Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma.

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

Engineering matrix-free laser desorption ionization mass spectrometry using glancing angle deposition films.

RATIONALE: Thin, nanoporous films fabricated using Glancing Angle Deposition (GLAD) technology are demonstrated for solid matrix laser desorption/ionization mass spectrometry (SMALDI-MS). GLAD allows facile engineering of nanoporosity, film thickness, post alignment, and material composition, as demonstrated here by the fabrication of Co-GLAD and Si-GLAD films for SMALDI, and by exploration of the SMALDI performance as a function of thickness, post density, and angle of the post relative to surface normal. METHODS: GLAD films were prepared by electron beam evaporation onto silicon substrates, using steep angles of incidence for the vacuum deposition, with computer controlled substrate rotation. LDI from the GLAD films was evaluated using an MDS-Sciex time-of-flight (TOF) MALDI mass spectrometer. RESULTS: Co-GLAD films give a limit of quantitation of 6 fmol for complex carbohydrate derivatives, and slanted-post Si-GLAD films show up to three times higher sensitivity than vertical post structures. Reproducibility of both Si and Co films is much higher than conventional MALDI methods for m/z below at least 2100 Da. Both reproducibility and detection limits are comparable to or better than other nano-structured materials. Co-GLAD films are significantly better in performance than Co powders or Co thin films on silicon substrates previously evaluated. CONCLUSIONS: The flexibility of GLAD for thin film fabrication of LDI materials is demonstrated by the range of nanoporous materials that can be grown, and the fine control over structural conformation, thickness and porosity. Copyright © 2017 John Wiley & Sons, Ltd.

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

Listeria monocytogenes (Lm)-LLO immunotherapies reduce the immunosuppressive activity of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment.

Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) are major components of the immune suppressive cells that potentially limit the effectiveness of an immunotherapy-based treatment. Both of these suppressive cell types have been shown to expand in tumor models and promote T-cell dysfunction that in turn favors tumor progression. This study demonstrates that Listeria monocytogenes (Lm)-LLO immunotherapies effect on the suppressive ability of MDSC and Treg in the tumor microenvironment (TME), resulting in a loss in the ability of these cells to suppress T cells. This alteration of immunosuppression in the TME was an inherent property of all Lm-LLO immunotherapies tested and was independent of the tumor model. The virtually total loss in the suppressive ability of these cells in the TME was linked to the reduction in the expression of arginase I in MDSC and IL-10 in Treg. The results presented here provide insight into a novel mechanism of Lm-LLO immunotherapies that potentially contributes to therapeutic antitumor responses.

Anti-PD-1 antibody significantly increases therapeutic efficacy of Listeria monocytogenes (Lm)-LLO immunotherapy.

BACKGROUND: One of the significant tumor immune escape mechanisms and substantial barrier for successful immunotherapy is tumor-mediated inhibition of immune response through cell-to-cell or receptor/ligand interactions. Programmed death receptor-1 (PD-1) interaction with its ligands, PD-L1 and PD-L2, is one of the important strategies that many tumors employ to escape immune surveillance. Upon PD-Ls binding to PD-1, T cell receptor (TCR) signaling is dampened, causing inhibition of proliferation, decreased cytokine production, anergy and/or apoptosis. Thus PD-Ls expression by tumor cells serves as a protective mechanism, leading to suppression of tumor-infiltrating lymphocytes in the tumor microenvironment. Lm-LLO immunotherapies have been shown to be therapeutically effective due to their ability to induce potent antigen-specific immune responses. However, it has been demonstrated that infection with Lm leads to up-regulation of PD-L1 on mouse immune cells that can inhibit effector T cells through PD-1/PD-L1 pathway. METHODS: Therapeutic and immune efficacy of Listeria-based vaccine (Lm-LLO-E7) in combination with anti-PD-1 antibody was tested in E7 antigen expressing TC-1 mouse tumor model. Tumor growth, survival, as well as peripheral and tumor-infiltrating immune cell profiles after immunotherapy were assessed. RESULTS: Here we demonstrate that the combination of an Lm-LLO immunotherapy with anti-PD-1 antibody that blocks PD-1/PD-L1 interaction, significantly improves immune and therapeutic efficacy of treatment in TC-1 mouse tumor model. Importantly, we show that in addition to significant reduction of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) in both spleen and tumor microenvironment that are mediated solely by the Lm-LLO immunotherapy, the addition of anti-PD-1 antibody to the treatment results in significant increase of antigen-specific immune responses in periphery and CD8 T cell infiltration into the tumor. As a result, this combinational treatment leads to significant inhibition of tumor growth and prolonged survival/complete regression of tumors in treated animals. We also demonstrate that in vitro infection with Lm results in significant upregulation of surface PD-L1 expression on human monocyte-derived dendritic cells suggesting the translational capacity of this finding. CONCLUSIONS: Our findings demonstrate that combination of Lm-LLO-based vaccine with blocking of PD-1/PD-L1 interaction is a feasible approach with clinical translation potential that can lead to overall enhancement of the efficacy of anti-tumor immunotherapy.

Combined immunotherapy with Listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer.

Radiation therapy (RT) is an integral part of prostate cancer treatment across all stages and risk groups. Immunotherapy using a live, attenuated, Listeria monocytogenes-based vaccines have been shown previously to be highly efficient in stimulating anti-tumor responses to impact on the growth of established tumors in different tumor models. Here, we evaluated the combination of RT and immunotherapy using Listeria monocytogenes-based vaccine (ADXS31-142) in a mouse model of prostate cancer. Mice bearing PSA-expressing TPSA23 tumor were divided to 5 groups receiving no treatment, ADXS31-142, RT (10 Gy), control Listeria vector and combination of ADXS31-142 and RT. Tumor growth curve was generated by measuring the tumor volume biweekly. Tumor tissue, spleen, and sera were harvested from each group for IFN-γ ELISpot, intracellular cytokine assay, tetramer analysis, and immunofluorescence staining. There was a significant tumor growth delay in mice that received combined ADXS31-142 and RT treatment as compared with mice of other cohorts and this combined treatment causes complete regression of their established tumors in 60 % of the mice. ELISpot and immunohistochemistry of CD8+ cytotoxic T Lymphocytes (CTL) showed a significant increase in IFN-γ production in mice with combined treatment. Tetramer analysis showed a fourfold and a greater than 16-fold increase in PSA-specific CTLs in animals receiving ADXS31-142 alone and combination treatment, respectively. A similar increase in infiltration of CTLs was observed in the tumor tissues. Combination therapy with RT and Listeria PSA vaccine causes significant tumor regression by augmenting PSA-specific immune response and it could serve as a potential treatment regimen for prostate cancer.

Lm-LLO-Based Immunotherapies and HPV-Associated Disease.

HPV infection is a direct cause of neoplasia and malignancy. Cellular immunologic activity against cells expressing HPV E6 and E7 is sufficient to eliminate the presence of dysplastic or neoplastic tissue driven by HPV infection. Live attenuated Listeria monocytogenes- (Lm-) based immunotherapy (ADXS11-001) has been developed for the treatment of HPV-associated diseases. ADXS11-001 secretes an antigen-adjuvant fusion (Lm-LLO) protein consisting of a truncated fragment of the Lm protein listeriolysin O (LLO) fused to HPV-16 E7. In preclinical models, this construct has been found to stimulate immune responses and affect therapeutic outcome. ADXS11-001 is currently being evaluated in Phase 2 clinical trials for cervical intraepithelial neoplasia, cervical cancer, and HPV-positive head and neck cancer. The use of a live attenuated bacterium is a more complex and complete method of cancer immunotherapy, as over millennia Lm has evolved to infect humans and humans have evolved to prevent and reject this infection over millennia. This evolution has resulted in profound pathogen-associated immune mechanisms which are genetically conserved, highly efficacious, resistant to tolerance, and can be uniquely invoked using this novel platform technology.

Cancer immunotherapy using recombinant Listeria monocytogenes: transition from bench to clinic.

Cancer immunotherapy has developed into a field of intense study as aspects of the immune system involved in the eradication of cancer have become delineated. Listeria monocytogenes is a gram-positive, facultative intracellular bacterium which infects antigen presenting cells (APC), and is being used as a cancer vaccine to deliver tumor antigens directly to the APC. This results in the generation of a strong immune response towards the tumor associated antigen and direct targeting of the tumor by the immune system. Advances in this field have led to the development of a series of L. monocytogenes-based cancer vaccines, which are currently in clinical trials. A phase I study has shown these vaccines can be safely administered and well-tolerated in terminal stage cancer patients and an efficacy signal was observed in patients who did not respond to other therapies. Additional data on the efficacy of these vaccines is expected in the near-term.

