(M1230-04-22) Novel Personalized Treatment for Multiple Myeloma: Multi-Antigen T Cell Hybridizers for T Cell Activation

Purpose: Multiple myeloma (MM) represents a heterogeneous hematological malignancy characterized by the clonal proliferation of plasma cells within the bone marrow [1]. Despite significant advancements in the therapeutic strategies, MM remains largely incurable, necessitating the development of innovative treatment modalities. One promising avenue is immunotherapy, particularly the use of T cell engaging therapies, which facilitates the recruitment and activation of T cells and harnesses their cytotoxic potential to eliminate cancer cells. B-cell maturation antigen (BCMA) has emerged as a promising target for MM immunotherapy due to its high expression on malignant plasma cells while exhibiting limited expression on healthy tissues [1]. Bispecific T cell engaging antibody constructs targeting BCMA have shown encouraging clinical results, such as teclistamab, highlighting the therapeutic potential of redirecting T cells to BCMA-expressing MM cells. However, the efficacy of these approaches is often limited by antigenic heterogeneity within MM tumors, as well as the potential for BCMA downregulation or loss, leading to treatment resistance and disease relapse. Multi-antigen T cell hybridizers (MATCH) is a novel and versatile approach to enhance the precision and efficacy of T cell-based immunotherapy for MM. Hybridization of complementary 25 base pair morpholino oligonucleotides, one bound to cancer targeting Fab’ (Fab’_BCMA-MORF1), the other to T cell directed Fab’ (Fab’_CD3-MORF2) mediates T cell recruitment and activation against malignant B cells. MATCH builds upon the concept of bispecific antibody constructs but offers the unique ability to target multiple antigens simultaneously, including BCMA and other MM-associated antigens, such as CD38 and CS1. Personalized therapy can be achieved by applying a cocktail of B cell targeting nanoconjugates (e.g., mixture of Fab’_BCMA-MORF1, Fab’_CD38-MORF1, Fab’_SLAM7-MORF1) based upon the antigen expression profile of patient cells. Targeting a broader range of antigens overcomes the limitations associated with single antigen targeting strategies and improves overall therapeutic outcomes.

Methods: Two complementary 25 base pair phosphonodiamidites MORFs were customized from Gene Tools. Fab’-MORF conjugates were synthesized as previously described [2]. The nanoconjugates were characterized by size exclusion chromatography (SEC) and SDS-PAGE to ensure correct assembly and integrity. The efficacy of MATCH in vitro was evaluated on the MM.1S and RPMI-8226 multiple myeloma cell lines, which express BCMA, CD38, and CS1 antigens. T cells were isolated from the whole blood of healthy donors using a T cell isolation kit provided by StemCell Technologies. The cytotoxic impact of T cells on tumor cells was quantitatively measured using the Cell Counting Kit-8 (CCK-8). Additionally, apoptosis was evaluated using the Annexin V/Propidium Iodide (PI) staining method, providing a detailed assessment of early and late apoptotic events within the tumor cells. To further dissect the underlying mechanisms of T cell-mediated cytotoxicity, we focused on two key pathways: degranulation and the Fas-FasL recognition pathway. Degranulation was assessed by measuring the surface expression of CD107a, a marker of T cell activation and degranulation, indicating the release of cytotoxic granules. The involvement of the Fas-FasL pathway, critical for inducing apoptosis in target cells, was evaluated through mitochondria depolarization and caspase 3/7. These investigations provided comprehensive insights into the functional efficacy and mechanism of action of the T cells when engaged by the MATCH therapy [3].

Results: Conjugates targeting cancer cells (Fab’_BCMA-MORF1) and T cells (Fab’_CD3-MORF2) were synthesized and their hybridization was demonstrated (Figure 1). The integrity and successful conjugation of these components were confirmed by shifts in the peaks observed in size exclusion chromatography (SEC) (Figure 1C) and distinct band positions in sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE) (Figure 1D). Preliminary in vitro experiments demonstrated that naïve T cells from healthy donors were robustly activated and could effectively kill MM.1S cells when engaged by MATCH configurations targeting BCMA, CD38, and CS1 antigens on MM.1S surface. This engagement significantly shifted IC50 values, indicating enhanced potency of trispecific MATCH compared to monospecific configurations (Figure 2C). The apoptosis induced by MATCH is shown in Figure 2D. Further analysis of the mechanisms underlying T cell activation and tumor cell killing showed substantial T cell degranulation (Figure 3B), mitochondrial depolarization (Figure 3D) and activation of caspases 3/7 (Figure 3C), which are critical markers of apoptosis. These results underline the potential for durable responses and highlight the effectiveness of MATCH in overcoming resistance mechanisms and enhancing T cell-mediated cytotoxicity. Furthermore, a rechallenge experiment with T cells and MM.1S cells showed that repeated exposure to MATCH-treated cells led to sustained T cell efficacy and tumor cell depletion, highlighting the potential for durable responses.

Conclusion: MATCH represents a significant advancement in the immunotherapeutic management of multiple myeloma, offering a versatile, potent, and customizable approach to target the complex antigenic landscape of MM tumors. This technology not only promises to improve response rates but also to provide a strategic platform for addressing treatment resistance and relapse, thereby potentially transforming the therapeutic landscape for MM and other hematological malignancies.

References: [1] N. Shah et al., Leukemia. 34, 985 (2020).
[2] L. Li et al., ACS Nano. 12, 3658 (2018).
[3] M.R. Betts et al., Method Cell Biol. 75, 497 (2004).

Acknowledgements: The research was supported by NIH grant R01 CA246716 (to JK) from the National Cancer Institute and by a grant from Huntsman Cancer Institute to (JY).


Figure 1. Synthesis and characterization of MATCH nanoconjugates. A) Structure of MORF1 and MORF2. B) Schematic of Fab’-MORF1 synthesis. (C) Size exclusion chromatography spectra showing the peak of the BCMA F(ab’)2, Fab’BCMA-MORF1, and hybridization of Fab’BCMA-MORF1 and Fab’CD3-MORF2. (D) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of anti-BCMA Ab, Fab’BCMA-MORF1 and Fab’BCMA.


Figure 2. Myeloma cell line antigen profiling and T cell activation. A) Target antigen expression profiles of BCMA, CS1 and CD38 on MM.1S and RPMI 8226 cells. B) Fluorescence imaging demonstrating the binding of Cy5-Fab’BCMA-MORF1 on the surface of MM.1S cells, illustrating target-specific attachment. C) Comparison of cell viability assays on MM.1S cells treated with monospecific MATCH therapies versus a trispecific combinatorial MATCH therapy incorporating BCMA, CS1 and CD38, highlighting the enhanced efficacy of the trispecific approach. D) Apoptosis induced by T cell activation on MM.1S using BCMA-target MATCH.


Figure 3. A) Schematic depiction of the MATCH targeting motif capable of hybridizing with complementary oligonucleotide strands on T cell engager molecules and inducing cell death by degranulation and Fas-FasL recognition. B) Quantification of degranulation of T cells measured by CD107a surface expression. C) Caspase 3/7 activation following treatment with BCMA-target MATCH therapy D) Mitochondrial depolarization after treatment of BCMA-targeting MATCH.