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Background: Keyhole limpet hemocyanin (KLH) is a suitable neo-antigen for studying adaptive immune responses, which can be utilized to investigate the pharmacological activity of novel immunomodulatory drugs on T and B cell-mediated immunity [1]. The use of KLH has been widely adapted in pre-clinical studies, though healthy volunteers (HV) studies could also benefit from the use of KLH to overcome the limitation of frequently lacking suitable biomarkers in this population. By eliciting a transient and controlled immune response, KLH could support the understanding of pathophysiological mechanisms of autoimmune diseases and the early clinical evaluation of effectiveness of novel immune-targeted treatments.
Objectives: To present applications and outcome measures of the human KLH challenge model used in clinical studies at the Centre for Human Drug Research (CHDR).
Methods: The KLH challenge model consists of an immunization period which is followed by an intradermal (i.d.) challenge. 1 to 3 intramuscular (i.m.) KLH injections are administered in the deltoid muscle with a 2 to 4 week interval, evoking a cellular immune response, which can be studied in vivo by circulating KLH-specific antibodies or by ex vivo analyses of KLH-driven cellular responses. The i.d. challenge is administered approximately 3 weeks after the final immunization, eliciting a local, cellular recall response. The local immune reaction can be quantified non-invasively by assessing the cutaneous blood flow as measured by Laser Speckle Contrast Imaging (LSCI) and erythema as measured by multispectral imaging. Minimally invasive assessments include cellular and molecular markers in skin biopsies and blister exudates.
Results: A randomized, double-blind, placebo-controlled study in HV showed that anti‐KLH antibody titers increased incrementally after KLH immunization. KLH-specific IFN-γ and IL-13 responses were demonstrated by ELISpot analyses. Maximal response after the skin challenge was observed at 24h after i.d. administration, quantified as an increase in erythema of 50% (95% CI 35 – 68%, p < 0.0001) and increased perfusion of 173% (95% CI 134 – 219%, p < 0.0001) compared to baseline. Blister fluid analysis showed cell influx (T cells; B cells; monocytes; dendritic cells) at 24h, largely decreasing at 48h, as confirmed by immunofluorescent staining of skin biopsies [2]. In a randomized, double-blind intervention study with an anti‐OX40L monoclonal antibody (KY1005, currently amlitelimab), the model was successfully applied. Amlitelimab dose-dependently decreased KLH-driven cutaneous blood perfusion and erythema quantified as average redness at doses from 0.45 mg/kg, compared to placebo. Furthermore, exposure‐response analysis showed a statistically significant treatment effect on KLH antibody levels and erythema skin response [3]. Based on the results of this study, the phase 2 dose was selected, which was shown to be effective in atopic dermatitis patients [4, 5]. Next steps include further exploration of the translation of the KLH model from HV to rheumatoid arthritis (RA) patients by investigating the KLH response in treatment naïve RA patients that are starting methotrexate (MTX) therapy in responders compared to non-responders to MTX therapy.
Conclusion: Early phase proof-of-mechanism studies help to elucidate the pharmacological mechanism of action of novel drugs in vivo , and establish the dose-response relationship. Our studies demonstrate that KLH drives an antigen-specific immune response in man in vivo , displaying Th1, Th2 and Th17 characteristics. Our KLH model allows evaluation of systemic antigen-specific responses but also in peripheral tissue. As demonstrated by the amlitelimab data, the KLH model can be used to establish proof-of-mechanism in early phase drug development with novel compounds targeting the adaptive immune system, and may facilitate effective dose selection for future phase 2 studies.
REFERENCES: [1] Saghari M, Jansen MAA, Grievink HW, Rissmann R, Moerland M. Characterization of KLH-driven immune responses in clinical studies: A systematic review. Frontiers in Drug Discovery [Internet]. 2022 Oct 25;2. Available from:
[2] Ronner, MN; Grievink, HW; Saghari, M; Osse, M; van den Bosch, TPP; Damman, J; de Kam, ML; Burggraaf, J; Klarenbeek, NB; Jansen, MAA; Moerland M. Profiling of a neo-antigen driven adaptive immune response in healthy volunteers: A randomized, double-blind, placebo-controlled study characterizing the human KLH challenge model. 2025 Jan. Submitted.
[3] Saghari M, Gal P, Gilbert S, Yateman M, Porter‐Brown B, Brennan N, et al. OX40L Inhibition Suppresses KLH‐driven Immune Responses in Healthy Volunteers: A Randomized Controlled Trial Demonstrating Proof‐of‐Pharmacology for KY1005. Clin Pharmacol Ther [Internet]. 2022 May;111(5):1121–32. Available from:
[4] Weidinger S, Blauvelt A, Papp KA, Reich A, Lee CH, Worm M, et al. Phase 2b randomized clinical trial of amlitelimab, an anti-OX40 ligand antibody, in patients with moderate-to-severe atopic dermatitis. Journal of Allergy and Clinical Immunology [Internet]. 2024 Nov; Available from:
[5] Weidinger S, Bieber T, Cork MJ, Reich A, Wilson R, Quaratino S, et al. Safety and efficacy of amlitelimab, a fully human nondepleting, noncytotoxic anti-OX40 ligand monoclonal antibody, in atopic dermatitis: results of a phase IIa randomized placebo-controlled trial. British Journal of Dermatology [Internet]. 2023 Oct 25;189(5):531–9. Available from:
Acknowledgements: NIL.
Disclosure of Interests: None declared.
© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (