IT Injection Research Articles

Effect of ignition timing and hydrogen injection phase on HDI engine combustion and NOx emissions

ABSTRACT In order to improve the ITE and reduce NOx emissions in direct-injected hydrogen (HDI) engines under at medium and high loads, the effects of IT and hydrogen injection phase on the combustion and NOx emission characteristics of the HDI engine at high rpm and medium load were investigated numerically. The base engine is a four-cylinder turbocharged direct-injected gasoline (GDI) engine, which is modified into a HDI engine with direct-injected hydrogen fuel. The simulation is performed using GT-Power software. The operating condition of HDI engine is set to 5500 rpm and 50% load. Firstly, IT is optimized, and then based on the IT optimization results, the hydrogen injection phase is optimized. The results show that as the IT is advanced, the maximum pressure rise rate (MPRR) shows a gradual increasing trend, while the combustion intensity and the indicated thermal efficiency (ITE) increases firstly and then decreases, and the former reaches a maximum value at 25° CA BTDC, while the latter reaches a maximum value of 40.5% at 18°CA BTDC. In addition, the nitrogen oxide (NOx) emissions decrease as the IT is delayed. Based on 18°CA BTDC of IT, the results indicated that the later the moment of hydrogen injection, the higher the MPRR, resulting in the greater the knock intensity. The ITE is less affected by the hydrogen injection phase. When the hydrogen injection phase is delayed, NOx emissions increase slightly.

Phase 1 study of AM003, a novel individualized immunotherapy, in a basket of advanced solid tumors.

TPS2692 Background: Aummune is developing a unique individualized immunotherapy (‘AM003’), designed to have three activities mediated by three different domains: (1) the ‘Variable Domain, identified de novo for each individual patient, based on its ability to specifically induce cell death on patient’s tumor cells; (2) the ‘General Domain’, designed to engage with immune T cells to cause tumor cell lysis; and (3) a ‘CpG-rich Domain’ that stimulates Antigen Presenting Cells. Aummune’s process begins with sampling the patient’s tumor (via biopsy) and healthy cells (PBMCs). The cancerous cells are grown in a 3D culture and once the cells propagate and reach a critical biomass, they are subjected to Aummune’s proprietary platform for the identification of the Variable Domain. Once a tumoricidal sequence is identified, it is manufactured and the drug product (DP) is assembled at the production facility. Following release, the DP is shipped to the medical site for treatment of the patient. Methods: AM003 is being evaluated as an immune-oncology agent in advanced/metastatic relapsed/refractory solid tumors. A First-In-Human Phase 1, open-label multicenter, dose-escalation study of AM003 is ongoing with the following cohorts: (1) 34mg (2) 68mg and (3) 136 mg of AM003 monotherapy administered intratumorally / local subcutaneously once per week for 4 weeks, followed by 6 doses administered every 2 weeks. Key inclusion criteria include (i) histologically confirmed locally advanced/metastatic solid tumors who received and progressed after, or were intolerant to, at least 1 prior systemic therapy and are not candidates for any therapy known to confer clinical benefit. (ii) lesions that are safely amenable to IT injection. The primary objective is to evaluate the safety and tolerability of AM003. Additional objectives include assessment of preliminary evidence of clinical benefit, pharmacokinetic and pharmacodynamics measurements as well as identification of biomarkers. Endpoints: AEs are assessed according to CTCAE v5, treatment response is determined according to RECIST 1.1 and iRECIST, changes in putative markers are evaluated based on multiple analyses methodologies. 10 patients were included in the dose-escalation part: 1(n=4), 2 (n=3), and 3 (n=3). Cohorts 1, 2 and 3 have been completed without DLTs. The DSMB last reviewed the escalation data in January 2024 and concluded that the trial can continue as planned. Enrollment of 136mg dose expansion is ongoing and completion of the trial will be mid 2024. Clinical trial information: MOH_2022-03-29_010690 .

Updated results from an ongoing phase 1/2a study of T3011, an oncolytic HSV expressing IL-12 and PD-1 antibody, administered via IT injection as monotherapy or combined with pembrolizumab in advanced solid tumors.

