Apr 01, 2025

Antibody-recruiting strategy
(Glycoforum. 2025 Vol.28 (2), A9)
DOI: https://doi.org/10.32285/glycoforum.28A9

Yoshiyuki Manabe / Koichi Fukase

真鍋 良幸

Yoshiyuki Manabe
Associate Professor, Graduate School of Science, Osaka University
Dr. Yoshiyuki Manabe received his Ph.D. in 2011 from Tohoku University under the supervision of Prof. Minoru Ueda. From 2011 to 2012, he researched natural product synthesis with Prof. Tohru Oishi at Kyushu University. In 2012, he became an assistant professor at Osaka University, started work on glycochemistry with Prof. Koichi Fukase, and was promoted to associate professor in 2024. His research interests include synthetic chemistry, chemical biology, glycoscience, and immunology.

深瀬 浩一

Koichi Fukase
Professor, Graduate School of Science, Osaka University
Dr. Koichi Fukase received his Ph.D. in 1987 from Osaka University under the supervision of Prof. Tetsuo Shiba. In 1988 after his postdoctoral tenure, he was appointed assistant professor at Osaka University. In 1996 he was promoted to lecturer, and in 1998, he was promoted to associate professor. In 2004, he was appointed professor in the same department. His research interests include synthetic chemistry, glycoscience, radiochemistry, medicinal chemistry, and immunology.

Abstract

Antigen glycans provoke acute immune responses due to the prevalence of abundant natural antibodies against these glycans in the body. Therefore, by labeling pathogenic sites with antigen glycans that lead to recruitment of natural antibodies against these glycans and subsequent stimulation of immune responses, diseases can be treated. This innovative therapeutic approach, termed the antibody-recruiting strategy, has attracted substantial attention. This paper introduces recent advancements in this antibody-recruiting strategy, with focus on our research efforts.

1. Introduction

Antigen glycans whose antibodies are abundantly present in the body play a critical role in immune responses (Figure 1)1-4. A well-known example is the ABO blood group antigens expressed on the surface of red blood cells5. When these glycans are recognized by their corresponding natural antibodies, they trigger blood agglutination, leading to rejection reactions in case of blood transfusion incompatibility. Furthermore, although the α-gal epitope is widely distributed in nature, humans cannot produce this glycan structure. Instead, humans possess high levels of antibodies against α-gal, resulting in rejection reactions during xenotransplantation involving pig-derived organs6-8. Similarly, humans possess substantial amounts of antibodies against the α-rhamnose (α-Rha) structure9-11. These antigen glycans hold potential for therapeutic applications.

図1
Figure 1. Structure of antigen glycans

The antibody-recruiting molecules, originally proposed by Spiegel et al., are bifunctional molecules designed to induce immune responses against pathogenic cells (Figure 2)12-14. These molecules consist of two functional components: a target-binding terminus that specifically recognizes pathogenic cells and an antibody-binding terminus that interacts with endogenous natural antibodies. These molecules trigger targeted immune responses against the pathogenic cells by recruiting natural antibodies to the pathogenic sites. The aforementioned antigen glycans represent highly promising candidates for the antibody-binding domain. Specifically, by employing antigen glycans as antibody-recruiting termini, their abundant natural antibodies can be harnessed to elicit robust immune responses against target pathogenic cells. Here, we introduce the antibody-recruiting strategy, an innovative therapeutic approach that leverages the potent immune responses elicited by these antigen glycans.

図2
Figure 2. Concept of an antibody-recruiting molecule

2. Antibody-based antibody recruiting molecules

We developed antibody-recruiting molecules that utilize an antibody as the target-binding terminus and α-gal as the antibody-binding terminus (Figure 3)15. Antibodies are widely used in the treatment of various diseases due to their high specificity for targets, and they also function as vehicles for drug delivery, such as in antibody-drug conjugates16-18. Therefore, we employed antibodies as target-binding terminus to achieve precise and highly specific targeting. Meanwhile, α-gal was selected as the antibody-binding terminus because of its ability to provoke acute immune responses, such as hyperacute rejection in xenotransplantation6-8, thus anticipating the high efficacy.

We initially synthesized and evaluated the first-generation α-gal-antibody conjugates (Figure 3b). In this approach, the reducing end of chemically synthesized α-gal19 was activated with N-hydroxysuccinimide (NHS) ester, which was subsequently conjugated to an anti-CD20 antibody, widely used in the treatment of B-cell lymphoma. By varying the α-gal concentration in the conjugation reactions, different numbers of α-gal epitopes were introduced onto the antibody. The immune response, specifically the complement-dependent cytotoxicity (CDC), induced by the resultant α-gal-antibody conjugates, was then assessed. As anticipated, significant CDC activity was observed, which increased in a dose-dependent manner with respect to the α-gal loading ratio. Notably, the potency of antibody was markedly enhanced by this simple modification, i.e., introduction of α-gal.

Furthermore, we designed and evaluated the second-generation α-gal-antibody conjugates (Figure 3c). Based on the α-gal-loading-ratio-dependent efficacy observed in the above experiments, we dendrimerized α-gal and conjugated the resulting α-gal dendrimer with the antibody. Specifically, we synthesized 8-mer and 16-mer α-gal dendrimers and selectively introduced them onto the cysteine residues of the hinge region of the half-antibody, obtained through the reduction of the antibody20-22. This approach enabled the preparation of homogeneous α-gal-antibody conjugates with a high level of α-gal. As expected, the second-generation α-gal-antibody conjugates induced CDC activity, with activity directly correlated to the number of introduced α-gal units. By employing glycan dendrimers, we successfully exploited the multivalent effect to enhance the efficiency of antibody-recruitment.

図3
Figure 3. Immune induction using α-gal-antibody conjugates. a) Experimental scheme of immune induction using α-gal-antibody conjugates. b) Complement-dependent cytotoxic (CDC) activity of 1st generation α-gal-antibody conjugates. c) CDC activity of 2nd generation α-gal-antibody conjugates.

3. Practical antibody recruiting by a combined metabolic glycan engineering and caging strategy

We further explored an antibody-recruiting strategy by presenting antigen glycans on the pathogenic cell surface via the metabolic glycan labeling23-25 (Figure 4)26. In this approach, an azide-containing sugar27,28 is metabolically incorporated into cell surface glycans, which are then conjugated to the azide via click chemistry29,30. These conjugates recruit natural antibodies to trigger the targeted immune responses31-35. This method covalently introduces the antigen glycan onto the cell surface, with the expectation of enhanced immune induction activity.

To develop a “practical” antibody-recruiting method, we integrated a caging strategy36. When applying the aforementioned antibody-recruiting strategy in promiscuous environments, such as in vivo systems, there is a concern that the antigen glycan could be captured by the abundant natural antibodies present in the body, potentially interfering with the glycan-introduction step via click chemistry. To address this issue, we employed a caging strategy. In this approach, the caged antigen glycan, whose activity is temporarily masked, is introduced onto the cell surface. The caged antigen glycan is then uncaged by external stimuli, such as light irradiation, to activate its function. Because the caged antigen glycan is not recognized by its antibodies, this method is expected to facilitate the efficient progression of click chemistry on the cell surface, even in the presence of its antibodies. Additionally, this approach enables the spatiotemporal control of immune induction, offering the potential to minimize the risk of side effects.

図4
Figure 4. Antibody-recruiting by combinatorial use of a metabolic glycan labeling and caging strategy

Here, we selected α-Rha9-11 as the antigen glycan and synthesized caged α-Rha (Figure 5a). To ensure the water solubility and precise activity control of caged α-Rha, we introduced a photolabile protective group onto only one of the three hydroxyl groups of α-Rha. After optimizing the reaction conditions, we achieved selective introduction of the protecting group at the 3-position of α-Rha using 4-dimethylaminopyridine (DMAP) as a catalyst. As intended, the synthesized caged α-Rha underwent smooth deprotection upon UV-irradiation.

