Dr. Yang Yi of Yantang Bio: Advancing Next-Generation Antibody-Drug Conjugates with the Proprietary YTConju™ Fucosyl Conjugation Platform

In recent years, an increasing number of bispecific or multispecific drugs have begun to play a significant role, ushering in the fourth revolutionary wave in the history of the modern pharmaceutical industry: the emergence of multispecific therapeutics. To date, several multispecific drugs have received regulatory approval, and this trend is rapidly gaining momentum.

In the booming XDC sector, the development of multispecific conjugate drugs is also progressing vigorously. However, as drug mechanisms become more complex, there is a greater need to deliver superior efficacy within an acceptable safety risk profile. In this process, mature technology platforms will be a critical factor influencing the development of multispecific drugs.

Glyco-therapy Biotechnology is leveraging its multispecific drug conjugation technology platform to empower partners in developing next-generation antibody-drug conjugates.

Glyco-therapy Biotechnology, established in July 2019, centers on its YTConju™ fucosyl conjugation technology platform. Through in-depth development and engineering of spacers, cleavable linkers, and payloads, the company has constructed a series of antibody-drug conjugates (ADCs) featuring precise drug-to-antibody ratios (DARs) of 2, 4, 8, 2+2, and 4+4, as well as diversified payloads. Compared with traditional conjugation technologies, this platform offers an efficient, rapid, and stable “one-pot” aqueous-phase reaction process. The resulting ADCs exhibit high homogeneity, hydrophilicity, and stability, do not interfere with antigen binding by the antibody, and demonstrate robust efficacy both in vitro and in vivo.

Recently, at the 2023 Second Global Innovation Summit on ADCs and Conjugated Drugs,Dr. Yang Yi, Founder and CEO of Glyco-therapy Biotechnology, delivered a keynote address titled “Construction of Multispecific Conjugate Drugs Using the One-Step Glycoconjugation Strategy via Enzymatic Chemistry (YTConju™),” providing a detailed overview of the current state of glycoconjugation strategies as well as the primary research and development directions of Glyco-therapy Biotechnology.

The following is a summary of the key points from his speech:

First, let us understand multispecific drugs. Compared with traditional small-molecule drugs or other large-molecule therapeutics, multispecific drugs emphasize the highly effective spatiotemporal integration of two distinct effectors, thereby achieving a synergistic therapeutic effect where 1+1>2. Antibody–drug conjugates (ADCs), multispecific antibodies, PROTACs, and targeted gene therapies all fall within the category of multispecific drugs.

While multispecific antibodies can be obtained through fusion expression, the vast majority of multispecific drugs cannot bypass a critical technology—conjugation. Taking antibody-drug conjugates (ADCs), a classic example of multispecific drugs, as an illustration, 15 ADC drugs have been approved for marketing to date. Among these, except for AstraZeneca’s moxetumomab pasudotox, which is a fusion protein, all others utilize amino acid residues for conjugation, representing the first generation of conjugation technology.Although the first-generation conjugation technology is highly mature, it has certain limitations. First, the conjugation sites are difficult to control, leading to heterogeneous drug-to-antibody ratio (DAR) values; second, there are certain safety concerns.

Therefore, based on the first-generation conjugation technology,Development of the Second-Generation Site-Specific Conjugation Technology: A Strategy Based on Fusion TagsBy introducing a module recognizable by specific enzymes or chemical reaction functional groups into antibodies through a specific peptide sequence or site-specific insertion of non-natural amino acids, payloads can be subsequently incorporated via enzyme catalysis or specific chemical labeling reactions. This strategy has significantly improved the homogeneity and stability of antibody-drug conjugates (ADCs).

However, this strategy requires genetic editing of the antibody, which entails extensive, time-consuming, and labor-intensive engineering efforts during the early stages of research and development. Furthermore, such modifications may significantly impact antibody yield to a certain extent.Thus, increasing attention has been directed toward whether naturally occurring groups on antibodies can be modified to circumvent these limitations, therebyGlycoconjugation Strategies Emerge.

The Fc region of antibodies typically contains a conserved N297 glycosylation site; however, due to variations in terminal modifications, multiple glycoforms often coexist, making glycan-editing-based modifications highly challenging. Consequently, many researchers have attempted to manipulate glycoforms by employing chemoenzymatic methods to functionally modify specific glycan structures, thereby achieving glycan conjugation.

The most extensively explored category to date is the glycosyltransferase-based conjugation strategy.Such strategies typically begin with in vitro enzymatic glycan remodeling to achieve glycoform homogeneity, while simultaneously transferring sugar units bearing bioorthogonal functional groups onto antibodies via glycosyltransferases. Subsequently, payloads equipped with complementary reactive groups are conjugated to the antibodies through a single bioorthogonal reaction (e.g., click chemistry). The most well-known example of this approach is GlycoConnect™ technology from Synaffix. Currently, glycosyltransferase-based conjugation strategies are limited by the enzyme’s substrate compatibility and catalytic activity, allowing only small-molecule reactive functional groups to be directly introduced onto antibodies. Consequently, these approaches are restricted to a “two-step” conjugation strategy.This strategy has the following drawbacks:1) The conjugation process is relatively complex; 2) Payloads bearing bioorthogonal reactive functional groups usually exhibit high hydrophobicity; 3) Residual functional groups from the bioorthogonal reaction are inevitably introduced into the conjugation products, which are typically highly hydrophobic and possess potential immunogenicity.

