Dennis J. Carlo

Characterization of a novel metabolic strategy used by drug-resistant tumor cells

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug‐resistant cells from drug‐sensitive cells. This strategy may serve to protect drug‐resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug‐resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug‐resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.—Harper, M.‐E., Antoniou, A., Villalobos‐Menuey, E., Russo, A., Trauger, R., Vendemelio, George, A. M., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., Newell, M. K. Characterization of a novel metabolic strategy used by drug‐resistant tumor cells. FASEB J. 16, 1550–1557 (2002)

Initial studies on active immunization of HIV-infected subjects using a gp120-depleted HIV-1 Immunogen: long-term follow-up.

In 1987, exploratory clinical studies were initiated to determine whether the development of AIDS in HIV-infected individuals might be delayed or prevented by immunization with an inactivated HIV preparation. Preclinical studies had shown the preparation to be safe and immunogenic. Twenty-three patients with biopsy-confirmed persistent generalized lymphadenopathy (CDC III) and two with asymptomatic HIV infection and CD4 lymphocyte counts between 135 and 769/mm3 were studied, of whom eight (32%) had additional HIV-related symptoms. Over a 3-year period, they received a median of eight open-label inoculations of 100 micrograms of inactivated gp 120-depleted HIV-1 Immunogen in incomplete Freunds adjuvant (IFA). Clinical, general laboratory, immunologic, and virologic parameters were followed for up to 6 years. No serious treatment-related adverse experiences were reported, nor was accelerated HIV disease progression seen. Twelve patients developed a delayed-type hypersensitivity response (HIV-DTH) to the immunogen and nine showed fourfold or greater increases in anti-p24 antibody titers. In the follow-up period, 10 of the 25 patients developed AIDS and one with Kaposis sarcoma (KS) at baseline progressed. Of the 12 patients who became HIV-DTH-responsive, one developed an opportunistic infection (OI), occurring approximately 5 years from study onset, and subsequently died. One additional HIV-DTH responder developed KS. Of the 13 patients who remained HIV-DTH-nonresponsive, nine (69%) progressed to AIDS and seven of these have died. Differences were also observed in terms of HIV-DNA copy number, CD4 percentages, and anti-p24 antibody patterns between the HIV-DTH-responsive and -nonresponsive groups, suggesting a more favorable clinical course in the former. HIV-1 Immunogen in IFA appears to be safe and immunogenic. Further studies are indicated to determine clinical efficacy of the HIV Immunogen as well as the significance of the apparent correlation between HIV-DTH responsivity and a more favorable clinical course.

HIV-1 immunogen induction of HIV-1-specific delayed-type hypersensitivity: results of a double-blind, adjuvant-controlled, dose-ranging trial.

ObjectiveTo investigate the capacity of an HIV-1 immunogen to induce or augment HIV-1-specific delayed-type hypersensitivity (DTH) over a range of doses in asymptomatic HIV-1-seropositive adults. DesignA single center, double-blind, adjuvant-controlled, dose-ranging trial involving 48 HIV-1 -seropositive asymptomatic patients. Each dose group consisted of 12 subjects, eight receiving HIV-1 immunogen and four incomplete Freunds adjuvant (IFA). The doses studied were 50, 100, 200, or 400 μg (total protein). The HIV-1 immunogen was administered intramuscularly every 4 weeks for 36 weeks, with dosing contingent on the lack of an HIV-1 immunogen DTH response. A maximum of six doses was permitted. MethodsImmunogenicity was assessed every 4 weeks by DTH skin testing to the inactivated HIV-1 antigen in saline with >9 mm induration representing a response to immunization. Changes in p24-antibody levels were determined by endpoint titration using an enzyme-linked immunosorbent assay and Western blot. ResultsAt doses of > 100 μg, all treated patients demonstrated significant differences in the ability to mount an HIV-1-specific cell-mediated response relative to adjuvant controls. Dose-related response patterns were observed in the period between doses and the occurrence of rises in HIV-1 DTH. Treatment appeared to increase p24-antibody titers as well as reactivities to other HIV-1 antigens as determined by Western blots. The HIV-1 immunogen was well tolerated. ConclusionsThe minimum dose of the HIV-1 immunogen in IFA required to induce HIV-1 DTH relative to the IFA control group was 100 μg in this patient population.

Viral load, CD4 percentage, and delayed-type hypersensitivity in subjects receiving the HIV-1 immunogen and antiviral drug therapy

Two trials of subjects inoculated with the inactivated, gp120-depleted HIV-1 Immunogen are reported. In one study, in which 19 subjects received ZDV and 8 subjects received ddI, treatment with the HIV-1 Immunogen did not affect the pharmacokinetic parameters of the antiviral drugs. In another study, 65 subjects who were previously immunized with the HIV-1 Immunogen over a mean period of 4.0 years (range, 1.2–5.4 years) received inoculations at 0 and 6 months. At some point during this 48-week study, 72% of the subjects (47/65) were receiving antiviral drug therapy. The HIV-1 DNA load in CD4 cells and CD4 percentage were found to be stable over the 48-week period. Delayed-type hypersensitivity to HIV-1 antigens increased after two inoculations with the HIV-1 Immunogen. In these two trials, no serious treatment-related adverse events were documented in the subjects. The two studies presented herein are the first to suggest that an immune-based therapy such as the HIV-1 Immunogen can be combined safely with antiviral drugs, supporting further study to evaluate the clinical utility of this approach.

Effect of HIV-specific immune-based therapy in subjects infected with HIV-1 subtype E in Thailand

Objective:To examine the effect of treatment with an inactivated, gp120-depleted, HIV-1 immunogen (Remune) in 30 Thai subjects infected with HIV-1 subtype E. Design:Sixty-week open-label study. Methods:Thirty HIV-positive volunteers with CD4 cell counts ≥ 300 × 106/l were given intramuscular injections of Remune into the triceps muscle on day 1 and then at weeks 4, 8, 12, 24, 36, 48 and 60. Results:Treatment with Remune was well-tolerated and augmented HIV-1-specific immune responses. Furthermore, subjects had a significant increase in CD4 cell count (P < 0.0001), CD4 cell percentage (P < 0.0001), CD8 cell percentage (P < 0.0001), and body weight (P < 0.0001) compared with pretreatment levels. Fourteen subjects with detectable viral load at day 1 showed a decrease at week 60 (P = 0.04). Retrospective Western blot analysis showed 23 subjects with increased intensity of antibody bands and 15 patients showed development of new reactivities to HIV proteins, especially towards p17 and p15. Conclusion:These results indicate that HIV-specific immune-based therapeutic approaches such as Remune should be further examined in countries with different clades of HIV-1 and where access to antiviral drug therapies is limited.

HIV-specific immunity during structured antiviral drug treatment interruption☆

The immunologic correlates associated with control of viremia in HIV disease are poorly understood. We hypothesized that structured antiviral drug treatment interruptions could be utilized to better understand the relationship between HIV-specific immunity and viral replication. We thus examined the effects of two 8 weeks antiviral structured treatment interruptions (STIs) in a cohort of HIV-1 chronically infected individuals on highly active antiretroviral treatment (HAART) with (n = 13) and without (n = 12) therapeutic HIV immunizations. In this study, we observed that p24 gag antigen (np24) stimulated MIP-1beta levels and T helper immune responses prior to antiviral drug discontinuation were associated with control of viremia. Stronger and earlier production of gag peptide stimulated gamma interferon was observed in the immunized group during the structured antiviral drug interruptions. These results support the concept that HIV-specific immune responses are associated with control of viremia. Further study of immune-based therapies that enhance HIV-specific immunity is warranted.

Long-term follow-up of HIV-1-infected Thai patients immunized with Remune monotherapy.

