Gabriela Plesa

Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV

BACKGROUNDnCCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (gene editing)--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.nnnMETHODSnWe enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.nnnRESULTSnOne serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.nnnCONCLUSIONSnCCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T Cells

Adoptively transferred chimeric antigen receptor T cells have stable stem cell–like persistence for at least a decade and more than 500 years of patient safety. Standing the Test of Time Retroviral vectors were once the mainstay of gene transfer because they could stably integrate into the host genome. However, some patients in early trials developed leukemia because of insertional mutagenesis. Now, Scholler et al. report that retroviral vector–mediated gene transfer in T cells may not have the same safety concerns, and that these cells may persist over a decade in patients. The authors followed patients from three clinical trials who received T cells transduced with gammaretroviruses carrying a chimeric antigen receptor. They found that these cells were present in recipients over a decade after infusion at levels higher than those induced by standard vaccines. These cells were still functional, had stable levels of engraftment, and did not require host immunosuppression before transplant. Moreover, the authors found no evidence of integration-induced immortalization, with no observable enrichment of integration sites near genes involved in growth control or transformation. Thus, the safety of retroviral vectors may be cell type–specific, opening up engineered T cells as a delivery platform for therapeutics. The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. We report long-term results from three clinical trials to evaluate gammaretroviral vector–engineered T cells for HIV. The vector encoded a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). CAR T cells were detected in 98% of samples tested for at least 11 years after infusion at frequencies that exceeded average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells; integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells had stable levels of engraftment, with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression before T cell transfer is not required to achieve long-term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient-years of follow-up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases.

Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies

Beatty, Haas, and colleagues report antitumor activity in two patients treated with autologous T cells transfected with mRNA encoding a chimeric antigen receptor that recognizes mesothelin and contains the CD3-ζ and 4-1BB costimulatory domains (CARTmeso). The short-lived CARTmeso cells induced novel antiself antibodies and a broadly directed epitope spreading. Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB costimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was shown in both patients, and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel antiself antibodies. These data show the potential of using mRNA-engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. Cancer Immunol Res; 2(2); 112–20. ©2013 AACR.

Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

T cells engineered to express affinity-enhanced TCRs directed to a MAGE A3 peptide cross-react with a similar, but unrelated, self-peptide. Cross-Reactive Adoptive Therapy Engineering T cells with enhanced affinity to cancer targets is a promising therapy. However, one key bottleneck in this strategy is the identification of targets that are expressed on cancer cells but not on normal healthy tissue. One way to identify these antigens is by looking at the family of cancer-testis antigens, which have restricted expression in normal tissue but are frequently up-regulated in tumors. Cameron et al. now report that a T cell engineered to target one such antigen—MAGE A3—cross-reacts with a peptide from a muscle protein, Titin. The authors developed a T cell that targeted a MAGE A3 antigen for use in adoptive immunotherapy. Although extensive preclinical investigations demonstrated no off-target antigen recognition, patients who received these T cells had serious adverse events, including fatal cardiac toxicity. The authors then used amino acid scanning to search for potential cross-reactivity of these T cells with an off-target peptide and identified a peptide derived from the muscle protein Titin. Because affinity-enhanced T cells are highly potent, this cross-reactivity was likely the cause of the off-target toxicity. This study highlights methods that may be used to prevent cross-reactivity in future trials of adoptive immunotherapy. MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)–A*01–restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

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

A chimeric antigen receptor redirects T cells to treat glioblastoma. CAR T cells drive glioblastoma therapy Immunotherapy with chimeric antigen receptor (CAR) T cells can successfully treat B cell malignancies, but expansion into solid tumors has been limited by the lack of availability of tumor-specific antigens. Now, Johnson et al. target CAR T cells to a variant III mutation of the epidermal growth factor receptor (EGFRvIII), which is thought to be enriched in glioblastoma stem cells. They found that a low-affinity single-chain variable fragment was specific for EGFRvIII over wild-type EGFR and that CAR T cells transduced with this fragment were able to target antigen-expressing cells in vitro and in vivo in multiple mouse xenograft models of human glioblastoma. These cells are currently being moved into the clinic in a phase 1 clinical trial. 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).

The Inducible Costimulator (ICOS) Is Critical for the Development of Human T H 17 Cells

