Stability Of Antibody-drug Conjugates Research Articles

The Journey of Antibody-Drug Conjugates: Lessons Learned from 40 Years of Development.

Antibody-drug conjugates (ADC) represent one of the most rapidly expanding treatment modalities in oncology, with 11 ADCs approved by the FDA and more than 210 currently being tested in clinical trials. Spanning over 40 years, ADC clinical development has enhanced our understanding of the multifaceted mechanisms of action for this class of therapeutics. In this article, we discuss key insights into the toxicity, efficacy, stability, distribution, and fate of ADCs. Furthermore, we highlight ongoing challenges related to their clinical optimization, the development of rational sequencing strategies, and the identification of predictive biomarkers. Significance: The development and utilization of ADCs have allowed for relevant improvements in the prognosis of multiple cancer types. Concomitantly, the rise of ADCs in oncology has produced several challenges, including the prediction of their activity, their utilization in sequence, and minimization of their side effects, that still too often resemble those of the cytotoxic molecule that they carry. In this review, we retrace 40 years of development in the field of ADCs and delve deep into the mechanisms of action of these complex therapeutics and reasons behind the many achievements and failures observed in the field to date.

Site-Specific Stability Evaluation of Antibody-Drug Conjugate in Serum Using a Validated Liquid Chromatography-Mass Spectrometry Method.

Antibody-drug conjugate (ADC) consists of engineered antibodies and cytotoxic drugs linked via a chemical linker, and the stability of ADC plays a crucial role in ensuring its safety and efficacy. The stability of ADC is closely related to the conjugation site; however, no method has been developed to assess the stability of different conjugation sites due to the low response of conjugated peptides. In this study, an integrated strategy was developed and validated to assess the stability of different conjugation sites on ADC in serum. Initial identification of the conjugated peptides of the model drug ado-trastuzumab emtansine (T-DM1) was achieved by the proteomic method. Subsequently, a semiquantitative method for conjugated peptides was established in liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (LC-QTRAP-MS/MS) based on the qualitative information. The pretreatment method of the serum sample was optimized to reduce matrix interference. The method was then validated and applied to evaluate the stability of the conjugation sites on T-DM1. The results highlighted differences in stability among the different conjugation sites on T-DM1. This is the first study to assess the stability of different conjugation sites on the ADC in serum, which will be helpful for the design and screening of ADCs in the early stages of development.

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

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

Over the past two decades, antibody-drug conjugates (ADCs) have emerged as a highly effective drug delivery technology. ADCs utilise a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types. 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. Legumain is abundantly present in lysosomes and is known to play a role in tumour 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. 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 tumours, 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.

Evaluating the Potential of Cyclodextrins in Reducing Aggregation of Antibody–Drug Conjugates with Different Payloads

Cyclodextrins (CDs) are versatile agents used to solubilize small drugs and stabilize proteins. This dual functionality may be particularly beneficial for antibody–drug conjugates (ADCs), as CDs may “mask” the hydrophobicity of the drug payloads. In this study, we explored the effect of CDs on the physical stability of ADCs composed of the same antibody but with different payloads (maytansinoid, auristatin, and fluorophore payloads). The aggregation of ADCs was evaluated under shaking stress conditions and elevated temperatures using size-exclusion chromatography, turbidity, and backgrounded membrane imaging. Our results showed that hydroxypropyl-(HP)-CDs effectively stabilized all ADCs during shaking stress, with increasing stabilization in the order of HPαCD < HPγCD < HPβCD at concentrations of 7.5 mM and (near) complete stabilization at 75 mM. Native CDs without surface activity also stabilized certain ADCs, although less effectively than HP-CDs under agitation stress. During quiescent incubation, the HP-CD effects were small for most ADCs. However, for an ADC with a fluorophore payload that rapidly aggregated after conjugation, HPγCD substantially reduced aggregate levels, in line with fluorescence data supporting CD–ADC interactions. In contrast, sulfobutylether-β-CD (SBEβCD) increased the aggregation rates in all ADCs under all stress conditions. In conclusion, this study highlights the potential of appropriate CD formulations to improve the physical stability of ADCs.

“Negative” Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody–Drug Conjugates

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Antibody drug conjugates as targeted cancer therapy: past development, present challenges and future opportunities.

