Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with nine ADCs approved for clinical use and two under review. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. To select antibodies, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes in an unconjugated form. For payload selection, we biotinylated toxins and conjugated these to streptavidin conjugated antibodies. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (trastuzumab-SB-DM1) to trastuzumab emtansine (T-DM1), which is approved for clinical use. We showed equivalent mechanism of action and comparable potency in reduction of breast cancer cell survival in vitro, and in growth restriction of HER2-expressing orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs, and this approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.
Ricarda M Hoffmann1, 2, Pablo Romero-Clavijo1, Silvia Mele1, Anthony Cheung1, 3, Daniel Larcombe-Young3, Gintare Bucaite4, 5, Eirini Sachouli1, Iva Zlatareva1, Hassan O J Morad1, Rebecca Marlow3, 6, James M McDonnell4, 5, Mariangela Figini7, Katie E Lacy1, Andrew J N Tutt3, 6, James F Spicer8, David E Thurston9, 10, Sophia N Karagiannis1, 2, 3, Silvia Crescioli11, 12
1 St. John's Institute of Dermatology, School of Basic & Medical Biosciences, King's College London, Tower Wing, 9th Floor, Guy's Hospital, London, SE1 9RT, United Kingdom.
2 NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, King's College London, London, United Kingdom.
3 Breast Cancer Now Research Unit, School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, United Kingdom.
4 Randall Centre for Cell and Molecular Biophysics, King's College London, London, SE1 1UL, United Kingdom.
5 Asthma UK Centre in Allergic Mechanisms of Asthma, King's College London, London, SE1 1UL, United Kingdom.
6 Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, London, United Kingdom.
7 Biomarker Unit, Department of Applied Research and Technology Development, Fondazione, IRCCS Istituto Nazionale dei Tumouri Milano, 20133, Milan, Italy.
8 School of Cancer & Pharmaceutical Sciences, King's College London, 3rd Floor, Guy's Hospital, London, United Kingdom.
9 Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King's College London, London, SE1 9NH, United Kingdom.
10 Femtogenix Ltd, Lawes Open Innovation Hub, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom.
Conjugation with reduced Cysteine of inter-chain disulfide bonds from IgG mAbs (mainly IgG1 & IgG4) dominates the conjugation strategy in ADC development. 6 out of 9 FDA approved ADCs are produced this way. In many occasions ADCs with 4 drug load (DAR4) give the best clinical performance, yet their productions by traditional way always result in heterogeneous products with wide DAR distributions: Efforts have been made to improve the homogeneity of ADCs, eg, by point mutation with Cysteine or introduction of non-natural amino acids, glycan re-modeling, enzyme involved conjugation. Here we introduce the WUXIDAR4 technology for ADC production resulting in high DAR4 population with linker-payloads at Fab region. It is achieved by chemical interference without changing IgG1 structure, or by a simple switching of IgG domains.
We introduce a novel linker technology for antibody-drug conjugates (ADCs) that enables site-specific payload conjugation to native (‘off-the-shelf’) antibodies with no prior engineering necessary. Our approach is based on novel hydrophilic peptide-linkers and the well-known microbial transglutaminase (MTG) – previously believed to require antibody engineering for payload conjugation. We will show that two or four payloads can directly and efficiently be conjugated using as little as 5 molar excess of the payload vs. the antibody resulting in ADCs with a very well-defined DAR2 or 4. The ADCs show an excellent stability in various sera incl. mouse, cyno and human with no linker cleavage and/or payload deconjugation detected after 14d of incubation while the reference ADC, based on conventional hinge-cysteine conjugation using the same antibody and payload showed significant payload loss. Interestingly, our linker is as efficiently cleaved by Cathepsin B (CatB) as the reference ADC which uses a CatB cleavable sequence in the linker. Furthermore, the ADCs show an excellent stability profile with no increase in aggregation compared to the reference after 14d of incubation at 37°C. In head-to-head cell-toxicity assays, our ADCs are as potent as the reference ADCs. Importantly, this conjugation approach can also be performed at gram-scale with no detectable loss of conjugation efficiency, with the ADCs being >98% monomeric, an ADC recovery rate of ~90% (under non-optimized conditions) and a final endotoxin-level below the detection limit.
