Antibody-drug Conjugates Linker Research Articles

Adverse events of patients treated with antibody-drug conjugates in phase 1 clinical trials at the Royal Marsden Drug Development Unit from 2014 to 2024.

e15020 Background: Antibody-drug conjugates (ADCs) are designed to improve therapeutic indices of cytotoxic agents. Nevertheless, they may induce unexpected toxicities not observed with parent payloads. This study aimed to characterize the adverse events (AEs) seen in patients treated with ADCs in Phase 1 trials at The Royal Marsden Drug Development Unit (DDU). Methods: This is a retrospective cohort analysis of consecutive patients who received at least one dose of an ADC in a phase I trial from January 2014 to January 2024 at The Royal Marsden DDU. Descriptive statistics of patient characteristics, ADCs, and treatment-related AEs were performed. Univariate and multivariate logistic regression will be utilized to assess correlations among patient and ADC characteristics and adverse events. P values of < 0.05 will be considered significant. Results: A total of 131 patients who participated in twelve ADC trials were included. The median age of participants was 63 years (range: 31-84) and 61% were females. Patients received a median of three prior lines of systemic treatment. The most common type of ADC target, antibody, linker and payload were receptor tyrosine kinase (51%), humanized IgG monoclonal antibody (69%), valine-citrulline (VC) cleavable linker (85%), and a tubulin-binding payload (59%), respectively. Most patients received an ADC dose of 1-3 mg/kg (76%) with each cycle given once every 3 weeks (64%). The median number of cycles was 3 with an average treatment duration of 63 days. Within the cohort, 89% of patients developed any grade AE and 17% experienced grade 3 or higher AEs. The major treatment-related AEs were fatigue (48%), nausea/vomiting (27%), ocular toxicity (26%), neuropathy (26%), skin rash (20%) and pneumonitis (8%). The median time to first onset of treatment-related adverse events were 37 days for ocular toxicity, 46 days for pneumonitis, and 54 days for neuropathy. ADC-related AEs led to treatment interruptions (51%), dose reductions (13%), and treatment discontinuations (28%). Two treatment-related grade 5 events occurred. The objective response rate was 18%, with 1 complete response and 22 partial responses. Conclusions: Despite being designed to minimize side effects, ADCs were associated with significant toxicities, including unexpected adverse events unrelated to target, antibodies or the payloads, limiting their therapeutic indices. Further analysis of the relationship between different components of the wide range of ADCs and AEs are warranted to better understand the potential mechanisms of ADC toxicity.

A comparison of the activity, lysosomal stability, and efficacy of legumain-cleavable and cathepsin cleavable ADC linkers

: 1. Over the past two decades antibody-drug conjugates (ADCs) have emerged as a highly effective drug delivery technology. ADCs utilize a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types. 2. Challenges such as premature linker cleavage and clearance due to linker hydrophobicity have adversely impacted the stability and safety of ADCs. While there are various solutions to these challenges, our team has focused on replacement of hydrophobic ValCit linkers (cleaved by CatB) with Asn-containing linkers that are cleaved by lysosomal legumain. 3. Legumain is abundantly present in lysosomes and is known to play a role in tumor microenvironment dynamics. Herein, we directly compare the lysosomal cleavage, cytotoxicity, plasma stability, and efficacy of a traditional cathepsin cleavable ADC to a matched Asn-containing legumain-cleavable ADC. 4. We demonstrate that Asn-containing linker sequences are specifically cleaved by lysosomal legumain and that Asn-linked MMAE ADCs are broadly active against a variety of tumors, even those with low legumain expression. Finally, we show that AsnAsn-linked ADCs exhibit comparable or improved efficacy to traditional ValCit-linked ADCs. Our study paves the way for replacement of the traditional ValCit linker technology with more hydrophilic Asn-containing peptide linker sequences.

Abstract 4689: 4HF CancerDataMiner platform accelerates target discovery and evaluation

Abstract For the management of metastasized cancer, tumor-targeted chemotherapies with improved therapeutic index are urgently needed. Over the last 25 years, antibody drug conjugates (ADCs) targeting cancer-associated proteins have emerged as a promising new treatment modality. While the first ADCs were registered for the treatment of acute leukemias and lymphomas, more recently at least six ADCs were approved by the FDA for solid tumors. Despite these successes, clinical development of ADCs remains a risky exercise as evident from the failure of more than 20 pivotal clinical phase III trials. A narrow therapeutic index due to excessive toxicity of potent payloads, little therapeutic activity, and difficulties in chemistry, manufacturing, and controls were among the limiting factors. Meanwhile, ADC technology and production have advanced substantially. More than 100 ADCs are currently in clinical development in approximately 600 clinical trials, with some of the investigated ADCs being close to approval. While issues related to ADC linkers and payloads have been extensively investigated and many problems have been solved, the choice of molecular targets for ADCs remains challenging. This is, in part, due to a lack of information about candidate targets: while knowledge about generally accepted cancer targets such as ERBB2 is abundant, data about many novel targets is still scarce. In particular, reliable information about expression in normal tissues, subcellular localization and protein function is often missing. 4HF Biotec has established the CancerDataMiner, an in-silico platform dedicated to cancer data mining by integrating large OMICS datasets that are connected and aligned. It allows molecular analyses of all human genes in >12,000 tumor samples and >14,000 normal tissues, using proprietary tools and widgets for visualization and statistics. Target evaluations are performed at the DNA, RNA and protein levels and employ complementary tools such as pathway analysis and literature search to obtain a comprehensive picture of candidate targets. Using this approach, we recently reviewed the molecular targets of all ADCs that either have already received approval or are in clinical development. As proof-of-concept for the CancerDataMiner, here we analyze targets of ADCs and compare our findings with the clinical data obtained for those ADCs. The profiles of selected ADC targets will be discussed, among them CD79B and CD33 for hematologic malignancies and FOLR1 and Claudin 18.2 for solid cancers. We will demonstrate that proteins with very diverse profiles can make good ADC targets. In conclusion, the 4HF CancerDataMiner is a platform which has proven its value for the identification and characterization of cancer-associated targets for ADC development for all tumor types. Citation Format: Heinz-Herbert Fiebig, Anne-Lise Peille, Mirko Schmitz, Vincent Vuaroqueaux, Thomas Metz. 4HF CancerDataMiner platform accelerates target discovery and evaluation [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 4689.

Abstract 5089: Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates

Abstract Background: Endometrial cancer is the most frequently occurring gynecologic cancer in developed countries and the third leading cause of cancer deaths among women. It remains one of the few malignant tumors with rapidly increasing mortality and morbidity. Patients with recurrent or metastatic endometrial cancer continue to experience poor outcomes. Antibody-drug conjugates (ADCs) represent a class of targeted therapeutic modalities that offer biomarker-based therapeutic options for patients with endometrial cancer. In this study, we proposed to identify a novel molecular target for the preclinical development of an ADC for endometrial cancer-targeted therapy. Methods: Firstly, to determine the level of target expression within endometrial cancer tissue components as well as differences, we performed immunohistochemical staining of human endometrial cancer and paracancerous tissue. Secondly, the expression of the target on the cell membrane surface was quantitated and qualitatively verified using flow cytometry and single-photon confocal microscopy. In the third step, an endocytosis assay was used to verify the endocytosis of the target antibody by EC cells. Confocal imaging can directly observe the endocytosis of the target antibody, and flow cytometry can be analyzed and quantified. Co-localization experiments with antibody fluorescence can verify the specific site where the target antibody is endocytosed into cancer cells. We then designed and characterized two targeted ADCs. Next, the in vitro inhibitory activities of these ADCs against endometrial cancer cell lines and normal cells were evaluated using the CCK8 assay. Finally, we established a cell line-derived xenograft (CDX) model of endometrial cancer to validate the in vivo efficacy and bio-safety of the ADCs. Results: We have determined a correlation between ICAM1 overexpression and EC, and IHC staining of patient tissues showed significantly higher levels of ICAM1 expression in endometrial cancer tissues than in paracancerous tissues. The high cell surface expression of ICAM1 was verified in a panel of various human endometrial cancer cell lines. Moreover, two ICAM1 ADCs were established using clinically-approved ADC linker and payload combinations. It was visually confirmed that ICAM1 ADCs selectively recognize endometrial tumors and efficiently enter endometrial cells via antigen-mediated endocytosis. We compared the anti-tumor activity of ICAM1 ADCs with standard-of-care chemodrugs, and ICAM1 ADCs outperformed chemodrugs in the CDX model of endometrial cancer without toxicity. Conclusion: We identified that ICAM1 is a promising ADC target for endometrial cancer and confirmed the potent anti-tumor activity and biosafety of ICAM1 ADCs in vitro and in vivo, warranting further pre-clinical and clinical investigations. Citation Format: Hanfei Xie, Lu Sun, ShiLi Yao, Peng Guo, YingLi Zhang. Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates [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 5089.

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.

Generation and Characterization of Iduronidase-Cleavable ADCs.

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

Lysosomal-Cleavable Peptide Linkers in Antibody-Drug Conjugates.

Antibody-drug Conjugates (ADCs) are a powerful therapeutic modality for cancer treatment. ADCs are multi-functional biologics in which a disease-targeting antibody is conjugated to an effector payload molecule via a linker. The success of currently used ADCs has been largely attributed to the development of linker systems, which allow for the targeted release of cytocidal payload drugs inside cancer cells. Many lysosomal proteases are over expressed in human cancers. They can effectively cleave a variety of peptide sequences, which can be exploited for the design of ADC linker systems. As a well-established linker, valine-citrulline-p-aminobenzyl carbamate (ValCitPABC) is used in many ADCs that are already approved or under preclinical and clinical development. Although ValCitPABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma, many studies have shown that they are susceptible to carboxylesterase 1C (Ces1C) in mouse and rat plasma, which hinders the preclinical evaluation of ADCs. Furthermore, neutropenia and thrombocytopenia, two of the most commonly observed dose-limiting adverse effects of ADCs, are believed to result from the premature hydrolysis of ValCitPABC by human neutrophil elastase. In addition to ValCitPABC, the GGFG tetrapeptidyl-aminomethoxy linker is also cathepsin-cleavable and is used in the highly successful ADC drug, DS8201a. In addition to cathepsin-cleavable linkers, there is also growing interest in legumain-sensitive linkers for ADC development. Increasing plasma stability while maintaining lysosomal cleavability of ADC linkers is an objective of intensive current research. This review reports recent advances in the design and structure-activity relationship studies of various peptide/peptidomimetic linkers in this field.

