During the 16th Annual PEGS Boston Virtual Conference & Expo, which took place between August 31 – September 4, 2020, Gregory Bleck, Ph.D., Global Head of R&D, Catalent Biologics, presented “GPEx® Boost – A Novel Approach for High-Expressing CHO Cell Line Engineering.

Catalent’s GPEx technology is used for engineering high-expressing Chinese hamster ovary (CHO) cells for the production of pharmaceuticals. The technology uses replication and retrovector technology in which genes are inserted into mammalian cell lines. To make genetically stable cell lines, the process does not require antibiotic selection nor the use of toxic compounds for gene amplification, so stability testing can be kept off of the critical path for some programs.

In his presentation, Bleck explained how the next generation of Catalent’s GPEx cell line development technology may result in highly specific productivities, titers, and improved cell growth characteristics for most protein products.

The presentation was part of PEGS’ “Optimizing Protein Expression” stream.

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SMARTag® technology platform
In a separate presentation, Robyn Barfield, Ph.D., Group Leader, Catalent Biologics discussed Catalent’s proprietary SMARTag® technology, which uses a simple, robust manufacturing process to generate stable, site-specific antibody-drug conjugates (ADCs).

In her presentation, titled “Novel SMARTag Linkers Enable Better-Tolerated ADCs,” Barfield explained how the company’s aldehyde-reactive non-cleavable, HIPS-maytansine linker/payload called RED-106 is well-tolerated and resistant to P-glycoprotein efflux, offering wider therapeutic windows as compared to other technologies. This advantage is illustrated by Catalent’s anti-HER2 RED-106 ADC in comparison to the related drug, ado-trastuzumab emtansine (T-DM1; Kadcyla®; Genentech/Roche).

During the virtual event, Barfield also presented the case study in her presentation as a poster, “A Novel HER2-Targeted Antibody-Drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-Equivalent Exposure Levels.”

Ado-trastuzumab emtansine targets human epidermal growth factor receptor 2 (HER2) via trastuzumab antibody coupled to emtansine, which inhibit microtubule formation. The drug is approved for the treatment of HER2-positive metastatic breast cancer after treatment with trastuzumab and a taxane.

In her presentation, Barfield explained that in some instances ado-trastuzumab emtansine may fail in the clinic may, which, in part, may be caused to limited systemic exposure achieved by ado-trastuzumab emtansine relative to trastuzumab because of toxicity-related dosing constraints on the ADC.[1]

Researchers at Catalent developed an anti-HER2 RED-106 trastuzumab-based ADC (called CAT-01-106) which was site-specifically conjugated to maytansine through a non-cleavable linker with a drug-to-antibody ratio (DAR) of 1.8. The DAR of this investigational agent is approximately half the average DAR of ado-trastuzumab emtansine, which comprises a mixture of antibodies variously conjugated with an average DAR of 3-4 (potentially ranging from 0 to 8 for individual species). [1]

The high DAR species present in ado-trastuzumab emtansine contribute to its toxicity and, as a result, limit its clinical dose. Catalent’s anti-HER2 RED-106 trastuzumab-based ADC showed superior in vivo efficacy compared with ado-trastuzumab emtansine at equal payload dosing and was equally or better tolerated compared with ado-trastuzumab emtansine at equal payload dosing.[1]

The presentation was part of the PEGS’s “Engineering Antibody-Drug Conjugates” stream.

Site-specific conjugation
Research has shown that site-specific conjugation approaches will help to optimize stability, pharmacokinetics, efficacy, and safety as well as improve manufacturing consistency. And while first-generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are using site-specific conjugation.

Using Catalent’s SMARTag® technology platform helps develop optimized ADCs and bioconjugates. The technology, originally developed by Redwood Bioscience (in 2014 acquired by Catalent), overcomes limitations associated with conventional protein chemistries that produce heterogeneous products with variable conjugate potency, toxicity, and stability.

By introducing a bioorthogonal aldehyde handle through the oxidation of a cysteine residue, embedded in a specific peptide sequence (CxPxR), to the aldehyde-bearing formylglycine (fGly), the SMARTag® technology platform, which uses enzymatic modification carried out by the formylglycine-generating enzyme (FGE) –  a copper-dependent posttranslational protein modifier – offers a practical and efficient chemoenzymatic solution for site-specific protein modifications. [2][3]

As a result, the technology enables site-specific, controlled drug-protein conjugation, and uses only naturally occurring modifications to proteins requiring minimal cell-line engineering.

The SMARTag® technology platform helps developers to precisely control conjugate configuration, generating ADCs with optimal efficacy, safety, and stability.

The technology has demonstrated compatibility with clinically validated ADC payloads, including maytansine, auristatin, pyrrolobenzodiazepine (PBD), and duocarmycin, couples proprietary aldehyde-based conjugation chemistry with proprietary linker chemistries that improve conjugate stability to prevent systemic drug loss, improve targeted delivery of payloads to increase potency, and are capable of counteracting multidrug resistance pathways.

Bleck G. GPEx® Boost: A Novel Approach for High-Expressing CHO Cell Line Engineering. [Presentation]
Barfield RM. Novel SMARTag Linkers Enable Better-Tolerated ADCs [Presentation]

P023: A Novel HER2-Targeted Antibody-Drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-Equivalent Exposure Levels [Poster]

[1] Barfield RM, Kim YC, Chuprakov S, et al. A Novel HER2-targeted Antibody-drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-equivalent Exposure Levels. Mol Cancer Ther. 2020;19(9):1866-1874. doi:10.1158/1535-7163.MCT-20-0190
[2] Liu J, Barfield RM, Rabuka D. Site-Specific Bioconjugation Using SMARTag® Technology: A Practical and Effective Chemoenzymatic Approach to Generate Antibody-Drug Conjugates. Methods Mol Biol. 2019;2033:131-147. doi:10.1007/978-1-4939-9654-4_10
[3] Rupniewski I, Rabuka D. Site-Specific Labeling of Proteins Using the Formylglycine-Generating Enzyme (FGE). Methods Mol Biol. 2019;2012:63-81. doi:10.1007/978-1-4939-9546-2_5

Featured image: PEGS Exhibition before the Pandemic – Bird’s Eye Overview. The 15th annual PEGS: The Essential Protein Engineering Summit brought more than 2,700 world-renowned experts, visionaries, and influencers from top pharma, biotech, academic, and government institutions to the Seaport World Trade Center in Boston, MA. The 15th annual PEGS was held April 8-12, 2019, setting record-breaking attendance. Photo courtesy: CHI/PEGS.

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