Drug-to-antibody Ratio Research Articles

Abstract 2622: TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans

Abstract TUB-040 is a novel Antibody-Drug Conjugate (ADC) targeting Napi2b, a highly overexpressed target in ovarian cancer and lung adenocarcinoma. Napi2b is a member of the solute-carrier transporter family with eight transmembrane domains and an antibody-accessible extracellular loop, that regulates sodium-dependent phosphate homeostasis. Ethynylphosphonamidate conjugation chemistry was used to build TUB-040 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 Napi2b using a cleavable linker system. In vitro, TUB-040 is characterized by sub-nanomolar affinity and specific binding to Napi2b, efficient target-mediated internalization and high cytotoxicity towards Napi2b-expressing ovarian and lung cancer cell lines as well as bystander activity against co-cultured target-negative cells. TUB-040 induces DNA damage and apoptosis in a dose-dependent fashion as well as markers of immunogenic cell death. In addition, no unspecific uptake and cytotoxicity in healthy, target-negative human cells could be detected, reducing the risk of target-independent toxicities. Pharmacokinetic analysis showed that - in contrast to maleimide-conjugated ADCs - TUB-040 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation, thus enabling most efficient and continued delivery of Exatecan to the tumor. Across a variety of cell line derived xenograft (CDX) and patient derived xenograft (PDX) models of ovarian and non-small cell lung cancer, including models with low target expression, single dose administration of TUB-040 results in long-lasting tumor growth inhibition with high complete remission rates in vivo. The minimally effective dose (MED) leading to complete remission is 1 mg/kg. Preliminary repeat-dose toxicological assessment of TUB-040 in the pharmacologically relevant species cynomolgus monkey demonstrated that TUB-040 is well tolerated with no signs of lung toxicity and thrombocytopenia. Allometric scaling and PK/PD modelling predicts a human serum half-life greater 10 days and a therapeutic index in humans up to 55. Based on these results, TUB-040, designed with differentiating technology for optimal efficacy and tolerability, is ready for testing in clinical trials. 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. TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans [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 2622.

Abstract 3121: Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers

Abstract To address the need for targeted therapy in the treatment of HER3-positive solid tumors, the human HER3 mAb, seribantumab, was evaluated in the context of an antibody-drug conjugate (ADC). As a proof-of-concept, seribantumab was conjugated with a cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) payload via the stochastic cysteine conjugation method to yield HER3-ADC1. A drug-antibody ratio of 4 was selected for preclinical assessment in HER3-expressing models. Methods: HER3-ADC1 was evaluated in vitro and in vivo, with patritumab deruxtecan (patri-DXd) as a comparator. Binding to BT474 breast carcinoma cells (HER3 high; immunohistochemical (IHC) staining intensity 3+) was measured by flow cytometry. Internalization properties were established using seribantumab and patritumab coupled to Fab-AF488 following 4 hours incubation in SK-BR-3 breast adenocarcinoma (HER3 high; IHC 3+) and HCC1569 breast carcinoma (HER3 low; IHC 0-1+) cell lines. In vitro cytotoxicity was evaluated for HER3-ADC1, isotype-MMAE and free MMAE payload as well as patri-DXd, isotype-DXd and free deruxtecan payload in BT474 cells, SK-BR-3 cells and NCI-H446 lung carcinoma (HER3 low; IHC 0-1+) cells. In vivo anti-tumor activity was assessed for HER3-ADC1, isotype-MMAE, patri-DXd and isotype-DXd in patient derived xenograft (PDX) models of pancreatic (HER3 high; IHC 3+) and breast cancer (HER3 low; IHC 0-1+). Results: HER3-ADC1 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on HER3 expression. In assays investigating antibody internalization, seribantumab displayed similar internalization capacity as patritumab. For both HER3 mAbs, internalization was greater in a HER3 high than a HER3 low cell line. In cytotoxicity assays, HER3-ADC1 and patri-DXd displayed HER3 expression level-dependent cell killing. HER3-ADC1 outperformed patri-DXd in HER3 high cell lines based on in vitro cytotoxicity. HER3-ADC1 and patri-DXd were ineffective against NCI-H466 cells with low HER3 expression. In a PDX model of pancreatic cancer (HER3 high), HER3-ADC1 induced tumor regression, while isotype-MMAE had only a moderate anti-tumor effect. Both patri-DXd and isotype-DXd had moderate anti-tumor effects, indicating a lack of HER3 target dependence. In a PDX model of breast cancer (HER3 low), HER3-ADC1 and isotype-MMAE lacked anti-tumor activity; isotype-DXd had a moderate anti-tumor effect and patri-DXd had a substantial anti-tumor effect. Conclusions: HER3-ADC1 demonstrated target-dependent in vitro cytotoxicity and in vivo anti-tumor activity in a HER3-expressing pancreatic cancer PDX model. Results from in vitro and in vivo studies highlight the promising therapeutic potential of a seribantumab-based ADC for patients with HER3-expressing cancers. Additional results on the optimization and characterization of HER3-ADC1 will be presented. Citation Format: Thomas O'Hare, Jaclyn Cleveland, Valerie M. Jansen, David Dornan. Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers [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 3121.

Abstract 1891: Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers such as urothelial, head-and-neck, breast, lung, pancreatic, ovarian, and cervical cancer, 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 and to maximize on the potential of Nectin-4 as a tumor target beyond urothelial cancers, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio of 8. ADRX-0706 is comprised of a novel fully human IgG1k monoclonal antibody linked to an optimized tubulin inhibitor payload AP052 through a proprietary cleavable linker. ADRX-0706 has strong binding affinity and selectively induces cytotoxicity in Nectin-4-expressing tumor cells. Importantly, ADRX-0706 is designed to have improved bystander effect, which allows tumor regression even in cases of heterogeneous Nectin-4 expression. This translates to superior in vivo efficacy of ADRX-0706 over EV in multiple mouse xenograft models of other indications beyond bladder cancer. In a mouse clinical trial of patient-derived cervical cancers with a range of Nectin-4 expression levels, ADRX-0706 reaches a 73% ORR. Since peripheral neuropathy is a main platform toxicity for this payload class, ADRX-0706 is assessed in an in vitro co-culture model to evaluate its neurotoxic potential. While EV displays a 75% decrease in neurite outgrowth, ADRX-0706 strikingly has no impact at concentrations up to 1mg/ml, suggesting a lower risk of peripheral neuropathy in humans for ADRX-0706. In preclinical repeat dose toxicology study, ADRX-0706 reaches an HNSTD of 18mg/kg in NHPs well above therapeutic exposures in mouse pharmacology studies, indicating a tremendous increase in the therapeutic index compared to EV. In summary, the highly selective and potent anti-tumor activity in multiple tumor types and wide preclinical therapeutic index 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: Andrew M. Hau, Maria Shahmoradgoli, Dong Jun Lee, Wes Sisson, Anna Wang, Peter P. Challita, Erin Nye, Mario M. Kuo, Alexis Mahloch, Monica Leung, Oscar Betancourt, Alexander Chu-Kung, Maojun Guo, Hui Li, Sabine Rottmann, Pia M. Challita-Eid. Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved 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 1891.

Peptide‐Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates

AbstractThe role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity‐peptide mediated functionalization of native off‐the‐shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug–antibody ratio (DAR) of 2. Here, we report the traceless, peptide‐directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off‐the‐shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide‐directing moiety.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

65 Recent developments in antibody-drug conjugates (ADC) in the treatment of head & neck cancer

Chemotherapy, targeted therapy and immunotherapy represent standard of care treatment options in patients with head and neck squamous cell carcinoma (HNSCC). Despite this armamentarium, the prognosis of patients with recurrent and/or metastatic disease remains poor.Antibody drug conjugates (ADC) are a novel class of anticancer drugs that are composed by an antibody, a linker and a payload consisting of anticancer drug. Additional important features include the Drug Antibody Ratio (DAR) and the stability of the linker. The aim of ADCs is to deliver conjugated drugs selectively to cancer cells while sparing normal cells.First ADCs were approved for clinical use more than two decades ago, initially for the treatment of hematological malignancies followed by metastatic breast cancer.The most obvious target for ADCs in HNSCC is EGFR, but other targets were investigated as well. Results of clinical trials investigating the safety and efficacy of ADCs in HNSCC will be presented.

Antibody and antibody fragments site-specific conjugation using new Q-tag substrate of bacterial transglutaminase

During the last few years Antibody-Drug Conjugates (ADCs) have become one of the most active and very promising therapeutic weapons. Lessons learned from the traditional chemical conjugations (via lysine or cysteine residues of the antibodies) and the clinical studies of the developed ADCs have recently paved the way to the improvement of the conjugation technologies. Use of site-specific conjugation is considered as the promising path for improving the design and development of homogeneous ADCs with controlled Drug-Antibody ratio (DAR). Moreover, some of these conjugations can be applied to antibody fragments such as Fab, scfv and VHH for which random and chemical conjugation showed significant limitations. In this study, we identified a novel small peptide substrate (Q-tag) with high affinity and specificity of bacterial transglutaminase which can be genetically fused to different formats of antibodies of interest for the development of enzymatic site-specific conjugation we named “CovIsolink” platform. We describe the synthesis of chemically defined drugs conjugation in which the site and stoichiometry of conjugation are controlled using a genetically encoded Q-tag peptide with specific amino acids which serves as a substrate of bacterial transglutaminase. This approach has enabled the generation of homogeneous conjugates with DAR 1,7 for full IgG and 0,8 drug ratio for Fab, scfv and VHH antibody fragments without the presence of significant amounts of unconjugated antibody and fragments. As a proof of concept, Q-tagged anti Her-2 (human IgG1 (Trastuzumab) and the corresponding fragments (Fab, scfv and VHH) were engineered and conjugated with different aminated-payloads. The corresponding Cov-ADCs were evaluated in series of in vitro and in vivo assays, demonstrating similar tumor cell killing potency as Trastuzumab emtansine (Kadcyla®) even with lower drug-to-antibody ratio (DAR).

Abstract B030: ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model

Abstract Background: ABC101-1 is an antibody-drug conjugate (ADC) targeting receptor tyrosine-protein kinase erbB-2, also known as HER2. ABC101-1 consists of a humanized IgG1 monoclonal antibody called trastuzumab conjugated to a microtubulin inhibitor, DM1, through interchain cysteines with a drug-to-antibody ratio (DAR) of 8. Our novel non-cleavable linker platform using maleimide as a connector enabled the construction of homogeneous ABC101-1 with DAR of 8, via a straightforward process resulting in high conjugation yield. Employing non-cleavable linkers may ensure that the cytotoxic drug is delivered stably to tumor cells, enhancing the therapeutic efficacy of ADC. Herein, we describe anti-breast cancer activity through in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and binding affinity of ABC101-1. Methods: In vitro cytotoxicity of ADC and linker-payload was evaluated by measuring cell viability using Cell Count Kit-8 (CCK8) analysis. In vivo anti-tumor activity was investigated using SKBR3 cell line-derived xenograft (CDX) model. A pharmacokinetic (PK) profile was assessed by performing indirect ELISA to determine concentrations of total mAb in the serum. The binding affinity of ABC101-1 to HER2 protein was assessed using the indirect ELISA measuring the concentrations of total mAb. Results: In this study, ABC101-1 exhibited potent in vitro cytotoxicity with picomolar activity on HER2-positive breast cancer cell line. In the CDX breast cancer model, ABC101-1 led to stronger tumor growth inhibition in both early and late phases than ado-trastuzumab emtansine (DAR 3.5) at the same dose. Also, the favorable pharmacokinetic profile of ABC101-1 was observed, along with the preserved antigen-binding ability upon the conjugation to linker-payload. Conclusions: ABC101-1 displayed preclinical efficacy in in vitro and in vivo studies as well as a favorable PK, thereby suggesting its therapeutic potential as an anti-tumor drug in breast cancer therapy. This study would reveal that our novel linker platform is a promising and efficient strategy to develop effective and homogeneous ADCs with DAR 8. Our finding may support the further pre-clinical evaluation of ABC101-1. Citation Format: Seo Woo Kim, Hana Yu, Geon Ho Lee, Tae Gyu Lim, Minji Jeong, Hyeonjoo Hwang, Jee Eun Moon, Yong Beom Lee, Sung-Ha Jin, Jin Hyun Jeong. ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B030.

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

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.

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 .

Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer

Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.

Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

Impact of drug-linker on method selection for analytical characterization and purification of antibody-drug conjugates.

In addition to traditional characterisation methods of hydrophobic interaction (HIC) and reverse phase (RP) chromatography, an anion exchange chromatography (AIEX) was developed to analyse and purify antibody drug conjugates (ADCs). Since different drug antibody ratio (DAR) species may impact biological activity, therapeutic index, PK parameters or even potential immunogenicity, homogenous ADC DAR demands have been significantly increasing. To accelerate linker designs, drug screening and ADC DAR purification for in vitro and in vivo studies, we built the analytical toolbox including HIC, RP, AIEX, icIEF, SEC, and MS for downstream ADC DAR purification using HIC and AIEX. The established analytical methods can quickly assess the quality of ADC DAR profiles and provide important information to select the proper ADC DAR purification method. Since drug-linker structures can significantly affect ADC physicochemical properties, and highly impact on selections of analytical methods, we applied both HIC and AIEX characterisation and purification platforms to achieve ADC DAR homogenous. Our experiments also implied that unlike HIC, AIEX could be used to separate DAR4 positional isomers.

Preparation of trastuzumab-DM1 conjugate with a high drug-to-antibody ratio for breast cancer therapy

Antibody-drug conjugates (ADCs) are important in active tumor-targeting therapy. However, improving the efficiency of payload delivery to tumors without affecting the tumor-targeting ability of the antigen-binding fragment (Fab) structure remains a challenge. In this study, a Fab-nondestructive ADC loaded with maytansine (DM1) (T-NPLG-DM135) was prepared with drug-to-antibody ratio (DAR) of up to 35 by utilizing the carboxyl groups on the side-chain of poly(L-glutamic acid) (PLG) as the payload binding sites. The use of the Fc-III-4C peptide and the preparation condition of aqueous environment were conducive to avoiding the impact of the Fab structure of monoclonal antibodies on tumor-targeting capability. T-NPLG-DM135 was recognized and rapidly internalized by HER2-expressing SKBR-3 tumor cells, with a 3.8-fold increase in accumulation compared to that of the non-tumor-targeting IgG-NPLG-DM135. T-NPLG-DM135 also showed excellent tumor inhibition and its therapeutic effect improved as the DAR increased. The successful development of T-NPLG-DM135 demonstrates the potential of this approach for future ADC design and development.

Validation of ADC Platform for Protein Concentration and the Drug-Antibody Ratio (DAR) using Variable Pathlength Technology

Antibody Drug Conjugates (ADCs) are biopharmaceutical drugs which are designed to treat cancer by targeting and killing the cancerous cells while leaving the healthy tissue intact. ADCs are comprised of an antibody connected to a cytotoxic drug by a linkerThe antibody targets a specific antigen which is present in cancerous cells but not healthy cells. The ADC binds to the cancer cell and becomes internalized where the cytotoxic drug is released and kills the cancer cell. Two important pieces of information required about the ADC are the protein concentration and the Drug-Antibody Ratio (DAR). The DAR is the number of drug molecules attached to the antibody. It is important during the manufacturing steps to ensure that the process is proceeding as expected and that there is not more or less drug present than required.

Abstract C121: Development of CRB-701 (SYS6002): A novel site-specific, Nectin-4 targeting ADC

Abstract CRB-701 (SYS6002) is a clinical stage, next generation ADC targeting Nectin-4 wherein two monomethyl auristatin E (MMAE) moieties are site-specifically conjugated to a novel Nectin-4 targeting IgG1 antibody via a cleavable linker. Nectin proteins are cell adhesion molecules belonging to the immunoglobulin (Ig) superfamily that participate in cell-to-cell interactions and form adhesion junctions between cells. Nectin-4 is widely expressed in epithelial cancers including bladder, breast, lung, colorectal, pancreatic, and ovarian. By contrast the expression level of Nectin-4 is very low in healthy adult human tissues. Due to this differential expression, Nectin-4 has emerged as a robust tumor associated antigen (TAA) that has been clinically validated in urothelial carcinoma by Enfortumab Vedotin (EV). While approved, EV carries associated toxicity concerns including skin rash, peripheral neuropathy and ocular toxicities that can lead to EV discontinuations and lasting adverse effects. CRB-701 (SYS6002) has demonstrated high affinity Nectin-4 binding (~2ng/ml) across species and has selectivity for Nectin-4 among the Nectin family of proteins (>5000 ng/ml). The antibody retains potent in vitro activity against FcgRI and C1q (<10nM) conferring antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) activity in vitro. Compared to EV, CRB-701 (SYS6002) reduces free MMAE exposure by 4.8-9.7-fold when adjusted to ADC exposure (AUC) and there is a 2.2-7.4-fold reduction in MMAE in circulation based on Cmax in rats and non-human primates. Collectively these data reflect lower free MMAE in plasma, reducing the risk of MMAE-induced toxicities observed with EV in the clinic. This improved PK and safety profile coincides with a longer half-life of the ADC in plasma potentially reducing the frequency of administration to achieve efficacy. The internalization rate of CRB-701 (SYS6002) was 2-fold faster than EV when assessed in nectin-4 expressing cells. These data may explain the statistically significant reduction in tumor growth observed in a BL0597 patient derived xenograft model (PDX) of bladder cancer with low Nectin-4 protein expression by IHC (H score =50) when compared to EV at the same dose and frequency. Collectively these data suggest that CRB-701 (SYS6002) could demonstrate an improved therapeutic index and PK profile relative to EV and as such could achieve higher tolerated concentrations at lower dose frequency. The stability of the site-specific linker and the reduction in the drug-antibody ratio (DAR) could lead to a differentiated profile clinically that enables higher doses, longer treatment duration and a greater potential in drug combinations in nectin-4 positive tumors. CRB-701 (SYS6002) is currently being explored clinically in a phase 1 dose escalation with an anticipated completion date mid-2024. Citation Format: Rachael Brake, Zhaopeng Sun, Mo Dan, Lu Lv, Congcong Niu, Yang Zhang, Mingyue Shen, XiXin Hu, Xiwu Hui, Andrew Kolodziej. Development of CRB-701 (SYS6002): A novel site-specific, Nectin-4 targeting ADC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C121.

IKS03, a Novel CD19-Targeted Antibody Drug Conjugate, Induces Target Dependent In Vivo Cell Killing of B-Cell Lymphoma Xenografts By DNA Crosslinking

CD19 is widely expressed in B-cell non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). IKS03 is a CD19-targeting antibody drug conjugate (ADC) with a novel linker payload design for tumor-selective payload release. Site-specific conjugation using farnesyltransferase-catalysed ConjuALL technology to a DNA crosslinking agent yields a homogenous ADC with drug-to-antibody ratio (DAR) of 2. To minimize systemic release of the highly potent payload in human plasma, IKS03 incorporates a linker specifically cleavable by the lysosomal enzyme beta-glucuronidase. In addition, the payload is a pro-drug with a protecting moiety cleavable by the same enzyme. Therefore, following CD19-dependent binding and uptake, IKS03 requires intracellular lysosomal processing in the target cell to both release and fully activate the free pyrrolobenzodiazepine (PBD) dimer payload which induces DNA crosslinking, blocks DNA replication and ultimately leads to tumor selective cell death. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. IKS03 was highly active against Farage and OCI-LY10 DLBCL xenografts as well as Granta-519 MCL xenografts in SCID mice with durable tumor regressions observed following treatment with a single dose of 0.1 to 0.3 mg/kg. Likewise, in a Ramos lymphoma model a single IKS03 dose of 0.3 mg/kg significantly inhibited tumor growth. IKS03 demonstrated greater in vivo efficacy than the clinical benchmark ADCs polatuzumab vedotin and loncastuximab tesirine approved for the treatment of patients with B-cell non-Hodgkin lymphomas. PBD cytotoxic agents have been described to induce DNA crosslinking leading to DNA damage. To demonstrate that IKS03 in vivo activity is the result of targeted payload release, Ramos xenografts were established in immunodeficient mice and treated with up to 1 mg/kg of IKS03 in comparison with a non-targeted ADC utilizing the same linker payload format. Tumors were excised at 1 day and 3 days post-treatment and assessed for the presence of phospho-H2AX positive foci, a marker of DNA damage, by immunohistochemistry. IKS03 treatment increased both frequency and staining intensity of nuclear phospho-H2AX foci in Ramos xenografts in a dose- and time-dependent manner. In contrast, the non-targeted control ADC did not result in any changes as compared to a vehicle control. This confirms the specific induction of DNA damage in CD19 positive lymphomas as a pharmacodynamic response to IKS03 treatment in vivo. In conclusion, IKS03 is highly effective in an array of preclinical MCL and DLBCL xenograft models at doses that are well tolerated. In vivo, IKS03 treatment increased the level of phospho-H2AX indicative of irreversible DNA damage in a target-dependent manner. This is consistent with the proposed mechanism of action of CD19-mediated delivery of a PBD payload specifically to B-cell tumors. Clinical investigation in patients with advanced B-Cell Non-Hodgkin Lymphomas is being initiated (NCT05365659).

Debio 1562M, a Next Generation Antibody Drug Conjugate (ADC) Targeting CD37 for AML and MDS Treatment

CD37 is a trans-membrane protein specifically expressed on normal hematopoietic tissues such as B cells, neutrophils and macrophages, and has demonstrated roles in adhesion, cell migration (de Winde et al, 2018; Wee et al, 2015), survival and apoptotic signals (Lapalombella et al, 2012). Increased expression of CD37 has been observed in various hematological cancers such as lymphomas, Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML) (Beckwith et al, 2014; Deckert et al, 2013; Pereira et al, 2015; Yan et al, 2021). Higher expression of CD37 was also associated with poor patient outcome in AML (Zhang et al, 2020; Larkin et al, ASH 2018). Debio 1562M is a second-generation novel ADC directed against CD37 and including Debiopharm's proprietary Multilink cleavable linker-payload technology. Multilink improves manufacturing to achieve homogenous high drug antibody ratio while retaining excellent solubility as well as favorable biologic characteristics with preserved internalization kinetics on target cells. Debio 1562M is conjugated to 8 molecules of a tubulin-binding agent and despite cleavable linker that ensure effective intracellular delivery of toxic payload, we show sustained plasma stability in vitro and in vivo with no premature extracellular release. In this work, we report Debio 1562M specificity and potent antitumor activity in multiple AML models both in vitro and in vivo. First, in our in vitro cytotoxicity assay, Debio 1562M displayed nano- to picomolar anti-proliferative activity in both AML cell lines and primary samples. Out of 30 primary samples tested, 20 were sensitive to Debio 1562M with IC50 ranging from 1 to 10nM, this regardless of their mutation status, subtype or medical history. Interestingly, we observed that AML cells have high internalization capability and therefore relatively low levels of surface CD37 is sufficient for cytotoxic activity. The specificity of Debio 1562M was demonstrated by using 3 different CD37 knock out AML cell lines which were generated by CRISPR-Cas9 technology. Parental and CD37 KO cells were treated for 72h with a dose range of Debio 1562M and viability was measured (Figure 1). While Debio 1562M has an IC50 below 1nM in all 3 parental cell lines, its activity is reduced by more than 2 log in the KO cell lines. In vivo, in 3 different AML cell line xenograft mouse models, a single injection of Debio 1562M at 5mg/kg induced complete tumor regression and all mice remained alive at study termination, more than 60 days after treatment, except one mouse in one model. In a patient derived xenograft (PDX) model, the same dose of Debio 1562M significantly reduced tumor burden in blood and bone marrow with sustained activity 42 days after treatment (37% and 98% of blasts in blood and bone marrow respectively in control animals versus 0% and 3% in treated mice, pvalue<0.001). Furthermore, we evaluated the activity of Debio 1562M over venetoclax and azacytidine combination in the MV4-11 Luciferase xenografted mouse model. NCG mice were inoculated with 1x10 7 cells in the tail vein and randomization in groups of 8 mice was performed 14 days later. Mice received either one administration of Debio 1562M at 5mg/kg or a combination of venetoclax (QDx14 100mg/kg) and azacitidine (QDx5 3,5mg/kg). Complete regression of the tumor was observed in all mice after Debio 1562M administration and all mice survived at study termination, 75 days after treatment while none survived after 56 days when treated with venetoclax and azacitidine combination (Figure 2). The toxicology profile of Debio 1562M in mice showed expected clinical signs related to the payload's off target toxicity but with a significant safety margin. Interestingly, no peripheral neuropathy symptoms were detected, as commonly observed for this class of payload. Additionally, Multilink TM-payload construct alone was not associated with any signs of toxicity. On-target toxicity profile couldn't be evaluated as Debio 1562M isn't cross reactive with mouse CD37, however 1 st generation CD37 ADC has already demonstrated safety and tolerability in Phase 1 and 2 clinical trials.Overall, our data confirm the relevance of CD37 targeting for AML treatment. The excellent antitumor activity and tolerability of Debio 1562M in several pre-clinical models now pave the way for upcoming clinical development.

TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

BackgroundPancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface antigen overexpressed in the tumors of more than half of pancreatic cancer patients, has been identified as a potential target for antibody–drug conjugates (ADCs). Almost all reported TROP2-targeted ADCs are of the IgG type and have been poorly studied in pancreatic cancer. Here, we aimed to develop a novel nanobody-drug conjugate (NDC) targeting TROP2 for the treatment of pancreatic cancer.ResultsIn this study, we developed a novel TROP2-targeted NDC, HuNbTROP2-HSA-MMAE, for the treatment of TROP2-positive pancreatic cancer. HuNbTROP2-HSA-MMAE is characterized by the use of nanobodies against TROP2 and human serum albumin (HSA) and has a drug-antibody ratio of 1. HuNbTROP2-HSA-MMAE exhibited specific binding to TROP2 and was internalized into tumor cells with high endocytosis efficiency within 5 h, followed by intracellular translocation to lysosomes and release of MMAE to induce cell apoptosis in TROP2-positive pancreatic cancer cells through the caspase-3/9 pathway. In a xenograft model of pancreatic cancer, doses of 0.2 mg/kg and 1 mg/kg HuNbTROP2-HSA-MMAE demonstrated significant antitumor effects, and a dose of 5 mg/kg even eradicated the tumor.ConclusionHuNbTROP2-HSA-MMAE has desirable affinity, internalization efficiency and antitumor activity. It holds significant promise as a potential therapeutic option for the treatment of TROP2-positive pancreatic cancer.Graphical

EGFR-targeted humanized single chain antibody fragment functionalized silica nanoparticles for precision therapy of cancer

The combination of highly specific targeting ability and potent killing effect has made antibody-drug conjugates (ADCs) a popular area of focus in the development of anti-cancer drugs. However, the large molecular weight of IgG antibodies (∼ 150 kDa) often faces challenges in penetrating capillaries and stroma in tumor tissue. Moreover, when the drug-antibody ratio (DAR) is too low (DAR < 2) or too high (DAR > 6) it decreases the effectiveness of the ADC and further increases the potential for aggregation, overall clearance of the early system payload, and release rate. In this study, an EGFR-based single-chain antibody fragment (husA)-human serum albumin (HSA)-coupled FITC-labeled mesoporous silica nanoparticle (FMSN-DOX-H-husA) was developed. Chinese hamster ovarian cells express the husA, which is a single chain antibody fragment of the EGFR that has been humanized. The small molecular weight of the single chain antibody allows for shorter penetration into solid tumors and the absence of adverse effects of the Fc fragment. The modification of HSA improves the safety of the antibody nanoparticle couples by both improving the biocompatibility of the nanoparticles, prolonging the circulation time of the nanoparticles, and avoiding early release of the payload. Also, the humanization substantially reduces the immunogenicity. More importantly, the ratio of drug antibodies on nanoparticles was experimentally and computationally derived to be 11.8, providing a more accurate guide for clinical trials. The results of both in vivo and in vitro experiments indicated promising antitumor activity and safety of FMSN-DOX-H-husA. Thus, this antibody-drug conjugate provided a hopeful option for cancer treatment.