Adoptive T-cell Immunotherapy Research Articles

IL-15 signaling promotes adoptive effector T-cell survival and memory formation in irradiation-induced lymphopenia.

BackgroundLymphopenia promotes naïve T-cell homeostatic proliferation and adoptive effector T-cell survival and memory formation. IL-7 plays a critical role in homeostatic proliferation, survival and memory formation of naïve T-cells in lymphopenia, and its underlying molecular mechanism has also been well studied. However, the mechanism for adoptively transferred effector T-cell survival and memory formation is not fully understood. Here, we transferred in vitro-activated transgenic OT-I CD8+ effector T-cells into irradiation (600 rads)-induced lymphopenic C57BL/6, IL-7 knockout (KO) and IL-15 KO mice, and investigated the survival and memory formation of transferred T-cells in lymphopenia.ResultsWe demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice, in a manner that depends on IL-15 signaling, but not IL-7. We determine that in vitro stimulation of naïve or effector T-cells with IL-7 and IL-15 reduces IL-7Rα, and increases and/or maintains IL-15Rβ expression, respectively. Consistent with these findings, the expression of IL-7Rα and IL-15Rβ is down- and up-regulated, respectively, in vivo on transferred T-cells in an early phase post T-cell transfer in lymphopenia. We further show that in vitro IL-15 restimulation-induced memory T-cells (compared to IL-2 restimulation-induced effector T-cells) and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice (compared to IL-15-deficient IL-15 KO mice) have increased mitochondrial content, but less NADH and lower mitochondrial potential (ΔΨm), and demonstrate greater phosphorylation of signal transducers and activators of transcription-5 (STAT5) and Unc-51-like kinase-1 (ULK1), and higher expression of B-cell leukemia/lymphoma-2 (Bcl2) and memory-, autophagy- and mitochondrial biogenesis-related molecules.ConclusionIrradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl2 pathway, enhances T-cell memory formation via IL-15 activation of the forkhead-box family of transcription factor (FOXO)/eomesodermin (Eomes) memory and ULK1/autophagy-related gene-7 (ATG7) autophagy pathways, and via IL-15 activation of the mitochondrial remodeling. Our data thus identify some important targets to consider when designing potent adoptive T-cell immunotherapies of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0098-2) contains supplementary material, which is available to authorized users.

T-cell adoptive immunotherapy using tumor-infiltrating T cells and genetically engineered TCR-T cells.

Immunotherapy has received the expectation that it should contribute to the therapy of cancer patients for >100 years. At long last, recent clinical trials of immunotherapy with immune checkpoint inhibitors and adoptive cell therapy with genetically engineered T cells have reported their remarkable efficacies. Nowadays, it is expected that T-cell adoptive immunotherapy can not only control tumor progression but even cure cancer in some patients. Conversely, severe adverse events associated with efficacy have frequently been reported in clinical trials, suggesting that the assessment and control of safety will be indispensable in the future development of the therapy. New approaches in T-cell adoptive immunotherapy such as reduction of adverse events, targeting of new antigens or utilization of allogeneic cells will open a new gate for less harmful and more effective immunological treatment of cancer patients.