We then performed the immune induction assay using the caged α-Rha (Figure 5b). Two assay systems were employed to compare the activities of α-Rha and caged α-Rha; α-Rha (or caged α-Rha) was introduced via click chemistry in the absence or presence of human serum containing anti-α-Rha antibodies (Figure 5b; Methods A and B: Method B simulates promiscuous conditions). When α-Rha was used, CDC activity was significantly reduced under Method B, indicating that, α-Rha was captured by its antibodies, thereby inhibiting its introduction onto the cell surface via click chemistry. In contrast, when caged α-Rha was employed, comparable CDC activity was observed under both conditions. Notably, when light irradiation was used in Method B, caged α-Rha exhibited higher activity than α-Rha. These results demonstrate that caged α-Rha evades sequestration by natural antibodies, facilitating the efficient α-Rha introduction onto the cell surface via click chemistry and thereby enhancing the practicality of an antibody-recruiting strategy.

図5
Figure 5. Immune induction using metabolic glycan labeling integrated with caging strategy. a) Synthesis and uncaging of caged α-Rha. b) Evaluation of the effect of caging strategy on antibody recruiting activity. α-Rha, α-rhamnose; DMAP, 4-dimethylaminopyridine; Pyr., pyridine; ManNAz, N-azidoacetylmannosamine tetraacylated; CDC, complement-dependent cytotoxicity.

4. Concluding remarks

The antibody-recruiting strategy represents a promising therapeutic approach that harnesses the patient’s own immune system for disease treatment. This strategy not only induces acute immune responses by recruiting endogenous natural antibodies to the pathogenic site but also holds the potential to stimulate acquired immunity11,37-42. Various modalities have been proposed as antibody-recruiting molecules. Here, we have highlighted methods using the antibody, and other reported approaches utilizing small molecules43-45, which may enable the development of orally administrable antibody-recruiting therapeutics. Furthermore, while we introduced glycan dendrimers as an efficient antibody binding terminus, recent studies have also explored the use of glycopolymers33,46-49. On the other hand, the primary concern in the practical application of antibody recruiting-strategies lies in mitigating adverse effects caused by interactions with the abundant natural antibodies present in the body. The caging strategy presented here offers a promising solution to this issue. Continued refinement of the molecular designs to maximize therapeutic efficacy while minimizing adverse effects is expected to facilitate the practical application of antibody-recruiting strategies.


