Association of TRIMCyp and TRIM5α from assam macaques leads to a functional trade-off between HIV-1 and N-MLV inhibition.

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A (TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera (amTRIMCyp), along with a TRIM5α allelic protein (amTRIM5α). Herein, we investigated the antiviral activity of amTRIMCyp and amTRIM5α individually, as well as their interaction and joint effects. amTRIMCyp showed a divergent restriction pattern from amTRIM5α. Although both proteins potently restricted the replication of HIV-1, only amTRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when amTRIMCyp and amTRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous amTRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from amTRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the amTRIMCyp-amTRIM5α interaction. In contrast, amTRIMCyp completely abrogated the anti-N-MLV activity mediated by amTRIM5α, showing a dominant-negative effect, indicating that the generation of amTRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.

CAR T cell immunotherapy for human cancer.

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αß heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.

CAR T Cells Releasing IL-18 Convert to T-Bethigh FoxO1low Effectors that Exhibit Augmented Activity against Advanced Solid Tumors.

Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells has achieved remarkable efficacy in the treatment of hematopoietic malignancies. However, eradicating large solid tumors in advanced stages of the disease remains challenging. We explored augmentation of the anti-tumor immune reaction by establishing an acute inflammatory reaction. Systematic screening indicates that IL-18 polarizes CAR T cells toward T-bethigh FoxO1low effectors with an acute inflammatory response. CAR T cells engineered with inducible IL-18 release exhibited superior activity against large pancreatic and lung tumors that were refractory to CAR T cells without cytokines. IL-18 CAR T cell treatment was accompanied by an overall change in the immune cell landscape associated with the tumor. More specifically, CD206- M1 macrophages and NKG2D+ NK cells increased in number, whereas Tregs, suppressive CD103+ DCs, and M2 macrophages decreased, suggesting that "iIL18 TRUCKs" can be used to sensitize large solid tumor lesions for successful immune destruction.

[T Lymphocytes with Modified Specificity in the Therapy of Malignant Diseases].

Immunotherapy is one of the most rapidly progressing and promising fields in antitumor therapy. It is based on the idea of using immune cells of patient or healthy donors for elimination of malignant cells. T lymphocytes play a key role in cell-mediated immunity including the response to tumors. Recently developed approaches of altering antigen specificity of T cells consist of their genetic modification (introduction of additional T cell receptor or chimeric antigen receptor), as well as the use of bispecific molecules that crosslink target and effector cells. These approaches are used to retarget T lymphocytes with arbitrary specificity against tumor antigens in the context of antitumor immunotherapy. The high potential of T cell immunotherapy was demonstrated in a number of clinical trials. In the future, it is possible to develop approaches to the therapy of a wide spectrum of tumors. The selection of the optimal antigen is the main challenge in successful T cell immunotherapy, as it largely determines the effectiveness of the treatment, as well as the risk of side effects. In this review we discuss potential methods of modification of T cell specificity and targets for immunotherapy.

Development of unique cytotoxic chimeric antigen receptors based on human scFv targeting B7H6.

As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.

Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent.

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.

Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma.

CD28 is the major costimulatory receptor on T cells regulating proliferation, survival and effector function. Acquired mutations in the extracellular domain of CD28 have been identified in patients with cutaneous T cell lymphoma, angioimmunoblastic T cell lymphoma and other T cell neoplasms, suggesting it may contribute to disease pathogenesis. We used a heterologous system in which mutant human CD28 was expressed on primary murine T cells deficient in CD28 to ascertain how specific mutations identified in a genetic screen of patients with cutaneous T cell lymphoma affected normal T cell function. All three mutant CD28 proteins examined enhanced CD28-dependent T cell proliferation and effector function. These data suggest that the mutant CD28 isoforms could accelerate tumor cell growth and increase tumor burden in affected patients. Interruption of CD28:ligand interactions may be an effective, targeted therapy for a subset of patients whose tumors bear the mutant CD28 receptor.

The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.

Chimeric Antigen Receptors: A Cell and Gene Therapy Perspective.

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T lymphocytes to target chosen antigens. The targeting of CD19, a cell surface molecule expressed in the vast majority of leukemias and lymphomas, has been successfully translated in the clinic, earning CAR therapy a special distinction in the selection of "cancer immunotherapy" by Science as the breakthrough of the year in 2013. CD19 CAR therapy is predicated on advances in genetic engineering, T cell biology, tumor immunology, synthetic biology, target identification, cell manufacturing sciences, and regulatory compliance-the central tenets of CAR therapy. Here, we review two of these foundations: the genetic engineering approaches and cell types to engineer.

Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers.

Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated CAR-T therapy targeting carcino-embryonic antigen (CEA)-positive colorectal cancer (CRC) patients with metastases to evaluate its safety and efficacy. Five escalating dose levels (DLs) (1 × 105 to 1 × 108/CAR+/kg cells) of CAR-T were applied in 10 CRC patients. Our data showed that severe adverse events related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced progressive disease (PD) in previous treatments had stable disease after CAR-T therapy. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level was apparent in most patients even in long-term observation. Furthermore, we observed persistence of CAR-T cells in peripheral blood of patients receiving high doses of CAR-T therapy. Importantly, we observed CAR-T cell proliferation especially in patients after a second CAR-T therapy. Taken together, we demonstrated that CEA CAR-T cell therapy was well tolerated in CEA+ CRC patients even in high doses, and some efficacy was observed in most of the treated patients.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor.

