Supplementary Figures S1-S5 from Antibody-Maytansinoid Conjugates Are Activated in Targeted Cancer Cells by Lysosomal Degradation and Linker-Dependent Intracellular Processing

Supplementary Table S1: Amounts of catabolites generated upon treatment of cancer cells with CX and SMCC ADCs.

Supplementary Methods (S1-S3). Supplementary Method S1: Synthesis of di-, tri-, tetra-glycyl, and valine-citrulline-glycine peptide linkers and catabolites. Supplementary Method S2: Test of in vitro cytotoxicity, cell-cycle inhibition, and bystander cytotoxic activity of ADCs. Supplementary Method S3: Catabolism of [3H]-labeled and non-radiolabeled CX and SMCC ADCs by cancer cells.

Supplementary Table 1 from Maytansine and Cellular Metabolites of Antibody-Maytansinoid Conjugates Strongly Suppress Microtubule Dynamics by Binding to Microtubules

Supplementary Tables 1-2, Figures 1-4 from Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance

Supplementary Figures (S1-S4). Supplementary Figure S1: In vitro cytotoxic activities of tetraglycyl (Gly4), triglycyl (Gly3), diglycyl (Gly2), and valine-citrulline-glycine (VCG)-linked anti-EGFR ADCs and a triglycyl-linked non-binding antibody ADC in PC-9, Ca9-22, HSC-2, H1975, A-431, and OSC-19 cells (3-4 DAR ADCs). Supplementary Figure S2: In vitro cytotoxic activities of anti-EpCAM CX and SMCC ADCs toward a low EpCAM antigen-expressing cell line, RPMI 8226 (~50,000 EpCAM per cell). The CX ADCs tested included a conjugate with a typical payload number (3.9 maytansinoid molecules per antibody molecule; 3.9 DAR) and conjugates with high payload numbers (8 and 9.6 DAR). The SMCC ADC had a typical payload number (4.3 DAR). Supplementary Figure S3: Binding-competition ELISA of catabolites of CX ADC (DM-CX1 and DM-CX2) and SMCC ADC (lysine-SMCC-DM1): inhibition by catabolites toward binding of anti-maytansine antibody to immobilized BSA-maytansinoid conjugate. Supplementary Figure S4: Test of bystander cytotoxic activity of anti-EGFR-CX-DM1 or SPDB-DM4 conjugate in mixed culture of EGFR-positive (Ca9-22) and EGFR-negative (Ramos) cells.

Abstract Folate Receptor alpha (FRα) is an attractive antibody drug conjugate (ADC) target due to its over expression in multiple epithelial malignancies including ovarian, endometrial, triple negative breast, and non-small cell lung cancer, with limited expression on normal tissues. IMGN853 (i.e., mirvetuximab soravtansine and M9346A-sulfo-SPDB-DM4), a FRα targeting ADC, is currently in phase III (MIRASOL) clinical evaluation as monotherapy in patients with platinum-resistant epithelial ovarian cancer with high levels of FRα expression. The MIRASOL study builds on the results from the prior randomized study, FORWARD I, which demonstrated that improved outcomes with IMGN853 correlated with FRα expression, with the strongest treatment effects for all efficacy endpoints in ovarian cancer patients with FRα-high disease (Moore, ESMO 2019). In order to address the unmet needs of additional patient populations, we sought to develop a next generation FRα-targeting ADC active against tumors with a broad range of FRα expression. Development of a new molecular entity with the desired antitumor properties included optimization of the antibody format and the linker-payload. The resulting lead ADC denoted IMGN151 comprises an asymmetric, bivalent, biparatopic antibody targeting two independent epitopes of FRα, linked to the highly potent maytansinoid derivative DM21 via a stable cleavable peptide linker. The average drug per antibody ratio is 3.5. The binding, internalization and processing of the biparatopic IMGN151 and the parent monospecific antibodies were compared using 3H-antibodies. In tumor cells with medium (JHOS4) and high (KB) FRα expression the biparatopic antibody boosted antibody binding events and processing by 100% and 170%, respectively. The plasma stability of IMGN151 was tested in a cynomolgus monkey pharmacokinetic study. The stable linker increased ADC half-life by 60 hours and conjugate exposure in vivo by 40%, as compared to IMGN853. IMGN151 activity was characterized against cell lines and xenograft models with a wide range of FRα expression and compared to IMGN853. In in vitro studies, both ADCs had similar activity against FRα-high KB cells; IMGN151 was up to 200 times more active against four FRα-medium cell lines. IMGN151 had also notably stronger bystander killing activity in a mixed culture of target-positive and negative cells. In vivo IMGN151 induced complete tumor regressions of human tumor xenograft models with high (KB, H-score of 300), medium (Igrov-1 and Ishikawa, H-score of 140 and 100, respectively) and low (Ov-90, H-score of 30) FRα expression. All tested doses were well tolerated with no body weight loss observed. With a novel biparatopic antibody and linker payload design, IMGN151 has shown potent antitumor activity against ovarian cancer models with a broad range of FRα expression, which warrants further development into the clinic for patients with tumors expressing FRα at a wide range of levels. Citation Format: Olga Ab, Laura M. Bartle, Leanne Lanieri, Jose F. Ponte, Qifeng Qiu, Surina Sikka, Juliet A. Costoplus, Wayne Deats, Nicholas C. Yoder, Wayne C. Widdison, Katherine Mucciarone, Kate Selvitelli, Ying Chen, Neeraj Kohli, Thomas Chittenden, Richard Gregory, Yulius Setiady, Eric H. Westin. IMGN151 - A next generation folate receptor alpha targeting antibody drug conjugate active against tumors with low, medium and high receptor expression [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 2890.

Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

A new type of antibody-drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC's linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate's cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC's cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.

Antibody-drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure-activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.

The maytansinoids are highly cytotoxic benzoansamacrolides that suppress microtubule dynamics to preferentially kill dividing versus quiescent cells, typically with concentrations that give 50% inhibition (IC50 values) in the sub-nanomolar range. Here, methods are described for preparing maytansinoids for incorporation into antibody–drug conjugates (ADCs) via coupling to antibodies that selectively target antigens on the surface of cancer cells. Maytansinoid ADCs kill antigen-positive cancer cells and, in some instances, release metabolites that can also kill other tumour cells, a mechanism termed “bystander killing”. In vitro and in vivo models comparing maytansinoid ADCs with different linker stabilities as well as those releasing metabolites that induce different degrees of bystander killing are presented. In vivo, non-specific cellular uptake and premature metabolite release are discussed as potential mechanisms leading to ADC systemic toxicity. In addition, the relative tolerance of slowly dividing cells to maytansinoids is presented as a potential tolerability advantage for this payload class compared with payloads that kill cells less discriminately.

ImmunoGen’s newest antibody-drug conjugate (ADC) design uses the novel maytansinoid linker-payload, DM21-C that bears a peptidase/protease cleavable linker. These ADCs show good bystander killing of proximal antigen negative cells, suggesting the generation of cell-permeable catabolites. The goal of this study was to identify the catabolites generated upon incubation in antigen-positive cancer cells (both cell pellet and media), in mouse plasma, as well as in in vitro catabolic systems. Upon incubation with a cysteine-conjugated DM21-C ADC, the small molecule fraction was extracted with an organic solvent and analyzed by high resolution mass spectrometry after chromatographic separation. Through comparison to reference standards, mass spectral signal extraction of possible products, and database search of unknown peaks, the potential in vitro catabolite products of DM21-C conjugate were identified. Information on linker-payload stability in these model systems, including plasma stability, in vitro metabolism of the ADC, and catabolic products that were both retained and effluxed from cancer cells was obtained. As a result, we identified DM51 (the thiol- resulting from self-immolation of the cleaved linker-payload) as a major catabolite of the DM21-C ADC. Citation Format: Janet Lau, Paulin Salomon, Kerstin Sinkevicius, Juliet Costoplus, Megan Fuller, Raymond Xu, Stuart Hicks, Ravi Chari, Wayne Widdison, Nicholas Yoder, Thomas Keating. LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 538.

