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BackgroundCellular immunotherapies for cancer represent a means by which a patient’s immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic engineering to ‘redirect’ peripheral T cells to tumor targets, showing remarkable potency in blood cancers. However, due to several resistance mechanisms, CAR-T cell therapies remain ineffective in solid tumors. We and others have shown the tumor microenvironment harbors a distinct metabolic landscape that produces a barrier to immune cell function. Further, altered differentiation of T cells within tumors induces defects in mitochondrial biogenesis, resulting in severe cell-intrinsic metabolic deficiencies. While we and others have shown murine T cell receptor (TCR)-transgenic cells can be improved through enhanced mitochondrial biogenesis, we sought to determine whether human CAR-T cells could be enabled through a metabolic reprogramming approach.Materials and methodsAnti-EGFR CAR-T cells were infused in NSG mice which bore A549 tumors. The tumor infiltrating lymphocytes were analyzed for exhaustion and metabolic deficiencies. Lentiviruses carrying PPAR-gamma coactivator 1α (PGC-1α), PGC-1αS571A and NT-PGC-1α constructs were used to co-transduce T cells with anti-EGFR CAR lentiviruses. We performed metabolic analysis via flow cytometry and Seahorse analysis in vitro as well as RNA sequencing. Finally, we treated therapeutically A549-carrying NSG mice with either PGC-1α or NT-PGC-1α anti-EGFR CAR-T cells. We also analyzed the differences in the tumor-infiltrating CAR-T cells when PGC-1α is co-expressed.ResultsHere, in this study, we show that an inhibition resistant, engineered version of PGC-1α, can metabolically reprogram human CAR-T cells. Transcriptomic profiling of PGC-1α-transduced CAR-T cells showed this approach effectively induced mitochondrial biogenesis, but also upregulated programs associated with effector functions. Treatment of immunodeficient animals bearing human solid tumors with these cells resulted in substantially improved in vivo efficacy. In contrast, a truncated version of PGC-1α, NT-PGC-1α, did not improve the in vivo outcomes.ConclusionsOur data further support a role for metabolic reprogramming in immunomodulatory treatments and highlight the utility of genes like PGC-1α as attractive candidates to include in cargo along with chimeric receptors or TCRs for cell therapy of solid tumors.

Gene transfer technology and its application to human gene therapy greatly expanded in the last decade. One area of investigation that appears particularly promising is the transfer of new genetic material into T cells for the potential treatment of cancer. Herein, we describe several core technologies that now yield high-efficiency gene transfer into primary human T cells. These gene transfer techniques include viral-based gene transfer methods based on modified Retroviridae and non-viral methods such as DNA-based transposons and direct transfer of mRNA by electroporation. Where specific examples are cited, we emphasize the transfer of chimeric antigen receptors (CARs) to T cells, which permits engineered T cells to recognize potential tumor antigens.

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8 + cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

PurposeExpression of the immune checkpoint protein PD-L1 constitutes a major mechanism of tumor immune evasion. Multiple clinical trials in solid tumors have demonstrated that inhibition of tumor PD-L1 or immune effector PD-1 via monoclonal antibodies (mAbs) can produce dramatic clinical responses in many cancer patients. The main function of these mAbs is to inhibit signaling induced by ligation of PD-L1 on tumor cells with PD-1 on tumor infiltrating immune effectors. Antibody-dependent cellular cytotoxicity (ADCC) represents an additional mechanism of action for mAbs of the IgG1 isotype. In the current study, we describe investigations of a novel anti-PD-L1 mAb of the IgG1 isotype (MSB0010718). This mAb is currently in Phase I clinical trials for patients with metastatic or locally advanced solid tumors at the NCI, and is the first such mAb with the capacity to induce ADCC of PD-L1 positive tumor cells. We sought to investigate MSB0010718's ability to induce ADCC and to determine factors affecting tumor cell sensitivity to this mechanism.ResultsUsing whole PBMCs as effectors in in vitro ADCC assays, we demonstrated that many cancer cell lines are sensitive to ADCC induced by MSB0010718. Sensitivity to ADCC positively correlated with PD-L1 MFI as determined by flow cytometry. Treatment of tumor cell lines with IFN-γ increased PD-L1 expression with concurrent increase in ADCC sensitivity in some cases. Isolation of NK cells for use as effectors significantly increased ADCC activity as compared to whole PBMCs, demonstrating NK cells as the major effectors of ADCC. ADCC activity could be significantly increased via activation of NK effectors with IL-12, suggesting potential synergy with IL-12 based therapeutics. Furthermore, a MUC1+ tumor cell line that was insensitive to CD8+ MUC1-specific CTL was demonstrated to be sensitive to the ADCC mechanism as a result of high surface PD-L1 expression.ConclusionsAs it is clear that not all patients respond to current PD-1 or PD-L1 based therapies, additional mechanisms of action may be needed to increase therapeutic efficacy. Our data demonstrate significant ADCC activity induced via MSB0010718. This mechanism of action represents a potential advantage for MSB0010718 over other anti-PD-L1 mAbs, as it can induce target cell lysis in the absence of an effective CD8+ CTL response. As cell surface PD-L1 density was an important predictor of ADCC sensitivity, this mechanism is expected to be most active against tumors with high density PD-L1 expression.

Antibodies that block PD-1/PD-L1 interactions have shown efficacy against both lung and skin cancers in early-stage clinical trials, and may also be effective in other tumor types, particularly bladder tumors. PD-L1 expression has been previously reported to correlate with high-grade tumors, a high recurrence rate, and reduced survival rate in patients with bladder cancer. These findings and the high frequency of somatic mutations found in bladder tumors indicate that bladder cancer patients may respond well to anti-PD-L1 therapy. Both murine (MB49, MBT-2) and human (J82, T24, TCCSUP) bladder cancer cell lines constitutively express PD-L1 as determined by flow cytometry. As expected, in vitro IFN-γ addition up-regulated PD-L1 expression levels on each of those tumor cells. A human IgG1 anti-PD-L1 antibody, MSB0010718C, induced ADCC activity in vitro against all three human bladder cancer cell lines following treatment with IFN-γ. In initial in vivo murine studies, the growth of s.c. MB49 tumors in syngeneic mice was significantly delayed following three i.p. injections of 400 µg of the MSB0010718C anti-PD-L1 antibody. An orthotopic bladder model consisting of the MB49 cells tagged with luciferase (MB49-luc) was also used to evaluate the antitumor efficacy of the anti-PD-L1 antibody. MB49-luc cells were instilled intravesically (bladder) in B6 mice. Beginning at 7 or 10 days post-instillation, three i.p. injections of the anti-PD-L1 antibody substantially reduced tumor volumes, as determined by intravital imaging, leading to long-term tumor-free survival for 40-60% of the treated mice. While initial immune cell subset depletion studies implicated both CD4+ and CD8+ T cells, continuing efforts will further define the cellular mechanisms responsible for the antitumor effects of the anti-PD-L1 antibody. Taken together, these results suggest that MSB0010718C therapy might be used to activate both innate and adaptive immune mechanisms to treat PD-L1-expressing bladder tumors. Furthermore, the MB49 tumor model can be used to evaluate the combined effects of anti-PD-L1 and other therapeutic agents, particularly ones that induce IFN-γ production and tumor PD-L1 up-regulation. MSB0010718C is currently being evaluated in a Phase I clinical trial (NCT01772004).

