Conventional Antibody-drug Conjugates Research Articles

Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and noncleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for noncleavable linker ADCs.

Breaking the Mold: Trailblazing Melanoma Therapy Beyond Checkpoint Through Innovative Approaches.

Melanoma has long been a difficult malignancy to treat with low response rates to standard chemotherapies. In recent years, the use of immune checkpoint inhibitors have demonstrated promising results, paving the way for the use of the rapidly developing novel immune targeting therapies. In this review, we look beyond immune checkpoint inhibitor treatments and summarize several emerging treatment strategies for melanoma, including neoantigen vaccines, conventional antibody drug-conjugates, and bispecific T-cell engager therapies.

Could a Long-Acting Prodrug of SN-38 be Efficacious in Sacituzumab Govitecan-Resistant Tumors?

We previously proposed that sacituzumab govitecan (SG, Trodelvy®) likely acts as a simple prodrug of systemic SN-38 as well as an antibody drug conjugate (ADC). In the present commentary, we assess whether a long-acting SN-38 prodrug, such as PLX038, might be efficacious in SG-resistant patients. We first describe possible mechanisms of action of SG, with new insights on pharmacokinetics and TROP2 receptor occupancy. We argue that SG is not an optimal conventional ADC and that the amount of systemic SN-38 spontaneously hydrolyzed from the ADC is so high it must have activity. Then, we describe the concept of time-over-target as related to the pharmacology of SG and PLX038 as SN-38 prodrugs. To be clear, we are not in any way suggesting that PLX038 or any SN-38 prodrug is superior to SG as an anticancer agent. Clearly, SG has the benefit over antigen-independent SN-38 prodrugs in that it targets cells with the TROP2 receptor. However, we surmise that PLX038 should be a more efficacious and less toxic prodrug of systemic SN-38 than SG. Finally, we suggest possible mechanisms of SG resistance and how PLX038 might perform in the context of each. Taken together, we argue that-contrary to many opinions-SG does not exclusively act as a conventional ADC, and propose that PLX038 may be efficacious in some settings of SG-resistance.

Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) play important roles in tumor therapy. However, traditional ADCs are limited by the extremely large molecular weight of the antibody molecules, which results in low permeability into solid tumors. The use of small ADCs may be expected to alleviate this problem, but this switch brings the new limitation of a greatly shortened blood circulation half-life. Here, we propose a new cleavable ADC design with excellent tumor tissue permeability and a long circulation half-life by fusing the small ADC ZHER2-MMAE with the Fc domain of the antibody for circulation half-life extension, and inserting a digestion sequence between them to release the small ADC inside tumors for better tumor penetration. The experimental results showed that the designed molecule Fc-U-ZHER2-MMAE has a significantly increased blood circulation half-life (7.1 h, 59-fold longer) compared to the small ADC ZHER2-MMAE, and significantly improved drug accumulation ability at tumor sites compared to the conventional full-length antibody-coupled ADC Herceptin-MMAE. These combined effects led to Fc-U-ZHER2-MMAE having significantly enhanced tumor treatment ability, as shown in mouse models of NCI-N87 gastric cancer and SK-OV-3 ovarian cancer, where Fc-U-ZHER2-MMAE treatment achieved complete regression of tumors in all or a portion of animals with no obvious side effects and an MTD exceeding 90 mg/kg. These data demonstrate the therapeutic advantages of this cleavable ADC strategy, which could provide a new approach for ADC design.

Preclinical antitumor activity of an anti-HER2/Trop-2 bispecific antibody-drug conjugate with a new DNA topoisomerase I inhibitor.

e15013 Background: In recent years, antibody-drug conjugates (ADCs) have made significant progress in cancer therapy and show a promising future. Among the approved ADCs, Enhertu, the HER2 directed ADC and Trodelvy, the Trop-2 directed ADC, have shown considerable clinical benefits. Bispecific antibody-drug conjugate represents a fast-growing class of next generation ADC due to its potentials in enhancing specificity, improving efficacy, and reducing toxicity. Additionally, targeting more than one molecule can be useful to avoid resistance to treatment and broaden the patient population. Methods: We generated a novel bispecific ADC (BIO-201) co-targeting HER2 and Trop-2 which are the two clinically validated tumor-associated antigens that are expressed on a wide variety of tumors. BIO-201 is consisted of a novel bispecific anti-HER2/Trop-2 antibody conjugated with a potent DNA topoisomerase I inhibitor (TOPO Ii) via a cleavable linker. TOPO Ii is a proprietary payload platform developed in house that exhibits similar characteristics including in vitro and in vivo anti-tumor activities and safety profile to DXd. The bispecific antibody was fully characterized and in vitro and in vivo anti-tumor activity of BIO-201 was assessed using preclinical models. Results: The bispecific antibody showed comparable or increased cell binding and internalization compared with the parental antibodies in various tumor cells that express different surface levels of HER2 and Trop-2. In the in vitro cytotoxicity assay, the bispecific ADC BIO-201 effectively killed cancer cells with IC50 in the sub nM range with BIO-201 being most potent against cancer cells that co-express HER2 and Trop-2. The anti-tumor activity of BIO-201 was further demonstrated in vivo in several tumor xenograft models. Treatment of BIO-201 induced significant tumor regression in HER2 or Trop-2 positive tumors, suggesting broader coverage of tumor types than conventional HER2 and Trop-2 ADCs. BIO-201 also showed similar anti-tumor activity to Herceptin-DXd. Conclusions: Taken together, these findings suggest that anti-HER2/Trop-2 bispecific ADC has potential as an effective therapy for a variety of cancers that co-express HER2/Trop-2 or express either of the targets. BIO-201 may be expected to have a similar clinical activity to Enhertu or Trodelvy but treat more patient sub-populations and indications.

Abstract 1541: NextGen conditionally active biologic (CAB) anti-Nectin-4-ADC with improved stability and safety

Abstract In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents. Today, development of a new ADC faces two major challenges: on-target, off-tumor toxicity based on target expression on healthy tissue and off-target, off-tumor toxicity caused by extracellular linker vulnerability and/or premature payload release. On-target, off-tumor toxicity can be effectively eliminated by using a Conditionally Active Biologics (CAB) antibody to specifically target tumor cells. CAB technology is a proprietary platform used to engineer antibodies that target antigens in the context of diseased tissues, but not normal tissues by exploiting the unique extracellular acidic conditions found on the surface of cancer cells (Chang et al. 2021). This platform has allowed us to generate CAB antibody drug conjugates (ADCs) specific to tumor associated antigens that are often expressed on healthy tissues. CAB selectively reduces on-target toxicity, as well as off-target toxicity by eliminating target mediated drug disposition. Conventional ADCs can induce unacceptable toxicities caused by binding to antigen expressed on normal cells, resulting in the undesired release of the payload. This can severely limit the therapeutic window of the drug candidate. Two CAB-ADCs (Mecbotamab Vedotin and Ozuriftamab Vedotin) using the well-established vedotin linker with MMAE as payload are currently in phase 2 clinical trials and show promising results with increased safety profiles, with currently no signs of on-target, off-tumor toxicity. BA3361, a CAB-ADC targeting Nectin-4, showed good in vitro and in vivo efficacy data demonstrating cytotoxicity against human Nectin-4-expressing tumor cell lines and tumor regression in xenograft models using the vedotin linker (AACR 2020, Poster #4560). To eliminate vedotin linker-related off-target toxicities, we developed a NextGen Nectin-4 CAB ADC with a novel cleavable linker that is highly stable in human serum and has increased solubility. In vivo efficacy data from CDX models as well as data from non-GLP tox studies in rats and cynomolgus monkeys will be presented. In conclusion, our data indicates that the CAB-Nectin-4-ADC with its optimized linker payload, has superior serum stability while maintaining the CAB target selectivity. Reference: 1. Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, Short JM. Proc Natl Acad Sci U S A. 2021 Mar 2;118(9):e2020606118. 2. Jing Wang, Charles Xing, Haizhen Liu, Ana Paula Cugnetti, Christina Wheeler, Mathew Lucas, Gerhard Frey, Cathy Chang, William J. Boyle, Jay M. Short. Novel conditionally active ADCs (CAB ADCs): A novel class of molecules targeting solid tumors. AACR Annual Meeting 2020, Abstract #7890, Poster #4560 Citation Format: Gerhard Frey, Jing Wang, Kyrie Johnson, Haizhen Liu, Christina Wheeler, Charles Xing, Ana Paula Cugnetti, Patricia McNeeley, Solmarie Joyner, Cathy Chang, William J. Boyle, Jay M. Short. NextGen conditionally active biologic (CAB) anti-Nectin-4-ADC with improved stability and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1541.

Abstract 5242: Rationally designing a radionuclide drug conjugate for heterogeneous genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) is one of common malignant tumors with a 5-year survival rate less than 10% for patients in advanced stages. Genomically stable gastric cancer (GSGC), featuring the worst prognosis of four subtypes of GC based on its molecular characteristics, receives limited benefits from recently approved HER2-targeted antibody drug conjugates (ADCs) due to the lack and heterogeneity of tumoral HER2 expression. Currently, radionuclide drug conjugates (RDCs) have attracted great attention in targeted therapy, owing to its unique advantages of radiation-associated direct DNA damages and relatively long radiation distances. These unique advantages enable RDCs to efficiently and broadly ablate both cancer cells and cancer-associated stromal cells in heterogeneous tumor microenvironment without being internalized by target cells, featuring a novel and more efficient “bystander killing” effect than linker-cleavable ADCs. Here, a RDC was developed by rationally screening GSGC-targeting antibodies and radionuclides for targeted treatment of GSGCs. Meanwhile, the “bystander killing” effect of the RDC will be quantitatively determined and compared with conventional linker-cleavable ADCs. Results: First, ICAM1 was identified as a more preferable molecular target relatively to HER2 in human GSGCs via flow cytometric analyses. Next, a RDC was constructed by covalently conjugating anti-ICAM1 antibody with radionuclide 131I via indogen chemistry. 131I, the RDC warhead emitting β-particles with an emission range of approximately 0.8 mm, is a clinically-used radionuclide with FDA approval for treating several tumors. Then, the half maximum inhibitory concentrations (IC50s) of constructed 131I conjugated ICAM1 antibody (131I-ICAM1) was evaluated in human GSGC cell lines. Its inherent bystander killing effect was validated by measuring the viability of ICAM1 negative cells co-incubated with ICAM1 positive cells in the treatment of 131I-ICAM1, in comparison with ADC of the same target. Ultimately, the anti-tumor efficacy of 131I-ICAM1 was verified in heterogeneous GSGC models. Simultaneously, the histopathological damages to normal organs by 131I-ICAM1 was minimized by optimizing its dose. Conclusions: In this study, we constructed a rationally designed 131I-ICAM1 by conjugating ICAM-1 antibody and clinical available radionuclide 131I and investigated the intrinsic bystander killing effect of this RDC in ablating heterogeneous GSGCs. Our research explored the potential of 131I-ICAM1 as a promising targeted therapeutic candidate for treating heterogeneous GSGCs. Citation Format: Le Yang, Rui Xu, Peng Guo, Heqing Yi, Xiangdong Cheng. Rationally designing a radionuclide drug conjugate for heterogeneous genomically stable gastric cancer [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 5242.