Site-specific antibody conjugation using the GlyCLICK technology has proven a promising platform for obtaining homogenous antibody drug conjugates (ADCs). The technology is based on enzymatic processing of the Fc-glycans on a native antibody and site-specific conjugation using click-chemistry. We have previously demonstrated GlyCLICK conjugated antibodies to stay longer in circulation and increase tumor uptake compared to randomly conjugated antibodies. In this work we combine enzymatic conjugation of antibodies at the Fc glycans with two-step cleavable glycopeptide linker-payloads and characterize the conjugation process as well as study the functional properties of the ADCs in vitro and in vivo. The glycopeptide cleavable linker relies on a both β-glucuronidase and cathepsin B for release, which reduces off target toxicity. As a model therapeutic, trastuzumab was conjugated with two glycopeptide linker-payloads with PNU and MMAE, respectively. The conjugation process was optimized for linker-payloads and the enzymatic steps were carefully studied using middle-level LC-MS and revealed DAR 2.0 conjugates for both payloads. The trastuzumab-PNU and trastuzumab-MMAE were further studied for in vitro efficacy using SK-BR-3 HER2+ cells and the antibody drug conjugates showed IC50-values in the pM range. Further, a nondisclosed antibody was conjugated with the PNU-linker using the same process and evaluated in a mouse B16-F10 melanoma model. Complete and durable responses were observed after single 5 mg/kg dose of PNU ADC in this difficult to treat cold tumor model.
In conclusion, the data demonstrates site-specific conjugation and generation of unique ADCs using a glycopeptide linker with potent payloads that results in DAR 2.0 conjugates with potent tumor killing activities in vitro and in vivo. This technology has potential to transform any native antibody into a potent ADC and has applications both for preclinical and clinical development of site-specific ADCs.
SPR binding assays allowed real time kinetic analysis of interactions between Fc receptors and mAb-based therapeutics including ADCs. We have demonstrated rapid analysis of different constructs using SPR which provides preliminary indication of linker selection on Fc binding activity. Our results suggested some loss in affinity for FcRn but a general increase in FcɣRIIIa binding activity for Tmab using PEG linkers. Further investigation is required to understand the impact on the linker conjugation to the sensitivity of target binding and effector function. The goal of ADC Express is to deliver research ready, ADC construct diversity sets to assist customers with the selection of appropriate candidates for clinical scale production. Working with our Millipore Sigma partners, we can provide any and all diversity concepts from varied mAb constructs to conjugation methodologies to linkers and payloads. These libraries can be used to promote confident, data-driven candidate selection at the pre-production development phase to maximize therapeutic outcomes in the clinic. Our aim is to offer a commercially viable diversity set(s) that can become the industry standard tool for pre-clinical candidate selection.
Yu-Ting Hsu, Jeffery Carroll, Jason Ramsay, and Lisa McDermott
Process and Analytical Development | Life Science | Process Solutions | Actives & Formulation | Merck KGaA, Darmstadt, Germany
Deepa Raghu, Pamela Hamill
BioReliance Product Characterization Services | Life Science | Process Solutions Services | Merck KGaA, Darmstadt, Germany
Presenter Name: Yu-Ting Hsu
Job Title: Senior Scientist, Process & Analytical Development
Antibody Drug Conjugates (ADCs) represent a significant area with clinical and economic growth for the biopharmaceutical market. ADCs combine the targeted specificity of a monoclonal antibody (mAb) with a cytotoxic small molecule. They are manufactured by attaching the monoclonal antibody to potent cytotoxic payload via a heterobifunctional linker. Here we consider the crossflow filtration process in ADC and evaluate the benefit of introducing Hydrosart® Cassettes to remove reaction related impurity to lowest level with fastest processing time.
In the world of Antibody Drug Conjugates (ADC), maleimide-drugs are often used because of their readiness to react with mAbs and form ADCs. However, the bioconjugation process involves several steps and for each step there are several parameters that may affect the final quality of the ADC. These issues are all the more likely to appear when scaling-up the process.
In this case study, the Drug to Antibody Ratio (DAR) was not fully controlled when manufacturing at larger scales. That is why, a scaling-down approach was used and several Designs of Experiments (DoE) were executed to identify the main parameters affecting the DAR. Then, a final DoE was performed to determine the optimal parameters for the bioconjugation process.
Finally, scaling-up was successfully carried out with a controlled DAR to target a value of 4.0.
Claire DEFAIX, Process Development Engineer, Novasep Seripharm, 1 rue Démocrite 72000 Le Mans, FRANCE
Melanie DERDE, Bioconjugation analytical manager, Novasep Seripharm, 1 rue Démocrite 72000 Le Mans, FRANCE
Bertrand COTTINEAU, PR&D Group Head, Novasep Seripharm, 1 rue Démocrite 72000 Le Mans, FRANCE
Presenter Name: Bertrand Cottineau
Job Title: Head of Potent Chemistry, Bioconjugation, Research & Development
This poster describes the use of design of experiments (DOE) to understand the impact of process parameters at the reactive stages on an ADC product quality attributes. A 24 full factorial design was performed and the results were analysed via the MODDE software, with Drug to Antibody ratio (DAR) results shown. The software enabled identification of safe manufacturing set points for the process parameters.
Eoin Gould (Piramal, Protein Scientist),
Erik Johansson (Sartorius, Principal Data Scientist),
Ian Schwartz (Sartorius, Global Technology Consultant),
Xavier Despinoy (Piramal, Process Technology Lead)
The chemical conjugation of antibodies in a site-directed manner remains an area of great interest and active efforts within the ADC community. One of the difficulties often encountered during the chemical modification of native non-engineered antibodies is the lack of selectivity that can be dialed in toward specific residues. In this poster, we present a novel platform named “AJICAP™” for the site-selective conjugation of native antibodies through the use of a class of IgG Fc-affinity reagents to install payload-compatible linkers to well-defined amino acid residues. Recent enhancements of the AJICAP™ platform (dubbed Second Generation AJICAP™) and conjugation at an additional Lys residue will be introduced. Therapeutic window enhancement will be also discussed.
Introduction: Patritumab deruxtecan is an investigational human epidermal growth factor receptor 3 (HER3)-directed antibody drug conjugate (ADC) with a topoisomerase I inhibitor payload, and it has shown preliminary antitumor activity in patients with previously treated metastatic or locally advanced epidermal growth factor receptor-mutated (EGFRm) non–small cell lung cancer (NSCLC) in an ongoing phase 1 study (NCT03260491). Here we present initial observations from comprehensive genomic profiling in tumor tissue and circulating tumor DNA (ctDNA), tumor HER3 expression and its association with prior treatment and with the clinical activity of patritumab deruxtecan, and dynamic changes in a ctDNA panel during study treatment.
Methods: We evaluated tumor tissue and peripheral blood samples from 56 patients with EGFR-mutated NSCLC treated with 5.6 mg/kg patritumab deruxtecan (intravenously once every 3 weeks) after failure of EGFR tyrosine kinase inhibitor (TKI). Blood samples for ctDNA were collected before each dose and at the end of treatment. Pre-treatment tumor samples were evaluated by immunohistochemistry for HER3 expression. Genomic profiling of pre-treatment tumor tissue and analyses of ctDNA were performed using the Oncomine™ Comprehensive Assay v3 and the GuardantOMNI™ panel, respectively. In addition, serial ctDNA samples were analyzed using the Biodesix platform to detect changes in the presence of four types of EGFR mutated alleles: Ex19Del, L858R, T790M, and C797S.
Rationale: Previous reports have demonstrated that EGFR TKI treatment is associated with dynamic changes in relative levels of EGFR mutant alleles in ctDNA samples. This presentation provides the first characterization of shifts in tumor heterogeneity during treatment with patritumab deruxtecan, and it describes such changes using a panel of EGFR mutant alleles in ctDNA during treatment with patritumab deruxtecan and at the time of progression. Moreover, detailed analyses of tumor and peripheral blood ctDNA genomic alterations in these patients will be presented with a focus on diverse TKI resistance mechanisms. A preliminary characterization of HER3 expression, as well as the relationship between antitumor activity and the presence of baseline tumor mutations and ctDNA clearance, will be presented. This study will provide important information about potential predictive and pharmacodynamic biomarkers associated with patritumab deruxtecan treatment, and these findings will be of significant interest to clinicians seeking an understanding of how molecular determinants might guide the use of patritumab deruxtecan in patients with previously treated EGFR-mutated NSCLC.
Pasi A. Jänne,1 Christina Baik,2 Hidetoshi Hayashi,3 Wu-Chou Su,4 Zhenhao Qi,5 Kei Enomoto,6 Maha Karnoub,5 Channing Yu,5 Yang Qiu,5 Helena Yu7
1Dana-Farber Cancer Institute, Boston, MA; 2Seattle Cancer Care Alliance, Seattle, WA; 3Kindai University Hospital, Osaka-Sayama City, Japan; 4National Cheng Kung University Hospital, Tainan, Taiwan; 5Daiichi Sankyo, Inc., Basking Ridge, NJ; 6Daiichi Sankyo Co., Ltd., Tokyo, Japan; 7Memorial Sloan Kettering Cancer Center, New York, NY
Presenter Name: Dr. David Sternberg
Job Title: Executive Director, Global Oncology R&D
Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.
Experimental Design: We evaluated FRα expression in breast cancers by genomic (n=3,414) and IHC (n=323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the anti-tumour functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft model.
Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumours, and in post-neoadjuvant chemotherapy–residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. RNA interference to deplete FRα decreased Src and ERK signalling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with Src inhibitor (A-419259) significantly restricted TNBC xenograft growth.
Conclusions: FRα is overexpressed in high-grade TNBC and post-chemotherapy residual tumours. It participates in cancer cell signalling and presents a promising target for therapeutic strategies such as antibody–drug conjugate (ADC).
Anthony Cheung1,2, Ricarda M. Hoffmann2, James Opzoomer2, Alicia Chenoweth1,2, Kristina M Ilieva1,2, Patrycja Gazinska3, Hasan Mirza1, Rebecca Marlow1, Silvia Crescioli2, Heng Sheng Sow2, Anita Grigoriadis1, Andrew N J Tutt1,3 and Sophia N Karagiannis1,2
1 Breast Cancer Now Research Unit, School of Cancer & Pharmaceutical Sciences, King’s College London, London, United Kingdom;
2 St. John’s Institute of Dermatology, School of Basic & Medical Biosciences, King’s College London, & NIHR Biomedical Research Centre at Guy’s and St. Thomas’ Hospitals and King’s College London, Guy’s Hospital, King’s College London, London, United Kingdom;
3 Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, London, United Kingdom
Recently, site-specific conjugation technologies for ADC development become more and more important. Here we report a novel site-specific conjugation platform by conjugating the linkers and payloads to antibody at glycan sites. This method can complete control the conjugation site and ratio and generate homogeneous ADCs. Moreover, it is capable of conjugating two different payloads to one antibody at the same time.
First, we generate the trimannosyl antibodies from normal naked antibodies by the sequential digestion of neuraminidase, β-1,4-galactosidase and N-acetylglucosaminidase S enzymes. Next, N-Acetyl glucosamine transferase I and N-Acetyl glucosamine transferase II were used as enzymes to transfer azido-GlcNAc (GlcNAz) to conjugate the trimannosyl antibodies. The glycol-azide modified antibodies are anailable to lable with Dibenzocyclooctyne (DBCO)-linked payload by strain-promoted azide-alkyne click chemistry (SPAAC) reaction to form a homogenous ADC (DAR=4A or 2A+2B). The trimannosyl ADCs posses similar binding affinity and PK profile to naked antibodies and show better in vivo efficacy than random conjugation ADCs.
Despite the recent resurgence, Antibody Drug Conjugates (ADCs) are failing to address challenging cancer indications due to 3 critical limitations: Low potency, ineffective solid-tumour penetration and poor tolerability. The industry is well-served by approaches where full-length Immunoglobulins are designed to carry defined numbers of payloads. However, antibody fragments (e.g. single-chain Fvs-scFvs), which have many advantages including rapid tumour penetration, faster clearance, inexpensive manufacture, have been technologically challenging to apply in oncology. Our novel approach enables scFvs to have a high Drug:Antibody loading ratio (DAR) whilst retaining effective binding and other favourable biophysical properties, leading to a new product class tailored for solid tumours.
Antikor has two ‘first-in-class’ FDC products in development for solid tumours: anti-HER2 FDC (ANT-043) a follow-up product against a second target (ANT-045) which will be disclosed during this presentation. ANT-043 has demonstrated excellent tumour ablation effects in breast, ovarian and gastric cancer xenograft models and superior tolerability compared to an ADC. Quantitative payload tumour uptake by mass spectrometry and fluorescent immuno-histological studies demonstrate superior solid tumour penetration across the entire tumour and diffusion from blood vessels and rapid kinetic uptake. In collaboration with our partners, Essex Biotechnology, ANT-043 is moving towards clinical development. Our new flagship product, ANT-045, which emerged from our proprietary FDC ‘discovery engine’, is progressing well and new data will illustrate how ANT-045 could have a broader patient benefit in gastro-intestinal cancers. Like ANT-043, ANT-045 has potent in vitro cell-kill properties, excellent stability and drug-like features and illustrates how linker-payload design is critical for the tailoring properties of this emerging format of ADC. This poster will focus on ANT-043 and ANT-045's remarkable development and show that these FDCs demonstrate superior efficacy and tolerability compared to an ADC when equated on an equi-mass, equi-molar and equi-payload basis.
Since its introduction, xenografts on the chicken embryo’s ChorioAllantoic Membrane (CAM) has proven extremely valuable for in vivo studies of tumor development, angiogenesis and malignant cell dissemination. The CAM’s ability to efficiently grow inoculated xenogenic tumor cells greatly simplify the analysis of human tumor cell metastasis. Here we demonstrate that our in ovo model is useful for rapidly testing and comparing efficacy of Antibody Drug Conjugates (ADCs) not only on tumors, but also on metastasic invasion.
Embryonated chicken eggs N87 gastric carcinoma cells grafted on the CAM were treated with different ADCs consisting of trastuzumab (Herceptin®) and 5 different payloads.
Tumor efficacy was measured via the reduction in tumor weight compared to the negative control group (treated with only the vehicle). Thus, we observed a dose-dependent effect for all ADCs and were able to rank them according to their efficacy from 0% (Ctrl ADC) to 60% reduction in tumor weight for the most effective.
In addition to efficacy on primary tumors, we also determined the effect of ADCs on the dissemination of cancer cells. Thus, two ADCs with the same efficacy on tumors showed two radically different effects on metastasis: one showed no particular efficacy while the second reduced metastasis to 20% of those observed in the negative control group.
Camptothecins form a powerful class of cytotoxic drugs in the field of oncology, for example in treatment of solid tumors with topotecan or irinotecan. Based on their ability to inhibit DNA topoisomerase I, camptothecins also stand out as a class of payloads with a unique mode-of-action for application in antibody-drug conjugates (ADCs), as exemplified by the recent market approval of Enhertu® and Trodelvy®, as well as six additional clinical programs. We have shown earlier that the native glycan of monoclonal antibodies is a priviliged conjugation site for ADCs (GlycoConnect™) and the highly polar spacer technology (HydraSpace™) enables the conjugation of any cytotoxic payload, leading to stable ADCs with significantly expanded therapeutic index (TI) versus mainstream ADC technologies. We here show that exatecan, a clinically validated and potent campothecin, is readily combined with HydraSpace™ technology, giving rise to a potent linker-payload (SYNtecan E™) for antibody conjugation using GlycoConnect™. Homogeneous and stable ADCs of different formats (drug loading, spacer lengths) were readily generated based on the HER2-targeting antibody trastuzumab. The resulting ADCs were compared head-to-head to ADCs containing deruxtecan, the linker-payload also applied in Enhertu® and other clinical programs, and were found to be of equal efficacy, both in vitro and in vivo. Most promisingly, complete tumor regression was observed in a mouse xenograft study (BT-474) after a single dose administration, thereby demonstrating the potential of SYNtecan E™ as a novel linker-payload from the Synaffix toxSYN™ platform that is now available for licensing