ABSTRACT
Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.
Subject(s)
Immunotherapy, Adoptive/methods , Interleukin-15/administration & dosage , Interleukin-15/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/transplantation , Animals , Disease Models, Animal , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute/immunology , Leukemia, Promyelocytic, Acute/therapy , Mice , Mice, Inbred NOD , Mice, SCID , Xenograft Model Antitumor AssaysSubject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cytotoxicity, Immunologic/drug effects , Immunomodulation/drug effects , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Leukemia, Myeloid, Acute/immunology , Protein Kinase Inhibitors/pharmacology , Pyridinium Compounds/pharmacology , Animals , Biomarkers , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line, Tumor , Cyclic N-Oxides , Disease Models, Animal , Humans , Immunophenotyping , Indolizines , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Protein Kinase Inhibitors/therapeutic use , Pyridinium Compounds/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
In rodent graft-versus-host disease (GVHD) models, anti-IL-21 neutralizing mAb treatment ameliorates lethality and is associated with decreases in Th1 cytokine production and gastrointestinal tract injury. GVHD prevention was dependent on the in vivo generation of donor-inducible regulatory T cells (Tregs). To determine whether the IL-21 pathway might be targeted for GVHD prevention, skin and colon samples obtained from patients with no GVHD or grade 2 to 4 GVHD were analyzed for IL-21 protein expression. By immunohistochemistry staining, IL-21 protein-producing cells were present in all gastrointestinal tract samples and 54% of skin samples obtained from GVHD patients but not GVHD-free controls. In a human xenogeneic GVHD model, human IL-21-secreting cells were present in the colon of GVHD recipients and were associated with elevated serum IL-21 levels. A neutralizing anti-human IL-21 mAb given prophylactically significantly reduced GVHD-associated weight loss and mortality, resulting in a concomitant increase in Tregs and a decrease in T cells secreting IFN-γ or granzyme B. Based on these findings, anti-IL-21 mAb could be considered for GVHD prevention in the clinic.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , Interleukins/antagonists & inhibitors , Lymphocytes/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Animals , Enzyme-Linked Immunosorbent Assay , Fetal Blood , Flow Cytometry , Graft vs Host Disease/mortality , Humans , Interleukins/immunology , Lymphocyte Count , Mice , Mice, Inbred NOD , Mice, SCID , Signal Transduction , Survival Rate , Weight LossABSTRACT
Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4(+)CD25(+) regulatory T cells (nT(regs)) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT(regs) has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT(regs) could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT(regs) in UCB units is limited, and average yield after expansion was only 1 × 10(9) nT(regs). Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT(regs). PB nT(regs) were purified under GMP conditions and expanded 80-fold to yield 19 × 10(9) cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 10(9) cells while maintaining Foxp3 expression and suppressor function. nT(reg) expansion was ~50 million-fold when flow sort-purified nT(regs) were restimulated four times with aAPCs. Indeed, cryopreserved donor nT(regs) restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nT(regs) could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.
