Heterogeneous Antibody-drug Conjugates Research Articles

Site-specific antibody-drug conjugates with variable drug-to-antibody-ratios for AML therapy

Random conjugations of chemotherapeutics to monoclonal antibodies result in heterogeneous antibody-drug conjugates (ADCs) with suboptimal pharmacological properties. We recently developed a new technology for facile generation of homogeneous ADCs by harnessing human CD38 catalytic domain and its dinucleotide-derived covalent inhibitor, termed ADP-ribosyl cyclase-enabled ADCs (ARC-ADCs). Herein we advance this technology by designing and synthesizing ARC-ADCs with customizable drug-to-antibody ratios (DARs). Through varying numbers and locations of CD38 fused to an antibody targeting human C-type lectin-like molecule-1 (hCLL-1), ARC-ADCs featuring DARs of 2 and 4 were rapidly generated via a single step with cytotoxic monomethyl auristatin F (MMAF) as payloads. In contrast to anti-hCLL-1 ARC-ADC carrying 2 drug molecules, anti-hCLL-1 ARC-ADC with a DAR of 4 shows highly potent activity in killing hCLL-1-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. This work provides novel ADC candidates for combating AML and supports ARC-ADC as a general and versatile approach for producing site-specific ADCs with defined DARs.

Antibody Glycoengineering and Homogeneous Antibody-Drug Conjugate Preparation.

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals in which cytotoxic agents are conjugated to monoclonal antibodies (mAbs), allowing targeted drug delivery. Present heterogeneous ADCs (conjugated in random variable positions) suffered from issues of stability, reproducibility, efficacy, etc. Recent advances have led to the development of homogeneous ADC preparations by site-specific conjugation, allowing the control of the drug-to-antibody ratio. These approaches have increased the therapeutic window, efficacy, and batch-to-batch consistency of the ADC preparations. Antibodies carry a pair of heterogeneous N-glycans in the Fc regions, which are critical for antibody function. Drug conjugation through glycoengineering has been achieved with different approaches, including the use of endo-β-N-acetylglucosaminidase (ENGases) and monosaccharyl transferase mutants. In this article, we summarize different glycoengineering approaches for antibody-drug conjugation, and discuss their advantages for the development of next-generation homogeneous ADCs.

Mechanistic Modeling of Intra-Tumor Spatial Distribution of Antibody-Drug Conjugates: Insights into Dosing Strategies in Oncology.

Antibody drug conjugates (ADCs) provide targeted delivery of cytotoxic agents directly inside tumor cells. However, many ADCs targeting solid tumors have exhibited limited clinical efficacy, in part, due to insufficient penetration within tumors. To better understand the relationship between ADC tumor penetration and efficacy, previously applied Krogh cylinder models that explore tumor growth dynamics following ADC administration in preclinical species were expanded to a clinical framework by integrating clinical pharmacokinetics, tumor penetration, and tumor growth inhibition. The objective of this framework is to link ADC tumor penetration and distribution to clinical efficacy. The model was validated by comparing virtual patient population simulations to observed overall response rates from trastuzumab‐DM1 treated patients with metastatic breast cancer. To capture clinical outcomes, we expanded upon previous Krogh cylinder models to include the additional mechanism of heterogeneous tumor growth inhibition spatially across the tumor. This expansion mechanistically captures clinical response rates by describing heterogeneous ADC binding and tumor cell killing; high binding and tumor cell death close to capillaries vs. low binding, and high tumor cell proliferation far from capillaries. Sensitivity analyses suggest that clinical efficacy could be optimized through dose fractionation, and that clinical efficacy is primarily dependent on the ADC‐target affinity, payload potency, and tumor growth rate. This work offers a mechanistic basis to predict and optimize ADC clinical efficacy for solid tumors, allowing dosing strategy optimization to improve patient outcomes.

