Approved Antibody-drug Conjugates Research Articles

Antibody-drug conjugates as a novel therapeutic modality to treat recurrent refractory germ cell tumors.

This review provides a rationale for using the Food and Drug Administration (FDA)-approved antibody-drug conjugates (ADCs) for implementing as therapy in recurrent refractory germ cell tumors similar to their position in the treatment of other types of chemoresistant solid tumors. Germ cell tumors (GCTs) originate from germ cells; they most frequently develop in ovaries or in the testes, while being the most common type of malignancy in young men. GCTs are very sensitive to cisplatin-based chemotherapy, but therapeutic resistance occurs in a considerable number of cases, which is associated with disease recurrence and poor patient prognosis. ADCs are a novel type of targeted antitumor agents that combine tumor antigen-specific monoclonal antibodies with chemically linked chemotherapeutic drugs (payload) exerting a cytotoxic effect. Several FDA-approved ADCs use as targeting moieties the antigens that are also detected in the GCTs, offering a benefit of this type of targeted therapy even for patients with relapsed/refractory testicular GCTs (rrTGCT) unresponsive to standard chemotherapy.

Application of the ESMO Magnitude of Clinical Benefit Scale to assess the clinical benefit of antibody drug conjugates in solid cancer: a systematic descriptive analysis of phase III and pivotal phase II trials

ObjectiveThe aim of this study was to assess the clinical benefit value of approved antibody drug conjugates (ADCs) for solid tumours using the European Society for Medical Oncology Magnitude of...

Abstract 1897: Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid cancers

Abstract Introduction: Antibody-drug conjugates (ADCs) seek to increase the therapeutic window of potent cytotoxic agents by linking them with monoclonal antibodies (mAbs) to selectively deliver cytotoxic payloads to tumor cells. The effectiveness and safety of ADCs rely on both the mAb specificity, and the linker-payload employed. Several approved ADCs using microtubule inhibitor payloads are impacted by ocular adverse events that have been observed both preclinically and in patients. A class of linker-payloads incorporating topoisomerase 1 inhibitors (TOP1i) has emerged recently as an effective alternative to tubulin inhibitor-based ADCs. To date, TOP1i ADCs have not been associated with dose-limiting ocular toxicity seen with microtubule inhibitor payloads. Here we report the preclinical development of an ADAM9 (a disintegrin and metalloprotease domain 9)-targeted ADC that incorporates a novel glycan-linked TOP1i. ADAM9, a member of the ADAM family of multifunctional type 1 transmembrane proteins, plays a role in tumorigenesis and cancer progression and is overexpressed in multiple cancers, making it an attractive target for cancer treatment. Methods: MGC028 incorporates the cleavable linker-payload, bicyclononyne carbamoyl sulfamide Val-Ala-PABC exatecan (SYNtecan E™), site-specifically conjugated at asparagine 297 of the heavy chain through enzymatic glycan remodeling and metal-free click chemistry using Synaffix’s GlycoConnect™ technology. In vivo efficacy studies were performed in immunodeficient mice with ADAM9-expressing human tumor cell-line (CDX) or patient-derived (PDX) xenografts. A non-human primate toxicology study was conducted in which MGC028 was administered by 15-minute IV infusion every two weeks at dose levels of 22.5 and 55 mg/kg for a total of two doses. Results: MGC028 exhibited specific, dose-dependent in vivo antitumor activity toward ADAM9-positive CDX models representing gastric, lung, pancreatic and colorectal cancer, and head and neck squamous cell carcinoma. Furthermore, MGC028 demonstrated antitumor activity toward ADAM9-positive PDX models of lung and pancreatic cancer, and cholangiocarcinoma. MGC028 was well tolerated in a repeat-dose non-human primate toxicology study, up to 55 mg/kg, the highest dose level tested. Observations were limited to mild, reversible increases in liver enzymes, without microscopic correlates, and decreased lymphoid cellularity in the thymus. In particular, ocular toxicities were not observed. Conclusions: MGC028 exhibited potent antitumor activity in in vivo models representing various solid cancer indications and was well tolerated in non-human primates at exposure levels exceeding those required for antitumor activity. Our findings support continued investigation of MGC028 as an ADC therapeutic for the treatment of ADAM9-expressing solid cancers. Citation Format: Juniper A. Scribner, Jennifer G. Brown, Thomas Son, Linda Jin, Carroll McKenzie, Viktoriya Nam, Curtis Bush, Dienis Quinonez, Delta Ford, Verlene Gonzalez, James Tamura, Sergey Gorlatov, Hua Li, Shelley Butler, Ezio Bonvini, Deryk Loo. Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid 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 1897.

