Site-Specific Conjugation of Cytotoxic Cytolysins Could Lead to Highly Effective Antibody-Drug Conjugates

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Recent collaboration offers more heterogeneous, stable antibody drug conjugates of synthetic tubulin-binding agents shown to rapidly trigger cancer cell death.

Targeted drug delivery remains a significant goal of companies and researchers developing next-generation therapies. Antibody-drug conjugates (ADCs) offer the potential to achieve this important goal. The attachment of a cytotoxic agent (the payload) to an antibody or antibody fragment that binds a specific tumor marker enables the targeted delivery of the drug to the site of interest and avoids damage to healthy cells. To be successful, in addition to using an effective payload, the linker that connects that payload to the antibody in an ADC must be stable during delivery to, and incorporation by, the target tumor cells, but then release the payload at the appropriate time.

The recent collaboration between PolyTherics Limited, which offers a portfolio of proprietary technologies to enable the development of better biopharmaceuticals including site-specific conjugation technologies, and TUBE Pharma, a developer of natural anticancer agents, is designed to address both of these requirements.

Highly potent payload
The wide range of natural tubulysins and synthetic tubulysin derivatives developed by TUBE (referred to by TUBE as cytolysins) inhibit polymerization (or microtubule formation) of the globular protein tubulin. Because this activity is arrested throughout the cell cycle, rapid cell death, or apoptosis, occurs. They have, according to Wolfgang Richter, managing director of TUBE Pharma, been shown to exhibit very high cytotoxic activity against tumor cells in in vitro and in vivo tumor models. They are particularly active against resistant tumor cell lines and are several orders of magnitude more potent than other marketed chemotherapeutics. In addition, structure-activity relationship studies of the cytolysins have indicated that there are many possible mechanisms for conjugating them to antibodies, proteins, peptides, polymers, forms of ribonucleic acid, T-cell receptors, aptamers, nanobodies, and more.

Robust site-specific linker chemistry
ThioBridge conjugation and linker technology from PolyTherics is designed to attach cytotoxic payloads to specific sites on whole antibodies, antibody fragments and other scaffolds through reaction with disulfide bonds. According to CEO John Burt, the ThioBridge conjugation process, therefore, avoids random conjugation of the ADC payload, which would result in multiple positional isomers with potentially different bioactivity, pharmacokinetics, distribution, immunogenicity, safety, and stability profiles.


Site-specific conjugation using ThioBridge is achieved in a two-step process. First, the disulfide bond between two cysteine thiols in the antibody is reduced. Secondly, bis-alkylation enables the introduction of a three-carbon bridge to which the ThioBridge linker, already carrying the cytotoxic payload, is covalently attached. More specifically, in the alkylation step, under the reaction conditions, a leaving group departs from each sulfur compound, and then sequential Michael addition reactions take place to recreate the disulfide linkage.

It is the reconstruction of the disulfide bond that sets the ThioBridge technology apart from other established site-specific technologies that target this bond, according to Burt. “With our technology, the tertiary structure of the protein is not disrupted, and as a result, the structural integrity is retained.  On the other hand, with maleimide chemistry, for example, the disulfide bond remains broken, and recently we have been able to show that, as a consequence, ADCs prepared using this chemistry are not as stable as those obtained using ThioBridge linkers,” he explains.

In different tests using trastuzumab as the exemplar antibody, ThioBridge conjugates were found to be stable in human serum while maleimide conjugates were observed to be unstable and undergo payload de-conjugation and cross-conjugation to serum proteins, and there was evidence of antibody fragmentation, according to Burt. In addition, when the two types of conjugates were incubated in sera from relevant preclinical species, the ThioBridge conjugate was stable, while degradation was detected for the maleimide conjugate.

PolyTherics believes that the excellent serum stability and reduced heterogeneity of ThioBridge conjugates will translate into improvements in ADC efficacy and tolerability.

Developing the best payload linker combinations
PolyTherics and TUBE have collaborated in order to leverage the different expertise that each company has to offer. “By working together, we can combine the highly potent novel cytolysin payloads with our site-specific conjugation technology to create a wide range of payload/linker combinations and screen them with a specific customer antibody to identify the best possible solutions,” Burt observes. Once the most promising candidates are selected, the customer then conducts the necessary in vitro and in vivo studies against their specific targets of interest.

Alternatively, TUBE and PolyTherics can prepare specific ThioBridge-cytolysin reagents for screening by their customers.  “We believe that these ThioBridge-cytolysin payloads provide a new and very attractive option for companies developing the next generation of antibody-drug conjugates.”

Burt adds that, by forming the partnership between TUBE and PolyTherics, the relationship with customers is simplified. Rather than needing to go through two separate rounds of legal discussions, customers can sign one joint agreement, and thus it is easier for customers to use the technology, and projects can move more quickly from the discussion stage to the development phase.

Generating real interest
“The formation of our partnership with TUBE Pharma and our recent comparative results for the ThioBridge and maleimide conjugation and linker technologies is attracting a lot of interest. I can’t provide any details at this point, but we are in the midst of commercial negotiations with a number of interested parties,” Burt states. “In addition, given our successes to date, I am very optimistic about our position in the ADC landscape,” he concludes.