Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.

Enhanced immune priming with spatial distribution of paracrine cytokine vaccines.

In preclinical models, tumor cells genetically modified to express cytokines or other costimulatory molecules can generate systemic antitumor immunity. In some cases, these tumor vaccines have been shown to eradicate micrometastases. These results have led to the initiation of numerous phase I clinical trials employing either autologous or allogeneic tumor vaccines genetically modified to express cytokines and other genes. In this report, we use our murine model to identify a number of parameters that may be critical for enhancing vaccine efficacy. In addition to antigen dose and cytokine level, the distribution of vaccine inoculation was found to have a significant impact on vaccine potency. These results require consideration in early clinical trials designed to evaluate cellular vaccine therapy.

Consistent chromosome abnormalities in adenocarcinoma of the pancreas.

Little is known about the somatic genetic changes which characterize pancreatic adenocarcinoma. The identification of acquired genomic alterations would further our understanding of the biology of this neoplasm. We have studied 62 primary pancreatic adenocarcinomas obtained from surgical resections using classical cytogenetics and fluorescent in situ hybridization methods. Clonally abnormal karyotypes were observed in 44 neoplasms. Karyotypes were generally complex (greater than three abnormalities) and included both numerical and structural chromosome abnormalities. Many tumors contained at least one marker chromosome. The most frequent whole chromosomal gains were chromosomes 20 (eight tumors) and 7 (seven tumors). Losses were much more frequent: chromosome 18 was lost in 22 tumors followed in frequency by chromosomes 13 (16 tumors), 12 (13 tumors), 17 (13 tumors), and 6 (12 tumors). Structural abnormalities were frequent. Two hundred nine chromosome breakpoints were identified. Excluding Robertsonian translocations, the chromosomal arms most frequently involved were 1p (12); 6q (11); 7q and 17p (9 each); and 1q, 3p, 11p, and 19q (8 each). Portions of the long arm of chromosome 6 appeared to be lost in nine tumors. To determine whether the apparent losses of portions of 6q are real, four tumors with 6q deletions were hybridized with a biotin-labeled microdissection probe from 6q24-ter. Loss of one copy of this region was verified in three of four tumors. In addition, double minute chromosomes were identified in eight cases. To our knowledge, these represent the first primary specimens of pancreatic adenocarcinoma with cytogenetic evidence of gene amplification.

Plasmodium yoelii: cellular immune responses in splenectomized and normal mice.

Spleen and lymph node cells from Plasmodium yoelii 17X-infected, C57BL/6 (B6), and DBA/2 (D2) mice were cultured in vitro with parasite antigens. The ability of these cells to proliferate was quantified by uptake of [3H]thymidine and ELISA was used to measure secretion of IFN-gamma and IL-5. B6 mice are relatively susceptible to P. yoelii 17X infection compared to D2 mice. Susceptible mouse strains develop higher levels of parasitemia, become more anemic, and take longer to resolve their infections than do resistant strains. Following splenectomy, D2 mice resisted P. yoelii 17X infections as well as did sham-operated controls, but splenectomized B6 mice failed to resolve their infections and all died. Spleen cells from infected mice of either strain were activated in vitro as evidenced by their proliferation in the absence of exogenous antigen. When malaria antigen was added to these cultures, cells from resistant D2 mice responded strongly with increased proliferation, whereas cells from susceptible B6 mice responded weakly, and on Day 14 postinfection, responses were actually suppressed. Mesenteric lymph node cells from infected B6 and D2 mice did not proliferate in the presence or absence of P. yoelii 17X antigen unless the spleen was removed. Following splenectomy, mesenteric lymph node cells from D2 mice, but not B6 mice, proliferated strongly compared to cells from sham-operated controls. IFN-gamma and IL-5 production from spleen and lymph node cells was measured following in vitro stimulation with P. yoelii 17X antigen. Spleen cells from D2 mice produced levels of IFN-gamma increased over those of cells from B6 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

High efficiency gene transfer into primary human tumor explants without cell selection.

Preclinical studies with murine tumor models have demonstrated that autologous tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. These results have engendered much interest in developing this strategy for gene therapy of human cancer. The major limitation to creating genetically modified autologous human tumor vaccines is efficient gene transfer into primary tumor explants, since the majority of human tumors fail to proliferate in long-term culture. Using the retroviral vector MFG in conjunction with short-term culture techniques, we have achieved, in the absence of selection, a mean transduction efficiency of 60% in primary renal, ovarian, and pancreatic tumor explants, and we have developed an autologous granulocyte-macrophage colony-stimulating factor secreting tumor vaccine for clinical trials.

IFN-gamma- and IL-5-producing cells compartmentalize to different lymphoid organs in Trichinella spiralis-infected mice.

The differential induction of cytokines associated with Th1 and Th2 subsets has recently been described during Trichinella spiralis infection. Increased levels of resistance appear to correlate with elevated levels of the Th1-associated cytokines, IFN-gamma and IL-2. In the present report, a filter immunoplaque assay is used to quantify the actual numbers of cells that secrete IFN-gamma and IL-5. It is demonstrated that, in T. spiralis-infected B10.Q mice, Th1- and Th2-associated responses are compartmentalized to different lymphoid organs. Thus, Ag-induced IFN-gamma-producing cells predominate in the spleen, whereas IL-5-producing cells prevail in the mesenteric lymph nodes (MLN). A corresponding compartmentalization of Ag-specific IgA and IgG1 antibody-secreting cells to the MLN is also noted. The virtual absence of Th1-associated responses in the MLN appears to be an Ag-associated phenomenon. MLN from either naive or T. spiralis-infected mice do have the capacity to secrete IFN-gamma if stimulated with Con A. The striking compartmentalization of Ag-driven cytokine responses seen in this parasite system may facilitate study of the mechanisms that regulate the induction of Th1 and Th2 subsets.

MHC-restricted antibody responses to Trichuris muris excretory/secretory (E/S) antigen.

Two panels of H-2 recombinant mice were used in a detailed serological study to analyse the role of H-2-linked genes in the control of the antibody response to excretory/secretory (E/S) antigens of Trichuris muris. An apparent H-2q (I-Aq) restriction on the early development of high levels of IgG1 antibody to E/S antigen was revealed by ELISA. No such restriction was demonstrated for the specific IgG2a response patterns. Recognition of two high molecular weight antigens (90-95 kDa, 105-110 kDa) by IgG antibodies was also shown to be almost exclusively H-2q restricted and may be related at least in part to the high antibody levels seen for H-2q strains of mice. Immune serum from resistant (B10.BRxB10.G) F1 hybrid mice (H-2q/k) containing high levels of IgG1 antibodies specific for T. muris E/S and IgG antibodies which recognized the 90-95 kDa and 105-110 kDa E/S antigens was effective in transferring protection to the non-responsive B10.BR mouse strain as seen on day 35 post-infection (p.i.). It is suggested that the IgG responses described for the generally very resistant H-2q mouse strains may contribute to, but not be an absolute requirement for, protective immunity, antibody-mediated damage facilitating a subsequent cellular attack in certain strains of mice.

The influence of genes mapping within the major histocompatibility complex on resistance to Trichuris muris infections in mice.

Two panels of H-2 recombinant strains of mice were used in an attempt to map the H-2-linked genes which control resistance to infection with Trichuris muris. Response phenotypes could be related to the presence of 'resistance' (q,b) or 'susceptibility' (k,d) alleles at I-A. The influence of these genes was modulated by other alleles, particularly q or d alleles, at the D end of the H-2. Absence of I-E molecules correlated with resistance to infection in some but not all strains studied. Thus the (B10.BR x B10.G) F1 strain which expressed I-Ek gene products was resistant to infection. A study of the time-course of infection in strains of mice expressing q alleles throughout the H-2 on 4 different genetic backgrounds (NIH, SWR, DBA and B10) revealed that most strains were resistant to infection. However, the DBA/1 strain exhibited differential responsiveness, 4 out of 6 individuals harbouring mature adult parasites on day 35 post-infection.