(M1130-01-04) XenoImmune™ Mouse Model, a Novel and Proprietary Animal Model that Recapitulates Clinical Immunogenicity Rates of Biologics

Purpose: The immunogenicity of therapeutic modalities such as biologics and viral vectors is a major challenge to the clinical development of such therapeutics. Early determination of immunogenicity risks is desirable during candidate selection, scale-up, and formulation development. To date, no quantitative non-clinical tool exists that can predict clinical immunogenicity rates. All available assays for immunogenicity prediction are qualitative in nature and do not reflect the clinical immunogenicity profile of the test article, Here, we report on a non-clinical animal model (XenoImmune™) that recapitulated the clinical immunogenicity rates of 3 biologics (FVIII, GAA, and IFN-β) as well as AAV9.

Methods: Human PBMCs were isolated from buffy coats (BioIVT) of healthy donors as reported previously. Animals: wild type C57BL/6 mice were purchased from CRL or JAX. XenoImmune™ mice were developed at LAPIX Therapeutics Inc using an optimized, novel, and proprietary protocol for the conditioning of mice for the engraftment of a healthy human donor immune system. Flow cytometry: 50 µl of blood was collected at the end of the conditioning and engraftment period of XenoImmune™ mice. Samples were stained for murine (m) or human (h) CD45, CD4, CD19, and viability dye. Samples were acquired by an Attune NxT Flowcytomer using Attune NxT AutoSampler. Data analyses were done in FlowJo v 10.7.1. Immunization: 1 µg/mouse of each of rhFVIII (LifeSpan BioSciences, Inc ), rhGAA (LifeSpan BioSciences, Inc ) and rhInterferon-β (MedChem Express), or 10⁸ empty capsid/mouse of AAV9 (Vigene Biosciences) were administered SC once a week for 2 weeks to XenoImmune™ mice or C57BL/6 mice. One week after the last administration, blood samples were collected for titer analysis by ELISA. Titer Analysis: Total human or mice anti-FVIII IgG, anti-GAA IgG, and anti-Interferon-β IgG antibody titers were measured using an ELISA assay. Titers were determined using a statistically significant cutoff as described by Frey et al [4]. Human Anti-AAV9 antibodies were detected by an anti-AAV9 antibody ELISA kit from creative diagnostics (Shirley, NY), and mouse anti-AAV9 antibodies were detected using the same plates from the human ELISA kit but using mouse-detection antibodies.

Results: Immune phenotyping of XenoImmune™ mice: Total lymphocytes from the blood of XenoImmune™ mice were Analyzed by flow cytometry. At the end of the conditioning and engrafting period, we were able to generate mice that were chimeric with respect to hematopoietic cells (i.e. CD45 cells), however, those mice have exclusively human T-cells and B-cells. The average %CD45 in 34 XenoImmune™ mice derived from 10 healthy human donors was 23.4% (min 17.2% and max 29.2%). The average %CD45+/CD4+ T-cells in the same mice was 25.5% (min 21.0% and max 30.4%). Finally, the average %CD19+/CD45+ B-cells in those mice was 9.5% (min 8.3% and max 11%). Stability of XenoImmune mice and duration of engraftment: percentage of human hematopoietic cells (CD45+) in 4 mice derived from 2 different human donors increased from 32% (SD = 5.2) on day 28 to 61.7% (SD = 2.4) on day 183. The percentage of human CD4+ T-cells in the human hematopoietic cells was maintained between days 28 and 183 (29.5% vs 29.7%). Similarly, the percentage of human CD19+ B-cells in the human hematopoietic cells was maintained between days 28 and 183 (9.6% vs 12%). ADA response in XenoImmune™ mice vs C57BL6 mice. Only human anti-drug antibodies were detected in XenoImmune™ mice. In C57BL6 mice, only mouse anti-drug antibodies were detected. The percent of XenoImmune™ mice that were positive for human anti-drug antibodies was 25% for each of rhFVIII (3/12) and rh-Interferon-β (2/8) and 100 % for each of rhGAA (14/14), and AAV9 (11/11). These same XenoImmune™ mice were all negative for mouse anti-drug antibodies. The percent of C57BL/6 mice that were positive for human anti-drug antibodies was 0% for rhFVIII, rhGAA, rhInterferon-β and AAV9 treated mice respectively. Those same mice were all positive for mouse anti-drug antibodies for rhFVIII, rhGAA, rhInterferon-β and AAV9 respectively.

Conclusion: We have shown a successful engraftment of human PBMCs into our XenoImmune™ mice. The immunogenicity of 3 biologics; rhFVIII, rhGAA, and rh-Interferon-β, as well as AAV9, in our XenoImmune™ model recapitulates clinical immunogenicity rates vs wild-type C57BL/6.

Acknowledgements: This work was fully funded by LAPIX Therapeutics Inc. Both Authors are employees of LAPIX Therapeutics Inc and hold stock in the company.