Abstract 4054: Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates

Abstract There are currently 5 antibody drug conjugates (ADC) on the market and over 80 other ADCs evaluated in the clinic. The major drawbacks of the commonly employed bioconjugation methods based on nucleophilic coupling to lysine and cysteine are the non-selectivity and the in vivo instability. These limitations may be overcome by using alternative bioconjugation chemistries. To avoid the need for antibody engineering, disulfide-bridging linkers have recently emerged for the generation of homogeneous and stable ADCs. Here, we report a novel maleimide-based linker for site-selective bioconjugation of drugs via covalent re-bridging of reduced interchain disulfide bonds on native IgG1 antibodies. After optimization of the maleimide and linker parts, as well as of the coupling process, the sulfomaleimide re-bridging (SMR) platform was used to conjugate tubulin and DNA-targeting cytotoxic agents to model antibodies. Compared to conventional maleimide (MC)-ADCs, the SMR-ADCs were highly homogeneous by HIC-HPLC and LC-MS analysis, with DAR 4 species as the major product, and highly stable in vitro, with no deconjugation observed after incubation in different types of serum. Then, IGF-1R-targeting SMR-ADCs were synthesized with Auristatin and PNU-159682 derivatives and evaluated in in vitro and in vivo models. These ADCs exhibited potent cytotoxicity and anti-tumoral activity in 2+ and 3+ IGF-1R-expressing models at different doses (0.3 to 3 mg/kg) and schedules of administration (Q1W to Q3W, i.v.). Significant differences in in vivo efficacy were observed between SMR- and MC-ADCS constructed with Auristatin derivatives. While they were comparable in a 3+ xenograft model (MCF-7), complete regressions were indeed obtained in the 2+ CaOV3 and NCI-H2122 models after single injections of the Auristatin-based SMR-ADC, whereas the corresponding MC-ADC induced only partial responses in the same conditions. To compare their in vivo stability and tolerability, these ADCs were injected in mice at doses of 3-30 mg/kg (Q2W, i.v.). No mortality and clinical signs were observed. Body weight evolution was not affected whatever the treatment. From a histopathological perspective, no difference between both constructs could be highlighted at this point. Toxicokinetic analyses carried out from satellite animals demonstrated that there was no major difference between both ADCs and that animal exposure increased proportionally with the dose. Nevertheless, serum exposure to the payload was 2-to-7-fold higher for MC-ADC than for SMR-ADC. Our results demonstrate that the SMR platform can be applied to native IgG1 antibodies, without antibody engineering, to yield highly homogeneous and stable ADCs via cysteine cross-linking. The resulting ADCs demonstrate a therapeutic index benefit compared to analogous conventional heterogeneous ADCs. Citation Format: Jean-Francois Haeuw, Frederic Marion, Cyrille Dreyfus, Barbara Akla, Laurence Zanna, Marie-Claire Janin, Charlotte Beau-Larvor, Marion Millet, Martine Farrie, Alain Batton, Celine Thuilliez, Alain Beck, Sarah Cianferani, Thierry Clerc, Philippe Maillos, Nathalie Corvaia. Development of a new site-specific sulfomaleimide rebridging platform for the generation of homogeneous, stable, potent and safe antibody drug conjugates [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 4054.

Application of Native Ion Exchange Mass Spectrometry to Intact and Subunit Analysis of Site-Specific Antibody-Drug Conjugates Produced by AJICAP First Generation Technology.

Antibody-drug conjugates (ADCs) are at the forefront of the next generation of oncology biopharmaceuticals. Conventional ADCs involve stochastic conjugation of the antibody to a cytotoxic drug, creating a highly heterogeneous product. The resulting stochastic distribution often leads to a narrow therapeutic index and makes it difficult to analyze the composition of heterogeneous ADCs. With the goal of overcoming these issues, we developed a site-specific conjugation technology, named AJICAP, for production of low heterogeneity ADCs. For analysis of these site-specific ADCs, we report herein strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS). Retention time reproducibility after SCX column equilibration enabled monitoring of important changes in product quality. SCX-UV-MS performed with MS-compatible mobile phases was conducted for intact native ADC analysis, allowing drug-antibody ratio characterization and charge variant characterization in single analysis. Furthermore, subunit analysis of the site-specific ADCs by native SCX-UV-MS confirmed the Fc site selectivity of ADCs generated by AJICAP conjugation.

