Payloads For Antibody-drug Conjugates Research Articles

Abstract 1832: Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy

Abstract Background: Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and modulate pre-mRNA splicing. Alterations in splicing machinery and mRNA splicing is common in cancer and represents a potential susceptibility that can be exploited by targeted delivery of a splicing modulator to tumors with antibody drug conjugates (ADCs). Results: PH1 payload is a novel derivative of Thailanstatin optimized for metabolic stability and anti-tumor activity. PH1 is an efficient modulator of splicing in vitro and in vivo and a poor substrate for the MDR1 transporter. Transcriptome analysis of PH1- treated NCI-N87 cells revealed multiple classes of mis-splicing events including elevated expression of transcripts with skipped exons (3364 genes/4x change) and retained introns (332 genes/2.5x change). Translation of transcripts with retained introns and novel exon-exon junctions results in generation of neoepitopes. Upon conjugation to Trastuzumab, Tras PH1 ADC exhibited nanomolar potency specific to HER2-expressing cancer cells, with durable anti-tumor response in vivo and favorable pharmacokinetic exposure in mice. Due to the potential for generation of neoepitopes, combination studies of Tras PH1 with a checkpoint inhibitor antibody was performed in a syngeneic MC38-HuHer2 model. Single agent and combination treatments induced tumor recruitment of CD11b+ positive myeloid cell subsets. While the combination treatment eradicated tumors, the treated animals also developed tumor-specific immunity. A toxicology study performed in cynomolgus monkeys confirmed favorable PK profile and a wide safety margin for Tras PH1 ADC. Conclusions: We present the development of a splicing modulator-based payload class with the ability to target tumor mRNA splicing, induce tumor neoepitopes, recruit myeloid cells and generate anti-tumor immunity. These findings support the development of ADCs using this novel class of immunostimulatory payload. Citation Format: Satyajit K. Mitra, Vasu Jammalamadaka, Jeffrey Kang, Teodora Losic, Greg Tuffy, Tony W. Liang, Kim Tipton, Adriana Lopez, Scott Savage, William Monteith, William E. Haskins, Melissa S. Jurica, Sanjeev Satyal, Mary Do. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832.

Abstract 1319: Rapid accumulation of cytotoxic payload in tumor tissue drives BT5528 activity in tumor models

Abstract BT5528 was developed as a Bicycle® toxin conjugate to deliver monomethyl auristatin E (MMAE) -payload to EphA2 overexpressing tumors. It consists of a bicyclic peptide targeting the tumor antigen EphA2, linked to the cytotoxin MMAE via a molecular spacer and cleavable linker. Administration of BT5528 results in rapid uptake of payload into EphA2 overexpressing xenograft tumors associated with persistent toxin levels in tumor tissue and limited systemic exposure of both parent drug and payload (1). BT5528 showed a favorable preclinical profile supporting the initiation of a first-in-human Phase I/II study (NCT04180371) to investigate safety and efficacy of BT5528 in indications with evidence of EphA2 expression including non-small-cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), gastric/upper gastrointestinal (GI), pancreatic and urothelial cancers (2). We have used EphA2 overexpressing tumor xenograft models in both mice and rats to elucidate the relative differences between toxin payload delivery and systemic exposure of BT5528. Comparison of payload-delivery and systemic exposure from EphA2 -targeted Bicycle toxin conjugate and antibody-drug conjugate (ADC) demonstrate the differentiation of these toxin-payload delivery platforms. ADC payload delivery depends upon a sustained plateau-like pharmacokinetic (PK) profile that exposes target and potentially nontarget organs to intact ADC for several days. In contrast, BT5528 achieves prolonged toxin delivery to tumors, following transient exposure in plasma of less than one hour. A Phase I/II study of BT5528 in patients with solid tumors is ongoing, which will investigate if this PK novel, differentiated pharmacokinetic profile, unique to Bicycle toxin conjugates, gives rise to a favorable balance of efficacy and safety.

Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency.

The development of antibody-drug conjugates (ADCs) has significantly been advanced in the past decade given the improvement of payloads, linkers and conjugation methods. In particular, linker design plays a critical role in modulating ADC stability in the systemic circulation and payload release efficiency in the tumors, which thus affects ADC pharmacokinetic (PK), efficacy and toxicity profiles. Previously, we have investigated key linker parameters such as conjugation chemistry (e.g., maleimide vs. disulfide), linker length and linker steric hindrance and their impacts on PK and efficacy profiles. Herein, we discuss our perspectives on development of integrated strategies for linker design to achieve a balance between ADC stability and payload release efficiency for desired efficacy in antigen-expressing xenograft models. The strategies have been successfully applied to the design of site-specific THIOMABTM antibody-drug conjugates (TDCs) with different payloads. We also propose to conduct dose fractionation studies to gain guidance for optimal dosing regimens of ADCs in pre-clinical models.

C3 ester side chain plays a pivotal role in the antitumor activity of Maytansinoids

Maytansinoids, the chemical derivatives of Maytansine, are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADC). Structure-activity-relationship studies had identified the C3 ester side chain as a critical element for antitumor activity of maytansinoids. The maytansinoids bearing the methyl group at C3 position with D configuration were about 100 to 400-fold less cytotoxic than their corresponding L-epimers toward various cell lines. The detailed mechanism of how chirality affects the anticancer activity remains elusive. In this study, we determined the high-resolution crystal structure of tubulin in complex with maytansinol, L-DM1-SMe and D-DM1-SMe. And we found the carbonyl oxygen atom of the ester moiety and the tail thiomethyl group at C3 side chain of L-DM1-SMe form strong intramolecular interaction with the hydroxyl at position 9 and the benzene ring, respectively, fixing the bioactive conformation and enhancing the binding affinity. Additionally, ligand-based and structure-based virtually screening methods were used to screen the commercially macrocyclic compounds library, and 15 macrocyclic structures were picketed out as putatively new maytansine-site inhibitors. Our study provides a possible strategy for the rational discovery of next-generation maytansine site inhibitors.

Axially Substituted Silicon Phthalocyanine Payloads for Antibody–Drug Conjugates

AbstractIR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody–drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phthalocyanine skeleton. Here, we report a new axially substituted SiPc payload that is more easily synthesized. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressing cancer cell lines.

Quantitative Evaluation of the Effect of Antigen Expression Level on Antibody-Drug Conjugate Exposure in Solid Tumor.

Antibody-drug conjugates (ADCs) rely on high expression of target antigens on cancer cells to effectively enter the cell and release a cytotoxic payload. Previous studies have shown that ADC efficacy is not always tied to antigen expression. However, our recent in vitro study suggests a linear relationship between antigen expression and the intracellular levels of the ADC payload. In this study, we have explored the relationship between antigen expression and intratumoral ADC exposure in vivo. Using trastuzumab-vc-MMAE (T-vc-MMAE) and four cell lines with varying expression of human epithelial growth factor receptor 2 (HER2), the pharmacokinetics of total trastuzumab, released ("free") MMAE, and total MMAE were evaluated in a tumor xenograft model. Nude mice were implanted with tumors originating from BT-474, MDA-MB-453, MCF-7, and MDA-MB-468 cell lines and dosed with 10 mg/kg or 1 mg/kg of ADC. Observed data were mathematically characterized using a mechanism-based PK model. A strong positive correlation was observed between antigen expression levels and free/total MMAE exposure (R2 ≥ 0.91) (total MMAE being the sum of released and conjugated MMAE) within the tumor, but not for total trastuzumab exposure. The PK model was able to recapitulate plasma PK through simulation; however, the tumor PK was overpredicted or underpredicted in some cases potentially due to differences in tumor vasculature or extracellular matrix conditions. Our results indicate a linear relationship between antigen expression and tumor exposure of free/total ADC payload in vivo, validating our previous finding in vitro, while also revealing the need to understand complex physiology of the tumor to predict tumor PK of ADC and its components. Our findings also support the concept of antigen expression screening in patients for targeted therapies like ADCs to achieve the maximum therapeutic benefit of the treatment.

Amino DSA analogues as payloads for antibody-drug conjugates with multiple sites for conjugation. Initial studies and solid phase synthesis

Duocarmycins are highly potent and promising anticancer payloads for ADC applications. They tolerate a range of chemical modifications which allow the chemist to modulate both their biophysical and pharmacological properties. The possibility to synthesize these payloads on resin and orthogonally add linkers while immobilized on the solid phase, would allow a combinatorial design of payload analogues with linkers, potentially aided by automation. Working towards this goal, we report a concise and high yielding synthesis of an alkylating unit suitable for solid phase synthesis (10, 9 steps, 34% yield) and demonstrate its applicability to the synthesis of duocarmycin SA analogues (19, 20). An intermediate for traditional solution phase synthesis (8) is also described in 7 steps and 44% yield. A side reaction with potential application to the stereoselective synthesis of these derivatives has also been described.

Combretastatin A4-derived payloads for antibody-drug conjugates.

We describe the use of natural product combretastatin A4 (CA4) as a versatile new payload for the construction of antibody-drug conjugates (ADCs). Cetuximab conjugates consisting of CA4 derivatives were site-specially prepared by disulfide re-bridging approach using cleavable and non-cleavable linkers. These ADCs retained antigen binding and internalization efficiency and exhibited high potencies against cancer cell lines invitro. The conjugates also demonstrated significant antitumor activities in EGFR-positive xenograft models without observed toxicities. CA4 appears to be a viable payload option for ADCs research and development.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

In-situ Reverse Phased HPLC Analysis of Intact Antibody-Drug Conjugates.

The field of oncology has recently seen an exponential growth in antibody-drug conjugates (ADCs) as a biopharmaceutical class with seven ADCs being launched onto the market in the last ten years. Despite the increase in the industrial research and development of these compounds, their structural complexity and heterogeneity continue to present various challenges regarding their analysis including reaction monitoring. Robust and simple reaction monitoring analysis are in demand in the view of at-line in-process monitoring, and can instill control, confidence and reliability in the ADC manufacturing process. Aiming at providing chromatographic methods for conjugation monitoring, we evaluated herein the potential of utilizing reverse phase HPLC analysis, without sample pretreatment, for characterization of traditional cysteine-based ADCs. This analysis can be used for estimation of drug antibody ratio (DAR), which has shown the same trends and results as other well-established HPLC techniques. This methodology was also applied to three ADCs derived from three different antibodies. Additionally, we analyzed unpurified ADC samples existing in a complex reaction matrix and separated ADC species and payload compounds. This investigation was conducted using three different ADCs based on different payloads. The results described herein indicate the potential application of this RP-HPLC methodology in reaction monitoring studies.

Design, Synthesis, and Biological Investigation of Thailanstatin A and Spliceostatin D Analogues Containing Tetrahydropyran, Tetrahydrooxazine, and Fluorinated Structural Motifs.

Thailanstatin A and spliceostatin D, two naturally occurring molecules endowed with potent antitumor activities by virtue of their ability to bind and inhibit the function of the spliceosome, and their natural siblings and designed analogues, constitute an appealing family of compounds for further evaluation and optimization as potential drug candidates for cancer therapies. In this article, the design, synthesis, and biological investigation of a number of novel thailanstatin A analogues, including some accommodating 1,1-difluorocyclopropyl and tetrahydrooxazine structural motifs within their structures, are described. Important findings from these studies paving the way for further investigations include the identification of several highly potent compounds for advancement as payloads for antibody-drug conjugates (ADCs) as potential targeted cancer therapies and/or small molecule drugs, either alone or in combination with other anticancer agents.

Challenges and Opportunities to Develop Enediyne Natural Products as Payloads for Antibody-Drug Conjugates.

Calicheamicin, the payload of the antibody-drug-conjugates (ADCs) gemtuzumab ozogamicin (Mylotarg®) and inotuzumab ozogamicin (Besponsa®), belongs to the class of enediyne natural products. Since the isolation and structural determination of the neocarzinostatin chromophore in 1985, the enediynes have attracted considerable attention for their value as DNA damaging agents in cancer chemotherapy. Due to their non-discriminatory cytotoxicity towards both cancer and healthy cells, the clinical utilization of enediyne natural products relies on conjugation to an appropriate delivery system, such as an antibody. Here, we review the current landscape of enediynes as payloads of first-generation and next-generation ADCs.

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.

Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing.

Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody‐drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non‐pathological cells proximal to the tumour (“bystander killing”). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz‐azastatin methyl ester, which included the C2‐elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76 % overall yield. While Cbz‐protected azastatin methyl ester (0.13–3.0 nM) inhibited proliferation more potently than MMAE (0.47–6.5 nM), removal of the Cbz‐group yielded dramatically increased IC50‐values (9.8–170 nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next‐generation ADC development.

Expeditious Total Synthesis of Hemiasterlin through a Convergent Multicomponent Strategy and Its Use in Targeted Cancer Therapeutics.

Hemiasterlin is an antimitotic marine natural product with reported sub‐nanomolar potency against several cancer cell lines. Herein, we describe an expeditious total synthesis of hemiasterlin featuring a four‐component Ugi reaction (Ugi‐4CR) as the key step. The convergent synthetic strategy enabled rapid access to taltobulin (HTI‐286), a similarly potent synthetic analogue. This short synthetic sequence enabled investigation of both hemiasterlin and taltobulin as cytotoxic payloads in antibody–drug conjugates (ADCs). These novel ADCs displayed sub‐nanomolar cytotoxicity against HER2‐expressing cancer cells, while showing no activity against antigen‐negative cells. This study demonstrates an improved synthetic route to a highly valuable natural product, facilitating further investigation of hemiasterlin and its analogues as potential payloads in targeted therapeutics.

Abstract 2907: Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy

Abstract Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) independent from cell proliferation. This opens up the possibility to target slowly growing tumors as well as resting antigen-positive tumor cells in addition to highly proliferative tumors. We developed a novel structural class of NAMPT inhibitors as ADC payloads to complement the currently available effector chemistries. An SAR-driven approach supported by available structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffold and linker led to highly potent effector chemistries which were conjugated to anti-C4.4a (LYPD3) or anti-B7H3 (CD276) antibodies and tested on antigen-positive and -negative cancer cell lines derived from solid and hematological tumor indications. Furthermore, tuning of the hydrophilicity of the linker and the conjugation method ensured low aggregation of the NAMPT inhibitor ADCs. Pharmacokinetic studies were performed in human plasma to assess the stability of the linkage of the NAMPT inhibitor payload to the antibody. Moreover, permeability studies of the payload metabolites helped to evaluate potential by-stander effects of the ADCs. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile versus the corresponding isotype control ADCs. In depth in vitro and in vivo studies on the internalization and the metabolism allowed analysis of the intracellular fate of the payload metabolites and revealed the formation of the phosphoribosylated catabolite adducts in C4.4a expressing A549-cells. Taken together, we hereby present the development of a new NAMPT inhibitor-based payload class applicable for conjugation to diverse antibodies with a good technical profile and high potency and selectivity in antigen-positive cancer models. Citation Format: Niels Böhnke, Markus Berger, Nils Griebenow, Anja Giese, Judith Günther, Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Rudolf Beier, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Antje Rottmann, Michael Erkelenz, Naomi Barak, Ulf Bömer, Dominik Mumber, Bertolt Kreft, Lars Linden, Carl Friedrich Nising, Hilmar Weinmann. Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy [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 2907.

Abstract 1807: Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications

Abstract 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.

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

Total Synthesis of α- and β-Amanitin.

α-Amanitin and related amatoxins have been studied for more than six decades mostly by isolation from death cap mushrooms. The total synthesis, however, remained challenging due to unique structural features. α-Amanitin is a potent inhibitor of RNA polymerase II. Interrupting the basic transcription processes of eukaryotes leads to apoptosis of the cell. This unique mechanism makes the toxin an ideal payload for antibody-drug conjugates (ADCs). Only microgram quantities of toxins, when delivered selectively to tumor sites through conjugation to antibodies, are sufficient to eliminate malignant tumor cells of almost every origin. By solving the stereoselective access to dihydroxyisoleucine, a photochemical synthesis of the tryptathion precursor, solid-phase peptide synthesis, and macrolactamization we obtained a scalable synthetic route towards synthetic α-amanitin. This makes α-amanitin and derivatives now accessible for the development of new ADCs.

UPLC-based assay to assess the hydrophobicity of Antibody-Drug Conjugate (ADC) payloads

Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area.