Immunological assessment and dose optimization of recombinant canine interleukin-15 following subcutaneous administration in Beagle dogs

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In veterinary medicine, recombinant canine IL-15 (rcIL-15) has emerged as a potential immunotherapeutic agent; however, limited information is available on its safety and efficacy when administered subcutaneously (SC) compared to the intravenous (IV) route. This study aimed to determine whether SC administration of rcIL-15 provides comparable safety and immunological effects to IV administration in Beagle dogs, and to identify an optimal SC dosing regimen. Three male Beagle dogs were sequentially administered rcIL-15 at 20 µg/kg/day via IV and SC routes for four consecutive days. Subsequently, SC doses of 40 and 60 µg/kg/day were tested in the same dogs. Clinical assessments were conducted throughout the study. Peripheral blood mononuclear cells were collected before treatment and on day 7 post-treatment for flow cytometric analysis of lymphocyte subsets. All dogs tolerated rcIL-15 well, with no adverse effects or injection site reactions observed, even at the highest SC dose (60 µg/kg/day), which was threefold higher than the standard IV dose. Flow cytometric analysis showed increased frequencies of CD3⁺CD5⁺ low , Nkp46⁺CD5⁺ low , and non-B non-T cells, indicating NK cell activation. CD8⁺CD5⁺ low NK-like T cells were consistently increased, whereas CD4⁺CD5⁺ T cell responses were variable. SC administration at 40 and 60 µg/kg/day elicited immunological responses comparable to those observed with IV administration. These findings suggest that SC delivery of rcIL-15 is well tolerated and induces immune activation similar to IV administration, suggesting the need for further investigation for its clinical applicability in canine immunotherapy. Recombinant canine interleukin-15 subcutaneous administration immune cell activation dose optimization immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Interleukin-15 (IL-15) is a key cytokine involved in the development, survival, and activation of natural killer (NK) cells and CD8⁺ cytotoxic T lymphocytes [ 1 ]. Although it shares receptor subunits and partial functional overlap with interleukin-2 (IL-2), IL-15 has distinct biological effects, particularly in promoting long-lived memory T cells and bridging innate and adaptive immunity [ 2 , 3 ]. Owing to these properties, IL-15 has been studied extensively as a potential immunotherapeutic agent in infectious diseases, autoimmune disorders, and cancer [ 4 ]. In veterinary medicine, the rising incidence of cancer in companion animals, driven in part by increased life expectancy, has highlighted the need for alternative therapeutic strategies beyond conventional chemotherapy [ 5 ]. However, the development of immune-based cancer therapies specifically for dogs remains limited. Most current protocols rely on the off-label use of human drugs, which may not be optimized for canine physiology [ 6 ]. In this context, recombinant canine IL-15 (rcIL-15) has emerged as a promising immunostimulatory agent that may enhance anti-tumor responses by activating NK cells and cytotoxic T cells [ 7 , 8 ]. Previous preclinical studies in Beagle dogs have demonstrated the immunological activity and safety of the rcIL-15 administered intravenously at 20 µg/kg/day, using both continuous and cyclic dosing regimens [ 7 , 8 ]. However, intravenous administration requires venous access and extended infusion times, which may be less practical for routine use, especially in small or geriatric patients. In contrast, subcutaneous (SC) administration is less invasive and more feasible in clinical practice. Nonetheless, there is limited data comparing the immunological effects and tolerability of SC versus IV administration in dogs [ 9 , 10 , 11 ]. The present study aimed to evaluate the safety, tolerability, and immune-stimulating effects of the rcIL-15 administered subcutaneously at various doses in comparison with standard intravenous administration, and to identify an appropriate SC dosing regimen for future clinical application in dogs. 2 Materials and Methods 2.1 Experimental animals Three healthy male Beagle dogs, 5 months old and weighing 6–8 kg, were purchased from Woojeong Bio Co., Ltd., a certified supplier of laboratory animals (Hwaseong-si, Gyeonggi-do, Republic of Korea). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Biomaterial R&BD Center, Chonnam National University (BMC-IACUC-2023-62(04)). Upon arrival, the dogs underwent quarantine and general health screening based on pathogen testing provided by the supplier. Only dogs with no clinical abnormalities during the quarantine and acclimatization period (minimum 7 days) were included in the study. The animals were housed individually in stainless steel cages within a controlled environment (temperature 20 ± 3°C; relative humidity 50 ± 10%; 10–15 air changes per hour). A 12-hour light/dark cycle was maintained. Each dog received 500 g/day of a standard commercial diet (LAB ANIMAL DIET − 38070, Purina, Cheongju, Republic of Korea), and had ad libitum access to filtered, UV-sterilized underground water via a reverse osmosis system. Following the completion of the study, euthanasia was performed under deep anesthesia to ensure complete unconsciousness. The Beagle dogs were initially anesthetized via intravenous injection of alfaxalone (Alfaxan ® Multidose, Zoetis Inc., USA; 1.5–2 mg/kg). After confirming the loss of consciousness, endotracheal intubation was performed, and anesthesia was maintained with isoflurane (Ifran, Hana Pharm Co., Ltd., Republic of Korea; 4–5%) via inhalation. Once a surgical plane of anesthesia was reached, with no response to noxious stimuli, euthanasia was performed by intravenous administration of T-61 ® . All procedures were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC). 2.2 Study Design and Dosing Protocols Three Beagle dogs received all four rcIL-15 dosing regimens (IV 20, SC 20, 40, and 60 µg/kg/day) in a crossover design. A minimum 17-day washout period was applied between treatment cycles to prevent carry-over effects. The order of treatments was randomly assigned to each dog to minimize sequence-related bias. Each regimen was administered once daily for four consecutive days. Blood samples were collected prior to the first dose (Day 0) and on day 7, which is three days after the final injection of each treatment, to assess immunological responses (Fig. 1 A and 1 B). The dosing regimens were selected based on previous IL-15 immunostimulatory studies and earlier rcIL-15 research in dogs. The intravenous dose of 20 µg/kg/day was adopted from a prior canine study [ 8 ], in which this regimen demonstrated good tolerability and immune-stimulatory activity. The same dose was administered subcutaneously to enable direct comparison between routes. For the SC route, two additional doses (40 and 60 µg/kg/day) were included to explore a potential dose–response relationship. The highest SC dose, 60 µg/kg/day, was chosen considering its clinical practicality and prior evidence from nonhuman primate studies showing that higher IL-15 doses (e.g., up to 100 µg/kg/day SC) can be well tolerated and immunologically active [ 12 ]. Pilot tolerability testing in dogs further supported the use of this dose as a feasible upper limit for evaluating enhanced immune activation. Due to the limited sample size, statistical analyses were not performed. Immunological data were summarized descriptively (mean ± SEM) and interpreted as exploratory trends. 2.3 Test Material The recombinant canine interleukin-15 (rcIL-15) was provided by VaxCell Biotherapeutics Co., Ltd. (Hwasun, Jeonnam, Republic of Korea) as a veterinary pharmaceutical formulation. Each vial contained 100 µg of rcIL-15 as the active component. The formulation included excipients such as 10 µL glycerin and 1 mL phosphate-buffered saline (PBS). The product was stored under refrigerated conditions (2–8°C) until use. 2.4 Administration Procedures For IV administration, the rcIL-15 solution was diluted to 20 µg/kg in 20 mL of sterile saline and infused slowly over 15–20 minutes using a syringe pump (Medifusion DS-3000, Daehwa Medical, Republic of Korea). The cephalic vein of the forelimb was the primary injection site; if unavailable, the saphenous vein of the hindlimb was used. Venous access was achieved with a 22 G or 24 G catheter following standard aseptic preparation with alcohol swabs. After infusion, the catheter was removed, and the injection site was compressed for one minute to ensure hemostasis. SC injections were administered in the dorsal or nuchal region by lifting the skin to form a tent and inserting the needle into the subcutaneous space. The site was disinfected prior to injection, and post-injection pressure was applied for one minute. For consecutive-day injections, the SC site was divided into four quadrants and rotated daily to minimize local tissue irritation. 2.5 Clinical Observation and Safety Monitoring Post-injection observations were performed at 2, 4, and 6 hours after each administration. Throughout the study period, all animals were monitored daily for general health status, gastrointestinal (GI) signs (vomiting, diarrhea), appetite, water intake, activity, and urination/defecation patterns. Injection sites were examined for erythema, swelling, or other abnormalities. Severe adverse reactions, such as persistent GI distress, dehydration, or anorexia, were considered grounds for animal withdrawal. If all animals within a group exhibited such effects, the experiment for that group was to be terminated. 2.6 Flow Cytometric Analysis Peripheral blood was collected from each dog via the jugular or cephalic vein into acid citrate dextrose (ACD) Vacutainer tubes (BD, USA). To minimize stress and ensure compliance during sample collection, gabapentin (Neurontin, 100 mg capsule; Pfizer Korea) was administered orally at 10 mg/kg on the morning of blood sampling and the rcIL-15 administration. To compensate for fluid loss following collection, 40 mL of normal saline was injected subcutaneously into two pelvic sites (20 mL per site). To isolate peripheral blood mononuclear cells (PBMCs), 6.5–7.5 mL of blood was diluted at a 1:2 ratio with Dulbecco’s Phosphate-Buffered Saline (DPBS; Gibco, USA). The diluted sample was layered over Histopaque®-1119 (Sigma-Aldrich, USA) and LymphoPrep™ (PROGEN, Germany) and centrifuged at 400 × g for 25 minutes at 25°C (acceleration 1, deceleration 0). The PBMC layer was collected, washed twice in DPBS (2,500 rpm, 5 minutes), and resuspended in calcium- and magnesium-free DPBS containing 1% bovine serum albumin (Biosesang, Korea). Cell concentrations were determined using the NucleoCounter NC-250 (ChemoMetec, Denmark). Cell viability was assessed using Solution 18 (ChemoMetec, Denmark), a fluorescent dye containing acridine orange (AO) and 4’,6-diamidino-2-phenylindole (DAPI), and only samples with viability above 95% were used for flow cytometry analysis. For immunophenotyping, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies against surface markers CD3, CD4, CD5, CD8, CD21, and an activated T cell cocktail, along with corresponding isotype controls (see Table 1 ). Surface staining was performed at 4°C for 15 minutes. For intracellular proliferation marker analysis, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ solution, then incubated with PE-Cy7-conjugated anti-human Ki-67 or isotype control for 30 minutes at room temperature. Flow cytometric analysis was conducted using a FACS Canto II cytometer (BD Biosciences, Sweden), and data were analyzed using FlowJo™ software version 10.10.0. Table 1 Antibodies used for flow cytometric analysis of canine peripheral blood mononuclear cells Antibody Clone Fluorochrome Supplier Mouse IgG1 Negative Control: FITC - FITC Bio-Rad Mouse Anti Dog CD3: FITC CA17.2A12 FITC Bio-Rad Rat Anti Dog CD4: RPE-Cy7 YKIX302.9 PE-Cyanine7 Bio-Rad Rat IgG2a Negative Control: APC - APC Bio-Rad CD5 Monoclonal Antibody (YKIX322.3), APC YKIX322.3 APC Invitrogen Rat Anti Dog CD8: RPE YCATE55.9 RPE Bio-Rad PB Anti-human CD14 Antibody M5E2 PB BioLegend Mouse Anti Canine CD21: RPE CA2.1D6 RPE Bio-Rad Mouse anti Canine CD21 CA2.1D6 - Bio-Rad Goat anti-mouse IgG (H + L) Crossed-Adsorbed Secondary antibody, PB - PB Invitrogen Ig Isotype Control Cocktail – A MOPC-21, G155-228 FITC, APC, PE BD Pharmingen Dog Activated T Lymphocyte Cocktail LSM8.358, LSM11.425, CTL2.58 FITC, APC, PE BD Pharmingen Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE-Cyanine7 P3.6.2.8.1 PE-Cyanine7 Invitrogen Anti-Human Ki-67, PE-Cyanine7 20Raj1 PE-Cyanine7 Invitrogen Anti-Canine NKp46 (CD335) Antibody, clone 48A 48A - Sigma-Aldrich Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody - PE Invitrogen Details of the gating strategy for identifying viable CD14⁻ lymphocytes and downstream subsets are provided in Supplementary Fig. 1. 3 Results 3.1 General Toxicity and Clinical Observations Across all dosing groups—including IV and SC administration of rcIL-15 at doses of at 20, 40, and 60 µg/kg/day—no adverse clinical signs were observed during or after treatment. Dogs maintained stable appetite, behavior, and hydration status. No local injection site reactions such as erythema, swelling, or pain were noted. Similarly, no systemic signs of toxicity were observed in any treatment group. Despite the use of higher doses, all physiological parameters, including body condition, urination, and defecation, remained within clinically normal limits. Body weight remained stable throughout the study, with no notable fluctuations (Fig. 2 A). Heart rate and respiratory rate, measured at 2, 4, and 6 hours post-injection, also remained within normal physiological ranges (Fig. 2 B), indicating overall tolerability of the treatment. 3.2 Immunophenotypic Changes in Lymphocyte Subsets Following rcIL-15 Administration Flow cytometric analysis of PBMCs revealed route- and dose-dependent alterations in lymphocyte subpopulations following rcIL-15 administration. The proportion of CD3⁺CD5⁺ low cells, a phenotype associated with NK-like populations, was markedly increased in all groups on day 7 compared to baseline, regardless of administration route or dose (Fig. 3 ). This suggests that both intravenous and subcutaneous delivery of rcIL-15 effectively promote NK cell activation in dogs. The magnitude of increase was comparable among the SC groups and similar to that observed with IV administration. Changes in non-B, non-T cells, a population encompassing innate lymphoid cells including NK cells, showed variable trends. In the 20 µg/kg SC group, two of the three dogs exhibited a decrease in non-B, non-T cell expression on day 7. In contrast, all animals in the 20 µg/kg IV, 40 µg/kg SC, and 60 µg/kg SC groups showed increased expression, indicating a more consistent immunostimulatory effect at higher doses or via IV delivery (Fig. 4 ). The population of Nkp46⁺CD5⁺ low cells, considered a specific marker for canine NK cells, increased in most animals following treatment. Marked increases were particularly evident in the 40 and 60 µg/kg SC groups, indicating dose-responsive expansion of activated NK cells. However, one animal each in the 20 µg/kg IV and SC groups did not show a measurable increase in this subset, suggesting individual variability at lower doses (Fig. 5 ). The CD4⁺CD5⁺ T cell population showed inconsistent responses. While a slight increase was noted in one dog from the 20 µg/kg IV group, the remaining animals—particularly in the SC groups—tended to show decreased expression by day 7. These findings suggest that rcIL-15 may not notably stimulate CD4⁺ T cell proliferation under the tested conditions (Fig. 6 ). Conversely, CD8⁺CD5⁺ low NK-like T cells were markedly increased in all groups on day 7 compared to baseline, regardless of administration route or dosage (Fig. 7 ). This suggests that rcIL-15 consistently enhances the expansion or activation of cytotoxic T cells in vivo. 4 Discussion This study evaluated the safety and immunomodulatory effects of rcIL-15 administered via SC and IV routes in Beagle dogs. The results demonstrate that rcIL-15 is well tolerated across a range of doses, including supratherapeutic levels, and that it effectively activates innate and adaptive immune cell populations. No adverse effects or clinical abnormalities were observed during or after administration, regardless of dose or route. Parameters such as body weight, vital signs, appetite, hydration status, and local