An Agent-Based Systems Pharmacology Model of the Antibody-Drug Conjugate Kadcyla to Predict Efficacy of Different Dosing Regimens.

The pharmaceutical industry has invested significantly in antibody-drug conjugates (ADCs) with five FDA-approved therapies and several more showing promise in late-stage clinical trials. The FDA-approved therapeutic Kadcyla (ado-trastuzumab emtansine or T-DM1) can extend the survival of patients with tumors overexpressing HER2. However, tumor histology shows that most T-DM1 localizes perivascularly, but coadministration with its unconjugated form (trastuzumab) improves penetration of the ADC into the tumor and subsequent treatment efficacy. ADC dosing schedule, e.g., dose fractionation, has also been shown to improve tolerability. However, it is still not clear how coadministration with carrier doses impacts efficacy in terms of receptor expression, dosing regimens, and payload potency. Here, we develop a hybrid agent-based model (ABM) to capture ADC and/or antibody delivery and to predict tumor killing and growth kinetics. The results indicate that a carrier dose improves efficacy when the increased number of cells targeted by the ADC outweighs the reduced fractional killing of the targeted cells. The threshold number of payloads per cell required for killing plays a pivotal role in defining this cutoff. Likewise, fractionated dosing lowers ADC efficacy due to lower tissue penetration from a reduced maximum plasma concentration. It is only beneficial when an increase in tolerability from fractionation allows a higher ADC/payload dose that more than compensates for the loss in efficacy from fractionation. Overall, the multiscale model enables detailed depictions of heterogeneous ADC delivery, cancer cell death, and tumor growth to show how carrier dosing impacts efficacy to design the most efficacious regimen.

Applications of SNAP-tag technology in skin cancer therapy.

BackgroundCancer treatment in the 21st century has seen immense advances in optical imaging and immunotherapy. Significant progress has been made in the bioengineering and production of immunoconjugates to achieve the goal of specifically targeting tumors.DiscussionIn the 21st century, antibody drug conjugates (ADCs) have been the focus of immunotherapeutic strategies in cancer. ADCs combine the unique targeting of monoclonal antibodies (mAbs) with the cancer killing ability of cytotoxic drugs. However, due to random conjugation methods of drug to antibody, ADCs are associated with poor antigen specificity and low cytotoxicity, resulting in a drug to antibody ratio (DAR) >1. This means that the cytotoxic drugs in ADCs are conjugated randomly to antibodies, by cysteine or lysine residues. This generates heterogeneous ADC populations with 0 to 8 drugs per an antibody, each with distinct pharmacokinetic, efficacy, and toxicity properties. Additionally, heterogeneity is created not only by different antibody to ligand ratios but also by different sites of conjugation. Hence, much effort has been made to find and establish antibody conjugation strategies that enable us to better control stoichiometry and site‐specificity. This includes utilizing protein self‐labeling tags as fusion partners to the original protein. Site‐specific conjugation is a significant characteristic of these engineered proteins. SNAP‐tag is one such engineered self‐labeling protein tag shown to have promising potential in cancer treatment. The SNAP‐tag is fused to an antibody of choice and covalently reacts specifically in a 1:1 ratio with benzylguanine (BG) substrates, eg, fluorophores or photosensitizers, to target skin cancer. This makes SNAP‐tag a versatile technique in optical imaging and photoimmunotherapy of skin cancer.ConclusionSNAP‐tag technology has the potential to contribute greatly to a broad range of molecular oncological applications because it combines efficacious tumor targeting, minimized local and systemic toxicity, and noninvasive assessment of diagnostic/prognostic molecular biomarkers of cancer.

