Immune cell tumor engagers (SITE) via morpholino-guided self-assembly for precision immunotherapy of multiple myeloma Free

Introduction: Bispecific T cell engagers have shown significant clinical efficacy and are currently FDA approved for the treatment of relapsed and refractory multiple myeloma (RRMM). However, challenges remain, including targeting flexibility, immune-related toxicity, and durability of response. To overcome these limitations, we developed Self-Assembled Immune Cell-Tumor Engagers (SITE), a two-component system that targets both immune cell and MM cell surface antigens; each component is tagged with a complementary morpholino oligonucleotide (M1 or M2). Upon administration, these components hybridize in situ via M1-M2 pairing to form bispecific complexes to redirect immune cells to MM cells. Utilization of this pre-targeting approach enables the sequential engagement of immune cells, including T cells, NK cells, and macrophages. Additionally, by simultaneously targeting of multiple antigens (i.e. BCMA, GPRC5D and CD38), this strategy minimizes the risk of antigen loss and immune escape. Herein, we report the efficacy of SITE across multiple models, including in vitro cell lines, ex vivo patient-derived samples, and in vivo mouse models, demonstrating its potential as an innovative, cost-effective platform for orchestrating a coordinated and robust anti-tumor immune response.

Methods: Anti-MM antibodies were digested enzymatically to F(ab')₂ fragments, further reduced with tris(2-carboxyethyl)phosphine to generate Fab'_MM-thiols. A pair of 25 bp M1/M2 was customized from Genetools. Following 3'-end maleimido modification, a panel of Fab'_MM-M1 targeting BCMA, GPRC5D and CD38 was obtained. Similarly, α-hCD3 and α-hCD314 were used to generate immune cell targeting Fab'_immune-M2. Stability of M1-M2 hybrids was assessed using circular dichroism (CD) spectroscopy in PBS, and size-exclusion chromatography (SEC) following incubation in mouse serum. Primary human T cells and NK cells were isolated from healthy donor blood. In vitro cytotoxicity and immune activation of SITE were assessed by flow cytometry on MM.1S and RPMI-8226 cells. To assess therapeutic efficacy, both patient-derived bone marrow mononuclear cells and a preclinical NRG mouse model were utilized. Mice were sub-lethally irradiated and i.v. injected with 3×10⁶ MM.1S-Luc cells. Human T cells (9×10⁶,3×10⁶, or1×10⁶) were administered to study the influence of T cells. Different dosing regimens (premix vs. consecutive) of T cell-specific SITE constructs (Fab'_BCMA-M1/Fab'_CD3-M2) were evaluated and compared with conventional teclistamab treatment. Cytokine production (IL-2, IFN-γ, TNF-α) was analyzed, and immune cell subsets were profiled by flow cytometry.

Results: Conjugates Fab'_MM-M1 and Fab'_immune-M2 were successfully synthesized; hybridization of Fab'_MM-M1/Fab'_immune-M2 upon 1:1 mixing was confirmed by a shift in elution profiles in SEC. Complexes remained stable in 10% mouse serum for over 24 h. CD spectra showed a positive peak at 260 nm and a negative minimum at 210 nm, indicating formation of A-form double helices under physiological conditions. In vitro, SITE constructs successfully engaged T cells and NK cells, triggering potent cytotoxicity towards MM.1S and RPMI-8226 cells. Flow cytometry demonstrated increased expression of activation markers and induction of apoptosis in target cells. Ex vivo studies using patient-derived samples confirmed significant clearance of MM cells by patient's own T cells. In vivo, NRG mice bearing MM.1S-Luc and treated with SITE-T cell therapy extended survival and inhibited tumor growth compared to teclistamab-treated groups. Flow cytometric analysis of bone marrow confirmed a marked reduction in MM.1S cell populations in SITE-treated mice. T cell-related toxicity was found to be associated with injected T-cell numbers: reducing the dose from 9×10⁶ to 3×10⁶ or lower, no body weight loss occurred, highlighting the advantageous flexibility of two-component SITE. Overall, SITE demonstrated a superior anti-tumor effect compared to teclistamab, highlighting its potent efficacy in controlling MM progression in vivo.

Conclusion: The SITE platform represents a promising advance in immunotherapy for MM, offering a versatile, potent, and modular approach to address the disease's complex antigenic landscape. By enabling multi-antigen targeting and the orchestration of diverse immune effectors, the platform effectively overcomes challenges such as antigenic heterogeneity, immune escape, and cytokine release syndrome.