γ-Secretase inhibitor in combination with BCMA chimeric antigen receptor T-cell immunotherapy for individuals with relapsed or refractory multiple myeloma: a phase 1, first-in-human trial.

BACKGROUND: γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. METHODS: We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50 × 106 CAR T cells, 150 × 106 CAR T cells, 300 × 106 CAR T cells, and 450 × 106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. FINDINGS: 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450 × 106 CAR+ cells, and the recommended phase 2 dose was not reached. INTERPRETATIONS: Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. FUNDING: Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.

Effective, safe, and sustained correction of murine XLA using a UCOE-BTK promoter-based lentiviral vector.

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (BTK). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in Btk and Tec double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human BTK promoter. Hematopoietic stem cells from Btk -/- Tec -/- mice transduced with this vector rescued lineage-specific expression and restored B cell function in Btk -/- Tec -/- recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eµ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy.

Anti-B-cell Maturation Antigen Chimeric Antigen Receptor T cell Function against Multiple Myeloma Is Enhanced in the Presence of Lenalidomide.

Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have shown promising clinical responses in patients with relapsed/refractory multiple myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, drives antimyeloma activity, and alters the suppressive microenvironment; these properties may effectively combine with anti-BCMA CAR T cells to enhance function. Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell function in a concentration-dependent manner. Lenalidomide increased CAR T effector cytokine production, particularly under low CAR stimulation or in the presence of inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced CAR T cytokine production, cytolytic activity, and activation profile relative to untreated CAR T cells in chronic stimulation assays. This unique potentiation of both short-term CAR T activity and long-term functionality during chronic stimulation prompted investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures from RNA sequencing and assay for transposase-accessible chromatin using sequencing indicated that pathways associated with T-helper 1 response, cytokine production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a murine, disseminated, multiple myeloma model indicated that lenalidomide increased CAR T cell counts in blood and significantly prolonged animal survival. In summary, preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in low-antigen or suppressive environments and delayed onset of functional exhaustion. These results support further investigation of lenalidomide and anti-BCMA CAR T cells in the clinic.

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell-derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells.

Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.

Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome.

The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)-deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.

WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity.

Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell-intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein-deficient (WASp(-/-)) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell-intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity.

Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders.

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin ß (Igß) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.

B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia.

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.

Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors.

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.

CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function.

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.