The HER2-directed antibody-drug conjugate DHES0815A in advanced and/or metastatic breast cancer: preclinical characterization and phase 1 trial results

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.

Meta-analysis of quality of life in patients with cancer treated with antibody-drug conjugates in randomized controlled trials.

Purpose: To evaluate the patient-reported outcomes of patients treated with commercially approved antibody-drug conjugates (ADC) reported in randomized controlled trials (RCT) published up toSeptember 2023. Methods: A meta-analysis of 6430 patients from 12randomized controlled trials wasconducted. Results: No significant change was observed between the groups from baseline to end of treatment and end of follow-up, with a standardized mean difference of -0.08 (95% CI: -0.27-0.12) and 0.01 (95% CI: -0.11-0.12), respectively. Treatment with ADCs delayed the deterioration of patients' clinical condition compared with treatment with non-ADCs, with a hazard ratioof 0.78 (95% CI: 0.67-0.92). Conclusion: ADCs have a good correlation with delay of clinical deterioration in patients with cancer.

Target Antigen Attributes and Their Contributions to Clinically Approved Antibody-Drug Conjugates (ADCs) in Haematopoietic and Solid Cancers.

Antibody drug conjugates (ADCs) are powerful anti-cancer therapies comprising an antibody joined to a cytotoxic payload through a chemical linker. ADCs exploit the specificity of antibodies for their target antigens, combined with the potency of cytotoxic drugs, to selectively kill target antigen-expressing tumour cells. The recent rapid advancement of the ADC field has so far yielded twelve and eight ADCs approved by the US and EU regulatory bodies, respectively. These serve as effective targeted treatments for several haematological and solid tumour types. In the development of an ADC, the judicious choice of an antibody target antigen with high expression on malignant cells but restricted expression on normal tissues and immune cells is considered crucial to achieve selectivity and potency while minimising on-target off-tumour toxicities. Aside from this paradigm, the selection of an antigen for an ADC requires consideration of several factors relating to the expression pattern and biological features of the target antigen. In this review, we discuss the attributes of antigens selected as targets for antibodies used in clinically approved ADCs for the treatment of haematological and solid malignancies. We discuss target expression, functions, and cellular kinetics, and we consider how these factors might contribute to ADC efficacy.

Targeted Drug Delivery and Theranostic Strategies in Malignant Lymphomas.

Simple SummaryThe concept of targeted drug delivery (TDD) represents an innovative and effective treatment approach, which was developed with an attempt to minimize damage toward healthy tissues. Radioimmunotherapy (RIT) with radioimmunoconjugates and TDD with antibody–drug conjugates (ADC) both represent drug delivery systems (DDS) based on monoclonal antibody-mediated delivery of toxic payloads toward the lymphoma tissue. Other modalities of TDD are based on new formulations of “old” cytostatic agents and their passive trapping in the tumor bulk by means of enhanced permeability and retention (EPH) effect. These comprise several clinically approved liposomal formulations of anthracyclines and many investigational nanomedicines including pegylated and non-pegylated liposomes, or polymer-based nanoparticles. Currently, the diagnostic and restaging procedures in aggressive lymphomas are based on nuclear imaging, predominantly on 2-[F18] fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). On a preclinical level, it has been repeatedly demonstrated that the assessment of response and therapy delivery can be fused. Such a theranostic approach that would combine the diagnostic or restaging imaging procedure with a targeted therapy represents an appealing innovative strategy in personalized medicine in hemato-oncology.Malignant lymphomas represent the most common type of hematologic malignancies. The first clinically approved TDD modalities in lymphoma patients were anti-CD20 radioimmunoconjugates (RIT) 131I-tositumomab and 90Y-ibritumomab-tiuxetan. The later clinical success of the first approved antibody–drug conjugate (ADC) for the treatment of lymphomas, anti-CD30 brentuximab vedotin, paved the path for the preclinical development and clinical testing of several other ADCs, including polatuzumab vedotin and loncastuximab tesirine. Other modalities of TDD are based on new formulations of “old” cytostatic agents and their passive trapping in the lymphoma tissue by means of the enhanced permeability and retention (EPR) effect. Currently, the diagnostic and restaging procedures in aggressive lymphomas are based on nuclear imaging, namely PET. A theranostic approach that combines diagnostic or restaging lymphoma imaging with targeted treatment represents an appealing innovative strategy in personalized medicine. The future of theranostics will require not only the capability to provide suitable disease-specific molecular probes but also expertise on big data processing and evaluation. Here, we review the concept of targeted drug delivery in malignant lymphomas from RIT and ADC to a wide array of passively and actively targeted nano-sized investigational agents. We also discuss the future of molecular imaging with special focus on monoclonal antibody-based and monoclonal antibody-derived theranostic strategies.

Abstract P062: The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1

Abstract Background The approved antibody-drug conjugate (ADC) sacituzumab-govitecan (SG) that exploits the topoisomerase I inhibitor SN38, represents a substantial advance in the ADC field. SN38 is the first payload with low nanomolar cytotoxicity. This deviates from current design principles, which utilize ultracytotoxic payloads. The CL2A linker has a short non-cleavable seven polyethylene glycol segment that enables a higher number of hydrophobic SN38 molecules to be attached to reduced cysteines. CL2A is also designed to permit the release of SN38 in an acidic environment via a pH-sensitive benzyl carbonate bond to SN38’s lactone ring. There is no cathepsin B cleavage site. Key to the performance of SG is the SN38 release half-life of 1-2 days. This also deviates from very stable linker systems that require full ADC internalization and lysosomal processing for payload release. As a result of these novel ADC properties, SG is given in higher and repeated doses, which results in slow release at the tumor site and enhanced uptake by target cancer cells and reduced toxicity on healthy cells due to low potency SN38 alone. Our goal is to further evaluate the CL2A-SN38 system when conjugated to trastuzumab. Material and methods Trastuzumab was conjugated to CL2A-SN38 to reduced cysteines (T-SN38). Monitoring the changes in DAR over time was performed to determine the stability of T-SN38 in PBS at pH values of 7.4, 5.72, and 4.58, to mimic circulating blood, early endosomes, and lysosomes, respectively. Cytotoxicity assays for 72 h on HER2-positive ovarian cancer SKOV3.ip and HER2-negative CHO cells were performed and compared to the approved anti-HER2 ADC trastuzumab-emtansine (T-DM1). Cytotoxicity assays were also performed between T-SN38 and T-DM1 at their respective IC50 values and the percent of cell survival determined daily from 24-96 h. Results T-SN38 was generated with a DAR of 6.97. Surprisingly, the half-life of SN38 release in acidic pH was extremely slow (t1/2=~120 h) and at the final evaluated time point (120 h) 69% of SN38 remained conjugated to trastuzumab. As anticipated, as a function of concentration T-DM1 was significantly more potent than T-SN38 (IC50=0.0013 vs 0.0832 mg/mL). However, when cytotoxicity was evaluated as a function of time T-SN38 switched to the more potent ADC at the late time points. T-SN38 killed SKOV3.ip cells 36% more effectively than T-DM1 at 96 h. Conclusions Our data from SN38 release experiments in acidic buffer suggest that pH-induced cleavage of the CL2A linker is not a contributable release mechanism of SN38 when bound to trastuzumab and is counterintuitive for what is currently described for SG. However, traceless release of hydrophobic SN38 capable of traversing cell membranes is only possible by hydrolysis of the benzyl carbonate bond. Our cytotoxicity studies over time suggest that pH-dependent release does occur. Further investigations are ongoing to more precisely determine the contribution of the carbonate bond for SN38 release and cytotoxic potency. Citation Format: Hardeep Singh, Victor Jeffrey Leyton. The CL2A-SN38 linker-payload system conjugated to trastuzumab results in improved cellular cytotoxicity over time relative to T-DM1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P062.

