Abstract A110: Proteasome inhibitor (PI) ADC: A novel broad spectrum payload platform

Ado-trastuzumab-emtansine or T-DM1, an antibody drug conjugate (ADC) targeting the well-characterized breast cancer oncogene HER2, has shown benefit for breast cancer patients. However, treatment is not indicated for patients whose tumors express low or intermediate levels of HER2. Thus, additional targets for ADC are needed in breast cancer. The lineage-restricted marker prolactin receptor (PRLR) is also expressed in a subset of breast cancers. Unexpectedly, we found that, unlike HER2, low levels of cell-surface PRLR are sufficient to mediate efficient ADC killing of breast ductal carcinoma cells, including T47D. To understand what properties of PRLR vs HER2 allow for efficient cell killing, we compared intracellular trafficking of these two receptors. We found that approximately 90% of a PRLR antibody was internalized by T47D cells within 1h after treatment, and the internalized PRLR Ab co-localized with the lysosomal marker, Lysotracker Red. In contrast, trastuzumab was restricted to the plasma membrane and did not co-localize with Lysotracker Red. Overnight incubation of T47D cells with PRLR Ab, but not trastuzumab, resulted in accumulation in a late lysosomal compartment, as detected using the pH-sensitive marker, pHrodo. Inhibiting protein synthesis with cycloheximide resulted in almost complete degradation of PRLR after 2h, whereas HER2 was degraded only slightly after 4h. The rapid turnover of PRLR was not significantly affected by adding exogenous ligand (prolactin), or by PRLR antibodies, or by proteasome inhibitors, but was blocked by lysosomal inhibitors including bafilomycin A1, and monensin. The signals for this constitutive PRLR internalization and degradation appear to be contained within its cytoplasmic domain, since substitution of the PRLR extracellular domain by that of HER2 still resulted in degradation rates similar to those of full length PRLR. Moreover, simultaneous substitution of two dileucine lysosomal sorting signals contained in the PRLR cytoplasmic domain (e.g. 283LL and 292LL) to alanine significantly diminished constitutive PRLR turnover. In accordance with these data, PRLR ADC, but not T-DM1, induced cell cycle arrest (proportional to PRLR ADC-induced cell killing) in T47D cells, which was completely abolished by lysosomal inhibitors. Taken together, these data indicate that rapid constitutive ligand-independent turnover of PRLR, but not Her2, can deliver high amounts of ADC to lysosomes, resulting in efficient tumor cell killing. Citation Format: Julian Andreev, NIthya Thambi, Frank Delfino, Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Douglas MacDonald, Nicholas Popadopoulos, Willian Olson, Gavin Thurston. Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC). [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 A131.

Toxicity and efficacy of antibody-drug conjugates in advanced solid tumors: a retrospective single-center analysis of clinical trials.

<div>Abstract<p>Topoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer antibody–drug conjugates (ADC) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are using camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker–payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl <i>p</i>-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker–payload structures, we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB–exatecan moiety. Furthermore, we demonstrate that the identified linker–payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison with Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability <i>in vitro</i> and <i>in vivo</i> and superior <i>in vivo</i> efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker–payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1 inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.</p></div>

Antibody drug conjugates (ADCs) are promising cancer therapeutics with minimal toxicity as compared to small cytotoxic molecules alone and have shown the evidence to overcome resistance against tumor and prevent relapse of cancer. The ADC has a potential to change the paradigm of cancer chemotherapeutic treatment. At present, 13 ADCs have been approved by USFDA for the treatment of various types of solid tumor and haematological malignancies. This review covers the three structural components of an ADC-antibody, linker, and cytotoxic payload-along with their respective structure, chemistry, mechanism of action, and influence on the activity of ADCs. It covers comprehensive insight on structural role of linker towards efficacy, stability & toxicity of ADCs, different types of linkers & various conjugation techniques. A brief overview of various analytical techniques used for the qualitative and quantitative analysis of ADC is summarized. The current challenges of ADCs, such as heterogeneity, bystander effect, protein aggregation, inefficient internalization or poor penetration into tumor cells, narrow therapeutic index, emergence of resistance, etc., are outlined along with recent advances and future opportunities for the development of more promising next-generation ADCs.

