Antibody–Drug Conjugates: The Past, Present and Future

Antibody-Drug Conjugates (ADCs) are rapidly growing in importance as a targeted cancer therapy, with over ten approved, and many currently undergoing clinical trials. ADCs comprise of a cytotoxic agent (i.e., the "payload") conjugated via a chemical linker to a tumor-targeting antibody. There is a demand for novel ADC payloads with unique mechanisms of action, enhanced tolerability profiles and improved physicochemical properties. Such payloads could lead to ADCs with therapeutic efficacy in patients resistant to other therapies, and payloads with reduced hydrophobicity should lead to improved conjugation, minimal aggregation, and higher Drug-Antibody Ratios (DARs). We report here, studies on a novel class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) payloads, designed through molecular modeling, which form cross-links between Guanine and Adenine bases within the DNA minor groove with defined sequence specificity. Eleven novel analogs will be described which have been structurally modified with various functional groups to allow tunable hydrophobicity, and new vectors through which antibodies may be attached. Most of these analogs are highly cytotoxic, with some exhibiting IC50 values in tumor cells down to the femtomolar range (e.g., FGX37-140, 0.9 pM in both RAJI and Jurkat cells, 72 h incubation). The DNA interactivity of these analogs has been studied using a range of methods such as gel-based DNA footprinting and cross-linking, FRET melting and Transcription Factor (TF) Array assays, the results of which will be described. Overall, the analogs appear to favor the formation of intrastrand rather than interstrand DNA cross-links and can inhibit the DNA binding of several key cancer-related transcription factors such as NFκB. To explore the potential of these analogs as ADC payloads, one member, FGX8-46, was conjugated to the EGFR-targeting antibody Cetuximab in stochastic fashion (DAR2) to produce Cetuximab-(FGX16-11). This ADC had an unexpectedly high Maximum Tolerated Dose (MTD) in a standard mouse model of at least 45 mg/kg, perhaps reflecting the novel mechanism of action of the payload. It was then evaluated in a Human Tumor Xenograft study based on BALB/c mice transplanted with the EGFR-expressing human colon cancer cell line SW-48 using single doses ranging from 1 mg/kg to 40 mg/kg. Cetuximab-(FGX16-11) was active at all dose levels including 1 mg/kg which provided tumor suppression out to approximately three weeks. Complete tumor suppression out to 50 days was seen with 20 mg/kg. Overall, the unique mechanism of action, potent cytotoxicity, and excellent in vivo tolerability and significant efficacy in ADC format, make the CBI-PDDs a promising new class of ADC payloads suitable for further development. Citation Format: George Procopiou, Paul J. Jackson, Daniella M. di Mascio, Jennifer L. Auer, Paolo Andriollo, Ilona Pysz, Khondaker M. Rahman, Keith R. Fox, David E. Thurston. A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads [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 1749.

