A critical review of biologic mesh use in ventral hernia repairs under contaminated conditions.

PURPOSE: We used an evidence-based approach to determine whether the promotions and claims of superiority of biologic mesh over synthetic mesh use in ventral hernia repairs (VHRs) under contaminated conditions were sound and valid. METHODS: We searched the Medline database to specifically identify review articles relating to biologic mesh and VHR and critically reviewed these studies using an evidence-based approach. RESULTS: For the past 45 years, four clinical reviews and one systematic review have included biologic meshes as part of a larger discussion on available prosthetics for VHR. All reviews supported biologic mesh use, especially in the setting of contaminated fields. Yet, the primary literature included in these reviews and served as the basis for these conclusions consisted entirely of case series and case reports, which have the lowest level of evidence in determining scientific validity. Furthermore, the FDA has neither cleared nor approved this particular use. CONCLUSIONS: The cumulative data regarding biologic mesh use in VHRs under contaminated conditions does not support the claim that it is better than synthetic mesh used under the same conditions. The highly promoted and at least moderately utilized practice of placing biologic mesh in contamination is being done outside of the original intended use, and a re-evaluation of or possible moratorium on biologic mesh use in hernia surgery is seriously warranted. Alternatively, an industry-sponsored national registry of patients in whom ventral hernia repairs involved biologic mesh would substantively add to our understanding regarding how these intriguing biomaterials are being used and their overall clinical efficacy.

Successful cancer vaccine therapy for carcinoembryonic antigen (CEA)-expressing colon cancer using genetically modified dendritic cells that express CEA and T helper-type 1 cytokines in CEA transgenic mice.

This study was designed to determine whether the vaccination of genetically modified dendritic cells (DCs) simultaneously expressing carcinoembryonic antigen (CEA), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 12 (IL-12) can overcome the peripheral T-cell tolerance to CEA and thereby elicit a therapeutic response in CEA transgenic mice. CEA transgenic mice were immunized once by subcutaneous injection with DCs adenovirally transduced with CEA and T helper-type 1 cytokine genes. The cytotoxic activity of spleen cells against CEA-expressing tumors, MC38-CEA, in the mice immunized with DCs expressing CEA (DC-AxCACEA) was higher than that in those immunized with DCs-AxCALacZ (p < 0.0001), and was augmented by the cotransduction with the GM-CSF/IL-12 gene (p < 0.05). The vaccination with DC-AxCACEA/GM-CSF/IL-12 could elicit a more potent therapeutic immunity than the vaccination with DC-AxCACEA in subcutaneous tumor models (p < 0.0001), and 4 of 5 mice showed a complete eradication of the subcutaneous tumors in these vaccination groups. Even in a large tumor model, this vaccination therapy completely eliminated the subcutaneous tumors in all mice. This antitumor activity mostly vanished with the depletion of CD8(+) T cells and NK cells in vivo and was completely abrogated with the depletion of CD4(+) T cells. A histopathological examination showed no evidence of an autoimmune reaction. No other adverse effects were observed. This vaccination strategy resulted in the generation of highly efficient therapeutic immune responses against MC38-CEA in the absence of autoimmune responses and demonstrated no adverse effects, and may therefore be useful for future clinical applications as a cancer vaccine therapy.

Adenovirus fiber shaft contains a trimerization element that supports peptide fusion for targeted gene delivery.

Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice.

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund's adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1-CpG-immunized mice showed an increased proliferative CD4(+) Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-gamma). This vaccine also induced MHC class I antigen-restricted CD8(+) T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1-CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1-QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.

DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor.

The interaction of NKG2D, a stimulatory receptor expressed on natural killer (NK) cells and activated CD8(+) T cells, and its ligands mediates stimulatory and costimulatory signals to these cells. Here, we demonstrate that DNA-based vaccines, encoding syngeneic or allogeneic NKG2D ligands together with tumor antigens such as survivin or carcinoembryonic antigen, markedly activate both innate and adaptive antitumor immunity. Such vaccines result in highly effective, NK- and CD8(+) T cell-mediated protection against either breast or colon carcinoma cells in prophylactic and therapeutic settings. Notably, this protection was irrespective of the NKG2D ligand expression level of the tumor cells. Hence, this strategy has the potential to lead to widely applicable and possibly clinically useful DNA-based cancer vaccines.

Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2.

Angiogenesis is a critical mechanism for tumor progression. Multiple studies have suggested that tumor growth can be suppressed if tumor angiogenesis can be inhibited using various types of antiangiogenic agents. Recent studies in mouse systems have shown that tumor angiogenesis can also be inhibited if cellular immune response could be induced against vascular endothelial growth factor receptor 2 (VEGFR2), which is one of the key factors in tumor angiogenesis. In this study, we examined the possibility of developing this novel immunotherapy in clinical setting. We first identified the epitope peptides of VEGFR2 and showed that stimulation using these peptides induces CTLs with potent cytotoxicity in the HLA class I-restricted fashion against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR2. In A2/Kb transgenic mice that express alpha1 and alpha2 domains of human HLA-A*0201, vaccination using these epitope peptides in vivo was associated with significant suppression of the tumor growth and prolongation of the animal survival without fatal adverse effects. In antiangiogenesis assay, tumor-induced angiogenesis was significantly suppressed with the vaccination using these epitope peptides. Furthermore, CTLs specific to the epitope peptides were successfully induced in cancer patients, and the specificities of the CTLs were confirmed using functional and HLA-tetramer analysis. These results in vitro and in vivo strongly suggest that the epitope peptides derived from VEGFR2 could be used as the agents for antiangiogenic immunotherapy against cancer in clinical settings.

Dendritic cells pulsed with an anti-idiotype antibody mimicking carcinoembryonic antigen (CEA) can reverse immunological tolerance to CEA and induce antitumor immunity in CEA transgenic mice.

In this report, we have studied the immunogenicity of the nominal antigen, carcinoembryonic antigen (CEA), and that of an anti-idiotype antibody, 3H1, which mimics CEA and can be used as a surrogate for CEA. We have demonstrated that immunization of CEA transgenic mice with bone marrow-derived mature dendritic cells (DC) loaded with anti-idiotype 3H1 or CEA could reverse CEA unresponsiveness and result in the induction of CEA-specific immune responses and the rejection of CEA-transfected MC-38 colon carcinoma cells, C15. Immunized mice splenocytes proliferated in an antigen-specific manner by a mechanism dependent on the functions of CD4, MHC II, B7-2, CD40, CD28, and CD25. However, immune splenic lymphocytes isolated from 3H1-DC-vaccinated mice when stimulated in vitro with 3H1 or CEA secreted significantly higher levels of Th1 cytokines than did CEA-DC vaccinated mice. DC vaccination also induced antigen-specific effector CD8+ T cells capable of expressing interleukin-2, IFN-gamma, and tumor necrosis factor (TNF)-alpha and displayed cytotoxic activity against C15 cells in an MHC class I-restricted manner. 3H1-DC vaccination resulted in augmented CTL responses and the elevated expression of CD69, CD25, and CD28 on CD8(+) CTLs. The immune responses developed in 3H1-DC-immunized mice resulted in rejection of C15 tumor cells in nearly 100% of experimental mice, whereas only 40% of experimental mice immunized with CEA-DC were protected from C15 tumor growth. These findings suggest that under the experimental conditions used, 3H1-DC vaccination was better than CEA-DC vaccination in breaking immune tolerance to CEA and inducing protective antitumor immune responses in this murine model transgenic for human CEA.

A novel transgenic mouse model for immunological evaluation of carcinoembryonic antigen-based DNA minigene vaccines.

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, CEA(691)-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 CEA(691) (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.

In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts.

The concept of antibody-mediated targeting of antigenic MHC/peptide complexes on tumor cells in order to sensitize them to T-lymphocyte cytotoxicity represents an attractive new immunotherapy strategy. In vitro experiments have shown that an antibody chemically conjugated or fused to monomeric MHC/peptide can be oligomerized on the surface of tumor cells, rendering them susceptible to efficient lysis by MHC-peptide restricted specific T-cell clones. However, this strategy has not yet been tested entirely in vivo in immunocompetent animals. To this aim, we took advantage of OT-1 mice which have a transgenic T-cell receptor specific for the ovalbumin (ova) immunodominant peptide (257-264) expressed in the context of the MHC class I H-2K(b). We prepared and characterized conjugates between the Fab' fragment from a high-affinity monoclonal antibody to carcinoembryonic antigen (CEA) and the H-2K(b) /ova peptide complex. First, we showed in OT-1 mice that the grafting and growth of a syngeneic colon carcinoma line transfected with CEA could be specifically inhibited by systemic injections of the conjugate. Next, using CEA transgenic C57BL/6 mice adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, we demonstrated that systemic injections of the anti-CEA-H-2K(b) /ova conjugate could induce specific growth inhibition and regression of well-established, palpable subcutaneous grafts from the syngeneic CEA-transfected colon carcinoma line. These results, obtained in a well-characterized syngeneic carcinoma model, demonstrate that the antibody-MHC/peptide strategy can function in vivo. Further preclinical experimental studies, using an anti-viral T-cell response, will be performed before this new form of immunotherapy can be considered for clinical use.

Murine dendritic cells pulsed with an anti-idiotype antibody induce antigen-specific protective antitumor immunity.

In this study, using the carcinoembryonic antigen (CEA)-expressing C15 murine colon carcinoma system in syngeneic C57BL/6 mice, we have evaluated the efficacy of bone marrow-derived dendritic cells (DCs) pulsed with the murine anti-idiotype antibody 3H1 as a tumor vaccine. Anti-idiotype 3H1 mimics a distinct and specific epitope of CEA and can generate anti-CEA immunity in mice, rabbits, monkeys, and humans when used with a conventional immune adjuvant. Our goal was to determine whether the use of DC as direct antigen-presenting cells would improve the potency of 3H1 as vaccine. Bone marrow-DC pulsed with 3H1 and injected into naïve mice induced both humoral and cellular anti-3H1, as well as anti-CEA immunity. Specific killing of C15 cells in in vitro antibody-dependent cellular cytotoxicity has been observed by immune sera. Immune-splenic lymphocytes when stimulated in vitro with 3H1 or CEA, showed increased proliferative CD4(+) Th1 type T-cell response and secreted significantly high levels of Th1 cytokines [IFN-gamma, interleukin (IL)-2] and low levels of Th2 cytokines (IL-4, IL-10). This vaccine also induced MHC class I antigen-restricted CD8(+) T-cell responses. The up-regulation of activation markers CD69 and CD25 on CD8(+) CTLs correlated with antigen-specific strong CTL responses in vitro. The immunity induced in mice resulted in a complete rejection of CEA-expressing C15 tumor cells in 100% of experimental mice, whereas no protection was observed when 3H1-pulsed DC-vaccinated mice were challenged with CEA-negative MC-38 cells. The tumor rejection in 3H1-pulsed DC-treated mice was associated with the induction of a memory response that helped those mice to survive a second challenge with a lethal dose of C15 cells.

Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice.

A carcinoembryonic antigen (CEA)-based DNA vaccine, adsorbed onto cationic microparticles of poly(DL-lactide-co-glycolide) (PLG) induced tumor-protective immunity against a lethal challenge of MC38-CEA colon carcinoma cells in CEA-transgenic mice that was more potent than that of the corresponding naked DNA vaccine. Boosting with a plasmid encoding murine GM-CSF increased the vaccine's efficacy leading to a complete rejection of tumor cells in 50% of mice. This effect was due to activation of MHC class I-restricted CD8(+) T cells coupled with an increased secretion of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-2. Also, specific activation of dendritic cells was indicated by a two-three-fold upregulation of their costimulatory CD80 and MHC class II molecules. This approach may be a promising new strategy for the rational design of cancer vaccines for future clinical applications.

A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice.

A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells. Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). These conclusions are supported by four lines of evidence. First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2. Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1. Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12. Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69. Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.