injection site condition remained within normal ranges. These findings align with previous studies in dogs [ 8 ] and in non-human primates, where recombinant IL-15 analogs were well tolerated at high SC doses [ 12 ], supporting the safety profile of rcIL-15 and its potential suitability for clinical use. Flow cytometry revealed consistent increases in CD3⁺CD5⁺ low , non-B non-T, and Nkp46⁺CD5⁺ low lymphocyte subsets following rcIL-15 administration, indicating effective NK cell activation. CD5 + low cells have previously been identified as a canine NK cell-enriched subset, characterized by high expression of NK-associated markers such as NKG2D, CD94, and CD16, and morphological features consistent with NK cell identity following cytokine stimulation [ 13 , 14 ]. The expansion of these subsets, particularly at higher SC doses, reinforces the role of rcIL-15 in enhancing NK cell activity [ 8 , 13 , 15 ]. Additionally, non-B non-T cells, which include NK and other innate lymphoid cells, also increased in frequency. Their capacity for antibody-dependent cellular cytotoxicity (ADCC) has been previously described [ 16 ], suggesting that rcIL-15 may augment both innate cytotoxicity and tumor surveillance. The study also demonstrated that CD8⁺CD5⁺ low NK-like T cells, a key cytotoxic T lymphocyte subset, were markedly increased following both SC and IV administration. In a previous study, daily IV administration of rcIL-15 led to a significant increase in the CD8⁺CD5⁺ low population, whereas the CD8⁺CD5⁺ high population remained unchanged in both treatment and control groups [ 8 ]. These findings suggest that the CD5⁺ low phenotype may define a cytotoxic T-cell subset that is particularly responsive to rcIL-15 stimulation. Moreover, the CD5⁺ low subset has been reported to include NK-like T cells with enhanced cytotoxic function [ 13 ]. Therefore, we specifically analyzed this population to more accurately capture the biologically relevant rcIL-15-induced changes in cytotoxic T cells. This is consistent with IL-15’s known role in promoting the survival and proliferation of memory and effector CD8⁺ T cells [ 3 , 8 ]. In contrast, the response of CD4⁺CD5⁺ T cells was more variable, with limited or decreased expression observed in several animals. IL-15 has been shown to influence CD4⁺ T cell subsets through trans-presentation via IL-15Rα rather than direct autocrine signaling [ 17 ]. In our study, no notable changes were observed in CD4⁺ T cell frequencies following rcIL-15 administration, suggesting that, under the tested conditions, rcIL-15 does not induce excessive CD4⁺ T cell activation. This is notable because overactivation of CD4⁺ T cells can carry a risk of exacerbating autoimmune conditions [ 18 , 19 ], highlighting the importance of dose optimization and further mechanistic studies. Notably, SC administration of rcIL-15 at 40 and 60 µg/kg/day induced immune responses comparable to those seen with IV administration. These findings suggest that SC injection is not only a feasible alternative but may offer practical advantages in veterinary practice, particularly for long-term or outpatient treatment protocols. This study has several limitations. One limitation is the absence of a subcutaneous vehicle-only control group. Although intravenous IL-15 at 20 µg/kg/day served as a positive control based on previous research, the lack of a negative control for the SC route raises the possibility that injection-related stress or formulation excipients may have contributed to the observed immune cell changes. This decision was made to reduce animal use in line with ethical guidelines, and future studies incorporating such controls will help clarify SC-specific effects. Another limitation is the lack of pharmacokinetic data comparing systemic exposure between SC and IV administration. Although a separate pharmacokinetic study using rcIL-15 is currently underway, its results were not available at the time of this study. Comparative analysis of blood IL-15 levels following different administration routes will be addressed in future investigations. Finally, as an exploratory preclinical study with only three animals, all flow cytometric analyses were performed on the same individuals across treatment cycles. This limited the assumptions of normality and statistical power, and group means may have been affected by inter-individual variability. Although a crossover design was employed to minimize this, the small sample size precluded repeated-measures statistical analysis. As a result, data were interpreted descriptively to highlight immunological trends. Further studies involving larger cohorts and fully powered statistical comparisons will be required to validate these findings. While this study provides meaningful insight into the immunological effects of rcIL-15, it is limited by the absence of clinical efficacy assessments. Future studies are required to incorporate disease models and evaluate relevant immunological and disease-associated biomarkers to assess the therapeutic potential of rcIL-15 in oncologic and inflammatory conditions. 5 Conclusion Subcutaneous administration of rcIL-15 was well tolerated and induced activation of NK cells and CD8⁺ T lymphocytes in Beagle dogs, with immunological effects comparable to intravenous delivery and no observed adverse effects. These findings support the clinical potential of rcIL-15 as an immunotherapeutic agent in dogs and support the feasibility of the SC route for future veterinary applications. Abbreviations IL-15 Interleukin-15 NK Natural killer rcIL-15 Recombinant canine IL-15 SC Subcutaneous IV Intravenous IL-2 Interleukin-2 PBS Phosphate-buffered saline GI Gastrointestinal ACD Acid citrate dextrose PBMCs Peripheral blood mononuclear cells DPBS Dulbecco’s Phosphate-Buffered Saline AO Acridine orange DAPI 4’,6-diamidino-2-phenylindole ADCC Antibody-dependent cellular cytotoxicity IL-15Rα Interleukin-15 receptor alpha chain Declarations Competing interests YS, JL, GK, HMK and JJL are employees of VaxCell Biotherapeutics Co., Ltd., the company that funded this study. HMP and MHK declare no competing interests. Author Contributions YS was responsible for data curation, formal analysis, validation, and original draft preparation. SL and HMK contributed to data curation. JL, GK and HMP were involved in the investigation. Validation was performed by YS and JL. JJL and MHK were responsible for conceptualization, project administration, and overall supervision of the research. All authors critically reviewed and approved the final version of the manuscript. Funding This study was financially supported by VaxCell Biotherapeutics Co., Ltd (Jeollanam-do, Republic of Korea). Availability of data and materials The raw data supporting the conclusions of this article are available from the corresponding author upon reasonable request. Ethics Statement This study was approved by the Institutional Animal Care and Use Committee of the Biomaterial R&BD Center, Chonnam National University (BMC-IACUC-2023-62(04)). The studies were conducted in accordance with the local legislation and institutional requirements. Acknowledgment This study was financially supported by VaxCell Biotherapeutics Co., Ltd. Consent for publication Not applicable. References Zhou Y, Husman T, Cen X, Tsao T, Brown J, Bajpai A, Li M, Zhou K, Yang L. Interleukin 15 in cell-based cancer immunotherapy. Int J Mol Sci. 2022;23:7311. 10.3390/ijms23207311 . 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Signalling mechanisms driving homeostatic and inflammatory effects of interleukin-15 on tissue lymphocytes. Discov Immunol. 2024;3:kyae002. 10.1093/discim/kyae002 . Additional Declarations No competing interests reported. Supplementary Files SuhAdditionalFile1BMCVeterinaryResearch.