IL-7 signaling imparts polyfunctionality and stemness potential to CD4+ T cells

ABSTRACTThe functional status of CD4+ T cells is a critical determinant of antitumor immunity. Polyfunctional CD4+ T cells possess the ability to concomitantly produce multiple Th1-type cytokines, exhibiting a functional attribute desirable for cancer immunotherapy. However, the mechanisms by which these cells are induced are neither defined nor it is clear if these cells can be used therapeutically to treat cancer. Here, we report that CD4+ T cells exposed to exogenous IL-7 during antigenic stimulation can acquire a polyfunctional phenotype, characterized by their ability to simultaneously express IFNγ, IL-2, TNFα and granzyme B. This IL-7-driven polyfunctional phenotype was associated with increased histone acetylation in the promoters of the effector genes, indicative of increased chromatin accessibility. Moreover, forced expression of a constitutively active (CA) form of STAT5 recapitulated IL-7 in inducing CD4+ T-cell polyfunctionality. Conversely, the expression of a dominant negative (DN) form of STAT5 abolished the ability of IL-7 to induce polyfunctional CD4+ T cells. These in-vitro-generated polyfunctional CD4+ T cells can traffic to tumor and expand intratumorally in response to immunization. Importantly, adoptive transfer of polyfunctional CD4+ T cells following lymphodepletive chemotherapy was able to eradicate large established tumors. This beneficial outcome was associated with the occurrence of antigen epitope spreading, activation of the endogenous CD8+ T cells and persistence of donor CD4+ T cells exhibiting memory stem cell attributes. These findings indicate that IL-7 signaling can impart polyfunctionality and stemness potential to CD4+ T cells, revealing a previously unknown property of IL-7 that can be exploited in adoptive T-cell immunotherapy.

The integrin αvβ6: a novel target for CAR T-cell immunotherapy?

Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding field. CARs are fusion molecules that couple the binding of a tumour-associated cell surface target to the delivery of a tailored T-cell activating signal. Re-infusion of such genetically engineered T-cells to patients with haematological disease has demonstrated unprecedented response rates in Phase I clinical trials. However, such successes have not yet been observed using CAR T-cells against solid malignancies and this is, in part, due to a lack of safe tumour-specific targets. The αvβ6 integrin is strongly up-regulated in multiple solid tumours including those derived from colon, lung, breast, cervix, ovaries/fallopian tube, pancreas and head and neck. It is associated with poorer prognosis in several cancers and exerts pro-tumorigenic activities including promotion of tumour growth, migration and invasion. By contrast, physiologic expression of αvβ6 is largely restricted to wound healing. These attributes render this epithelial-specific integrin a highly attractive candidate for targeting using immunotherapeutic strategies such as CAR T-cell adoptive immunotherapy. This mini-review will discuss the role and expression of αvβ6 in cancer, as well as its potential as a therapeutic target.

A stochastic nominal control optimizing the adoptive immunotherapy for cancer using tumor-infiltrating lymphocytes

In this work, a stochastic nominal optimal control approach is proposed with a view to treating cancer using the adoptive T-cell immunotherapy based on the administration of tumor-infiltrating lymphocytes. Indeed, the stochastic analysis is relevant and appropriate for studying the possible tumor-immune cellular interactions in order to better understand both dynamics of immune system and cancer evolution process. However, in the presence of an additional initial concentration of tumor cells, the therapeutic enhancement of immune response is considered using an appropriate neighboring treatment as a supplement to the adopted nominal therapy. Moreover, the Pontryagin-type stochastic maximum principle and the Pontryagin’s procedure are used to provide the explicit formulations of optimal controls. Finally, an adapted iterative method is implemented to solve numerically the optimal systems.

