Non-clinical Evaluation of 4D-310 in Combination with Rituximab/Sirolimus - A Translational Study to Support Adoption of a Novel Prophylactic Immunomodulation Regimen in Clinical Trials in Adults with Fabry Disease

Sep 11, 2026

Non-clinical evaluation assessed safety, anti-capsid antibody responses, biodistribution, complement activation, and transgene expression after intravenous administration of 4D-310 to nonhuman primates. The study compared rituximab/sirolimus versus prednisolone across multiple dosing cohorts and measured humoral immune and complement biomarkers, including total and IgM antibodies to the C102 capsid and complement factors such as C3a, C4a, Bb, C3, C4, and sC5b–9. Rituximab/sirolimus efficiently depleted CD40+ B cells, attenuated treatment-emergent antibody responses, and maintained complement activity near normal.

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Background and Rationale

Methods

Study Design

Study Outcomes

Results

B Cell Depletion

Humoral Immune Response

Complement Activity

Safety and Tolerability

\#047 Non-clinical Evaluation of 4D-310 in Combination with Rituximab/Sirolimus A Translational Study to Support Adoption of a Novel Prophylactic Immunomodulation Regimen in Clinical Trials in Adults with Fabry Disease Melissa A. Calton, Joshua Holter, Caralee Schaefer, Kathryn Yoh, Ted Sullivan, Samantha Jensen, Edward Kim, Christian Vettermann, David Kirn, An Song 4D Molecular Therapeutics Inc., Emeryville, California Background and Rationale ▪ 4D-310 is a clinical-stage genetic medicine that employs a cardiotropic AAV vector (C102) to deliver a codon-optimized human GLA transgene to cardiomyocytes for cell-autonomous 􀁄-galactosidase A (AGA) production via intravenous administration; activity is augmented by cross-correction from AGA secreted into blood. C102 Ubiquitous Promoter coh-GLA Transgene Figure 1. 4D-310 gene therapy product design. ▪ Thrombotic microangiopathy (TMA) and atypical hemolytic uremic syndrome (aHUS) caused by antibody-dependent activation of the classical complement pathway are known class effects of systemic AAV-based gene therapy. 1–4 ▪ Published reports have shown that prophylactic administration of rituximab/sirolimus attenuates humoral response to the AAV capsid following systemic AAV9-based gene therapy.5 ▪ To support the adoption of rituximab/sirolimus as a prophylactic immunomodulatory regimen in the ongoing 4D-310 INGLAXA Phase 1/2 clinical trial (NCT04519749), we performed a 10-week nonhuman primate study to evaluate safety, anti-capsid antibody response, biodistribution, and transgene expression following administration of 4D-310 with either rituximab/sirolimus or prednisolone. Methods Study Design Cohort 1 Cohorts 2 and 3 | Cohort | N | 4D-310 | Immunomodulatory Regimen | | --- | --- | --- | --- | | 1 | 2 | 1 × 1013 vg/kg | Prednisolone 3 mg/kg/d (beginning on day –1)* | | 2 | 3 | 1×1013 vg/kg | IV rituximab 750 mg/m2 (days –15 and 0)†
Oral sirolimus 0.5–8 mg/m2/d (beginning on day –3)‡ | | 3 | 3 | 3 × 1012 vg/kg | | 4D-310 Prednisolone Sirolimus Rituximab Rituximab Day -15 0 56 * Protocol-specified prophylactic immunomodulatory therapy in the initial dose cohort in the INGLAXA Phase 1 clinical trial evaluating 4D-310. †Diphenhydramine 4 mg/m2 administered via intramuscular injection prior to rituximab ‡Dose titrated to maintain a 24-hour trough concentration of 2–4 ng/mL. Study Outcomes | Outcome | Marker(s) | | --- | --- | | Humoral immune response | Serum antibodies (Total and IgM), B cells | | Complement activation | Serum/plasma complement factors (C3a, C4a, Bb, C3, C4, sC5b–9) | | 4D-310 biodistribution | Viral DNA (qPCR), GLA mRNA (ISH), AGA protein (IHC) | | Transgene protein expression | Tissue AGA activity | | Safety and tolerability | In-life examination, clinical pathology, anatomic pathology | Results B Cell Depletion CD40 + B ce l ls (% ) 100 80 60 40 20 0 Day 1x1013 vg/kg + pred 1x1013 vg/kg + R/S 3x1012 vg/kg + R/S -21 1 15 29 43 57 -21 1 15 29 43 57 -21 1 15 29 43 57 Rituximab/sirolimus results in efficient depletion of CD40+ B cells. Flow cytometry immunophenotyping and quantification of CD40+ B cells following administration of 4D- 310 with either rituximab/sirolimus (n=3 animals per dose group) or prednisolone (n=2 animals) . CD40+ cells were used to quantify B cells owing to the potential for rituximab to interfere with detection of CD20+ cells by flow cytometry. A >95% reduction in CD40+ B cells was observed on Day 1 compared to Day-21 in both rituximab/sirolimus groups. Data are shown as mean +SEM. Humoral Immune Response A B Ant i-C 102 tota l Ab t iter 2×105 1.5×105 1×105 5×104 0 1x1013 vg/kg + prednisolone 1x1013 vg/kg + R/S 3x1012 vg/kg + R/S -16 1 3 5 7 15 22 29 36 43 57 Day Ant i-C 102 IgM t iter 6×104 5×104 4×104 3×104 2×104 1×104 0 1x1013 vg/kg + prednisolone 1x1013 vg/kg + R/S 3x1012 vg/kg + R/S -16 1 3 5 7 15 22 29 36 43 57 Day Ri

FAQs

What was the main purpose of this non-clinical study of 4D-310?

To evaluate safety and key immunological and biological responses after administering 4D-310, supporting adoption of a rituximab/sirolimus prophylactic immunomodulation regimen for clinical trials in adults with Fabry disease.

How were the immunomodulatory regimens compared in the study?

Animals received 4D-310 with either rituximab/sirolimus or prednisolone, with multiple cohorts differing by 4D-310 dose and treatment schedule.

What did the results show regarding humoral responses and B-cell depletion?

Rituximab/sirolimus produced efficient depletion of CD40+ B cells and attenuated treatment-emergent total and IgM antibody responses to the C102 capsid compared with prednisolone.

Sep 11, 2026

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