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 19 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor assigned by journal 11 Aug, 2025 Editor invited by journal 18 Jul, 2025 Submission checks completed at journal 18 Jul, 2025 First submitted to journal 18 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6955583","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502745448,"identity":"45eb8a2b-5d8e-43ac-aec6-9609dcc572ae","order_by":0,"name":"Yooran Suh","email":"","orcid":"","institution":"Chonnam National University Hwasun Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yooran","middleName":"","lastName":"Suh","suffix":""},{"id":502745449,"identity":"ccf1d437-6091-482f-87b5-226c92118a0b","order_by":1,"name":"Jaeil Lee","email":"","orcid":"","institution":"VaxCell Biotherapeutics Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Jaeil","middleName":"","lastName":"Lee","suffix":""},{"id":502745450,"identity":"11aca23b-944d-4f6f-91cc-3a16c20daa8d","order_by":2,"name":"Gyeyoung Koh","email":"","orcid":"","institution":"VaxCell Biotherapeutics Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Gyeyoung","middleName":"","lastName":"Koh","suffix":""},{"id":502745451,"identity":"2ae02f1a-5041-4409-b8f9-b5e2d72652d7","order_by":3,"name":"Sejin Lee","email":"","orcid":"","institution":"VaxCell Biotherapeutics Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Sejin","middleName":"","lastName":"Lee","suffix":""},{"id":502745452,"identity":"06bc6e00-4e80-4ae3-b884-179a63b0fba7","order_by":4,"name":"Hyun-Min Kang","email":"","orcid":"","institution":"VaxCell Biotherapeutics Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hyun-Min","middleName":"","lastName":"Kang","suffix":""},{"id":502745453,"identity":"b0871011-6273-47ca-b07f-24114e592007","order_by":5,"name":"Hee-Myung Park","email":"","orcid":"","institution":"Konkuk University","correspondingAuthor":false,"prefix":"","firstName":"Hee-Myung","middleName":"","lastName":"Park","suffix":""},{"id":502745454,"identity":"e0def084-020f-4110-b668-9d24c2baa8bf","order_by":6,"name":"Je-Jung Lee","email":"","orcid":"","institution":"Chonnam National University Hwasun Hospital","correspondingAuthor":false,"prefix":"","firstName":"Je-Jung","middleName":"","lastName":"Lee","suffix":""},{"id":502745456,"identity":"b32df001-7d99-4af1-bce9-cfcdcc8b469c","order_by":7,"name":"Min-Hee Kang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACCTYQacOMEOEhTksa6VoOI4kQ0qI7uy3xc8Gv8+z8s9svfi5gsJNn4Dn7AK8WszvHDkvP7LvNLHHnTLH0DIZkwwbedgP8Wm6kN0jz9txmZriRkyDNw8CcwMDPht9hQC3Nv3l7zjHL38hJ/s3DUE+MlrRj0jw/DjAb3EgHMhgOJzDwthHUkmbN25DMbHgjh82ax+C4YRvPMYJajG/z/LFLlruR/vg2T0W1PD9PGn4tYMDYxpAMjA9gQAERAZ/AwB8GOwYG9gfEKR4Fo2AUjIIRBwB/Ez+CxqJHYAAAAABJRU5ErkJggg==","orcid":"","institution":"Jangan University","correspondingAuthor":true,"prefix":"","firstName":"Min-Hee","middleName":"","lastName":"Kang","suffix":""}],"badges":[],"createdAt":"2025-06-23 10:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6955583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6955583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89573168,"identity":"e56b3e29-6c97-47cd-85e0-84e7a2f141d9","added_by":"auto","created_at":"2025-08-21 12:39:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77879,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for rcIL-15 administration and blood collection. (A) Intravenous (IV) group: Beagle dogs received intravenous rcIL-15 at 20 µg/kg/day for four consecutive days (Days 0-3). Blood samples were collected on Day 0 (prior to the first dose) and Day 7 (four days after the final dose). A minimum 17-day washout period was observed before the next treatment cycle. (B) Subcutaneous (SC) groups: The same cohort of dogs received subcutaneous rcIL-15 at 20, 40, or 60 µg/kg/day for four consecutive days in separate treatment cycles. Blood collection and washout periods were identical to those in (A).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/46397fdbc97e9523d8b89c71.png"},{"id":89573159,"identity":"ec9c976f-ece6-41fd-a3db-08d8d7634fd2","added_by":"auto","created_at":"2025-08-21 12:39:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":420287,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological parameters in Beagle dogs following rcIL-15 administration. (A) Body weight changes throughout the study period. (B) Heart rate and respiratory rate following rcIL-15 administration. Heart rate (top row) and respiratory rate (bottom row) were measured at 2, 4, and 6 hours after daily SC or IV administration of rcIL-15 from Day 0 to Day 3. Data are presented as mean ± standard deviation (SD) from three dogs (n=3) per group. No notable changes were observed across time points or between treatment groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/faa556810606c61a2e68dd68.jpeg"},{"id":89573153,"identity":"c8487bcd-cd1f-4451-9b49-c579ec96b23a","added_by":"auto","created_at":"2025-08-21 12:39:16","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":769336,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in CD3⁺CD5⁺\u003csup\u003elow \u003c/sup\u003ecell populations in canine PBMCs following rcIL-15 administration. (A) Representative contour plots from flow cytometry analysis showing CD3⁺CD5⁺\u003csup\u003elow \u003c/sup\u003ecell populations before (Day 0) and after (Day 7) rcIL-15 treatment. (B) Quantification of CD3⁺CD5⁺\u003csup\u003elow \u003c/sup\u003ecells (% of lymphocytes) in each treatment group at Day 0 and Day 7. Data are presented as mean ± standard error of the mean (SEM) from three dogs (n=3) per group. All groups, including IV and SC administration at different doses, showed an increase in CD3⁺CD5⁺low cell frequencies, indicative of NK-like cell activation.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/ac2d58c588ed82f79b9fd922.jpeg"},{"id":89573154,"identity":"273f7a13-76f4-464b-adae-31942750c81e","added_by":"auto","created_at":"2025-08-21 12:39:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":876283,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in non-B, non-T cell populations in canine PBMCs following rcIL-15 administration. (A) Representative contour plots from flow cytometry analysis showing non-B, non-T (double negative) cell populations before (Day 0) and after (Day 7) rcIL-15 treatment. These cells were defined as negative for both T-cell and B-cell markers (red rectangle). (B) Quantification of non-B, non-T cells (% of lymphocytes) in each treatment group at Day 0 and Day 7. Data are presented as mean ± standard error of the mean (SEM) from three dogs (n=3) per group. Most groups exhibited increased frequencies, except for two dogs in the 20 μg/kg SC group who showed a decrease on day 7.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/4cd1de7b44ffe603f21d0ab4.jpeg"},{"id":89573173,"identity":"1005aaf5-d37c-4c57-9db6-16b829539900","added_by":"auto","created_at":"2025-08-21 12:39:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":687062,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of NKp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells populations after rcIL-15 administration. (A) Representative contour plots from flow cytometry analysis showing NKp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cell populations before (Day 0) and after (Day 7) rcIL-15 treatment. (B) Quantification of NKp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells (% of lymphocytes) in each treatment group at Day 0 and Day 7. Data are presented as mean ± standard error of the mean (SEM) from three dogs (n=3) per group. Increased frequencies were observed in all groups, with more notable increases in the 40 and 60 μg/kg SC groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/5fa03190315f1443efbd64a8.jpeg"},{"id":89573172,"identity":"bd7873c3-d389-4606-9b09-c7780d80bf64","added_by":"auto","created_at":"2025-08-21 12:39:23","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":899885,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations in CD4⁺CD5⁺ cell populations following rcIL-15 administration. (A) Representative contour plots from flow cytometry analysis showing CD4⁺CD5⁺ cell populations in PBMCs before (Day 0) and after (Day 7) rcIL-15 treatment. (B) Quantification of CD4⁺CD5⁺ cells (% of lymphocytes) in each treatment group at Day 0 and Day 7. Data are presented as mean ± standard error of the mean (SEM) from three dogs (n=3) per group. The frequency of CD4⁺CD5⁺ cells remained relatively stable or showed slight decreases, indicating minimal effect on this subset.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/a17e0264c67eff7775a6e693.jpeg"},{"id":89573170,"identity":"37e89da2-ff5e-4f36-9bf2-4ef63b44a8ad","added_by":"auto","created_at":"2025-08-21 12:39:23","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":817280,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cell populations following rcIL-15 administration. (A) Representative contour plots from flow cytometry analysis showing CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cell populations in PBMCs before (Day 0) and after (Day 7) rcIL-15 treatment. (B) Quantification of CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells (% of lymphocytes) in each treatment group at Day 0 and Day 7. Data are presented as mean ± standard error of the mean (SEM) from three dogs (n=3) per group. All treatment groups exhibited increased proportions of CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells, with the most prominent expansion observed in the 60 μg/kg SC group.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/1e3fd57488cbe5bd5ef65476.jpeg"},{"id":89575312,"identity":"b4a78832-4f23-4d52-9d58-8e9d2df38683","added_by":"auto","created_at":"2025-08-21 12:55:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5240541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/c6812e95-4591-4cfc-a266-7177e96dc6ee.pdf"},{"id":89573152,"identity":"4439f836-dc28-46d7-9be7-b4fa4d44414e","added_by":"auto","created_at":"2025-08-21 12:39:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":169426,"visible":true,"origin":"","legend":"","description":"","filename":"SuhAdditionalFile1BMCVeterinaryResearch.docx","url":"https://assets-eu.researchsquare.com/files/rs-6955583/v1/709631d98dc0aec7df27144b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunological assessment and dose optimization of recombinant canine interleukin-15 following subcutaneous administration in Beagle dogs","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eInterleukin-15 (IL-15) is a key cytokine involved in the development, survival, and activation of natural killer (NK) cells and CD8⁺ cytotoxic T lymphocytes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although it shares receptor subunits and partial functional overlap with interleukin-2 (IL-2), IL-15 has distinct biological effects, particularly in promoting long-lived memory T cells and bridging innate and adaptive immunity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Owing to these properties, IL-15 has been studied extensively as a potential immunotherapeutic agent in infectious diseases, autoimmune disorders, and cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn veterinary medicine, the rising incidence of cancer in companion animals, driven in part by increased life expectancy, has highlighted the need for alternative therapeutic strategies beyond conventional chemotherapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the development of immune-based cancer therapies specifically for dogs remains limited. Most current protocols rely on the off-label use of human drugs, which may not be optimized for canine physiology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this context, recombinant canine IL-15 (rcIL-15) has emerged as a promising immunostimulatory agent that may enhance anti-tumor responses by activating NK cells and cytotoxic T cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious preclinical studies in Beagle dogs have demonstrated the immunological activity and safety of the rcIL-15 administered intravenously at 20 \u0026micro;g/kg/day, using both continuous and cyclic dosing regimens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, intravenous administration requires venous access and extended infusion times, which may be less practical for routine use, especially in small or geriatric patients. In contrast, subcutaneous (SC) administration is less invasive and more feasible in clinical practice. Nonetheless, there is limited data comparing the immunological effects and tolerability of SC versus IV administration in dogs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study aimed to evaluate the safety, tolerability, and immune-stimulating effects of the rcIL-15 administered subcutaneously at various doses in comparison with standard intravenous administration, and to identify an appropriate SC dosing regimen for future clinical application in dogs.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e\u003cp\u003eThree healthy male Beagle dogs, 5 months old and weighing 6\u0026ndash;8 kg, were purchased from Woojeong Bio Co., Ltd., a certified supplier of laboratory animals (Hwaseong-si, Gyeonggi-do, Republic of Korea). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Biomaterial R\u0026amp;BD Center, Chonnam National University (BMC-IACUC-2023-62(04)). Upon arrival, the dogs underwent quarantine and general health screening based on pathogen testing provided by the supplier. Only dogs with no clinical abnormalities during the quarantine and acclimatization period (minimum 7 days) were included in the study.\u003c/p\u003e\u003cp\u003eThe animals were housed individually in stainless steel cages within a controlled environment (temperature 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C; relative humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%; 10\u0026ndash;15 air changes per hour). A 12-hour light/dark cycle was maintained. Each dog received 500 g/day of a standard commercial diet (LAB ANIMAL DIET \u0026minus;\u0026thinsp;38070, Purina, Cheongju, Republic of Korea), and had \u003cem\u003ead libitum\u003c/em\u003e access to filtered, UV-sterilized underground water via a reverse osmosis system.\u003c/p\u003e\u003cp\u003eFollowing the completion of the study, euthanasia was performed under deep anesthesia to ensure complete unconsciousness. The Beagle dogs were initially anesthetized via intravenous injection of alfaxalone (Alfaxan\u003csup\u003e\u0026reg;\u003c/sup\u003e Multidose, Zoetis Inc., USA; 1.5\u0026ndash;2 mg/kg). After confirming the loss of consciousness, endotracheal intubation was performed, and anesthesia was maintained with isoflurane (Ifran, Hana Pharm Co., Ltd., Republic of Korea; 4\u0026ndash;5%) via inhalation. Once a surgical plane of anesthesia was reached, with no response to noxious stimuli, euthanasia was performed by intravenous administration of T-61\u003csup\u003e\u0026reg;\u003c/sup\u003e. All procedures were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Study Design and Dosing Protocols\u003c/h2\u003e\u003cp\u003eThree Beagle dogs received all four rcIL-15 dosing regimens (IV 20, SC 20, 40, and 60 \u0026micro;g/kg/day) in a crossover design. A minimum 17-day washout period was applied between treatment cycles to prevent carry-over effects. The order of treatments was randomly assigned to each dog to minimize sequence-related bias. Each regimen was administered once daily for four consecutive days. Blood samples were collected prior to the first dose (Day 0) and on day 7, which is three days after the final injection of each treatment, to assess immunological responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dosing regimens were selected based on previous IL-15 immunostimulatory studies and earlier rcIL-15 research in dogs. The intravenous dose of 20 \u0026micro;g/kg/day was adopted from a prior canine study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], in which this regimen demonstrated good tolerability and immune-stimulatory activity. The same dose was administered subcutaneously to enable direct comparison between routes. For the SC route, two additional doses (40 and 60 \u0026micro;g/kg/day) were included to explore a potential dose\u0026ndash;response relationship. The highest SC dose, 60 \u0026micro;g/kg/day, was chosen considering its clinical practicality and prior evidence from nonhuman primate studies showing that higher IL-15 doses (e.g., up to 100 \u0026micro;g/kg/day SC) can be well tolerated and immunologically active [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Pilot tolerability testing in dogs further supported the use of this dose as a feasible upper limit for evaluating enhanced immune activation.\u003c/p\u003e\u003cp\u003eDue to the limited sample size, statistical analyses were not performed. Immunological data were summarized descriptively (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) and interpreted as exploratory trends.