Antibody-drug Conjugates Pharmacokinetics Research Articles

Exploring the Role of Target Expression in Treatment Efficacy of Antibody-Drug Conjugates (ADCs) in Solid Cancers: A Comprehensive Review.

Antibody-drug conjugates (ADCs) offer a promising path for cancer therapy, leveraging the specificity of monoclonal antibodies and the cytotoxicity of linked drugs. The success of ADCs hinges on precise targeting of cancer cells based on protein expression levels. This review explores the relationship between target protein expression and ADC efficacy in solid tumours, focusing on results of clinical trials conducted between January 2019 and May 2023. We hereby highlight approved ADCs, revealing their effectiveness even in low-expressing target populations. Assessing target expression poses challenges, owing to variations in scoring systems and biopsy types. Emerging methods, like digital image analysis, aim to standardize assessment. The complexity of ADC pharmacokinetics, tumour dynamics, and off-target effects emphasises the need for a balanced approach. This review underscores the importance of understanding target protein dynamics and promoting standardized evaluation methods in shaping the future of ADC-based cancer therapies.

Generation and Characterization of Iduronidase-Cleavable ADCs.

A crucial design feature for the therapeutic success of antibody-drug conjugates (ADCs) is the linker that connects the antibody with the drug. Linkers must be stable in circulation and efficiently release the drug inside the target cell, thereby having a fundamental impact on ADC pharmacokinetics and efficacy. The variety of enzymatically cleavable linkers applied in ADCs is limited, and some are believed to be associated with unwanted side effects due to the expression of cleavage-mediating enzymes in nonmalignant cells. Based on a bioinformatic screen of lysosomal enzymes, we identified α-l-iduronidase (IduA) as an interesting candidate for ADC linker cleavage because of its low expression in normal tissues and its overexpression in several tumor types. In the present study, we report a novel IduA-cleavable ADC linker using exatecan and duocarmycin as payloads. We showed the functionality of our linker system in cleavage assays using recombinant IduA or cell lysates and compared it to established ADC linkers. Subsequently, we coupled iduronide-exatecan via interchain cysteines or iduronide-duocarmycin via microbial transglutaminase (mTG) to an anti-CEACAM5 (aCEA5) antibody. The generated iduronide-exatecan ADC showed high serum stability and similar target-dependent tumor cell killing in the subnanomolar range but reduced toxicity on nonmalignant cells compared to an analogous cathepsin B-activatable valine-citrulline-exatecan ADC. Finally, in vivo antitumor activity could be demonstrated for an IduA-cleavable duocarmycin ADC. The presented results emphasize the potential of iduronide linkers for ADC development and represent a tool for further balancing out tumor selectivity and safety.

First-in-human study of ABBV-011, a seizure-related homolog protein 6 (SEZ6)–targeting antibody-drug conjugate, in patients with small cell lung cancer.