An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice.

A DNA vaccine encoding human carcinoembryonic antigen (CEA) broke peripheral T-cell tolerance toward this tumor self-antigen expressed by Lewis lung carcinoma stably transduced with CEA in C57BL/6J mice transgenic for CEA. This vaccine, delivered by oral gavage with an attenuated strain of Salmonella typhimurium (SL7207), and boosted with an antibody-IL2 fusion protein, induced tumor-protective immunity mediated by MHC class I antigen-restricted CD8(+) T cells, resulting in eradication of subcutaneous tumors in 100% of mice and prevention of experimental pulmonary metastases in 75% of experimental animals. Both CTL and antigen-presenting dendritic cells were activated as indicated by a decisive increase in their respective activation markers CD2, CD25, CD28 as well as CD48 and CD80. The antitumor effects of this CEA-based DNA vaccine obtained in prophylactic settings, suggest that this approach could lead to the rational design of effective treatment modalities for human lung cancer.

Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice.

Peripheral T-cell tolerance toward human carcinoembryonic self-antigen (CEA) was broken in CEA-transgenic C57BL/6J mice by an oral CEA-based DNA vaccine. This vaccine, delivered by the live, attenuated AroA- strain of Salmonella typhimurium (SL7207), induced tumor-protective immunity mediated by MHC class I-restricted CD8+ T cells. Activation of these T cells was indicated by increased secretion of proinflammatory cytokines IFN-gamma, interleukin (IL)-12 and granulocyte/macrophage-colony stimulating factor, as well as specific tumor rejection and growth suppression in vaccinated CEA-transgenic mice after a lethal challenge with murine MC38 colon carcinoma cells. These tumor cells were double transfected with CEA and the human epithelial cell adhesion molecule (Ep-CAM)/KSA and consequently served as a docking site for a recombinant antibody-IL2 fusion protein (KS1/4-IL2) recognizing KSA. Importantly, the efficacy of the tumor-protective immune response was markedly increased by boosts with this antibody-IL2 fusion protein, resulting in more effective tumor rejection coupled with increased expression of costimulatory molecules B7.2/B7.2 and intercellular adhesion molecule 1 (ICAM-1) on dendritic cells and intensified release of proinflammatory cytokines IFN-gamma, IL-12, and granulocyte/macrophage-colony stimulating factor from T cells of successfully vaccinated CEA-transgenic C57BL/6J mice. Increased T-cell activation mediated by boosts with KS1/4-IL2 fusion protein after tumor cell challenge was further indicated by expanded expression of T-cell activation markers CD25, CD28, CD69, and LFA-1. The application of such CEA-based DNA vaccines and its further improved versions may ultimately prove useful in combination therapies directed against human carcinomas expressing CEA self-antigens.

Targeting and therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibody-interleukin 2 fusion protein.

The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic antigen (CEA) antibody and an interleukin 2 (IL-2) fusion protein derivative for selective tumor targeting of cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody [single-chain variable fragment joined to the crystallizable fragment, Fc (scFvFc)]. The fusion protein (scFvFc.IL-2) consisted of mouse IL-2-fused to the COOH-terminal end of the scFvFc. The engineered proteins were assembled as complete molecules and were similar to the intact anti-CEA monoclonal antibody (Mab) in antigen-binding properties. Based on IL-2 content of the fusion protein, its ability to support proliferation of CTLL-2 cells was identical with that of IL-2. Despite a molecular size similar to that of the intact Mab, the blood clearance of the fusion protein was markedly faster than that of the intact Mab or scFvFc. Incubation of radiolabeled scFvFc.IL-2 but not the intact or scFvFc antibodies in mouse serum was accompanied by the appearance of complexes, suggesting that the latter may contribute to the accelerated clearance of the fusion protein. Biodistribution and tumor targeting studies were carried out in CEA-transgenic mice bearing CEA-positive murine tumors as well as the antigen-negative parental tumor. The bivalent anti-CEA scFvFc had tumor localization properties similar to those of the intact Mab. Although fusion of IL-2 to the COOH-terminal end of the bivalent scFvFc altered its pharmacokinetic properties, the fusion antibody was able to target tumors specifically. Maximum uptake of the intact Mab, scFvFc, and scFvFc.IL-2 in CEA-positive tumors was 29.3 +/- 5.0, 19.5 +/- 2.1, and 6.6 +/- 0.9% injected dose/g, respectively. Maximum tumor localization ratios (CEA-positive/CEA-negative tumor) were similar for all three antibody types (4.6-6.0), demonstrating the antigen specificity of the tumor targeting. Significant antigen-specific targeting to CEA-positive normal tissues of transgenic mice was not observed. Although the tumor-targeting properties of the fusion protein were low, the growth of CEA-expressing (P = 0.01) but not antigen-irrelevant (P = 0.22) syngeneic tumor cells was inhibited after treatment of transgenic mice with the anti-CEA-IL-2 antibody. Therapy of CEA-expressing tumors was improved after i.v. administration of the fusion protein (P = 0.0001). These studies indicate that anti-CEA antibody-directed cytokine targeting may offer an effective treatment for CEA-expressing carcinomas. The availability of an immunocompetent CEA transgenic mouse model will also help to determine the immunotherapeutic properties of these fusion proteins.

Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen.

In order to develop immunotherapy strategies that are based on eliciting immune responsiveness to the self-antigen, human carcinoembryonic antigen (CEA), we examined whether cytotoxic T lymphocyte (CTL) activity against CEA could be elicited in CEA-transgenic and nontransgenic mice. CEA-transgenic [C57BL/ 6-TGN(CEAGe)18FJP] and nontransgenic mice were primed with CEA-transfected syngeneic fibroblasts in combination with Corynebacterium parvum. Spleen cells from immunized mice were cultured with irradiated syngeneic MC-38 colon carcinoma cells transfected with CEA (MC-38.CEA) as stimulators prior to the measurement of CTL activity. Primed nontransgenic spleen cells showed augmented CTL activity against MC-38.CEA cells as compared with control parental MC-38 cells, nontransfected or transfected with vector only. Moreover, primed CEA transgenic spleen cells showed augmented CTL activity against MC-38.CEA cells that was similar to that observed in nontransgenic mice. All CTL clones derived from either transgenic or nontransgenic mice showed cross-reactivity with MC-38 cells expressing the CEA-related antigen, nonspecific cross-reacting antigen, but not biliary glycoprotein. CEA-specific CTL clones were not identified. Adoptive transfer of cloned CTL resulted in inhibition of MC-38.CEA but not MC-38.BGP tumor growth. Tumor cures were elicited in mice treated with a combination of cloned CTL and cyclophosphamide. Histopathological examination of CEA-expressing colons from either immunized mice or recipients of cloned CTL did not reveal any autoimmune reactions. These studies demonstrate that CTL recognizing cross-reactive class I epitopes on the CEA molecule can be induced in transgenic mice. The expression of these epitopes on tumor cells creates effective targets for CTL in vivo without inducing adverse reactions in CEA-expressing normal tissues. Since anti-CEA CTL have been generated in humans, CEA-transgenic mice may be a useful model to study vaccines that are based on CTL effector mechanisms.

Tumor targeting with radiolabeled antibodies in a human carcinoembryonic antigen transgenic mouse model.

Mice transgenic for the carcinoembryonic (CEA) gene were used to study the biodistribution and tumor targeting of a radioiodinated monoclonal antibody (MAb), T84.66. The specificity of antibody uptake in tumors was assessed in mice bearing a CEA-transfected syngeneic tumor as well as the antigen-negative parental tumor. With high CEA-expressing tumors, the percent injected dose per gram (%ID/g) approached 30% at 48 hr. Tumor uptake in antigen-positive tumors was 5-8-fold higher than that observed in the antigen-negative parental tumors. Only antigen-positive tumors were visualized by immunoscintigraphy. The tumor targeting obtained in athymic nude mice bearing human tumor xenografts was similar to that observed with CEA-expressing murine tumors implanted in either athymic nude or transgenic mice. The degree of localization of CEA-transfected murine tumors was related with the level of antigen expression. Circulating antigen-radio-antibody complexes were not detected while blood clearance of radio-antibody was similar between transgenic and non-transgenic mice. With the exception of the large bowel, the distribution of radioiodinated MAb in normal tissues was similar in both CEA transgenic and non-transgenic mice. Increased localization of intact antibody was observed in the large bowel from transgenic mice, suggesting specific targeting to antigen-positive normal tissues. These results suggest that the CEA transgenic mouse model will be useful in the development of antibodies for radio-immunodetection and treatment of carcinomas expressing CEA.

Dose escalation trial of indium-111-labeled anti-carcinoembryonic antigen chimeric monoclonal antibody (chimeric T84.66) in presurgical colorectal cancer patients.

UNLABELLED: Chimeric T84.66 (cT84.66) is a high-affinity (1.16x10(11) M(-1)) IgG1 monoclonal antibody against carcinoembryonic antigen (CEA). The purpose of this pilot trial was to evaluate the tumor-targeting properties, biodistribution, pharmacokinetics and immunogenicity of 111In-labeled cT84.66 as a function of administered antibody protein dose. METHODS: Patients with CEA-producing colorectal cancers with localized disease or limited metastatic disease who were scheduled to undergo definitive surgical resection were each administered a single intravenous dose of 5 mg of isothiocyanatobenzyl diethylenetriaminepentaacetic acid-cT84.66, labeled with 5 mCi of 111In. Before receiving the radiolabeled antibody, patients received unlabeled diethylenetriaminepentaacetic acid-cT84.66. The amount of unlabeled antibody was 0, 20 or 100 mg, with five patients at each level. Serial blood samples, 24-hr urine collections and nuclear images were collected until 7 days postinfusion. Human antichimeric antibody response was assessed up to 6 mo postinfusion. RESULTS: Imaging of at least one known tumor site was performed in all 15 patients. Fifty-two lesions were analyzed, with an imaging sensitivity rate of 50.0% and a positive predictive value of 76.9%. The antibody detected tumors that were not detected by conventional means in three patients, resulting in a modification of surgical management. Interpatient variations in serum clearance rates were observed and were secondary to differences in clearance and metabolic rates of antibody and antibody:antigen complexes by the liver. Antibody uptake in primary tumors, metastatic sites and regional metastatic lymph nodes ranged from 0.4% to 134% injected dose/kg, resulting in estimated 90Y-cT84.66 radiation doses ranging from 0.3 to 193 cGy/mCi. Thirteen patients were evaluated 1-6 mo after infusion for human antichimeric antibody, and none developed a response. No major differences in tumor imaging, tumor uptake, pharmacokinetics or organ biodistribution were observed with increasing protein doses, although a trend toward increasing blood uptake and decreasing liver uptake was observed with increasing protein dose. CONCLUSION: Chimeric T84.66 demonstrated tumor targeting comparable to other radiolabeled intact anti-CEA monoclonal antibodies. Its immunogenicity after single administration was lower than murine monoclonal antibodies. These properties make 111In-cT84.66, or a lower molecular weight derivative, attractive for further evaluation as an imaging agent. Yttrium-90 dosimetry estimates predict potentially cytotoxic radiation doses to select tumor sites, which makes 90Y-cT84.66 also appropriate for further evaluation in Phase I radioimmunotherapy trials. Although clinically important changes in biodistribution, pharmacokinetics and tumor targeting with increasing protein doses of 111In-cT84.66 were not demonstrated, the results do suggest that antibody clearance from the blood is driven by hepatic uptake and metabolism, with more rapid blood clearance seen in patients with liver metastases. These patients with rapid clearance and potentially unfavorable biodistribution for imaging and therapy may, therefore, be a more appropriate subset in which to evaluate the role of administering higher protein doses. This underscores the need to further identify, characterize and understand those factors that influence the biodistribution and clearance of radiolabeled anti-CEA antibodies, to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.