Expression of inducible factors reprograms CAR-T cells for enhanced function and safety.

Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.

Folate Receptor Beta as a Direct and Indirect Target for Antibody-Based Cancer Immunotherapy.

Folate receptor beta (FRß) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRß is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRß is a potential target for both direct and indirect cancer therapy. We demonstrate that FRß is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRß (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRß and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRß using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRß-expressing cancers.

The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCbeta.

Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCbeta isoforms. Pharmacological inhibition of PKCbeta resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCbeta. To test this hypothesis, bone marrow-derived macrophages (BMMf) obtained from PKCbeta knockout (PKCbetaKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild-type L. monocytogenes escapes from the phagosome of PKCbetaKO BMMf as well as from C57Bl/6 BMMf. However, in PKCbetaKO BMMf, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCbeta.

Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis.

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

Involvement of Listeria monocytogenes phosphatidylinositol-specific phospholipase C and host protein kinase C in permeabilization of the macrophage phagosome.

We have previously shown that phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Listeria monocytogenes activates a host protein kinase C (PKC) cascade which promotes escape of the bacterium from a macrophage-like cell phagosome. Here, we provide evidence linking bacterial PI-PLC and host PKC beta to phagosome permeabilization, which precedes escape.

Induction of myasthenia gravis in HLA transgenic mice by immunization with human acetylcholine receptors.

We utilized HLA transgenic mice to identify the dominant epitopes on the human (H)-AChR alpha subunit. The cytoplasmic H-AChR peptide alpha320-337 was the dominant T cell epitope for DQ8, DR3, and DQ8xDQ6 F1 mice. The H-AChR-immunized HLA-DQ8, DR3, DQ8xDR3 F1 and DQ8xDQ6 F1 mice developed clinical EAMG, whereas HLA-DQ6 mice were less susceptible.

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis.

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.

Suppressed clinical experimental autoimmune myasthenia gravis in bm12 mice is linked to reduced intracellular calcium mobilization and IL-10 and IFN-gamma release by acetylcholine receptor-specific T cells.

Class II MHC mutant bm12 mice have an increased resistance to experimental autoimmune myasthenia gravis (EAMG) compared to C57BL/6 mice. In vitro, this relative resistance was mainly associated with a reduced cytokine response to acetylcholine receptor (AChR) and its dominant pathogenic peptide alpha 146-162, whereas the response to the epitope alpha 111-126 remained intact. Calcium mobilization after stimulation of AChR-immune T cells with AChR or alpha 146-162 peptide, but not alpha 111-126 peptide, was decreased in bm12 compared to C57BL/6. Thus, the reduced incidence of clinical EAMG in bm12 is linked to lower IFN-gamma and IL-10 release, and intracellular calcium mobilization by alpha 146-162-specific T cells.

Role of IL-5 during primary and secondary immune response to acetylcholine receptor.

To analyze the role of the Th2 cytokine interleukin-5 (IL-5) in experimental autoimmune myasthenia gravis (EAMG) pathogenesis, we induced clinical EAMG in C57BL/6 and IL-5 gene-knockout (KO) mice in the C57BL/6 background. IL-5 KO mice had a significantly reduced incidence and severity of EAMG. Despite their increased resistance to EAMG, IL-5 KO mice displayed intact secondary antibody and lymphoproliferative responses to acetylcholine receptor (AChR) after immunization with this molecule. However, the relative resistance of IL-5 KO mice was associated with a reduced primary lymphocyte response to AChR, and reduced C3 levels in muscle extracts compared to those in C57BL/6 mice.

Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice.

Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8xDQ6 and DQ8xDR3 F(1) transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (alpha320-337, alpha304-322, and alpha419-437), of which the response to alpha320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR- and alpha320-337 peptide-specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG(2b), and IgG(2c) Ab's and reduced IL-2 and IFN-gamma secretion by H-AChR- and peptide alpha320-337-specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence alpha320-337 as a dominant trait.

Tumor necrosis factor receptor p55 and p75 deficiency protects mice from developing experimental autoimmune myasthenia gravis.

The precise pathogenic role of proinflammatory cytokines belonging to the tumor necrosis factor (TNF) family has not been investigated yet in antibody-mediated myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG). In this study we report that TNF receptor p55(-/-) p75(-/-) mice were resistant to the development of clinical EAMG induced by acetylcholine receptor (AChR) immunizations. The resistance was associated with reduced serum levels of IgG, IgG(1), IgG(2a), and IgG(2b) anti-AChR antibody isotypes. However, IgM anti-AChR antibodies were not reduced, suggesting defective anti-AChR IgG class switching in TNF receptor p55(-/-) p75(-/-) mice. We thus demonstrate the genetic evidence for the role of TNF receptor p55 and p75 in EAMG pathogenesis, and their requirement for the generation of anti-AChR IgG antibodies.