Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine.

BACKGROUND: Human carcinoembryonic antigen (CEA) is a 180-kd glycoprotein expressed in human colorectal, gastric, pancreatic, breast, and non-small-cell lung carcinomas. Previous studies have demonstrated enhanced immune responses to other antigens presented with vaccinia virus proteins via a recombinant vaccinia virus construct. In addition, we have developed a recombinant CEA-vaccinia virus construct, designated rV(WR)-CEA, and have demonstrated humoral anti-CEA responses in mice after immunization with that virus. PURPOSE: The goals of this study were (a) to construct a recombinant CEA-vaccinia vaccine in a less virulent vaccinia strain that is potentially safe and effective for treatment of patients whose tumors express CEA and (b) to evaluate the ability of the recombinant CEA-vaccinia vaccine to prevent and reverse tumor growth in mice and to elicit cell-mediated and humoral anti-CEA immune responses. METHODS: Using the New York City strain of vaccinia virus, which is used in smallpox vaccination and is more attenuated for humans than rV(WR), we derived a recombinant CEA-vaccinia construct, designated rV(NYC)-CEA. The ability of this construct to induce antitumor immunity was evaluated in mice receiving subcutaneous injections of murine colon adenocarcinoma cells expressing the human CEA gene. RESULTS: Administration of rV(NYC)-CEA in mice induced strong anti-CEA antibody responses, as well as CEA-specific cell-mediated responses, including delayed-type hypersensitivity, lymphoproliferative, and cytotoxic responses. Vaccination of mice with the rV(NYC)-CEA rendered them resistant to the growth of subsequently transplanted CEA-expressing tumors. Moreover, when mice were vaccinated 7 days after tumor cell injection, tumor growth was either greatly reduced or eliminated. No toxic effects were observed in any of the mice. CONCLUSION: These studies demonstrate that antitumor activity can be induced with the use of a recombinant CEA-vaccinia virus construct derived from an attenuated vaccinia strain, and they reveal the range of cell-mediated and humoral responses induced by this recombinant vaccine.     

Vector-based vaccine/cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity

Many antigens associated with human tumors are overexpressed in tumor cells as compared with normal tissues; these "self" tumor-associated antigens are also expressed during fetal development, and it is, thus, not surprising that they are either weakly immunogenic or functionally nonimmunogenic in the tumor-bearing host. In the studies reported here, we have used different vaccines and vaccine strategies in an attempt to develop antitumor immunity in a stringent animal model. The tumor antigen used was human carcinoembryonic antigen (CEA). The model used was CEA transgenic mice, in which the human CEA transgene is under the control of the endogenous CEA promoter; CEA is expressed in fetal tissues and normal gastrointestinal tissues, and CEA protein is found in sera. Previous studies have shown these CEA transgenic mice to be tolerant to the induction of CEA immunity using CEA protein in adjuvant as an immunogen. CEA-expressing tumor cells were implanted 14 days before vaccine therapy. The vaccines used were recombinant vaccinia virus containing the transgenes for CEA and three T-cell costimulatory molecules [B7-1, ICAM-1, and LFA-3, designated recombinant vaccinia (rV)-CEA/TRICOM], with each transgene under the control of individual poxvirus promoters, and a replication-defective avipox virus (fowlpox; rF) containing the same four transgenes (designated rF-CEA/TRICOM). The results demonstrate that (a) continued boosting with vaccine is required to maintain CEA-specific T-cell responses, and boosting with rF-CEA/TRICOM is superior to boosting with rF-CEA; (b) a diversified vaccination protocol consisting of primary vaccination with rV-CEA/TRICOM followed by boosting with rF-CEA/TRICOM is more efficacious than homogeneous vaccination with rF-CEA/TRICOM in the induction of both CEA-specific T-cell responses and antitumor activity; and (c) the use of cytokines, local granulocyte macrophage colony-stimulating factor (GM-CSF) and low-dose systemic interleukin 2, in combination with vaccine is essential in inducing antitumor activity, as compared with the use of cytokines alone, or the use of vaccines without cytokine. Both GM-CSF and interleukin 2 were shown to contribute to the induction of CEA-specific T-cell responses. These studies thus provide a "proof of concept" that potent vaccines and vaccine strategies, in combination with cytokines, may be essential to obtain the level of T-cell responses directed against a self-antigen that is necessary to achieve antitumor responses.     

癌胚抗原重组痘苗病毒转染树突状细胞诱导CEA特异性T细胞免疫

 目的　研究人癌胚抗原重组痘苗病毒 (rV CEA)转染树突状细胞 (DC)在体外诱导CEA特异性的T细胞免疫。方法　将rV CEA转染外周血单个核细胞来源的DC后用于激发自体的T细胞 ,通过对T细胞的增殖及杀伤功能的检测 ,与野生型痘苗病毒 (V 76 1)转染的DC激发的T细胞进行比较。结果　经rV CEA转染的DC激活的T细胞增殖力强 ,对CEA分泌性肿瘤细胞具特异性杀伤作用。结论　rV CEA转染的DC可以诱导CEA特异性T细胞活性。     

A recombinant vaccinia virus expressing human prostate-specific antigen (PSA): Safety and immunogenicity in a non-human primate

Prostate-specific antigen (PSA) is. a serine protease secreted by prostatic epithelial cells and is widely used as a marker for prostate cancer. The tissue specificity of PSA makes it a potential target for active specific immunotherapy, especially in prostate cancer patients who have undergone prostatectomy and in whom the only PSA-expressing tissue in the body resides in metastatic deposits. We report here the cloning, construction and immunological consequences of immunization of rhesus monkeys with a recombinant vaccinia virus expressing human PSA (designated rV-PSA). The prostate gland of the rhesus is structurally and functionally similar to the human prostate. While rodent and other mammalian species do not share homology with human PSA, there is 94% homology between the amino acid sequences of rhesus and human PSA. Immunization of rhesus monkeys with wild-type vaccinia virus or rV-PSA elicited the usual low-grade constitutional symptoms of vaccinia virus infection. There was no evidence of any adverse effects in any immunized monkeys. A short-lived PSA-specific IgM antibody response was noted in all rV-PSA immunized monkeys regardless of dose level. All monkeys receiving the 10⁸pfu dose of rV-PSA demonstrated PSA-specific T-cell responses that were maintained up to 270 days. No differences in anti-PSA immune responses or toxicity were observed in animals that received prostatectomy prior to immunization. Our results thus demonstrate the safety and immunogenicity of rV-PSA in a non-human primate and have implications for potential specific immunotherapy protocols using PSA as a target.     

Monkey antisera with increased specificity to carcino-embryonic antigen (CEA).

Three Macaca irus monkeys were immunized with purified human CEA. One received unmodified CEA and two were immunized with haptenated (4-hydroxy-5-iodo-3-nitrophenyl acetyl) CEA. All three monkeys formed precipitating antibodies to CEA. The antisera did not precipitate the CEA-related normal glycoprotein antigen (NCA). In contrast, CEA-immunized rabbits, sheep and goats invariably form antibodies against NCA. Radio-immunoassays showed the monkey antisera to contain trace amounts of antibodies to NCA but the titers were about 10-1,000 times lower than those in rabbit and sheep sera with comparable anti-CEA titers. These results show that monkeys produce antibodies against CEA, but respond weakly to a normal, CEA-related antigen. The weak response to NCA suggests that monkeys may possess an NCA-related antigen. Antisera lacking reactivity to normal CEA-related antigens may be helpful in establishing a more specific diagnostic test for CEA.     

Phase I study of sequential vaccinations with fowlpox-CEA(6D)-TRICOM alone and sequentially with vaccinia-CEA(6D)-TRICOM, with and without granulocyte-macrophage colony-stimulating factor, in patients with carcinoembryonic antigen-expressing carcinomas.

PURPOSE: Our previous clinical experience with vaccinia and replication-defective avipox recombinant carcinoembryonic antigen (CEA) vaccines has demonstrated safety and clinical activity with a correlation between CEA-specific immune response and survival. Preclinical evidence demonstrated that the addition of the transgenes for three T-cell costimulatory molecules (B7-1, ICAM-1, LFA-3, designated TRICOM) results in a significant improvement in antigen-specific T-cell responses and antitumor activity. We describe here the first trial in humans of the CEA-TRICOM vaccines (also including an enhancer agonist epitope within the CEA gene). PATIENTS AND METHODS: Fifty-eight patients with advanced CEA-expressing cancers were accrued to eight cohorts that involved vaccinations with the following: replication-defective fowlpox recombinant (rF)-CEA(6D)-TRICOM; primary vaccination with recombinant vaccinia (rV)-CEA(6D)-TRICOM plus rF-CEA(6D)-TRICOM booster vaccinations; and rV-CEA(6D)-TRICOM and then rF-CEA(6D)-TRICOM, plus granulocyte-macrophage colony-stimulating factor (GM-CSF) with vaccines, or with divided doses of vaccine with GM-CSF. Vaccines were administered every 28 days for six doses and then once every 3 months. Reverting to treatments every 28 days was allowed if patients progressed on the 3-month schedule. RESULTS: In this phase I study, no significant toxicity was observed. Twenty-three patients (40%) had stable disease for at least 4 months, with 14 of these patients having prolonged stable disease (> 6 months). Eleven patients had decreasing or stable serum CEA, and one patient had a pathologic complete response. Enhanced CEA-specific T-cell responses were observed in the majority of patients tested. CONCLUSION: We demonstrated that the CEA-TRICOM vaccines are safe and can generate significant CEA-specific immune responses, and they seem to have clinical benefit in some patients with advanced cancer.     

A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA)

Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.     

Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses

Cancer vaccine regimens use various strategies to enhance immune responses to specific tumor-associated antigens (TAAs), including the increasing use of recombinant poxviruses [vaccinia (rV) and fowlpox (rF)] for delivery of the TAA to the immune system. However, the use of replication competent vectors with the potential of adverse reactions have made attenuation a priority for next-generation vaccine strategies. Modified vaccinia Ankara (MVA) is a replication defective form of vaccinia virus. Here, we investigated the use of MVA encoding a tumor antigen gene, carcinoembryonic antigen (CEA), in addition to multiple costimulatory molecules (B7-1, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3 designated TRICOM). Vaccination of mice with MVA-CEA/TRICOM induced potent CD4+ and CD8+ T-cell responses specific for CEA. MVA-CEA/TRICOM could be administered twice in vaccinia na?ve mice and only a single time in vaccinia-immune mice before being inhibited by antivector-immune responses. The use of MVA-CEA/TRICOM in a diversified prime and boost vaccine regimen with rF-CEA/TRICOM, however, induced significantly greater levels of both CD4+ and CD8+ T-cell responses specific for CEA than that seen with rV-CEA/TRICOM prime and rF-CEA/TRICOM boost. In a self-antigen tumor model, the diversified MVA-CEA/TRICOM/rF-CEA/ TRICOM vaccination regimen resulted in a significant therapeutic antitumor response as measured by increased survival, when compared with the diversified prime and boost regimen, rV-CEA/TRICOM/rF-CEA/TRICOM. The studies reported here demonstrate that MVA, when used as a prime in a diversified vaccination, is clearly comparable with the regimen using the recombinant vaccinia in both the induction of cellular immune responses specific for the "self"-TAA transgene and in antitumor activity.     

载体对癌胚抗原DNA疫苗在小鼠体内表达CEA及诱导体液免疫应答的影响

目的　比较用pcDNA3和pCI-neo做载体构建的癌胚抗原 (CEA)真核表达质粒在小鼠体内表达CEA及诱导体液免疫应答的差别。方法　将两套重组质粒分别肌注BABL/c小鼠 ,ELISA法检测二者在小鼠体内表达CEA及抗 -CEA水平。结果　以pCI -CEA免疫的小鼠体内表达CEA量高于pcDNA3-CEA组 ,二者OD值分别为 0 65和 0 35 ;抗体量的表达前者OD值为 0 89,后者为0 70 ,比较差异有显著性 (P <0 .0 5 )。结论　表达载体的结构对抗原的表达及抗体的产生具有重要作用     

Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses.