Antibody drug conjugates (ADCs) are designed to target surface antigen(s) expressed at higher levels on cancer cells compared to normal cells. ADCs are internalized and the antibody component is subsequently degraded in catabolic vesicles to release cytotoxic metabolites that can kill the cell. Membrane permeable metabolites can also diffuse into and kill neighboring cells (also called bystander cells) via a mechanism known as bystander killing, resulting in greater tumor cell killing. Non-antigen-mediated mechanisms of antibody and ADC uptake are also known to occur, and the maximum tolerated dose for most ADCs is driven by target independent delivery. DM21 is a peptide-cleavable immolative maytansinoid payload that was designed to allow ADCs to efficiently release hydrophobic metabolites better than conjugates utilizing the disulfide linked maytansinoid DM4. DM21 ADCs typically have similar direct in vitro cytotoxicity as DM4 ADCs against antigen positive cells, but have much greater bystander killing activity in assays where antigen positive cells are mixed with antigen negative cells. To evaluate the toxicity of DM21 as an ADC, it was conjugated to the non-targeting, chimeric anti-soybean trypsin inhibitor antibody (chKTI), and administered to cynomolgus monkeys. Two groups of 5 male cynomolgus monkeys received a single intravenous dose of chKTI-DM21 at dose levels of 11 and 22 mg/kg (204 and 408 µg/kg DM21), while a concurrent group of 5 male monkeys was administered the formulation buffer as a control group. Three monkeys/group were sacrificed on Day 5 (terminal necropsy) to assess acute toxicity, and the remaining two monkeys/group were sacrificed on Day 29 (recovery necropsy) to assess the recovery, persistence, or progression of any effects. Toxicity was determined based upon clinical observations, body weights, ophthalmic examinations, and clinical and anatomic pathology. Plasma and serum samples were also collected to evaluate the toxicokinetic (TK) profile. chKTI-DM21 was well tolerated at both doses. There was no effect on body weight gain, and clinical observations were limited to reddened/darkened skin, scabbing, and soft/liquid feces. Effects noted on clinical pathology parameters included alterations in erythroid and leukocyte parameters, increased platelet counts and fibrinogen, and transient increases in ALT and AST without histopathologic correlates. The target organ noted at the terminal necropsy was the large intestine (cecum, colon, and rectum), but all findings were resolved by the recovery necropsy indicating reversibility. Toxicokinetic analysis of the samples showed that chKTI-DM21 has dose proportional exposure and apparent stability of the peptide linkage in cynomolgus plasma. In conclusion, DM21 is a promising maytansinoid payload with a high-degree of bystander activity and a favorable toxicity profile. Citation Format: Wayne Deats, Wayne Widdison, Juliet Costoplus, Bahar Matin, Nicole McBrine, Laura Bartle, Olga Ab, Richard Gregory, Jan Pinkas. Preclinical evaluation of DM21, a next-generation maytansinoid payload with a stable peptide linker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3898.

AIM Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. METHODOLOGY Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. CONCLUSION The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.