PurposeThe MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated VNTR region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, numerous studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types.Experimental designWe report here the identification of seven potential CD8+ cytotoxic T lymphocyte epitopes of MUC1: five in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2 and A3 MHC class I alleles, which encompass two thirds of the population.ResultsThe agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner.ConclusionsThe agonist epitopes described here can be incorporated in various vaccine platforms and for ex vivo generation of human T cells. These studies thus provide the rationale for the T-cell-mediated targeting of the oncogenic C-terminus of MUC1, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

PurposeThe T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. A phase I clinical trial is ongoing at the NCI using a recombinant yeast Brachyury vaccine. We have previously demonstrated that agonist epitopes of tumor-associated antigens are more effective than native epitopes at activating antigen-specific T cell responses. The current study sought to identify an agonist of the Brachyury HLA-A2 epitope in order to increase T cell activation and tumor lysis.Experimental designA novel agonist epitope of Brachyury was generated by residue substitution of the native epitope. Characterization of this epitope as an agonist included; comparison of HLA-A2 binding affinity and stability, interferon-γ production by epitope-specific T cell lines, FACS analysis of activation markers, as well as Brachyury and HLA-A2 specific lysis of tumor cells. The presence of Brachyury-specific T cells in cancer patients that recognize the agonist peptide was determined by ELISPOT.ResultsThe agonist epitope was shown to bind HLA-A2 with higher avidity and stability than the native. PBMC from colon and ovarian cancer patients reacted to the agonist peptide in an ELISPOT assay. Tetramer staining revealed Brachyury agonist-specific T cells in the PBMC of a prostate cancer patient after in vitro stimulation with the agonist peptide. T cell lines generated from both the native and agonist epitopes produced higher levels of interferon-γ in response to stimulation with the agonist epitope, and they also expressed higher levels of intracellular Ki-67 and perforin. The agonist-specific T cell line lysed a variety of Brachyury-expressing tumor cells more efficiently.ConclusionsAn agonist epitope for Brachyury has been identified that, as compared to the native epitope, increased T cell activation and cytotoxic activity against tumor cells expressing native Brachyury. A T cell line generated with the agonist peptide displayed increased activation (Ki67, perforin) and cytotoxic activity upon subsequent stimulation with the peptide. This study supports the use of the Brachyury agonist epitope as a cancer vaccine strategy.

microRNAs (miRNAs) are small non-coding RNAs that function as master regulators of gene expression in animals ranging from C. elegans to Homo sapiens. Recent work has indicated a major role for deregulation of miRNA signaling in the process of tumorigenesis, with widespread miRNA downregulation being sufficient to drive tumor formation in mouse models. The let-7 family of miRNAs was identified via its ability to control developmental timing in C. elegans, and the human let-7 family contains 13 members distributed across 8 chromosomes. Loss of let-7 expression has been demonstrated in a wide variety of human tumors and is a marker of less differentiated and highly aggressive cancers. Here, I demonstrate that Insulin-like growth factor II mRNA binding protein 1 (IMP-1) is a specific let-7 target gene whose upregulation contributes to typical properties of cancer in cancer cell lines. Let-7 regulates the expression of IMP-1 protein in cancer cell lines, and the IMP-1 3'-UTR is specifically targeted by endogenous let-7. Expression of IMP-1 contributes to proliferation and motility in the let-7 low A549 lung cancer cell line, as reduction of IMP-1 protein inhibited both of these cellular functions. I also demonstrate that let-7 expression is not a general chemosensitizer of cancer cell lines, as modulation of let-7 levels in either direction did not affect sensitivity to Taxol or carboplatinum in multiple cell lines. A link between let-7 and chemosensitivity does exist, however, in the ADR-RES cell line that expresses both IMP-1 and the ABC drug pump MDR1. Forced expression of let-7g sensitized this cell line to the MDR1 substrate Taxol due to specific loss of IMP-1 protein and concurrent loss of MDR1 mRNA and protein expression. A hypothesis is presented that outlines a potential signaling network in let-7 low tumor-initiating cells (T-ICs) that includes IMP-1, MDR1 and the T-IC marker (and IMP-1 target) CD44. Future work will focus on demonstrating whether a significant relationship exists between IMP-1 expression and MDR1 expression in treatment-refractory disease and on further elucidation of a potential role for IMP-1 expression in the maintenance of a stem-like phenotype in tumor-initiating cells with low let-7 expression and expression of MDR1 and CD44.