Another commonly used strategy involves the use of endoglycosidases and their mutants.Endoglycosidases can recognize the biantennary structure on antibodies and cleave the glycan chain after the core N-acetylglucosamine. By mutating key sites critical for endoglycosidase activity, the enzyme can be engineered to possess glycosyltransferase activity, enabling it to recognize and transfer a polysaccharide or disaccharide substrate onto an antibody that retains only the core N-acetylglucosamine. The synthesis of polysaccharide substrates in this approach is relatively complex and therefore presents certain challenges. Furthermore, current conjugation strategies are limited in the diversity of drug-to-antibody ratio (DAR) design, as each glycan substrate can typically be used to prepare ADCs with only a specific DAR value (usually DAR2).

To address the issues associated with existing glycosylation coupling technologies,YanTang Biotech has independently developed a fucosyltransferase-based antibody-drug conjugation technology platform, which enables the “one-step” direct conjugation of payloads of various molecular sizes onto antibodies. Furthermore, by utilizing a glycan substrate, this approach facilitates the construction of molecules with diverse drug-to-antibody ratios (DARs).

Next, I will discuss the specific research and attempts undertaken by Glyco-therapy Biotechnology.

Glyco-therapy Biotechnology has independently developed the multi-specific drug conjugation technology platform YTConju™, which enables efficient and stable conjugation of small molecules (ASC), peptides and proteins (APC), oligonucleotides (AOC), and other drug moieties to antibodies featuring an Fc region via a “one-step” enzymatic catalysis reaction.

Under this platform, we first attempted to base on G₂Design of the DAR4 Structure for F. Following structural optimization of fucose derivatives and engineering of fucosyltransferase activity, Glyco-therapy Biotechnology significantly enhanced the rate and efficiency of the conjugation reaction, thereby yielding a homogeneous DAR4 product. Furthermore, we aimed to construct DAR2 molecules based on the same substrate structure. To this end, we employed in vitro enzymatic chemical editing to install two core LacNAc structures (termed A-LacNAc) in situ on the asparagine residues of the antibody. This approach enabled the construction of DAR2 molecules. Unexpectedly and gratifyingly, the efficiency of fucosyltransferase was markedly improved on the A-LacNAc structure. In G₂For the DAR4 structure of F, complete conversion may require 8–16 hours, whereas for the DAR2 structure of A-LacNAc, complete conversion was achieved within 5 minutes.

Currently, both the DAR4 and DAR2 molecular constructs of Glyco-therapy Biotechnology can be manufactured using a “one-pot, one-step” process. This procedure does not require the addition of organic solvents, making it a highly streamlined and stable manufacturing process.

Furthermore, by branching fucose derivatives, we can further increase the drug payload, thereby enabling the construction of antibody-drug conjugates (ADCs) with high drug-to-antibody ratios (DAR). For instance, ADC molecules with a DAR of 4+4 (8) can be constructed based on a DAR4 scaffold, and those with a DAR of 2+2 (4) can be built upon a DAR2 scaffold. Due to the presence of highly hydrophilic functional groups in the fucose derivatives, the branched fucose derivatives retain excellent water solubility, thus preventing aggregate formation.

Under this design, the antibody conjugates we obtained all exhibit highly homogeneous, hydrophilic, and stable characteristics, along with excellent in vitro and in vivo efficacy.

Dual payloads are also a key area of focus for Glyco-therapy Biotechnology., as it represents a crucial strategy for overcoming tumor drug resistance in the future. In fact, the concept of dual payloads has been explored for quite some time, but its implementation poses significant challenges, particularly from the perspective of drug construction. First, combining different payloads within the same molecular system increases the difficulty of conjugation due to hydrophobicity issues; second, the resulting conjugates tend to be less stable.

On the YTConju™ platform, the presence of highly hydrophilic structures on fucose derivatives provides a distinct advantage for developing dual-payload antibody-drug conjugates (ADCs). Accordingly, we designed a DAR2+2 ADC molecule based on a DAR2 architecture, incorporating two microtubule inhibitors and two topoisomerase inhibitors. Characterization data demonstrate that this dual-payload ADC exhibits high homogeneity and high hydrophilicity, with minimal hydrophobic shift even compared to the naked antibody. Furthermore, this ADC molecule displays excellent plasma stability.

To enable more pharmaceutical industry professionals to access Glyco-therapy’s YTConju more easily^TMTechnology, to experience the product strength and convenience our technology brings to everyone in early-stage R&D,We jointly launched AGLink with ACROBiosystems.^TMSeries of Conjugation Kits.Currently, we have launched MMAE-based kits for DAR2 and DAR4. Although these kits utilize an earlier version of our manufacturing process, they still enable the construction of ADC molecules via a “one-pot, one-step” approach. Our current process offers shorter processing times and greater operational convenience, and we look forward to continuously updating and iterating with superior products and technologies in the future.

Next, in addition to the conjugation of small-molecule payloads and antibodies in the traditional sense,We have partnered with Professor Li Jie’s team at Nanjing University to further expand the YTConju™ technology platform to the conjugation of antibodies and biological macromolecules.We achieved efficient conjugation of antibodies with peptides and proteins, thereby preparing non-fused bispecific T-cell engagers with unique spatial structures that simultaneously target CD3 and Her2. Furthermore, we accomplished efficient and homogeneous conjugation of antibodies with oligonucleotides of varying lengths (20 nt to 106 nt), and the related research findings were published in Agnew (https://doi.org/10.1002/anie.202308174）。

In conclusion, the YTConju™ technology platform not only enables efficient conjugation via a “one-pot” approach but also yields products with superior hydrophilicity, homogeneity, thermal stability, plasma stability, and efficacy. Furthermore, it offers greater flexibility in DAR selection and molecular design. Glyco-therapy Biotechnology aims to leverage the YTConju™ platform to help more partners develop highly competitive multispecific drugs more efficiently and conveniently.