Randomized controlled phase IIa clinical trial of safety, pharmacokinetics and pharmacodynamics of tenofovir and tenofovir plus levonorgestrel releasing intravaginal rings used by women in Kenya.

Introduction: Globally, many young women face the overlapping burden of HIV infection and unintended pregnancy. Protection against both may benefit from safe and effective multipurpose prevention technologies. Methods: Healthy women ages 18-34 years, not pregnant, seronegative for HIV and hepatitis B surface antigen, not using hormonal contraception, and at low risk for HIV were randomized 2:2:1 to continuous use of a tenofovir/levonorgestrel (TFV/LNG), TFV, or placebo intravaginal ring (IVR). In addition to assessing genital and systemic safety, we determined TFV concentrations in plasma and cervicovaginal fluid (CVF) and LNG levels in serum using tandem liquid chromatography-mass spectrometry. We further evaluated TFV pharmacodynamics (PD) through ex vivo CVF activity against both human immunodeficiency virus (HIV)-1 and herpes simplex virus (HSV)-2, and LNG PD using cervical mucus quality markers and serum progesterone for ovulation inhibition. Results: Among 312 women screened, 27 were randomized to use one of the following IVRs: TFV/LNG (n = 11); TFV-only (n = 11); or placebo (n = 5). Most screening failures were due to vaginal infections. The median days of IVR use was 68 [interquartile range (IQR), 36-90]. Adverse events (AEs) were distributed similarly among the three arms. There were two non-product related AEs graded >2. No visible genital lesions were observed. Steady state geometric mean amount (ssGMA) of vaginal TFV was comparable in the TFV/LNG and TFV IVR groups, 43,988 ng/swab (95% CI, 31,232, 61,954) and 30337 ng/swab (95% CI, 18,152, 50,702), respectively. Plasma TFV steady state geometric mean concentration (ssGMC) was <10 ng/ml for both TFV IVRs. In vitro, CVF anti-HIV-1 activity showed increased HIV inhibition over baseline following TFV-eluting IVR use, from a median of 7.1% to 84.4% in TFV/LNG, 15.0% to 89.5% in TFV-only, and -27.1% to -20.1% in placebo participants. Similarly, anti-HSV-2 activity in CVF increased >50 fold after use of TFV-containing IVRs. LNG serum ssGMC was 241 pg/ml (95% CI 185, 314) with rapid rise after TFV/LNG IVR insertion and decline 24-hours post-removal (586 pg/ml [95% CI 473, 726] and 87 pg/ml [95% CI 64, 119], respectively). Conclusion: TFV/LNG and TFV-only IVRs were safe and well tolerated among Kenyan women. Pharmacokinetics and markers of protection against HIV-1, HSV-2, and unintended pregnancy suggest the potential for clinical efficacy of the multipurpose TFV/LNG IVR. Clinical Trial Registration: NCT03762382 [https://clinicaltrials.gov/ct2/show/NCT03762382].

A phase I study to assess safety, pharmacokinetics, and pharmacodynamics of a vaginal insert containing tenofovir alafenamide and elvitegravir.

Background: New multi-purpose prevention technology (MPT) products are needed to prevent human immunodeficiency virus (HIV) and herpes simplex virus type 2 (HSV2). In this study, we evaluated a fast-dissolve insert that may be used vaginally or rectally for prevention of infection. Objective: To describe the safety, acceptability, multi-compartment pharmacokinetics (PK), and in vitro modeled pharmacodynamics (PD) after a single vaginal dose of an insert containing tenofovir alafenamide (TAF) and elvitegravir (EVG) in healthy women. Methods: This was a Phase I, open-label, study. Women (n=16) applied one TAF (20mg)/EVG (16mg) vaginal insert and were randomized (1:1) to sample collection time groups for up to 7 days post dosing. Safety was assessed by treatment-emergent adverse events (TEAEs). EVG, TAF and tenofovir (TFV) concentrations were measured in plasma, vaginal fluid and tissue, and TFV-diphosphate (TFV-DP) concentration in vaginal tissue. PD was modeled in vitro by quantifying the change in inhibitory activity of vaginal fluid and vaginal tissue against HIV and HSV2 from baseline to after treatment. Acceptability data was collected by a quantitative survey at baseline and post treatment. Results: The TAF/EVG insert was safe, with all TEAEs graded as mild, and acceptable to participants. Systemic plasma exposure was low, consistent with topical delivery, while high mucosal levels were detected, with median TFV vaginal fluid concentrations exceeding 200,000 ng/mL and 1,000 ng/mL for up to 24 hours and 7 days post dosing, respectively. All participants had vaginal tissue EVG concentrations of > 1 ng/mg at 4 and 24 hours post dosing. The majority had tissue TFV-DP concentrations exceeding 1000 fmol/mg by 24 - 72 hours post dosing. Vaginal fluid inhibition of HIV-1 and HSV-2 in vitro significantly increased from baseline and was similarly high at 4 and 24 hours post dosing. Consistent with high tissue TFV-DP concentrations, p24 HIV antigen production from ectocervical tissues infected ex vivo with HIV-1 significantly decreased from baseline at 4 hours post dosing. HSV-2 production from tissue also decreased post treatment. Conclusions: A single dose of TAF/EVG inserts met PK benchmarks, with PK data supporting an extended window of high mucosal protection. PD modeling supports mucosal protection against both HIV-1 and HSV-2. The inserts were safe and highly acceptable. Clinical trial registration: ClinicalTrials.gov, identifier NCT03762772.

Randomized, placebo controlled phase I trial of the safety, pharmacokinetics, pharmacodynamics and acceptability of a 90 day tenofovir plus levonorgestrel vaginal ring used continuously or cyclically in women: The CONRAD 138 study.

Multipurpose prevention technologies (MPTs), which prevent sexually transmitted infection(s) and unintended pregnancy, are highly desirable to women. In this randomized, placebo-controlled, phase I study, women used a placebo or tenofovir (TFV) and levonorgestrel (LNG) intravaginal ring (IVR), either continuously or cyclically (three, 28-day cycles with a 3 day interruption in between each cycle), for 90 days. Sixty-eight women were screened; 47 were randomized to 4 arms: TFV/LNG or placebo IVRs used continuously or cyclically (4:4:1:1). Safety was assessed by adverse events and changes from baseline in mucosal histology and immune mediators. TFV concentrations were evaluated in multiple compartments. LNG concentration was determined in serum. Modeled TFV pharmacodynamic antiviral activity was evaluated in vaginal and rectal fluids and cervicovaginal tissue ex vivo. LNG pharmacodynamics was assessed with cervical mucus quality and anovulation. All IVRs were safe with no serious adverse events nor significant changes in genital tract histology, immune cell density or secreted soluble proteins from baseline. Median vaginal fluid TFV concentrations were >500 ng/mg throughout 90d. TFV-diphosphate tissue concentrations exceeded 1,000 fmol/mg within 72hrs of IVR insertion. Mean serum LNG concentrations exceeded 200 pg/mL within 2h of TFV/LNG use, decreasing quickly after IVR removal. Vaginal fluid of women using TFV-containing IVRs had significantly greater inhibitory activity (87-98% versus 10% at baseline; p<0.01) against HIV replication in vitro. There was a >10-fold reduction in HIV p24 antigen production from ectocervical tissues after TFV/LNG exposure. TFV/LNG IVR users had significantly higher rates of anovulation, lower Insler scores and poorer/abnormal cervical mucus sperm penetration. Most TFV/LNG IVR users reported no change in menstrual cycles or fewer days of and/or lighter bleeding. All IVRs were safe. Active rings delivered high TFV concentrations locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. Trial registration: ClinicalTrials.gov #NCT03279120.