Defective cross-presentation of viral antigens in GILT-free mice.

Gamma-interferon-inducible lysosomal thiolreductase (GILT) promotes major histocompatibility complex (MHC) class II-restricted presentation of exogenous antigens containing disulfide bonds. Here, we show that GILT also facilitates MHC class I-restricted recognition of such antigens by CD8+ T cells, or cross-presentation. GILT is essential for cross-presentation of a CD8+ T cell epitope of glycoprotein B (gB) from herpes simplex virus 1 (HSV-1) but not for its presentation by infected cells. Initiation of the gB-specific CD8+ T cell response during HSV-1 infection, or cross-priming, is highly GILT-dependent, as is initiation of the response to the envelope glycoproteins of influenza A virus. Efficient cross-presentation of disulfide-rich antigens requires a complex pathway involving GILT-mediated reduction, unfolding, and partial proteolysis, followed by translocation into the cytosol for proteasomal processing.

GILT is a critical host factor for Listeria monocytogenes infection.

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen that can cause severe illness in humans and animals. On infection, it is actively phagocytosed by macrophages; it then escapes from the phagosome, replicates in the cytosol, and subsequently spreads from cell to cell by a non-lytic mechanism driven by actin polymerization. Penetration of the phagosomal membrane is initiated by the secreted haemolysin listeriolysin O (LLO), which is essential for vacuolar escape in vitro and for virulence in animal models of infection. Reduction is required to activate the lytic activity of LLO in vitro, and we show here that reduction by the enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT, also called Ifi30) is responsible for the activation of LLO in vivo. GILT is a soluble thiol reductase expressed constitutively within the lysosomes of antigen-presenting cells, and it accumulates in macrophage phagosomes as they mature into phagolysosomes. The enzyme is delivered by a mannose-6-phosphate receptor-dependent mechanism to the endocytic pathway, where amino- and carboxy-terminal pro-peptides are cleaved to generate a 30-kDa mature enzyme. The active site of GILT contains two cysteine residues in a CXXC motif that catalyses the reduction of disulphide bonds. Mice lacking GILT are deficient in generating major histocompatibility complex class-II-restricted CD4(+) T-cell responses to protein antigens that contain disulphide bonds. Here we show that these mice are resistant to L. monocytogenes infection. Replication of the organism in GILT-negative macrophages, or macrophages expressing an enzymatically inactive GILT mutant, is impaired because of delayed escape from the phagosome. GILT activates LLO within the phagosome by the thiol reductase mechanism shared by members of the thioredoxin family. In addition, purified GILT activates recombinant LLO, facilitating membrane permeabilization and red blood cell lysis. The data show that GILT is a critical host factor that facilitates L. monocytogenes infection.

Immunoediting sculpts tumor epitopes during immunotherapy.

Immunoediting of tumor-associated antigens occurs in response to immune pressure. We show that the mutation of residues within epitopes of HER-2/neu leads to the outgrowth of autochthonous tumors after immunizing HER-2/neu transgenic mice with Listeria monocytogenes therapeutic vaccines expressing fragments of HER-2/neu. Three of these vaccines target the extracellular domain (LmLLO-EC1, LmLLO-EC2, and LmLLO-EC3), and two of these vaccines target the intracellular domain (Lm-LLO-IC1 and Lm-LLO-IC2). Mutations occurred in the regions of the HER-2/neu molecule targeted by the Listeria strain expressing that region, which suggests that the rate of generation of escape mutants was a significant factor in the efficacy of each vaccine. A longer delay in the onset of tumors after immunotherapy occurred with the vaccine that targeted the kinase domain. We verified that the mutations in this domain occurred within novel CD8(+) T-cell epitopes, and that the mutation of these residues abrogated CTL responses to these epitopes. The long delay in the onset of tumors after immunotherapy targeting the kinase domain may be because this region of HER-2/neu cannot undergo extensive mutations without impairing its ability to signal cell growth.

In the FVB/N HER-2/neu transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by Listeria monocytogenes-based vaccines.

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8(+) T cells and the presence of CD4(+)CD25(+) regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8(+) T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8(+) T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.

