Khaled A. Tolba

HSV ICP0 recruits USP7 to modulate TLR-mediated innate response

Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-kappaB and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKKgamma, thus terminating TLR-mediated NF-kappaB and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits this physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence.

Adoptively Transferred Tumor-Specific T Cells Stimulated Ex vivo Using Herpes Simplex Virus Amplicons Encoding 4-1BBL Persist in the Host and Show Antitumor Activity In vivo

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.

Efficacy and Toxicity of Chemoradiotherapy with Carboplatin and Irinotecan Followed by Consolidation Docetaxel for Unresectable Stage III Non-small Cell Lung Cancer

Introduction: In 2003, consolidation docetaxel was a promising concept for unresectable stage IIIA/B nonsmall cell lung cancer (NSCLC). To test the hypothesis that chemoradiotherapy with carboplatin and irinotecan followed by consolidation docetaxel would be feasible and clinically active, we conducted a phase II study. Methods: Thirty-two patients with unresectable stage IIIA/B NSCLC received irinotecan (30 mg/m2) and carboplatin dosed to a target area under the concentration curve of 2, each administered weekly for 7 weeks. Concurrent radiotherapy was administered more than 7 weeks to a total dose of 63 Gy in 35 fractions. Consolidation docetaxel (75 mg/m2) was administered every 3 weeks for 3 doses 4 weeks after chemoradiotherapy. The primary end point was objective response rate by RECIST. Results: Complete responses occurred in 4 patients and partial responses occurred in 14, for an objective response rate of 56.3% (95% confidence interval [CI], 37.7-73.6%). Median progression-free survival was 6.5 months (95% CI, 4.6-13.5); median duration of survival was 14.8 months (95% CI, 6.9-27.3). The most common hematologic toxicity was leukopenia, which were grade 3 or 4 in 16 patients (50%). Radiation pneumonitis (grade ≥2) occurred in 13 of 31 treated patients (42%). Conclusions: These findings suggested that concurrent chemoradiotherapy with carboplatin and irinotecan followed by consolidation docetaxel is clinically active based on median survival in patients with unresectable stage III NSCLC; however, the 42% incidence of clinical radiation pneumonitis was unexpected and warrants further investigation to determine the mechanism and preventive strategies.

Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

Yersinia pestis evades immune responses in part by injecting into host immune cells several effector proteins called Yersinia outer proteins (Yops) that impair cellular function. This has been best characterized in the innate effector cells, but much less so for cells involved in adaptive immune responses. Dendritic cells (DC) sit at the crossroads between innate and adaptive immunity, and can function to initiate or inhibit adaptive immune responses. Although Y. pestis can target and inactivate DC, the mechanism responsible for this remains unclear. We have found that injection of Y. pestis YopJ into DC progenitors disrupts key signal transduction pathways and interferes with DC differentiation and subsequent function. YopJ injection prevents up‐regulation of the NF‐κB transcription factor Rel B and inhibits MAPK/ERK activation – both having key roles in DC differentiation. Furthermore, YopJ injection prevents costimulatory ligand up‐regulation, LPS‐induced cytokine expression, and yields differentiated DC with diminished capability to induce T cell proliferation and IFN‐γ induction. By modulating DC function through YopJ‐mediated disruption of signaling pathways during progenitor to DC differentiation, Yersinia may interfere with the adaptive responses necessary to clear the infection as well as establish a tolerant immune environment that leads to chronic infection/carrier state in the surviving host.

Concurrent radiotherapy with carboplatin and cetuximab for the treatment of medically compromised patients with locoregionally advanced head and neck squamous cell carcinoma

Background: Cetuximab (Cx) + radiation therapy (RT) is well-tolerated and has improved survival in patients (pts) with locoregionally advanced head and neck squamous cell carcinomas (LA-HNSCC). However, its efficacy when compared to HD-DDP + RT has been questioned. At our institution, low-dose weekly carboplatin is added to Cx + RT for patients unsuitable for HD-DDP. Methods: We reviewed records of 16 patients with LA-HNSCC treated with definitive Cx + carboplatin + RT at the University of Miami from 2007 to 2011. Median follow-up was 24 months (range: 1–69 months). Results: Median age: 71.5 years (range: 57–90 years); 15 male, 1 female. ECOG PS 0 = 15, 1 = 1. TNM staging was: T1 = 1, T2 = 5, T3 = 8, T4 = 2; N stage: N0 = 8, N1 = 5, N2a = 2, N2b = 1. All patients received weekly carboplatin (AUC 1.5–2), Cx given conventionally and daily conventionally fractionated RT. Median total weeks of concurrent systemic therapy = 7 (range: 3–8 weeks). RT was delivered to a median total dose of 70 Gy (range 30–74 Gy). Of the 15 evaluable patients, there were: 12 CR, 2 PR, and 1 PD. There were three local in-field failures, two regional failures, and three distant failures. At last follow-up, 8/15 patients remained with NED. Three-year locoregional recurrence was 28.3% (95% CI: 7.7–53.9%). Mean percentage of weight loss was 14% (range: 6–26%). Two patients required systemic therapy dose reduction. Three patients experienced a treatment delay and three did not finish RT as planned including a patient who received only 30 Gy due to death secondary to MI during treatment. Conclusion: In this small retrospective series, carboplatin/Cx/RT was well-tolerated and efficacious in patients unsuitable for HD-DDP having LA-HNSCC. Acute toxicities were similar to Cx + RT, likely due to the non-overlapping toxicity profiles of the two systemic agents. We hypothesize that the addition of a well-tolerated cytotoxic chemotherapy agent may improve the therapeutic ratio of Cx + RT in patients who are poor candidates for more aggressive therapies and warrants evaluation in a prospective manner.

Augmentation of anti-tumor responses of adoptively transferred CD8+T cells in the lymphopenic setting by HSV amplicon transduction

Treatment of cancer with cytotoxic agents may induce lymphopenia. Adoptively transferred T cells have been reported to display enhanced anti-tumor efficacy in the lymphopenic setting. We reasoned that the anti-tumor effects of adoptively transferred cells in the lymphopenic host could be further augmented through local provision of an innate stimulus in the tumor bed. Utilizing a model in which mice were irradiated to induce lymphopenia, with limited shielding to allow tumor growth, we demonstrate that “triple” therapy consisting of radiation-induced lymphopenia, adoptive transfer of naïve CD8+ T cells, and intra-tumoral HSV amplicon injection resulted in reduced tumor growth compared to the combination of any two of the aforementioned interventions. To gain insight into the mechanism underlying this effect we studied the effects of HSV amplicon transduction into tumors on cytokine expression and on anti-tumor specific T cells. HSV amplicon transduction specifically induced several cytokine mRNAs including IFN-γ, and IP-10. Adoptively transferred transgenic OT-1 T cells directed against Ovalbumin were more effective against Ovalbumin-expressing tumors when combined with intra-tumoral HSV amplicon injections in the lymphopenic host. Following intra-tumoral HSV-amplicon injections, anti-tumor T cells secreted higher levels of interferon-γ in response to in-vitro re-stimulation with tumor cells, implying that HSV amplicon injection provided a strong signal for T cell activation. Combining adoptive transfer of naïve T cells in the lymphopenic setting with local T cell stimulation may facilitate expansion and activation of anti-tumor T cell populations in vivo, resulting in enhanced anti-tumor responses without the need to resort to prolonged in vitro T cell culture and/or manipulation.

Radiaton pneumonitis with the combination of irinotecan and carboplatin concomitantly with radiation for stage III non-small cell lung cancer

7582 Background: We evaluated the efficacy and safety of carboplatin (C) and irinotecan (I) concomitantly with radiation therapy followed by docetaxel (D) in patients (pts) with stages IIIA/B NSCLC. Methods: We enrolled 32 pts. The daily dose of radiation was 1.8 Gy, 5 days a week for 5 weeks, 25 fractions, (45 Gy) to the primary tumor and mediastinum (primary planning target volume: PPTV. After 45 Gy, the primary tumor and involved nodal metastasis (secondary planning target volume: SPTV) was boosted at 2 Gy per day to 18 Gy in 9 fractions. The total dose given was 63 Gy in 35 fractions over 7 weeks. C was given with an AUC=2 and I was given at 30mg/m2 both of them weekly for 7 weeks. D was given at 75mg/m2 as consolidation chemotherapy for 3 cycles. Results: Median age was 55 years (range: 42–78), most pts were females (74%), 57% were Caucasian and 57% were Hispanic. The most common histologies were poorly differentiated and squamous cell carcinomas. Half of the pts were stage IIIB. The overall response...

HSV amplicon mediated augmentation of antitumor effects induced by tumor-specific T cells in the lymphopenic host

2559 Background: Adoptive transfer of T cells has been used in combination with chemotherapy or radiation in order to decrease tumor growth. The specific role of lymphopenia on adoptively transferred T cells in the setting of established tumors is not known. We studied the adoptive transfer of transgenic ovalbumin-specific OT-I T cells into irradiated immunocompetent mice or RAG-1 mice bearing LLC tumor expressing ovalbumin (LLC-ova). In addition, we evaluated the antitumor effects induced by intratumoral injection of HSV amplicons encoding beta-galactosidase (HSV-LacZ), or the combination of HSV-SLC and HSV-CD40L (HSV-S/C). METHODS C57BL/6 mice were injected with LLC-ova cells in the right flank and treated with 550 cGy when the tumor size reached approximately 5mm in diameter. Tumor implants were protected from direct irradiation by a 5 half-value layer lead shield. One day after irradiation, mice were injected intratumorally with PBS, HSV-LacZ, or HSV-S/C every 2 days times 4. RAG-1 mice were injected in both flanks with LLC-ova and right-sided tumors received intratumoral injection of PBS or HSV-LacZ. OT-I T cells were adoptively transferred one day after the first intratumoral injection in both experiments. RESULTS On day 21, mean tumor size for mice treated with radiation, OT-I T cells, and HSV-LacZ was significantly smaller than for mice treated with radiation, OT-I T cells, and PBS (40 mm2 vs 190 mm2 respectively, p < 0.01). Injection of HSV-S/C did not result in additional improvement compared to HSV-LacZ (77 mm2 versus 40 mm2, p = 0.51). Tumor response in mice treated with HSV-LacZ required the presence of OT-I T cells (178 mm2 vs 40 mm2, p < 0.01). In the RAG-1 model, bilateral tumor responses were observed in mice injected with HSV-LacZ. CONCLUSIONS In the lymphopenic environment, adoptively transferred OT-I T cells require an additional stimulus for optimal antitumor effect. Contralateral tumor response in the RAG-1 experiment indicates a systemic effect. These results suggest a major role for the innate immune system in activating adoptively transferred T cells. Supported by ASCO 2003 YIA. No significant financial relationships to disclose.