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

The concept of an antibody-drug conjugate (ADC) is deceptively simple: a monoclonal antibody against a tumor-specific antigen delivers a potent cytotoxic payload directly to the malignant cells, resulting in targeted killing. However, this apparent simplicity belies a complex product with multiple interacting components. Working to optimize these components, we have identified three important concepts that impact the therapeutic potential of ADCs. First, we have learned that stability of an ADC can vary according to the specific conjugation site on the antibody. During the development of engineered cysteine antibodies (EC-mAbs), we evaluated various cysteine substitutions and found that S239C on the IgG heavy chain is particularly stable when conjugated using standard maleimide chemistry. Alternatively, we have devised a method for producing stable conjugation to native cysteine residues by designing maleimides that catalyze their own ring-opening after coupling to the antibody. These modifications in ADC stability appear to result in both improved activity and decreased toxicity in preclinical models. Second, we have observed that modulation of ADC hydrophobicity results in profound changes in both biodistribution and antitumor activity. When utilizing typical drug-linkers, ADCs with a high drug-to-antibody ratio (DAR >4) appear to be cleared from circulation more rapidly by reticuloendothelial cells. By reducing drug-linker hydrophobicity, we can circumvent this clearance pathway and it becomes possible to increase the DAR, resulting in more potent ADCs that retain desirable pharmacokinetic properties. We have achieved a similar result with the use of short, discrete PEG oligomers to mask the drug linker, thereby reducing the apparent hydrophobicity of the ADC. Finally, the chemical properties of the released payload have a major impact on both efficacy and toxicity. The difference between polar and nonpolar payloads will be discussed, including bystander activity, potential mechanisms of resistance, and adverse events observed in clinical trials. A new class of DNA crosslinking payloads is represented by the pyrrolobenzodiazepines (PBD) dimer, which is several logs more potent than auristatins and has the ability to overcome multi-drug resistance phenotypes. These examples demonstrate how rapidly the ADC field is evolving and provide hope that the next generation of ADCs will be even more effective and well-tolerated in the fight against cancer. Citation Format: Jonathan G. Drachman. The next generation of ADCs. [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 SY35-01. doi:10.1158/1538-7445.AM2015-SY35-01

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

Antibody-drug conjugates (ADCs) have drawn a lot of attention in the field of cancer therapy due to their improved efficacy and reduced side effects compared to traditional therapeutic antibodies or chemotherapeutics. However, cancer patients still develop resistance against ADCs and there is an urgent need for the development of strategies to reinforce ADC efficacy. Radiolabeling of an antibody with therapeutic radioisotopes (e.g., 177Lu or 225Ac) can be considered an option. Herein, we synthesized radiolabeled ADCs and evaluated their potential therapeutic efficacies in vitro and in vivo for cancer therapy. New trastuzumab-based ADCs utilizing fendiline or gemifloxacin as drug moieties were developed, and they were decorated with therapeutic radionuclide 177Lu or 225Ac. Anticancer effects of radiolabeled ADCs were evaluated using human epidermal growth factor receptor 2 (HER2) expressing cancer cells and compared to that of cold ADCs. Radiolabeled versions of newly synthesized ADCs showed significantly improved anticancer efficacy compared to unlabeled ADCs, especially when they were armed with 225Ac, demonstrating the great potential of radiolabeled ADCs in cancer therapy. This study offers an effective strategy for improving the therapeutic efficacy of ADCs by fortifying them with radionuclides.

<div>Abstract<p>Antibody–drug conjugates (ADC) have become important scaffolds for targeted cancer therapies. However, ADC exposure–response correlation is not well characterized. We demonstrated that intratumor payload exposures correlated well with the corresponding efficacies of several disulfide-linked ADCs, bearing an DNA alkylating agent, pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD), in HER2-expressing xenograft models. The correlation suggests that a threshold concentration of intratumor payload is required to support sustained efficacy and an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy (“Plateau” effect). In contrast to tumor PBD concentrations, related assessments of systemic exposures, plasma stability, and drug-to-antibody ratio changes of related ADCs did not consistently rationalize the observed ADC efficacies. A minimal efficacious dose could be determined by ADC dose-fractionation studies in the xenograft models. Mechanistic investigations revealed that both linker immolation and linker disulfide stability are the key factors that determine intratumor PBD concentrations. Overall, this study demonstrates how a linker design can impact ADC efficacy and that the intratumor exposure of a payload drug as the molecular mechanism quantitatively correlate with and predict the antitumor efficacy of ADCs. <i>Mol Cancer Ther; 17(3); 677–85. ©2018 AACR</i>.</p></div>

At a time of high prevalence of cancer and painful chemotherapy, the effective and patient-friendly cancer therapy is necessary. The use of antibody-drug conjugates (ADCs) to develop anti-cancer drugs has become a hotspot. ADCs typically consist of a monoclonal antibody (mAb) linked to a cytotoxic payload via a linker. It can not only target cancer cells specifically and precisely, but also kill and eliminate cancer cells efficiently. Although there are now three generations of ADC drugs active in the treatment of cancer, and some drugs have been approved by the FDA, there are still some limitations. Whether prevalent adverse reactions or drug resistance will affect the safety and efficacy of ADC drugs. To investigate the optimal efficacy of ADC after optimization, researches are constantly being promoted. Both the improvement of ADC structure and combination therapy can be beneficial to obtain more treatable ADC drugs. Therefore, this review discussed how ADCs attacked cancer cells from the mechanism of the different ways in which ADCs work in order to gain a deeper understanding of how ADCs are effective in treating cancer. At the same time, for the purpose of obtaining better drugs, on the basis of briefly introducing the development history of ADCs and summarizing the three generations of ADCs, the drug resistance of ADCs was analyzed and the future improvement was prospected.