Antibody drug conjugates (ADCs) are promising cancer therapeutics with minimal toxicity as compared to small cytotoxic molecules alone and have shown the evidence to overcome resistance against tumor and prevent relapse of cancer. The ADC has a potential to change the paradigm of cancer chemotherapeutic treatment. At present, 13 ADCs have been approved by USFDA for the treatment of various types of solid tumor and haematological malignancies. This review covers the three structural components of an ADC-antibody, linker, and cytotoxic payload-along with their respective structure, chemistry, mechanism of action, and influence on the activity of ADCs. It covers comprehensive insight on structural role of linker towards efficacy, stability & toxicity of ADCs, different types of linkers & various conjugation techniques. A brief overview of various analytical techniques used for the qualitative and quantitative analysis of ADC is summarized. The current challenges of ADCs, such as heterogeneity, bystander effect, protein aggregation, inefficient internalization or poor penetration into tumor cells, narrow therapeutic index, emergence of resistance, etc., are outlined along with recent advances and future opportunities for the development of more promising next-generation ADCs.

Abstract 1538: Revisiting the dogma of antibody drug conjugates (ADCs): Emerging data challenge the benefit of linker stability and the primacy of payload delivery

Abstract Antibody drug conjugates (ADCs) are an effective class of cancer therapeutics which have become more prominent after eight FDA approvals in the past three years. ADCs are envisioned to enable the selective delivery of a potent drug to cells that express the antibody target while sparing normal tissues, and thus to improve the therapeutic window by both increasing the maximum tolerated dose (MTD) and decreasing the minimum efficacious dose (MED) of the conjugated drug. Mounting clinical evidence does not support this simplified paradigm [1]. Herein we focus on three prominent issues in ADC development: 1. Alternative mechanisms over and above direct tumor targeting are likely to contribute to ADC therapeutic benefits in the clinic [2]. The responses to certain ADCs in patients with different levels of target antigen expression reveal some target-independent antitumor effects, suggesting the possibility that circulating payload may contribute to efficacy. 2. The preclinical optimization of ADCs is based on efficacy and tolerability in non-human animal studies. Fundamental differences in biology and translatability between species potentially overemphasize the importance of linker stability. 3. Clinical data, although limited, does not support the idea that increased ADC stability drives substantial therapeutic benefit. None of the 13 approved ADCs are completely ‘stable’ in circulation, and more stable drug-linkers have been associated with alternative toxicity profiles and no significant improvement in MTD. Novel meta-analyses and case studies assembled and integrated from &amp;gt;40 approved or clinically active ADCs and &amp;gt;70 discontinued ADCs will be presented in support of these three perspectives. The ADCs included in the analysis differ in terms of payload class, linker type, conjugation chemistry, drug-to-antibody ratio, and antibody target. Gaining a better understanding of the mechanism(s) of action of effective ADCs and the interrelationship between ADC structural components and their pharmacokinetics and pharmacodynamics is critical to inform the next generation of ADCs.

Stability assessment of Polatuzumab vedotin and Brentuximab vedotin using different analytical techniques

Antibody-drug conjugates (ADC) are considered to be fast-growing innovative biopharmaceuticals. The science used for conjugating potent cytotoxic payload to the targeted monoclonal antibody through a chemical linker has played a great value in the area of oncology treatment. In this study; Polatuzumab vedotin (POLA) and Brentuximab vedotin (SGN-35) were subjected to various stress conditions enclosing different pH, thermal stress, agitation, and successive cycles of freeze and thaw in order to produce potential degradation by-products and guarantee the appropriateness of the applied testing protocol. Different analytical techniques were established and validated to be used in the quantitation of the degraded products from different perspectives. The formation of ADC aggregates and fragments was monitored using SE-HPLC as well as dynamic light scattering (DLS). The drug antibody ratio (DAR) and ADC conjugation profile were determined using hydrophobic interaction chromatography (HIC-HPLC). In addition to performing a statistical interpretation of HIC-HPLC results by principal component analysis (PCA) to explicate the obtained data. Also, the quantity of the unconjugated toxic drug was quantified using RP-HPLC. Testing the binding activity of ADC to their target receptor ADC was conducted using ELISA. Results presented that used assay protocol had worked as a complementary design for characterization and stability assessment of the used ADC. Variances in the stability profile of both products were observed which could be attributed to the usage of different formulation buffers. This highlighted the importance of using multiple techniques for the assessment of the quality attributes of such sophisticated products. The analytical assay protocol should be used for the evaluation of the quality and stability of several ADC.