9535 Background: T3011 is a recombinant HSV-1 oncolytic virus expressing both IL-12 and anti-human PD-1 antibody. Upon injection, locally produced IL-12 induces IFN-γ production, enhances the oncolytic activity of NK cells and cytotoxic T lymphocytes, promotes anti-angiogenesis and inhibits tumor growth. PD-1 antibodies act as immune checkpoint inhibitors to augment tumor-killing activity of T-cells. Here we report the updated results from an open-label, dose escalation and expansion study of T3011 administered via intratumoral injection as monotherapy or in combination with intravenous pembrolizumab. Methods: Pts with pathologically confirmed recurrent or metastatic malignancy after failure of SOC were enrolled and received T3011 monotherapy (1*106-5*107 PFU/mL Q2W) or in combination with pembrolizumab (Lead-in T3011 1*106 PFU/mL Q2W monotherapy for 2 cycles followed by combination therapy Q3W). Pts with documented progression on T3011 monotherapy may cross over to receive combination therapy at the discretion of the investigator. The primary endpoints were safety and tolerability. Key secondary endpoints included confirmed ORR, DCR, DOR, PFS by investigator per RECIST1.1; OS; PD and PK. Results: As of 17 Jan 2023, 29 pts received T3011 alone or in combination with pembrolizumab, the median follow-up was 14.1 (1.2-27.6) months. No DLTs were reported. Any treatment-related adverse events (TRAEs) occurred in 75.9% (22/29) of pts (≥G3 in 10.3%). Treatment-related SAEs occurred in 3.4% (1/29). Most frequent TRAEs (≥10%) were pyrexia (27.6%), fatigue (20.7%) and chills, injection site pain, arthralgia, nausea, headache (10.3% each). No additional safety signals were observed with combination therapy. 12 advanced melanoma pts failing prior PD-1 or PD-1/CTLA-4 combination treatment received 5*107 PFU/mL of T3011 monotherapy. The confirmed ORR and DCR were 25.0% (3/12) and 33.3% (4/12), respectively. 12-month PFS rate was 36.4%. 6 of these pts were re-challenged with immunotherapy with T3011 (5*107 PFU/mL) combined with pembrolizumab after progressing on T3011 monotherapy, 1 pt achieved PR after 4 months lasting > 8 months at data cutoff. 15 pts had tumor tissues for PD analysis. Increased CD8+ cells were observed in 46.7% (7/15) of all pts and 50% (5/10) of melanoma pts. It appeared to be more pronounced in pts with PR and SD, 66.7% (6/9) in all pts and 80% (4/5) in melanoma pts, respectively. Notably, the increment of CT8+ cells in 2 melanoma pts with PR was over 15 folds. Conclusions: Both T3011 IT monotherapy and combination therapy with pembrolizumab were safe and tolerable. The efficacy of T3011 in immune-resistant melanoma was encouraging. Our data suggest T3011 may modify the tumor microenvironment and overcome immune resistance. Clinical trial information: NCT04370587 .

Intratumoral MPL Induces Th1 Polarization and Augments In Situ Vaccination Generated by Radiation and Checkpoint Blockade

<h3>Purpose/Objective(s)</h3> Radiation therapy (RT) has been demonstrated to generate an in situ vaccination effect in murine models and in cancer patients, however this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the in situ vaccination regimen consisting of combination RT and ICI. <h3>Materials/Methods</h3> We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Mice bearing B78 or Myc-CaP tumors were randomized to receive RT (12 Gy), RT + anti-CTLA4 (C4; 100 µg IP injection days 3, 6, 9 after RT), MPL (20 µg IT injection days 5, 7, 9 after RT), RT+C4+MPL, or PBS control. To model metastatic disease, 250,000 B16 melanoma cells (parental to B78) were injected via tail vein immediately following RT. To evaluate the effect of MPL on the irradiated tumor microenvironment, a separate cohort of B78 tumor bearing mice from each treatment group were sacrificed 15 days following RT, and tumor together with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling and serum was collected for analysis of anti-tumor antibody populations. <h3>Results</h3> Combination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared to RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment towards M1 macrophage and Th1 CD4 T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1 and M1 associated proinflammatory cytokines. T cells co-cultured with MPL-stimulated macrophages significantly increased expression of the activation marker CD69 in CD8 T cell populations and the Th1 polarization marker CXCR3 in CD4 T cell populations. MPL treatment significantly increased production of Th1-associated, IgG2c anti-tumor antibodies which were required for and predictive of anti-tumor response to RT and MPL and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic anti-tumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4⁺ but not CD8⁺ T cells. <h3>Conclusion</h3> We report the potential for MPL to augment the in situ vaccination effect of combination RT+C4 through FcγR, macrophage, and CD4 dependent mechanisms. To our knowledge this is the first report describing generation of a CD8⁺ T cell-independent, Th1 polarized, systemic anti-tumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

Local TLR4 stimulation augments in situ vaccination induced via local radiation and anti-CTLA-4 checkpoint blockade through induction of CD8 T-cell independent Th1 polarization

BackgroundRadiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced...

Piglets' acute responses to local anesthetic injection and surgical castration: Effects of the injection method and interval between injection and castration.

Although applied in some countries, efficacy of local anesthetics based on procaine to mitigate acute responses to piglet castration remains questioned. This paper presents results from a factorial study examining the effects of two methods of injection of a procaine-based drug (intra-funicular, IF, vs. intra-testicular, IT), and four intervals between drug injection and castration (2.5, 5, 10, and 30 min) on acute responses of 3–4 day old piglets. The study involved 597 male piglets, and 13 treatments: surgical castration without anesthesia (CC), local anesthesia followed by castration involving all combinations of injection method and interval, and sham handling separated by the same four intervals (SH). Responses of piglets to drug injection, castration and sham handling were evaluated based on quantification of intra-procedural vocalizations and leg movements, as well as saliva cortisol concentration in samples taken before and after castration. No differences were found between IF and the simpler IT injection method. Intervals of 2.5 or 30 min led to stronger piglet responses than the other intervals. Overall, treatments involving anesthesia led to significantly stronger responses than sham handling, during both injection and castration. All treatments, even sham handling, led to a significant increase in saliva cortisol, with no differences between anesthesia treatments and controls. Based on these results, castration 5–10 min after intra-testicular injection of procaine seems to be preferable as compared to the other treatments tested. However, piglets still showed measurable signs of pain and stress during both injection and castration, while handling alone (including the use of a castration bench) triggered a noticeable stress response. In light of these findings, the overall benefit of the procedure in terms of piglet welfare remains arguable.

