Currently, the Aptacure is cooperating with the members of HKAP by focusing on developing core technologies, including the technologies for aptamer drug discovery, the long-lasting modification technologies of aptamers, and the high-affinity modification technologies of aptamers to their targets.

Low success rate in the screening of high-affinity aptamer sequences, short elimination half-life and weak interaction of aptamers to their targets could limit the druggability of aptamers (Figure 1 and 2). Therefore, the development of the technologies to facilitate identifying aptamer candidates with high binding affinity is considered to be the rudimentary research in the aptamer drug discovery (Yu et al., Int J Mol Sci 2016; Chen et al., Int J Mol Sci 2021). The aptamer drug design against the targets are absolutely concerned with the development of two important modification technologies viz., i) long-lasting modification technology and (ii) high-affinity modification technology. (Ni et al., ACS Appl Mater Interfaces 2020; Yu et al., Acta Pharm Sin B 2022). Indeed, through cumulative technological efforts, the Aptacure has developed the following technologies, including the technologies for aptamer drug discovery, the proprietary technologies of long-lasting modification of aptamers, and the proprietary technologies of high-affinity modification technologies of aptamers to their targets.

The technology for aptamer drug discovery: The Aptacure employs experimental SELEX in combination with the artificial intelligence (AI) prediction in the screening of high-affinity aptamer sequences. Nucleic acid aptamers could be selected against particular targets from an initial random library (~10¹⁵ single-stranded oligonucleotide sequences) by SELEX to obtain an enriched pool with high affinity sequences. The enriched pool could be analyzed using high-throughput next generation sequencing (300,000 single-stranded oligonucleotide sequences). However, it is impossible to perform affinity characterization for all candidate sequences by experimental methods. Only a small number of representative sequences could be selected for affinity characterization. Unfortunately, the selection has great limitations and a high risk of missing potential sequences with high affinity, which is one of the reasons for the increased failure rate in aptamer drug screening. Affinity prediction of candidate sequences by AI algorithms could overcome the selection limitations of the representative sequences (Figure 3). The AI model could be trained using massive established data of high affinity aptamer sequences from SELEX and then used for identifying and generating new potential candidate sequences with high affinity, which could reduce the number of sequences for experimental synthesize and validation, reduce time and economic costs and thereby improve screening efficiency. With the iteration of three generations of screening technologies developed by the Aptacure, especially with AI, the sequencing depth of the enriched pool from SELEX has been significantly increased. In addition, the number of candidate sequences which could be characterized with high affinity has also been significantly increased (Figure 4).

