Synaffix, a Netherlands-based biotechnology company exclusively focusing on the continued advancement of best-in-class and industry leading antibody-drug conjugate (ADC) technology platforms, has competed a new set of preclinical studies that further supports the potential for technology developed by the company to enable safer and more effective targeted cancer therapeutics.

Antibody-drug conjugates use monoclonal antibodies (mAb) to deliver highly potent cytotoxic chemotherapeutic agents targeted to specific tumor cells.  Compared to unconjugated cytotoxic chemotherapy, antibody-drug conjugates improve the therapeutic index or TI, the relationship between (the highest) exposure of a cytotoxic agent and the therapeutic dose, of an anti-cancer agent.[1]  In drug discovery and development, this pharmacodynamic parameter is relevant because it establishes how safe (or toxic) a specific drug is, leading to an appropriately balanced safety–efficacy profile for a given indication.

…[New] experimental findings… highlight the potential…  to address the persistent unmet medical need across a wide variety of cancer types…

Traditional conjugation process
Today, the majority of antibody-drug conjugates in the clinic are based on the conjugation of payload to naturally available amino acid side-chains (i.e. lysine, cysteine). While the majority of these ADCs use a linkage to the thiol of cysteines on the antibody, a lesser number of these drug candidates utilize chemistry to surface lysines of the antibody. This generally results in a stochastic distribution of drug−antibody ratio or DAR between 0 and 8. [1][2]

Figure 1.0: With a suite of advanced tools for the design and development of antibody-drug conjugates Synaffix offers scientists a technology that can be used to develop ADCs targeted against a wide variety of cancer types. Courtesy: © Synaffix BV, The Netherlands.  Used with permission.

Emerging technologies
A better understanding of conjugation chemistry and the underlying biologies have helped scientists advance the technologies used in the development of first- and second-generation antibody-drug conjugates, leading to new approaches, including considerable emphasis on site-specific conjugation, to ensure homogenous ADCs with well-defined DARs. [3][4]

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New site-specific approaches being developped not only increase the homogeneity of antibody-drug conjugates, they also enable novel bio-orthogonal chemistries* based on reactive moieties other than thiol or amine.  In turn, these novel approaches broaden the diversity of linkers used, leading to better linker design for future generations of ADCs. [2][8]

Among these emerging technologies is Synaffix’s novel, proprietary, approach, which, for example, includes a robust, generally applicable, nongenetic technology designed to convert monoclonal antibodies into stable and homogeneous ADCs.[4]

The data in Synafix’ preclinical studies, based on the company’s latest R&D efforts, demonstrates that the company’s proprietary platform technologies, GlycoConnect™ and HydraSpace™, generate ADCs with significantly improved therapeutic index when directly compared to brentuximab vedotin (Adcetris®; Seattle Genetics/Takeda) and ado-trastuzumab emtansine (Kadcyla®; Genentech/Roche), two approved ADCs for the treatment of multiple lymphoma indications and HER2-positive breast cancer, respectively.[5]

Figure 2.0: GlycoConnect™ chemoenzymatic antibody remodeling and conjugation process developed by Synaffix. Courtesy: © Synaffix BV, The Netherlands.  Used with permission.

Technology Platforms
GlycoConnect, Synaffix’ robust chemoenzymatic technology platform, is a site-specific and stable antibody conjugation technology that allows for efficient antibody-to-ADC conversion by anchoring a payload to the antibody’s glycan at asparagine-297 (Asn-297).

Human immunoglobulins, including Immunoglobulin G (IgG), the most prominent antibody in humans, are mainly glycosylated at the asparagine residue at position 297 (Asn 297) of the heavy chain CH2 domain.

Synaffix’ process involves proprietary enzymatic N-glycan remodeling with one azide, forming an anchor point for the copper-free click attachment of a cytotoxic payload.

The antibody remodeling includes a two-step approach.  In the first step the enzyme trims the glycan. In the second step the enzyme installs a proprietary small molecule substrate bearing a functional handle, which will be used during subsequent conjugation. In this process, the proprietary azide-based enzymatic substrate preserves the high reactivity and selectivity afforded by conjugation via click chemistry.

Since aromatically stabilized structures typically come with an order of magnitude higher stability than non-aromatic structures, the aromatically-stabilized triazole linker, formed during the second reaction, offers improved safety and efficacy profiles. As a result, a higher level of cytotoxic payloads reach the targeted cancer cell, increasing efficacy, while minimizing the potential of premature detachment in circulation and improving the safety profile.

Enhancing therapeutic index
GlycoConnect was shown to be capable of significantly enhancing the therapeutic index of an antibody-drug conjugate on its own.

Using novel proprietary linkers, the highly polar properties of HydraSpace-technology, an ADC-enhancing spacer technology which enables conjugation of highly hydrophobic payloads, improves the solubility and stability of the payload.

One of the challenges in the development of antibody-drug conjugates, is that most of the cytotoxic payloads are hydrophobic. As a result, linking them to a monoclonal antibody with an additional hydrophobic moiety may create problems due to aggregation. [6]

Since ADC aggregates are insoluble, such aggregates can limit achievable drug loading onto the antibody. In addition, some studies suggest that ADC aggregates are sequestered in the liver, leading to hepatotoxicity and is linked to increased immunogenicity.[6]

However, in contrast to standard approaches, the highly polar properties of HydraSpace-technology enables the most challenging hydrophobic payloads to be efficiently conjugated to antibodies.

According to Synaffix’ s scientists, the result is the generation of stable ADCs at the desired drug-to-antibody ratio (a homogeneous DAR 2 or DAR 4).  This novel technology makes it possible to increase drug loading using two different drugs with varying mechanism of action (MOA) to be incorporated into a single therapeutic ADC (DAR 2+2 “dual-payload” ADCs) by a single conjugation event. [4][7]

Metal-free Click Chemistry
Metal-free click chemistry is widely used by researchers in pharma, biotech and academia.  This chemistry offers a unique capability for rapid, selective and stable conjugation of complex macromolecules.

Synaffix’ scientists have extensively optimized metal-free click chemistry between cyclooctynes and azides in conjunction with GlycoConnect for conjugation of potent cytotoxins site-specifically to antibodies.

The resulting antibody-drug conjugates feature an aromatically stabilized triazole. Given its high stability, the resulting chemistry provides a powerful alternative to cysteine-maleimide conjugation chemistry currently used in the majority of antibody-drug conjugates in the clinic.

Furthermore, pre-clinical studies have demonstrated the versatility of GlycoConnect across a range of different antibody isotypes and linker-to-payload combinations as well as excellent in vivo efficacy and high tolerability.  This approach paves the way for the next generation of antibody-drug conjugates with an improved therapeutic index.[7]

Synaffix’ scientists were also able to seamlessly upscale GlycoConnect, further confirming manufacturability.

Key metric
“Improvement in the therapeutic index is a key metric in the quest for superior ADCs. What is exciting about our technology is that we can now consistently demonstrate in preclinical models of both liquid and solid tumors that if we connect the same antibody and payload from each [of the] commercially-available ADC products using our proprietary technology, we are able to increase the efficacy of the drug as well as its safety and tolerability,” noted Floris van Delft, PhD, co-founder and Chief Scientific Office at Synaffix.

“Our experimental findings to date … highlights the potential of our technology to address the persistent unmet medical need across a wide variety of cancer types,” Van Delft added.

Note: * Coined by Carolyn R. Bertozzi, the term bio-orthogonal chemistries refer to chemical reactions occurring inside of living systems without interfering. These reactions neither interact with nor interfere with native biochemical processes. [8]


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