90Y-daclizumab, an anti-CD25 monoclonal antibody, provided responses in 50% of patients with relapsed Hodgkin's lymphoma.

Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong ß emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.

Oral Tranexamic Acid Reduces Transfusions in Total Knee Arthroplasty.

BACKGROUND: Tranexamic acid (TXA) reduces intraoperative blood loss and transfusions in patients undergoing total knee arthroplasty. Although numerous studies demonstrate the efficacy of intravenous and topical TXA in these patients, few demonstrate the effectiveness and appropriate dosing recommendations of oral formulations. METHODS: A retrospective cohort study was performed to evaluate differences in transfusion requirements in patients undergoing primary unilateral total knee arthroplasty with either no TXA (n = 866), a single-dose of oral TXA (n = 157), or both preoperative and postoperative oral TXA (n = 1049). Secondary outcomes included postoperative hemoglobin drop, total units transfused, length of stay, drain output, and cell salvage volume. RESULTS: Transfusion rates decreased from 15.4% in the no-oral tranexamic acid (OTA) group to 9.6% in the single-dose OTA group (P < .001) and 7% in the 2-dose group (P < .001), with no difference in transfusion rates between the single- and 2-dose groups (P = .390). In addition, postoperative hemoglobin drop was reduced from 4.2 g/dL in the no-OTA group to 3.5 g/dL in the single-dose group (P < .01) and to 3.4 g/dL in the 2-dose group (P < .01), without a difference between the single- and 2-dose groups (P = .233). CONCLUSION: OTA reduces transfusions, with greater ease of administration and improved cost-effectiveness relative to other forms of delivery.

Phase I Trial of Anti-PSMA Designer CAR-T Cells in Prostate Cancer: Possible Role for Interacting Interleukin 2-T Cell Pharmacodynamics as a Determinant of Clinical Response.

BACKGROUND: Chimeric antigen receptor (CAR)-modified "designer" T cells (dTc, CAR-T) against PSMA selectively target antigen-expressing cells in vitro and eliminate tumors in vivo. Interleukin 2 (IL2), widely used in adoptive therapies, was proven essential in animal models for dTc to eradicate established solid tumors. METHODS: Patients under-went chemotherapy condi-tion-ing, followed by dTc dosing under a Phase I escalation with continuous infusion low dose IL2 (LDI). A target of dTc escalation was to achieve ≥20% engraftment of infused activated T cells. RESULTS: Six patients enrolled with doses prepared of whom five were treated. Patients received 10(9) or 10(10) autologous T cells, achieving expansions of 20-560-fold over 2 weeks and engraftments of 5-56%. Pharmacokinetic and pharmacodynamic analyses established the impact of conditioning to promote expansion and engraftment of the infused T cells. Unexpectedly, administered IL2 was depleted up to 20-fold with high engraftments of activated T cells (aTc) in an inverse correlation (P < 0.01). Clinically, no anti-PSMA toxicities were noted, and no anti-CAR reactivities were detected post-treatment. Two-of-five patients achieved clinical partial responses (PR), with PSA declines of 50% and 70% and PSA delays of 78 and 150 days, plus a minor response in a third patient. Responses were unrelated to dose size (P = 0.6), instead correlating inversely with engraftment (P = 0.06) and directly with plasma IL2 (P = 0.03), suggesting insufficient IL2 with our LDI protocol to support dTc anti-tumor activity under optimal (high) dTc engraftments. CONCLUSIONS: Under a Phase I dose escalation in prostate cancer, a 20% engraftment target was met or exceeded in three subjects with adequate safety, leading to study conclusion. Clinical responses were obtained but were suggested to be restrained by low plasma IL2 when depleted by high levels of engrafted activated T cells. This report presents a unique example of how the pharmaco-dynamics of "drug-drug" interactions may have a critical impact on the efficacy of their co-application. A new Pilot/Phase II trial is planned to test moderate dose IL2 (MDI) together with high dTc engraftments for anticipated improved therapeutic efficacy. Prostate 76:1257-1270, 2016. © 2016 Wiley Periodicals, Inc.

Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T.

Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.

T cell exhaustion and Interleukin 2 downregulation.

T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigen-rich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion.

Advanced generation anti-prostate specific membrane antigen designer T cells for prostate cancer immunotherapy.

BACKGROUND: Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. METHODS: A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. RESULTS: The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. CONCLUSIONS: Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy.

A novel real-time CTL assay to measure designer T-cell function against HIV Env(+) cells.

BACKGROUND: To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. METHODS: We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors. CD4-CAR-transduced T cells were added to Env(+) 293T cells at E:T of 1:1. Killing of target cells was measured as reduced impedance. RESULTS: We observed gene expression in 60-70% of rhesus CD3(+) CD8(+) T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR-transduced populations specifically killed Env(+) cells. CONCLUSIONS: In these studies, we showed that designer T cells were redirected to kill Env(+) cells. Control of viremia without HAART would revolutionize treatment for HIV patients.

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor.

BACKGROUND: Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells. METHODS: Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions. RESULTS: We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates. CONCLUSIONS: We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.

Autoimmunity associated with immunotherapy of cancer.

In this age of promise of new therapies for cancer, immunotherapy is emerging as an exciting treatment option for patients. Vaccines and cytokines are being tested extensively in clinical trials, and strategies using monoclonal antibodies and cell transfer are mediating dramatic regression of tumors in patients with certain malignancies. However, although initially advocated as being more specific for cancer and having fewer side effects than conventional therapies, it is becoming increasingly clear that many immunotherapies can lead to immune reactions against normal tissues. Immunotoxicities resulting from treatment can range from relatively minor conditions, such as skin depigmentation, to severe toxicities against crucial organ systems, such as liver, bowel, and lung. Treatment-related toxicity has correlated with better responses in some cases, and it is probable that serious adverse events from immune-mediated reactions will increase in frequency and severity as immunotherapeutic approaches become more effective. This review introduces immunotherapeutic approaches to cancer treatment, provides details of toxicities arising from therapy, and discusses future potential ways to avoid or circumvent these side effects.

Obstructive jaundice expands intrahepatic regulatory T cells, which impair liver T lymphocyte function but modulate liver cholestasis and fibrosis.

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4(+)CD25(-) T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.

Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/- mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2-/- tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/- heterozygous (>60 weeks) mice that carry spontaneous Tsc2-/- tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

Cell density plays a critical role in ex vivo expansion of T cells for adoptive immunotherapy.

The successful ex vivo expansion of a large numbers of T cells is a prerequisite for adoptive immunotherapy. In this study, we found that cell density had important effects on the process of expansion of T cells in vitro. Resting T cells were activated to expand at high cell density but failed to be activated at low cell density. Activated T cells (ATCs) expanded rapidly at high cell density but underwent apoptosis at low cell density. Our studies indicated that low-cell-density related ATC death is mediated by oxidative stress. Antioxidants N-acetylcysteine, catalase, and albumin suppressed elevated reactive oxygen species (ROS) levels in low-density cultures and protected ATCs from apoptosis. The viability of ATCs at low density was preserved by conditioned medium from high-density cultures of ATCs in which the autocrine survival factor was identified as catalase. We also found that costimulatory signal CD28 increases T cell activation at lower cell density, paralleled by an increase in catalase secretion. Our findings highlight the importance of cell density in T cell activation, proliferation, survival and apoptosis and support the importance of maintaining T cells at high density for their successful expansion in vitro.

Author Correction: Engineering Axl specific CAR and SynNotch receptor for cancer therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HITM-SIR: phase Ib trial of intraarterial chimeric antigen receptor T-cell therapy and selective internal radiation therapy for CEA+ liver metastases.

Effective chimeric antigen receptor-modified T-cell (CAR-T) therapy for liver metastases (LM) will require innovative solutions to ensure efficient delivery and minimization of systemic toxicity. We previously demonstrated the safety of CAR-T hepatic artery infusions (HAI). We subsequently conducted the phase 1b HITM-SIR trial, in which six patients (pts) with CEA+ LM received anti-CEA CAR-T HAIs and selective internal radiation therapy (SIRT). The primary endpoint was safety with secondary assessments of biologic activity. Enrolled pts had a mean LM size of 6.4 cm, 4 pts had >10 LM, and pts received an average of two lines of prior systemic therapy. No grade 4 or 5 toxicities were observed, and there were no instances of severe cytokine-release syndrome (CRS) or neurotoxicity. The mean transduction efficiency was 60.4%. Following CAR-T HAI, reduced levels of GM-CSF-R, IDO, and PD-L1 were detected in LM, and serum CEA levels were stable or decreased in all subjects. Median survival time was 8 months (mean 11, range 4-31). Anti-CEA CAR-T HAI with subsequent SIRT was well tolerated, and biologic responses were demonstrated following failure of conventional therapy. HAI of CAR-T was once again confirmed not to be associated with severe CRS or neurotoxicity.

HITM-SURE: Hepatic immunotherapy for metastases phase Ib anti-CEA CAR-T study utilizing pressure enabled drug delivery.

In recent years, cell therapy technologies have resulted in impressive results in hematologic malignancies. Treatment of solid tumors with chimeric antigen receptor T-cells (CAR-T) has been less successful. Solid tumors present challenges not encountered with hematologic cancers, including high intra-tumoral pressure and ineffective CAR-T trafficking to the site of disease. Novel delivery methods may enable CAR-T therapies for solid tumor malignancies. A patient with liver metastases secondary to pancreatic adenocarcinoma received CAR-T targeting carcinoembryonic antigen (CEA). Previously we reported that Pressure-Enabled Drug Delivery (PEDD) enhanced CAR-T delivery to liver metastases 5.2-fold. Three doses of anti-CEA CAR-T were regionally delivered via hepatic artery infusion (HAI) using PEDD technology to optimize the therapeutic index. Interleukin-2 was systemically delivered by continuous intravenous infusion to support CAR-T in vivo. HAI of anti-CEA CAR-T was not associated with any serious adverse events (SAEs) above grade 3 and there were no on-target/off-tumor SAEs. Following CAR-T treatment, positron emission tomography-CT demonstrated a complete metabolic response within the liver, which was durable and sustained for 13 months. The response was accompanied by normalization of serum tumor markers and an abundance of CAR+ cells found within post-treatment tumor specimens. The findings from this report exhibit biologic activity and safety of regionally infused CAR-T for an indication with limited immune-oncology success to date. Further studies will determine how HAI of CAR-T may be included in multidisciplinary treatment plans for patients with liver metastases. ClinicalTrials.gov number, NCT02850536.

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo.

Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.

Second-generation anti-carcinoembryonic antigen designer T cells resist activation-induced cell death, proliferate on tumor contact, secrete cytokines, and exhibit superior antitumor activity in vivo: a preclinical evaluation.

PURPOSE: This report describes the development and preclinical qualification tests of second-generation anti-carcinoembryonic (CEA) designer T cells for use in human trials. EXPERIMENTAL DESIGN: The progenitor first-generation immunoglobulin-T-cell receptor (IgTCR) that transmits Signal 1-only effectively mediated chimeric immune receptor (CIR)-directed cytotoxicity, but expressor T cells succumbed to activation-induced cell death (AICD). The second-generation CIR (termed "Tandem" for two signals) was designed to transmit TCR Signal 1 and CD28 Signal 2 to render T cells resistant to AICD and provide prolonged antitumor effect in vivo. RESULTS: A CIR was created that combines portions of CD28, TCRzeta, and a single chain antibody domain (sFv) specific for CEA into a single molecule (IgCD28TCR). As designed, the gene-modified Tandem T cells exhibit the new property of being resistant to AICD, showing instead an accelerated proliferation on tumor contact. Tandem T cells are more potent than first generation in targeting and lysing CEA+ tumor. Tandem T cells secrete high levels of interleukin-2 and IFNgamma on tumor contact that first-generation T cells lacked, but secretion was exhaustible, suggesting a need for interleukin-2 supplementation in therapy even for these second-generation agents. Finally, second-generation T cells were more effective in suppressing tumor in animal models. CONCLUSION: An advanced generation of anti-CEA designer T cells is described with features that promise a more potent and enduring antitumor immune response in vivo. These preclinical data qualify the human use of this agent that is currently undergoing trial in patients with CEA+ cancers.

STAT3 inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived suppressor cells.

Immunosuppressive myeloid-derived suppressor cells (MDSC) subvert antitumor immunity and limit the efficacy of chimeric antigen receptor T cells (CAR-T). Previously, we reported that the GM-CSF/JAK2/STAT3 axis drives liver-associated MDSC (L-MDSC) proliferation and blockade of this axis rescued antitumor immunity. We extended these findings in our murine liver metastasis (LM) model, by treating tumor-bearing mice with STAT3 inhibitors (STATTIC or BBI608) to further our understanding of how STAT3 drives L-MDSC suppressive function. STAT3 inhibition caused significant reduction of tumor burden as well as L-MDSC frequencies due to decrease in pSTAT3 levels. L-MDSC isolated from STATTIC or BBI608-treated mice had significantly reduced suppressive function. STAT3 inhibition of L-MDSC was associated with enhanced antitumor activity of CAR-T. Further investigation demonstrated activation of apoptotic signaling pathways in L-MDSC following STAT3 inhibition as evidenced by an upregulation of the pro-apoptotic proteins Bax, cleaved caspase-3, and downregulation of the anti-apoptotic protein Bcl-2. Accordingly, there was also a decrease of pro-survival markers, pErk and pAkt, and an increase in pro-death marker, Fas, with activation of downstream JNK and p38 MAPK. These findings represent a previously unrecognized link between STAT3 inhibition and Fas-induced apoptosis of MDSCs. Our findings suggest that inhibiting STAT3 has potential clinical application for enhancing the efficacy of CAR-T cells in LM through modulation of L-MDSC.

YY1 Upregulates Checkpoint Receptors and Downregulates Type I Cytokines in Exhausted, Chronically Stimulated Human T Cells.

T cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-?). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28?p38MAPK/JNK?YY1? exhaustion.

Engineering Axl specific CAR and SynNotch receptor for cancer therapy.

Axl is a tyrosine kinase receptor that is commonly overexpressed in many cancers. As such, Axl represents an attractive therapeutic target. The transfer of engineered T cell expressing chimeric antigen receptor (CAR) is an exciting cancer therapeutic approach that shows high efficacy against cancers in clinical trials, especially for B cell malignancies. Furthermore, recently developed synthetic Notch (synNotch) receptor has demonstrated potential in enhancing the specificity of CAR T cell therapy and delivering therapeutic payloads to tumors in an antigen-dependent manner. Therefore, a CAR or synNotch against Axl could be a valuable therapeutic reagent against many cancers. Here, we develop a single-chain variable fragment from a humanized monoclonal antibody against Axl. The scFv is attached to CD3ζ, CD28, and 4-1BB signaling domains to generate an anti-Axl CAR. When introduced into human primary T cells, the anti-Axl CAR can lead to cytokine production and cell killing in response to tumor cells expressing Axl. Moreover, an anti-Axl synNotch generated using the same scFv can be activated with Axl expressing tumor cells. Given the fact that Axl is an important cancer therapeutic target, these receptors could be valuable reagents for developing anti-Axl therapies.