Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.
XenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.
Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.
Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.
The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct “off-the-shelf” products could be engineered to engender functionally potent immune responses against true “self”-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15 × 106 transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA.
Radiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression.
Murine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA+) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA+ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM).
In vitro, sublethal hyperthermia of MC38-CEA+ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4+ T-cell immune responses to CEA and eradicated both primary CEA+ and distal CEA– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.
Genetically modified Salmonella typhimurium VNP20009 (VNP) is a useful vehicle for cancer therapy and vaccine development but exhibits limited tumor targeting in vivo. We engineered a novel VNP derivative that expressed carcinoembryonic antigen (CEA)-specific single chain antibody fragments (scFv) on the cell surface to increase tumor-specific targeting. There was significant scFv cell surface display visualized by flow cytometry and confocal microscopy when cells were probed with fluorescently labeled CEA. Atomic force microscopy (AFM) measurements on whole bacteria confirmed binding of unlabelled CEA to the displayed scFv. The modified VNP strain exhibited increased localization in the upper gastrointestinal tract of CEA transgenic mice and accumulated in CEA-expressing tumors. Furthermore, treatment with a single dose of the VNP derivative inhibited growth of MC38CEA tumors and was associated with local accumulation of CD3+ T cells and CD11b+ macrophages. The display of antibody fragments on the surface of VNP represents a novel strategy for both targeting CEA-expressing tumors and increasing the immunogenicity of Salmonella-based vaccines for cancer.
Salmonella; scFv display; cancer therapy; vaccine vector; carcinoembryonic antigen
This study investigated the immunomodulatory effects of sunitinib in order to rationally design combinational platforms with immunotherapies for the treatment of solid tumors. Using a mouse model, we studied the effects of sunitinib given for 4 weeks at concentrations comparable to 37.5–50 mg/day in humans, followed by 2 weeks off the drug (sunitinib 4/2). We assessed the effect of differently timed combinations of sunitinib and a poxvirus-based vaccine encoding carcinoembryonic antigen (CEA) plus 3 costimulatory molecules on immune responses in CEA-transgenic (CEA-Tg) mice. Antitumor studies were performed in CEA-Tg mice bearing CEA-transfected MC38 murine colon carcinomas (MC38-CEA), treated either concurrently or sequentially with sunitinib and vaccine. In vitro, sunitinib inhibited PDGFR phosphorylation on MC38-CEA cells at concentrations similar to those biologically available during human treatment. In vivo, one cycle of sunitinib 4/2 caused bimodal immune effects: (a) decreased regulatory cells during the 4 weeks of treatment and (b) an immune-suppression rebound during the 2 weeks of treatment interruption. In a model using CEA-Tg mice bearing CEA+ tumors, continuous sunitinib followed by vaccine increased intratumoral infiltration of antigen-specific T lymphocytes, decreased immunosuppressant T regulatory cells and myeloid-derived suppressor cells, reduced tumor volumes, and increased survival. The immunomodulatory activity of continuous sunitinib administration can create a more immune-permissive environment. In combination with immunotherapies, sunitinib treatment should precede vaccine, in order to precondition the immune system, to maximize the response to vaccine-mediated immune enhancement.
sunitinib; vaccine; immunotherapy; TKI; cancer
Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells.
HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR α and β chains to improve recognition of CEA.
Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1+ CEA+ human colon cancer cells. Both CD8+ and CD4+ human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8+ lymphocytes specifically recognized HLA-A2.1+ CEA+ colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the α chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8+ lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4+ T cells.
The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.
Radiation is a primary modality in cancer treatment. Radiation can also reduce tumor growth outside the treatment field, often referred to as the abscopal effect. The mechanisms and therapeutic potential of the abscopal effect have not been fully elucidated. We evaluated the role of vaccination directed against a tumor-associated antigen (TAA) in the induction and amplification of radiation induced abscopal effects. Active-specific immunotherapy with a TAA-specific vaccine regimen was used to induce and potentiate T-cell responses against carcinoembryonic antigen (CEA) in combination with local irradiation of subcutaneous tumors. We examined the potential synergy of a poxvirus-based CEA vaccine regimen in CEA-transgenic (Tg) mice in combination with either external beam radiation or brachytherapy of local tumors. The induction of CD8+ T cells specific for multiple TAAs not encoded by the vaccine was observed after the combination therapy. In two tumor models, the antigen cascade responses induced by vaccine and local irradiation mediated the regression of antigen negative metastases at distal subcutaneous or pulmonary sites. Clinically, local control of the primary tumor is necessary and can sometimes prevent metastases; however, irradiation generally fails to control preexisting metastases. These studies suggest that by coupling tumor irradiation with immunotherapy, the abscopal effect can transcend from anecdotal observation to a defined mechanism that can be exploited for the treatment of systemic disease.
abscopal; radiation; vaccine; modulation; Fas; immunotherapy
While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA “cancer vaccines” using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established  which takes advantage of the ability of the Factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC) . To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations.
Cancer Vaccines; exosomes; C1C2 domain; tumor antigens; adenovirus vectors; CEA; HER2
Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross- reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.
Tumor-associated antigens are weakly immunogenic. Human carcinoembryonic antigen (CEA) is overexpressed on a wide range of human carcinomas and represents an attractive target for cancer immunotherapy. This study analyzes the ability of a Saccharomyces cerevisiae vector containing the transgene encoding CEA (yeast-CEA) to activate human dendritic cells (DCs) and stimulate CEA-specific T-cell responses. We demonstrate for the first time that treatment with yeast-CEA can activate human DCs, resulting in increases in surface expression of CD80, CD83, CD54, CD58, and MHC class II, and increased production by DCs of IL-12p70, TNF-α, IFN-γ, IL-8, IL-2, IL-13, IL-10, and IL-1β. We also show that human DCs treated with yeast-CEA can activate CEA-specific T-cell lines and can act as antigen-presenting cells (APCs) to generate CEA-specific T-cell lines capable of lysing CEA+ human tumor cells. Gene expression profiles of human DCs treated with yeast-CEA show increased expression of numerous genes involved in the production of chemokines and cytokines and their receptors, and genes related to antigen uptake, antigen presentation, and signal transduction.
Saccharomyces cerevisiae; yeast-CEA; human dendritic cell
Saccharomyces cerevisiae, a nonpathogenic yeast, has previously been used as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumorburden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity.
CEA-transgenic mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and subcutaneous pancreatic tumor models.
These studies demonstrate that recombinant yeast can break tolerance and that a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and subcutaneous pancreatic tumor models.
Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-transgenic mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.
Saccharomyces cerevisiae; yeast; CEA; tumor immunity
Carcinoembryonic antigen (CEeA) is a glycosylated cell surface antigen known to be highly overexpressed in several adenocarcinomas, including colorectal cancer, while demonstrating limited expression in normal tissues. Prior work has shown that the plasma clearance of T84.66, a monoclonal anti-CEA antibody, is enhanced by several-fold in a CEA-expressing xenograft mouse model, suggesting the presence of a target mediated elimination pathway. purpose of this study is to investigate the influence of tumor volume on the plasma clearance of and test the hypothesis that the plasma pharmacokinetics of T84.66 may be used as a sensitive and selective test for the diagnosis of CEA-positive tumors. plasma pharmacokinetics were studied following intravenous (iv) administration of a 1 mg/kg dose in animals without tumor and mice bearing low (20–75 mm3), medium (400–570 mm3), and high volume (800–1,200 mm3) LS174T xenografts.
Based on comparison of the disposition of in non-tumor bearing mice and mice bearing low-volume tumors, it was predicted that a single plasma concentration of obtained seven days after dosing, would provide a sensitive and selective means of determining the presence of tumor in mice. A blinded follow-up study was conducted using athymic mice with or without intraperitoneal LS174T xenografts. 1 mg/kg of 125I-T84.66 was administered iv, and plasma samples were collected on day 7. Comparison of the observed concentration of 125I-T84.66 to the pre-determined threshold value (7.63 nM) enabled identification of tumor bearing mice with a sensitivity of 93.3% and specificity of 100%.
carcinoembryonic antigen (CEA); target mediated disposition (TMD); T84.66; anti-CEA IgG; screening test; sensitivity; specificityin
Sensitive antibody-based tumor targeting has the potential not only to image metastatic and micrometastatic disease, but also to be the basis of targeted therapy. The vast majority of pancreas cancers express carcinoembryonic antigen (CEA). Thus, we sought to evaluate the potential of CEA as a pancreatic cancer target utilizing a rapidly clearing engineered anti-CEA scFv-Fc antibody fragment with a mutation in the Fc region [anti-CEA scFv-Fc H310A].
Immunohistochemistry (IHC) with the antibody fragment was used to confirm expression of CEA on human pancreas cancer specimens. In vivo tumor targeting was evaluated by tail vein injection of I124-labeled anti-CEA scFv-Fc(H310A) into mice harboring CEA-positive and -negative xenografts. MicroPET/CT imaging was performed at successive time intervals. Radioactivity in blood and tumor was measured after the last time point. Additionally, unlabeled anti-CEA scFv-Fc(H310A) was injected into CEA-positive tumor bearing mice and ex vivo IHC was performed to identify the presence of the antibody to define the microscopic intratumoral pattern of targeting.
Moderate to strong staining by IHC was noted on 84% of our human pancreatic cancer specimens and was comparable to staining of our xenografts. Pancreas xenograft imaging with the radiolabeled anti-CEA scFv-Fc(H310A) antibody demonstrated average tumor/blood ratios of 4.0. Immunolocalization demonstrated peripheral antibody fragment penetration of one to five cell diameters (0.75 to 1.5 μm).
We characterized a preclinical xenograft model with respect to CEA expression that was comparable to human cases. We demonstrated that the anti-CEA scFv-Fc(H310A) antibody exhibited antigen-specific tumor targeting and shows promise as an imaging and potentially therapeutic agent.
imaging; pancreas cancer; CEA; antibody
Although tumors express potentially immunogenic tumor-associated antigens (TAAs), cancer vaccines often fail because of inadequate antigen delivery and/or insufficient activation of innate immunity. The engineering of non-pathogenic bacterial vectors to deliver TAAs of choice may provide an efficient way of presenting TAAs in an immunogenic form. In this study, we used genes of Salmonella Pathogenicity Island 2 (SPI2) to construct a novel cancer vaccine, where a TAA, survivin was fused to SseF effector protein and placed under control of SsrB, the central regulator of SPI2 gene expression. This construct uses the type III secretion system (T3SS) of Salmonella and allows preferential delivery of tumor antigen into the cytosol of antigen-presenting cells for optimal immunogenicity. In a screen of a panel of attenuated strains of Salmonella we found that a double-attenuated strain of Salmonella typhimurium, MvP728 (purD/htrA) was not toxic to mice and effectively expressed and translocated survivin protein inside cytosol of murine macrophages. We also found that a ligand for CD1d-reactive Natural Killer T (NKT) cells, α-Glucuronosylceramide (GSL1) enhanced MvP728-induced IL-12 production in human DCs and that in vivo co-administration of a NKT ligand with MvP728-Llo or MvP728-survivin enhanced effector-memory CTL responses. Furthermore, combined use of MvP728-survivin with GSL1 produced anti-tumor activity in mouse models of CT26 colon carcinoma and orthotopic DBT glioblastoma. Therefore, the use of TAA delivery via SPI-2-regulated T3SS of Salmonella and NKT ligands as adjuvants may provide a foundation for new cancer vaccines.
Cancer vaccine; Salmonella; Salmonella Pathogenicity Island 2 (SPI2); Natural Killer T (NKT) cells; Survivin
IFN-α is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-α with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen.
The phenotypic and functional effects of IFN-α were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-α administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-α and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice.
We identified a dose and schedule of IFN-α that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8+) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-α distal to the site of vaccination. The combination of IFN-α and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA+ adenocarcinomas. In mice with pancreatic tumors, IFN-α slowed tumor growth, induced CTL activity, and increased CD8+ tumor-infiltrating lymphocytes.
These data suggest that IFN-α can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.
Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant >1010 M−1, no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1, 4, 7-tris(carboxymethyl)-10-(vinylsulfone)-1, 4, 7, 10-tetraazacyclododecane) that binds a range of radiometals including 111In for gamma-ray imaging and 64Cu for Positron Emission Tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins.
Athymic mice peripherally xenografted with CEA-positive human colon tumors (LS-174T) were injected with 111In-labeled or 64Cu-labeled SH-DO3A-VS-M5A, NH-DO3A-VS-M5A, or DOTA-M5A and sacrificed at various time points for biodistribution measurements. Other mice injected with 64Cu-labeled SH-DO3A-VS-M5A or NH-DO3A-VS-M5A were imaged serially with small animal PET from 1 to 48 h post injection and then sacrificed for biodistribution measurements.
Virtually identical biodistributions were obtained for SH- and NH-DO3A-VS-M5A or DOTA-M5A whether radiolabeled with 111In or 64Cu. Rapid tumor uptake of radiolabel was observed, reaching 40% injected dose/gram or more by 48 h. Importantly, excellent PET images of tumor were obtained as early as 22 h after injection of 64Cu-labeled SH- or NH-DO3A-VS-M5A.
Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.
carcinoembryonic antigen; bifunctional chelate; radioimmunoimaging; positron emission tomography
Active-specific immunotherapy used as an adjuvant therapeutic strategy is rather unexplored for cancers with poorly characterized tumor antigens like gastric cancer. The aim of this study was to augment a therapeutic immune response to a low immunogenic tumor cell line derived from a spontaneous gastric tumor of a CEA424-SV40 large T antigen (CEA424-SV40 TAg) transgenic mouse.
Mice were treated with a lymphodepleting dose of cyclophosphamide prior to reconstitution with syngeneic spleen cells and vaccination with a whole tumor cell vaccine combined with GM-CSF (a treatment strategy abbreviated as LRAST). Anti-tumor activity to subcutaneous tumor challenge was examined in a prophylactic as well as a therapeutic setting and compared to corresponding controls.
LRAST enhances tumor-specific T cell responses and efficiently inhibits growth of subsequent transplanted tumor cells. In addition, LRAST tended to slow down growth of established tumors. The improved anti-tumor immune response was accompanied by a transient decrease in the frequency and absolute number of CD4+CD25+FoxP3+ T cells (Tregs).
Our data support the concept that whole tumor cell vaccination in a lymphodepleted and reconstituted host in combination with GM-CSF induces therapeutic tumor-specific T cells. However, the long-term efficacy of the treatment may be dampened by the recurrence of Tregs. Strategies to counteract suppressive immune mechanisms are required to further evaluate this therapeutic vaccination protocol.
The relationship of serum carcinoembryonic antigen (CEA) levels to tumour size and antigen content was studied in artificially immune-deprived mice bearing human colonic, breast and lung tumour xenografts. Size was measured as in vivo volume and tumour weight at post-mortem. A multiple implant technique combined with early harvest was used to minimize centrilobular tumour necrosis. CEA was extracted from resected tumours with perchloric acid. A radioimmunoassay using chemical precipitation was used to estimate CEA in blood samples. A correlation was found between CEA blood levels and tumour size in half the tumour lines, in contrast to a recent report (Lewis & Keep, 1981). The CEA content was found to be constant for one tumour line but not another. The possibility that central necrosis in xenograft tumours may account for the discrepancies is discussed. There may be serious limitations for the use of xenograft tumour models for studying the biology of CEA.
Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called '3G7'. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.
Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.
We created an anti-tumor vaccine by using adenovirus as a vector which contains a cytomegalovirus early promoter-directed human carcinoembryonic antigen gene (AdCMV-hCEA). In an attempt to develop the skin patch vaccine, we epicutaneously vaccinated Balb/c mice with AdCMV-hCPA. After nine weeks post-immunization, vaccinated mice evoked a robust antibody titer to CEA and demonstrated the capability of suppressing in vivo growth of implanted murine mammay adenocarioma cell line (JC-hCEA) tumor cells derived from a female Balb/c mouse. Proteomic analysis of the tumor masses in the non-vaccinated naïve and vaccinated mice reveal that six proteins change their abundance in the tumor mass. The levels of adenylate kinase 1, β-enolase, creatine kinase M chain, hemoglobin beta chain and prohibitin were statistically increased whereas the level of a creatine kinase fragment, which is undocumented, was decreased in the tumor of vaccinated mice. These proteins may provide a vital link between early-stage tumor suppression and immune response of skin patch vaccination.
Adenovirus; Carcinoembryonic antigen; Proteome; Tumors; Vaccine
A lack of relevant animal models has hampered preclinical screening and critical evaluation of the efficacy of human vaccines in vivo. Carcinoembryonic antigen–A2Kb (CEA–A2Kb) double transgenic mice provide a biologically relevant model for preclinical screening and critical evaluation of human CEA vaccine efficacy in vivo, particularly because such animals are peripherally tolerant of CEA. We established the utility of this model by demonstrating that an oral DNA minigene vaccine induces effective HLA-A2–restricted, CEA-specific antitumor CTL responses. This finding is supported by three lines of evidence: (a) an effective HLA-A2–restricted, CEA691-specific CTL response; (b) specific in vitro killing of CEA-A2Kb transduced MC-38 colon carcinoma cells; and (c) protective immunity induced in vaccinated mice against challenges of these tumor cells. Importantly, peripheral T cell tolerance against CEA in CEA-A2Kb double transgenic mice was broken by the CEA691 (IMIGVLVGV) minigene vaccine. In conclusion, CEA-A2Kb double transgenic mice were demonstrated to be good candidates for in vivo testing of human CEA–based vaccines. This result suggests a potential for these vaccines in future human vaccine development. The feasibility of using nonmutated self-antigens as targets for therapeutic vaccinations was indicated, provided that such antigens are presented in an immunogenic context; that is, as a DNA minigene in a bacterial carrier system.
A 3-layer immunoperoxidase technique was used to demonstrate carcinoembryonic antigen (CEA) in colonic polyps from patients with or without previous or concurrent malignancy. CEA was demonstrated in a higher percentage of the polyps received as fresh specimens that were rapidly frozen and fixed in ethanol, than in formalin-fixed, paraffin-embedded sections. Tissue CEA content of both colonic carcinomas and polyps was determined by radioimmunoassay, and it was found that benign colonic tumours had levels of tissue CEA comparable to colonic cancer, indicating that CEA concentration in a tumour does not reflect its grade of malignancy. In fact, in one case in which both colonic cancer and polyps were removed, the polyps had the higher quantities of tissue CEA. Further, tissue CEA concentration of a polyp was not dependent on its size or location. Studying the titres of circulating CEA in these patients revealed an elevation of plasma CEA in one-third of the patients with only colonic polyps, whilst the patients with cancer all had increased titres.
The prognostic and predictive value of pretreatment serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) were assessed in advanced non-small cell lung cancer (NSCLC) patients treated with gefitinib or erlotinib.
Materials and Methods
Pretreatment CEA and CYFRA 21-1 were measured in 123 advanced NSCLC patients receiving gefitinib or erlotinib. High CEA levels (h-CEA) were significantly associated with females, patients with adenocarcinoma, and non-smokers.
Low CYFRA 21-1 levels (l-CYFRA) were significantly associated with a good performance status (ECOG PS 0-1). The overall response rate (RR) was 27.6%, and higher RR was associated with adenocarcinoma, h-CEA, and epidermal growth factor receptor (EGFR) mutation. Patients with h-CEA had significantly longer progression-free survival (PFS) (p=0.021). Patients with l-CYFRA had significantly longer PFS and overall survival (p=0.006 and p<0.001, respectively). Of note, h-CEA and l-CYFRA had good prognosis in patients with unknown EGFR mutation status or patients with squamous cell carcinoma (p=0.021 and p=0.015, respectively). A good ECOG PS (HR=0.45, p=0.017), h-CEA (HR=0.41, p=0.007), l-CYFRA 21-1 (HR=0.52, p=0.025), and an EGFR mutation (HR=0.22, p<0.001) were independently predictive of a longer PFS.
h-CEA and l-CYFRA 21-1 may be prognostic and predictive serum markers for higher response and longer survival in patients with advanced NSCLC receiving gefitinib or erlotinib, especially in patients with unknown EGFR mutation status or patients with squamous cell carcinoma.
Carcinoma; non-small-cell lung; biological markers; carcinoembryonic antigen; CYFRA 21-1; tyrosine kinase inhibitor
The prognostic and predictive value of pre-treatment serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) were assessed in advanced non-small cell lung cancer (NSCLC) patients treated with gefitinib or erlotinib. Pre-treatment CEA and CYFRA 21-1 levels were measured in 123 advanced NSCLC patients receiving gefitinib or erlotinib. High CEA levels (h-CEA) were significantly associated with females, patients with adenocarcinoma and non-smokers. Low CYFRA 21-1 levels (l-CYFRA 21-1) were significantly associated with a good performance status (ECOG PS 0-1). The overall response rate (RR) was 27.6%, and a higher RR was associated with adenocarcinoma, h-CEA, and epidermal growth factor receptor (EGFR) mutation. Patients with h-CEA had significantly longer progression-free survival (PFS) (P=0.021). Patients with l-CYFRA 21-1 had significantly longer PFS and overall survival (OS) (P=0.006 and P<0.001, respectively). Notably, h-CEA and l-CYFRA 21-1 levels were associated with good prognosis in patients with unknown EGFR mutation status or patients with squamous cell carcinoma (P=0.021 and P=015, respectively). A good ECOG PS (HR=0.45, P=0.017), h-CEA (HR=0.41, P=0.007), l-CYFRA 21-1 (HR=0.52, P=0.025), and an EGFR mutation (HR=0.22, P<0.001) were independently predictive of a longer PFS. A good ECOG PS (HR=0.52, P=0.018), l-CYFRA 21-1 (HR=0.36, P=0.004), and EGFR mutation (HR=0.53, P=0.051) were independently predictive of longer OS. h-CEA and l-CYFRA 21-1 may be prognostic and predictive serum markers for higher response and longer survival in patients with advanced NSCLC receiving gefitinib or erlotinib, particularly in patients with unknown EGFR mutation status or patients with squamous cell carcinoma.
carcinoma; non-small cell lung cancer; biological markers; carcinoembryonic antigen; cytokeratin-19 fragments; tyrosine kinase inhibitor