Intratumoral administration of CD1c (BDCA-1)+ and CD141 (BDCA-3)+ myeloid dendritic cells in combination with talimogene laherparepvec in immune checkpoint blockade refractory advanced melanoma patients: a phase I clinical trial

BackgroundIntratumoral (IT) myeloid dendritic cells (myDCs) play a pivotal role in initiating antitumor immune responses and relicensing of anti-tumor cytotoxic T lymphocytes within the tumor microenvironment. Talimogene laherparepvec (T-VEC) induces...

Abstract CT183: First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors

Abstract Background: IL-12 is a key mediator of antitumor immune response. In preclinical models, IT IL-12 mRNA led to IFNγ release and CD8+ T cell-dependent tumor regression and potentiated PD-L1 blockade. MEDI1191, a lipid nanoparticle-formulated mRNA encoding IL-12 delivered by IT injection, drives IL-12 production and enhances antitumor immune response with improved tolerability. We hypothesized that combining MEDI1191 with PD-L1 blockade would augment antitumor immunity in vivo. Here we report updated results from the dose-escalation phase of the first-in-human study of IT MEDI1191 and IV durvalumab (D; anti-PD-L1) for advanced/metastatic solid tumors (NCT03946800). Methods: In this multicenter, open-label study, MEDI1191 was dosed sequentially (seq, Part 1A) or concurrently (conc, Part 1B) with D. In Part 1A, MEDI1191 was given IT on Days 1 and 22 followed by D 1500 mg on day 43 and then Q4W IV. In Part 1B, MEDI1191 was given IT on Days 1, 29, 57 and then Q8W, along with D on Day 1 and then Q4W. Treatment continued until progression or unacceptable toxicity for up to 2 years. Eligible adult pts had any solid tumor with cutaneous or subcutaneous lesions suitable for IT injection and progression on standard therapy for recurrent/metastatic disease. Primary objectives were safety and tolerability and determination of maximum tolerated dose (MTD); secondary objectives included preliminary antitumor activity by RECIST v1.1. Results: Starting in May 2019, 31 pts received seq MEDI1191 with D (Part 1A cohorts 0.1-12 μg; n=20) or conc MEDI1191 with D (Part 1B cohorts 1.0-3.0 μg; n=11); 23 pts had received prior anti-PD-1/PD-L1 therapy. Most common tumor types were melanoma, n=8; head and neck cancer, n=4; and breast cancer, n=4. At the data cutoff of Dec 7, 2021, there were no dose-limiting toxicities and no MTD was identified. One pt (3.2%) had a Gr ≥3 MEDI1191-related AE (Gr 3 pyrexia, resolved within 24 hr) and 1 (3.2%) had a MEDI1191-related serious AE (SAE; Gr 2 confusion). Two pts had Gr 3 D-related AEs (6.5%, pyrexia also related to MEDI1191 and pruritus; each n=1); none had a D-related SAE. There were no Gr 4 related AEs. 3 pts had partial responses (PR): 1 with head and neck cancer (unconfirmed, off study) and 2 with anti-PD-1 resistant melanoma (1 confirmed, &amp;gt;12 months on treatment; 1 unconfirmed, off study); 10 pts had stable disease (including the 2 unconfirmed PRs). Among pts with available biomarker data (up to 3 μg in Part 1A and 1 μg in Part 1B), MEDI1191 increased serum IL-12 in 15/17 pts, increased CD8+ T cell tumor infiltration by &amp;gt;2-fold in 8/14 pts and increased tumor PD-L1 expression in 6/14 pts. Conclusions: IT MEDI1191 plus systemic anti-PD-L1 was safe and feasible. Preliminary antitumor efficacy and pharmacodynamics, including tumor CD8+ T cell recruitment, were consistent with expected mechanism of action. Pts with injectable deep visceral and superficial lesions are being recruited. Citation Format: Benedito A. Carneiro, Dmitriy Zamarin, Thomas Marron, Inderjit Mehmi, Sandip P. Patel, Vivek Subbiah, Anthony El-Khoueiry, David Grand, Kirema Garcia-Reyes, Sanjay Goel, Phillip Martin, Jixin Wang, Yuling Wu, Steven Eck, Benjamin Ridgway, Nairouz Elgeioushi, Jim Eyles, Nicholas Durham, Analia Azaro, Omid Hamid. First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT183.