IKS03, a Next Generation CD19-Targeted Antibody Drug Conjugate, Shows Potent Activity in Preclinical Models of Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma (NHL). Although many patients respond to frontline chemoimmunotherapy, and treatment options for patients with relapsed or refractory DLBCL have improved with the success of CAR T cell therapies, clinical outcome for many patients is still suboptimal. CD19 is widely expressed in DLBCL tumor cells, with normal tissue expression limited to B-cells, making it an attractive target for targeted therapies including antibody drug conjugates (ADC). IKS03 is a CD19-targeting ADC with a PBD dimer pro-drug payload that induces DNA crosslinking and blocks DNA replication ultimately leading to cell death. Following tumor selective binding and uptake, IKS03 requires intracellular lysosomal processing of beta-glucuronidase protecting groups to fully activate the payload which minimizes systemic release of the PBD dimer in human plasma. IKS03 is derived from a human antibody that binds with nanomolar affinity to human and cynomolgus monkey CD19. It is generated by site-specific bioconjugation yielding a homogeneous conjugate with a drug to antibody ratio of 2. In vitro, IKS03 induces specific and dose dependent cell killing of diverse CD19+ lymphoma cell lines with sub-nanomolar EC50 values. Conversely, in vitro cytotoxicity of the conjugate against normal human primary peripheral blood mononuclear cells was attenuated with an EC50 of approximately 1 μM, similar to a non-PBMC binding ADC bearing the same PBD pro-drug. The unconjugated payload more potently reduced PBMC viability with a sub-nanomolar EC50. This is consistent with low non-specific payload release from the conjugate underscoring the improved properties of the PBD pro-drug design. Pharmacokinetics of IKS03 was assessed in a repeat dose toxicity study in monkeys and the plasma concentrations of total ADC and total antibody were determined by LC-MS/MS across the 3-week dosing interval. Overall mean half-life for total ADC was approximately 8 days and clearance rate was slow (mean 0.2 mL/h/kg). Total ADC peak concentration, total plasma exposure and half-life were comparable to that of total antibody demonstrating favorable ADC stability in plasma with limited systemic payload release. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. Granta-519 cells contain the CCND1 t(11;14) translocation often present in mantle cell lymphoma (MCL). IKS03 was highly active against Granta-519 xenografts in SCID mice with complete tumor regressions observed with a single dose of 0.1 mg/kg. Efficacy was further evaluated in CD19-positive, low passage DLBCL patient-derived xenograft (PDX) models of known genomic profiles. IKS03 was highly active yielding complete tumor regressions following a single dose of 0.3 mg/kg in a refractory lymphoma Bcl2+ PDX model derived from a patient with no response to prior CHOP treatment. Post tumor collection, this patient was also refractory to EPOCH chemotherapy. A second xenograft model was derived from a treatment naïve high-grade 'triple hit' lymphoma patient (BCL2+; BCL6+; MYC+; XP01 amplified). Post tumor collection, this patient was refractory to R-CHOP therapy with disease progression within 2 months. Tumors rapidly progressed in this model, yet single dose treatment with IKS03 resulted in significant tumor growth inhibition at 0.3 mg/kg and complete tumor regressions at 1 mg/kg. Likewise, in a pre-treated MYC-amplified GCB lymphoma model, a single IKS03 dose of 0.3 mg/kg inhibited tumor growth and 1 mg/kg yielded complete inhibition of tumor growth. In conclusion, IKS03 is highly effective in preclinical MCL and DLBCL xenograft models that contain some of the genetic alterations commonly found in relapsed/refractory NHL patients in need of treatment. Preclinical data further demonstrates that IKS03's advanced ADC design results in specific target-dependent in vitro activity, excellent plasma stability and allows for effective targeting of B-cell tumors while sparing normal CD19-negative cells.

Enhancing the Safety and Efficacy of PSMA-Based Small-Molecule Drug Conjugates by Linker Stabilization and Conjugation to Transthyretin Binding Ligand.