Abstract P196: Novel hydrophilic drug linkers enable exatecan-based antibody-drug conjugates with promising physiochemical properties and in vivo activity

Abstract The physiochemical properties of an antibody-drug conjugate (ADC) can impact 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. However, the need of incorporating lipophilic payloads with enhanced bystander activity has posed significant challenges to ADC and linker design, especially at higher drug-antibody ratios (DAR). Here we present novel hydrophilic linkers that can greatly improve the hydrophilicity of ADCs conjugated to lipophilic payloads such as exatecan. Exatecan is camptothecin analogue that has failed as a small molecule drug in clinical trials due to lack of therapeutic window but retains considerable promise as an ADC payload because of its high potency and resistance to Pgp efflux. By introducing highly polar PEG, polyhydroxyl and/or polycarboxyl groups, we generated hydrophilic linkers that enable site specific, highly homogeneous conjugation of exatecan to multiple prototypical monoclonal antibodies at DAR8. The linkers may also allow for other DARs such as DAR4 and DAR16. These exatecan-based ADCs with novel hydrophilic linkers were evaluated for their binding affinity and stability and compared with corresponding naked antibodies and deruxtecan-based and conventional vedotin-based ADCs for hydrophilicity, pharmacokinetics, in vitro and in vivo anti-tumor activities. Safety assessments in cynomolgus monkeys have also been planned. ADCs conjugated with the novel hydrophilic linkers were stable at 37oC for 15 days, after 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 affinity to target-positive cell lines was similar to corresponding naked antibodies. At DAR8, these ADCs were more hydrophilic than deruxtecan-based ADCs and DAR2 species of conventional vedotin-based ADCs. These ADCs also demonstrated potent in vitro cell growth inhibition and induced comparable or stronger tumor regression with single or multiple dosing in multiple mouse xenograft models. The pharmacokinetics profiles of these ADCs are similar to those of the naked antibodies. In summary, our novel hydrophilic linkers can enable conjugation of exatecan-based ADCs at high DARs with favorable physiochemical properties, which result in robust stability, pharmacokinetics, potency, and the potential for a meaningful therapeutic window. Citation Format: Haidong Liu, Lei Wang, Julia Gavrilyuk, Tae Han, Baiteng Zhao, Xiao Shang. Novel hydrophilic drug linkers enable exatecan-based antibody-drug conjugates with promising physiochemical properties and in vivo activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P196.

Chromatographic analysis of site-specific antibody-drug conjugates produced by AJICAP first-generation technology using a recombinant FcγIIIa receptor-ligand affinity column

Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods.

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.

A Purification Strategy Utilizing Hydrophobic Interaction Chromatography to Obtain Homogeneous Species from a Site-Specific Antibody Drug Conjugate Produced by AJICAP™ First Generation.

In recent years, site-specific antibody drug conjugates (ADC)s have been in great demand because they have an expanded therapeutic index compared with conventional ADCs. AJICAP™ technology is a chemical conjugation platform to obtain site-specific ADCs through the use of a class of Fc-affinity compounds. Promising results from early technology development studies led to further investigation of AJICAP™ ADC materials to obtain site-specific and homogeneous drug antibody ratio (DAR) ADCs. Here we report site-specific conjugation followed by a preparative hydrophobic interaction chromatography (HIC) purification strategy to obtain purified “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADC materials. Optimization of the mobile phase conditions and resin achieved a high recovery rate. In vitro biological assay demonstrated the target selective activity for purified homogeneous DAR ADCs. These results indicate the ability of a HIC purification strategy to provide “DAR = 1.0” and “DAR = 2.0” AJICAP™ ADCs with considerable potency and target selectivity.

Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody-Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis.

Conventional antibody-drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug-antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25-100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.

Homogeneous Antibody-Drug Conjugates via Glycoengineering.

Conventional antibody-drug conjugates (ADCs) randomly assemble small-molecule drugs onto Lys or Cys residues of a tumor-targeting antibody, featured with heterogeneity in payload numbers and conjugation positions. Glycosite-specific ADCs (gsADCs) link payload drugs onto IgG Fc N-glycans with high homogeneity that facilitates structural optimization and quality control for ADC drug development. In this protocol, we report two strategies for preparation of homogeneous ADCs via chemoenzymatic glycoengineering. First, an azido-tagged unnatural N-glycan substrate is transferred onto Fc glycosites of a therapeutic antibody through Endo-S-catalyzed glycoremodeling, followed by click reaction with an alkyne-tagged payload drug to give a well-defined gsADC. In an alternative way, glycoengineering of antibody with a natural sialylated N-glycan and successive selective oxidation of sialic acid moieties using sodium periodate provided an aldehyde handle on the glycans for conjugation with an aminooxy-assembled payload. These two strategies both enable gsADCs with high homogeneity in their conjugation sites, payload numbers, and glycoforms, which are characterized of a single mass under mass-spectral detection.

Development, Optimization, and Structural Characterization of an Efficient Peptide-Based Photoaffinity Cross-Linking Reaction for Generation of Homogeneous Conjugates from Wild-Type Antibodies.

Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.

Efficient and Selective Bioconjugation Using Surfactants.

The surfactant sodium decanote is used in the drug substance process of Besponsa, an antibody drug conjugate (ADC), to facilitate bioconjugation between activated calicheamicin derivative (linker payload) and inotuzumab (monoclonal antibody). Under the normal conjugation process conditions, sodium decanoate forms micelles and the micelle formation was shown to be critical for the efficient conjugation reaction. Further screening studies indicated that sodium dodecyl sulfate, sodium deoxycholate, and dodecyltrimethylammonium bromide were also able to facilitate the conjugation reaction. While the choice of surfactant and its concentration in the reaction impact the conjugation efficiency, the charge of surfactant and the choice of linker payload influence the conjugated lysine site selectivity. Eight major conjugated lysine sites are observed in Besponsa, as compared to approximately 80 conjugated lysine sites typically observed in conventional lysine-based ADCs.

Abstract 746: Development of novel formats of anti-AXL ADCs

Abstract AXL is a member of the Tyro3- AXL -Mer (TAM) receptor tyrosine kinase subfamily which has been associated with several cellular functions including growth, migration, aggregation, and anti-inflammation (Li Y et al., 2009) via activation through its ligand, Gas6. Originally identified as an oncogene in chronic myelogenous leukemia, expression of Axl has been reported to be upregulated in a variety of cancers including breast, gastric, prostate, ovarian, and lung. AXL expression has been shown to be negatively associated with patient survival and implicated in epithelial-to-mesenchymal transition, a process closely linked with invasive motility and metastasis of malignant cells. Several recent studies have reported that AXL is overexpressed during resistance to chemotherapy and other molecularly targeted therapies, as well as during hypoxia (Schoumacher M et al 2017). Consequently, Axl is promising therapeutic target for development into ADCs for the first or second line treatment of cancer. At the National Research Council (NRC) of Canada, we have selected mouse monoclonal (mAbs) or llama derived single domain antibodies (sdAbs) that are selective for the extracellular domain of human Axl and which have been shown to be internalized into Axl expressing cells for antibody drug conjugate (ADC) development using a surrogate ADC screen. These antibodies were also characterized based on their binding domains and ability to compete with Gas6, the extracellular ligand for Axl. A panel of Axl-specific ADCs was subsequently directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. Axl-DM1 ADCs were shown to efficiently induce cytotoxicity in vitro, in Axl expressing cells including breast (MDA-MB 231), lung (NCI-H292) and ovarian (SKOV3) cell lines with sub nM potencies achieved with both sdAb and conventional mAb-based ADCs. When tested in vivo using ovarian (SKOV3) and breast cancer (MDA-MB 231) xenograft models, Axl-DM1 ADCs were shown to have potent anti-tumor activity. Biparatopic antibodies (i.e. those which target two non-overlapping epitopes) directed against HER2 have been previously shown to induce target clustering and promote robust internalization, lysosomal trafficking, and degradation (LI JY et al 2016). Since these properties are important in the mechanism of action of ADCs, we have further tested anti-Axl antibodies in various configurations and have identified potent biparatopic antibodies for further development into ADCs. Citation Format: Maria L. Jaramillo, Myriam Banville, Alma Robert, Luc Meury, Maurizio Acchione, Anne Marcil, Christine Gadoury, Cunle Wu, Yves Fortin, Kevin Henry, Joseph Schrag, The-Minh Tu, Binbing (Erica) Ling, Jacqueline Slinn, Maria Moreno. Development of novel formats of anti-AXL ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 746.

Abstract 2780: Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens

Abstract Our laboratory has developed a unique platform for the delivery of the highly pro-apoptotic serine protease granzyme B (GrB). The construct contains enzymatically active GrB and therefore does not require release from the targeting carrier by a fragile linker in contrast to conventional antibody-drug conjugates (ADCs). We employed two scFvs targeting the CD33 antigen found on AML and the juxtamembrane epitope of CEA found on numerous solid tumors. These human scFvs (designated HU33 and CEA respectively) were fused to human GrB through an engineered IgG heavy-chain fragment (hinge to Fc and designated Fc) which contains a dimerization domain. The GrB-Fc-HU33 and GrB-Fc-CEA constructs were cloned into the pSECTag vector containing an upstream 5kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells and grown at 37°C for 3 days in 5% CO2. The molecules were isolated from culture media and purified by IMAC followed by EK cleavage and SP chromatography to remove the free tag. The yields were 6-10 mg/L for both molecules. Specific binding of each molecule to the antigen target is undergoing Biacore and/or ELISA assessment. The cytotoxicity of the GrB-Fc-HU33 against HL-60 cells showed a specific cytotoxic effect with an IC50 of 120 nM. Tests of this agent against a panel of cells expressing various levels of CD33 are ongoing. The cytotoxic effects of the GrB-Fc-CEA were assessed against HT29, HT1080-CEA and A431 cells and demonstrated IC50 values of 80, 110 and 95 nM respectively. Based on IC50 values, these data demonstrate that the fusion construct targeting CD33 is efficiently internalized and effectively delivers active serine protease to the cytosol even at relatively low antigen expression levels. In addition, the juxtamembrane epitope of the CEA antibody also allows efficient internalization for delivery of GrB to the cytosol. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2780.

Abstract 745: Development of a novel ron receptor targeted antibody-drug conjugates using cysteine bridging technology for potential treatment of pancreatic cancer

Abstract Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody-drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis-alkylating linker (BL), attached to a lysosomal protease-cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL-MMAE was then conjugated to Zt/g4 (anti-RON mAb) through cysteine bridging technology to produce Zt/g4-BL-MMAE with a homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4-BL-MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared through maleimide based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4-BL-MMAE specifically targeted RON-expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4-BL-MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 µg/ml. We conclude that Zt/g4-BL-MMAE is a novel anti-RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON-targeted ADCs in pancreatic cancer treatment in the future. Citation Format: Sreedhar Reddy Suthe, Hang-Ping Yao, Paul C. Trippier, Ming-Hai Wang. Development of a novel ron receptor targeted antibody-drug conjugates using cysteine bridging technology for potential treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 745.

Development of A Novel RON Targeted Antibody‐Drug Conjugates using Cysteine Bridging Technology for Potential Treatment of Pancreatic Cancer

Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody‐drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis‐alkylating linker (BL), attached to a lysosomal protease‐cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL‐MMAE was then conjugated to Zt/g4 (anti‐RON mAb) through cysteine bridging technology to produce Zt/g4‐BL‐MMAE with a relatively homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4‐BL‐MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared by maleimide‐based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4‐BL‐MMAE specifically targeted RON‐expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4‐BL‐MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 μg/ml. Therapeutic studies using mouse xenograft models are currently underway to determine the efficacy of Zt/g4‐BL‐MMAE for pancreatic and other epithelial cancers. We conclude that Zt/g4‐BL‐MMAE is a novel anti‐RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON‐targeted ADCs in pancreatic cancer treatment in the future.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.