Vaginal microbiota and mucosal pharmacokinetics of tenofovir in healthy women using tenofovir and tenofovir/levonorgestrel vaginal rings.

Recent data support that the vaginal microbiota may alter mucosal pharmacokinetics (PK) of topically delivered microbicides. Our team developed an intravaginal ring (IVR) that delivers tenofovir (TFV) (8-10 mg/day) alone or with levonorgestrel (LNG) (20 ug/day). We evaluated the effect of IVRs on the vaginal microbiota, and describe how the vaginal microbiota impacts mucosal PK of TFV. CONRAD A13-128 was a randomized, placebo controlled phase I study. We randomized 51 women to TFV, TFV/LNG or placebo IVR. We assessed the vaginal microbiota by sequencing the V3-V4 regions of 16S rRNA genes prior to IVR insertion and after approximately 15 days of use. We measured the concentration of TFV in the cervicovaginal (CV) aspirate, and TFV and TFV-diphosphate (TFV-DP) in vaginal tissue at the end of IVR use. The change in relative or absolute abundance of vaginal bacterial phylotypes was similar among active and placebo IVR users (all q values >0.13). TFV concentrations in CV aspirate and vaginal tissue, and TFV-DP concentrations in vaginal tissue were not significantly different among users with community state type (CST) 4 versus those with Lactobacillus dominated microbiota (all p values >0.07). The proportions of participants with CV aspirate concentrations of TFV >200,000 ng/mL and those with tissue TFV-DP concentrations >1,000 fmol/mg were similar among women with anaerobe versus Lactobacillus dominated microbiota (p = 0.43, 0.95 respectively). There were no significant correlations between the CV aspirate concentration of TFV and the relative abundances of Gardnerella vaginalis or Prevotella species. Tissue concentrations of TFV-DP did not correlate with any the relative abundances of any species, including Gardnerella vaginalis. In conclusion, active IVRs did not differ from the placebo IVR on the effect on the vaginal microbiota. Local TFV and TFV-DP concentrations were high and similar among IVR users with Lactobacillus dominated microbiota versus CST IV vaginal microbiota. Trial registration: ClinicalTrials.gov NCT02235662.

Use of contraceptive depot medroxyprogesterone acetate is associated with impaired cervicovaginal mucosal integrity.

BACKGROUND: Injectable depot medroxyprogesterone acetate (DMPA) is one of the most popular contraception methods in areas of high HIV seroprevalence. Evidence is accumulating that use of DMPA might be associated with an increased risk of HIV-1 acquisition by women; however, mechanisms of this association are not completely understood. The goal of this study was to gain insight into mechanisms underlying the possible link between use of DMPA and risk of HIV-1 acquisition, exploring transcription profiling of ectocervical tissues. METHODS: Healthy women received either DMPA (n = 31) or combined oral contraceptive (COC), which has not been linked to an increased risk of HIV acquisition (n = 32). We conducted a comparative microarray-based whole-genome transcriptome profiling of human ectocervical tissues before and after 6 weeks of hormonal contraception use. RESULTS: The analysis identified that expression of 235 and 76 genes was significantly altered after DMPA and COC use, respectively. The most striking effect of DMPA, but not COC, was significantly altered expression (mostly downregulation) of many genes strategically involved in the maintenance of mucosal barrier function; the alterations, as indicated by Ingenuity Pathway Analysis (IPA), were most likely due to the DMPA-induced estrogen deficiency. Furthermore, IPA predicted that transcriptome alterations related to ectocervical immune responses were in general compatible with an immunosuppressive effect of DMPA, but, in some women, also with an inflammatory-like response. CONCLUSION: Our results suggest that impairment of cervicovaginal mucosal integrity in response to DMPA administration is an important mechanism contributing to the potential increased risk of HIV-1 acquisition in DMPA users. TRIAL REGISTRATION: ClinicalTrials.gov NCT01421368. FUNDING: This study was supported by the United States Agency for International Development (USAID) under Cooperative Agreement GPO-A-00-08-00005-00.

Differences in Local and Systemic TFV PK Among Premenopausal Versus Postmenopausal Women Exposed to TFV 1% Vaginal Gel.

OBJECTIVE: We describe and compare the local and systemic pharmacokinetics (PK) of tenofovir (TFV) and TFV-diphosphate (TFV-DP) in healthy premenopausal (PRE) and postmenopausal (POST) women using TFV 1% gel and correlate local PK with other mucosal end points. METHODS: PRE (n = 20) and POST (n = 17) women used 2 doses of TFV 1% vaginal gel, separated by 2 hours. Blood and cervicovaginal samples were obtained 3 and 23 hours after the second dose. PRE women used gel in the follicular and luteal phases of the menstrual cycle. POST women used gel at baseline and again after approximately 2 months of treatment with 0.01% vaginal estradiol (E2) cream. RESULTS: Median TFV concentrations in cervicovaginal aspirate (ng/mL) and vaginal tissue (ng/mg) were significantly higher in PRE (4.3E10, 49.8) versus POST women (2.6E10, 2.2). POST women had significantly higher median molecular ratios of TFV-DP to TFV (3.7%) compared with PRE (0.19%). After vaginal E2 treatment, the local and systemic PK end points in POST women were generally similar to PRE women (all P values > 0.05). Importantly, median vaginal tissue TFV-DP concentrations (fmol/mg) among PRE, POST, and POST women after E2 therapy were similar (292.5, 463.3, and 184.6, respectively). Vaginal tissue TFV concentrations were significantly positively correlated with vaginal epithelial thickness, whereas vaginal tissue TFV-DP concentrations were positively correlated with density of vaginal CD4 and CD8 immune cells. CONCLUSIONS: The state of the cervicovaginal mucosa has a significant impact on local and systemic PK of a topically applied microbicide.

Comparison of Mucosal Markers of Human Immunodeficiency Virus Susceptibility in Healthy Premenopausal Versus Postmenopausal Women.

The objective of this study was to characterize cervicovaginal (CV) mucosal factors modulating susceptibility to human immunodeficiency virus (HIV) acquisition in healthy premenopausal (PRE) and postmenopausal (POST) women before and after treatment with estradiol (E2). We compared CV mucosal epithelial histology and immune cells, vaginal microbiota, antimicrobial activity of and soluble mucosal protein concentrations in the CV fluid lavage (CVL), and p24 antigen production after ex vivo infection of ectocervical tissues with HIV-1BaL among PRE women (n = 20) in the follicular and luteal phases of the menstrual cycle and POST women (n = 17) at baseline and after ∼1 month of treatment with 0.01% vaginal E2 cream. Compared to PRE women, we measured higher levels of p24 antigen after ex vivo infection in tissues from POST women. POST women had a significantly thinner vaginal epithelium with decreased tight junction proteins and a higher density of mucosal immune T cells and lower levels of CD1a antigen-presenting cells, antimicrobial peptides, and inflammatory cytokines in the CVL (p values <.05). POST women had higher vaginal pH and lower vaginal Lactobacilli (p values <.05) than PRE women. After vaginal E2 therapy, CV endpoints and ex vivo HIV replication in POST tissues were similar to those observed in PRE tissues. The CV mucosa in POST women is thinned and compromised, with increased HIV-target immune cells and decreased antimicrobial factors, being more susceptible to HIV infection. After POST women receive topical E2 treatment, mucosal endpoints are similar to PRE levels.

Comparison of Follicular and Luteal Phase Mucosal Markers of HIV Susceptibility in Healthy Women.