3002 Background: Small cell lung cancer (SCLC) has a dismal prognosis and new therapies are urgently needed. SEZ6 is a transmembrane protein expressed in SCLC tumors that may be used as a therapeutic target. ABBV-011 is an antibody-drug conjugate (ADC) targeting SEZ6 with a calicheamicin payload, which has shown antitumor activity in preclinical models of SCLC. Preliminary results from the monotherapy dose-escalation and -expansion cohorts of the first-in-human ABBV-011 study are presented. Methods: Phase 1, open-label, multicenter study (NCT03639194) of ABBV-011 alone or in combination with budigalimab, a programmed cell death 1 inhibitor. Primary objectives were to assess the safety and tolerability and to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose of ABBV-011. Adults (≥18 years) with relapsed/refractory SCLC (1–3 lines of prior therapy) were enrolled. Dose escalation was guided by Bayesian continual reassessment method. ABBV-011 was administered intravenously at doses from 0.3 to 2.0 mg/kg once every 3 weeks. Dose expansion was conducted in SEZ6-selected patients. Results: At data cutoff on August 22, 2022, 99 patients were treated with ABBV-011 monotherapy. Median age was 63 years (range, 41–79), 50% of patients were male, and 68% had received ≥2 prior therapies. ABBV-011 ADC pharmacokinetics were approximately dose-proportional with an elimination half-life of 4.6 days across the dose range of 0.3–2.0 mg/kg. In dose escalation (n=26), 1 patient had a dose-limiting toxicity of grade (G) 3 fatigue at 2.0 mg/kg. We report safety and efficacy results for 40 patients in the dose-expansion 1.0-mg/kg ABBV-011 cohort. Median duration of treatment was 12 weeks (range, 1.9–63.3). Treatment-emergent adverse events (TEAEs) occurred in 39 (98%) patients, the most frequent being fatigue (48%), nausea (45%), anorexia (38%), thrombocytopenia (38%), and vomiting (35%). G3 TEAEs occurred in 18 (45%) patients, the most frequent being fatigue, thrombocytopenia, and neutropenia (10% each); 1 G4 TEAE of dyspnea was reported. Seven patients died due to malignant neoplasm/disease progression (n=6) or respiratory distress (n=1); none were related to ABBV-011. Hepatotoxicity was observed, including G≥2 TEAEs of hyperbilirubinemia (18%), increased gamma-glutamyltransferase (8%), ascites (5%), veno-occlusive liver disease (3%), and portal hypertension (3%). Confirmed objective response rate was 25% (10 partial responses [PR]), with median duration of response of 4.2 months (95% CI: 2.6, 6.7). Clinical benefit rate (CBR) was 65% (10 PR and 16 stable disease) and CBR lasting >12 weeks was 43%. The median progression-free survival was 3.5 months. Conclusions: The MTD was not reached and ABBV-011 was well tolerated at 1.0 mg/kg with promising antitumor activity observed. Further evaluation of ABBV-011 is ongoing. Clinical trial information: NCT03639194 .

Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

Effect of Intrinsic and Extrinsic Factors on the Pharmacokinetics of Antibody-Drug Conjugates (ADCs).

Antibody–drug conjugates (ADCs) are complex molecules wherein a monoclonal antibody is linked to a biologically active drug (a small molecule), forming a conjugate. Initially, most of the ADCs were developed and are being developed for the treatment of cancer; however, with time, it has been realized that ADCs can also be developed to manage or cure other diseases. Pharmacokinetics (PK) plays an important role in modern-day drug development and the knowledge of PK is crucial in designing a safe and efficacious dose to treat a wide variety of diseases. There are several factors that can alter the PK of a drug; as a result, one has to adjust the dose in a patient population. These factors can be termed ‘intrinsic’ or ‘extrinsic’. For small molecules, the impact of both intrinsic and extrinsic factors is well established. The impact of age, gender, disease states such as renal and hepatic impairment, drug–drug interaction, food, and in many cases alcohol on the PK of small molecules are well known. On the other hand, for macromolecules, the impact of these factors is not well established. Since the ADCs are a combination product of a monoclonal antibody linked to a small molecule, both the small molecule and the monoclonal antibody of the ADCs may be subjected to many intrinsic and extrinsic factors. This review summarizes the impact of intrinsic and extrinsic factors on the PK of ADCs and the payloads.

Capturing the Magic Bullet: Pharmacokinetic Principles and Modeling of Antibody-Drug Conjugates.

Over the past two decades, antibody-drug conjugates (ADCs) have emerged as a promising class of drugs for cancer therapy and have expanded to nononcology fields such as inflammatory diseases, atherosclerosis, and bacteremia. Eight ADCs are currently approved by FDA for clinical applications, with more novel ADCs under clinical development. Compared with traditional chemotherapy, ADCs combine the target specificity of antibodies with chemotherapeutic capabilities of cytotoxic drugs. The benefits include reduced systemic toxicity and enhanced therapeutic index for patients. However, the heterogeneous structures of ADCs and their dynamic changes following administration create challenges in their development. The understanding of ADC pharmacokinetics (PK) is crucial for the optimization of clinical dosing regimens when translating from animal to human. In addition, it contributes to the optimization of dose selection and clinical monitoring with regard to safety and efficacy. This manuscript reviews the PK characteristics of ADCs and summarizes the diverse approaches for PK modeling that can be used to evaluate an ADC at the preclinical and clinical stages to support their successful development. Despite the numerous available options, fit-for-purpose modeling approaches for the PK and PD of ADCs should be critically planned and well-thought-out to adequately support the development of an ADC.