Combination of radiomic and biomarker signatures as exploratory objective in a phase II trial with intratumoral BO-112 plus pembrolizumab for advanced melanoma.

TPS9586 Background: Intratumoral immunotherapies are gaining interest in oncology, particularly in melanoma. These therapies, however, have faced some issues. For instance, standard response criteria do not accurately describe tumor burden, and responses may differ for injected/non injected lesions. Besides, target lesions may become non evaluable. Biomarkers provide interesting information for these therapies. In addition, some radiomic signatures have been associated with CD-8 infiltration. BO-112 is a double stranded synthetic RNA formulated with polyethyleneimine (PEI) that mimics a viral infection, mobilizing the immune system and changing tumor microenvironment. Clinical data are available from a first-in-human study, which showed ORR of 11% and DCR of 46% in patients who had developed progressive disease on immunotherapy. In patients with melanoma, this ORR was 20%. A phase 2 clinical study of BO-112 with pembrolizumab in patients with liver metastases from digestive tumors is ongoing. Both studies brought up data regarding how some biomarkers are increased after a single dose of BO-112 and correlated with responses. In this phase II study in patients with pretreated melanoma (NCT04570332), we will prospectively assess CD-8 and PD-L1 by immunohistochemistry, which will be compared with multi-parametric radiologic findings and correlated with clinical benefit. In addition, retrospective DNA sequencing will be performed. This kind of exploratory analysis in intratumoral immunotherapies might be key to identify predictive and prognostic factors. Methods: Phase 2, single arm, open label study of BO-112 with pembrolizumab in patients with advanced melanoma. BO-112 is administered once weekly (QW) in 1 to 8 tumor lesions, total dose 1-2 mg (depending on the number of injected lesions), for the first 7 weeks and then once every three weeks (Q3W); pembrolizumab 200 mg will be administered Q3W. Key eligibility criteria: advanced cutaneous or mucosal melanoma; patients must have progressed on or after treatment with an antiPD-1/L1 mAb; at least one measurable lesion amenable for weekly IT injection. Primary efficacy variable is ORR by RECIST 1.1, assessed by independent central radiologist (QUIBIM Precision platform). A 1-sided alpha of 4.19% and power of 81.8% are used. If less than 8 patients out of 40 have ORR, the study will not meet the statistical bar. Secondary endpoints include clinical activity by RECIST1.1 and iRECIST, overall survival, safety and PKs. Exploratory objectives include itRECIST and evaluation of CD-8 and PD-L1 expression by immunohistochemistry (Pangaea laboratory), which will be correlated with radiomic signatures (first order and second order) from standard-of-care computed tomography (CT) images. Enrollment is open and 1 of planned 40 patients has been enrolled. Nineteen sites are planned to participate. Clinical trial information: NCT04570332.

Comparison of the effect Of three treatment interventions for the control of Meniere\u2019s disease: a randomized control trial

BackgroundIntratympanic (IT) dexamethasone, with or without ventilation tube, is widely used to control vertigo in Meniere’s disease (MD) patients. Some authors believe that the tube on its own has a role in the control of MD symptoms. The authors’ goal was to compare the effects of them on multiple sets of patients. A three arms randomized control trial was conducted on 60 patients ( both males and females; age range, 30–60 years; mean age, 47 years) who were diagnosed with MD according to the 1995 American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) Foundation guidelines. The study was conducted at the Otolaryngology Department between May 2016 and May 2018. They were divided into three groups, with 20 patients in each group. The first group received direct IT dexamethasone, the second group received IT dexamethasone through a grommet tube, and the third received saline through grommet.ResultsNo treatment modality showed statistically significant improvement regarding both hearing and vertigo control at follow-up periods. However, comparison of patients with disease duration of 1 year and patients with duration of 5 years in the total sample of patients showed that patients with 1-year duration showed statistically significant higher percentage of improvement in pure tone audiometry (PTA) and vertigo control at all follow-up periods.ConclusionsNo golden standard in MD treatment; however, initial improvement may be explained as a result of the anti-inflammatory effect of steroids and/or the pressure release due to tube insertion; yet, the progressive course of the disease may diminish that improvement over time. Thus, early intervention either by direct IT injection or through grommet tube or by grommet placement alone can affect treatment outcomes.

Middle Ear Administration of a Particulate Chitosan Gel in an in vivo Model of Cisplatin Ototoxicity.

BackgroundMiddle ear (intratympanic, IT) administration is a promising therapeutic method as it offers the possibility of achieving high inner ear drug concentrations with low systemic levels, thus minimizing the risk of systemic side effects and drug-drug interactions. Premature elimination through the Eustachian tube may be reduced by stabilizing drug solutions with a hydrogel, but this raises the secondary issue of conductive hearing loss.AimThis study aimed to investigate the properties of a chitosan-based particulate hydrogel formulation when used as a drug carrier for IT administration in an in vivo model of ototoxicity.Materials and MethodsTwo particulate chitosan-based IT delivery systems, Thio-25 and Thio-40, were investigated in albino guinea pigs (n = 94). Both contained the hearing protecting drug candidate sodium thiosulfate with different concentrations of chitosan gel particles (25% vs. 40%). The safety of the two systems was explored in vivo. The most promising system was then tested in guinea pigs subjected to a single intravenous injection with the anticancer drug cisplatin (8 mg/kg b.w.), which has ototoxic side effects. Hearing status was evaluated with acoustically evoked frequency-specific auditory brainstem response (ABR) and hair cell counting. Finally, in vivo magnetic resonance imaging was used to study the distribution and elimination of the chitosan-based system from the middle ear cavity in comparison to a hyaluronan-based system.ResultsBoth chitosan-based IT delivery systems caused ABR threshold elevations (p < 0.05) that remained after 10 days (p < 0.05) without evidence of hair cell loss, although the elevation induced by Thio-25 was significantly lower than for Thio-40 (p < 0.05). Thio-25 significantly reduced cisplatin-induced ABR threshold elevations (p < 0.05) and outer hair cell loss (p < 0.05). IT injection of the chitosan- and hyaluronan-based systems filled up most of the middle ear space. There were no significant differences between the systems in terms of distribution and elimination.ConclusionParticulate chitosan is a promising drug carrier for IT administration. Future studies should assess whether the physical properties of this technique allow for a smaller injection volume that would reduce conductive hearing loss.

Role of spinal cord TRAF6/NF-κB signaling pathway in development of diabetic neuropathic pain in rats

Objective To evaluate the role of spinal cord tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa B(NF-κB)signaling pathway in the development of diabetic neuropathic pain(DNP)in rats. Methods Clean-grade healthy adult male Sprague-Dawley rats, aged 2 months, weighing 180-230 g, in which IT catheters were implanted, were used in this study.Streptozotocin 60 mg/kg was intraperitoneally injected after IT catheterization to establish the model of DNP.Twelve DNP rats were divided into 2 groups (n=6 each) by a random number table method: DNP group and DNP plus TRAF6 inhibitor group (group DTR). Another 6 age-matched Sprague-Dawley rats were used as normal control group(group NC). The rats in group DC and group DTR received IT injection of dimethyl sulfoxide 10 μl and TRAF6 inhibition 10 μg, respectively, once a day for 7 consecutive days starting from day 21 after establishing the model.The mechanical paw withdrawal threshold (MWT) was determined before establishing the model(T1), on 7, 14 and 21 days after establishing the model(T2-4), and on 1, 4 and 7 days after IT injection (T5-7). The rats were sacrificed after the last MWT measurement, and the L3-5 segments of the spinal cord were removed for determination of the expression of TRAF6 and NF-κB p65 by Western blot. Results Compared with group NC, the MWT at T3-7 in group DC and at T3-6 in group DTR was significantly decreased, and the expression of spinal TRAF6 and NF-κB p65 was up-regulated in DC and DTR groups (P<0.05). Compared with group DC, the MWT was significantly increased at T6-7, and the expression of spinal TRAF6 and NF-κB p65 was down-regulated in group DTR (P<0.05). Conclusion Spinal cord TRAF6/NF-κB signaling pathway is involved in the development of DNP in rats. Key words: Spinal cord; Tumor necrosis factor receptor-associated peptides and proteins; NF-kappa B; Diabetes mellitus; Neuralgia

Effect of intrathecal botulinum toxin A injection on sheep's cystomanometric parameters: A comparative study

Detrusor overactivity is a public health issue in patients with central neurological disorders, in particular spinal cord injury. Sheep are valuable animal models because the bladder and urethral pressure measures are similar to humans’. Intrathecal botulinum toxin A (IT BoNT-A) injections have already proved efficacy on radicular pain, without motor nor postural adverse effect in rat and mice. But the possible effects on the urinary tract functioning are not yet known. To evaluate the effect of an IT BoNT-A injection on sheep urodynamic parameters and to evaluate the potentially motor adverse effects. Five 2-year old sheep were used in this experiment. After a familiarizing procedure, a first urodynamic exam was done at T0. A unique IT injection of BoNT-A (100 Units-Xeomin ® ) was done 3 weeks later. Cystomanometric parameters were again recorded 5 (T1) and 12 weeks (T2) after the IT injection. A control group of 7 sheep with no IT BoNT-A injection were used to compare the results. Maximum cystometric capacity (MCC) and post-void residual urine (PVRU) increased significantly in the group receiving IT BoNT-A at T1 (MCC at T0 = 35.0 mL ± 16.4; MCC at T1 = 105.6 mL ± 16.10; P < 0.05) (PVRU at T0 = 2.8 mL ± 4.1; PVRU at T1 = 93.5 ± 12.6; P < 0.01). Bladder contractions appeared during the filling phase between T1 and T2 in the IT BoNT-A group. No motor problems induced by IT BoNT-A injection have been recorded. IT BoNT-A injections influence the urinary tract functioning, and particularly increase the bladder capacity at T1, but induce small bladder contractions at T1 and T2. If it is confirmed by larger studies, this result should be taken into consideration for further use of IT BoNT-A in patients with detrusor overactivity after a spinal cord injury.

Phase I/II dose escalation and expansion cohort safety and efficacy study of image guided intratumoral CD40 agonistic monoclonal antibody APX005M in combination with systemic pembrolizumab for treatment naive metastatic melanoma.

TPS3133 Background: Checkpoint blockade has become a major modality in the treatment of metastatic melanoma (MM). However, durable remission rates remain low. CD40 activation on antigen presenting cells (APCs) initiates their ability to prime and activate CD8+ T-cells through upregulation of co-stimulatory molecules (ie. CD80, CD86) as well as expression of effector cytokines. Furthermore, CD40 activation cause macrophages to develop a tumoricidal phenotype and increase MHC I expression on tumor cells. Direct IT immune modulation utilizes the tumor as a “vaccine site” to generate a tumor specific immune response. We hypothesize that IT injection of a CD40 Agonist (A), will “immunize” patients (Pts) against melanoma neoantigens through “licensing” of APCs for tumor specific T-cell priming and activation. In mouse models, we have shown that IT administration of the recombinant adenovirus encoding CD40L induces CD8+ T-cell-mediated activity against B16 melanoma and, importantly, also augmented the activity of anti-PD-1. Methods: This phase I/II trial (NCT02706353) evaluates the safety, efficacy, and immunological impact of IT administration of APX005M (CD40 A mAb) in combination with pembrolizumab in pts with MM. The phase I escalation is an accelerated 3+3 design. Pts will receive IT APX005M every 3 weeks for a total of 4 doses. Image guidance will allow for injection of visceral, nodal, and soft tissue metastases. The single-arm phase 2 expansion will evaluate the overall response rate (ORR) 12 weeks after treatment initiation. Key inclusion criteria: confirmed, measurable, metastatic cutaneous or mucosal melanoma; and at least 2 injectable lesions. Key exclusion criteria: prior immunotherapy, uveal melanoma, or active autoimmune disease. 26 pts will have 75% power to detect an improvement from a null ORR of 33% to 55%, using a one group chi-square test and assuming a one-sided α–level of 5%. Immune analysis will be performed on pre and on-treatment tumor/liquid biopsies both in injected and non-injected tumors. Dose level 3 of escalation phase has completed with 5 pts enrolled. Clinical trial information: NCT02706353.

Docetaxel-titanate nanotubes enhance radiosensitivity in an androgen-independent prostate cancer model.

Around 40% of high-risk prostate cancer patients who undergo radiotherapy (RT) will experience biochemical failure. Chemotherapy, such as docetaxel (DTX), can enhance the efficacy of RT. Multidrug resistance mechanisms often limit drug efficacy by decreasing intracellular concentrations of drugs in tumor cells. It is, therefore, of interest to develop nanocarriers of DTX to maintain the drug inside cancer cells and thus improve treatment efficacy. The purpose of this study was to investigate the use of titanate nanotubes (TiONts) to develop a TiONts-DTX nanocarrier and to evaluate its radiosensitizing in vivo efficacy in a prostate cancer model. In vitro cytotoxic activity of TiONts-DTX was evaluated using an MTS assay. The biodistribution of TiONts-DTX was analyzed in vivo by single-photon emission computed tomography. The benefit of TiONts-DTX associated with RT was evaluated in vivo. Eight groups with seven mice in each were used to evaluate the efficacy of the nanohybrid combined with RT: control with buffer IT injection ± RT, free DXL ± RT, TiONts ± RT and TiONts-DXL ± RT. Mouse behavior, health status and tumor volume were monitored twice a week until the tumor volume reached a maximum of 2,000 mm3. More than 70% of nanohybrids were localized inside the tumor 96 h after administration. Tumor growth was significantly slowed by TiONts-DTX associated with RT, compared with free DTX in the same conditions (P=0.013). These results suggest that TiONts-DTX improved RT efficacy and might enhance local control in high-risk localized prostate cancer.

TNF-α increases in the CSF of children with acute lymphoblastic leukemia before CNS relapse

There is scarce information regarding the concentration of cytokines in cerebrospinal fluid (CSF) of children with acute lymphoblastic leukemia (ALL) and their clinical association with CNS status. A prospective analysis of 40 patients <18years with newly diagnosed ALL was performed. Human cytokine magnetic bead panel assay values of IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1, TNF-α in CSF at diagnosis, end of induction to remission, and 6months after diagnosis were determined. IL-6 and MCP-1 values showed a significant increment at the end of induction. From the whole group 4 (10.0%), patients relapsed to the CNS at a median of 11.48months. A significantly higher value of TNF-α at third determination in these CNS-relapsed patients was documented, 7.48 vs. 2.86pg/mL in 36 children without relapse (p=0.024). TNF-α concentration increased at a median 5.48months before CNS relapse. By receiver-operating characteristic curve (ROC) analysis, the best cut-off point of TNF-α concentration that better predicted CNS relapse was ≥1.79pg/mL. In conclusion an increase in TNF-α concentration on CSF preceded CNS relapse in children with ALL. An increase in MCP-1 and IL-6 was not associated to CNS relapse and appears to result from an inflammatory response after IT injection of chemotherapy.

Abstract 2322: Intratumoral treatment with a highly interferogenic TLR9 agonist reverts tumor escape from PD-1 blockade

Abstract The induction of tumor specific T cell exhaustion by the PD-1/PDL-1 pathway is an important mechanism of immune escape in cancer. Therapies blocking or reversing T cell exhaustion can produce durable responses in metastatic cancers including melanoma, NSCLC, renal carcinoma and bladder cancer. However, only a small subset of patients experience response upon anti-PD-1/PDL-1 treatment, whereas many others are refractory, showing no response at all. Thus, a key challenge is transforming a non-permissive tumor microenvironment into an immunologically competent one, in order to increase the frequency of patients with durable responses to PD-1/PDL-1 blockade. We have characterized a mouse tumor model with a reproducible dichotomous response to anti-PD-1 treatment analogous to that observed in human cancers. We show that the ability of tumors to be rejected in response to anti-PD-1 treatment is highly correlated with degree of T cell infiltration, expression of Type I IFNs and B cell activation signatures, in whole tumors and in tumor infiltrating leukocytes. We then evaluate whether immunomodulation by intratumoral vaccination with a TLR9 agonist, SD-101, would affect rate of response to anti-PD-1 blockade. SD-101, is a CpG-C class oligonucleotide, optimized to induce both very high levels of IFN-alpha as well as maturation of dendritic cells and B cells. Intratumoral injection of SD-101 in tumor bearing mice undergoing anti-PD-1 therapy leads tumor rejection in 100% of treated mice versus a 20% tumor rejection in mice treated with anti-PD-1 blockade alone. SD-101 alone, although efficacious in reducing tumor growth, did not lead to durable tumor rejection. Immune cell subset depletion studies implicate CD8, but not CD4, T cells in the rejection of tumors treated with anti-PD1 plus SD-101. T cell response generated in one flank by IT injection of SD-101 synergizes with systemic PD-1 blockade to induce rejection of unvaccinated distal tumors. In addition, we show that mice receiving systemic anti-PD-1 plus SD-101 have a higher percentage of CD8+ T cells infiltrating the tumors when compared to anti-PD-1 responders, anti-PD-1 non-responders or SD-101 given alone. Infiltrating CD4 and CD8 T cells in tumors with combination treatment, show a higher proliferative capacity and a higher frequency of cells producing both TNF-a and IFN-g than tumors responding to anti-PD-1 monotherapy. This demonstrates the potential for SD-101 to significantly enhance the frequency of responders to anti-PD-1 therapy by increasing the number and functionality of anti-tumor T cells within the tumor. Citation Format: Shu Wang, Jose Campos, Edwina Naik, Crain Chad, Gallotta Marilena, Coffman Robert L., Cristiana Guiducci. Intratumoral treatment with a highly interferogenic TLR9 agonist reverts tumor escape from PD-1 blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2322.

Assessment of complications due to intratympanic injections.

ObjectiveThe purpose of the study is to report and to analyze the complications following intratympanic injections (ITI) of steroids. The occurrence rate of complications at different ITI sites, four quadrants of eardrum, was also compared.MethodsA retrospective clinical review in a medical center. Each patient received ITI twice in a week for 2–3 consecutive weeks as a salvage therapy for sudden sensorineural hearing loss. Post-injection complications, especially transient dizziness and vertigo, were recorded. Patients with acute or chronic vertigo episodes in 1 month were excluded.ResultsA total of 59 patients with sudden sensorineural hearing loss and a total of 278 times of ITI were performed in 1 year. The post-injection complications included pain, tongue numbness, transient dizziness, vertigo, tinnitus, and a small persistent perforation. There was no significant difference in the occurrence of these complications between the injections sites on the 4 quadrants of the tympanic membrane. However, there was statistical significance in the post-injection vertiginous episode after IT injections to posterior-inferior quadrant (Q3) and posterior-superior quadrant (Q4) compared to anterior-superior quadrant (Q1) and anterior-inferior quadrant (Q2) (P = 0.0113).ConclusionIT injection is recommended to be applied to the Q2 since the Q1 and Q4 injections are more likely to induce the adverse effect of tongue numbness, while the Q3 and Q4 areas are more likely to induce post-injection vertigo.

