Charles Wiseman

Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC class II molecules in LS1034 colorectal carcinoma cell line

BackgroundThe expression of major histocompatibility complex class II (MHCII) antigens in both mouse and human tumors is rare, and these antigens are not easily inducible by IFN-gamma (IFNg). Since MHCII may play an important role in the development of host antitumor immune response, we explored the possibility of restoring MHCII inducibility in several IFNg-resistant tumor cell lines using protein kinase C (PKC) agonists phorbol myristate acetate (PMA) or Bryostatin.ResultsTumor cells were co-cultured with various concentrations of PMA and IFNg for 48 hr. The expression of MHCII antigens and receptors IFNgR1 and IFNgR2 was determined by flow cytometry. We showed that the presence of as little as 0.1 ng/ml of PMA in tissue culture restored the ability of weakly inducible LS1034 colon carcinoma cells to express MHCII in response to IFNg (100 – 10,000 IU/ml) in a dose-dependent manner. Likewise, Bryostatin 1, as low as 10 ng/ml produced a 5–6 fold upregulation of MHCII. The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM). Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.ConclusionIn this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists. Bryostatin 1 may be considered for further investigation of IFNg-dependent MHCII induction in resistant tumors in vivo.

Objective Clinical Regression of Metastatic Breast Cancer in Disparate Sites after Use of Whole-Cell Vaccine Genetically Modified to Release Sargramostim

Abstract:  A patient with recurrent breast cancer metastases following initial response to chemotherapy and hormonal maintenance was treated with a whole‐cell tumor vaccine, resulting in a prompt objective complete remission of a lung lesion on computed tomography (CT) scans and near‐complete regression of multiple breast lesions on magnetic resonance imaging (MRI). Three months after completion of the protocol, metastases were again found in the breast and lung, with new lesions in the brain and liver. Reinstitution of vaccine inoculation resulted in major regression of the brain and breast lesions, improvement in all other areas, and no indication of new lesions. Therapy consisted of inoculation of 20 × 106 SV‐BR‐1‐GM cells, a unique breast cancer cell line transfected to release sargramostim (granulocyte macrophage colony‐stimulating factor [GM‐CSF]). Following lethal irradiation to 200 cGy, vaccine was injected intradermally in four divided doses to the back and thighs, every 2 weeks × 3, then every month × 3. Each treatment was preceded 48 hours earlier with low‐dose cyclophosphamide 300 mg/m2 to abrogate regulatory T‐cell activity. Interferon (IFN)‐α, 20,000 IU, was injected into each inoculation site at 48 and 96 hours postinoculation to provide an additional “danger signal.” The patient developed positive delayed‐type hypersensitivity responses and also antibody reactivity to the vaccine cells.

Phase I Study with SV-BR-1 Breast Cancer Cell Line Vaccine and GM- CSF: Clinical Experience in 14 Patients

We evaluated the safety and feasibility of breast cancer vaccine therapy using a new cell line, SV-BR-1, in conjunction with repeated injections of GM-CSF, as a prototype for a subsequent genetically engineered vaccine. Also addressed with this preliminary trial were monitoring effects on quality of life, screening for potential immune responses, and evaluating any clinical effects on tumor progression and patient survival. Fourteen patients with metastatic breast cancer were treated with SV-BR-1 tumor cells. The cell line strongly overexpresses HER2/neu, and has an unusual variety of cytogenetic abnormalities. Irradiated whole-cell suspensions (median dose 14 x 10 6 viable cells) were inoculated via intradermal injection or intralymphatic cannulation, the former usually including an equal amount of irradiated autologous peripheral blood lymphocytes (PBL). Vaccine was initially given at 2-week intervals x3, then monthly. Low-dose cyclophosphamide 300mg/m 2 was given 48-72 hrs prior to vaccine, and GM-CSF (125 mcg) was given subcutaneously immediately prior and for 8 days subsequently. The Kaplan-Meier mean survival for all 14 patients was 17.2 months (CI 5.8-28.5 months) and was 21.1 months (95% CI 5.0-37.2 months) for nine patients treated with the tumor cell/PBL admixture, although there were no clinical regressions. SV-BR-1 as a whole-cell vaccine appears feasible and without obvious major toxicity. The overall survival compares favorably with current Phase I studies. Based on these observations, we have initiated a clinical trial using the SV-BR1 cell line transfected to release GM-CSF in situ with four patients to date. One case responded with clear evidence of tumor regressions in multiple sites and the median survival of 35.0 months is three times longer than usual expectations for salvage programs.