The steadily growing number of HER2-targeted antibody-drug conjugates (ADCs) in preclinical development is a testament to the success of the original anti-HER2 ADC, ado-trastuzumab emtansine (T-DM1; Kadcyla®; Genentech/Roche), which comprises the trastuzumab antibody appended through a non-cleavable linker to the potent microtubule inhibitor payload, maytansine. [1] T-DM1 was the first approved ADC against a solid tumor indication, and its efficacy showed the promise of HER2-targeted therapies; follow-on molecules have been designed with the goal of improving upon this first-generation drug and thereby continue to raise the bar for patient outcomes. [2] However, the sheer number of follow-on candidates makes it difficult to keep track of the state-of-the-art, and for those not immersed in the field, it can be hard to differentiate among the many iterations in order to find the true innovations in the crowd.

Important innovations include trastuzumab-conjugates with payloads that confer different mechanisms of action, such as the recently approved drug, fam-trastuzumab deruxtecan (DS-8201; Enhertu®; Daiichi Sankyo and AstraZeneca) [3], which carries a topoisomerase inhibitor, and the clinical-stage therapeutic candidate BDC-1001*, [4] which bears a toll-like receptor (TLR) agonist. Also differentiating are conjugates built on substantial improvements to conjugation technology and linker chemistry. These fundamental ADC components are inherently connected and govern both the ease of manufacturing and the biophysical properties that determine a molecule’s in vivo efficacy and safety. Catalent Biologics’ SMARTag® conjugation platform is a great example of this latter form of innovation, and due to its modularity can be broadly applied to various disease contexts according to need.

The Catalent Biologics team recently published in Molecular Cancer Therapeutics the results of preclinical studies on their HER2-targeted ADC, CAT-01-106 [5]. The ADC is site-specifically conjugated to RED-106, a proprietary non-cleavable linker carrying a maytansine payload. CAT-01-106, which has a drug-to-antibody ratio (DAR) of 1.8, showed favorable efficacy and tolerability when compared in vivo to T-DM1, which has a DAR of 3.5. Specifically, at equal payload doses, CAT-01-106 offered improved efficacy against two xenograft models and showed equal or better tolerability in rat and cynomolgus monkey models. The pharmacokinetic analysis suggested that CAT-01-106 had better in vivo exposure compared to T-DM1 at the same antibody dose, underscoring the importance of optimized biophysical properties on in vivo outcomes. Together, the data suggested the possibility that CAT-01-106 might be tolerated in patients at 7.2 mg/kg—or twice the T-DM1 clinical dose—leading to systemic ADC exposure levels similar to those achieved by trastuzumab alone. In this scenario, the Catalent ADC might simultaneously enable both trastuzumab and maytansine mechanisms of action, potentially eliminating the need for the systemic taxane chemotherapy and thus improving patient quality of life.

In May 2020, Triphase Accelerator Corp., together with Catalent, announced interim results from a Phase 1 clinical trial of TRPH-222, the first SMARTag ADC to enter clinical studies. [6] TRPH-222 is an anti-CD22 targeted ADC with a DAR of 1.8 that carries the same RED-106 linker-payload found on CAT-01-106. Because ADC toxicities are most commonly driven by linker-payload rather than target antigen [7], the clinical tolerability observed with TRPH-222 is highly likely to correlate well with the clinical tolerability that could be achieved with CAT-01-106. The TRPH-222 Phase 1 study (NCT03682796) targeted heavily pretreated patients with relapsed/refractory B-cell lymphoma. TRPH-222 was safely dosed up to 7.5 mg/kg with plans to continue dose escalation to 10 mg/kg. Early signs of efficacy were observed, with five confirmed complete responses out of 19 evaluable patients. Importantly, preclinical studies of TRPH-222 (formerly called CAT-02-106) aligned well with its emerging promising clinical tolerability and efficacy profile, with no significant adverse events noted in cynomolgus monkeys after repeat doses up to 60 mg/kg [8]. Collectively, these data support the conclusions drawn from the HER2-targeted CAT-01-106 preclinical studies, underscoring the high likelihood that CAT-01-106 would be well-tolerated clinically and could be dosed at trastuzumab-equivalent exposure levels.

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The RED-106 non-cleavable maytansine linker-payload employed on TRPH-222 and CAT-01-106 confers additional therapeutically-relevant functions to an ADC. The linker-payload is resistant to efflux by target cells overexpressing P-glycoprotein [8] and can induce hallmarks of immunogenic cell death selectively on antigen-positive target cells in vitro [9]. These features suggest that RED-106 conjugates may be efficacious against tumors that have become refractory to treatment due to the upregulation of multidrug efflux transporters (a common adaptive response to chemotherapy (10)), and that the ADCs may pair well with immuno-oncology-based drugs as part of a combination therapy. In this regard, the high tolerability of RED-106 conjugates is particularly attractive, since combination therapy dosing is limited by the collective toxicities of the individual component drugs.

Catalent’s SMARTag ADC technology employs site-specific conjugation enabled through the insertion of a six amino acid consensus sequence termed the “aldehyde tag” (11). This tag contains a cysteine residue that is recognized in the context of the consensus sequence by a naturally-occurring human enzyme, formylglycine generating enzyme (FGE). FGE oxidizes the thiol in the cysteine residue to an aldehyde, thereby converting the cysteine to a formylglycine residue. The aldehyde moiety is chemically orthogonal to other reactive groups in the protein, thus serving as a bioorthogonal handle for site-specific conjugation. Catalent’s proprietary HIPS (Hydrazino-iso-Pictet Spengler) chemistry specifically reacts with the aldehyde, forming a stable carbon-carbon bond [12]. This conjugation chemistry is unique to Catalent’s platform and, together with the aldehyde tag, forms the foundation of a SMARTag ADC.

Generating a SMARTag ADC is simple. The tag sequence is genetically-encoded into the desired location within the antibody constant region. A favored location is the heavy chain C-terminus, though various placement options are available [13]. To yield higher DAR species, multiple tags can be incorporated into a single antibody [14]. The tagged antibody is produced in a cell line overexpressing human FGE, which achieves the cysteine to formylglycine conversion step cotranslationally. The antibody is secreted with the aldehyde installed at the desired location and is purified using conventional, Protein A-based affinity chromatography. Using this approach, titers of up to 5 g/L have been achieved with cysteine-to-formylglycine conversion yields of 95-98% [14]. The purified antibody is ready for the 1-step conjugation process, which consists of adding the HIPS-based linker-payload to the antibody in a buffered solution. After conjugation, the remaining unconjugated linker-payload is removed by tangential filtration, and the ADC preparation is complete. Interrogation of the DAR by hydrophobic interaction chromatography (HIC) reveals the efficiency of the SMARTag system, where incorporation of one aldehyde tag site (present twice in the antibody due to the dimeric nature of light/heavy chain composition in an IgG) routinely yields DARs of 1.8-1.9 without the need for enrichment. Furthermore, the production system is highly scalable, delivering the same results from the laboratory bench to the production line.

By virtue of its manufacturability, ease of analytics, and critical ability to deliver efficacy coupled with good tolerability, the SMARTag platform, and Catalent’s HER2 ADC stand out in the crowd. To learn more about the technology and Catalent’s SMARTag ADC pipeline, contact Penelope Drake or Steven Worsley.

SMARTag® is a registered trademark of Catalent, Inc. or its affiliates or subsidiaries in the United States and other countries.
Kadcyla® is a registered trademark of Genentech USA, Inc.
Enhertu® is a registered trademark of Daiichi Sankyo Company, Limited
*BDC-1001 was developed by Bolt Biotherapeutics, Inc.

Clinical trials
Study of TRPH-222 in Patients With Relapsed and/or Refractory B-Cell Lymphoma – NCT03682796

Highlights of Prescribing Information
Trastuzumab emtansine (T-DM1; Kadcyla®; Genentech/Roche)[Prescribing Information]
Trastuzumab deruxtecan (DS-8201; Enhertu®; Daiichi Sankyo and AstraZeneca)[Prescribing Information]

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[3] Cavallo J. SABCS 2019: [Fam-] Trastuzumab Deruxtecan in Patients With Pretreated HER2-Positive Metastatic Breast Cancer [Internet]. The ASCO Post. 2019 [cited 2020 Feb 3]. Available from:
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[6] Triphase Accelerator and Catalent Announce Interim Results of a Dose Escalation Phase 1 Clinical Trial of TRPH-222 in Patients with Non-Hodgkin’s Lymphoma [Internet]. [cited 2020 May 18]. Available from:
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[9] Bauzon M, Drake PM, Barfield RM, Cornali BM, Rupniewski I, Rabuka D. Maytansine-bearing antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in antigen-positive target cells. OncoImmunology. 2019;00:1–11.
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[12] Agarwal P, Kudirka R, Albers AE, Barfield RM, de Hart GW, Drake PM, et al. Hydrazino-Pictet-Spengler Ligation as a Biocompatible Method for the Generation of Stable Protein Conjugates. Bioconjug Chem. 2013;24:846–51.
[13] Huang BCB, Kim YC, Banas S, Barfield RM, Drake PM, Rupniewski I, et al. Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions. MAbs. 2018;10:1182–9.
[14] York D, Baker J, Holder PG, Jones LC, Drake PM, Barfield RM, et al. Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II). BMC Biotechnol. 2016;1–11.

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