Abstract B099: Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro

Abstract Introduction: Adoptive T-cell immunotherapy represents a promising approach, as a mean of cancer immunotherapy, for management of different types of malignant tumors. This immunotherapeutic approach has been used with significant clinical benefits in EBV-associated lymphoid disorders, myeloma, and leukemia, in addition to a group of solid tumors. Different studies have demonstrated the probability of generating cytotoxic T-lymphocytes (CTLs) lines to high manufacturing standards, as well as the tolerability and therapeutic benefit of the approach. In our previous studies we verified that the EBV-associated B lymphoblastoid cell line (LCL), HMy2, fusion to hematologic tumor cells and cell lines in vitro produced stable hybridoma cells that grew spontaneously and immortally in tissue culture, expressed relevant tumor partner antigens, and showed an enhanced ability to stimulate allogeneic and semi-autologous T cell responses. Moreover, these studies prospered in the induction of hematologic tumor antigen-specific CTLs in peripheral blood lymphocytes (PBL) from healthy donors, and semi-autologous cancer patients as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. In the current study, we aimed to further investigate newly generated solid/HMy2 hybrids for their abilities to stimulate naive HLA-A2+ T cells of healthy donors to induce new relevant TAA-specific, HLA-A2-restricted T cell clones of therapeutic value in management of hepatocellular and pancreatic adenocarcinomas. Materials and methods: Two tumor cell lines (HepG2; HCC cell line, and Panc-1; pancreatic tumor cell line) were used separately in generation of two distinct hybrids by fusion to HMy2, using PEG/ DMSO as fusogen and fusion protocol was followed as mentioned before. Fusion cells' (FCs) growth was selected physically using deferential detachment capabilities and then chemically by 3 days incubation in HAT supplemented growth media (RPMI-1640). Moreover, relevant CD markers and surface TAAs expression of the FCs were investigated to evaluate the fusion efficiency. Hybrid cells' viability under different tissue culture conditions, phenotypic properties (including CD markers, HLAs, and co-stimulatory molecules expression) and relevant TAAs expression profile were assessed using flowcytometric analysis and RT-PCR respectively. Two immunogenic, HLA-A2-restricted and high expressed antigens were selected (GPC3 and CEA for HCC and Panc-1 hybrids respectively) and targeted to induce T cell response against. Subsequently, depending on their phenotype, two FC clones were selected for stimulation of naïve allogeneic T cells, isolated from healthy HLA-A2+ donors, in vitro for several rounds, and the outgrowing T cells were phenotyped and analysed regarding T cell subtype, differentiation pattern, & γ-IFN release, cellular cytotoxic avidity and efficacy as well as HLA-A2-restricted peptide-specificity. Results: The generated hybrid cell lines grew continuously in tissue culture in loosely adherent pattern and expressed CD40, CD80, CD86, HLA class I and HLA class II and the level of expression were higher in the hybrid cell lines than in the parent tumor cells. T cell proliferation assays showed enhanced ability of both fusion clones to stimulate both CD4 and CD8 subtypes of both central and effector cells, however, with slight dominance of effector than central counterparts Two HLA-A2-restricted peptide-specific cytotoxic T cell clones (against HLA-A*02:01-GPC3 144-152 and HLA-A*02:01-CEA 694-702) were produced for the first time using this technique and demonstrated abundant & γ-IFN production and cytotoxic activity upon recognition of their target cells. Conclusion: Hybrid cell lines, such as those described, could be used as cost effective and feasible in vitro stimulators of semi-autologous, antigen-specific CTLs for adoptive T cell immunotherapy for not only hematologic but also for certain solid tumors. Citation Format: Yehia Mohamed, Michael Browning. Immortalized HMy2/solid tumor hybridomas induce two different tumor antigen-specific T cell clones in vitro. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B099.

Abstract B016: Allogenic CAR T-cells for adoptive immunotherapy

Abstract Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR's to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRβα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. We have adapted this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells of patients with Acute Myeloid Leukemia as well as CS1 a glycoprotein highly expressed on tumor cells of patients with multiple myeloma. We will present both in vitro and in vivo data demonstrating specific anti-tumor activity of engineered CAR T cells against cell lines expressing the targeted antigen. Citation Format: Roman Galetto, Celine Lebuhotel, Isabelle Chion-Sotinel, Patricia Francon, Agnes Gouble, Julianne Smith. Allogenic CAR T-cells for adoptive immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B016.

Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity.

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy.

Antigen-specific immunotherapies for acute myeloid leukemia.

Antigen-specific immunotherapies have emerged as important components of curative treatment algorithms for many cancers. In acute myeloid leukemia (AML), success has been less obvious. Nonetheless, among the few drugs shown to improve survival in recent randomized trials is the CD33 antibody-drug conjugate gemtuzumab ozogamicin. Significant antileukemic activity is also well documented for radioimmunoconjugates targeting CD33, CD45, or CD66. These therapeutics can intensify conditioning before hematopoietic cell transplantation, but their effect on patient outcomes needs clarification. Emerging data now suggest clinical antileukemic activity of several novel antibodies and perhaps some adoptive T-cell immunotherapies and vaccines. In parallel, numerous other agents targeting a wider variety of antigens are currently being explored. However, the antigenic heterogeneity characteristic of AML is a considerable limitation for all these therapeutics, and many important questions related to the ideal target antigen(s), disease situation in which to use these therapies, most suitable patient populations, exact treatment modalities, and details of supportive care needs remain open. Addressing such questions in upcoming studies will be required to ensure that antigen-directed therapies become an effective tool in AML, a disease for which outcomes with standard "3 + 7"-based chemotherapy have remained unsatisfactory in many patients.