The technology of long-lasting modification of aptamers: In practical application, aptamer is generally conjugated with polyethylene glycol (PEG) to form an average mass above the cut-off threshold of renal filtration (30–50 kDa) for extending the half-life (2~3 days). However, PEG moiety accounts for a large proportion within the PEG-aptamer conjugate, making it hard to increase the subcutaneous dosage for the aptamer moiety at a fixed subcutaneous administration volume, which dramatically limits the therapeutic potential. Meanwhile, repeated subcutaneous injections at a short interval could largely reduce the treatment compliance of the aptamer drugs. Human serum albumin (HSA), which accounts for 60% of total plasma protein, has a molecular weight of 67 kDa. HSA has large hydrophobic interface cages, which could bind some special small molecules to form molecular complexes. Mechanistically, some mentioned-above special small molecules could be used to modify aptamers to form the conjugated aptamers, which could bind HSA to form the molecular complexes with an average mass above the cut-off threshold of renal filtration for extending the half-life (Zhang et al., Front Cell Dev Biol 2022). Recently, the company research teamscreened out two small molecules with different structures (the fourth generation of long-lasting modification technology). Both the molecules were used to conjugate the sclerostin aptamer to form the conjugated sclerostin aptamer (Figure 5). According to the results of the pharmacokinetics study,the half-life of the mono-modified sclerostin aptamer is 8 days in healthy rats (The third generationof long-lasting modification technology is composed of  single molecule with high binding affinity to HSA), and dual modification could further extend the half-life of the conjugated sclerostin aptamer from 8 days to 12 days in healthy rats (Number of patent application: PCT/CN2022/082996). Besides, in healthy rats, the half-life of the PEGylated sclerostin aptamer and the marketed therapeutic sclerostin antibody romosozumab were merely 3 days. Therefore, the half-life of the fourth generation long-lastingsclerostin aptamer in healthy rats is at least 4-fold longer than either the half-life of the PEGylated sclerostin aptamer or the marketed therapeutic sclerostin antibody romosozumab (Florio et al., Nat Commun 2016)(Figure 6). The iteration of four generations ofvlong-lasting modification technology could not only increase the proportion of aptamer within the conjugate and increase the therapeutic effect at a fixed subcutaneous administration volume, but also increase the treatment compliance of the aptamer drugs (Figure 7). According to the results of the pharmacodynamics study, both the mono-modifiedsclerostin aptamer and the PEGylated sclerostin aptamer could increase bone mass and improve bone microarchitecture integrity in ovariectomy-induced osteoporotic rats at the same dosage and dosing interval. Importantly, the mono-modified sclerostin aptamer demonstrated significantly better bone anabolic potential than the PEGylated sclerostin aptamer (Figure 8). This indicates that the prolonged half-life and high proportion of aptamer within the conjugate contribute to therapeutic activity of the modified aptamer.In the fourth generation of long-lasting modification technology, which is mainly characterized by dual modification,both two small molecule derivatives for the dual modifications are too huge to be synthesized due to the high cost in both labor and time.To develop the fifth generation of long-lasting modification, Aptacure is employing its high-throughput DNA chip automation to attain a large number of binding affinity data for AI, generative and combination-optimized models (Figure 9). 

The technology of high-affinity modification of aptamers to their targets: DNA aptamers are composed of four deoxyribonucleosides (A/G/C/T), whereas antibodies are composed of twenty amino acids. The chemical diversity of DNA aptamer is much less than that of antibody, indicating the existing optimization space for the binding affinity of the aptamer screened by systematic evolution of ligands by exponential enrichment (SELEX). Hydrophobicity plays an important role in protein folding and the interactions of small molecules to receptors. Since DNA/RNA polymerase is well-tolerant to C5 modification in deoxyuridine (dU-C5), it is skillful to incorporate hydrophobic molecules into dU-C5 of the aptamer for enhancing the affinity to the target protein. In the early stage, the company selected a novel hydrophobic molecule in dU-C5 with high affinity to the protein (first generation). Then, a modified aptamer against sclerostin protein was obtained by pre-SELEX (second generation). To simulate the high-affinity, high-specificity interactions between antibody and antigen, this aptamer was further faked with amino acids by post-SELEX without interfering the intrinsical interactions between nucleobase and protein (third generation)(Figure 10). From pre-SELEX to post-SELEX, the renewal of the modification platform endows higher binding affinity of the sclerostin aptamer to sclerostin protein, which is almost 10-times higher than sclerostin antibody (Figure 11). A patent application has been filed for the aptamer high affinity modification technology (Patent is being processed by NTD Patent & Trademark Agency Ltd.). The third generation of high affinity modification technology emphasize on hydrophobic site and amino acid modification which leads to construct a number of possible types and sites in aptamers. It is impossible to characterize all the interactions of the modified aptamers to the target manually. But, by means ofthe established high-throughput DNA chip technology, the Aptacure could obtain binding ability data of aptamers with different types of modifications at different sites. Based on these data, the Aptacure could further build and train AI models to predict the affinity of the modified aptamers to their targets (Figure 12).

Reference

1. Yu, Y., Liang, C., Lv, Q., Li, D., Xu, X., Liu, B., Lu, A. & Zhang, G. (2016) Molecular selection, modification and development of therapeutic oligonucleotide aptamers. Int J Mol Sci17(3):358.
   

2. Chen, Z., Hu, L., Zhang, B., Lyu, A., Wang, Y., Yu, Y., Zhang, G. (2021) Artificial Intelligence in aptamer-target binding prediction. Int J Mol Sci. Mar 30;22(7):3605.

3. Ni, S., Zhuo, Z., Pan, Y., Yu, Y., Li, F., Liu, J., Wang, L., Wu, X., Li, D., Wan, Y., Zhang, L., Yang, Z., Zhang, BT. & Zhang, G. (2020). Recent progress in aptamer discoveries and modifications for therapeutic applications. ACS Appl Mater Interfaces 13: 9500-9519.

4. Yu, S., Li, D., Zhang, N., Ni, S., Sun, M., Wang, L., Xiao, H., Liu, D., Liu, J., Yu, Y., Zhang, Z., Yang, SYY., Zhang, S., Lu, A., Zhang, Z., Zhang, BT. & Zhang, G. (2022). Drug discovery of sclerostin inhibitors. Acta Pharm Sin B. 12(5):2150-2170.

5. Zhang, Y., Zhang, H., Chan, D W H., Ma, Y., Lu, A., Yu, S., Zhang, B., Zhang, G. (2022) Strategies for developing long-lasting therapeutic nucleic acid aptamer targeting circulating protein: the present and the future. Front Cell Dev Biol DOI: 10.3389/fcell.2022.1048148.

6. Florio, M., K. Gunasekaran, M. Stolina, X. Li, L. Liu, B. Tipton, H. Salimi-Moosavi, F. J. Asuncion, C. Li, B. Sun, H. L. Tan, L. Zhang, C. Y. Han, R. Case, A. N. Duguay, M. Grisanti, J. Stevens, J. K. Pretorius, E. Pacheco, H. Jones, Q. Chen, B. D. Soriano, J. Wen, B. Heron, F. W. Jacobsen, E. Brisan, W. G. Richards, H. Z. Ke & M. S. Ominsky. (2016). A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair. Nat Commun 7: 11505.

Figure 1. Main technical bottlenecks in aptamer drug screening

Figure 2. The bottlenecks of druggability of aptamer drugs. 

Note: MW_a is short for the molecular weight of the above mentioned unmodified aptamer; TV_r is short for the cut-off threshold of the renal filtration.

Figure 3.The Artificial intelligence algorithm aided generation of potential aptamer sequence with high affinity. 

Note: Flow 1: Model training; Flow 2: Prediction.

Figure 4. Schematic diagram of the iteration of aptamer screening technology.

Note: With the iteration of three generations of screening technologies developed by Aptacure, especially with AI, the sequencing depth of the enriched pool from SELEX has been significantly increased, and the number of candidate sequences which could be characterized with high affinity has been significantly increased.

Figure 5. The fourth generation of the long-lasting modification technology. 

Note: MW_c is short for the molecular weight of the above mentioned molecular complex; TV_r is short for the cut-off threshold of the renal filtration.

Figure 6. The four generations of long-lasting modification technology to facilitate extending the elimination half-life of the modified sclerostin aptamer. 

Note: The first generation: PEGylation; The second generation: mono-molecular modification (mono modification); The third generation: mono-molecular modification; The fourth generation: dual-molecular modification (dual modification).

Figure 7. Different modification technologies resulted in different aptamer proportion in the drug

Figure 8.The third generation long-lasting sclerostin aptamershowed significantly higher efficiency in promoting bone formation in ovariectomy-induced osteoporotic rats, in comparison the PEGylated aptamer.

Note:Themono-modified sclerostin aptamer:25 mg/kg dosage, twice weekly for eight weeks; The PEGylated aptamer: 25 mg/kg dosage, twice weekly for eight weeks.

Figure 9. Artificial intelligence-aided generation of both two derivatives and prediction of the most promising dual combinations.

Figure 10. The high-affinity modification technology of aptamers to their targets

Figure 11. The renewal Aptacure platform of the high-affinity modification technologies of aptamers to their targets. 

Notes: The binding affinity of the modified sclerostin aptamer to sclerostin protein is higher with the renewal Aptacure platform of aptamer high-affinity modification. Lower K_d value represented higher affinity.

Figure 12. The schematic diagram of AI-aided Aptacure platform for aptamer high-affinity modification.

T₀ represents hydrophobic modification; G₁, G₂ et al. represent different types of amino acid modifications.