Background: Antibody-drug conjugates (ADC) are increasingly utilized to treat a variety of solid tumor and hematologic malignancies. Fam-trastuzumab deruxtecan-nxki (T-DXd) is an ADC that combines the anti-HER2 monoclonal antibody, trastuzumab, to a highly potent topoisomerase I inhibitor payload. It is indicated in the treatment of metastatic HER2-positive and HER2-low and ultralow breast cancer. While veno-occlusive disease (VOD) is commonly described in patients with hematologic malignancies, it is rarely seen in patients with solid tumors but has been documented with other ADCs. We present a rare case of VOD that occurred in a patient with breast cancer receiving T-DXd. Case Presentation: A 45-year-old female with triple-negative breast cancer developed a rapid metastatic recurrence following neoadjuvant chemo-immunotherapy, then rapid progression while on first-line sacituzumab govitecan. Given the tumor was HER2-low (IHC 2+, FISH negative), she received second-line T-DXd. After cycle 4 of T-DXd, she developed rapid elevations of total and direct bilirubin as well as transaminases. Abdominal imaging was negative for progression in the liver. Due to further elevations in bilirubin, a liver biopsy was conducted and revealed venous outflow obstruction consistent with VOD. Defibrotide was considered for treatment, however the patient ultimately pursued hospice care. Discussion: VOD results from damage to the small blood vessels in the liver, leading to obstruction of blood flow, volume overload, and potentially liver failure. It can rapidly progress to multiorgan failure if untreated. VOD presents with tender hepatomegaly, rapid weight gain, ascites, and jaundice, often accompanied by elevations in total and direct bilirubin, as well as transaminases. VOD occurs most commonly after hematopoietic stem cell transplantation, and more rarely can occur secondary to treatment with ADCs. The two most implicated ADCs are gemtuzumab ozogamicin and inotuzumab ozogamicin, utilized in hematologic malignancies. Management of VOD includes defibrotide, a thrombolytic agent that stabilizes the endothelium. On review of the literature, there are reports of VOD occurring after treatment with trastuzumab emtansine, another ADC used in breast cancer. However, to our knowledge, there are no reports of VOD occurring after treatment with T-DXd in breast cancer patients. The most commonly reported adverse effects of T-DXd include edema, cytopenias, GI upset, peripheral neuropathy, alopecia, and elevated liver function tests. Rarely, T-DXd can cause decreased ventricular function, pneumonitis, and febrile neutropenia. Our patient received prior chemotherapy in the neoadjuvant and adjuvant setting, which is a well-described risk factor in the development of VOD. VOD is likely underrecognized in the breast cancer population given overlapping symptoms with the metastatic disease itself, such as elevations in serum liver function tests, ascites, jaundice, pain, and weight fluctuations. There is limited data regarding the optimal management and prognosis of patients with breast cancer who develop VOD after treatment with an ADC. Conclusion: We present a rare case of VOD after receipt of T-DXd in a heavily pretreated patient with metastatic breast cancer. Citation Format: K. N. Koehler, K. Noce, S. Kelly, G. Bader, M. Cherian, A. Davenport, K. C. Johnson, N. Lopetegui-Lia, A. Roy, D. Quiroga, S. Sardesai, D. Stover, R. Wesolowski, N. Williams, S. Vasu, M. E. Gatti-Mays. A case report of veno-occlusive disease (VOD) in a patient with metastatic triple-negative breast cancer receiving trastuzumab deruxtecan [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-06-07.

The most effective treatments for locally advanced cancers rely on non-targeted chemotherapies given with radiotherapy. Advances in cancer biology have identified vulnerabilities amenable to precision oncology approaches including antibody drug conjugates (ADCs). In theory, ADCs combine specificity of cancer cell receptor antibody targeting with potent cytotoxins. However, toxicities and resistance limit ADC clinical efficacy. Delivering ADCs with radiotherapy can improve their therapeutic index. Here, the combination of ADC payloads (anti-tubulin monomethyl auristatin E (MMAE) or topoisomerase I inhibitors DXd and SN-38) with radiotherapy is reported in immune-competent murine models. To directly compare ADC payload effects and remove targeting bias, the payloads are tested as free drugs and as tumor-targeted ADC or peptide-drug conjugates in combination with ionizing radiation. Both DXd and MMAE induce anti-tumor immune response that block re-challenge tumor growth. As monotherapy, DXd is more potent than MMAE at inhibiting tumor formation. In contrast when combined with ionizing radiation at subtherapeutic doses, MMAE but not DXd radiosensitizes resulting in improved tumor control and greater immune activation with MMAE. The differential effects of anti-tubulin versus topoisomerase I inhibitors when combined with ionizing radiation and immunotherapies can inform and optimize clinical development of ADC based chemo-radio-immunotherapy combinations for cancer patients.

Purpose:Antibody–drug conjugates (ADC) harboring topoisomerase I (TOP1) inhibitor payloads have improved survival for patients with metastatic breast cancer. However, knowledge of ADC resistance mechanisms and potential impact on the sequential use of ADCs is limited. In this study, we report the incidence and characterization of TOP1 mutations arising in the setting of ADC resistance in metastatic breast cancer.Experimental Design:Patients with metastatic breast cancer treated with ADCs with available posttreatment plasma-based genotyping were included. TOP1 mutation incidence, mutant allele frequency, and functional characterization were assessed, and incidence was compared with that in patients with metastatic breast cancer not receiving ADC treatment and in The Cancer Genome Atlas.Results:Plasma-based genotyping identified distinct TOP1 mutations (S57C, R364H, W401C, and G359E) in 12.9% of patients (4/31) at the time of disease progression on ADC, compared with 0.7% (3/420) in non–ADC-treated patients with metastatic breast cancer and 0.5% in The Cancer Genome Atlas. The appearance of mutations was associated with clinical cross-resistance, as median duration on the first ADC was 455 versus 52 days for the second ADC. The functional characterization of three novel TOP1-mutant proteins demonstrated that all exhibited reduced enzymatic activity, attenuated covalent DNA binding, and resistance to TOP1 inhibitor ADC payloads SN38 and deruxtecan.Conclusions:We describe the recurrent emergence of functionally altered, resistance-associated TOP1 mutations in vivo under selective pressure from ADCs and the potential impact on mediating cross-resistance to sequential ADCs. TOP1 mutation may represent a biomarker of resistance in this setting, and additional work is needed to optimize biomarkers and ADC payload design to improve outcomes for the sequential use of ADCs.See related commentary by Gwin and Hurvitz, p. 1824

<div>AbstractPurpose:<p>Antibody–drug conjugates (ADC) harboring topoisomerase I (TOP1) inhibitor payloads have improved survival for patients with metastatic breast cancer. However, knowledge of ADC resistance mechanisms and potential impact on the sequential use of ADCs is limited. In this study, we report the incidence and characterization of <i>TOP1</i> mutations arising in the setting of ADC resistance in metastatic breast cancer.</p>Experimental Design:<p>Patients with metastatic breast cancer treated with ADCs with available posttreatment plasma-based genotyping were included. <i>TOP1</i> mutation incidence, mutant allele frequency, and functional characterization were assessed, and incidence was compared with that in patients with metastatic breast cancer not receiving ADC treatment and in The Cancer Genome Atlas.</p>Results:<p>Plasma-based genotyping identified distinct <i>TOP1</i> mutations (S57C, R364H, W401C, and G359E) in 12.9% of patients (4/31) at the time of disease progression on ADC, compared with 0.7% (3/420) in non–ADC-treated patients with metastatic breast cancer and 0.5% in The Cancer Genome Atlas. The appearance of mutations was associated with clinical cross-resistance, as median duration on the first ADC was 455 versus 52 days for the second ADC. The functional characterization of three novel TOP1-mutant proteins demonstrated that all exhibited reduced enzymatic activity, attenuated covalent DNA binding, and resistance to TOP1 inhibitor ADC payloads SN38 and deruxtecan.</p>Conclusions:<p>We describe the recurrent emergence of functionally altered, resistance-associated <i>TOP1</i> mutations <i>in vivo</i> under selective pressure from ADCs and the potential impact on mediating cross-resistance to sequential ADCs. <i>TOP1</i> mutation may represent a biomarker of resistance in this setting, and additional work is needed to optimize biomarkers and ADC payload design to improve outcomes for the sequential use of ADCs.</p><p><a href="https://aacrjournals.org/clincancerres/article-abstract/doi/10.1158/1078-0432.CCR-25-0166" target="_blank"><i>See related commentary by Gwin and Hurvitz, p. 1824</i></a></p></div>

Background: About half of the currently FDA-approved antibody drug conjugates (ADCs) are based on microtubule inhibitors, making them effective only against actively dividing cells. Non-dividing tumor stem cells in contrast cannot be eradicated and may cause resistance and tumor relapse. Hence, there is a high demand for novel payloads with unique modes of action to overcome the current limitations. Inhibition of NAMPT, the rate-limiting enzyme in the salvage biosynthetic pathway of NAD+ starting from nicotinamide, induces cell death due to energy shortage in both dividing and non-dividing cell populations. Herein, we present encouraging in vitro and in vivo results of ADCs based on in silico designed NAMPT inhibitors (NAMPTi) as payloads, highlighting the potential of NAMPT inhibition as an emerging mode of action in ADC technology. Materials and methods: In Silico modelling: PDB published co-crystals of NAMPT with NAMPTi were used for database generation, compound enumeration, virtual screening and lead optimization of candidates in a multistep process using Schrödinger Suite. In vitro viability cell lines: L540 (CD30+); MDA-MB-453 (HER2+) ADC: The cysteine-reactive NAMPTi linker-payload constructs were synthesized at Heidelberg Pharma and conjugated to both interchain and engineered cysteine residues of anti-CD30 or anti-HER2 antibodies yielding ADCs with an average DAR of 10. Animal models: The anti-tumor efficacy of NAMPTi-based ADCs was evaluated in female NXG mice intravenously inoculated with L540 tumor cells, and female NMRI-nu mice inoculated subcutaneously with NCI-N87 tumor cells. Animals were treated with single or multiple doses of anti-CD30 and anti-HER2 ADCs, respectively. Results: Computational models allowed the discovery of unexploited features within NAMPT catalytic pocket with the potential to be used for a new generation of NAMPTi. This new generation of NAMPTi was accessed through a new in silico campaign leading to NAMPTi optimized for the use as ADC payloads, translating in excellent in vitro and in vivo anti-tumor efficacy. Single and multiple dose treatment resulted in complete remission of NCI-N87 tumors and survival of L540 disseminated tumor-bearing animals was significantly prolonged upon treatment. Conclusions: Targeted drug delivery to CD30+ and HER2+ cell lines was achieved using ADCs with in silico optimized NAMPTi linker-payloads. The use of computational approaches in the optimization of NAMPTi enabled to address unexploited features of the catalytic pocket resulting in more potent, selective and stable inhibitors, which were optimized for the targeted delivery by ADC technology. The use of anti-CD30 and HER2 ADCs loaded with NAMPTi in the therapy of CD30+ Hodgkin lymphoma and HER2+ breast cancer models, highlights this new modality as a promising approach in cancer therapy. Citation Format: Pablo Ruedas Batuecas, Hendrik Gruss, Sarah Jane Neuberth, Alexander Hempelmann, Anikó Pálfi, Marija Vranic, Andreas Pahl, Torsten Hechler. In silico optimized NAMPT inhibitor for targeted delivery by antibody drug conjugates as novel therapeutic modality for treatment of liquid and solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1162.

Synthetic analogs based on the DNA bis-intercalating natural product peptides sandramycin and quinaldopeptin were investigated as antibody drug conjugate (ADC) payloads. Synthesis, biophysical characterization, and in vitro potency of 34 new analogs are described. Conjugation of an initial drug-linker derived from a novel bis-intercalating peptide produced an ADC that was hydrophobic and prone to aggregation. Two strategies were employed to improve ADC physiochemical properties: addition of a solubilizing group in the linker and the use of an enzymatically cleavable hydrophilic mask on the payload itself. All ADCs showed potent in vitro cytotoxicity in high antigen expressing cells; however, masked ADCs were less potent than payload matched unmasked ADCs in lower antigen expressing cell lines. Two pilot in vivo studies were conducted using stochastically conjugated DAR4 anti-FRα ADCs, which showed toxicity even at low doses, and site-specific conjugated (THIOMAB) DAR2 anti-cMet ADCs that were well tolerated and highly efficacious.

Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive subtype of prostate cancer (PC) without curative treatments. Antibody-drug conjugates (ADCs) emerged as promising cancer therapeutics that selectively deliver cytotoxic agents (payloads) to the tumors. Although ADCs have been successfully applied in the treatment of hematological and solid tumors, ADC monotherapy has not demonstrated durable responses in mCRPC and the mechanisms of PC resistance to ADCs have not been thoroughly investigated. Our study aimed to improve ADC efficacy using a new integrated approach for custom ADC design and multiplexing. To nominate rational combinations of ADC targets and ADC payloads, we (1) examined protein co-expression of three clinically relevant surface antigens—B7 homolog 3 (B7-H3), prostate specific membrane antigen (PSMA), and six-transmembrane epithelial antigen of prostate-1 (STEAP1)—in a series of human mCRPC samples and (2) screened established ADC payloads and their combinations in mCRPC cell lines with different phenotypes. We identified synergistic interactions between DNA-damaging payloads and Bcl-xL inhibitor A-1331852 as well as their coordinated induction of the intrinsic apoptosis pathway. The functional relevance of isolated p53 loss and impaired PC responses to three genotoxic ADCs (B7-H3-seco-DUBA, PSMA-SG3249, and STEAP1-DXd) and their combinations with A-1331852 was established using genetic knockout models. Lastly, we found enhanced in vivo antitumor activity in mCRPC by combining the clinically relevant agents B7-H3-seco-DUBA (vobramitamab duocarmazine) and A-1331852. Collectively, our findings provide rationale for the development of ADC therapies combining genotoxic payloads with Bcl-xL inhibitors for mCRPC.

Antibody–drug conjugates: Recent advances in payloads

Antibody-drug conjugates (ADCs) combine the targeting specificity of antibodies with powerful cytotoxic payloads to direct antigen-specific tumor cell killing. Multiple ADCs have shown single-agent clinical activity against solid and hematological cancers and are currently being evaluated in combination with immune checkpoint inhibitors (CPIs). Therapeutic benefit from CPIs relies on the presence and quality of responding antitumor T cells, both in the tumor and from new T cell priming and expansion outside the tumor. Select cytotoxic ADC payloads, like monomethyl auristatin E (MMAE), have the potential to amplify T cell responses through immunogenic cell death (ICD) and, as such, may be optimal combination partners with CPIs. However, ADC payloads released in the tumor (bystander drug) or released systemically also have the potential to negatively impact T cell viability, proliferation, and priming. The compatibility of different ADC payload chemotypes (Auristatins, camptothecins (CPTs), Pyrrolobenzodiazepine (PBDs), Tubulysins) with CPIs may depend on both the direct and indirect effects of payloads and their associated free-drugs on immune cells. Here, we determined the relative potencies of ADC payload free-drugs MMAE and DXd on activated human and murine T cells in vitro and in a model of xeno-graft versus host disease (xeno-GVHD) to evaluate the impact of ADC bystander effects on human immune cell expansion and function in vivo. In vitro, ADC payloads showed a range of potencies on activated T cells, with IC50 values skewing lower for CD4 compared to CD8 T cells. For each payload, murine T cells were significantly less sensitive than human T cells, which may have implications for modeling combinations of ADCs and CPIs. Mouse CD8 T cells were ~10-fold less sensitive to MMAE and 20-25-fold less sensitive to clinical CPT payloads compared to human T cells. In an in vivo model of human immune cell activation, xeno-GVHD, bystander effects from a non-targeted MMAE-ADC did not impair the expansion of human immune cells, however, a non-targeted DXd-ADC reduced CD4 T cells and robustly reduced populations of non-T cells in spleens. Consistent with reduced immune cell expansion, the DXd-ADC significantly slowed the xeno-GVHD disease course compared to the MMAE-ADC treated and control groups. These data suggest that the most commonly used ADC payload classes, MMAE and DXd, may have different direct effects on proliferating human immune cells as bystander free-drugs, which may have clinical relevance for combinations of ADCs with CPIs. Continued research exploring the balance between ADC potency on tumor cells, direct and indirect effects of payloads on immune cells, and dose timing and sequencing could help maximize the potential of ADCs in combination with immunotherapies. Citation Format: Reice D. James, Michelle L. Ulrich, J Hartsuyker, Alyson J. Smith, Ryan A. Heiser. Evaluating the potency of antibody-drug conjugate (ADC) free-drugs on T cells [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 2604.

TPS3157 Background: PRO1184 is an antibody-drug conjugate (ADC) directed to folate receptor alpha (FRα), a cell surface antigen overexpressed in multiple cancers including ovarian, endometrial, lung, mesothelioma, and breast cancer. PRO1184 consists of a human monoclonal antibody that selectively binds FRα, a novel cleavable hydrophilic linker, and a topoisomerase 1 inhibitor payload, exatecan. Previous studies demonstrated that the hydrophilic linker confers excellent physicochemical properties and pharmacokinetic (PK) profiles across a range of payload mechanisms and is superior to conventional linkers on these critical parameters for ADCs. In addition, exatecan is broadly active in many tumor types, is membrane permeable, and is not a substrate of multidrug resistance efflux pumps. It may thus lend a robust bystander effect and induce deeper or more durable responses in refractory tumors. Preclinical studies further established that PRO1184 exerts potent antitumor activity in mouse xenograft models with high, moderate, and low FRα expression, consistent with the inherent potency and expected bystander activity of the exatecan payload. PRO1184 is stable in plasma and retains the excellent PK properties and bioactivity of the unconjugated parent antibody. The preliminary safety profile of PRO1184 was more favorable than a benchmarking deruxtecan-based ADC in cynomolgus monkeys. PRO1184 is thus a promising development candidate with a potentially large therapeutic index to benefit a broad population of patients with FRα-expressing solid tumors. Methods: PRO1184-001 is an ongoing, phase 1/2, open-label dose escalation and expansion study. Eligible patients are adults with metastatic or unresectable solid tumors, including ovarian, endometrial, non-small cell lung, breast cancer, or mesothelioma. Patients must have measurable disease per the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, or mRECIST 1.1 for pleural mesothelioma. Patients must also have previously received therapies known to confer clinical benefit unless considered ineligible, refused by the patient, or not available in the region. PRO1184 is given by intravenous infusion on Day 1 of a 21-day cycle and treatment may continue until disease progression, unacceptable toxicity, or other reason for discontinuation. The primary objectives are to evaluate the safety and tolerability of PRO1184 and to identify the maximum tolerated dose, if reached, and recommended phase 2 dose (RP2D). Part A of the study consists of a dose escalation phase and Part B consists of 4 FRα-expressing tumor specific expansion cohorts treated at the RP2D. PK, immunogenicity, and antitumor activity will also be evaluated. The study is currently enrolling at sites in the US, with future enrollment in China planned (NCT05579366). Clinical trial information: NCT05579366 .

