaluated by Elevation Oncology for the treatment of patients with advanced or metastatic solid tumors likely to express CLDN18.2, particularly gastric and GEJ adenocarcinomas. Methods: This is a Phase I, open-label, multi-center, dose escalation and expansion study to investigate the safety, tolerability, pharmacokinetics (PK) and preliminary anti-tumor activity of EO-3021 in patients with solid tumors likely to express CLDN18.2 (NCT05980416). Patients whose tumors have progressed on or after standard therapy, or who are intolerable for available standard therapy are eligible for participation. Approximately 120 patients will be enrolled in this study. There are 4 planned dose levels in the dose finding portion of the study. Patients will receive EO-3021 intravenously (IV) once every three weeks until disease progression or unacceptable toxicity. The expansion cohort will evaluate EO-3021 in patients with gastric/GEJ adenocarcinoma. Provision of tumor samples (archived and fresh biopsy) is required for study enrollment. CLDN18.2 expression in tumor tissue via will be assessed by retrospective evaluation via central immunohistochemistry assay; results will be explored for possible correlation with tumor response. Enrollment in the dose escalation portion of the study began in August 2023. 1. Dan M, et al. Cancer Res. 83, 2023. 2. Wang Y, et al. J Clin Oncol. 41, 2023. Clinical trial information: NCT05980416 .

Leveraging TROP2 Antibody-Drug Conjugates in Solid Tumors.

Antibody-drug conjugates (ADCs) have become the cornerstone of effective therapeutics in solid and hematological malignancies by harnessing potent cytotoxic payloads with targeted tumoricidal delivery. Since the monumental shift occurred with HER2-targeted ADCs, the discovery of the TROP2 antigen has revolutionized the landscape of ADC development. Moving beyond the traditional ADC design, multiple novel ADCs have successfully shaped and improved survival outcomes in patients with various tumor histologies. Here we review and contrast the clinical impact of the well-known TROP2 ADCs currently in clinical use. We also shed light on upcoming investigational TROP2 ADCs showing promise with novel ADC platforms.

Abstract LB214: A first-in-class bispecific antibody-drug conjugate (DM002) targeting HER3 and the juxtamembrane domain of MUC1

Abstract Despite the entry of new anti-cancer drugs into the market, there were more than 10 million deaths from cancer globally in 2021, with cancers of the lung, stomach, breast, and pancreas contributing to the most cancer-related deaths in China and the USA. Accordingly, there is an urgent need for improved therapeutic interventions. Antibody-drug conjugates (ADC) are novel drugs that exploit the specificity of a monoclonal antibody for target antigens expressed on cancer cells in order to achieve targeted delivery of a potent cytotoxic payload. More recently, bispecific ADCs (BsADC) targeting two tumor-associated antigens (TAA) have been developed to further amplify tumor cell specificity while minimizing toxicity to normal tissue, thus allowing for broader therapeutic windows. HER3 and MUC1 are two TAAs that are commonly expressed/co-expressed on multiple tumor types, including lung, gastric, breast and pancreatic cancer; however, neither antigen has been successfully targeted by effective drugs: of the candidates targeting HER3 antigen being evaluated in human clinical studies, most are well-tolerated, but with limited efficacy. On the other hand, high levels of MUC1 in diseased tissues can undergo auto-proteolysis, so that drugs targeting the MUC1-N region will be neutralized before reaching the tumor tissues, thereby limiting the recognition of tumor cells. To overcome these challenges, we generated fully human bispecific antibody candidates with human-monkey cross-species reactivity that target HER3 and the juxtamembrane domain of MUC1. These bsAbs exhibit higher endocytic activity in HER3-low tumor cell lines compared with other currently available HER3 mAb. These bsAbs were subsequently conjugated with monomethyl auristatin E (MMAE) via a protease-cleavable linker to obtain first-in-class BsADC candidates, DM002. DM002 candidates showed robust anti-tumor activity in multiple CDX and PDX models of lung, breast, gastric and pancreatic cancer; most notably, DM002 candidates outperformed benchmark ADCs in BP0508 lung PDX models. Together, these data indicate that DM002 will be a promising therapeutic drug for patients with HER3 and MUC1 co-expressing tumors. Citation Format: Yifu Zhang, Chenzhang Shang, Nannan Wang, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. A first-in-class bispecific antibody-drug conjugate (DM002) targeting HER3 and the juxtamembrane domain of MUC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB214.