Choice of Better T-cells for Adoptive Immunotherapy

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. The definition of optimal targets and antigen receptors as well as the differentiation status of transferred T cells are emerging as crucial parameters for generating cell products with predictable efficacy and safety profiles. Our laboratory has demonstrated that defined subsets within the memory CD8+ T cell compartment fulfill all key characteristics of adult tissue stem cells and are essential for robust and long-term maintained responses upon adoptive transfer. We have developed clinical multi-parameter enrichment technologies to purify these memory stem cells for clinical applications. In my presentation I will report on the status of ongoing clinical trials using such purified cell products either as a primary T cell population for the treatment of infections upon allogeneic stem cell transplantation or after genetic modification with a CD19 CAR for the treatment of malignancies (collaboration with Stan Riddell, FHCC/Seattle). Infusing small numbers of T cells within a memory stem cell product can be highly effective therapeutically, but bears some risk of toxicity. Therefore, safeguards that allow selective depletion of transferred cells in the case of un-tolerable side effects may be needed to further improve adoptive immunotherapy. I will present results exploring the capacity of a truncated version of EGFR (EGFRt) co-expressed with T cells expressing a CD19-CAR. In pre-clinical mouse models we demonstrate that application of Cetuximab, which binds to EGFRt, confers selective depletion of adoptively transferred CAR-T cells in vivo. Long-term B cell aplasia, which is a main side effect of CD19-CAR T cell therapy, can be completely reverted with this strategy. Vaccination studies upon B cell recovery demonstrate full functionality of antigen-specific antibody formation. EGFRt co-expressing CD19-CAR T cells have been successfully transferred into first human patients, providing the option to test for the first time in a clinical setting whether treatment of B cell aplasia after long-term leukemia remission can be achieved by selective depletion. DisclosuresBusch:STAGE cell therapeutics: Other: I was share holder of STAGE cell therapeutics, a company that was recently bought by Juno therapeutics.. Off Label Use: CD19 CAR T cells.

Comprehensive T-Cell Receptor Repertoire Analysis Using Deep Sequencing and Single Cell Cloning Reveals Extreme Oligoclonality of Ex Vivo Expanded Cytomegalovirus-Reactive Cytotoxic T-Cells

