Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.

Purpose Glioblastoma (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target and we determined if HER2-specific T cells can be generated from patients with disease that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. Experimental design HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor (CAR). The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Results Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-γ and IL-2 in a HER2-dependent manner. Patients’ HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of established autologous GBM xenografts established in the brain of SCID mice. Conclusions Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T-cells may be a promising immunotherapeutic approach for GBM.

Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.

Outcomes for patients with glioblastoma (GBM) remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)-13Rα2, epidermal growth factor receptor variant III (EGFRvIII), or human epidermal growth factor receptor 2 (HER2) has shown promise for the treatment of gliomas in preclinical models and in a clinical study (IL-13Rα2). However, targeting IL-13Rα2 and EGFRvIII is associated with the development of antigen loss variants, and there are safety concerns with targeting HER2. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) has emerged as an attractive target for the immunotherapy of GBM as it is overexpressed in glioma and promotes its malignant phenotype. To generate EphA2-specific T cells, we constructed an EphA2-specific CAR with a CD28-ζ endodomain. EphA2-specific T cells recognized EphA2-positive glioma cells as judged by interferon-γ (IFN-γ) and IL-2 production and tumor cell killing. In addition, EphA2-specific T cells had potent activity against human glioma-initiating cells preventing neurosphere formation and destroying intact neurospheres in coculture assays. Adoptive transfer of EphA2-specific T cells resulted in the regression of glioma xenografts in severe combined immunodeficiency (SCID) mice and a significant survival advantage in comparison to untreated mice and mice treated with nontransduced T cells. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive GBM.

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T cell function in immunosuppressive tumors are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumor microenvironment. We devised an in vivo pooled shRNA screen in which shRNAs targeting negative regulators became highly enriched in tumors by releasing a block on T cell proliferation upon tumor antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumor or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family: In tumors, Ppp2r2d knockdown inhibited T cell apoptosis and enhanced T cell proliferation as well as cytokine production. Key regulators of immune function can thus be discovered in relevant tissue microenvironments.

IntroductionImmunotherapy with antigen-specific T cells has shown promise in the treatment of hematologic malignancies in preclinical models and in phase 1/2 clinical studies. [1][2][3] One attractive strategy to generate tumor-specific T cells is by genetic modification with chimeric antigen receptors (CARs), which consist of an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain derived from the TCR CD3-chain often linked to costimulatory molecule endodomains. 4,5 CARs targeting CD19 and CD20 antigens for the treatment of hematologic malignancies have been explored extensively, but this approach is limited to B cell-derived malignancies and may produce prolonged impairment of humoral immunity because of the potentially long life span of T cells. 6,7 It is therefore desirable to prepare CARs directed against alternative antigens that could broaden the spectrum of potentially treatable tumors and/or potentially reduce damage to normal cells.CD70 is the membrane-bound ligand of the CD27 receptor, which belongs to the tumor necrosis factor receptor superfamily. 8,9 CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkin lymphoma, Waldenström macroglobulinemia, and multiple myeloma, and by human T-lymphotropic virus type 1-and EBV-associated malignancies. [10][11][12][13][14] In addition, CD70 is expressed by nonhematologic malignancies such as renal cell carcinoma and glioblastoma. 15,16 Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells. Whereas CD70/CD27 costimulation plays a role in T-cell activation, CD70/ CD27 signaling is not essential for the development and maintenance of a functional immune system, because CD27-knockout mice have no overt immunodeficiency and recover from influenza virus infection within the same time frame as wild-type mice. 17,18 Targeting CD70-positive malignancies with CD70-specific monoclonal antibodies has shown promise in preclinical animal models, 14,19,20 and we have now evaluated whether T cells can be redirected to CD70 by forced expression of the appropriate CAR. Because CARs consist of an extracellular antigen-recognition domain derived from murine monoclonal antibodies, they may induce human anti-mouse antibody on infusion unless fully humanized. 21,22 One potential strategy to overcome this limitation is to engineer the antigen-recognition domain using endogenous protein ligands or receptors rather than monoclonal antibodies. 23,24 To target CD70 with T cells, we took advantage of the physiologic CD70/CD27 interaction and generated a CD70-specific CAR, which consists of full-length CD27 as the antigen-recognition domain fused to the intracellular domain of the CD3-chain. Engagement of chimeric CD27-by tumor targets expressing the CD70 ligand resulted in T-cell activation and CD27 costimulation, which was dependent on the presence of the TRAF2-binding site within the cytoplasmic tail of CD27. CD70-specifi...

Glioblastoma (GBM) is the most common primary brain cancer in adults and is virtually incurable. Recent studies have shown that CMV is present in the majority of GBMs. To evaluate if the CMV antigens pp65 and IE1, which are expressed in GBMs, can be targeted with CMV-specific T cells, we measured the frequency of T cells targeting pp65 and IE1 in the peripheral blood of a cohort of 11 sequentially-diagnosed CMV-seropositive GBM patients, and evaluated whether it was feasible to expand autologous CMV-specific T cells for future clinical studies. All 11 CMV-seropositive GBM patients had T cells specific for pp65 and IE1 in their peripheral blood assessed by IFNγ ELIspot assay. However, the precursor frequency of pp65-specific T cells was decreased in comparison to healthy donors (p=0.001). We successfully reactivated and expanded CMV-specific T cells from 6 out of 6 GBM patients using antigen presenting cells transduced with an adenoviral vector encoding pp65 and IE1. CMV-specific T-cell lines contained CD4-positive as well as CD8-positive T cells, recognized pp65- and IE1-positive targets and killed CMV-infected autologous GBM cells. Infusion of such CMV-specific T-cell lines may extend the benefits of T-cell therapy to patients with CMV-positive GBMs.

Background aims Hematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein–Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens. Methods To expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9–12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector. Results After 18 days in culture nine CTL lines expanded from less than 1.5 × 107 PBMC to a mean of 6.1 × 107 T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47–995] and IE-1 (median 154 SFC, range 11–505), the Ad antigens hexon (median 153 SFC, range 26–465) and penton (median 37 SFC, range 1–353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9–301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets. Conclusions CTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.