Abstract Folate receptor α (FRα) is an antigen that is overexpressed on the cell surface of solid tumors including ovarian cancer. The differential expression on cancer cells makes FRα an attractive target for antibody-drug conjugates (ADCs), and an ADC targeting FRα, Mirvetuximab soravtansine, has demonstrated promising activity and safety profiles in the clinic. Here, we employed a new linker (NL) to enhance the bystander activity of ADCs, which is the ability of ADCs to generate cell-permeable catabolites that can diffuse into and kill proximal cancer cells with little or no target expression. With the goal of improving the potency of anti-FRα ADC in solid tumors with heterogeneous FRα expression, we constructed the M9346A-NL-DM. M9346A-NL-DM is a conjugate of the tubulin-disrupting maytansinoid (DM), linked via a novel linker to M9346A, a humanized antibody that binds FRα with high affinity. After cellular uptake and catabolism of the ADC, the new linker undergoes lysosomal cleavage followed by self-immolation to generate free DM that can readily penetrate neighboring cancer cells. Correspondingly, M9346A-NL-DM showed enhanced bystander cytotoxic activity against proximal antigen-negative cells in vitro. In the xenograft tumor models in vivo, M9346A-NL-DM demonstrated enhanced efficacy against tumors with heterogeneous expression of FRα. Interestingly, M9346A-NL-DM also showed improved antitumor activity against a tumor model with homogeneous expression of FRα, possibly due to better tumor penetration of the cell-permeable catabolite. In summary, M9346A-NL-DM is a novel ADC with enhanced bystander activity and antitumor activity that can target tumors with heterogeneous expression of FRα. Citation Format: Qifeng Qiu, Rui Wu, Leanne Lanieri, Erin Maloney, Anna Skaletskaya, Shan Jin, Lintao Wang, Olga Ab, Joe Ponte, Yulius Setiady, Wayne Widdison, Thomas Keating, Ravi Chari, Richard Gregory, Erica Hong. Bystander activity and in vivo efficacy of a folate receptor α (FRα)-targeting antibody-drug conjugate with a novel peptide linker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 71. doi:10.1158/1538-7445.AM2017-71

Antibodies targeting surface antigens on cancer cells typically have progressively lower access to tumor cells that are further removed from blood vessels. Also, the antibody will not bind to cells in the tumor mass that do not express antigen, including stromal cells of the tumor, many of which reportedly aid in the survival or metastasis of cancer cells. ADCs can bind to antigen positive cancer cells, after which they are internalized and catabolized to release one or more cytotoxic metabolite(s) that can kill the targeted cell. Metabolites that are membrane permeable may also diffuse into and kill neighboring cells, often called bystander cells, that would normally be less accessible. The goal of this work was to design ADCs that would have increased bystander activity, which could result in greater killing of cancer cells and stromal cells in the tumor environment. We have prepared a new type of peptide-cleavable immolative ADC (PCI-ADC) that efficiently releases membrane permeable cytotoxic maytansinoid metabolites upon cleavage of the peptide linker, followed by immolation. Several PCI-ADCs were prepared that release metabolites having different degrees of hydrophobicity. As the hydrophobicity of the metabolite increased, the PCI-ADCs’ bystander activity also increased. The lead PCI-ADC generally displayed a similar degree of in vitro cytotoxicity as maytansinoid ADCs that utilize disulfide linkers, however the PCI-ADC induced significantly more bystander killing. In mice bearing large tumor xenografts (250 mm3) or tumor xenografts that express the target antigen heterogeneously, PCI-ADCs were found to be more efficacious than maytansinoid ADCs that use disulfide linkers, as well as our recently reported peptide-para-anilino maytansinoid ADCs. The nature of the amino acid residues in the peptide linker of the PCI-ADC was also altered so that the tolerability of the ADCs in mice could be increased without impeding efficacy. In conclusion, we have developed a promising new type of maytansinoid ADC, one that provides a high degree of bystander killing, improved activity in homogeneous and heterogeneous tumor models in vivo, and has a different mechanism of metabolite release than current maytansinoid based ADCs. Citation Format: Wayne C. Widdison, Juliet A. Costoplus, Jose F. Ponte, Leanne Lanieri, Yulius Setiady, Ling Dong, Anna Skaletskaya, Rui Wu, Qifeng Qiu, Yelena Kovtun, Ravi V. Chari. Peptide-cleavable maytansinoid (ADCs) induce high bystander killing leading to improved anti-tumor activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2186. doi:10.1158/1538-7445.AM2017-2186

AbstractA triglycyl peptide linker (CX) was designed for use in antibody–drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5–100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo. Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311–20. ©2016 AACR.

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.