Background: Identification of functional T-cell receptors (TCRs) to their cognate antigens is the key to the development of effective anti-viral or anti-tumor T-cell therapy. Deep sequencing of rearranged complementarity-determining region 3 (CDR3) regions of TCRA and TCRB gene segments is an emerging technology that facilitates high-throughput and semi-quantitative analysis of TCR repertoire with high resolution and accuracy. However, this method alone does not yield data for correct pairs of TCRA and TCRB sequences required for the structural determination of TCRαβ heterodimers expressed in a single T cell. "Human TCR efficient cloning within 10 days" (hTEC10) is a powerful novel technology that enables concurrent sequencing of paired TCRA and TCRB gene segments at a single cell level (Nat Med. 2013;19:1542-6). In this study, we attempted to elucidate the comprehensive TCR repertoire of cytomegalovirus (CMV)-reactive cytotoxic T-cells (CTLs) by combining quantitative deep sequencing and hTEC10.Methods: 20 ml peripheral blood samples were collected from healthy adult volunteers who gave written informed consent for the study. All donors were screened for CMV serostatus and typed for HLA-A. CMV-specific CD8+ T-cells were isolated and flow-sorted from peripheral blood (PB) mononuclear cells using an HLA-tetramer, NLV/A2, specific for HLA-A2-restricted CMV pp65-derived epitope (NLV peptide: NLVPMVATV). In some experiments, sorted NLV/A2-positive cells underwent one or two rounds of expansion with autologous PB mononuclear cells depleted of CD8+ and CD4+ T cells in the presence of NLV peptide. Using these samples, massive parallel sequencing of TCRA and TCRB V-D-J segments was performed after unbiased amplification of the target sequences by adaptor-ligation PCR. Concurrent TCRA and TCRB sequencing of TCRs expressed in a single sorted cell was performed by the hTEC10 protocol.Results: Of 20 donors who participated in the study, 8 were found to be CMV-seropositive and HLA-A2-positive (CMV+A2+). The samples obtained from 2 of CMV+A2+ donors, V001 and V004, were subjected to comprehensive TCR analysis by quantitative deep sequencing and single cell cloning: the respective frequencies of NLV/A2 tetramer-positive cells in PB of these donors were 0.36% and 0.25% of a CD8+ T-cell fraction. After two rounds of NLV-peptide stimulation, NLV/A2-tetramer-positive cells obtained from V001 and V004 were enriched to 84.3% and 90.5% of CD8+ T-cells, respectively. By deep sequencing analysis, the total number of unique TCRA/TCRB reads in PB samples from these donors was 1472/5787 in V001 and 2054/9179 in V004, while that of the enriched NLV/A2 tetramer-positive fractions was 178/62 in V001 and 100/104 in V004. However, in both donors, TCR repertoire of the expanded tetramer-positive cells was extremely skewed and the number of the most abundant top 3 reads comprised more than 90-95% of total reads for both TCRA and TCRB.To confirm the correct pairing of these TCRA and TCRB clonotypes at a single cell level, we also examined TCR sequences of the enriched tetramer-positive cells by hTEC10. A total of 180 cells each obtained from V001 and V004 were subjected for analysis. We identified 3 TCRA and TCRB clonotypes in samples from V001 and 6 clonotypes in those from V004. Importantly, 7 of 9 clones identified by hTEC10 were not listed in the clonotype determined by deep sequencing. To evaluate the effector functions of the cloned TCRs, paired TCRA and TCRB gene segments obtained by hTEC10 were transduced into PHA blasts established from T cells derived from CMV seronegative HLA-A2-negative donor. We confirmed that these PHA blasts transduced with HLA-A2-restricted CMV-specific TCRA and TCRB genes were NLV/A2-tetramer positive. Furthermore, these cells secreted INF-γ in response to NLV peptide as measured by ELISA.Conclusions: Ex vivo expanded anti-CMV CTLs reactive with NLV/A2 tetramers were extremely oligoclonal and consisted of only a few dominant clones. Determination of CDR3 sequences of these clones was feasible by quantitative deep sequencing combined with single cell cloning of TCRA and TCRB gene segments, although the results were complementary and not identical. This method could be useful for the efficient screening of the highly functional TCRs for adoptive T-cell immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Ovarian cancer chemokines may not be a significant barrier during whole tumor antigen dendritic-cell vaccine and adoptive T-cell immunotherapy

ABSTRACTTumor barriers preventing T-cell homing and engraftment should be neutralized during cancer immunotherapy. We recently discovered that ovarian cancer expresses quasi-universal chemokines that can support T-cell homing. Furthermore, T cells elicited by whole tumor antigen dendritic-cell vaccines express cognate chemokine receptors which are upregulated by CD3/CD28 costimulation.

Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies.

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals

Directed T-Cell Therapies for Leukemia and Lymphoma After Hematopoietic Stem Cell Transplant: Beyond Chimeric Antigen Receptors

This review focuses on the recent advances utilizing adoptive T-cell immunotherapies for patients after hematopoietic stem cell transplant using T cells after autologous transplant to treat the highest risk patients. The particular emphasis is the use of T cells to treat leukemias and lymphomas with gene transfer and nongene transfer approaches to direct specificity to tumor associated antigens. In this review, we will highlight how these novel therapeutics can be successfully used to prevent or treat high-risk patients who relapse after hematopoietic stem cell transplant.

