Inducible Caspase-9 Selectively Modulates the Toxicities of CD19-Specific Chimeric Antigen Receptor-Modified T Cells.

Immunotherapy with T cells expressing the chimeric antigen receptor (CAR) specific for the CD19 antigen (CD19.CAR-Ts) is a very effective treatment in B cell lymphoid malignancies. However, B cell aplasia and cytokine release syndrome (CRS) secondary to the infusion of CD19.CAR-Ts remain significant drawbacks. The inclusion of safety switches into the vector encoding the CAR is seen as the safest method to terminate the effects of CD19.CAR-Ts in case of severe toxicities or after achieving long-term sustained remissions. By contrast, the complete elimination of CD19.CAR-Ts when CRS occurs may jeopardize clinical responses as CRS and antitumor activity seem to concur. We have demonstrated, in a humanized mouse model, that the inducible caspase-9 (iC9) safety switch can eliminate CD19.CAR-Ts in a dose-dependent manner, allowing either a selective containment of CD19.CAR-T expansion in case of CRS or complete deletion on demand granting normal B cell reconstitution.

Chronic Activation of Innate Immunity Correlates With Poor Prognosis in Cancer Patients Treated With Oncolytic Adenovirus.

Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P < 0.001), granulocyte macrophage colony-stimulating factor (GM-CSF) signaling (P < 0.001), and leukocyte extravasation signaling (P < 0.001), in patients surviving a shorter time than their controls. In immunohistochemical analysis, markers CD4 and CD163 were significantly elevated (P = 0.020 and P = 0.016 respectively), in patients with shorter than expected survival. Interestingly, T-cell exhaustion marker TIM-3 was also found to be significantly upregulated (P = 0.006) in patients with poor prognosis. Collectively, these data suggest that activation of several functions of the innate immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

GMCSF-armed vaccinia virus induces an antitumor immune response.

Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumor-associated hypermetabolism was armed with human granulocyte-macrophage colony-stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene-carrying viruses was similar to unarmed virus. The hGMCSF-encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co-culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF-virus-treated tumors. These findings help clarify the mechanism of action of GMCSF-armed vaccinia viruses undergoing clinical trials.

Serotype chimeric oncolytic adenovirus coding for GM-CSF for treatment of sarcoma in rodents and humans.

Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.

Oncolytic adenovirus with temozolomide induces autophagy and antitumor immune responses in cancer patients.

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.

An oncolytic adenovirus enhanced for toll-like receptor 9 stimulation increases antitumor immune responses and tumor clearance.

Oncolytic viruses represent a multifaceted tool for cancer treatment. In addition to specific killing of cancer cells (oncolysis), these agents also provide danger signals prompting the immune system to stimulate an antitumor immune response. To increase adenovirus adjuvancy, we engineered the genome of Ad5D24 by inserting 18 immunostimulatory islands (Ad5D24-CpG). The toxicity and immunogenicity profile of Ad5D24-CpG showed that the safety of the maternal virus was retained. The efficacy of the CpG-enriched virus was assessed in a xenograft model of lung cancer where a significant increase in antitumor effect was seen in comparison with controls. When the experiment was repeated in animal depleted of natural killer (NK) cells, Ad5D24-CpG lost its advantage. The same was seen when Toll-like receptor (TLR)9 was blocked systemically. In a syngeneic model of melanoma (B16-OVA), we observed a significant increase of OVA-specific T cells and a decrease of activation of myeloid-derived suppressor cells in Ad5D24-CpG-treated mice. In conclusion, we have generated the first genetically modified oncolytic adenovirus backbone able to enhance TLR9-stimulation for increased antitumor activity.

Ad3-hTERT-E1A, a fully serotype 3 oncolytic adenovirus, in patients with chemotherapy refractory cancer.

Twenty-five patients with chemotherapy refractory cancer were treated with a fully serotype 3-based oncolytic adenovirus Ad3-hTERT-E1A. In mice, Ad3 induced higher amounts of cytokines but less liver damage than Ad5 or Ad5/3. In humans, the only grade 3 adverse reactions were self-limiting cytopenias and generally the safety profile resembled Ad5-based oncolytic viruses. Patients that had been previously treated with Ad5 viruses presented longer lasting lymphocytopenia but no median increase in Ad3-specific T-cells in blood, suggesting immunological activity against antigens other than Ad3 hexon. Frequent alterations in antitumor T-cells in blood were seen regardless of previous virus exposure. Neutralizing antibodies against Ad3 increased in all patients, whereas Ad5 neutralizing antibodies remained stable. Treatment with Ad3-hTERT-E1A resulted in re-emergence of Ad5 viruses from previous treatments into blood and vice versa. Signs of possible efficacy were seen in 11/15 (73%) patients evaluable for tumor markers, four of which were treated only intravenously. Particularly promising results were seen in breast cancer patients and especially those receiving concomitant trastuzumab. Taken together, Ad3-hTERT-E1A seems safe for further clinical testing or development of armed versions. It offers an immunologically attractive alternative, with possible pharmacodynamic differences and a different receptor compared to Ad5.

Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors.

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.

Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus.

Oncolytic adenoviruses are an emerging experimental approach for treatment of tumors refractory to available modalities. Although preclinical results have been promising, and clinical safety has been excellent, it is also apparent that tumors can become virus resistant. The resistance mechanisms acquired by advanced tumors against conventional therapies are increasingly well understood, which has allowed development of countermeasures. To study this in the context of oncolytic adenovirus, we developed two in vivo models of acquired resistance, where initially sensitive tumors eventually gain resistance and relapse. These models were used to investigate the phenomenon on RNA and protein levels using two types of analysis of microarray data, quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. Interferon (IFN) signaling pathways were found upregulated and Myxovirus resistance protein A (MxA) expression was identified as a marker correlating with resistance, while transplantation experiments suggested a role for tumor stroma in maintaining resistance. Furthermore, pathway analysis suggested potential therapeutic targets in oncolytic adenovirus-resistant cells. Improved understanding of the antiviral phenotype causing tumor recurrence is of key importance in order to improve treatment of advanced tumors with oncolytic adenoviruses. Given the similarities between mechanisms of action, this finding might be relevant for other oncolytic viruses as well.

Immunological effects of low-dose cyclophosphamide in cancer patients treated with oncolytic adenovirus.

Patients with advanced solid tumors refractory to and progressing after conventional therapies were treated with three different regimens of low-dose cyclophosphamide (CP) in combination with oncolytic adenovirus. CP was given with oral metronomic dosing (50 mg/day, N = 21), intravenously (single 1,000 mg dose, N = 7) or both (N = 7). Virus was injected intratumorally. Controls (N = 8) received virus without CP. Treatments were well tolerated and safe regardless of schedule. Antibody formation and virus replication were not affected by CP. Metronomic CP (oral and oral + intravenous schedules) decreased regulatory T cells (T(regs)) without compromising induction of antitumor or antiviral T-cell responses. Oncolytic adenovirus given together with metronomic CP increased cytotoxic T cells and induced Th1 type immunity on a systemic level in most patients. All CP regimens resulted in higher rates of disease control than virus only (all P < 0.0001) and the best progression-free (PFS) and overall survival (OS) was seen in the oral + intravenous group. One year PFS and OS were 53 and 42% (P = 0.0016 and P < 0.02 versus virus only), respectively, both which are unusually high for chemotherapy refractory patients. We conclude that low-dose CP results in immunological effects appealing for oncolytic virotherapy. While these first-in-human data suggest good safety, intriguing efficacy and extended survival, the results should be confirmed in a randomized trial.

Characteristics and Trends of COVID-19 Infection in a Tertiary Hospital in Romania: A Retrospective Study.

(1) Background: The outbreak of the COVID-19 pandemic represented a real challenge for all of humanity. Characterized by a complex spectrum of signs and symptoms, by various severity degrees, the disease spread rapidly around the world. After more than two and half years since the beginning of COVID-19 pandemic, in the context of a paradoxical, enigmatic, and relentless COVID-19, the objective of the current study was to evaluate the characteristics and evolution of patients with SARS-CoV-2 infection, hospitalized in "St. Parascheva" Clinical Hospital of Infectious Diseases (Iasi, Romania). (2) Methods: This is a retrospective study that used the medical database recorded between July and November 2021 in order to highlight the characteristics of SARS-CoV-2 infection in patients from the northeastern region of Romania. (3) Results: We enrolled in the study a total of 1732 SARS-CoV-2 infected patients, mean age 67 ± 3.4 years, the female gender predominating (987 cases; 56.98%) as well as patients from the urban environment (982 patients; 56.69%). Moderate form of the disease predominated (814 cases; 47%), pulmonary imaging changes were found in 1042 (60.16%) cases, and 1242 (71.71%) patients had at least one underlying disease. After a median length of hospitalization of 9.5 days, 1359 (78.46%) patients were discharged cured, 48 (2.77%) were transferred to other services by decompensating the associated pathologies, 302 (17.43%) patients needed extensive support in the intensive care unit and there were 325 (18.76%) deaths. (4) Conclusions: The epidemiological characteristics of SARS-CoV-2 infection recorded in our study were mostly the same as characteristics of COVID-19 from all over the world.

Antiangiogenic arming of an oncolytic vaccinia virus enhances antitumor efficacy in renal cell cancer models.

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.

Treatment of cancer patients with a serotype 5/3 chimeric oncolytic adenovirus expressing GMCSF.

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.

Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy.

BACKGROUND: The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. METHODS: An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. RESULTS: A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. CONCLUSION: The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

Human adenovirus replication in immunocompetent Syrian hamsters can be attenuated with chlorpromazine or cidofovir.

BACKGROUND: Adenoviruses can cause severe toxicity in children and in immunocompromised adults, and therefore a means to abrogate replication would be useful. With regard to cancer treatment, replication competent oncolytic adenoviruses have been safe in humans, although their efficacy has been variable. Therefore, more effective agents are now entering clinical testing and, consequently, replication-associated side effects remain a concern. Preclinical analysis of replication related toxicity has been hampered by a lack of permissive models. Therefore, it has been difficult to study modulation of human adenovirus replication in immune competent animals. METHODS: We investigated four different hamster carcinoma cell lines for transduction and cell killing potency in vitro and in vivo. Gene transfer was assessed using replication-deficient adenoviruses expressing luciferase. Cell killing was studied in vitro and in vivo using an oncolytic adenovirus that kills tumor cells by viral replication. After the most promising animal model had been selected, abrogation of virus replication was assessed in vitro and in vivo using a TCID(50) assay. RESULTS: The results obtained suggest wild-type adenovirus replication in all four tested Syrian hamster cell lines and also normal organs. Virus replication could be abrogated with chlorpromazine, cidofovir and cytosine arabinoside, and the effect occurred subsequent to nuclear delivery of the viral genome. Attenuation of virus replication also was seen in vivo both in tumors and the liver. CONCLUSIONS: Syrian hamsters may comprise a valuable immune competent model for evaluating anti-adenoviral drugs. Furthermore, chlorpromazine or cidofovir might be useful in case of adenovirus replication-associated symptoms in humans.

Adenoviruses with an αvß integrin targeting moiety in the fiber shaft or the HI-loop increase tumor specificity without compromising antitumor efficacy in magnetic resonance imaging of colorectal cancer metastases.

BACKGROUND: Colorectal cancer is often a deadly disease and cannot be cured at metastatic stage. Oncolytic adenoviruses have been considered as a new therapeutic option for treatment of refractory disseminated cancers, including colorectal cancer. The safety data has been excellent but tumor transduction and antitumor efficacy especially in systemic administration needs to be improved. METHODS: Here, the utility of αvß integrin targeting moiety Arg-Gly-Asp (RGD) in the Lys-Lys-Thr-Lys (KKTK) domain of the fiber shaft or in the HI-loop of adenovirus serotype 5 for increased tumor targeting and antitumor efficacy was evaluated. To this end, novel spleen-to-liver metastatic colorectal cancer mouse model was used and the antitumor efficacy was evaluated with magnetic resonance imaging (MRI). RESULTS: Both modifications (RGD in the HI-loop or in the fiber shaft) increased gene transfer efficacy in colorectal cancer cell lines and improved tumor-to-normal ratio in systemic administration of the vector. CONCLUSIONS: Antitumor potency was not compromised with RGD modified viruses suggesting increased safety profile and tumor specificity.

T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.

PURPOSE: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells. PATIENTS AND METHODS: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant. RESULTS: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19+ B cells. Five patients remain in remission at 42-60+ months. CONCLUSIONS: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.

MUC1 in endometriosis and ovarian cancer.

Endometriosis is a chronic, debilitating disease, associated with pelvic pain and infertility. Recent epidemiological studies suggest that women with endometriosis are at increased risk for ovarian cancer. Although the causative factors for both endometriosis and ovarian cancer remain largely unknown, several similarities between the proposed etiology of ovarian cancer and the observed pathophysiology of endometriosis have been reported. MUC1 glycoprotein is present in endometriotic lesions and overexpressed in epithelial ovarian tumors. We are currently studying immunity to MUC1 antigen in newly emerging preclinical models for endometriosis and ovarian cancer and exploring the potential for immune therapy/prevention with MUC1 in both diseases.

Immunological effects of a tumor necrosis factor alpha-armed oncolytic adenovirus.

For long it has been recognized that tumor necrosis factor alpha (TNFa) has anticancer characteristics, and its use as a cancer therapeutic was proposed already in the 1980s. However, its systemic toxicity has limited its usability. Oncolytic viruses, selectively cancer-killing viruses, have shown great potency, and one of their most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local versus systemic concentrations. Therefore, the overall magnitude of tumor cell killing results from the combination of oncolysis, transgene-mediated direct effect such as TNFa-mediated apoptosis, and, perhaps most significantly, from activation of the host immune system against the tumor. We generated a novel chimeric oncolytic adenovirus expressing human TNFa, Ad5/3-D24-hTNFa, whose efficacy and immunogenicity were tested in vitro and in vivo. The hTNFa-expressing adenovirus showed increased cancer-eradicating potency, which was shown to be because of elevated apoptosis and necrosis rates and induction of various immune responses. Interestingly, we saw increase in immunogenic cell death markers in Ad5/3-d24-hTNFa-treated cells. Moreover, tumors treated with Ad5/3-D24-hTNFa displayed enhanced presence of OVA-specific cytotoxic T cells. We thus can conclude that tumor eradication and antitumor immune responses mediated by Ad5/3-d24-hTNFa offer a new potential drug candidate for cancer therapy.