Abstract 1987: Delivery of potent cytotoxic camptothecin derivatives to tumors by OncoFAP-based small molecule-drug conjugates

Abstract Small Molecule-Drug Conjugates (SMDCs) are anti-cancer pro-drugs composed by a tumor-targeting small organic ligand, a linker structure and a potent cytotoxic payload. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal protein overexpressed in multiple types of solid human malignancies. Nuclear medicine studies validated the applicability of the OncoFAP technology for tumor-targeting applications in a wide variety of tumor types. We have recently reported the development of SMDC products in which OncoFAP is used to deliver Monomethyl Auristatin E (MMAE) to tumors, exploiting linkers that are selectively cleaved by FAP. Here we describe the generation and in vivo characterization of novel OncoFAP-based SMDC products bearing potent camptothecin derivatives acting as topoisomerase I inhibitors. Those drug payloads have been already included in approved ADC products (EnhertuTM and TrodelvyTM). The FAP-cleavable linker technology was directly compared against linker strategies used for approved ADCs. Therapy results in preclinical xenograft models of solid tumors revealed that SMDC products activated by FAP show superior anti-cancer activity and deserve industrial development for the treatment of various human solid tumors. Citation Format: Matilde Bocci, Samuele Cazzamalli, Dario Neri, Andrea Galbiati. Delivery of potent cytotoxic camptothecin derivatives to tumors by OncoFAP-based small molecule-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1987.

Abstract 1548: CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells

Abstract The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short, and overcoming therapeutic resistance has proven difficult. The presence of chemoresistant leukemic stem cells (LSCs), among other mechanisms, has been implicated as a driver in the observed relapse. Antibody-drug conjugates (ADC) are targeted cancer therapeutics that leverage antigen specificity to deliver a potent cytotoxic payload. Recently, ADCs have shown promise in the clinic, but can also present safety and efficacy concerns. Mylotarg, an FDA approved CD33-directed ADC used to treat Acute Myeloid Leukemia, was the first ADC to be approved. The therapeutic is active against most AML tumors but can lead to severe myelotoxicity given the expression of CD33 on normal myeloid progenitor cells. We leveraged a pharmaco-pheno-multiomic analysis of primary AML tumors to uncover a previously unexplored target expressed on both LSCs and AML blasts, but not normal hematopoetic progenitor cells or other normal tissue. The target also showed capacity to rapidly internalize and has activity of well-established oncogenic kinases associated with expression. A novel monoclonal antibody (IgG1) with favorable binding and biophysical characteristics was established against this novel target and conjugated to a highly potent cytotoxic agent, creating a novel ADC. This ADC shows strong binding across AML tumors with effective internalization and highly potent killing of AML tumors. Citation Format: Garima Kaushik, Bandana Vishwakarma, Marina Bell, Kakajan Komurov. CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1548.

Stability assessment of Polatuzumab vedotin and Brentuximab vedotin using different analytical techniques

