In vivo CAR T-cell generation in non-human primates using lentiviral vectors displaying a multi-domain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high cost and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVecTM platform, a lentiviral vector capable of generating CAR T-cells in vivo. Here we describe the incorporation of T cell activation and costimulatory signals onto the surface of VivoVecTM particles (VVPs) in the form of a multi-domain fusion protein and show enhanced in vivo transduction and improved CAR-T cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into non-human primates resulted in the robust generation of anti-CD20 CAR T-cells and the complete depletion of B cells for more than 10 weeks. These data validate the VivoVecTM platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60.

Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by NK cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors, however, is limited because of adverse side effects. Using high-throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding, but rather suppressed posttranslational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3.

TOX2 regulates human natural killer cell development by controlling T-BET expression.

Thymocyte selection-associated high mobility group box protein family member 2 (TOX2) is a transcription factor belonging to the TOX family that shares a highly conserved high mobility group DNA-binding domain with the other TOX members. Although TOX1 has been shown to be an essential regulator of T-cell and natural killer (NK) cell differentiation in mice, little is known about the roles of the other TOX family members in lymphocyte development, particularly in humans. In this study, we found that TOX2 was preferentially expressed in mature human NK cells (mNK) and was upregulated during in vitro differentiation of NK cells from human umbilical cord blood (UCB)-derived CD34(+) cells. Gene silencing of TOX2 intrinsically hindered the transition between early developmental stages of NK cells, whereas overexpression of TOX2 enhanced the development of mNK cells from UCB CD34(+) cells. We subsequently found that TOX2 was independent of ETS-1 but could directly upregulate the transcription of TBX21 (encoding T-BET). Overexpression of T-BET rescued the TOX2 knockdown phenotypes. Given the essential function of T-BET in NK cell differentiation, TOX2 therefore plays a crucial role in controlling normal NK cell development by acting upstream of TBX21 transcriptional regulation.

Aberrant antibody affinity selection in SHIP-deficient B cells.

The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B-cell survival and selection in the GC. Here, we have used mice with B-cell-specific deletion of the 5'-inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag- and interferon (IFN)-induced activation, resulting in augmented negative selection in the BM and enhanced B-cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen-egg lysozyme Ag in the MDA4 transgenic model. SHIP-deficient B cells spontaneously produce isotype-switched antibodies; however, they are poor responders in immunization and infection models. While SHIP-deficient B cells form GCs and undergo mutation, they are not properly selected for high-affinity antibodies. These results illustrate the importance of negative regulation of B-cell responses, as lower thresholds for B-cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation.

Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability.

Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.

Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine 245.

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon gamma production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domain-containing protein tyrosine phosphatase 2 and beta-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection.

Differential roles for the inositol phosphatase SHIP in the regulation of macrophages and lymphocytes.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) negatively regulates antigen, cytokine, and Fc receptor signaling pathways in immune cells. Our knowledge of the function of SHIP largely derives from in vitro studies that utilized SHIP-deficient cell lines and immune cells isolated from SHIP null mice. To avoid the pleiotropic effects observed in mice with germline deletion of SHIP, we have used the Cre-lox system to generate SHIP conditional knockout mice with deletion in specific immune cell populations. In this review we summarize our observations from mice with deletion of SHIP in lymphocyte and macrophage lineages and contrast them with earlier data gathered by the analysis of SHIP null mice.

Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization.

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells.

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

Modulation of NKG2D ligand expression and metastasis in tumors by spironolactone via RXRγ activation.

Tumor metastasis and lack of NKG2D ligand (NKG2DL) expression are associated with poor prognosis in patients with colon cancer. Here, we found that spironolactone (SPIR), an FDA-approved diuretic drug with a long-term safety profile, can up-regulate NKG2DL expression in multiple colon cancer cell lines by activating the ATM-Chk2-mediated checkpoint pathway, which in turn enhances tumor elimination by natural killer cells. SPIR can also up-regulate the expression of metastasis-suppressor genes TIMP2 and TIMP3, thereby reducing tumor cell invasiveness. Although SPIR is an aldosterone antagonist, its antitumor effects are independent of the mineralocorticoid receptor pathway. By screening the human nuclear hormone receptor siRNA library, we identified retinoid X receptor γ (RXRγ) instead as being indispensable for the antitumor functions of SPIR. Collectively, our results strongly support the use of SPIR or other RXRγ agonists with minimal side effects for colon cancer prevention and therapy.

The inositol 5'-phosphatase SHIP-2 negatively regulates IgE-induced mast cell degranulation and cytokine production.

Aggregation of the high-affinity IgE receptor (FcepsilonRI) on mast cells initiates signaling pathways leading to degranulation and cytokine release. It has been reported that SHIP-1 negatively regulates FcepsilonRI-triggered pathways but it is unknown whether its homologous protein SHIP-2 has the same function. We have used a lentiviral-based RNA interference technique to obtain SHIP-2 knockdown bone marrow-derived mast cells (BMMCs) and have found that elimination of SHIP-2 results in both increased mast cell degranulation and cytokine (IL-4 and IL-13) gene expression upon FcepsilonRI stimulation. Elimination of SHIP-2 from BMMCs has no effect on FcepsilonRI-triggered calcium flux, tyrosine phosphorylation of MAPKs or in actin depolymerization following activation. Rather, we observe that absence of SHIP-2 results in increased activation of the small GTPase Rac-1 and in enhanced microtubule polymerization upon FcepsilonRI engagement. Coimmunoprecipitation experiments in rat basophilic leukemia (RBL 2H3) cells show that SHIP-2 interacts with the FcepsilonRI beta-chain, Gab2 and Lyn and that unlike SHIP-1, it does not associate with SHC in mast cells. Our results report a negative regulatory role of SHIP-2 on mast cell activation that is calcium independent and distinct from the regulation by SHIP-1.

Antigenicity and immunogenicity of the N-terminal 33-kDa processing fragment of the Plasmodium falciparum merozoite surface protein 1, MSP1: implications for vaccine development.

The Plasmodium falciparum merozoite surface protein 1 (MSP1), MSP1-42 and MSP1-19 are protective malaria vaccines. MSP1-42 is cleaved to form MSP1-33 and MSP1-19. The role of MSP1-33 in immunity is unclear. We investigated the antibody responses to MSP1-33; and to MSP1-33Trunc, in which major conserved sequences were excised. While anti-MSP1-33 antibodies were subdominant in the anti-MSP1-42 responses, immunizations with MSP1-33 or MSP1-33Trunc induced high levels of antibodies reactive with MSP1-42 or whole merozoites. Anti-MSP1-33 and anti-MSP1-33Tunc antibodies crossreacted with both allelic forms of MSP1-42. Anti-MSP1-33 sera were ineffective in inhibiting parasite growth in vitro; but they significantly enhanced the activities of sub-optimal concentrations of the inhibitory anti-MSP1-42 sera. Thus, immunization strategies with MSP1-based vaccines may benefit from co-induction of anti-MSP1-33 responses to enhance efficacy and potency.