The inducible costimulator ICOS is critical for the differentiation and expansion of human TH17 cells and promotes the antitumor capacity of these cells. Jack of All Trades Although some immune cells are very specialized, others are thought to do it all. These cells fight infection, contribute to autoimmunity, and engage cancer cells. Paulos et al. explored one such multitasking immune cell type—the T helper 17 (TH17) subset of CD4+ T cells. T helper cells, which express the molecule CD4, are master regulators of the immune system. Although T helper cells do not directly kill infected cells or produce antibodies to fight bacterial and viral invaders, they are involved in activating and directing the other immune cells that perform these functions. The absence of these cells, as seen in HIV-infected patients, can severely inhibit the immune response. T helper cells are classified into different subsets based on the molecules they secrete and their subsequent functional responses. The classical paradigm divided these cells into TH1 and TH2 subsets, which activate cellular and humoral immunity, respectively. Further studies indicated that this division was an oversimplification of T helper cell functions, and new T helper cell subsets were identified based on cytokine secretion profiles. One such T helper cell subset consists of TH17 cells. TH17 cells, which secrete the molecule interleukin-17, are developmentally distinct from TH1 and TH2 cells. TH17 cells are thought to be involved in inflammatory processes, having a pathogenic role in the development of autoimmune disease and a protective role in infection and cancer. Paulos et al. examined cell surface costimulatory molecules, which provide a nonspecific second signal for T cell activation, in the development of TH17 cells. They found that the inducible costimulator (ICOS) was critical for TH17 cell differentiation and that ICOS and not CD28, a costimulatory molecule frequently used to expand TH17 cells, was necessary for the optimal expansion and function of TH17 T cells. Moreover, CD28 stimulation blocked ICOS stimulation, resulting in decreased secretion of TH17-specific cytokines. Indeed, TH17-polarized T cells stimulated with ICOS resulted in higher levels of tumor regression in a mouse xenotransplantation model of mesothelioma than TH17-polarized T cells stimulated with CD28. Therefore, targeting ICOS may provide new therapeutic options for treating cancer and infection as well as inhibiting autoimmunity mediated by TH17 cells, a jack of all trades. Human T helper 17 (TH17) cells regulate host defense, autoimmunity, and tumor immunity. Although cytokines that control human TH17 cell development have been identified, the costimulatory molecules important for TH17 cell generation are unknown. Here, we found that the inducible costimulator (ICOS) was critical for the differentiation and expansion of human TH17 cells. Human cord blood contained a subset of CD161+CD4+ T cells that were recent emigrants from the thymus, expressed ICOS constitutively, and were imprinted as TH17 cells through ICOS signaling. ICOS stimulation induced c-MAF, RORC2, and T-bet expression in these cells, leading to increased secretion of interleukin-21 (IL-21), IL-17, and interferon-γ (IFN-γ) compared with cells stimulated with CD28. Conversely, CD28 ligation abrogated ICOS costimulation, dampening RORC2 expression while promoting the expression of the aryl hydrocarbon receptor, which led to reduced secretion of IL-17 and enhanced production of IL-22 compared with cells stimulated with ICOS. Moreover, ICOS promoted the robust expansion of IL-17+IFN-γ+ human T cells, and the antitumor activity of these cells after adoptive transfer into mice bearing large human tumors was superior to that of cells expanded with CD28. The therapeutic effectiveness of ICOS-expanded cells was associated with enhanced functionality and engraftment in vivo. These findings reveal a vital role for ICOS signaling in the generation and maintenance of human TH17 cells and suggest that components of this pathway could be therapeutically targeted to treat cancer or chronic infection and, conversely, that interruption of this pathway may have utility in multiple sclerosis and other autoimmune syndromes. These findings have provided the rationale for designing new clinical trials for tumor immunotherapy.

Monoclonal TCR-redirected tumor cell killing

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

A trial of autologous T cells redirected to a specific mutation in glioblastoma patients illustrates mechanisms of resistance. Speeding toward CAR T cell therapy for glioblastoma Chimeric antigen receptor (CAR) T cells have been successfully implemented for treating leukemia and are now being investigated for solid tumors. O’Rourke et al. conducted a phase 1 safety study of autologous CAR T cells targeted to EGFR variant III in glioblastoma patients. Treatment seemed to be well tolerated, which is critical because other CAR T cell products have been implicated in devastating central nervous system complications. Of the 10 patients enrolled, 7 had surgical intervention, allowing for some analysis of the tumors and T cells in patients’ brains. The results of this trial indicate that CAR T cell therapy is a viable option for treating glioblastoma. We conducted a first-in-human study of intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by a chimeric antigen receptor (CAR). We report our findings on the first 10 recurrent glioblastoma (GBM) patients treated. We found that manufacturing and infusion of CAR-modified T cell (CART)–EGFRvIII cells are feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome. One patient has had residual stable disease for over 18 months of follow-up. All patients demonstrated detectable transient expansion of CART-EGFRvIII cells in peripheral blood. Seven patients had post–CART-EGFRvIII surgical intervention, which allowed for tissue-specific analysis of CART-EGFRvIII trafficking to the tumor, phenotyping of tumor-infiltrating T cells and the tumor microenvironment in situ, and analysis of post-therapy EGFRvIII target antigen expression. Imaging findings after CART immunotherapy were complex to interpret, further reinforcing the need for pathologic sampling in infused patients. We found trafficking of CART-EGFRvIII cells to regions of active GBM, with antigen decrease in five of these seven patients. In situ evaluation of the tumor environment demonstrated increased and robust expression of inhibitory molecules and infiltration by regulatory T cells after CART-EGFRvIII infusion, compared to pre–CART-EGFRvIII infusion tumor specimens. Our initial experience with CAR T cells in recurrent GBM suggests that although intravenous infusion results in on-target activity in the brain, overcoming the adaptive changes in the local tumor microenvironment and addressing the antigen heterogeneity may improve the efficacy of EGFRvIII-directed strategies in GBM.

Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC) class I and II molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV) glycoprotein (GP) was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection.