Jan Zaloudik

Up-regulation of Fas (CD95) in human p53wild-type cancer cells treated with ionizing radiation.

Fas is a cell‐surface protein which belongs to the tumor‐necrosis‐factor‐receptor family. Signals through Fas are able to induce apoptosis in sensitive cells, and thus modalities for regulating the level of Fas expression on tumor cells are needed. We have studied cellular responses to gamma irradiation. The level of p53 tumor‐suppressor protein was found to be elevated 3 hr after irradiation of p53wild‐type MCF‐7 breast‐carcinoma cells. Interestingly, accumulation of p53 was followed by up‐regulation of surface Fas levels between 4 and 8 hr after irradiation. The level of Fas up‐regulation was dependent on dose and, whereas elevation in the level of p53 was transient, enhancement of Fas expression was stable. Fas up‐regulation occurred coincidentally with induction of G1 cell‐cycle arrest, a post‐irradiation phenomenon known to be dependent on wild‐type‐p53 activity. We studied 9 other tumor lines, 2 with wild‐type p53, 5 with mutant p53, and 2 expressing no p53. All lines expressing wild‐type p53 were found to arrest in G1 and to up‐regulate Fas after irradiation. In contrast, all 7 p53null and p53mutant lines failed not only to arrest their cell cycles in G1 phase, but also to up‐regulate Fas levels in response to treatment. These findings demonstrate a direct correlation between wild‐type‐p53 activity and Fas up‐regulation after treatment with ionizing radiation, strongly suggesting that post‐irradiation Fas up‐regulation is dependent on wild‐type‐p53 activity. Since low doses of radiation were sufficient to modulate Fas expression, up‐regulation of the Fas death receptor may have clinical implications following radiotherapy. Int. J. Cancer 73:757–762, 1997.

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

Abstract In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freunds complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freunds adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector.

Detection and Long-Term In Vivo Monitoring of Individual Tumor-Specific T Cell Clones in Patients with Metastatic Melanoma

We investigated the presence of individual melanoma-specific T cell clones in patients with metastatic melanoma. Ten patients were examined for the presence of melanoma-reactive T cells using dendritic cells loaded with autologous tumor cells. Their specificity was tested using nonradioactive cytotoxicity test. Individual immunodominant T cell clones were identified by the clonotypic assay that combines in vitro cell culture, immunomagnetic sorting of activated IFN-γ+ T cells, TCRβ locus-anchored RT-PCR, and clonotypic quantitative PCR. All patients had detectable melanoma-reactive T cells in vitro. Expanded melanoma-reactive T cells demonstrated specific cytotoxic effect against autologous tumor cells in vitro. Three patients experienced objective responses, and their clinical responses were closely associated with the in vivo expansion and long-term persistence of individual CD8+ T cell clones with frequencies of 10−6 to 10−3 of all circulating CD8+ T cells. Five patients with progressive disease experienced no or temporary presence of circulating melanoma-reactive T cell clones. Thus, circulating immunodominant CD8+ T cell clones closely correlate with clinical outcome in patients with metastatic melanoma.

Inhibition of tumor growth by recombinant vaccinia virus expressing GA733/CO17-1A/EpCAM/KSA/KS1-4 antigen in mice.

The human colorectal carcinoma (CRC)-associated GA733 antigen (Ag), also named CO17-1A/EpCAM/KSA/KS1-4, has been a useful target in passive immunotherapy of CRC patients with monoclonal antibody (mAb) and in active immunotherapy with anti-idiotypic antibodies or with recombinant protein. These approaches have targeted single epitopes (monoclonal anti-GA733 antibodies and anti-idiotypic antibodies) or extracellular domain epitopes (recombinant protein), primarily by B cells. To determine whether a reagent that induces immunity to a larger number of both B- and T-cell epitopes might represent a superior vaccine, we analyzed the capacity of full-length GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant vaccinia virus (VV GA733-2) to induce humoral, cellular, and/or protective immunity in mice. VV GA733-2 induced Ag-specific antibodies that reacted predominantly to unknown epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with murine peritoneal macrophages as effector cells. Immunized mice developed Ag-specific, proliferative and delayed-type hypersensitive lymphocytes. VV GA733-2 inhibited growth of ras-transformed syngeneic tumor cells expressing the human GA733 Ag in mice. These results suggest the potential of VV GA733-2 as a candidate vaccine for patients with CRC, possibly in combination with recombinant GA733-2–expressing adenovirus, which has been shown to induce cytolytic antibodies and T cells as well as tumor protective effects in mice. The combined vaccine approach may be superior to the use of either vaccine alone in patients who are pre-immune to both viruses.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Abstract. Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2+ tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4–/– mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Colorectal Cancer Vaccines: Antiidiotypic Antibody, Recombinant Protein, and Viral Vector

Abstract: The colorectal cancer antigen GA733 (also termed CO17‐1A, KSI‐4, Ep‐CAM, KSA) has proved to be a useful target in passive immunotherapy with monoclonal antibody and in active immunotherapy with antiidiotypic antibodies in cancer patients. The GA733 antigen was molecularly cloned and expressed in baculovirus (BV), adenovirus (AV), and vaccinia virus (VV). Recombinant BV‐, VV‐, and AV‐GA733 induced antigen‐specific cytotoxic antibodies and proliferative and delayed‐type hypersensitive lymphocytes. However, only the AV recombinant induced antigen‐specific cytolytic T lymphocytes and regression of established tumors. Cured mice were protected against challenge with antigen‐negative tumors, indicating antigen spreading of immune responses. In a model of active immunotherapy against the murine homologue of the human GA733 antigen, murine epithelial glycoprotein (mEGP), BV‐derived mEGP protein in various adjuvants did not protect mice against a challenge with mEGP‐positive tumors. AV mEGP, only when combined with interleukin‐2, significantly inhibited growth of established mEGP‐positive tumors. This is in contrast to the same vaccine expressing the human antigen that was effective without interleukin‐2. AV GA733, in combination with interleukin‐2, is a candidate vaccine for colorectal cancer patients.

Monoclonal anti-idiotypic antibody functionally mimics the human gastrointestinal carcinoma epitope GA733

Anti‐idiotypic antibodies (Ab2) that bind to the antigen‐combining region of anti‐tumor antibodies (Ab1) may functionally, and even structurally, mimic tumor antigen. We have previously demonstrated that polyclonal goat Ab2 directed against anti‐human gastrointestinal carcinoma Ab1 GA733 induces anti‐anti‐idiotypic antibodies (Ab3) in animals that are Ab1‐like in their binding specificity and idiotope expression. To obtain more defined Ab2 vaccines with potentially increased specificity and efficacy, a monoclonal Ab2 (FG1) was produced against Ab1 GA733 in rats. The monoclonal Ab2 FG1, similar to the polyclonal Ab2 described previously, induced Ab3 in rabbits that were Ab1‐like in their idiotope expression and binding specificity to tumor cells and antigen. Antigen‐specific Ab3 induced by Ab2 FG1 were easily detected in unprocessed rabbit sera, whereas the demonstration of such Ab3 after polyclonal Ab2 immunization required purification of the Ab3 from the rabbit sera. In addition, Ab2 FG1 induced antigen‐specific humoral and cellular immunity in mice. Murine Ab3 bound specifically to antigen‐positive tumor cells. Ab2‐immunized mice showed antigen‐specific delayed‐type hypersensitivity (DTH) reaction, and cultured splenocytes from the immune mice demonstrated specific proliferation and cytokine (interferon‐γ and interleukin‐4) secretion upon stimulation with GA733 antigen. However, immune mice were not protected against a challenge with syngeneic GA733 antigen‐expressing colon carcinoma cells.