Abstract 5293: Rationally developing antibody drug conjugates targeting genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) ranks the fifth most common cancer and the fourth most frequent cause of cancer-related deaths. Genomically stable gastric cancers (GSGCs) represent 20% of all GCs and GSGC patients have the worst prognosis among all GC subtypes. Although two HER2-targeted antibody-drug conjugates (ADCs), RC48 and DS8201, have shown promising clinical efficacies, most GSGC patients do not benefit from these ADCs due to the infrequent and heterogeneous HER2 expression. Therefore, there remains a significant and unmet medical need for developing novel ADCs targeting GSGC tumors. Methods: In this presented study, a quantitative and unbiased approach was first used to identify potential targets for GSGC in a panel of human GSGC cell lines in reference with two normal human gastric epithelial cells. Cell membrane localizations of potential targets on GSGC cell lines were determined by immunofluorescent (IF) staining. To correlate it with clinically-relevant GSGC tumor samples, immunohistochemical (IHC) staining of potential targets were performed in human diffuse GC tumor tissues along with normal gastric tissues. Next, GSGC cellular internalization capability of antibodies against potential targets was evaluated. Then, an optimized target was selected for developing GSGC-targeted ADCs. A series of ADC formulations with different linkers and payloads were constructed and characterized. Their anti-tumor activities against GSGC cell lines were subsequently screened in order to identify the optimized ADC formulation. Eventually, the anti-tumor activity and biosafety profile of the optimized ADC formulation were determined in suppressing tumor growth, progression and metastasis by using established orthotopic and peritoneal metastasis models of GSGC. Results: We first identified CD54 as a potential molecular target for human GSGCs using comparative flow cytometric analysis. We next evaluated its potential as an ADC target for GSGC-targeted therapy by determining its overexpression levels, cell membrane localizations, and cell internalization activity in GSGC cells. Furthermore, we constructed and characterized a panel of CD54-targeted ADCs and identified an optimized ADC formulation by quantitatively determining their half maximum inhibitory concentrations (IC50s) and validated its anti-tumor efficacy against GSGC tumors. Meanwhile, there was no evidence to indicate histopathological damages to normal organs in the optimized ADC treated group. Conclusions: In this study, we identified a molecular target for GSGC using an unbiased and quantitative screening assay and explored its potential application in developing GSGC-targeted ADCs. In order to maximize its efficacy, we optimized the ADC formulations against GSGC cells using different ADC linkers and payloads. Our study provides a promising targeted therapeutic candidate for the clinical treatment of GSGC. Citation Format: Rui Xu, Li Yuan, Xiao-Qing Guan, Shi-Li Yao, Bing Zhu, Tong Yang, Chun Liu, Peng Guo, Jiang-Jiang Qin, Xiang-Dong Cheng. Rationally developing antibody drug conjugates targeting genomically stable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5293.

Abstract 6174: Developing a novel antibody drug conjugate against cervical cancer

Abstract Background: Cervical cancer ranks fourth in incidence and mortality among female tumors, with over 0.6 million patients diagnosed worldwide in 2020. A large percentage of cervical cancer patients will develop advanced diseases (stage IV or recurrent disease) and the 5-year survival rate of these patients was only 15-20% due to the lack of efficacy and adverse effects of first line chemodrugs. Recently, antibody drug conjugates (ADCs) emerge as a novel class of targeted therapeutics that selectively ablate tumor cells without damaging normal organs and tissues, which has shown promising efficacy in treating solid tumors. In this study, we sought to develop a cervical cancer-targeted ADC using a novel molecular target. Methods: Firstly, human cervical cancer and paracancerous tissue were stained by immunohistochemistry (IHC) in order to determine the difference of the expression level of a molecular target. Three human cervical cancer cell lines (C-33A, Si-Ha and Ca-Ski) and one normal cervical cell line (HCerEpiC) were used to determine the subcellular location of an ADC target on the cells by immunofluorescence (IF) staining, flow cytometry and single photon confocal microscopy. Secondly, endocytosis capability of target antibodies was visualized by confocal imaging and was quantified by flow cytometric analysis. Furthermore, a panel of ADC formulations with different chemical linkers and payloads were designed, constructed and characterized as ADC candidates for cervical cancer therapy. We next evaluated in vitro inhibitory activity of these ADC candidates on three cervical cancer cell lines by CCK8 assay. Finally, orthotopic and lung metastasis animal models of cervical cancer were established to determine the in vivo inhibitory activity and biosafety of constructed ADC candidates. Results: IHC staining of clinical specimens showed that the expression level of CD54 in cervical tumor tissues was significantly higher than those of paracancerous tissues. Fluorescence imaging and flow cytometry further determined the overexpression level, plasma membrane location and antigen-mediated endocytosis activity of CD54 in multiple human cervical cancer cell lines. Among several ADC formulations, an optimized CD54-targeted ADC linker and warhead combination was identified by comparing its IC50 with other ADC candidates and standard-of-care chemodrugs for cervical cancer. The optimized CD54 ADC exhibited promising anti-tumor activity on primary and lung metastatic cervical tumor models. Conclusion: In our study, we identified CD54 as a novel ADC target for cervical cancer. An optimized CD54-targeted ADC formulation was determined and its anti-tumor activity and biosafety profile were determined in multiple human cervical cancer cell lines in vitro and orthotopic and lung metastatic cervical tumor models in vivo, providing a promising targeted therapeutic candidate for the targeted treatment of cervical cancer. Citation Format: Shili Yao, Bing Zhu, Xinyan Wang, Rui Xu, Tong Yang, Peng Guo, Huarong Tang, Tao Zhu. Developing a novel antibody drug conjugate against cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6174.

Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry.

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitatively assess the cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits a dramatically increased cellular signal due to an improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ∼50× brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or nonhindered disulfides, an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

Enzyme-Agnostic Lysosomal Screen Identifies New Legumain-Cleavable ADC Linkers.

Over the past two decades, antibody drug conjugates (ADCs) and small molecule drug conjugates (SMDCs) have widely employed valine-citruline and related cathepsin-cleavable linkers due to their stability in plasma and their rapid cleavage by lysosomal cathepsins. However, a number of recent studies have illustrated that these linkers are subject to cleavage by exogenous enzymes such as Ces1C and neutrophil elastase, thus resulting in off-target release of drug. As such, there is a need to diversify the portfolio of ADC linkers in order to overcome nonspecific drug release. Rather than targeting cathepsins, we began with an "enzyme agnostic" screen in which a panel of 75 peptide FRET pairs were screened for cleavage in lysosomal extracts and in plasma. Unexpectedly, a series of Asn-containing peptides emerged from this screen as being cleaved far more quickly than traditional ValCit-type linkers while retaining excellent stability in plasma. Catabolism studies demonstrated that these linkers were cleaved by legumain, an asparaginyl endopeptidase that is overexpressed in a variety of cancers and is known to be present in the lysosome. MMAE-containing ADCs that incorporated these new linkers were shown to exhibit highly potent and selective cytotoxicity, comparable to analogous ValCit ADCs. Importantly, the Asn-containing linkers were shown to be completely stable to human neutrophil elastase, an enzyme thought to be responsible for the neutropenia and thrombocytopenia associated with ValCitPABC-MMAE ADCs. The legumain-cleavable ADCs were shown to have excellent stability in both mouse and human serum, retaining >85% of the drug after 1 week of incubation. Moreover, the corresponding small molecule FRET pairs exhibited <10% cleavage after 18 h in mouse and human serum. On the basis of these results, we believe that these new linkers (AsnAsn in particular) have significant potential in both ADC and SMDC drug delivery applications.

Recent Advances in Antibody Drug Conjugates for Lymphoma

Antibody-drug conjugates (ADCs) comprise a unique class of chemoimmunotherapy agents, incorporating cytotoxic payloads covalently linked to a monoclonal antibody via specialized linkers. This strategy attempts to exploit antibody-antigen specificity to selectively deliver a potent 'warhead' payload to tumor cells (Figure), while sparing nontumor antigen-negative cells. Decades of development have culminated in the recent approvals of a handful of ADCs across multiple tumor types. ADCs for the treatment of lymphoma are particularly attractive due in part to the favorable spectrum of cell surface markers uniquely expressed on lymphocytes compared with other tissues. Here we discuss general principles of ADC design, including antigen/antibody, payload, and linker selection. We highlight the clinical successes of the 2 approved ADCs for treatment of lymphomas: brentuximab vedotin (Adcetris) and polatuzumab vedotin (Polivy). Finally, we describe several ADC agents currently under development for lymphoma, including emerging efficacy and toxicity data from early-stage clinical trials.