Antibody-drug conjugates (ADC) are considered to be fast-growing innovative biopharmaceuticals. The science used for conjugating potent cytotoxic payload to the targeted monoclonal antibody through a chemical linker has played a great value in the area of oncology treatment. In this study; Polatuzumab vedotin (POLA) and Brentuximab vedotin (SGN-35) were subjected to various stress conditions enclosing different pH, thermal stress, agitation, and successive cycles of freeze and thaw in order to produce potential degradation by-products and guarantee the appropriateness of the applied testing protocol. Different analytical techniques were established and validated to be used in the quantitation of the degraded products from different perspectives. The formation of ADC aggregates and fragments was monitored using SE-HPLC as well as dynamic light scattering (DLS). The drug antibody ratio (DAR) and ADC conjugation profile were determined using hydrophobic interaction chromatography (HIC-HPLC). In addition to performing a statistical interpretation of HIC-HPLC results by principal component analysis (PCA) to explicate the obtained data. Also, the quantity of the unconjugated toxic drug was quantified using RP-HPLC. Testing the binding activity of ADC to their target receptor ADC was conducted using ELISA. Results presented that used assay protocol had worked as a complementary design for characterization and stability assessment of the used ADC. Variances in the stability profile of both products were observed which could be attributed to the usage of different formulation buffers. This highlighted the importance of using multiple techniques for the assessment of the quality attributes of such sophisticated products. The analytical assay protocol should be used for the evaluation of the quality and stability of several ADC.

Stable, Site-Specific ADC Conjugation with SMARTag® Technology and Innovative Linker

Antibody-drug conjugates have achieved commercial success over the last 10 years with 15 therapies launched globally to date, including 11 with U.S. FDA approval, and two biosimilars. With their ability to direct highly potent cytotoxic payloads selectively to target cells, ADCs offer the prospect of more precise treatments and a reduction in off-target effects.

Modular Smart Molecules for PSMA-Targeted Chemotherapy.

New targeted chemotherapeutics are urgently needed to minimize off-target toxicity and reduce the high-mortality rate associated with metastatic prostate cancer. Herein, we report on the modular synthesis, pharmacokinetics, and efficacy of two small-molecule-drug conjugates (SMDC) targeted to prostate-specific membrane antigen (PSMA) incorporating either: (i) a cathepsin-B-cleavable valine-citrulline (Val-Cit), or (ii) an acid-cleavable phosphoramidate linker. Crucial components used in the design of the conjugates include: (i) CTT1298, a nanomolar affinity ligand that binds irreversibly to PSMA and has proven in past studies to rapidly internalize and shuttle payloads into PSMA-expressing prostate cancer cells, (ii) MMAE, a known potent cytotoxic payload, and (iii) an albumin-binder, proven to improve residence time of drug conjugates. At dose of 0.8 mg/kg (∼250 nmol/kg), the two SMDCs showed significant efficacy in a PSMA(+) PC3-PIP mouse model of human prostate cancer compared with controls, without inducing systemic toxicity. Though localization of the SMDCs was observed in tissues apart from the tumor, release of MMAE was observed predominantly in tumor tissue, at levels that were 2-3 orders of magnitude higher than non-target tissues. Furthermore, SMDC2, which incorporated a novel pH-responsive phosporamidate linker, demonstrated significantly improved efficacy over SMDC1 that has a Val-Cit linker, with a 100% survival over 90 days and 4 out of 8 mice showing complete tumor growth inhibition after 6 weekly doses of 0.8 mg/kg (244 nmol/kg). Our findings demonstrate the potential of irreversible PSMA inhibitors combined with pH-responsive linkers as a way to specifically deliver chemotherapeutic drugs to prostate cancer tumors with minimal toxicity.

A Review of Tisotumab Vedotin-tftv in Recurrent or Metastatic Cervical Cancer.

To evaluate the safety and efficacy of tisotumab vedotin-tftv (TV), a first-in-class vectorized anti-tissue factor (TF) antibody-drug conjugate (ADC), for the treatment of recurrent or metastatic cervical cancer. A literature search of ClinicalTrials.gov, Embase, and PubMed was conducted using the terms tisotumab vedotin AND cervical cancer from inception to June 30, 2022. All applicable publications, package inserts, meeting abstracts, and clinical trials involving TV in the treatment of cervical cancer were reviewed. TV is a fully human TF-specific monoclonal antibody conjugated to monomethyl auristatin E, which serves as a highly potent cytotoxic payload. In the pivotal phase II InnovaTV 204 clinical trial, TV demonstrated an objective response rate of 24% (95% confidence interval [CI], 16%-33%). The mean duration of response was 8.3 months. Common toxicities included abdominal pain, alopecia, conjunctivitis, constipation, decreased appetite, diarrhea, dry eye, epistaxis, nausea/vomiting, and peripheral neuropathy. Unique and/or serious adverse events warranting careful monitoring include ocular complications, hemorrhaging, peripheral neuropathies, fetal-embryo toxicity, pneumonitis, and immunogenicity. Recurrent or metastatic cervical cancer remains a high-risk disease with limited treatment options. Using ADCs to target tumors with aberrant expression of TF appears to be a viable treatment strategy. TV is the first Food and Drug Administration-approved TF-directed ADC. With a manageable safety profile and promising anticancer activity, TV warrants consideration as a novel targeted agent for the treatment of recurrent or metastatic cervical cancer. Further studies are required to determine the optimal place in therapy for TV.

C3 ester side chain plays a pivotal role in the antitumor activity of Maytansinoids

Maytansinoids, the chemical derivatives of Maytansine, are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADC). Structure-activity-relationship studies had identified the C3 ester side chain as a critical element for antitumor activity of maytansinoids. The maytansinoids bearing the methyl group at C3 position with D configuration were about 100 to 400-fold less cytotoxic than their corresponding L-epimers toward various cell lines. The detailed mechanism of how chirality affects the anticancer activity remains elusive. In this study, we determined the high-resolution crystal structure of tubulin in complex with maytansinol, L-DM1-SMe and D-DM1-SMe. And we found the carbonyl oxygen atom of the ester moiety and the tail thiomethyl group at C3 side chain of L-DM1-SMe form strong intramolecular interaction with the hydroxyl at position 9 and the benzene ring, respectively, fixing the bioactive conformation and enhancing the binding affinity. Additionally, ligand-based and structure-based virtually screening methods were used to screen the commercially macrocyclic compounds library, and 15 macrocyclic structures were picketed out as putatively new maytansine-site inhibitors. Our study provides a possible strategy for the rational discovery of next-generation maytansine site inhibitors.

The clinical development of antibody-drug conjugates - lessons from leukaemia.

Advances in our understanding of cancer biology have enabled drug development to progress towards better targeted therapies that are both more effective and safer owing to their lack of off-target toxicities. In this regard, antibody-drug conjugates (ADCs), which have the potential to combine the selectivity of therapeutic antibodies with the cytotoxicity of highly toxic small molecules, are a rapidly developing drug class. The complex and unique structure of an ADC, composed of a monoclonal antibody conjugated to a potent cytotoxic payload via a chemical linker, is designed to selectively target a specific tumour antigen. The success of an ADC is highly dependent on the specific properties of its components, all of which have implications for the stability, cytotoxicity, pharmacokinetics and antitumour activity of the ADC. The development of therapeutic ADCs, including gemtuzumab ozogamicin and inotuzumab ozogamicin, provided great knowledge of the refinements needed for the optimization of such agents. In this Review, we describe the key components of ADC structure and function and focus on the clinical development and subsequent utilization of two leukaemia-directed ADCs - gemtuzumab ozogamicin and inotuzumab ozogamicin - as well as on the mechanisms of resistance and predictors of response to these two agents.

Antibody-Drug Conjugates for Cancer Therapy.

Antibody–drug conjugates (ADCs) are novel drugs that exploit the specificity of a monoclonal antibody (mAb) to reach target antigens expressed on cancer cells for the delivery of a potent cytotoxic payload. ADCs provide a unique opportunity to deliver drugs to tumor cells while minimizing toxicity to normal tissue, achieving wider therapeutic windows and enhanced pharmacokinetic/pharmacodynamic properties. To date, nine ADCs have been approved by the FDA and more than 80 ADCs are under clinical development worldwide. In this paper, we provide an overview of the biology and chemistry of each component of ADC design. We briefly discuss the clinical experience with approved ADCs and the various pathways involved in ADC resistance. We conclude with perspectives about the future development of the next generations of ADCs, including the role of molecular imaging in drug development.

