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

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

ValCitGlyPro-dexamethasone antibody conjugates selectively suppress the activation of human monocytes.

Glucocorticoids (GCs) are effective in treating autoimmune and inflammatory disorders but come with significant side effects, many of which are mediated by non-immunological cells. Therefore, there is rapidly growing interest in using antibody drug conjugate (ADC) technology to deliver GCs specifically to immune cells, thereby minimizing off-target side effects. Herein, we report the study of anti-CD11a, anti-CD38, and anti-TNFα ADCs to deliver dexamethasone to monocytes. We found that anti-CD11a and anti-CD38 were rapidly internalized by monocytes, while uptake of anti-TNFα depended on pre-activation with LPS. Using these antibodies were attached to a novel linker system, ValCitGlyPro-Dex (VCGP-Dex), that efficiently released dexamethasone upon lysosomal catabolism. This linker relies on lysosomal cathepsins to cleave after the ValCit sequence, thereby releasing a GlyPro-Dex species that undergoes rapid self-immolation to form dexamethasone. The resulting monocyte-targeting ADCs bearing this linker payload effectively suppressed LPS-induced NFκB activation and cytokine release in both a monocytic cell line (THP1) and in human PBMCs. Anti-TNFα_VCGP-Dex and anti-CD38_VCGP-Dex were particularly effective, suppressing ∼60-80% of LPS-induced IL-6 release from PBMCs at 3-10 µg mL-1 concentrations. In contrast, the corresponding isotype control ADC (anti-RSV) and the corresponding naked antibodies (anti-CD38 and anti-TNFα) resulted in only modest suppression (0-30%) of LPS-induced IL-6. Taken together, these results provide further evidence of the ability of glucocorticoid-ADCs to selectively suppress immune responses, and highlight the potential of two targets (CD38 and TNFα) for the development of novel immune-suppressing ADCs.

Evaluation of an ester-linked immunosuppressive payload: A case study in understanding the stability and cleavability of ester-containing ADC linkers.

In spite of their value in prodrug applications, the use of esters in antibody-drug-conjugate (ADC) payloads and linkers has generally been avoided due to the ubiquitous and promiscuous nature of human esterases. ADCs generally have a long circulating half life (3-7 days) that makes them susceptible to esterase-mediated metabolism. Moreover, it is largely unclear whether lysosomal and cytosolic esterases cleave ester-containing linkers upon ADC internalization. Due to our interest in the targeted delivery of immune-modulators, our team has recently prepared a series of ester-linked dexamethasone ADCs. Herein, we report our studies of the functional activity of these ADCs, with a particular focus on their catabolism in various biological milieu. We found that esters are selectively but inefficiently cleaved upon cellular uptake, likely by cytosolic esterases. Lysosomal catabolism studies indicate that, in spite of the strong proteolytic activity, very little cleavage of ester-containing linkers occurs in the lysosome. However, ADCs bearing the ester-linked payloads are active in various immune-suppressive assays, suggesting that cytosolic cleavage is taking place. This was confirmed through LCMS quantitation of the payload following cell lysis. Finally, the stability of the ester linkage was evaluated in mouse and human plasma. We found, similar to other reports, there is a significant site-dependence on the cleavage. Esters attached at highly exposed sites, such as 443C, were rapidly cleaved in plasma while esters at more hindered sites, such at 334C, were not. Together, these results help to unravel the complexities of ester-incorporation into ADC linkers and pave a path forward for their utility in ADC applications.

Chemical and Physical Stability of an Admixture Containing Cefepime and Vancomycin in Lactated Ringer Solution.

Purpose: To evaluate the chemical and physical stability of an admixture containing cefepime and vancomycin in a single volume of lactated Ringer solution at refrigerated temperatures. Methods: Cefepime 2000 mg and vancomycin 1000 mg were, respectively, reconstituted with 10 and 20 mL of sterile water for injection (SWFI) per manufacturer instructions. This resulted in cefepime and vancomycin concentrations of 200 and 50 mg/mL, respectively. The resulting cefepime and vancomycin solutions at 10 and 20 mL, respectively, were drawn up and injected into 1000 mL lactated Ringer solution. Aliquot samples were obtained on days 0 to 9, visually inspected for gross incompatibility, and then stored at -80°C. Samples were thawed on the day of the analysis and run through ultraperformance liquid chromatography. Area under the concentration-time curve (AUC) on each day was compared with baseline AUC values. Chemical stability was defined as an AUC more than 93% of the baseline value. Results: No evidence of gross physical incompatibility was observed by visual inspection. Cefepime and vancomycin replicants were more than 94.5% and 98% of baseline AUC values. Therefore, all sample replicants were found to be more than 93% of their baseline AUC value. Conclusion: An admixture containing cefepime 2000 mg and vancomycin 1000 mg in 1000 mL lactated Ringer solution appears to be chemically and physically stable at refrigerated temperatures for up to 9 days.

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

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

Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering.

Site-specific conjugation technology frequently relies on antibody engineering to incorporate rare or non-natural amino acids into the primary sequence of the protein. However, when the primary sequence is unknown or when antibody engineering is not feasible, there are very limited options for site-specific protein modification. We have developed a transglutaminase-mediated conjugation that incorporates a thiol at a "privileged" location on deglycosylated antibodies (Q295). Perhaps surprisingly, this conjugation employs a reported transglutaminase inhibitor, cystamine, as the key enzyme substrate. The chemical incorporation of a thiol at the Q295 site allows for the site-specific attachment of a plethora of commonly used and commercially available payloads via maleimide chemistry. Herein, we demonstrate the utility of this method by comparing the conjugatability, plasma stability, and in vitro potency of these site-specific antibody-drug conjugates (ADCs) with analogous endogenous cysteine conjugates. Cytotoxic ADCs prepared using this methodology are shown to exhibit comparable in vitro efficacy to stochastic cysteine conjugates while displaying dramatically improved plasma stability and conjugatability. In particular, we note that this technique appears to be useful for the incorporation of highly hydrophobic linker payloads without the addition of PEG modifiers. We postulate a possible mechanism for this feature by probing the local environment of the Q295 site with two fluorescent probes that are known to be sensitive to the local hydrophobic environment. In summary, we describe a highly practical method for the site-specific conjugation of genetically nonengineered antibodies, which results in plasma-stable ADCs with low intrinsic hydrophobicity. We believe that this technology will find broad utility in the ADC community.