Adoptive T-cell therapies for refractory/relapsed leukemia and lymphoma: current strategies and recent advances.

Despite significant advancements in the treatment and outcome of hematologic malignancies, prognosis remains poor for patients who have relapsed or refractory disease. Adoptive T-cell immunotherapy offers novel therapeutics that attempt to utilize the noted graft versus leukemia effect. While CD19 chimeric antigen receptor (CAR)-modified T cells have thus far been the most clinically successful application of adoptive T immunotherapy, further work with antigen specific T cells and CARs that recognize other targets have helped diversify the field to treat a broad spectrum of hematologic malignancies. This article will focus primarily on therapies currently in the clinical trial phase as well as current downfalls or limitations.

Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.

Fine-tuning Tumor Immunity with Integrin Trans-regulation.

Inefficient T-cell homing to tissues limits adoptive T-cell immunotherapy of solid tumors. αLβ2 and α4β1 integrins mediate trafficking of T cells into tissues via engagement of ICAM-1 and VCAM-1, respectively. Inhibiting protein kinase A (PKA)-mediated phosphorylation of α4 integrin in cells results in an increase in αLβ2-mediated migration on mixed ICAM-1-VCAM-1 substrates in vitro, a phenomenon termed "integrin trans-regulation." Here, we created an α4(S988A)-bearing mouse, which precludes PKA-mediated α4 phosphorylation, to examine the effect of integrin trans-regulation in vivo. The α4(S988A) mouse exhibited a dramatic and selective increase in migration of lymphocytes, but not myeloid cells, to sites of inflammation. Importantly, we found that the α4(S988A) mice exhibited a marked increase in T-cell entry into and reduced growth of B16 melanomas, consistent with antitumor roles of infiltrating T cells and progrowth functions of tumor-associated macrophages. Thus, increased α4 trans-regulation of αLβ2 integrin function biases leukocyte emigration toward lymphocytes relative to myeloid cells and enhances tumor immunity.

Generation of specific polyclonal and polyfunctional CD4+ T-helper1 cells against WT-1, MAGE-A3, Survivin and ROR-1 for adoptive T-cell immunotherapy.

e14025 Background: Adoptive T cell therapy (ACT) is a promising option to treat cancer. Th1 cells have been shown effective against viremia and cancer and are mediators of senescence in tumor cells...

TLR5 Ligand-Secreting T Cells Reshape the Tumor Microenvironment and Enhance Antitumor Activity.

The tumor microenvironment counters antitumor T-cell responses, in part, by blunting their activation and infiltration. Ligands that engage Toll-like receptors (TLR) on T cells and antigen-presenting cells can act as potent immune adjuvants. In this study, we show how tumor-reactive T cells engineered to secrete bacterial flagellin, a TLR5 ligand (TLR5L), can engender a costimulatory signal that augments antitumor activity. Human T cells engineered to express TLR5L along with DMF5, a T-cell receptor that recognizes the melanoma antigen MART-127-35 (DMF5(TLR5L) T cells), displayed increased proliferation, cytokine production, and cytolytic activity against melanoma cells. In a xenogenetic model, adoptive transfer of DMF5(TLR5L) T cells reduced tumor growth kinetics and prolonged mouse survival. In a syngeneic model, similarly engineered melanoma-reactive T cells (pmel(TLR5L)) displayed a relative increase in antitumor activity against established tumors, compared with unmodified T cells. In this model, we documented increased T-cell infiltration associated with increased levels of CCR1 and CXCR3 levels on T cells, a reduction in PD-1(+)Lag3(+) T cells and CD11(+)Gr1(+) myeloid-derived suppressor cells, and changes in the chemokine/cytokine profile of tumors. Our findings show how T cell-mediated delivery of a TLR agonist to the tumor site can contribute to antitumor efficacy, in the context of adoptive T-cell immunotherapy.