A narrative review of the current landscape and future perspectives of HER2-targeting antibody drug conjugates for advanced breast cancer

Objective: To (I) provide an update of approved antibody drug conjugates (ADCs) and those registered with NMPA, (II) give an overview of HER2-targeting ADCs as a mono- or combination therapy in clinical development for breast cancer and the molecular mechanism of payloads of these molecules, (III) describe the pharmacokinetic profiles of ADCs using mostly exploited vcMMAE platform as an example, (IV) discuss future development strategies for ADC therapeutics in addressing substantially unmet medical needs, such as brain metastasis (BM) and drug resistance in patients with HER2-expressing advanced breast cancer.

An Insight into FDA Approved Antibody-Drug Conjugates for Cancer Therapy.

The large number of emerging antibody-drug conjugates (ADCs) for cancer therapy has resulted in a significant market ‘boom’, garnering worldwide attention. Despite ADCs presenting huge challenges to researchers, particularly regarding the identification of a suitable combination of antibody, linker, and payload, as of September 2021, 11 ADCs have been granted FDA approval, with eight of these approved since 2017 alone. Optimism for this therapeutic approach is clear, despite the COVID-19 pandemic, 2020 was a landmark year for deals and partnerships in the ADC arena, suggesting that there remains significant interest from Big Pharma. Herein we review the enthusiasm for ADCs by focusing on the features of those approved by the FDA, and offer some thoughts as to where the field is headed.

Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)-A Necessity for Future ADC Research and Development.

Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways (endosomal sorting (recycling, lysosome localization) and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept of targeted chemotherapy by coupling inhibitory antibodies to cytotoxic payloads. Significant advances in ADC technology and format, and target biology have hastened the FDA approval of nine ADCs (four since 2019). Although the links between aberrant endocytic machinery and cancer are emerging, the impact of dysregulated internalization processes of ADC targets and response rates or resistance have not been well studied. This is despite the reliance on ADC uptake and trafficking to lysosomes for linker cleavage and payload release. In this review, we describe what is known about all the target antigens for the currently approved ADCs. Specifically, internalization efficiency and relevant intracellular sorting activities are described for each receptor under normal processes, and when complexed to an ADC. In addition, we discuss aberrant endocytic processes that have been directly linked to preclinical ADC resistance mechanisms. The implications of endocytosis in regard to therapeutic effectiveness in the clinic are also described. Unexpectedly, information on endocytosis is scarce (absent for two receptors). Moreover, much of what is known about endocytosis is not in the context of receptor-ADC/antibody complexes. This review provides a deeper understanding of the pertinent principles of receptor endocytosis for the currently approved ADCs.

Abstract 920: Harnessing multiple mechanisms of action to target Nectin-4 as anti-tumor therapeutics

Abstract Nectin-4 belongs to an immunoglobulin superfamily (IgSF) of cell adhesion molecules that mediate cell-cell adhesion through homophilic or heterophilic interactions. In addition to the role in cell-cell adhesion, the Nectin family plays important role in regulating a diverse range of physiologic cellular activities, in viral entry, and in immune modulation. Nectin-4 has been reported to be upregulated in various epithelial cell cancers, such as breast cancer, lung cancer, and bladder cancer. The therapeutic value of Nectin-4 as an anti-tumor target has been demonstrated by a recently approved antibody-drug conjugate (ADC), Enfortumab vedotin. To fully exploit the potential of Nectin-4 as an anti-tumor target, we discovered a panel of monoclonal antibodies. These antibodies not only bind specifically to human Nectin-4 with high affinity, but also show differentiating characteristics including cell internalization, blockage of interaction with Nectin-1, blockage of interaction with TIGIT, and ADC-independent cytotoxicity. Our results suggest that in addition to ADC, other mechanisms targeting Nectin-4 can be utilized for therapeutics, such as immunomodulation and antibody- or immune cell-mediated tumor cell killing. Citation Format: Yi Pei, Thomas Pohl, Ahmed Hassan, Anna Przybyla, Raymond Yu, Huarui Lu, Fangqiang Tang, Kevin Chen, Wei Jin, Krizia Dominguez, Haichun Huang, Ming Lei, Zhong Liu, Han Li. Harnessing multiple mechanisms of action to target Nectin-4 as anti-tumor therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 920.

Natural Product Based Antibody Drug Conjugates: Clinical Status as of November 9, 2020.

As of early November 2020, there are 10 approved antibody drug conjugates (ADCs) plus two others that are not usually listed. In addition, there are 70 ADCs at stages from phase I to phase III and 23 that are at the preclinical stage. The warheads of all of these drugs and drug candidates have their origins in natural product structures. The sources and modifications are discussed in general and then specifically commented on in each case with either the generic name if known and/or the ADC's ID names. Interestingly, almost all warheads listed are from microbial sources though initially a number were thought to have been from plants. The latest NCT numbers from Clintrials.gov of all phase I to phase III candidates are also given. Three unusual ADCs are also discussed, two of which (an antitumor agent and one directed against autoimmune diseases) are not usually listed as ADCs, with the third being an anti-infective.

Merck to buy ADC developer VelosBio

Merck & Co. will pay $2.75 billion to acquire the private San Diego–based cancer firm VelosBio, which is developing antibody drugs that target tyrosine kinase–like orphan receptor 1 (ROR1). VelosBio’s most advanced candidate is an antibody-drug conjugate (ADC) that targets ROR1—found on some cancer cells but not healthy adult cells—and unleashes a toxic agent called monomethyl auristatin E. Three approved ADCs use the same toxic agent. VelosBio began a Phase 2 trial of its ADC in October. The firm also has a preclinical bispecific antibody that targets ROR1 and recruits immune cells to destroy the tumor.

Abstract 4829: Improved safety profile of HER2-KSPi-ADCs compared to T-DM1 in in vitro megakaryocyte assay predictive of thrombocytopenia

Abstract KSP inhibitors (KSPis) are a versatile new payload class for the generation of highly potent and selective antibody-drug conjugates (ADCs) against different targets. For HER2 (c-ERBB2)- and TWEAKR (Fn14/ TNFRSF12A)-KSPi-ADCs, we have previously shown that they have potent and selective anti-proliferative activity and induce apoptosis in HER2- or TWEAKR-positive cancer cell lines in vitro. Moreover, TWEAKR-KSPi-ADCs induced strong and long-lasting anti-tumor efficacy and complete tumor regression in cell line- and patient-derived xenograft models. Shortcomings of clinically tested and marketed ADC payload classes are the off-target / on-toxophore dose-limiting toxicities observed in the clinic, in particular neutropenia and thrombocytopenia. Thrombocytopenia is a common side effect of 3 of 4 approved ADCs (including T-DM1, Kadcyla). Thrombocytes are generated by proliferation, differentiation and fragmentation of specific megakaryocyte (MK) progenitors. As ADCs can be taken up by differentiating hematopoietic stem cells the released toxic payload can inhibit MK proliferation/differentiation and prevent generation of platelets resulting in thrombocytopenia. The potential to induce thrombocytopenia of KSPi-based ADCs with different effector chemistries (ECs) was compared with the clinically approved ADC T-DM1 (with a non-cleavable SMCC linker) by evaluating their impact on in vitro megakaryocyte differentiation. To this end, the HemaToxTM MK assay (Stemcell, Cologne, Germany) was used which allows to assess the impact of compounds on proliferation/differentiation of CD34+ stem cells into GPIIb/IIIa (CD41) and CD45 double positive megakaryocytes by flow cytometry. Whereas the anti-HER2 Ab trastuzumab had no impact on MK differentiation, T-DM1 elicited MK toxicity in vitro. HER2-targeted or isotype control KSPi-ADCs with a legumain (LGMN) cleavable EC showed a comparably benign profile in the MK assay as trastuzumab, and a further improved profile versus KSPi-ADCs with a non-cleavable EC. Both features, the LGMN specific cleavage of KSPi-ADCs after internalization and cellular trafficking to the lysosome, and the released, non-cell-permeable KSPi payload, may contribute to an improved safety profile compared to T-DM1. This is further supported by the safety profile of a KSPi-ADC with a LGMN cleavable EC was also studied in vivo: In a repeat dose Cynomolgus study with the IL3RA-KSPi-ADC (BAY-943) dosed up to 10 mg/kg (QWx3), thrombocytopenia was not observed. These results indicate that KSPi-ADCs with specifically designed effector chemistries containing LGMN cleavable linkers have an improved safety profile in the MK assay with first evidence that this may translate also in a better safety profile with regard to lack of induction of thrombocytopenia in vivo. Citation Format: Anette Sommer, Pascale Buchmann, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Christian Bertling, Jenny Thoennes, Sandra Johanssen, Christoph Schatz, Dominik Mumberg. Improved safety profile of HER2-KSPi-ADCs compared to T-DM1 in in vitro megakaryocyte assay predictive of thrombocytopenia [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 4829.

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology.

Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second-generation of clinically approved antibody-drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide-type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re-bridging.

Abstract A011: HER2-positive PDX model of breast cancer brain metastasis obtained from patient prior to T-DM1 therapy

Abstract Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is approved for treatment of high HER2-expressing (3+), trastuzumab-resistant breast and gastric cancers. While this agent is effective, HER2-positive breast cancer patients have a high risk of brain relapse, seen in up to 37% of patients of whom half die as a result of failure to control the intracranial disease. The aim of this study was to develop a model system that reflects the breast cancer brain metastases seen in patients undergoing T-DM1 therapy. We collected brain metastasis tissue from an HER2-positive breast cancer patient prior to receiving T-DM1 therapy. Methods: The high HER2-expressing (3+) breast cancer brain metastasis model designated ST3338 was established subcutaneously in athymic nude mice using biopsy material from a brain metastasis obtained from a 42-year-old breast cancer patient. The patient was on T-DM1 therapy following sample collection and had received prior treatment with trastuzumab, bevacizumab, and lapatinib. Immunohisotochemistry of both clinical and PDX tissues was applied to assess the estrogen (ER), progesterone (PR), and HER2 receptor levels. Drug sensitivity studies were performed in the ST3338 model established subcutaneously in athymic nude mice to single-agent T-DM1 administered intravenously as a single dose at 10 mg/kg. The ST3338 was also established orthotopically by stereotactic intracranial inoculation of enzymatically digested ST3338 tumor tissue. Tumor development was monitored by T2-weighted MR imaging and the study endpoint was survival by humane endpoints. Results: Immunohistochemistry of PDX tissues was scored as ER (0), PR (0), and HER2 (3+), which corresponded to the clinical brain metastasis sample: ER (0), PR (0), and HER2 (3+). A single dose of T-DM1 at 10 mg/kg showed tumor regression compared with Day 0 (T/C = 0%) in the subcutaneous setting. The ST3338 model was successfully established orthotopically determined by longitudinal T2-weighted MR imaging, and efficacy data of T-DM1 in the orthotopic/intracranial setting are pending. Conclusion: We have established a model system for evaluating drug sensitivity in HER2-positive breast cancer brain metastases. Treatment response to T-DM1 was observed in the subcutaneous setting. With the combination of subcutaneous and intracranial PDX models of breast cancer brain metastases, new drugs can be tested in preclinical models that more closely mimic the microenvironment and the challenges of drug delivery across the blood-brain barrier in breast cancer patients. Citation Format: Carsten H. Nielsen, Michael J. Wick, Lizette Gamez, Camilla S. Knudsen, Mette M. Jense, Melissa Rundle, Kyriakos P. Papadopoulos, Andreas Kjaer. HER2-positive PDX model of breast cancer brain metastasis obtained from patient prior to T-DM1 therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A011.

Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy.

Antibody-drug conjugates (ADCs) are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

Abstract C74: Establishment and characterization of a HER2-positive, TDM1-resistant PDX breast model

Abstract Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is approved for treatment of high HER2 expressing (3+), trastuzumab resistant breast and gastric cancers. While this agent is effective, resistance often develops. To better understand the mechanisms for resistance to TDM1 in high HER2 expressing breast cancer we collected tissue from a patient following response and progression to TDM1 and established a xenograft model for preclinical drug screening. The resulting model ST1360B was genomically characterized and screened in vivo using high dose TDM1; anti-tumor activity was compared with two trastuzumab-resistant but TDM-1 sensitive HER2 (3+) breast PDX models designated ST225 and ST340. Methods: The high HER2 expressing (3+) TDM1 resistant breast PDX model ST1360B was established in athymic mice using pleural fluid collected from a thoracentesis. Establishment of the HER2 (3+) ST225 and ST340 models has been previously described. Clinical and PDX tissues were subjected to genomic analysis; for each model, DNA was extracted and subjected to comparative genomic hybridization and exon sequencing of known oncogenes; growth factor receptor and ligand densities was interrogated using immunohistochemistry. Drug sensitivity studies were performed evaluating sensitivity of models to single agent T-DM1, administered intravenously at 10 mg/kg on Day 0; weekly TDM1 dosing at 5 mg/kg was also evaluated in the ST1360B model Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Genomic analysis of ST1360B identified amplified HER2 and TOP2A; interestingly CD44 was found highly amplified, suggesting a possible mechanism for TDM1 resistance in this model. Mutations were not identified in PIK3CA or other relevant genes. In efficacy studies TDM1 was confirmed active towards ST225 and ST340 model including tumor regressions and durable complete responses in both models (TGI>100%). However TDM1 was not effective in ST1360B, with only transient tumor growth inhibition even with weekly drug administration. Conclusion: We have established a model of TDM1 HER2 3+ resistant breast cancer and have characterized the model using comparative genomic hybridization and exon sequencing of known oncogenes, identifying amplified CD44 as a potential mechanism for drug resistance. This model can be utilized to better understand and to develop novel therapies to TDM1 resistance. Citation Format: Michael J. Wick, Teresa L. Vaught, Justin Meade, Monica Farley, Armando Diaz, Anthony W. Tolcher, Drew W. Rasco, Amita Patnaik, Murali Beeram, Kyriakos P. Papadopoulos. Establishment and characterization of a HER2-positive, TDM1-resistant PDX breast model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C74.

Abstract 2466: Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy

Abstract The mainstay of HER2+ breast cancer treatment has been monoclonal antibody therapy with Trastuzumab, a humanized antibody that targets HER-2. However, the efficacy of Trastuzumab monotherapy is only 10-15%. Therefore, strategies are being developed to enhance its therapeutic efficacy. Our previous study demonstrated that stimulation of NK cells with an anti-CD137 agonistic mAb enhanced Trastuzumab-mediated Antibody Dependent Cellular Cytotoxicity (ADCC). Anti-CD137 agonistic mAb enhanced anti-breast cancer activity of Trastuzumab in vivo in a xenotransplanted human breast cancer model (Kohrt et al. J Clin. Invest, 2012, 22:3). We have developed a treatment regimen consisting of sequential administration of Trastuzumab followed by CD137 antibody as three weekly injections. This regimen demonstrated more potent antitumor activity than administration of anti-CD137 mAb followed by Trastuzumab. Our combination therapy was superior to Traztuzumab alone in a primary HER2-overexpressing-breast tumor xenotransplant model but was ineffective in low HER-2 expressing breast cancer model. Next, we evaluated the therapeutic efficacy of FDA approved antibody drug conjugate Trastuzumab Emtansine (T-DM1) in combination with CD137 antibody. We used HER2 over-expressing HER18 xenograft model and an SU-258 primary breast cancer model in our studies. Following tumor inoculation, mice received either T-DM1 on day 3 (for HER18 xenograft), or day 30 (for SU-258) and anti-CD137 antibody on day 4(or HER18) or day 31(for SU-258) with each treatment repeated weekly for a total of three weeks. Sequential antibody strategy with T-DM1 and CD137 significantly improved survival compared to T-DM1 alone or with Trastuzumab and CD137 antibody. Our results demonstrate that T-DM1 retains its potency and demonstrates synergistic activity with anti-CD137 mAb therapy against HER2-overexpressing breast cancer models. Our results support a novel, sequential antibody approach against HER-2+breast cancer, by targeting first the tumor with an antibody drug conjugate and then the host immune system. Clinical investigations are now planned to determine the clinical outcome of our immunotherapy strategy. Citation Format: Suparna Dutt, Narendiran Rajasekaran, Aurelien Marabelle, Roch Houot, Mohith Sadaram, Jonathan Hebb, Idit Sagiv-Barfi, Sid Ambulkar, Amanda Rajapaksa, Cariad Chester, Erin Waller, Holbrook Kohrt. Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy. [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 2466. doi:10.1158/1538-7445.AM2015-2466