Nicotinamide phosphoribosyl-transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway, generating nicotinamide adenine dinucleotide (NAD) from nicotinamide (NAM). Inhibition of intracellular NAMPT activity represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) as it is not dependent on cell proliferation. Thus, NAMPT inhibitor-based ADCs have the potential to target both proliferating and resting tumor cells. We therefore developed a novel structural class of NAMPT inhibitors (NAMPTi) as a potent ADC payload class and characterized NAMPTi and NAMPTi-ADCs in vitro and in vivo in preclinical tumor models. We profiled the small molecule NAMPTi BAY-346 in comparison to the kinesin spindle protein inhibitor (KSPi) BAY-331 on a panel of 350 cancer cell lines from various tumor indications showing a differential sensitivity profile of BAY-346 vs BAY-331, with cell lines characterized by low NAMPT mRNA levels as being very sensitive to BAY-346. NAMPTi BAY-346, but not KSPi BAY-331, reduced the viability of quiescent HaCat cells as well as of serum starved non-proliferating NCI-N87 cells. In vitro treatment with NAMPTi BAY-346, which bears a pyridine warhead that can be phosphoribosylated by NAMPT, resulted in IC50 values in the nanomolar to subnanomolar range (2.8 nM to 0.01 nM) in cell lines derived from solid and hematologic tumor indications (e.g., THP-1, MV-4-11, U-251, NCI-H292, MDA-MB-453, LoVo, KPL4, HT1197 and BxPC3). BAY-346 was 100-fold more potent than the small molecule NAMPTi BAY-248, which cannot be phosphoribosylated. A BAY-346 derived NAMPTi was conjugated as a payload to a series of antibodies targeting different tumor-associated antigens: C4.4a (LYPD3), HER2, B7H3 (CD276), and TWEAKR (Fn14/ TNFRSF10A). The resulting NAMPTi-ADCs were tested in proliferation and cellular mechanistic in vitro assays. NAMPTi-ADCs depleted NAD+ in tumor cells and showed potent growth inhibitory activity with IC50 values in the subnanomolar- to nanomolar range in a target-dependent manner. A C4.4a-NAMPTi-ADC and a HER2-NAMPTi-ADC were tested In the C4.4a- and HER2-expressing MDA-MB-453 cell line derived subcutaneous breast cancer model xenografted on NOD/SCID mice. Both NAMPTi-ADCs showed highly potent anti-tumor Efficacy: The C4.4a-NAMPTi-ADC induced complete responses (in 3 of 8 mice) and stable diseases (in 5 of 8 mice) and the HER2-NAMPTi-ADC achieved complete tumor regression in all treated animals. In addition, in the THP-1 acute myeloid leukemia (AML) subcutaneous in vivo model, a B7H3-NAMPTi-ADC induced complete tumor responses in 7 of 8 treated animals. Taken together, we identified a new series of NAMPT inhibitors as a novel class of ADC payloads exhibiting strong in vivo efficacy in various preclinical xenograft models. Citation Format: Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Niels Boehnke, Markus Berger, Nils Griebenow, Andreas Steffen, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Anja Giese, Maria Quanz, Zhengzheng Bao, Xiuli Wu, Hilmar Weinmann, Lars Linden, Bertolt Kreft, Dominik Mumberg. Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1807.

Background: M9140 is the first clinical-stage TOP1 inhibitor payload ADC directed against CEACAM5, which is expressed in colorectal cancer (CRC) and other cancer indications. M9140 is composed of a CEACAM5-specific antibody conjugated to eight, highly stable and hydrophilic ß-glucuronide-exatecan linker-payloads. After linker cleavage, the highly potent and membrane permeable TOP1 inhibitor exatecan is released. Methods: M9140 potency was tested in viability assays using cancer cell lines with different levels of CEACAM5 expression. Potency shift viability assays were conducted using two CRC cell lines, with zosuquidar used as multidrug resistance-1 (MDR-1) inhibitor. The M9140 bystander effect was assessed in co-culture experiments with CEACAM5-negative and -positive cancer cells. Antitumor efficacy and safety were evaluated in patient-derived xenograft (PDX) mouse models and in cynomolgus monkeys, respectively. Results: M9140 demonstrated specific binding to CEACAM5 and selective killing of target-positive cancer cells with potencies between 0.09 nM and 0.62 nM. A potent bystander effect of M9140 was demonstrated in co-culture experiments indicating the potential to treat tumors with heterogeneous target expression. Unlike other ADC payloads such as monomethyl auristatin E (MMAE) and the maytansine derivative DM4, exatecan potency was not affected by MDR-1 inhibition, indicating that this drug efflux resistance mechanism may not be relevant for exatecan. A single treatment with 10 mg/kg M9140 caused strong antitumor effects in all 14 CRC PDX models tested, with tumor stasis in 10 models and tumor regression in four models as the best response. The TOP1 inhibitor-based ADC M9140 caused strong antitumor efficacy with a tumor volume reduction of 74% and 88% as best response in two CRC PDX models where a maytansine-based CEACAM5 ADC was not effective. In cynomolgus monkeys, M9140 showed dose-dependent hematolymphoid and intestinal effects, consistent with exatecan payload, with no indication of any other toxicities such as interstitial lung disease (ILD) or ocular toxicity. Conclusions: M9140 demonstrated high potency, strong antitumor activity, and bystander effect in preclinical models. The side effect profile in monkeys was favorable and in line with the expected toxicity of exatecan. Notably, ILD and ocular toxicity, which are known adverse effects of deruxtecan and maytansine-based ADCs respectively, were absent. A first-in-human study to evaluate the safety, tolerability, pharmacokinetics, and preliminary clinical activity of M9140 in advanced solid tumors is ongoing (NCT05464030). Citation Format: Sabine Raab-Westphal, Felix Hart, Willem Sloot, Min Shan, Nicolas Rasche, Christiane Amendt, Jan Anderl. Preclinical efficacy and safety of M9140, a novel antibody-drug conjugate (ADC) with topoisomerase 1 (TOP1) inhibitor payload targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)-expressing colorectal tumors [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 2362.

e15011 Background: Antibody-drug conjugates (ADCs) are attractive targeted agents in anticancer treatment due to their unique mechanism of action and reduced toxicity compared to other anti-cancer drugs. Yet little is still known about the adverse events profiles associated with ADCs. Methods: A systematic review was performed including randomized controlled trials (RCTs) of anticancer treatment that evaluated the efficacy of ADCs. PubMed and EMBASE were systematically searched for studies from January 1990 to December 2022. RCTs that were registered in ClinicalTrials.gov and had final reports of comprehensive adverse events were also included. We included studies for patients with both solid and hematologic tumors. The odds ratios (ORs) of treatment-related symptoms, and hematologic, hepatic, ocular, cardiovascular, renal, and respiratory toxicities for patients treated with ADCs and those without ADCs were examined. A random effects model was developed to estimate the pooled ORs of each adverse event associated with ADC use. Subgroup analyses were also performed based on the cancer type (solid tumors versus hematologic malignancy) and ADC regimen (ADC versus ADC plus chemotherapy). Results: 20 studies involving 10,075 patients (5,745 patients with ADC and 4,330 patients without ADC) were included. Among the included trials, 14 (70.0%) were trials for solid tumors and 6 (30.0%) were trials for hematological tumors. Among the included trials, ADC monotherapy was used in 15 (75.0%) experimental arms, and combination therapy of ADC with any other chemotherapy was used in 7 (35.0%). ADCs were associated with a higher risk for all-grade fatigue (OR 1.25, 95% CI 1.08-1.45), anorexia (OR 1.36, 1.09-1.69), nausea (OR 1.46, 1.09-1.97) and sensory neuropathy (OR 2.18, 1.27-3.76). Patients treated with ADCs had a significantly lower risk of all-grade febrile neutropenia (OR 0.46, 0.22-0.96), leukopenia (OR 0.47, 0.29-0.77), and neutropenia (OR 0.56, 0.31-1.01). Whereas, they had a higher risk of all-grade increased alanine aminotransferase (OR 2.51, 1.84-3.40), increased aspartate aminotransferase (OR 2.83, 2.04-3.93), cataract (OR 4.69, 1.86-11.81), blurry vision (OR 3.12, 1.02-9.55), pericardial effusion (OR 4.27, 2.71-6.72), and epistaxis (OR 2.45, 1.50-4.01). In the subgroup analysis, a similar toxicity profile was observed when comparing the solid tumors versus hematologic malignancy groups and the ADC versus ADC plus chemotherapy groups, except for hematologic adverse events, which showed an increased risk in patients with solid tumors and those treated with both chemotherapy and ADCs. Conclusions: This comprehensive profile of adverse events associated with ADC-based treatment shows increased treatment-related symptoms, hepatic, ocular, and cardiovascular toxicities although no increase of high-grade are seen.