Chimeric antigen receptors (CARs) combine T cell activation with antibody-mediated tumor antigen specificity, bypassing the need for T cell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using T cells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ T cells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

Adoptive Cellular Therapy (ACT) for Cancer Treatment.

Adoptive cellular therapy (ACT) with various lymphocytes or antigen-presenting cells is one stone in the pillar of cancer immunotherapy, which relies on the tumor-specific T cell. The transfusion of bulk T-cell population into patients is an effective treatment for regression of cancer. In this chapter, we summarize the development of various strategies in ACT for cancer immunotherapy and discuss some of the latest progress and obstacles in technical, safety, and even regulatory aspects to translate these technologies to the clinic. ACT is becoming a potentially powerful approach to cancer treatment. Further experiments and clinical trials are needed to optimize this strategy.

How antigen specificity directs regulatory T-cell function: self, foreign and engineered specificity.

Regulatory T cells (Tregs) are a suppressive subset of T cells that have important roles in maintaining self-tolerance and preventing immunopathology. The T-cell receptor (TCR) and its antigen specificity play a dominant role in the differentiation of cells to a Treg fate, either in the thymus or in the periphery. This review focuses on the effects of the TCR and its antigen specificity on Treg biology. The role of Tregs with specificity for self-antigen has primarily been studied in the context of autoimmune disease, although recent studies have focused on their role in steady-state conditions. The role of Tregs that are specific for pathogens, dietary antigens and allergens is much less studied, although recent data suggest a significant and previously underappreciated role for Tregs during memory responses to a wide range of foreign antigens. The development of TCR- or chimeric antigen receptor (CAR)-transduced T cells means we are now able to engineer Tregs with disease-relevant antigen specificities, paving the way for ensuring specificity with Treg-based therapies. Understanding the role that antigens play in driving the generation and function of Tregs is critical for defining the pathophysiology of many immune-mediated diseases, and developing new therapeutic interventions.

Design of chimeric antigen receptors with integrated controllable transient functions.

The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug.

Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis.

Old World monkey TRIM5α strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophilin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between α-helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4+ MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5α-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5α without substantial fitness cost when propagated in MT4 cells expressing CM TRIM5α. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5α recognize the HIV-1 capsid in different manners.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

PROKARYOTIC EXPRESSION AND BIOACTIVITY EVALUATION OF THE CHIMERIC GENE DERIVED FROM THE GROUP 1 ALLERGENS OF DUST MITES.

BACKGROUND: we successfully reconstituted the gene from group 1 allergens of dust mites, and obtained a body of shuffled genes. In order to verify the prediction on the chimeric gene, we tentatively cloned R8 into the vector that was prokaryoticly expressed, purified and assessed for its bio-activities. METHODS: the expressed product was detected by SDSPAGE and the target protein was purified. The purified protein R8 was detected by ELISA. 75 BALB/c mice were divided into 5 groups, namely PBS, rDer f1, rDer p1, R8 and asthma group. The mice were treated with dust mite allergens at 0, 7, 14 day by intraperitoneal injection and inhaled challenge as aerosol on day 21 for 7 days. Specific allergen immunotherapy was performed using rDer f1, rDer p1 and R8 allergens respectively. The level of IFN and IL- 4 in BALF was detected by ELISA. RESULTS: the chimeric gene R8 was expressed with a band of approximately Mr 35000. Compared with groups of rDer f 1 and rDer p 1 [(80.44±15.50) and (90.79±10.38) µg/ml respectively], IgE binding capacity of the protein R8 (37.03±12.46) µg/ml was statistically lower (P<0.001). The level of IFN in sera of R8 group [(343.43±38.79) pg/ml] was higher than that of the PBS and asthma groups [(393.93±50.68) and (208.44±46.11) pg/ml respectively] (P<0.01), but no statistical difference to that of the rDer f 1 and rDer p 1 groups (P>0.05). IL-4 level in R8 group was lower markedly than the others (P<0.05 or P<0.01). CONCLUSIONS: chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.

Antecedentes: se reconstituyó con éxito el gen del grupo 1 alérgenos de los ácaros del polvo, y obtuvo un conjunto de genes barajadas. Con el fin de verificar la predicción en el gen quimérico, hemos clonado tentativamente R8 en el vector que se expresó prokaryoticly, purificó y se evaluó por sus actividades-bio. Métodos: el producto expresado se detectó por SDS-PAGE y la proteína diana se purificó. La proteína purificada R8 se detectó por ELISA. Setenta y cinco ratones BALB/ c se dividieron en 5 grupos, a saber: PBS, rDer f1, rDer p1, R8 y el grupo de asma. Los ratones fueron tratados con alérgenos de ácaros del polvo a los 0, 7, 14 días mediante inyección intraperitoneal y inhaladas desafío como aerosol en día 21 durante 7 días. La inmunoterapia específica para el alérgeno se realizó utilizando rDer f1, rDer p1 y alérgenos R8, respectivamente. El nivel de IFN e IL-4 en BALF se detectó por ELISA. Resultados: el gen quimérico R8 se expresó con una banda de aproximadamente Mr 35000. En comparación con los grupos de rDer f 1 y rDer p 1 [(80,44 ± 15,50) y (90,79 ± 10,38) µg/ml, respectivamente], la capacidad de unión a IgE de la proteína R8 (37,03 ± 12,46) µg/ml fue estadísticamente inferior (P.

Adoptive T-cell therapy for cancer: The era of engineered T cells.

Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T-cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR-modified T cells and chimeric antigen receptor gene-modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments.