Page 118 - Haematologica Vol. 109 - July 2024

ARTICLE - Cytotoxic reprogramming for BiTE immunotherapy M. Casey et al.
cytokines (IL-2, IL-7, IL-15, and IL-21) and interferon (IF- N)-γ-inducing cytokines (IL-12 and IL-18) are recognized as potent adjuvants for cancer immunotherapy by their ability to support either T-cell proliferation or modulating effector functions.12-14 Currently, therapeutic administration of these recombinant cytokines is widely being tested in combination with immune checkpoint inhibitors and other immunotherapy.12 While cytokine-based therapy can be a possible combination partner for T-cell redirection thera- py, overstimulation of T cells can drive activation-induced cell death and increase the risk of severe immune-related adverse events, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syn- drome. Activated T cells release pro-inflammatory cyto- kines including IFN-γ, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM- CSF), all of which are known to fuel the vicious cycle of CRS by stimulating monocytes/macrophages to produce IL-1b and IL-6.15-17 Above all, IFN-γ plays a crucial role in the immunopathology of CRS, as blockade or deletion of IFN-γ can ameliorate cytokine-related toxicities in CAR T-cell therapy.18,19 By contrast, the perforin/granzyme-mediated cytotoxic granule pathway negatively regulates macro- phage activation syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH) by killing activated monocytes/ macrophages. Indeed, familial HLH is seen in patients with mutations in the perforin gene (PRF1) or genes related to cytotoxic granule exocytosis.20,21 More recently, Ishii et al. showed that adoptive transfer of perforin-deficient CAR T cells, but not wild-type CAR T cells, triggers the MAS/ HLH-like preclinical phenotype with the excessive release of IL-1 family cytokines.22 These results provide evidence that the cytotoxic granule pathway negatively regulates aberrant macrophages inflammatory responses during T-cell redirection immunotherapy. Importantly, accumulated evidence supports that IFN-γ is dispensable for the efficacy of CAR T therapy against hematological malignancies, but not solid malignancies.18,19,23 Thus, we hypothesize that a cytokine-based therapy selectively harnessing the cytotoxic granule pathway might be an ideal strategy to achieve better responses by T-BsAb therapy. By performing a functional screening of immunostimulatory cytokines, this study aims to determine the optimal approach for cytokine-mediated priming of effector T cells and to establish proof-of-con- cept for improving the efficacy of T-BsAb therapy against multiple myeloma.
Methods
Details on experimental procedures and materials are in- cluded in the Online Supplementary Appendix.
Multiple myeloma cell lines
Human multiple myeloma cell lines (JJN-3 cells, KMS-11
cells, and RPMI8226)24 were cultured in RPMI1640 media supplemented with sodium pyruvate, non-essential amino acids, penicillin/streptomycin, and 10% heat-inactivated fetal bovine serum (complete RPMI [cRPMI]) Luciferase was transduced by retrovirus transfection of the MSCV- mCherry-IRES-luc2 vector, as described previously.25 My- coplasma negativity was confirmed by the Mycoplasma Detection Kit. The transplantable Vk14451 myeloma cells expressing enhanced green fluorescent protein (EGFP) were maintained as previously described.11,26
Primary cells and clinical samples
Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from healthy donors using LymphoprepTM density gradient medium. CD8 T cells were magnetically isolated by EasySepTM Human CD8+ T Cell Isolation Kit. Clinical samples from patients with newly diagnosed mul- tiple myeloma were collected at the Princess Alexandra Hospital, Brisbane. The study was approved by the QIMR Berghofer Human Research Ethics Committee (P2125) and by the Metro South Hospital and Health Service Human Research Ethics Committee (9448).
Screening assays
PBMC (4×105) from healthy donors and patients with newly diagnosed multiple myeloma were co-cultured with JJN-3 myeloma cells (1×105) in the presence of anti-BCMA T-BsAb (0.1 μg/mL) and one of the following recombinant human immunostimulatory cytokines: rIL-2 (500 U/mL), rIL-7, rIL-12, rIL-15, rIL-18 and rIL-21 (50 ng/mL). The levels of granzyme B, perforin, IFN-γ, TNF-α, and GM-CSF in culture supernatants were determined by enzyme-linked immunosorbant assay (ELISA) 3 days after co-culture. Results were calculated as fold changes relative to control (T-BsAb stimulation in the absence of immunostimulatory cytokines).
T-cell-engaging bispecific antibody assays
For T-cell proliferation assays, PBMC were labeled with CellTraceTM Violet (CTV) and co-cultured with JJN-3 my- eloma cells (1×105) in cRPMI supplemented with recombi- nant IL-2 (rIL-2, 100 U/mL) at the effector-to-target ratio (E: T ratio) of 4:1. These cells were stimulated with the indicated concentrations of rIL-21 and anti-BCMA T-BsAb for 3 days, and CTV-dilution in live CD8 T cells was mea- sured by flow cytometry. In some experiments, unlabeled PBMC were used to examine expression levels of CD226 and CD28 in CD8 T cells. The levels of cytotoxic granules (granzyme B and perforin) and cytokines (IFN-γ, TNF-α, and GM-CSF) in culture supernatants were measured by ELISA. For ex vivo stimulation in primary samples, bulk bone marrow (BM) mononuclear cells were reconstituted in cRPMI supplemented with rIL-2 (50 U/mL) (8×105/mL). These cells were stimulated with anti-BCMA T-BsAb (0.2 μg/mL) in the presence or absence of rIL-21 (100 ng/mL) for 3 days.
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