References

  1. Dotan, N.; Altstock, R. T.; Schwarz, M.; Dukler, A., Anti-glycan antibodies as biomarkers for diagnosis and prognosis. Lupus 200615 (7), 442-450.
  2. Huflejt, M. E.; Vuskovic, M.; Vasiliu, D.; Xu, H.; Obukhova, P.; Shilova, N.; Tuzikov, A.;  Galanina, O.; Arun, B.; Lu, K.; Bovin, N., Anti-carbohydrate antibodies of normal sera: Findings, surprises and challenges. Mol. Immunol. 200946 (15), 3037-3049.
  3. Bovin, N.; Obukhova, P.; Shilova, N.; Rapoport, E.; Popova, I.; Navakouski, M.;  Unverzagt, C.; Vuskovic, M.; Huflejt, M., Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies? Biochim. Biophys. Acta - Gen. Subj. 20121820 (9), 1373-1382.
  4. Kappler, K.; Hennet, T., Emergence and significance of carbohydrate-specific antibodies. Genes Immun. 202021 (4), 224-239.
  5. Landsteiner, K., Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Z. Bakteriol. 190027, 357-362.
  6. Galili, U.; Rachmilewitz, E. A.; Peleg, A.; Flechner, I., A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J. Exp. Med. 1984160 (5), 1519-1531.
  7. Galili, U.; Shohet, S. B.; Kobrin, E.; Stults, C. L.; Macher, B. A., Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J. Biol. Chem. 1988263 (33), 17755-17762.
  8. McMorrow, I. M.; Comrack, C. A.; Sachs, D. H.; DerSimonian, H., Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(α1,3)Galactose epitope. Transplantation 199764 (3), 501-510.
  9. White-Scharf, M. E.; Rosenberg, L. T., Evidence that L-rhamnose is the antigenic determinant of hyporesponsiveness of BALB/c mice to Klebsiella pneumoniae type 47. Infect. Immun. 197822 (1), 18-21.
  10. Prakobphol, A.; Linzer, R.; Genco, R. J., Purification and characterization of a rhamnose-containing cell wall antigen of Streptococcus mutans B13 (serotype d). Infect. Immun. 198027 (1), 150-157.
  11. Chen, W.; Gu, L.; Zhang, W.; Motari, E.; Cai, L.; Styslinger, T. J.; Wang, P. G., L-rhamnose antigen: A promising alternative to α-gal for cancer immunotherapies. ACS Chem. Biol. 20116 (2), 185-191.
  12. Zhang, A. X.; Murelli, R. P.; Barinka, C.; Michel, J.; Cocleaza, A.; Jorgensen, W. L.; Lubkowski, J.; Spiegel, D. A., A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules. J. Am. Chem. Soc. 2010132 (36), 12711-12716.
  13. McEnaney, P. J.; Parker, C. G.; Zhang, A. X.; Spiegel, D. A., Antibody-recruiting molecules: An emerging paradigm for engaging immune function in treating human disease. ACS Chem. Biol. 20127 (7), 1139-1151.
  14. Achilli, S.; Berthet, N.; Renaudet, O., Antibody recruiting molecules (ARMs): Synthetic immunotherapeutics to fight cancer. RSC Chem. Biol. 20212 (3), 713-724.
  15. Sianturi, J.; Manabe, Y.; Li, H.-S.; Chiu, L.-T.; Chang, T.-C.; Tokunaga, K.; Kabayama, K.; Tanemura, M.; Takamatsu, S.; Miyoshi, E.; Hung, S.-C.; Fukase, K., Development of α-gal–antibody conjugates to increase immune response by recruiting natural antibodies. Angew. Chem. Int. Ed. 201958 (14), 4526-4530.
  16. Wu, A. M.; Senter, P. D., Arming antibodies: Prospects and challenges for immunoconjugates. Nat. Biotechnol. 200523 (9), 1137-1146.
  17. Ducry, L.; Stump, B., Antibody−drug conjugates: Linking cytotoxic payloads to monoclonal antibodies. Bioconjug. Chem. 201021 (1), 5-13.
  18. Chari, R. V. J.; Miller, M. L.; Widdison, W. C., Antibody–drug conjugates: An emerging concept in cancer therapy. Angew. Chem. Int. Ed. 201453 (15), 3796-3827.
  19. Tsutsui, M.; Sianturi, J.; Masui, S.; Tokunaga, K.; Manabe, Y.; Fukase, K., Efficient synthesis of antigenic trisaccharides containing N-acetylglucosamine: Protection of NHAc as NAc2. Eur. J. Org. Chem. 20202020 (12), 1802-1810.