Abstract Purpose: The purpose of this 2-year follow-up study was to investigate the long-term effect of Remune as monotherapy for HIV-1 infection. BACKGROUND: Participants previously enrolled in the phase II double-blind, randomized, adjuvant-controlled study of the HIV-1 Immunogen (Remune) were followed for 2 years. Open-label immunization with Remune monotherapy was given to each participant every 12 weeks. Remune, a gp 120-depleted HIV-1 that was inactivated in beta-propiolactone and irradiation, was emulsified with mineral oil (incomplete Freund’s adjuvant). Method: In Study 2101B, the effect of four doses of Remune given every 12 weeks over 40 weeks was compared to placebo in 297 asymptomatic type E HIV-infected patients [Churdboonchart et al., 2000]. A group of 17 volunteers were separated into a subset study and another 57 were excluded from analysis due to discontinuation or addition of other treatments. This 2-year follow-up study continued with open-label dosing of HIV-1 Immunogen every 12 weeks for the remaining 223 patients. Changes in CD4+ cells, CD8+ cells, and body weight were monitored at each patient visit. Results: Overall, immunizations were safe; common adverse events were tolerable injection site reactions. CD4+ T-cell counts remained stable over the 132-week observation period for this cohort with a slight increase of 36.01 cells/μL. CD8+ T-cell counts showed an increase from baseline uring the follow-up period (415.21 cells/μL). Furthermore, we also observed an increase in body weight from baseline (1.08 kg) at week 132. In addition, baseline CD4 count appeared to predict CD4 count at week 132 (slope = 0.31, p < .0001). Conclusion: These results suggest that long-term treatment of HIV-1 infection with Remune monotherapy is safe and results in a stabilization of CD4+ counts. Furthermore, it is likely that HIV-1 therapeutic immunization may show its greatest clinical benefit in participants with higher CD4+ cell counts. Such an approach may have important ramifications in developing countries where access to antiviral drugs is limited and also in early chronic HIV-1 infection when CD4+ cells are still over 300 cells/μL in order to limit the cost and toxicity.

RADIOIMMUNODETECTION STUDIES OF PROSTATE, COLON AND T-CELL LYMPHOMA TUMORS USING In-111 LABELED MONOCLONAL ANTITUMOR ANTIBODIES (In-111-MoAb); PRELIMINARY STUDIES

The purpose of these pilot studies was to determine if prostate carcinoma (PC), colon carcinoma (CC), and cutaneous T-Cell lymphoma (CTCL) could be detected using the In-111-MoAbs described below. Murine IgG MoAbs targeted against prostatic acid phosphatase (PAP), carcinoembryonic antigen (CEA), and an antibody that often recognizes CTCL (MAT-65) were labeled with In-111, and administered (Ad) intravenously to patients (PT) with PC, CC and CTCL respectively. One mg or less of MoAb was labeled with 1.5 – 5.0 mCI of In-111 in a total MoAb dose of 1–5 mg of anti-PAP, 0.5 mg of anti CEA, and 50 mg of MAT-65. All the MoAbs were infused over a two hour period. In one CTCL case, In-111-MoAb was Ad prior to, and a few weeks later, after a 50 mg dose of unlabeled MoAb. The infusion of In-111-MoAb followed a 50 mg infusion of unlabeled MoAb in the second CTCL case. Normal prostate tissue was visualized in 3 of 5 PT and 2 of the bone metastases imaged. Metastases from CC were visualized in 1 of 3 PTS. Outstanding definition of lymph nodes was achieved in CTCL, and the sequence of Ad markedly altered in the in-vivo kinetics of the In-111-MoAb. Some toxicity was observed in CTCL PTS, however, anti PAP and anti CEA were not toxic. We conclude that the above MoAbs will target tumor, and that further clinical trails with higher quantities of anti-PAP and CEA protein are warranted.

RADIOIMMUNODETECTION OF MELANOMA USING In-111???96.5 MONOCLONAL ANTIBODY (In-111???96.5 MoAb)

The purpose of this study was to evaluate In-111–96.5 MoAb as a radiopharmaçeuticl (R) for the detection of melanoma (mel). The 96.5 MoAb targets a 97 kilodalton surface antigen on the mel cells. Labeling was by a bifunctional chelation technique, and resulted in 3–5 mCi of In-111 chelated to 1 mg of antibody (A). The R was administered (Ad) intravenously through a 30–120 minute period. Twenty-two studies were performed in 21 patients (PT), with one PT studied twice. In four PT, unlabeled A was Ad prior to the R. Other PT received from 2–19 mg of unlabeled A mixed with the R. Blood (B) was drawn at multiple times following the infusion to observe R kinetics, and to determine if serum chemistries indicated toxicity (tox). There was no evidence of tox from the R or carrier A. Increasing protein mass slowed the loss of In-111 from B, and appeared to improve lesion detection. 66% of the 73 lesions 1.5 cm or larger were detected. Eight metastases were detected which were not clinically suspected. Five metastases imaged were in the 0.5–1.0 cm size range. Two were scalp metastases, and three were lymph nodes in the anterior cervical triangle of the neck. Liver uptake was a major cause of failure of the R as lesions could not be resolved if they occurred in the liver. We conclude that In-111–96.5 MoAb shows promise as a R for the detection of mel, and warrants further study.