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Veterinary sciences2026; 13(4); 343; doi: 10.3390/vetsci13040343

In Vitro Characterization of an Equinized Anti-PD-L1 Antibody for Cancer Immunotherapy in Horses.

Abstract: Horses develop spontaneous tumors, typically in old age. Although local tumor control can be achieved using conventional therapies, systemic therapies are required to treat recurrent and/or metastatic tumors. Immune checkpoint inhibitors, such as anti-PD-L1 antibodies, have been approved for the treatment of various tumor types in humans; however, little is known about the immunosuppressive roles of the PD-1/PD-L1 pathway in horses, and the therapeutic potential of these inhibitors remains to be elucidated. Previously, we reported that the rat monoclonal anti-PD-L1 antibody 6C11-3A11 cross-reacts with horse PD-L1 to block the PD-1/PD-L1 interaction. To further develop antibodies for therapeutic purposes, their immunogenicity must be reduced to maximize efficacy and safety. To this end, we designed an equinized (equine-ized) anti-PD-L1 antibody, Eq6C11, using the complementarity-determining regions of 6C11-3A11. Eq6C11 had antigen-binding properties comparable to those of 6C11-3A11 and inhibited equine PD-L1 binding to PD-1 in a recombinant protein-based assay. Treatment with Eq6C11 significantly increased IFN-γ and IL-2 production in equine peripheral blood mononuclear cell cultures, suggesting its stimulatory activity on T-cell activation. Although further studies are needed to clarify its immunogenicity and clinical activity, these results encourage further development of Eq6C11 as a candidate immune checkpoint inhibitor for cancer immunotherapy in horses.
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Publication Date: 2026-04-01 PubMed ID: 42076715PubMed Central: PMC13119938DOI: 10.3390/vetsci13040343Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Objective Overview

  • This study focuses on developing and characterizing a horse-specific (equinized) antibody that blocks PD-L1, a protein that helps tumors evade the immune system, to enhance cancer immunotherapy options for horses.

Background and Rationale

  • Horses naturally develop tumors, often in old age, and while local tumors can be treated with conventional therapies, systemic treatments are needed for recurrent or metastatic cancers.
  • Immune checkpoint inhibitors like anti-PD-L1 antibodies have proven effective in treating cancers in humans by blocking interactions that suppress the immune response, particularly the PD-1/PD-L1 pathway.
  • The role of the PD-1/PD-L1 pathway in equine immune suppression is not well understood, nor is the effectiveness of immune checkpoint inhibitors in horses.
  • Previous research identified a rat-derived monoclonal antibody (6C11-3A11) that can bind to and inhibit horse PD-L1, demonstrating potential for immune system activation against tumors.
  • However, using a rat antibody in horses could provoke immune reactions, reducing treatment efficacy and safety, highlighting the need for an equinized antibody version.

Development of the Equinized Antibody (Eq6C11)

  • Researchers engineered Eq6C11 by grafting the complementarity-determining regions (CDRs) — the parts of the antibody responsible for antigen recognition — from the rat antibody 6C11-3A11 onto a horse antibody framework.
  • This “equinization” aimed to reduce immunogenicity and improve compatibility within the horse immune system, potentially enhancing treatment safety and effectiveness.

In Vitro Characterization of Eq6C11

  • Binding Activity: Eq6C11 retained antigen-binding properties similar to the original rat antibody, confirming that it could effectively recognize and bind to equine PD-L1.
  • Functional Assay: In tests using recombinant proteins, Eq6C11 blocked the interaction between horse PD-L1 and PD-1, a critical immune checkpoint that tumors exploit to avoid immune attack.
  • Cellular Immune Response: Treating equine peripheral blood mononuclear cells (PBMCs) with Eq6C11 led to significant increases in the production of IFN-γ and IL-2, cytokines associated with T-cell activation and immune stimulation.

Implications and Future Directions

  • The ability of Eq6C11 to block PD-1/PD-L1 interaction and to stimulate T-cell cytokine production in vitro suggests that this equinized antibody has potential as a therapeutic immune checkpoint inhibitor for horses with cancer.
  • While these results are promising, further studies are necessary to evaluate Eq6C11’s immunogenicity in vivo (how much the horse’s immune system might recognize it as foreign) and its clinical effectiveness in treating tumors.
  • Successful development of such therapeutics could expand systemic treatment options for equine oncology, improving outcomes for horses with metastatic or recurrent tumors.

Cite This Article

APA
Horikawa T, Maekawa N, Okagawa T, Tiyamanee W, Ganbaatar O, Nakamura H, Ikehata M, Inoue M, Nakanishi T, Tachibana T, Kato Y, Yamamoto K, Suzuki Y, Murata S, Ohashi K, Konnai S. (2026). In Vitro Characterization of an Equinized Anti-PD-L1 Antibody for Cancer Immunotherapy in Horses. Vet Sci, 13(4), 343. https://doi.org/10.3390/vetsci13040343

Publication

Veterinary sciences
ISSN: 2306-7381
NlmUniqueID: 101680127
Country: Switzerland
Language: English
Volume: 13
Issue: 4
PII: 343

Researcher Affiliations

Horikawa, Takeru
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Maekawa, Naoya
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
  • Cancer Research Unit, One Health Research Center, Hokkaido University, Sapporo 060-0818, Japan.
  • Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo 001-0021, Japan.
Okagawa, Tomohiro
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
  • Business Development Unit, FASMAC Co., Ltd., Atsugi 243-0021, Japan.
Tiyamanee, Wisa
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Ganbaatar, Otgontuya
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Nakamura, Hayato
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Ikehata, Mari
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Inoue, Maho
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Nakanishi, Takeshi
  • Department of Chemistry and Bioengineering, Division of Science and Engineering for Materials, Chemistry and Biology, Graduate School of Engineering, Osaka Metropolitan University, Osaka 558-8585, Japan.
Tachibana, Taro
  • Department of Chemistry and Bioengineering, Division of Science and Engineering for Materials, Chemistry and Biology, Graduate School of Engineering, Osaka Metropolitan University, Osaka 558-8585, Japan.
Kato, Yukinari
  • Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
Yamamoto, Keiichi
  • Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo 001-0021, Japan.
Suzuki, Yasuhiko
  • Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo 001-0021, Japan.
  • Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan.
  • Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo 060-0808, Japan.
Murata, Shiro
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
  • Veterinary Research Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0021, Japan.
Ohashi, Kazuhiko
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
  • Veterinary Research Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0021, Japan.
  • International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Konnai, Satoru
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
  • Cancer Research Unit, One Health Research Center, Hokkaido University, Sapporo 060-0818, Japan.
  • Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo 001-0021, Japan.
  • Veterinary Research Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo 001-0021, Japan.

Grant Funding

  • JP23K23768, JP23KK0124, JP25K02162, and JP25K02163 / Japan Society for the Promotion of Science
  • JP223fa627005 and JP25ama121008 / Japan Agency for Medical Research and Development

Conflict of Interest Statement

T.O. is an employee of FASMAC Co., Ltd. and contributed to the roles shown above. The funder did not have any additional role in the study design, data interpretation, preparation of the manuscript, or decision to publish. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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