Abstract IA10: Effective immunotherapy regimens incorporating highly active human STING-activating cyclic dinucleotide derivatives

Abstract Innate immune sensing in the tumor microenvironment is a critical step in promoting tumor infiltrating lymphocytes (TILs) and spontaneous anti-tumor T cell priming. Transcriptional profiling analysis of melanoma patients has revealed that tumors with a T cell-inflamed immunophenotype are characterized by a type I IFN (IFN) transcriptional signature. Studies in mice support the notion that IFN produced by tumor-resident dendritic cells (DCs) plays a critical role in spontaneous T cell priming against tumor antigens, which is dependent upon the host Stimulator of Interferon Genes (STING) pathway. STING mediates host innate defense by responding to cytosolic nucleic acids, either through direct binding of cyclic dinucleotides (CDNs) produced by bacteria, or through binding of a structurally distinct CDN produced by host cyclic GMP-AMP synthetase in response to cytosolic double-stranded DNA. While CDN adjuvants have been explored previously in mice, we sought to develop compounds that activate human STING. We therefore synthesized a panel of cyclic dinucleotides (CDNs) that varied by purine nucleotide base, internucleotide phosphate bridge linkage, or by substitution of non-bridging oxygen atoms in the phosphate bridge with sulfur. We screened and selected from among these compounds based on their capacity to activate all known human STING alleles expressed in stably transformed reporter cell lines, stimulate the activation of human PBMCs, and impact significant antitumor efficacy in several mouse tumor models, without significant local or systemic toxicity. Strikingly, direct IT injection of particular CDN derivative molecules into two-week established flank B16 melanoma, CT26 colon, or 4T1 breast tumors profoundly inhibited tumor growth that was durable and correlated with induction of lasting systemic antigen-specific CD8+ T cell immunity that conferred complete protection against tumor re-challenge, or significantly inhibited the growth of distal untreated tumors. Induction of cytokines, tumor antigen-specific immunity, and antitumor efficacy was entirely STING-dependent. We selected dithio-[Rp,Rp]-c[A(2',5')pA(3',5')p], a synthetic CDN molecule that has significantly higher activity than natural STING ligands, as the lead molecule for continued development. We next tested whether direct activation of STING within the tumor microenvironment would enhance the absocopal effect resulting from irradiating the CDN-treated tumor. Treatment of one tumor in mice bearing established bilateral Panc02 flank tumors, with a suboptimal dose of 10 Gy of radiotherapy (RT) in combination with IT CDN injection resulted in rapid local and systemic induction of inflammatory mediators, and vascular damage that spread through the injected tumor without causing detectable damage to normal tissues. Compared to RT alone, CDN injection resulted in significantly enhanced adaptive-immune mediated control of the contralateral tumor. The synthetic CDN molecule described here was significantly more potent than IT TLR ligands, indicating its high translational potential as an approach to elicit effective unbiased T cell priming against an individual's unique tumor antigen repertoire. Citation Format: Laura Hix Glickman, Leticia Corrales, Sarah M. McWhirter, David B. Kanne, Kelsey E. Sivick, Jason R. Baird, Edward Lemmens, Justin J. Leong, Ken Metchette, Mark Crittenden, Michael Gough, Thomas F. Gajewski, Thomas W. Dubensky, Jr.. Effective immunotherapy regimens incorporating highly active human STING-activating cyclic dinucleotide derivatives. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA10.

Preclinical development of a docetaxel nanocarrier to enhance prostate cancer radiosensitivity.

41 Background: From 30% to 50% of high risk prostate cancer patients who undergo radiation therapy (RT) will have a biochemical failure. Combining chemotherapy, such as Docetaxel (DXL), with RT can enhance its efficiency. Multidrug resistance mechanisms often limit drug efficacy by decreasing tumor cell intracellular concentration of drugs. There is interest to develop nanocarrier of DXL to maintain drug inside cancer cells by improving its efficacy. The purpose of this study was to develop a titanate-nanotube (TiONt)-DXL nanocarrier (nanohybrid) and to evaluate its in vivobiodistribution as well as its radiosensitizing efficacy in association with RT on a hormone-independent prostate cancer model. Methods: DXL molecules were grafted on TiONts using PEG-3000 molecules to generate the nanohybrid. In vitrocytotoxic activity of the nanohybrid was evaluated on PC-3 cell line using MTS assay. After intratumoral injection, biodistribution analysis was performed by SPEC-CT imaging of mice bearing subcutaneous PC-3 human prostate tumors. To evaluate the benefit of nanohybrid and RT association, tumors were irradiated using 3 fractions of 4Gy administrated after the injection of nanohybrids. Nine groups of 7 mice were used to evaluate nanohybrid and RT association efficacy : untreated, control with buffer IT injection, +/- RT, free DXL, +/- RT, TiONt +/- RT and TiONt-DXL +/- RT. Mice behavior, health status and tumor volume were monitored twice a week until tumor growth recovery. Results: Biodistribution kinetics showed that more than 70% of nanohybrids were localized into the tumor 96 hours after injection. Mice receiving nanohybrid-RT exhibited a significant tumor growth delay to reach a volume of 1,000mm3compared to mice receiving free DXL-RT: 73.7 days (median, [58.9-89]) vs 56 days (median, [31.5-74]) (p=0.0127). Conclusions: TiONt-DXL improves RT efficacy compared to free DXL. These results suggest that local control might be enhanced by TiONt-DXL. TiONt-DXL can be injected as a radiosensitizer in men harboring high risk localized prostate cancer with needles during prostate brachytherapy procedure.