Listeria monocytogenes as a vector for tumor-associated antigens for cancer immunotherapy.

As a facultative intracellular bacterium, Listeria monocytogenes has adapted to live within the cytosol of the host cell. It is actively taken up by antigen-presenting cells through phagocytosis, and as Listeria survive within these cells, it is an ideal vector for the delivery of antigens to be processed and presented through both the class I and II antigen-processing pathways. Once phagocytosed, Listeria produces virulence factors within the phagolysosome of the host cell, which allows it to break out of this organelle and live in the host cytosol. It is possible that these virulence factors can enhance the immunogenicity of tumor-associated antigens, which are poorly immunogenic. Recent progress in the development of this bacterium as a vaccine vector for tumor-associated antigens is discussed in the context of bacterial vectors in general. In several mouse models, Listeria-based vaccines have been demonstrated to be an effective method of influencing tumor growth and eliciting potent antitumor immune responses. Safety issues and the transition of Listeria into human clinical trials will also be discussed in this review.

Vaccination strategy determines the emergence and dominance of CD8+ T-cell epitopes in a FVB/N rat HER-2/neu mouse model of breast cancer.

The HER-2/neu oncogene has >25 HLA epitopes, yet only one FVB/N mouse CD8(+) T-cell epitope has been mapped to date. This epitope has been termed the immunodominant epitope for the FVB/N mouse, but we propose that the vaccination strategy determines the dominance of epitopes. Using a series of overlapping peptides, we have mapped another CD8(+) T-cell epitope that emerges in the FVB/N mouse following vaccination with Listeria monocytogenes-based vaccines that express fragments of HER-2/neu. Following the identification of this novel H-2K(q)-restricted epitope, we sought to compare the T-cell response to this epitope with the previously identified PDSLRDLSVF epitope. This newly identified epitope and the previously identified epitope lie within fragments contained in different vaccines, the PDSLRDLSVF epitope in Lm-LLO-EC2 and the newly identified PYNYLSTEV epitope in Lm-LLO-EC1; thus, it has been possible to compare the responses of these epitopes independent of any competing response between the epitopes. CTL analysis of individual peptide-pulsed target cells and intracellular cytokine stain for IFN-gamma produced by splenocytes from Lm-LLO-EC1 compared with Lm-LLO-EC2 vaccinated FVB/N mice shows that there is no difference between the responses generated to either of these epitopes. We also show that the avidity of the CD8(+) T cells for either of these epitopes is similar based on the concentration of peptide necessary to mediate similar levels of lysis of target cells. In addition, HER-2/neu DNA vaccination followed by CTL analysis further showed that both of these peptides can emerge as epitopes.

Fusion to Listeriolysin O and delivery by Listeria monocytogenes enhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse.

Five overlapping fragments of rat HER-2/neu have been expressed in recombinant Listeria monocytogenes. Each fragment of HER-2/neu is secreted as a fusion protein with a truncated, nonhemolytic form of listeriolysin O (LLO). Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3 overlap the extracellular domain of HER-2/neu, whereas Lm-LLO-IC1 and Lm-LLO-IC2 span the intracellular domain. All five strains controlled the growth of established NT-2 tumors, a rat HER-2/neu-expressing tumor line derived from a spontaneously arising mammary tumor in a FVB/N HER-2/neu-transgenic mouse. The antitumor effect of each of these vaccine constructs was abrogated by the in vivo depletion of CD8(+) T cells, although only one known epitope has been defined previously and is present in Lm-LLO-EC2. Anti-HER-2/neu CTL responses were generated by each of the rLm vaccine constructs. With the use of a panel of 3T3 cell lines expressing overlapping fragments of HER-2/neu, regions of HER-2/neu with potential CD8(+) T cell epitopes have been defined. DNA vaccines expressing either a fragment or full-length HER-2/neu were constructed in LLO-fused and non-LLO-fused forms. CTL analysis of the DNA vaccines revealed a broadening in the regions of HER-2/neu recognizable as targets when the target Ag is fused to LLO. These studies show the efficacy of L. monocytogenes-based HER-2/neu vaccines in a murine model of breast cancer and also that the immunogenicity of self-Ags can be increased by fusion to LLO and delivery by L. monocytogenes revealing subdominant epitopes.