Osteosarcoma (OS), the most common bone tumor in children and adolescents, has high rates of metastasis leading to poor survival. Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein whose expression is modulated by TGFβ, was recently shown to be highly expressed on the surface of OS tumor cells. Here, we evaluate a novel antibody-drug conjugate (ADC) targeting LRRC15 in OS human cell lines and murine xenografts. We compare this new ADC, which is conjugated to the anthracycline derivative PNU-159682 (PNU), to a previously studied LRRC15 ADC that is conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE), since anthracyclines are standard of care in OS. We evaluated LRRC15 expression in OS cells using Western blots and flow cytometry, and analyzed the epigenetic landscape of the LRRC15 locus using chromatin immunoprecipitation. Efficacy of ADCs on cell growth was analyzed by IncuCyte live cell imaging. Intramuscular xenograft tumor growth was assessed by bioluminescence imaging and hematoxylin and eosin staining. LRRC15-PNU is more effective at inhibiting growth in vitro and in vivo than an isotype antibody control or the LRRC15-MMAE ADC in two high LRRC15 expressing OS cell lines. Low expressing cell lines are not sensitive to either ADC. Importantly, cells with low LRRC15 expression are amenable to re-expression after TGFβ treatment, suggesting a potential to sensitize insensitive OS cells to LRRC15 ADC treatment. In vivo, LRRC15-PNU had cure rates of 40-100% in OS xenograft models. Overall, LRRC15-directed ADCs are a promising new avenue for OS treatment.

Background: Antibody-drug conjugates (ADCs) have shown clinical success, but resistance, often driven by the payload, can limit long-term efficacy. To address this, Hexagon Bio is mining its natural product library to discover new payloads suitable for mono- or dual-payload ADCs. We report on a ribosome assembly inhibitor (RAI) with strong antitumor activity when conjugated to the anti-TROP2 antibody sacituzumab, both as a mono payload and in combination with the topoisomerase inhibitor Dxd (dual payload). The RAI also shows promise when conjugated to other antibodies, including those targeting low-abundance antigens. Materials and methods: By mining small molecules produced by Hexagon Bio’s 120,000-strain microbial library, several potent cytotoxic compounds were identified. The lead RAI was evaluated as a free agent in cell-free translation assays and as an ADC. It was conjugated to sacituzumab at drug-to-antibody ratios (DARs) of 4 and 8 and tested in:In vitro cytotoxicity (CellTiter-Glo, 120 hrs) and immunogenic cell death (ICD) assays (detection of hallmarks of ICD: extracellular ATP, secreted high mobility group box 1 and cell surface calreticulin, 120-168 hrs)In vivo efficacy models (KPL4, NCI-N87, HT1376)Pharmacokinetics/pharmacodynamic (PK/PD) studiesRodent tolerability studiesTo assess broader applicability, the RAI was also conjugated to other antibodies (anti-HER3, -Nectin4, -ITGB6, -B7H3, -B7H4), and cytotoxicity was evaluated. A dual-payload ADC combining the RAI on sacituzumab was also generated and tested. Results: As a free agent, the RAI potently inhibited protein translation in cell-free assays (EC50∼ 15 nM). When conjugated to sacituzumab, the ADC exhibited strong in vitro cytotoxicity (sub- to single-digit nM) across TROP2-expressing cell lines, including those that have low antigen levels and are resistant to Dxd. The ADC also induced hallmarks of ICD, suggesting it may combine well with checkpoint inhibitors. In vivo, RAI-based ADCs delivered complete responses at ≥ 5 mg/kg (DAR 4) in both Dxd-sensitive (KPL4, NCI-N87) and Dxd-resistant (HT1376) models, outperforming sacituzumab-Dxd (DAR 8). PK profiles were favorable, with circulating t1/2 in line with that of naked sacituzumab. In preliminary tolerability studies, the DAR 4 ADC was well tolerated in mice and rats, suggesting a reasonable therapeutic index.RAI-conjugated ADCs remained cytotoxic when paired with other antibodies, including those targeting antigens expressed as low as 20 K receptors per cell, supporting its translatability. The dual-payload ADC (RAI + Dxd) outperformed the Dxd-only ADC in vitro, including in Dxd-resistant cell lines, demonstrating its potential to broaden efficacy and overcome resistance. Conclusions: Our findings highlight the promise of a novel RAI as a versatile ADC payload, demonstrating potent antitumor activity alone or in combination with Dxd. Its compatibility with diverse antibodies, and favorable PK/tolerability profiles support its further evaluation as an approach to overcome resistance and improve patient outcomes. Citation Format: Tara L. Arvedson. Disrupting ribosome assembly to block protein translation: A novel ADC payload with strong antitumor activity in mono- and dual-payload formats [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A106.

In recent years, remarkable breakthroughs have been reported on antibody-drug conjugates (ADCs), with 15 ADCs successfully entering the market over the past decade. This substantial development has positioned ADCs as one of the fastest-growing domains in the realm of anticancer drugs, demonstrating their efficacy in treating a wide array of malignancies. Nonetheless, there is still an unmet clinical need for wider application, better efficacy, and fewer side effects of ADCs. An ADC generally comprises an antibody, a linker and a payload, and the combination has profound effects on drug structure, pharmacokinetic profile and efficacy. Hence, optimization of the key components provides an opportunity to develop ADCs with higher potency and fewer side effects. In this review, we comprehensively reviewed the current development and the prospects of ADC, provided an analysis of marketed ADCs and the ongoing pipelines globally as well as in China, highlighted several ADC platforms and technologies specific to different pharmaceutical enterprises and biotech companies, and also discussed the new related technologies, possibility of next-generation ADCs and the directions of clinical research.

<div>Abstract<p>The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼10<sup>6</sup> surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. <i>Mol Cancer Ther; 16(4); 681–93. ©2017 AACR</i>.</p></div>

CEACAM5 is an oncofetal protein that is expressed in a number of tumors of epithelial origin such as colorectal cancer (CRC), lung and gastric cancer. Differential expression of CEACAM5 protein in tumor tissues compared to normal tissues make this target attractive for targeting with an antibody-drug conjugate (ADC). The ADC that is characterized in this study contains a specific anti-CEACAM5 antibody and the maytansinoid derivative DM4, a potent antimitotic agent that inhibits microtubule assembly. DM4 is covalently bound to the humanized IgG1 (hIgG) antibody through a N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB) linker that is stable in plasma and cleavable inside cells. After binding and internalization into CEACAM5-positive cancer cells, this ADC is degraded, releasing cytotoxic DM4 metabolites. In the present study the Pharmacokinetic (PK), Pharmacodynamic (PD) and efficacy relationships of this ADC were investigated against subcutaneous colon Patient-Derived-Xenograft (PDX) in severe combined immunodeficient (SCID) mice. The modulation of several biomarkers after a single administration of this compound at 3 dose levels were quantified in the tumor by immunoassay (phosphorylated Histone H3 (pHH3), a marker of cells arrested in mitosis and free human CEACAM5 expression). The spatial localization in the tumor of the biomarkers (pHH3, free human CEACAM5, DM4 and hIgG) was analyzed by immunohistochemistry (IHC). A population pharmacokinetic analysis of the ADC was performed first, followed by population PK/PD analysis for pHH3 induction and tumor growth inhibition (TGI). This study has shown both by immunoassay and IHC that a single administration of this anti-CEACAM5 ADC induced in the xenografted tumor a dose dependent increase of pHH3 expression compared to control, with a maximal induction observed at 24-48 hours. The levels of DM4 and hIgG detected by IHC in the tumor were dose dependent and progressively decreased with time. DM4 and hIgG part of the ADC were essentially immunodetected at the membrane level of tumor cells and sub-localization was observed in vesicular intracellular compartments at later time point which confirms ADC tumor cell penetration. The PK/PD relationship analysis indicated that compound concentration producing 50% of the maximum pHH3 stimulation was 2.3 μg/mL and the threshold concentration for tumor eradication was 8 μg/mL in this colon PDX model. Both immunoassay and IHC assessment confirmed that single administration of the ADC had no impact on CEACAM5 expression at the tumor cell membrane. Overall these data have confirmed mechanism of action of this anti-CEACAM5 ADC and its attractiveness as a potent compound to induce dose dependent CEACAM5 expressing tumor cell death. This compound is now being explored in a phase 1 clinical trial in cancer patients. Citation Format: Christophe Henry, Céline Nicolazzi, Céline Amara, Anne-Marie Lefebvre, Christelle Larois, Florence Attenot, Claire Brillac, Marion Bouillon-Pichault, Nathalie Fagniez, Stéphanie Decary, Cécile Combeau, Pierre-François Berne, Souâd Naimi, Marielle Chiron, Véronique Blanc. PK/PD evaluation of an anti-CEACAM5 antibody drug conjugate, in a colon patient-derived-xenografted mice 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 B145.