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The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8 N/C EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8 N/C EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8 N/C EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. Cancer therapy using systemically administrated 4-1BB-targeting antibodies is often associated with severe toxicity due to the nonspecific activation of autoreactive T cells. Here, the authors have developed a trimeric antibody targeting both 4-1BB and EGFR, which activates T cells effectively and shows negligible cytotoxicity.

Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, andB15R). We found that these genes act together to inhibit the NFκB signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus in-fection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Nα and Nβ) to the infection site. Nβ cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design.

BackgroundMultiple barriers in the tumor microenvironment (TME) have hampered development of CAR-T cell therapies for solid tumors. These challenges include an immunosuppressive TME, poor trafficking of CAR-T cells to the tumor and shortage of highly expressed tumor-specific antigens. We recently demonstrated that Tumor-Specific Immuno-Gene (T-SIGn) viral vectors encoding immunostimulatory cytokines, costimulators and chemokines can reprogram the TME towards a pro-inflammatory phenotype resulting in a markedly increased therapeutic efficacy of CAR-T cells in a A549 human tumor xenograft and lung metastasis model.1 Here we further explored the potential of the T-SIGn platform in combination with CAR-T cell therapy by developing and characterizing a T-SIGn viral vector that simultaneously expresses immunostimulatory cytokines/chemokines and a secreted bispecific protein incorporating a CAR-T cell target antigen. This secreted ligand binds to (“spray paints”) tumor cells to enable recognition by CAR-T cells.MethodsWe used a T-SIGn virus (NG-1125) expressing a secreted anti-HER2-CD19 fusion protein (saH2-19), as a model “spray paint” antigen, encoded together with IFNa and CXCL9 as example cytokine/chemokines. In vitro, human tumor cell lines were used to assess the ability of T-SIGn viruses to induce tumor-specific expression and activity of saH2-19 as CAR-T cell target antigen. We quantified T-SIGn vector-encoded CD19 expression on tumor cell surfaces using flow cytometry and CAR-T mediated killing via xCELLigence. In vivo, expression of CD19 fusion protein and transferred CD19 CAR-T cells in tumors were assessed by flow cytometry analysis and immunohistochemistry of A549 tumor xenografts.ResultsUsing in vitro human cell culture models, the NG-1125 vector led to efficient expression of saH2-19 on tumor cell surfaces, both on vector-infected and non-infected or non-permissive cells. This enabled effective antigen-specific tumor cytotoxicity by CD19-specific CAR-T cells. Using an in vivo human A549 lung tumor xenograft model adoptively transferred with human CD19-specific CAR-T cells, NG-1125 induced tumor-specific CD19 expression on both vector infected and non-infected cells (demonstrating antigen “spray painting”) together with accumulation of activated T cells. This accumulation of T cells was not seen with a vector only expressing the saH2-19 transgene.ConclusionsTogether, our data provide a proof of concept that T-SIGn vectors can be designed to deliver TME-modifying immunomodulators together with “spray paint” antigens that effectively enable tumor cell recognition and destruction by CAR-T cells specific for target antigens not endogenously expressed by tumors. Further studies are exploring the full potential of this “spray painting” approach to enable CAR-T cell therapy for of solid tumors.ReferenceSonzogni O, Zak DE, Sasso MS, Lear R, Muntzer A, Zonca M, West K, Champion BR, Rottman JB. T-SIGn tumor reengineering therapy and CAR T cells synergize in combination therapy to clear human lung tumor xenografts and lung metastases in NSG mice. Oncoimmunology 2022; 11. https://doi.org/10.1080/2162402X.2022.2029070

Human adipose-derived stem cells (hASC) are mesenchymal stem cells with reduced immunogenicity and the ability to modulate immune responses. APRIL and BAFF proteins are overexpressed in inflammatory and autoimmune diseases for which allogeneic hASC therapy is currently under clinical investigation. Modification of hASC properties by the tissue microenvironment could be a critical factor in patient outcome and is still not well understood. Our aim was to characterize the APRIL/BAFF system in hASC by analyzing the ligand and receptor expression patterns, the effects mediated by APRIL and BAFF on hASC, and the underlying signaling. We found that hASC express the tumor necrosis factor proteins APRIL (a proliferation-inducing ligand) and BAFF (B cell-activator factor) as well as their receptors TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), BCMA (B cell maturation antigen) and the BAFF-specific receptor (BAFF-R). APRIL and BAFF secretion was differentially enhanced by CXCL12 and interferon (IFN)-γ, implicated in hASC-mediated migration and immunosuppression, respectively. In addition, APRIL and BAFF induced rapid phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt kinases and promoted an increase in hASC proliferation, without affecting the immunosuppressive capacity of these cells. The use of specific chemical inhibitors indicated that the PI3K transduction pathway is involved in hASC basal growth and that APRIL- and BAFF-mediated effects are ERK-dependent. These results provide new information about the molecular mechanisms that underlie APRIL and BAFF secretion and signaling in hASC, and are of special relevance for the use of allogeneic hASC as therapeutic tools.

Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, and B15R). We found that these genes act together to inhibit the NF...B signaling pathway. Triple ablation in modified virus restored NF...B function in macrophages. After virus infection of mice, NF...B pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Na and Nβ) to the infection site. Nβ cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design. (ProQuest: ... denotes formulae/symbols omitted.)