  20. Tanaka, K.; Siwu, E. R. O.; Minami, K.; Hasegawa, K.; Nozaki, S.; Kanayama, Y.; Koyama, K.; Chen, W. C.; Paulson, J. C.; Watanabe, Y.; Fukase, K., Noninvasive imaging of dendrimer-type N-glycan clusters: In vivo dynamics dependence on oligosaccharide structure. Angew. Chem. Int. Ed. 201049 (44), 8195-8200.
  21. Farabi, K.; Manabe, Y.; Ichikawa, H.; Miyake, S.; Tsutsui, M.; Kabayama, K.; Yamaji, T.; Tanaka, K.; Hung, S.-C.; Fukase, K., Concise and reliable syntheses of glycodendrimers via self-activating click chemistry: A robust strategy for mimicking multivalent glycan–pathogen interactions. J. Org. Chem. 202085 (24), 16014-16023.
  22. Manabe, Y.; Tsutsui, M.; Hirao, K.; Kobayashi, R.; Inaba, H.; Matsuura, K.; Yoshidome, D.; Kabayama, K.; Fukase, K., Mechanistic studies for the rational design of multivalent glycodendrimers. Chem. Eur. J. 202228 (61), e202201848.
  23. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R., Chemical remodelling of cell surfaces in living animals. Nature 2004430 (7002), 873-877.
  24. Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R., In vivo imaging of membrane-associated glycans in developing zebrafish. Science 2008320 (5876), 664-667.
  25. Wang, H.; Mooney, D. J., Metabolic glycan labelling for cancer-targeted therapy. Nat. Chem. 202012 (12), 1102-1114.
  26. Milawati, H.; Manabe, Y.; Matsumoto, T.; Tsutsui, M.; Ueda, Y.; Miura, A.; Kabayama, K.; Fukase, K., Practical antibody recruiting by metabolic labeling with caged glycans. Angew. Chem. Int. Ed. 202362 (25), e202303750.
  27. Saxon, E.; Bertozzi, C. R., Cell surface engineering by a modified staudingerfeaction. Science 2000287 (5460), 2007-2010.
  28. Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C. R., Investigating cellular metabolism of synthetic azidosugars with the staudinger ligation. J. Am. Chem. Soc. 2002124 (50), 14893-14902.
  29. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R., A strain-promoted [3 + 2] azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 2004126 (46), 15046-15047.
  30. Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L., Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem. Commun. 201046 (1), 97-99.
  31. Li, S.; Yu, B.; Wang, J.; Zheng, Y.; Zhang, H.; Walker, M. J.; Yuan, Z.; Zhu, H.; Zhang, J.; Wang, P. G.; Wang, B., Biomarker-basedmetabolic labeling for redirected and enhanced immune response. ACS Chem. Biol. 201813 (6), 1686-1694.
  32. Lin, B.; Wu, X.; Zhao, H.; Tian, Y.; Han, J.; Liu, J.; Han, S., Redirecting immunity via covalently incorporated immunogenic sialic acid on the tumor cell surface. Chem. Sci. 20167 (6), 3737-3741.
  33. Uvyn, A.; De Coen, R.; De Wever, O.; Deswarte, K.; Lambrecht, B. N.; De Geest, B. G., Cell surface clicking of antibody-recruiting polymers to metabolically azide-labeled cancer cells. Chem. Commun. 201955 (73), 10952-10955.
  34. Goyard, D.; Diriwari, P. I.; Berthet, N., Metabolic labelling of cancer cells with glycodendrimers stimulate immune-mediated cytotoxicity. RSC Med. Chem. 202213 (1), 72-78.
  35. Dzigba, P.; Rylski, A. K.; Angera, I. J.; Banahene, N.; Kavunja, H. W.; Greenlee-Wacker, M. C.; Swarts, B. M., Immune targeting of mycobacteria through cell surface glycan engineering. ACS Chem. Biol. 202318 (7), 1548-1556.
  36. Ellis-Davies, G. C. R., Caged compounds: Photorelease technology for control of cellular chemistry and physiology. Nat. Methods 20074 (8), 619-628.
  37. LaTemple, D. C.; Henion, T. R.; Anaraki, F.; Galili, U., Synthesis of alpha-galactosyl epitopes by recombinant alpha1,3galactosyl transferase for opsonization of human tumor cell vaccines by anti-galactose. Cancer Res. 199656 (13), 3069-3074.
  38. LaTemple, D. C.; Abrams, J. T.; Zhang, S. Y.; Galili, U., Increased immunogenicity of tumor vaccines complexed with anti-Gal: Studies in knockout mice for alpha1,3galactosyltransferase. Cancer Res. 199959 (14), 3417-3423.
  39. Rossi, G. R.; Mautino, M. R.; Unfer, R. C.; Seregina, T. M.; Vahanian, N.; Link, C. J., Effective treatment of preexisting melanoma with whole cell vaccines expressing α(1,3)-galactosyl epitopes. Cancer Res. 200565 (22), 10555-10561.
  40. Deguchi, T.; Tanemura, M.; Miyoshi, E.; Nagano, H.; Machida, T.; Ohmura, Y.;  Kobayashi, S.; Marubashi, S.; Eguchi, H.; Takeda, Y.; Ito, T.; Mori, M.; Doki, Y.; Sawa, Y., Increased immunogenicity of tumor-associated antigen, mucin 1, engineered to express alpha-gal epitopes: A novel approach to immunotherapy in pancreatic cancer. Cancer Res. 201070(13), 5259-5269.
  41. Sarkar, S.; Lombardo, S. A.; Herner, D. N.; Talan, R. S.; Wall, K. A.; Sucheck, S. J., Synthesis of a single-molecule L-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-L-rhamnose antibodies. J. Am. Chem. Soc. 2010132 (48), 17236-17246.
  42. Sarkar, S.; Salyer, A. C. D.; Wall, K. A.; Sucheck, S. J., Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into L-rhamnose displaying liposomes. Bioconjug. Chem. 201324 (3), 363-375.
  43. Rullo, A. F.; Fitzgerald, K. J.; Muthusamy, V.; Liu, M.; Yuan, C.; Huang, M.; Kim, M.; Cho, A. E.; Spiegel, D. A., Re-engineering the immune response to metastatic cancer: Antibody-recruiting small molecules targeting the urokinase receptor. Angew. Chem. Int. Ed. 201655 (11), 3642-3646.
  44. Liet, B.; Laigre, E.; Goyard, D.; Todaro, B.; Tiertant, C.; Boturyn, D.; Berthet, N.; Renaudet, O., Multifunctional glycoconjugates for recruiting natural antibodies against cancer cells. Chem. Eur. J. 201925 (68), 15508-15515.
  45. Sasaki, K.; Harada, M.; Miyashita, Y.; Tagawa, H.; Kishimura, A.; Mori, T.; Katayama, Y., Fc-binding antibody-recruiting molecules exploit endogenous antibodies for anti-tumor immune responses. Chem. Sci. 202011 (12), 3208-3214.
  46. Uvyn, A.; De Coen, R.; Gruijs, M.; Tuk, C. W.; De Vrieze, J.; van Egmond, M.; De Geest, B. G., Efficient innate immune killing of cancer cells triggered by cell-surface anchoring of multivalent antibody-recruiting polymers. Angew. Chem. Int. Ed. 201958 (37), 12988-12993.
  47. De Coen, R.; Nuhn, L.; Perera, C.; Arista-Romero, M.; Risseeuw, M. D. P.; Freyn, A.; Nachbagauer, R.; Albertazzi, L.; Van Calenbergh, S.; Spiegel, D. A.; Peterson, B. R.; De Geest, B. G., Synthetic rhamnose glycopolymer cell-surface receptor for endogenous antibody recruitment. Biomacromolecules 202021 (2), 793-802.
  48. Mu, W.; Chen, Y.; Zhong, Z.; Louage, B.; Lauwers, H.; Devoogdt, N.; Haustraete, J.; De Geest, B. G., HER2 nanobody-poly(rhamnose) conjugates efficiently recruit anti-rhamnose antibodies from serum to the surface of HER2-expressing cells. Chem. Mater. 202436 (20), 10113-10124.
  49. Ou, C.; Prabhu, S. K.; Zhang, X.; Zong, G.; Yang, Q.; Wang, L.-X., Synthetic antibody-rhamnose cluster conjugates show potent complement-dependent cell killing by recruiting natural antibodies. Chem. Eur. J. 202228 (16), e202200146.
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