LILRB4-targeting Antibody-Drug Conjugates for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.

Antibody-Drug Conjugates: The New Frontier of Chemotherapy.

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.

Rationally Designed Antibody Drug Conjugates Targeting the Breast Cancer-Associated Endothelium.

The promise of antiangiogenic therapy for the treatment of breast cancer has been limited by the inability to selectively disrupt the established tumor vasculature. Here, we report the development of rationally designed antibody drug conjugates (ADCs) that can selectively recognize and attack breast tumor-associated endothelial cells (BTECs), while sparing normal endothelial cells (NECs). We first performed a quantitative and unbiased screening of a panel of cancer-related antigens on human BTECs and identified CD105 as the optimal ADC target on these cells. We then used clinically approved ADC linkers and cytotoxic drugs to engineer two CD105-targeted ADCs: CD105-DM1 and CD105-MMAE and evaluated their in vitro efficacy in human BTECs and NECs. We found that both CD105-DM1 and CD105-MMAE exhibited highly potent and selective cytotoxicity against BTECs with IC50 values of 3.2 and 3.7 nM, respectively, significantly lower than their IC50 values on NECs (8-13 fold). Our proof-of-principle study suggests that CD105-targeted ADCs are promising antiangiogenic agents that have the potential to be used to inhibit the established tumor vasculature of breast tumors in a safe and precise manner.

Bioorthogonal Prodrug–Antibody Conjugates for On-Target and On-Demand Chemotherapy

Current antibody–drug conjugates (ADCs) suffer from low tissue penetration and significant side effects, largely due to the permanent linkage and/or premature release of cytotoxic payloads. Herein, we developed a prodrug–antibody conjugate (ProADC) strategy by conjugating a bioorthogonal-activatable prodrug with an antibody that allowed on-target release and on-demand activation of cytotoxic drugs at a tumor site. The bioorthogonal-caged prodrug exhibited an enhanced permeability into and on-demand activation within cancer cells, while the pH-sensitive ADC linker allowed on-target release of the anticancer agent. Together, the ProADCs showed enhanced tumor penetration and alleviated side effects for use as an on-target and on-demand chemotherapy agents.

Novel Chemical Linkers for Next-generation Antibody-drug Conjugates(ADCs)

Antibody-drug conjugates (ADCs), monoclonal antibodies conjugated with highly potent drugs (payloads) through chemical linkers, are an emerging class of therapeutic agents for cancer chemotherapy. Their clinical success has been demonstrated by the 4 ADCs already approved by the U.S. Food and Drug Administration (FDA), and more than 60 promising ADCs now in clinical trials. Further advancement of this novel molecular platform could potentially revolutionize current strategies and regimens for treating cancers. The linker structure and antibody-linker conjugation modality critically contribute to ADC homogeneity, circulation stability, pharmacokinetic profiles, tolerability, and overall treatment efficacy. Despite extensive efforts to improve these parameters, most ADC linkers used to date possess linear structures, and therefore accommodate only single payloads. The clinical potential of branched ADC linkers, enabling the installation of two payload molecules, remains unexplored because of the lack of efficient conjugation methods. In addition, according to a recent report, the stability of enzymatically cleavable linkers in mouse circulation is another crucial factor for the successful evaluation of ADCs in preclinical studies. In this review, I present my research group's effort to develop both branched linkers and efficient conjugation methods for constructing dual-loading ADCs with high homogeneity and enhanced potency. I also present a novel tripeptide ADC linker with enhanced stability in mouse circulation. Multidisciplinary experience, approaches, and collaboration are key to successfully advancing our ADC research programs. I herein describe how my experience in the U.S. has helped to develop and manage complex biomedical research projects in a small academic laboratory setting.

Toward a Ferrous Iron-Cleavable Linker for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs.

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

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

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.