The purpose of this study was to evaluate differences in vaginal immune cell populations, vaginal tissue gene expression, antimicrobial activity of the cervicovaginal (CV) lavage (CVL), vaginal flora, and p24 antigen production from CV tissues after ex vivo human immunodeficiency virus (HIV) infection between follicular (FOL) and luteal (LUT) phases of the menstrual cycle. CV tissue biopsies, CV secretions, and blood samples were obtained as part of two longitudinal clinical trials of healthy women (CONRAD D11-119 and A12-124 studies). Participants (n = 39) were HIV-seronegative women not using exogenous hormone supplementation, with normal menstrual cycles, who were screened to exclude sexually transmitted and reproductive tract infections. Serum levels of estradiol and progesterone were significantly higher in the LUT versus the FOL phase of the menstrual cycle. Controlling for race, reported contraceptive use/sexual practices, and clinical trial, we found no differences in vaginal tissue immune cell populations and activation status, transcriptomes, inhibition of HIV, herpes simplex virus type 2 and Escherichia coli by the CVL, vaginal pH or Nugent score, or production of p24 antigen after ex vivo infection by HIV-1BaL between CV samples obtained in the FOL phase versus the LUT phase of the menstrual cycle. There were no significant correlations between serum estradiol and progesterone levels and CV endpoints. The hypothesis that the LUT phase of the menstrual cycle represents a more vulnerable stage for mucosal infection with HIV was not supported by data from samples obtained from the lower genital tract (ectocervix and vagina) from these two clinical trials.

Gene Expression Profiling of Human Vaginal Cells In Vitro Discriminates Compounds with Pro-Inflammatory and Mucosa-Altering Properties: Novel Biomarkers for Preclinical Testing of HIV Microbicide Candidates.

BACKGROUND: Inflammation and immune activation of the cervicovaginal mucosa are considered factors that increase susceptibility to HIV infection. Therefore, it is essential to screen candidate anti-HIV microbicides for potential mucosal immunomodulatory/inflammatory effects prior to further clinical development. The goal of this study was to develop an in vitro method for preclinical evaluation of the inflammatory potential of new candidate microbicides using a microarray gene expression profiling strategy. METHODS: To this end, we compared transcriptomes of human vaginal cells (Vk2/E6E7) treated with well-characterized pro-inflammatory (PIC) and non-inflammatory (NIC) compounds. PICs included compounds with different mechanisms of action. Gene expression was analyzed using Affymetrix U133 Plus 2 arrays. Data processing was performed using GeneSpring 11.5 (Agilent Technologies, Santa Clara, CA). RESULTS: Microarraray comparative analysis allowed us to generate a panel of 20 genes that were consistently deregulated by PICs compared to NICs, thus distinguishing between these two groups. Functional analysis mapped 14 of these genes to immune and inflammatory responses. This was confirmed by the fact that PICs induced NFkB pathway activation in Vk2 cells. By testing microbicide candidates previously characterized in clinical trials we demonstrated that the selected PIC-associated genes properly identified compounds with mucosa-altering effects. The discriminatory power of these genes was further demonstrated after culturing vaginal cells with vaginal bacteria. Prevotella bivia, prevalent bacteria in the disturbed microbiota of bacterial vaginosis, induced strong upregulation of seven selected PIC-associated genes, while a commensal Lactobacillus gasseri associated to vaginal health did not cause any changes. CONCLUSIONS: In vitro evaluation of the immunoinflammatory potential of microbicides using the PIC-associated genes defined in this study could help in the initial screening of candidates prior to entering clinical trials. Additional characterization of these genes can provide further insight into the cervicovaginal immunoinflammatory and mucosal-altering processes that facilitate or limit HIV transmission with implications for the design of prevention strategies.

Depot medroxyprogesterone acetate increases immune cell numbers and activation markers in human vaginal mucosal tissues.