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Test Material\u003c/h2\u003e\u003cp\u003eThe recombinant canine interleukin-15 (rcIL-15) was provided by VaxCell Biotherapeutics Co., Ltd. (Hwasun, Jeonnam, Republic of Korea) as a veterinary pharmaceutical formulation. Each vial contained 100 \u0026micro;g of rcIL-15 as the active component. The formulation included excipients such as 10 \u0026micro;L glycerin and 1 mL phosphate-buffered saline (PBS). The product was stored under refrigerated conditions (2\u0026ndash;8\u0026deg;C) until use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Administration Procedures\u003c/h2\u003e\u003cp\u003eFor IV administration, the rcIL-15 solution was diluted to 20 \u0026micro;g/kg in 20 mL of sterile saline and infused slowly over 15\u0026ndash;20 minutes using a syringe pump (Medifusion DS-3000, Daehwa Medical, Republic of Korea). The cephalic vein of the forelimb was the primary injection site; if unavailable, the saphenous vein of the hindlimb was used. Venous access was achieved with a 22 G or 24 G catheter following standard aseptic preparation with alcohol swabs. After infusion, the catheter was removed, and the injection site was compressed for one minute to ensure hemostasis.\u003c/p\u003e\u003cp\u003eSC injections were administered in the dorsal or nuchal region by lifting the skin to form a tent and inserting the needle into the subcutaneous space. The site was disinfected prior to injection, and post-injection pressure was applied for one minute. For consecutive-day injections, the SC site was divided into four quadrants and rotated daily to minimize local tissue irritation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.5 Clinical Observation and Safety Monitoring\u003c/b\u003e\u003c/h2\u003e\u003cp\u003ePost-injection observations were performed at 2, 4, and 6 hours after each administration. Throughout the study period, all animals were monitored daily for general health status, gastrointestinal (GI) signs (vomiting, diarrhea), appetite, water intake, activity, and urination/defecation patterns. Injection sites were examined for erythema, swelling, or other abnormalities. Severe adverse reactions, such as persistent GI distress, dehydration, or anorexia, were considered grounds for animal withdrawal. If all animals within a group exhibited such effects, the experiment for that group was to be terminated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Flow Cytometric Analysis\u003c/h2\u003e\u003cp\u003ePeripheral blood was collected from each dog via the jugular or cephalic vein into acid citrate dextrose (ACD) Vacutainer tubes (BD, USA). To minimize stress and ensure compliance during sample collection, gabapentin (Neurontin, 100 mg capsule; Pfizer Korea) was administered orally at 10 mg/kg on the morning of blood sampling and the rcIL-15 administration. To compensate for fluid loss following collection, 40 mL of normal saline was injected subcutaneously into two pelvic sites (20 mL per site).\u003c/p\u003e\u003cp\u003eTo isolate peripheral blood mononuclear cells (PBMCs), 6.5\u0026ndash;7.5 mL of blood was diluted at a 1:2 ratio with Dulbecco\u0026rsquo;s Phosphate-Buffered Saline (DPBS; Gibco, USA). The diluted sample was layered over Histopaque\u0026reg;-1119 (Sigma-Aldrich, USA) and LymphoPrep\u0026trade; (PROGEN, Germany) and centrifuged at 400 \u0026times; g for 25 minutes at 25\u0026deg;C (acceleration 1, deceleration 0). The PBMC layer was collected, washed twice in DPBS (2,500 rpm, 5 minutes), and resuspended in calcium- and magnesium-free DPBS containing 1% bovine serum albumin (Biosesang, Korea). Cell concentrations were determined using the NucleoCounter NC-250 (ChemoMetec, Denmark). Cell viability was assessed using Solution 18 (ChemoMetec, Denmark), a fluorescent dye containing acridine orange (AO) and 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI), and only samples with viability above 95% were used for flow cytometry analysis.\u003c/p\u003e\u003cp\u003eFor immunophenotyping, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies against surface markers CD3, CD4, CD5, CD8, CD21, and an activated T cell cocktail, along with corresponding isotype controls (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Surface staining was performed at 4\u0026deg;C for 15 minutes. For intracellular proliferation marker analysis, cells were fixed and permeabilized using BD Cytofix/Cytoperm\u0026trade; solution, then incubated with PE-Cy7-conjugated anti-human Ki-67 or isotype control for 30 minutes at room temperature. Flow cytometric analysis was conducted using a FACS Canto II cytometer (BD Biosciences, Sweden), and data were analyzed using FlowJo\u0026trade; software version 10.10.0.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibodies used for flow cytometric analysis of canine peripheral blood mononuclear cells\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibody\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClone\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFluorochrome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSupplier\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse IgG1 Negative Control: FITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse Anti Dog CD3: FITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCA17.2A12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRat Anti Dog CD4: RPE-Cy7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYKIX302.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePE-Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRat IgG2a Negative Control: APC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD5 Monoclonal Antibody (YKIX322.3), APC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYKIX322.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRat Anti Dog CD8: RPE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYCATE55.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRPE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePB Anti-human CD14 Antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM5E2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse Anti Canine CD21: RPE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCA2.1D6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRPE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse anti Canine CD21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCA2.1D6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBio-Rad\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGoat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Crossed-Adsorbed Secondary antibody, PB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIg Isotype Control Cocktail \u0026ndash; A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMOPC-21,\u003c/p\u003e\u003cp\u003eG155-228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFITC, APC, PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBD Pharmingen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDog Activated T Lymphocyte Cocktail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLSM8.358,\u003c/p\u003e\u003cp\u003eLSM11.425,\u003c/p\u003e\u003cp\u003eCTL2.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFITC, APC, PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBD Pharmingen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE-Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP3.6.2.8.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePE-Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-Human Ki-67, PE-Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20Raj1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePE-Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-Canine NKp46 (CD335) Antibody, clone 48A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSigma-Aldrich\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGoat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDetails of the gating strategy for identifying viable CD14⁻ lymphocytes and downstream subsets are provided in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 General Toxicity and Clinical Observations\u003c/h2\u003e\u003cp\u003eAcross all dosing groups\u0026mdash;including IV and SC administration of rcIL-15 at doses of at 20, 40, and 60 \u0026micro;g/kg/day\u0026mdash;no adverse clinical signs were observed during or after treatment. Dogs maintained stable appetite, behavior, and hydration status. No local injection site reactions such as erythema, swelling, or pain were noted. Similarly, no systemic signs of toxicity were observed in any treatment group. Despite the use of higher doses, all physiological parameters, including body condition, urination, and defecation, remained within clinically normal limits.\u003c/p\u003e\u003cp\u003eBody weight remained stable throughout the study, with no notable fluctuations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Heart rate and respiratory rate, measured at 2, 4, and 6 hours post-injection, also remained within normal physiological ranges (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating overall tolerability of the treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Immunophenotypic Changes in Lymphocyte Subsets Following rcIL-15 Administration\u003c/h2\u003e\u003cp\u003eFlow cytometric analysis of PBMCs revealed route- and dose-dependent alterations in lymphocyte subpopulations following rcIL-15 administration.\u003c/p\u003e\u003cp\u003eThe proportion of CD3⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells, a phenotype associated with NK-like populations, was markedly increased in all groups on day 7 compared to baseline, regardless of administration route or dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that both intravenous and subcutaneous delivery of rcIL-15 effectively promote NK cell activation in dogs. The magnitude of increase was comparable among the SC groups and similar to that observed with IV administration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eChanges in non-B, non-T cells, a population encompassing innate lymphoid cells including NK cells, showed variable trends. In the 20 \u0026micro;g/kg SC group, two of the three dogs exhibited a decrease in non-B, non-T cell expression on day 7. In contrast, all animals in the 20 \u0026micro;g/kg IV, 40 \u0026micro;g/kg SC, and 60 \u0026micro;g/kg SC groups showed increased expression, indicating a more consistent immunostimulatory effect at higher doses or via IV delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe population of Nkp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e cells, considered a specific marker for canine NK cells, increased in most animals following treatment. Marked increases were particularly evident in the 40 and 60 \u0026micro;g/kg SC groups, indicating dose-responsive expansion of activated NK cells. However, one animal each in the 20 \u0026micro;g/kg IV and SC groups did not show a measurable increase in this subset, suggesting individual variability at lower doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe CD4⁺CD5⁺ T cell population showed inconsistent responses. While a slight increase was noted in one dog from the 20 \u0026micro;g/kg IV group, the remaining animals\u0026mdash;particularly in the SC groups\u0026mdash;tended to show decreased expression by day 7. These findings suggest that rcIL-15 may not notably stimulate CD4⁺ T cell proliferation under the tested conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConversely, CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e NK-like T cells were markedly increased in all groups on day 7 compared to baseline, regardless of administration route or dosage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This suggests that rcIL-15 consistently enhances the expansion or activation of cytotoxic T cells in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study evaluated the safety and immunomodulatory effects of rcIL-15 administered via SC and IV routes in Beagle dogs. The results demonstrate that rcIL-15 is well tolerated across a range of doses, including supratherapeutic levels, and that it effectively activates innate and adaptive immune cell populations.\u003c/p\u003e\u003cp\u003eNo adverse effects or clinical abnormalities were observed during or after administration, regardless of dose or route. Parameters such as body weight, vital signs, appetite, hydration status, and local injection site condition remained within normal ranges. These findings align with previous studies in dogs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and in non-human primates, where recombinant IL-15 analogs were well tolerated at high SC doses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], supporting the safety profile of rcIL-15 and its potential suitability for clinical use.\u003c/p\u003e\u003cp\u003eFlow cytometry revealed consistent increases in CD3⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e, non-B non-T, and Nkp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e lymphocyte subsets following rcIL-15 administration, indicating effective NK cell activation. CD5\u003csup\u003e+\u0026thinsp;low\u003c/sup\u003e cells have previously been identified as a canine NK cell-enriched subset, characterized by high expression of NK-associated markers such as NKG2D, CD94, and CD16, and morphological features consistent with NK cell identity following cytokine stimulation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The expansion of these subsets, particularly at higher SC doses, reinforces the role of rcIL-15 in enhancing NK cell activity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, non-B non-T cells, which include NK and other innate lymphoid cells, also increased in frequency. Their capacity for antibody-dependent cellular cytotoxicity (ADCC) has been previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting that rcIL-15 may augment both innate cytotoxicity and tumor surveillance.\u003c/p\u003e\u003cp\u003eThe study also demonstrated that CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e NK-like T cells, a key cytotoxic T lymphocyte subset, were markedly increased following both SC and IV administration. In a previous study, daily IV administration of rcIL-15 led to a significant increase in the CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e population, whereas the CD8⁺CD5⁺\u003csup\u003ehigh\u003c/sup\u003e population remained unchanged in both treatment and control groups [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings suggest that the CD5⁺\u003csup\u003elow\u003c/sup\u003e phenotype may define a cytotoxic T-cell subset that is particularly responsive to rcIL-15 stimulation. Moreover, the CD5⁺\u003csup\u003elow\u003c/sup\u003e subset has been reported to include NK-like T cells with enhanced cytotoxic function [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, we specifically analyzed this population to more accurately capture the biologically relevant rcIL-15-induced changes in cytotoxic T cells. This is consistent with IL-15\u0026rsquo;s known role in promoting the survival and proliferation of memory and effector CD8⁺ T cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In contrast, the response of CD4⁺CD5⁺ T cells was more variable, with limited or decreased expression observed in several animals. IL-15 has been shown to influence CD4⁺ T cell subsets through trans-presentation via IL-15Rα rather than direct autocrine signaling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our study, no notable changes were observed in CD4⁺ T cell frequencies following rcIL-15 administration, suggesting that, under the tested conditions, rcIL-15 does not induce excessive CD4⁺ T cell activation. This is notable because overactivation of CD4⁺ T cells can carry a risk of exacerbating autoimmune conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], highlighting the importance of dose optimization and further mechanistic studies.\u003c/p\u003e\u003cp\u003eNotably, SC administration of rcIL-15 at 40 and 60 \u0026micro;g/kg/day induced immune responses comparable to those seen with IV administration. These findings suggest that SC injection is not only a feasible alternative but may offer practical advantages in veterinary practice, particularly for long-term or outpatient treatment protocols.\u003c/p\u003e\u003cp\u003eThis study has several limitations. One limitation is the absence of a subcutaneous vehicle-only control group. Although intravenous IL-15 at 20 \u0026micro;g/kg/day served as a positive control based on previous research, the lack of a negative control for the SC route raises the possibility that injection-related stress or formulation excipients may have contributed to the observed immune cell changes. This decision was made to reduce animal use in line with ethical guidelines, and future studies incorporating such controls will help clarify SC-specific effects. Another limitation is the lack of pharmacokinetic data comparing systemic exposure between SC and IV administration. Although a separate pharmacokinetic study using rcIL-15 is currently underway, its results were not available at the time of this study. Comparative analysis of blood IL-15 levels following different administration routes will be addressed in future investigations. Finally, as an exploratory preclinical study with only three animals, all flow cytometric analyses were performed on the same individuals across treatment cycles. This limited the assumptions of normality and statistical power, and group means may have been affected by inter-individual variability. Although a crossover design was employed to minimize this, the small sample size precluded repeated-measures statistical analysis. As a result, data were interpreted descriptively to highlight immunological trends. Further studies involving larger cohorts and fully powered statistical comparisons will be required to validate these findings.\u003c/p\u003e\u003cp\u003eWhile this study provides meaningful insight into the immunological effects of rcIL-15, it is limited by the absence of clinical efficacy assessments. Future studies are required to incorporate disease models and evaluate relevant immunological and disease-associated biomarkers to assess the therapeutic potential of rcIL-15 in oncologic and inflammatory conditions.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eSubcutaneous administration of rcIL-15 was well tolerated and induced activation of NK cells and CD8⁺ T lymphocytes in Beagle dogs, with immunological effects comparable to intravenous delivery and no observed adverse effects. These findings support the clinical potential of rcIL-15 as an immunotherapeutic agent in dogs and support the feasibility of the SC route for future veterinary applications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-15\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-15\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNatural killer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ercIL-15\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRecombinant canine IL-15\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSubcutaneous\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntravenous\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate-buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGastrointestinal\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAcid citrate dextrose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBMCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePeripheral blood mononuclear cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDPBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s Phosphate-Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAcridine orange\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e4\u0026rsquo;,6-diamidino-2-phenylindole\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eADCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAntibody-dependent cellular cytotoxicity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL-15Rα\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin-15 receptor alpha chain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS, JL, GK, HMK and JJL are employees of VaxCell Biotherapeutics Co., Ltd., the company that funded this study. HMP and MHK declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS was responsible for data curation, formal analysis, validation, and original draft preparation. SL and HMK contributed to data curation. JL, GK and HMP were involved in the investigation. Validation was performed by YS and JL. JJL and MHK were responsible for conceptualization, project administration, and overall supervision of the research. All authors critically reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by VaxCell Biotherapeutics Co., Ltd (Jeollanam-do, Republic of Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Animal Care and Use Committee of the Biomaterial R\u0026amp;BD Center, Chonnam National University (BMC-IACUC-2023-62(04)). The studies were conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by VaxCell Biotherapeutics Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhou Y, Husman T, Cen X, Tsao T, Brown J, Bajpai A, Li M, Zhou K, Yang L. Interleukin 15 in cell-based cancer immunotherapy. 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Discov Immunol. 2024;3:kyae002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/discim/kyae002\u003c/span\u003e\u003cspan address=\"10.1093/discim/kyae002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Recombinant canine interleukin-15, subcutaneous administration, immune cell activation, dose optimization, immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6955583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6955583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInterleukin-15 (IL-15) is a cytokine critical to the activation and maintenance of natural killer (NK) cells and cytotoxic T lymphocytes. In veterinary medicine, recombinant canine IL-15 (rcIL-15) has emerged as a potential immunotherapeutic agent; however, limited information is available on its safety and efficacy when administered subcutaneously (SC) compared to the intravenous (IV) route. This study aimed to determine whether SC administration of rcIL-15 provides comparable safety and immunological effects to IV administration in Beagle dogs, and to identify an optimal SC dosing regimen. Three male Beagle dogs were sequentially administered rcIL-15 at 20 \u0026micro;g/kg/day via IV and SC routes for four consecutive days. Subsequently, SC doses of 40 and 60 \u0026micro;g/kg/day were tested in the same dogs. Clinical assessments were conducted throughout the study. Peripheral blood mononuclear cells were collected before treatment and on day 7 post-treatment for flow cytometric analysis of lymphocyte subsets. All dogs tolerated rcIL-15 well, with no adverse effects or injection site reactions observed, even at the highest SC dose (60 \u0026micro;g/kg/day), which was threefold higher than the standard IV dose. Flow cytometric analysis showed increased frequencies of CD3⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e, Nkp46⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e, and non-B non-T cells, indicating NK cell activation. CD8⁺CD5⁺\u003csup\u003elow\u003c/sup\u003e NK-like T cells were consistently increased, whereas CD4⁺CD5⁺ T cell responses were variable. SC administration at 40 and 60 \u0026micro;g/kg/day elicited immunological responses comparable to those observed with IV administration. These findings suggest that SC delivery of rcIL-15 is well tolerated and induces immune activation similar to IV administration, suggesting the need for further investigation for its clinical applicability in canine immunotherapy.\u003c/p\u003e","manuscriptTitle":"Immunological assessment and dose optimization of recombinant canine interleukin-15 following subcutaneous administration in Beagle dogs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 12:39:02","doi":"10.21203/rs.3.rs-6955583/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"185996536504451073677634555717956066604","date":"2025-08-19T19:48:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T17:52:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T16:17:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-18T07:31:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-18T07:13:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-07-18T07:09:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2aacbfb-ec35-40ac-9a73-13112db3fb03","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-21T12:39:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 12:39:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6955583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6955583","identity":"rs-6955583","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}