Targeting the Somatostatin Receptor 2 with the Miniaturized Drug Conjugate, PEN-221: A Potent and Novel Therapeutic for the Treatment of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma with a 95% mortality rate with no improvement to treatment in decades, and new therapies are desperately needed. PEN-221 is a miniaturized peptide-drug conjugate (∼2 kDa) designed to target SCLC via a Somatostatin Receptor 2 (SSTR2)-targeting ligand and to overcome the high proliferation rate characteristic of this disease by using the potent cytotoxic payload, DM1. SSTR2 is an ideal target for a drug conjugate, as it is overexpressed in SCLC with limited normal tissue expression. In vitro, PEN-221 treatment of SSTR2-positive cells resulted in PEN-221 internalization and receptor-dependent inhibition of cellular proliferation. In vivo, PEN-221 exhibited rapid accumulation in SSTR2-positive SCLC xenograft tumors with quick clearance from plasma. Tumor accumulation was sustained, resulting in durable pharmacodynamic changes throughout the tumor, as evidenced by increases in the mitotic marker of G2-M arrest, phosphohistone H3, and increases in the apoptotic marker, cleaved caspase-3. PEN-221 treatment resulted in significant antitumor activity, including complete regressions in SSTR2-positive SCLC xenograft mouse models. Treatment was effective using a variety of dosing schedules and at doses below the MTD, suggesting flexibility of dosing schedule and potential for a large therapeutic window in the clinic. The unique attributes of the miniaturized drug conjugate allowed for deep tumor penetration and limited plasma exposure that may enable long-term dosing, resulting in durable tumor control. Collectively, these data suggest potential for antitumor activity of PEN-221 in patients with SSTR2-positive SCLC.

Abstract 222: Novel protein drug conjugates targeting ROR1 through the development and exploitation of a drug discovery platform based on small, engineered VNAR domains

Abstract Whilst antibody drug conjugates (ADCs) have been successfully used to target highly potent cytotoxic agents to tumours, there is a continued emphasis on developing technologies for improving the therapeutic effectiveness of this class of agents. The vast majority of ADCs that have entered the clinic have been based on large intact immunoglobulins, however, there is significant interest in exploiting small protein domains as the payload delivery vehicle. These offer a number of potential benefits over full length antibodies including increased tumour penetration, amenability to protein engineering and site-specific conjugation, and improved tolerability. To this end, we have developed a therapeutics platform based on shark Variable New Antigen Receptor (VNAR) proteins. Shark VNAR domains are the smallest naturally occurring antigen binding domain (~11 kDa), with a binding mechanism distinct from traditional antibodies. Here we report the application of this platform to the development of homogenous VNAR-drug conjugates targeting the onco-embryonic receptor tyrosine kinase ROR1. ROR1 expression has been observed across a broad range of solid tumours and haematological malignancies, and is reported to correlate with poor clinical outcome for a number of cancer indications, but is largely absent from normal adult tissue. As such, the expression pattern of ROR1, coupled with its functional role in tumourogenesis and disease progression, make it an attractive PDC target. Using a combination of direct immunisation and synthetic VNAR library screening, we have generated high affinity binders to the extracellular domain of ROR1 which show species cross-reactivity, do not bind the closely related family member ROR2, and can target distinct non-overlapping regions of the protein. By exploiting the ability of VNAR domains to be flexibly reformatted, the lead sequences have been configured as a series of bivalent Fc fusions, VNAR multimers and novel bi-paratopic binders, and constructs identified that show further enhanced binding to ROR1 and which can induce internalisation and lysosomal trafficking in ROR1 expressing cancer cell-lines. Subsequent attachment of highly potent DNA-damaging cytotoxic payloads yielded homogenous VNAR-drug conjugates which demonstrated extremely potent (sub-nanomolar) in vitro cytotoxicity against selected cancer cell-lines in a receptor mediated fashion. Studies to assess the in vivo efficacy of these conjugates are in progress. These ROR1 VNAR constructs have also been successfully fused to sdAbs and scFv directed to other cell-surface protein targets, demonstrating the power of the platform and opening the way for novel bi-specific approaches for targeting both solid and haematological tumours. Citation Format: Graham Cotton, Jennifer Thom, Paul Trumper, Stacey Bell, Andrei Kamenski, Mark Wappett, Caroline Barelle, Marina Kovaleva, John Steven, Andy Porter, Estelle McLean, Chiara Saladino, Aidan McCann, Aaron Cranston, Tim Harrison. Novel protein drug conjugates targeting ROR1 through the development and exploitation of a drug discovery platform based on small, engineered VNAR domains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 222.

Abstract 229: Utilizing a mouse cross-reactive model system to better understand antibody-drug conjugate pharmacokinetics, biodistribution and efficacy

Abstract Antibody-drug conjugates (ADCs) are designed to deliver a potent cytotoxic payload directly to tumors, thus limiting exposure in normal tissues. However, target antigen expression on normal tissues can lead to lower systemic ADC exposures, resulting in sub-efficacious concentrations at the tumor site as well as heterogeneous distribution within tumors. Traditional preclinical efficacy studies performed in rodent models using ADCs with non-cross-reactive antibodies have been of limited translational relevance, and a better understanding of the factors that impact ADC dose and activity remains to be elucidated. To examine relationships between variables that could influence ADC efficacy, we generated a cross-reactive model system that utilized a chimeric anti-murine folate receptor α (FRα) antibody (designated rmFR1-12) that binds both mouse and human FRα, and can be conjugated to either maytansinoid (DM) or indolinobenzodiazepine (IGN) payloads. This model system was predicted to have substantial target-mediated drug disposition (TMDD) due to normal tissue expression of FRα. An rmFR1-12-s-SPDB-DM4 ADC was made using tritium-labeled DM4 and administered to tumor-bearing mice in order to assess ADC pharmacokinetics (PK), biodistribution, and efficacy. This approach allowed tracking of the ADC at multiple levels – whole animal, organ, tumor, and cell. Studies were undertaken that assessed the impact of xenograft antigen expression, ADC dose, and ADC drug-to-antibody ratio (DAR) on the PK, biodistribution, and efficacy of the rmFR1-12-s-SPDB-DM4 conjugate. The results showed that TMDD significantly impacted the PK, biodistribution, and activity of the conjugate relative to a non-cross-reactive ADC, with lower ADC doses being more severely impacted than higher doses. Antigen expression positively correlated with local ADC exposure and efficacy. Decreasing the DAR (by co-dosing naked antibody with the ADC) increased systemic exposure. A positive correlation between systemic exposure and dose of naked antibody was observed. Factors that impacted local exposure included: the type of tissue (normal vs tumor), presence or absence of FRα expression on normal tissue, and dose of naked antibody. Of note, in the naked antibody plus ADC study, efficacy did not correlate with local exposure - suggesting that ADC distribution within tumors is of similar importance as the amount of ADC delivered. Overall, these findings underscore the importance of accounting for site, and extent of, normal tissue target expression with respect to ADC PK/PD, and the data generated from these studies are currently being used to build a multiscale physiologically based PK model of a cross-reactive ADC. Citation Format: Leanne Lanieri, Rassol Laleau, Bahar Matin, Jenny Lee, Steven Boule, Paulin Salomon, Luke Harris, Michael Miller, Nicholas C. Yoder, Yulius Setiady, Neeraj Kohli, Thomas A. Keating, Jan Pinkas, Richard Gregory. Utilizing a mouse cross-reactive model system to better understand antibody-drug conjugate pharmacokinetics, biodistribution and efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 229.

Abstract 4835: LY3076226, a novel anti-FGFR3 antibody drug conjugate exhibits potent and durable anti-tumor activity in tumor models harboring FGFR3 mutations or fusions

Abstract Fibroblast growth factor receptor 3 (FGFR3) has been shown to be constitutively activated in bladder, multiple myeloma, non-small cell lung cancer (NSCLC), glioblastoma multiforme, and cervical cancer through overexpression, point mutations, and translocations. In bladder cancer, FGFR3 is typically found to be activated by mutation or over-expression. TCGA data suggests that ~48% of bladder cancer patients harbor FGFR3 mutations such as S249, Y373C and others, as well as fusion proteins of FGFR3 involving transforming acidic coiled-coil gene (TACC3) and FGFR3-BAIAP2L1. LY3076226 is an antibody-drug conjugate (ADC) comprising a fully human anti-FGFR3 antibody, IMC-D11, conjugated to a cytotoxic payload, the maytansine-derivative DM4, via a cleavable linker (sulfo-SPDB). The anti-FGFR3 Ab IMC-D11 recognizes and binds not only wild-type receptor, but also constitutively active mutant receptors and fusion proteins of FGFR3. Preclinical in vitro pharmacodynamics studies have demonstrated that LY3076226 binds to the human FGFR3 protein with high affinity, and following internalization can deliver the potent cytotoxic payload into tumor cells resulting in cell cycle arrest and cell death. In vivo studies with LY3076226 demonstrated robust antitumor efficacy resulting in tumor stasis or partial or complete tumor responses after 4 weekly doses at 5 mg/kg in bladder cancer cell lines: SW780, RT112 and UMUC-14 that harbor FGFR3-BAIAP2L1, FGFR3-TACC3 fusion or S249C mutation, respectively. We also evaluated over 90 PDXs models using a Modiplex assay and IHC to determine mutational and FGFR3 expression levels. We identified a number of bladder PDX models with R248C, S249 and G370 mutations and a single NSCLC with FGFR3-TACC3, and tested LY3076226 efficacy in these models. Interestingly the incidence of mutations and fusions in these PDX models confirmed prior TCGA results, and confirm that PDX models represent a valuable translational platform for patient tailoring. Similar to RT-112 cell line, LY3076226 significantly inhibited tumor growth in lung PDX model (LXF-2226) with FGFR3-TACC3 fusion resulting in durable complete response in 100% of mice. LY3076226 was also efficacious after 4 weekly treatments at 5 mg/kg, resulting in tumor growth inhibition, tumor stasis or tumor regressions in bladder PDX models with G370C, S249C or R248C FGFR3 mutations, respectively. Additionally, LY3076226 exhibited superior efficacy compared to the naked IMC-D11 Ab or chKTI-ADC controls. These data support the conclusion that therapy with FGFR3 targeted antibody-drug conjugate LY3076226 may confer tumor control in patients that are FGFR3 positive and harbor FGFR3 mutations or fusion proteins. Citation Format: David Surguladze, Anthony Pennello, Xiaodi Ren, Tim Mack, Alan Rigby, Paul Balderes, Elizabeth Navarro, Nelusha Amaladas, Scott Eastman, Michael Topper, Yung-mae Yao, Chris Moxham, Gregory Plowman, Dale Ludwig. LY3076226, a novel anti-FGFR3 antibody drug conjugate exhibits potent and durable anti-tumor activity in tumor models harboring FGFR3 mutations or fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4835.

Sortase A Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are highly potent targeted anticancer therapies. They rely on the linking of a selectively targeting antibody moiety with potent cytotoxic payloads to effect antitumoral activity. In recent years, one focus in the ADC field was to create novel methods for site-specifically conjugating payloads to antibodies. The method presented here is based on the S. aureus sortase A-mediated transpeptidation reaction. This method requires antibodies to be engineered in such a way that they possess the sortase recognition pentapeptide motif LPETG on the C-terminus of the immunoglobulin heavy and/or light chains. In addition, the toxin must contain an oligoglycine motif in order to make it a suitable substrate for sortase A. Here we describe a detailed method to conjugate a pentaglycine-modified toxin to the C-termini of LPETG-tagged antibody heavy and light chains using sortase-mediated antibody conjugation (SMAC-Technology™). Highly homogenous, site-specifically conjugated ADCs with controlled drug to antibody ratio and improved overall properties can be obtained with this method.