The relationship between exogenous contraceptive hormones and permissiveness of the female genital tract to human immunodeficiency virus type 1 (HIV-1) is the subject of renewed debate. To better characterize the effect of depot medroxyprogesterone acetate (DMPA) on HIV-1 cellular targets and epithelial integrity in the vagina, we compared leukocyte populations, markers of activation and proliferation, and the density of intercellular junctional proteins in the vaginal epithelium of women during the follicular and luteal phases of the menstrual cycle and approximately 12 weeks after receiving a DMPA injection. This prospective cohort study involved 15 healthy women. Vaginal biopsies were obtained in the follicular and luteal phases of the menstrual cycle, and approximately 12 weeks following a 150-mg intramuscular injection of DMPA. Leukocyte populations, activation phenotype, and epithelial tight junction and adherens proteins were evaluated by immunohistochemistry. After receiving DMPA, the numbers of CD45, CD3, CD8, CD68, HLA-DR, and CCR5 bearing immune cells were significantly (p<0.05) increased in vaginal tissues, compared to the follicular and/or luteal phases of untreated cycles. There were no significant differences in immune cell populations between the follicular and luteal phases of the control cycle. There were also no statistically significant differences in epithelial thickness and density of epithelial tight junction and adherens proteins among the follicular, luteal, and post-DMPA treatment sampling points. In this pilot study, vaginal immune cell populations were significantly altered by exogenous progesterone, resulting in increased numbers of T cells, macrophages, and HLA-DR- and CCR5-positive cells.

Induction of cyclooxygenase (COX)-2 in human vaginal epithelial cells in response to TLR ligands and TNF-α.

PROBLEM: Mucosal inflammation caused by infections of the female lower genital tract is considered to be an important cofactor for HIV transmission. We hypothesize that COX-2, a key inflammation-related enzyme, is involved in these responses and is upregulated by microbial ligands and pro-inflammatory cytokines. METHOD OF STUDY: Human vaginal epithelial cells (VK-2/E6E7) and ectocervical biopsy tissues were stimulated with TLR ligands and the cytokine TNF-α, used as surrogates of vaginal infections, and assessed for COX-2 expression and activity by microarray, real-time RT-PCR, immunoblotting, immunohistochemistry, and ELISA. RESULTS: TLR agonists and TNF-α induce transcriptional and translational expression of COX-2 in vaginal cells. TLR ligands, MALP2, Pam3CSK4, LTA, and imiquimod induced high epithelial COX-2 expression, while zymosan and poly dI:dC induced very low enzyme expression. Induced mRNA and protein expression correlated with increased COX-2 activity, which led to increased levels of PGE(2) in the cell culture supernatant. These cell-based findings were confirmed in primary cervicovaginal tissue explants. CONCLUSION: Induction of COX-2 expression and activity and the consequent increased levels of prostaglandins are common inflammatory pathways in human cervicovaginal epithelial cells and tissues in response to diverse TLR ligands and pro-inflammatory cytokines. These findings are relevant to the understanding of genital mucosal inflammation, its potential treatment, and its possible relationship with increased tissue susceptibility to HIV-1 infection.

Regulated expression of CCL21 in the prostate tumor microenvironment inhibits tumor growth and metastasis in an orthotopic model of prostate cancer.

Currently there are no curative therapies available for patients with metastatic prostate cancer. Thus, novel therapies are needed to treat this patient population. Immunotherapy represents one promising approach for the elimination of occult metastatic tumors. However, the prostate tumor microenvironment (TME) represents a hostile environment capable of suppressing anti-tumor immunity and effector cell function. In view of this immunosuppressive activity, we engineered murine prostate cancer cells with regulated expression (tet-on) of CCL21. Prostate tumor cells implanted orthotopically produced primary prostate tumors with predictable metastatic disease in draining lymph nodes and distant organs. Expression of CCL21 in the prostate TME enhanced survival, inhibited tumor growth and decreased the frequency of local (draining lymph node) and distant metastasis. Therefore, these studies provide a strong rationale for further evaluation of CCL21 in tumor immunity and its use in cancer immunotherapy.

Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon.

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.

Prostate tumor microenvironment alters immune cells and prevents long-term survival in an orthotopic mouse model following flt3-ligand/CD40-ligand immunotherapy.

A novel orthotopic metastatic model of mouse prostate cancer was developed using MHC-negative TRAMP-C1P3 (transgenic adenocarcinoma of mouse prostate) cells derived by serial passage of the parental TRAMP-C1 line in mouse prostate glands. TRAMP-C1P3 cells grew efficiently in mouse prostate glands and reproducibly metastasized to draining lymph nodes. Using this model, we show that Fms-like tyrosine kinase-3 ligand (flt3-L) dramatically inhibited growth of preexisting orthotopic TRAMP-C1P3 tumors and the development of metastatic disease. Mice remained in remission for several months following termination of flt3-L treatment but eventually relapsed and died of progressive disease. flt3-ligand treatment induced a pronounced mixed inflammatory cell infiltrate that consisted of CD8alpha-CD4- dendritic cells (CD11c+), macrophages, granulocytes (Gr-1+) and to a lesser extent T cells (CD4+ and CD8+). Dendritic cells isolated from TRAMP-C1P3 tumors were phenotypically immature (CD11c+ B7.2-I-A-CD40-), and this phenotype was also predominant in peripheral organs of mice treated with flt3-L alone or in combination with the DC maturation factor, CD40-L. Diminished expression of TCR-beta, CD3-epsilon, and CD3-zeta was also observed on intratumoral T cells, although these signaling proteins were reexpressed following in vitro culture with IL-2. The TCR/CD3 complex remained intact on peripheral T cells except in mice treated with flt3-L where CD3-zeta loss was observed. In contrast to alphabeta-T cells, tumor-infiltrating gammadelta-T cells maintained expression of their antigen receptors but not CD3epsilon. Thus, TRAMP-C1P3 tumors quickly establish a microenvironment that profoundly diminishes expression of molecules critical for normal dendritic cell and T cell function, thus limiting the efficacy of flt3-L and CD40-L immunotherapy. Overall, these data suggest that long-term cures of established MHC-negative tumors may not be achieved until therapeutic interventions are engineered to overcome this immunosuppressive microenvironment.

Orthotopic treatment model of prostate cancer and metastasis in the immunocompetent mouse: efficacy of flt3 ligand immunotherapy.

We established an orthotopic treatment model of prostate cancer to generate reproducible primary and metastatic carcinoma in immunocompetent C57BL/6 mice. Using an in vivo selection scheme of intraprostatic implantation of TRAMP-C1 cells, primary prostate tumors were cultured and recycled three times by intraprostatic injection resulting in the selection and establishment of the recycled cell line TRAMP-C1P3. Prostate tumors were detected approximately 30 days post-implantation with periaortic lymph node metastasis in 19/20 (95%) of mice. Tissue culture amplification, DNA ploidy and PCR amplification of the SV40 transgene were used to detect metastatic TRAMP-C1P3 in lymph node specimens. Tissue culture amplification and DNA ploidy were as sensitive as SV40 transgene amplification by PCR in detection of early metastatic disease in draining lymph nodes. To establish the use of the orthotopic model of prostate cancer for immunotherapy, mice were injected orthotopically with TRAMP-C1P3 cells and 7 days post-implantation treated daily for 28 days with either flt3L or carrier control. Carrier-treated mice had clinically detectable prostate tumors, lymph node metastasis and were moribund at 29-35 days, whereas flt3L therapy markedly suppressed primary TRAMP-C1P3 growth and lymph node metastasis, and prolonged survival. In summary, we have established a reproducible and clinically relevant orthotopic treatment model of prostate cancer in immunocompetent mice with application to a variety of therapeutic strategies. We demonstrate that flt3L treatment suppressed orthotopic prostate tumor growth and lymph node metastasis reinforcing a role for flt3L as an immunotherapeutic strategy for prostate cancer.