This work describes the enhancement of a novel antitumor therapeutic platform that combines advantages from small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Valine-citrulline (VCit) dipeptide linkers are commonly used cathepsin B cleavable linkers for ADCs. However, the instability of these linkers in mouse serum makes translating efficacy data from mouse to human more challenging. Replacing the VCit linker with glutamic acid-valine-citrulline (EVCit) has been reported to enhance the stability of ADCs in mouse serum. However, the effect of EVCit linker on the stability of SMDCs has not been reported. Here, we report that incorporating the EVCit linker in prostate-specific membrane antigen-targeting SMDCs, equipped with the transthyretin ligand AG10, resulted in conjugates with lower toxicity, an extended half-life, and superior therapeutic efficacy to docetaxel in a xenograft mouse model of prostate cancer. This should make SMDCs' preclinical toxicity and efficacy data from mice more reliable for predicting human results.

Abstract 1405: Novel ADC platform delivers promising in vivo activity and safety

Abstract The physiochemical properties of an antibody-drug conjugate (ADC) are one key design attribute that can impacts its stability and pharmacokinetics/pharmacodynamics and are one of the key design attributes. Typically, ADCs with better hydrophilicity are less prone to aggregation, and have lower systemic clearance, and greater anti-tumor activities and thus a larger therapeutic index. However, the need to incorporate lipophilic payloads with an enhanced bystander effects has posed significant challenges to ADC and linker design, especially at drug-antibody ratios of 8 and even 16higher. Here we present a novel ADC platform that can greatly improveimproves the hydrophilicity, physiochemical stability, and pharmacokinetics of ADCs conjugated to lipophilic payloads such as exatecan, MMAE, SN38 and EErribulin. All payloads which are allare expect to exhibit strong bystander effects.By introducing PEG, polyhydroxyl and/or polycarboxyl groups, we generated hydrophilic linkers that enable site specific and, highly homogeneous conjugation of payloads to multiple prototypical antibodies at DAR 4, 8 and even 16. Compared. These ADCs were evaluated for their binding affinity, hydrophilicity, physiochemical stability, in vitro and in vivo anti-tumor activities, tolerability, and pharmacokinetics. In addition, comparisons were made with with theirthe corresponding naked unconjugated parent antibodies and conventional vedotin- and deruxtecan-based ADCs., these ADCs were evaluated for their binding affinity, hydrophilicity, physiochemical stability, in vitro and in vivo anti-tumor activities, tolerability, and pharmacokinetics. These novel ADCs were found to be stable at 37℃ for up to 30 days, after for at least 5 cycles of freeze-thaw, and at concentrations as high as 100 mg/mL, as assessed by visual inspection, hydrophobic interaction chromatography and size exclusion chromatography. Binding affinities of these ADCs to target-positive cell lines were similar to their corresponding naked unconjugated parent antibodies. These ADCs demonstrated strong tumor growth inhibition with single or repeated dosing in multiple tumor xenograft mouse models. The pharmacokinetic profiles of these ADCs are similar to those of the naked respective parent unconjugated antibodies. Assessments of ADCs using these new platform linkers in cynomolgus monkeys demonstrated showed better tolerability than ADCs using the traditional drug linkers.In summary, our novel ADC platform enables conjugation of hydrophobic payloads at high DARs as high as 8 or 16 with favorable physiochemical properties, results in robust pharmacokinetics, strong potency in vivo, and better tolerability. Therefore the potential for a meaningful therapeutic window may be realized through this novel ADC platform. Citation Format: Haidong Liu, Wenke Qi, Lei Wang, Guobao Wang, Xinyue Chen, Julia Gavrilyuk, Tae Han, Baiteng Zhao, Xiao Shang. Novel ADC platform delivers promising in vivo activity and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1405.

Abstract 1765: Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead

Abstract The cell-surface glycoprotein B7-H4 is overexpressed in a range of solid tumors including breast cancer, ovarian serous carcinoma, endometrial carcinoma, and cholangiocarcinoma, yet has limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). This presentation describes for the first time the development of AZD8205, a B7-H4 targeted ADC incorporating a novel topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 which was designed to exploit the full potential of B7-H4 as an ADC target. Initially, we investigated a series of more than 35 TOP1i compounds as warheads and achieved activity in a clinically relevant nM range. We further optimized the conjugation site and chemistry to reduce the potential for aggregation while maintaining potency, overcoming major synthetic challenges to deliver a robust synthetic route amenable to scale-up. Finally, with a series of optimized linker-warheads, we explored the impact of linker-warhead design on ADC hydrophobicity, stability, efficacy, pharmacokinetics and tolerability culminating in the development of AZD8205. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. AZD8205 drove bystander killing of target negative cells in mixed cultures in vitro, which is further supported by robust antitumor activity observed in in vivo studies with patient-derived xenograft (PDX) tumors with heterogeneous target expression, representing multiple tumor indications. In a study of 26 human TNBC PDX tumors, a single IV administration of 3.5 mg/kg AZD8205 provided an overall response rate of 69% (tumor regression of 30% or greater from baseline) and complete responses observed in 9/26 (36%) of models. To understand the biology underlying antitumor response, we conducted a multiparametric analysis including genomics, proteomics and computational pathology and found that deeper antitumor activity was observed in models with elevated B7-H4 expression as well as in models with defects in DNA damage repair (DDR). To further exploit the DNA damage elicited by the TOP1i warhead, we examined combinations of AZD8205 with small molecules, including a novel PARP1 selective inhibitor, in a BRCA wild type MDA-MB-468 model. These data suggest that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase 1 study in patients with advanced solid tumors is currently ongoing (NCT05123482). Citation Format: Krista Kinneer, Niall J. Dickinson, Luke Masterson, Thais Cailleau, Ian Hutchinson, Balakumar Vijayakrishnan, Nazzareno Dimasi, R. James Christie, Mary McFarlane, Kathryn Ball, Arthur Lewis, Sofia Koch, Lee Brown, Yue Huang, Anton I. Rosenbaum, Jiaqi Yuan, Si Mou, Noel R. Monks, Jon Chesebrough, Ravinder Tammali, Judith Anderton, Darrin Sabol, Frances Anne Tosto, Philipp Wortmann, Zachary A. Cooper, Pauline Ryan, John Hood, Carlos Fernandez Teruel, Carlos Serra Traynor, Andy Pike, Michael Davies, Elisabetta Leo, Kimberly Cook, Nadia Luheshi, Philip W. Howard, Puja Sapra. Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1765.

Abstract 5284: M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression

Abstract Mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are co-expressed at high prevalence in multiple cancer indications such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma, triple negative breast cancer and ovarian cancer. Tumor-associated MUC1 is hypoglycosylated and exposes peptide epitopes within its extracellular domain, which are not accessible to antibodies in normal epithelial tissue. MUC1 co-localizes with EGFR as a result of loss of cell polarity in tumor cells. M1231 is a bispecific antibody-drug conjugate (ADC) based on a SEED (strand-exchange engineered domain)-antibody scaffold targeting tumor-associated MUC1 and EGFR. Sutro Biopharma’s cell free expression system Xpress CF+™ enables incorporation of non-natural amino acids at precise positions in the SEED-antibody for site-specific linker-payload conjugation. The cytotoxic payload of M1231 (SC209) is a hemiasterlin-related microtubule inhibitor. The cleavable linker (ValCit-PABA) maintained ADC stability in human plasma in vitro, ensuring low systemic payload release. Pre-clinical activity of M1231 was assessed in vitro in cancer cell lines and in vivo in NSCLC and ESCC patient-derived xenograft (PDX) models. M1231 inhibited tumor cell viability in vitro at sub-nanomolar IC50 values. The bispecific MUC1xEGFR antibody has demonstrated superior internalization and lysosomal trafficking compared to monospecific bivalent antibodies. Efficient ADC uptake resulted in superior antitumor activity of M1231 in PDX models compared to monospecific bivalent ADCs. M1231 demonstrated durable and dose-dependent antitumor activity in PDX models. Strong antitumor activity with complete regression was observed in NSCLC and ESCC PDX models following a single treatment of M1231. M1231 antitumor activity was associated with high target expression in NSCLC PDX models and was observed across a range of target expression levels in ESCC models. In summary, efficient payload delivery to tumor cells and strong antitumor activity of M1231 indicate that M1231 may have the potential to deliver an efficacy benefit to patients with tumors co-expressing MUC1 and EGFR. Citation Format: Christine Knuehl, Lars Toleikis, Julia Dotterweich, Jianguo Ma, Seema Kumar, Edith Ross, Claudia Wilm, Martina Schmitt, Hans Juergen Grote, Christiane Amendt. M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5284.

Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody–drug conjugates (ADCs) with payloads attached to the C’E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

Dual intra- and extracellular release of monomethyl auristatin E from a neutrophil elastase-sensitive antibody-drug conjugate

Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.

Polyethylene glycol-based linkers as hydrophilicity reservoir for antibody-drug conjugates

Antibody-drug conjugates (ADCs) are an established therapeutic entity in which potent cytotoxic drugs are conjugated to a monoclonal antibody. In parallel with the great emphasis put on novel site-specific bioconjugation technologies, future advancements in this field also rely on exploring novel linker-drug architectures that improve the efficacy and stability of ADCs. In this context, the use of hydrophilic linkers represents a valid strategy to mask or reduce the inherent hydrophobicity of the most used cytotoxic drugs and positively impact the physical stability and in vivo performance of ADCs. Here, we describe the use of linkers containing monodisperse poly(ethylene glycol) (PEG) moieties for the construction of highly-loaded lysine-conjugated ADCs. The studied ADCs differ in the positioning of PEG (linear or pendant), the bonding type with the antibody (amide or carbamate), and the drug-to-antibody ratio (DAR). These ADCs were first evaluated for their stability in solution under thermal stress, showing that both the drug-linker-polymer design and the nature of the antibody-linker bonding are of great importance for their physical and chemical stability. Amide-coupled ADCs bearing two pendant 12-unit poly(ethylene glycol) chains within the drug-linker structure were the best performing conjugates, distancing themselves from the ADCs obtained with a conventional linear 24-unit PEG oligomer or the linker of Kadcyla®. The pharmacokinetic profiles of amide-linked ADCs, with a linear or pendant configuration of the PEG, were tested in mice in comparison to Kadcyla®. Total antibody pharmacokinetics paralleled the trends in aggregation tendency, with slower clearance rates for the ADCs based on the pendant drug-linker format. The above-mentioned findings have provided important clues on the drug-linker design and revealed that the positioning and configuration of a PEG unit have to be carefully tuned to achieve ADCs with improved stability and pharmacokinetics.

Site-Specific Quantitation of Drug Conjugations on Antibody-Drug Conjugates (ADCs) Using a Protease-Assisted Drug Deconjugation and Linker-like Labeling (PADDLL) Method.

Antibody-drug conjugates (ADCs) are biopharmaceuticals for the targeted delivery of antitumor agents. ADCs can be highly heterogeneous with various drug-to-antibody ratio (DAR) species, conjugation sites, and occupancy levels. The conjugation site can modulate the ADC stability and efficacy and therefore can be considered to be a critical quality attribute (CQA) during development. Traditional mass spectrometry (MS)-based peptide mapping methods cannot accurately quantify site-specific conjugations due to a significant ionization discrepancy between unconjugated native peptides and conjugated peptides. Here, we developed a novel protease-assisted drug deconjugation and linker-like labeling (PADDLL) method to quantify the levels of linker payload at specific conjugation sites. We utilized optimized papain digestion to deconjugate the drug payload and labeled unoccupied conjugation sites with a linker-like structure to provide comparable ionization efficiency for MS-based quantitation. This method was successfully applied on two cysteine-linked, protease-cleavable ADCs, and the method demonstrated good linearity and reliability, reaching a limit of quantitation of below 1%. The calculated DARs were comparable with the results from intact mass analysis. The lot-to-lot variation in conjugation distribution and inferior conjugation stability at HC Cys225 to other interchain cysteines were observed. This method provides a valuable tool for ADC design and product development. To the best of our knowledge, this is the first analytical method developed to accurately quantify site-specific linker-drug payload conjugations for ADCs.

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.

Cysteine-Based Coupling: Challenges and Solutions.

Antibody-drug conjugates (ADCs) have attracted great attention in recent years in the wake of an accelerated FDA approval rate and several large-scale acquisitions. To date, there are ten ADC drugs on the market and more than 70 in various stages of clinical trials. Yet, due to the complicated nature of ADC molecules, considerations need to cover many aspects for the success of ADCs, including target specificity, linker-payload stability, tumor permeability, and clearance rate. This topical review summarizes and discusses current methods used to increase stability and homogeneity of ADCs of cysteine conjugation. We believe that they will lead to improvement of efficacy and pharmacokinetics (PK) of ADC drugs.