Site-Specific Conjugation of Thiol-Reactive Cytotoxic Agents to Nonnative Cysteines of Engineered Monoclonal Antibodies.

Antibody-drug conjugates (ADCs) have been proven to be a successful therapeutic concept, allowing targeted delivery of highly potent active pharmaceutical ingredients (HPAPIs) selectively to tumor tissue. So far, HPAPIs have been mainly attached to the antibody via a chemical reaction of the payload with lysine or cysteine side chains of the antibody backbone. However, these conventional conjugation technologies result in formation of rather heterogeneous products with undesired properties. To overcome the limitations of heterogeneous ADC mixtures, several site-specific conjugation technologies have been developed over the last years. Originally pioneered by scientist from Genentech with their work on THIOMABs, several engineered cysteine mAb ADCs (ECM-ADCs) are now investigated in clinical trials. Here, we describe in detail how to engineer additional cysteines into antibodies and efficiently use them as highly site-specific conjugation sites for HPAPIs.

Abstract 75: Comparison of site-specific and lysine-linked indolino-benzodiazepine antibody-drug conjugates (ADCs)

Abstract ADCs are a promising modality for cancer therapy enabled by chemical conjugation of potent cytotoxic compounds to monoclonal antibodies. While many ADCs in clinical evaluation employ heterogeneous conjugation chemistries where the payload is linked through lysine or endogenous cysteine residues, there has recently been considerable interest in site-specific conjugation. ADCs prepared using site-specific methods are believed to have a wider therapeutic index compared to heterogeneous ADCs. We have previously shown that site-specific ADCs incorporating the maytansinoid-based tubulin inhibitor DM1 were less efficacious in vivo when compared to analogous lysine-linked conjugates (Yoder et al AACR 2015 Abstract #545). More recently, we presented results from the evaluation of 2.5-3.0 drug-to-antibody ratio (DAR) heterogeneous lysine-linked and 2 DAR site-specific ADCs using antibodies with engineered cysteines at position 442 in the heavy chains (known as CYSMABTM). These ADCs used the peptide-linked indolino-benzodiazepine DNA alkylator DGN549 (also known as IGN-P1) as the effector. Unlike the DM1 case, site-specific DGN549 ADCs were at least as active in vivo when compared to lysine-linked ADCs (Yoder et al AACR 2016 Abstract #2960). We have made further pharmacological comparisons between CYSMAB and lysine-linked DGN549 ADCs at matched payload doses using two different antibodies targeting distinct cell surface receptors. In the case of mAb1, CYSMAB and lysine-linked ADCs were comparably active. For mAb2, the CYSMAB ADC was more active than the lysine-linked ADC in some models and similarly active in others. The mAb1 CYSMAB ADC exhibited a significantly higher maximum tolerated dose (MTD) compared to the lysine-linked ADC. In contrast, the MTDs of the mAb2 ADCs were similar. However, the mAb2 CYSMAB conjugate was better tolerated in terms of median lethal dose. The mechanism for the improved tolerability of the mAb1 CYSMAB conjugate is not apparent. In an effort to understand whether it is a consequence of conjugation chemistry or DAR, we compared the tolerability of the mAb1 CYSMAB conjugate to that of a ~4 DAR site-specific analog and found that both factors contribute. To determine if our observations can be rationalized in terms of in vivo disposition, we compared the pharmacokinetics of mAb1 CYSMAB and lysine-linked ADCs at matched antibody doses. Intriguingly, the CYSMAB ADC showed slightly greater exposure. These results, along with our previous work on site-specific DM1 ADCs, indicate that in some cases site-specific conjugation can lead to improved efficacy and tolerability. However, generalizations cannot be made across different combinations of antibody, linker, and payload. We conclude that the advantages and disadvantages of site-specific conjugation should be carefully considered for every candidate ADC. Citation Format: Chen Bai, Nicholas C. Yoder, Alan Wilhelm, Sharlene Adams, Kathleen Whiteman, Jenny Lee, Katie O'Callaghan, Erin Maloney, Manami Shizuka, Yelena Kovtun, Thomas A. Keating. Comparison of site-specific and lysine-linked indolino-benzodiazepine antibody-drug conjugates (ADCs) [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 75. doi:10.1158/1538-7445.AM2017-75

RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System

Antibody drug conjugates (ADCs) harness the target specificity of a monoclonal antibody (mAb) and the high cytotoxicity of a small molecule, enabling improved delivery of a potent antitumor agent compared to traditional chemotherapy for cancer therapy. Only two ADCs have been marketed, both of which are produced via nonsite-specific conjugation of the cytotoxic drug to either interchain cysteine (Adcetris) or lysine (Kadcyla). A growing body of evidence suggests that site-specific ADCs, because of their payload homogeneity, will improve pharmacokinetics and have wider therapeutic windows when compared to heterogeneous ADCs. Previously, we have demonstrated the use of a cell free expression system (Xpress CF+) for rapid production of site-specific ADCs. Here we report the generation of a variety of ADCs via conjugation between a site-specific incorporated non-natural amino acid (nnAA), para-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene glycol)4 (DBCO-(PEG)4) linked payloads using this platform. We developed a reversed phase HPLC method for drug to antibody ratio (DAR) characterization, which is applicable to both reduced and intact ADCs. We demonstrate that these ADCs are of near complete conjugation and exhibit potent cell killing activity and in vitro plasma stability. Moreover, we generated an ADC conjugated at both light and heavy chains, resulting in a DAR close to 4. With the increased number of payloads, the resultant DAR 4 ADC is potentially more efficacious than its DAR 2 counterparts, which could further improve its therapeutic index. These studies have demonstrated the competency of Xpress CF+ for site-specific ADC production and improved our understanding of the site-specific ADCs in general.

ChemInform Abstract: Processes for Constructing Homogeneous Antibody Drug Conjugates

AbstractReview: 91 refs.

Processes for Constructing Homogeneous Antibody Drug Conjugates

Antibody drug conjugates (ADCs) are synthesized by conjugating a cytotoxic drug or “payload” to a monoclonal antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react with lysines or cysteines on the antibody surface. A typical antibody contains over 60 lysines and up to 12 cysteines as potential conjugation sites. The desired DAR (drugs/antibody ratio) depends on a number of different factors and ranges from two to eight drugs/antibody. The discrepancy between the number of potential conjugation sites and the desired DAR, combined with use of conventional conjugation methods that are not site-specific, results in heterogeneous ADCs that vary in both DAR and conjugation sites. Heterogeneous ADCs contain significant fractions with suboptimal DARs that are known to possess undesired pharmacological properties. As a result, new methods for synthesizing homogeneous ADCs have been developed in order to increase their potential as therapeutic agents. This article will review re...

Hydrolytically Stable Site-Specific Conjugation at the N-Terminus of an Engineered Antibody.

Antibody-drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combine the target specificity of antibodies with the killing activity of anticancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogeneous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the N-terminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition.

Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases.

The therapeutic cargo molecules conjugated to a specific site on a monoclonal antibody (mAb), called antibody-drug conjugates (ADCs), are becoming powerful tools in cancer treatment. Generally, the cargo molecules conjugate at the cysteine or lysine residue of the mAb, which generally results in a highly heterogeneous ADC. Therapeutic cargo molecules need to be conjugated in a site-specific manner to the mAb so that the bioefficacy of these molecules is not compromised. The mAb (IgG1) are N-glycosylated at the conserved residue Asn(297), which is present in each heavy chain of the IgG1, near the CH2 domain of the Fc fragment. The mutant or wild-type glycosyltransferases transfer sugars with a chemical handle to the glycan molecule of IgG1, making the site-specific linking of cargo molecules possible via the chemical handle, and thus making the process an invaluable technique for the production of homogeneous ADCs.

Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody–Drug Conjugates

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.