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 4266: Computational exploration of mechanistic determinants of ADC pharmacokinetics using QSP modeling strategies

Abstract The pharmacokinetics of antibody drug conjugate (ADC) therapeutics typically show a discrepancy between the PK of total antibody (conjugated and unconjugated antibody) and that of conjugated antibody, carrying one or more payload molecules. This discrepancy is often attributed strictly to deconjugation (Kamath, 2014), however recent evidence suggests that the underlying mechanisms may be more complex. This work employs a computational quantitative systems pharmacology (QSP) approach that generates a hypotheses to better understand the impact of drug antibody ratio (DAR) and the resulting changes in molecular properties on overall PK and relative payload disposition as observed in preclinical and clinical studies. Our work establishes the benefit of using computational models to design novel ADCs and to optimize the discovery and development of existing ADCs to better enable clinical trial design.

The Chemical Design and Synthesis of Linkers Used in Antibody Drug Conjugates.

Antibody Drug Conjugates (ADCs) use targeting ability of monoclonal antibodies to deliver potent cytototoxic payloads to their intended target. The linker encompasses a conjugating functionality suitable for attachment to the antibody, a spacer unit that typically incorporates a hydrophilic element and a trigger which releases the potent cytototoxic warhead. Understanding the conflicting requirements of ADC design, providing stability in systemic circulation but efficient payload release once the ADC reaches its intended target, is crucial to effective linker development. ADC linker design has been approached in a variety of different ways, with increasingly elegant solutions continuing to be reported as understanding of the intricate design complexities increases. This review focuses on the synthetic approaches used in ADC linkers, and the impact of linker design on antibody conjugation, ADC pharmacokinetics and payload release. Linker approaches utilized in commercial ADCs as well as ADCs currently in clinical, pre-clinical and early stage development are discussed.

Pharmacokinetic Considerations for Antibody-Drug Conjugates against Cancer.

Antibody-drug conjugates (ADCs) are ushering in the next era of targeted therapy against cancer. An ADC for cancer therapy consists of a potent cytotoxic payload that is attached to a tumour-targeted antibody by a chemical linker, usually with an average drug-to-antibody ratio (DAR) of 3.5-4. The theory is to deliver potent cytotoxic payloads directly to tumour cells while sparing healthy cells. However, practical application has proven to be more difficult. At present there are only two ADCs approved for clinical use. Nevertheless, in the last decade there has been an explosion of options for ADC engineering to optimize target selection, Fc receptor interactions, linker, payload and more. Evaluation of these strategies requires an understanding of the mechanistic underpinnings of ADC pharmacokinetics. Development of ADCs for use in cancer further requires an understanding of tumour properties and kinetics within the tumour environment, and how the presence of cancer as a disease will impact distribution and elimination. Key pharmacokinetic considerations for the successful design and clinical application of ADCs in oncology are explored in this review, with a focus on the mechanistic determinants of distribution and elimination.

Abstract 4061: Computational exploration of mechanistic determinants of antibody drug-conjugate pharmacokinetics using quantitative systems pharmacology modeling strategies

Abstract The pharmacokinetics of antibody drug conjugate (ADC) therapeutics typically show a discrepancy between the PK of total antibody (conjugated and unconjugated antibody) and that of conjugated antibody, carrying one or more payload molecules. This discrepancy is often attributed to deconjugation (Kamath, 2014), however recent evidence suggests that the underlying mechanisms may be more complex. This work employs a computational quantitative systems pharmacology (QSP), or mechabistic PK/PD approach to understand the impact of drug antibody ratio (DAR) and the resulting changes in molecular properties on overall PK and relative payload disposition as observed in preclinical and clinical studies. Our work establishes the benefit of using computational models to design novel ADCs and to optimize the discovery and development of existing ADCs. Citation Format: A. Katharina Wilkins, Andrew Matteson, Lore Gruenbaum, Jennifer Park, John M. Burke, Joshua Apgar. Computational exploration of mechanistic determinants of antibody drug-conjugate pharmacokinetics using quantitative systems pharmacology modeling strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2017-4061

OA05.03 Single-Agent Rovalpituzumab Tesirine, a Delta-Like Protein 3 (DLL3)-Targeted Antibody-Drug Conjugate (ADC), in Small-Cell Lung Cancer (SCLC)

SCLC is one of the most deadly malignancies. Rovalpituzumab tesirine (SC16LD6.5, Rova-T) is a first-in-class ADC directed against DLL3, a novel target identified in tumor initiating cells and expressed in over 80% of SCLC cases. Seventy-four patients with progressive SCLC after at least one previous systemic therapy were enrolled in a first-in-human study (NCT01901653), irrespective of DLL3 expression, including 68 at active doses of 0.2-0.4 mg/kg administered intravenously every 3 or 6 weeks. Available archived tumor tissue (n=48) was assessed retrospectively by immunohistochemistry for DLL3. Among 60 evaluable subjects, active dose levels resulted in a confirmed objective response rate (ORR) of 18% and a confirmed clinical benefit rate (CBR; stable disease or better) of 68%. Among 26 evaluable subjects with DLL3 expression in at least 50% of tumor cells (DLL3-high), confirmed ORR and CBR were 39% and 89%, respectively. Median duration of response was 5.6 months. One-year survival rates among all and DLL3-high subjects were 18% and 32%, respectively. Among primary sensitive relapse patients, confirmed ORR and CBR among all subjects were 24% (8/33) and 67% (22/33); and among DLL3-high subjects were 53% (8/15) and 100% (15/15), with one-year survival rates of 17% and 33%, respectively. Among primary resistant/refractory relapse patients, confirmed ORR and CBR among all subjects were 12% (3/25) and 72% (18/25); and among DLL3-high subjects were 18% (2/11) and 73% (8/11), with one-year survival rates of 21% and 29%, respectively. The most common grade 3 or higher toxicities included thrombocytopenia (12%), serosal effusions (11%), and skin reactions (8%). ADC pharmacokinetics were linear with a terminal half-life of 10 - 14 days and anti-therapeutic antibodies did not develop. Rovalpituzumab tesirine demonstrates encouraging single-agent anti-tumor activity with a manageable safety profile, including among patients with disease resistant or refractory to primary chemotherapy. Further development of rovalpituzumab tesirine in SCLC is warranted.

A Phase 1 Study of Denintuzumab Mafodotin (SGN-CD19A) in Relapsed/Refactory B-Lineage Non-Hodgkin Lymphoma

BackgroundDenintuzumab mafodotin (SGN-CD19A) is a novel antibody-drug conjugate (ADC) composed of a humanized anti-CD19 monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) via a maleimidocaproyl linker. CD19 is a B-cell-specific marker expressed in the vast majority of patients (pts) with B-cell non-Hodgkin lymphoma (NHL).MethodsAn ongoing phase 1, dose-escalation study is investigating the safety, tolerability, pharmacokinetics (PK), and antitumor activity of denintuzumab mafodotin in pts with relapsed or refractory (R/R) B-cell NHL (NCT 01786135). Eligible pts were ≥12 yrs of age and were R/R to ≥1 prior systemic regimens; pts with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma grade 3 (FL3) also received intensive salvage therapy ± autologous stem cell transplant (ASCT), unless they refused or were ineligible. Denintuzumab mafodotin was administered IV every 3 weeks (q3wk; 0.5-6 mg/kg) for dose escalation and every 6 weeks (q6wk; 3 mg/kg) in a subsequent expansion cohort. A modified continual reassessment method was used for dose allocation and maximum tolerated dose (MTD) estimation in the q3wk dosing schedule. Archived tissue was collected to assess potential biomarkers of response.ResultsTo date, 62 pts have been treated, including 53 pts (85%) with DLBCL (of whom 16 had transformed DLBCL), 5 (8%) with mantle cell lymphoma, and 3 (5%) with FL3. Median age was 65 yrs (range, 28-81). Pts had received a median of 2 prior systemic therapies (range, 1-6); 15 pts (24%) had prior ASCT. Thirty-seven pts (60%) were refractory to the most recent prior therapy. Fifty-two pts were treated in the q3wk schedule (0.5-6 mg/kg), and 10 pts were treated with 3 mg/kg q6wk. Five pts remain on treatment (2 q3wk pts, 3 q6wk pts).Overall, 20 (33%) of 60 efficacy-evaluable pts achieved objective responses, including 13 (22%) with CRs. Eighteen of the 20 objective responses were achieved by the end of Cycle 2 (15 q3wk pts, 3 q6wk pts). TableQ3wk Dosing (N=51)Q6wk Dosing (N=9)RelapsedaN=22RefractorybN=29RelapsedaN=3RefractorybN=6Best clinical response, n (%)Complete remission (CR)7 (32)3 (10)3 (100)-Partial remission (PR)4 (18)3 (10)--Stable disease (SD)6 (27)7 (24)-3 (50)Progression5 (23)16 (55)-3 (50)ORR (CR+PR), % (95% CI)50 (28, 72)21 (8, 40)100 (29, 100)-CR rate, % (95% CI)32 (14,55)10 (2, 27)100 (29, 100)-ORR=objective response rateaBest response of CR/PR with most recent prior therapybBest response of SD/PD with most recent prior therapyMedian duration of objective response in the q3wk schedule was 39 wks for relapsed pts (95% CI: 11.6, - [range, 0.1+ to 73+ wks]) and 41 wks for refractory pts (95% CI: 13.7, 67 [range, 13.7 to 67 wks]); this included 2 pts who maintained their responses for >15 mos. Data for the q6wk schedule are not yet mature.The MTD was not reached at 0.5-6 mg/kg q3wk, and only 1 DLT was observed (G3 keratopathy at 3 mg/kg). Toxicity profiles were similar across both dosing schedules; the most frequently reported adverse events (AEs) were blurry vision (65%), dry eye (52%), fatigue and keratopathy (35% each), constipation (29%), photophobia (27%), and nausea (26%). Ocular symptoms and corneal exam findings consistent with superficial microcystic keratopathy were observed in 52 pts (84%); symptoms were less severe than the associated corneal exam findings. Keratopathy was managed with topical steroids and dose modifications, and improved/resolved within a median of ~5 wks (range, 1-17) in pts for whom there was sufficient follow-up. ADC PK demonstrated a mean terminal half-life of ~2 wks, and accumulation was observed following multiple dose administrations in both schedules.ConclusionsDenintuzumab mafodotin is generally well tolerated and demonstrates encouraging activity with durable responses in heavily pre-treated pts with B-cell NHL. In relapsed pts, 56% achieved objective responses with a CR rate of 40% across both the q3wk and q6wk schedules. The low rate of myelosuppression and neuropathy suggests that denintuzumab mafodotin could be incorporated into novel combination regimens in earlier lines of therapy. A randomized phase 2 trial is being initiated to evaluate RICE (rituximab, ifosfamide, carboplatin, etoposide) ± denintuzumab mafodotin pre-ASCT as second-line treatment for pts with DLBCL. DisclosuresMoskowitz:Seattle Genetics, Inc.: Consultancy, Research Funding; Merck: Research Funding; Genentech: Research Funding. Off Label Use: Denintuzumab mafodotin (SGN-CD19A) is not approved for use.. Fanale:Seattle Genetics, Inc.: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Shah:Janssen: Speakers Bureau; Seattle Genetics: Research Funding; DeBartolo Institute for Personlaized Medicine: Research Funding; Rosetta Genomics: Research Funding; Acetylon Pharmaceuticals, INC: Membership on an entity's Board of Directors or advisory committees; Plexus Communications: Honoraria; Spectrum: Speakers Bureau; Pharmacyclics: Speakers Bureau; Bayer: Honoraria; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SWOG: Consultancy; NCCN: Consultancy. Chen:Genentech: Consultancy, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding, Speakers Bureau. Kim:Bayer: Consultancy; Seattle Genetics, Inc.: Consultancy, Research Funding; Eli Lilly: Consultancy. Kostic:Seattle Genetics, Inc.: Employment, Equity Ownership. Liu:Seattle Genetics, Inc.: Employment, Equity Ownership, Other: Travel expenses. Peng:Seattle Genetics, Inc.: Employment, Equity Ownership. Forero-Torres:Seattle Genetics, Inc.: Research Funding.

Abstract 648: Optimization of a PEGylated glucuronide-auristatin linker for antibody-drug conjugates

Abstract The clinical success of antibody-drug conjugates (ADCs) brentuximab vedotin and ado-trastuzumab emtansine has led to an increased effort to identify new antibody payloads and develop improved linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC resulting in accelerated plasma clearance and diminished in vivo activity, particularly for conjugates with high drug to antibody ratios (DAR). Consequently, drug-linkers have been designed to minimize or mask hydrophobicity, enabling a higher DAR and improved pharmacokinetic properties, translating to enhanced in vivo potency. We recently reported on the incorporation of a discrete PEG polymer as a side chain in the β-glucuronide-monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. The PEG-glucuronide-MMAE lead has now been optimized by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linker constructs were prepared with PEG size varying from 0 to 24 ethylene oxide units, and uniform DAR 8 ADCs were evaluated. PEG size had small but variable effects in vitro, with diminished activity observed in some cell lines. In contrast, a clear relationship was observed between PEG length and plasma pharmacokinetics. Longer PEG chains resulted in slower clearance, with a ‘threshold’ length beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize ADC plasma clearance provided a wider therapeutic window relative to conjugates bearing shorter PEGs. Thus, sufficient masking of drug hydrophobicity is a viable strategy to provide uniform DAR 8 ADCs that preserve ADC pharmacokinetics and increase potency through higher drug loading. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Svetlana O. Doronina, Nicole M. Okeley, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. Optimization of a PEGylated glucuronide-auristatin linker for antibody-drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2015-648

Abstract 2059: In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer

Abstract Antibody drug conjugates (ADCs) are clinically validated as a modality for targeted therapy of solid and hematological cancer due to advancements in target selection, conjugation chemistry and linker technology. However, much about mechanism of action (MoA) is yet to be fully understood. Our goal was to interrogate ADC pharmacokinetics and pharmacodynamics establishing proof of mechanism (PoM) of drug action with a diverse panel of ADCs. Herein, we describe the development of novel immunohistochemical (IHC) methods for in situ visualization of ADCs binding to target expressing cells and their cognate downstream biomarkers of response in formalin fixed paraffin embedded cells/tissues. We demonstrate specific binding of 4 different ADCs spanning 2 solid tumor targets and an endothelial cell target using IHC with anti-human IgG in human tumor xenograft models expressing the respective targets. ADC binding to target is observed as early as 20 minutes after a single dose of ADC at 3 mg/kg. Utilizing an anti-microtubule inhibitor (MTI) payload-specific antibody we additionally detect ADC binding to tumor cells by monitoring the cytotoxic payload. The cell type where the antibodies and payload localized was identified by double and triple IHC. Pharmacodynamic biomarkers of response for two payload classes (DNA damaging agents and MTIs) were detected with antibodies against phospho-Histone H2AX and phospho-Histone H3, respectively - confirming the expected ADC MoAs. Downstream apoptosis of target cells was detected with cleaved caspase 3 IHC. The kinetics of biomarker response and downstream cellular impact was quantified via image analysis with biomarkers evident as early as 24 hours after a single dose for both tumor cell and vascular targets. Furthermore, we observed a correlation between biomarkers of response and efficacy of the ADCs as measured by statistically significant tumor growth inhibition for the 4 ADCs we studied. These data suggest that IHC interrogations of drug action should be used to further the clinical development of ADCs via demonstration of pharmacodynamic activities at the cellular level, establishing PoM data, and enabling predictive preclinical oncology models in order to reduce clinical attrition of ADCs. Citation Format: Jonathon Golas, Andrea T. Hooper, Justin Lucas, Heather Jones, Timothy Nichols, Kiran Khandke, Manoj Charati, Roger Conant, Michael Cinque, Judy Lucas, Marc Damelin, Ken Geles, Caiazzo Teresa, Frank Loganzo, Puja Sapra, Hans-Peter Gerber, Chad May. In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2014-2059

Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates

Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.