PURPOSE: This trial sought to determine, for the first time, the validity in human vaccinations of using two different recombinant vaccines in diversified prime-and-boost regimens to enhance T-cell responses to a tumor antigen. PATIENTS AND METHODS: Eighteen patients with advanced tumors expressing carcinoembryonic antigen (CEA) were randomized to receive either recombinant vaccinia (rV)-CEA followed by three avipox-CEA vaccinations, or avipox-CEA (three times) followed by one rV-CEA vaccination. Subsequent vaccinations in both cohorts were with avipox-CEA. Immunologic monitoring was performed using a CEA peptide and the enzyme-linked immunospot assay for interferon gamma production. RESULTS: rV-CEA followed by avipox-CEA was superior to the reverse order in the generation of CEA-specific T-cell responses. Further increases in CEA-specific T-cell precursors were seen when local granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose interleukin (IL)-2 were given with subsequent vaccinations. The treatment was extremely well tolerated. Limited clinical activity was seen using vaccines alone in this patient population. Antibody production against CEA was also observed in some of the treated patients. CONCLUSION: rV-CEA was more effective in its role as a primer of the immune system; avipox-CEA could be given up to eight times with continued increases in CEA T-cell precursors. Future trials should use rV-CEA first followed by avipox-CEA. Vaccines specific to CEA are able to generate CEA-specific T-cell responses in patients without significant toxicity. T-cell responses using vaccines alone may be inadequate to generate significant anticancer objective responses in patients with advanced disease. Cytokines such as GM-CSF and IL-2 may play a key role in generating such responses.     

突变N-ras基因重组痘苗病毒rV-N-ras/61的构建

目的：构建Ｎ－ｒａｓ基因第６１位密码子突变基因（Ｎ－ｒａｓ／６１）的重组痘苗病毒,通过痘苗病毒载体所产生的蛋白的强烈免疫原性来提高变异Ｎ－ｒａｓ蛋白有肿瘤抗原性。方法：利用双ＰＣＲ方法得到第６１位突变的Ｎ－ｒａｓ／６１基因片段（５００ｂｐ）,将该基因生段插入痘苗病毒表达载体Ｐ１１０８,通过Ｃｅｌｌ ＦＥＣＴＩＮ转染ＣＶ－１细胞,以ＰＣＲ,Ｗｅｓｔｅｒｎ ｂｌｏｔ方法鉴定重组痘苗病毒。结果：通过ｔｋ＾－筛选、ＰＣＲ鉴定,得了第６１位突变Ｎ－ｒａｓ／６１基因的重组痘苗病毒ｒＶ－Ｎ－ｒａｓ／６１,Ｗｅｓｔｅｒｎ ｂｌｏｔ检测其在ＣＶ－１细胞中有表达。结论：目前对突变ｒａｓ基因的研究多着重于机理方面,本研究对研制有效的突变ｒａｓ基因疫苗是一个有益的尝试,其免疫治疗作用有待于进一步研究。     

Immunohistochemical analysis of pulmonary and pleural neoplasms using a monoclonal antibody (47D10) which reacts with nonspecific cross-reacting antigen

A series of 251 human pulmonary carcinomas were analyzed immunohistochemically for the antigens recognized by a new monoclonal antibody (MAb) 47D10. These antigens are part of a complex family of substances similar to, yet distinct from carcinoembryonic antigen (CEA), and are termed 'nonspecific cross-reacting antigens' (NCAs). The NCA epitope recognized by the MAb 47D10 is expressed on the cell surface and has previously been shown to be distinct from epitopes detected by several anti-CEA MAbs, as well as by MAbs 19-9 and Du-PAN-2. The NCA epitope recognized by MAb 47D10 is well preserved in formalin-fixed and paraffin-embedded tissues. Using immunohistochemistry, this epitope has been shown to have a limited biodistribution in normal tissues, and to be expressed by adenocarcinomas arising in the pancreas, colon, breast, ovary, prostate and lung. The frequency and pattern of NCA expression in human pulmonary neoplasms was found to correlate with the known distribution of CEA: and was often present in the non-small-cell carcinomas. In addition, the expression of CEA relative to NCA was evaluated in a select group of non-small-cell carcinoma cases using several anti-CEA MAbs, to directly compare the expression of CEA to NCA. In general, the NCA reaction pattern is more intense and expressed on more cells within the tumors than that of CEA expression.     

Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists

Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role.     

Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates

Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients.     

Nonspecific crossreacting antigen (NCA) is a major member of the carcinoembryonic antigen (CEA)-related gene family expressed in lung cancer.

Carcinoembryonic antigen (CEA) is one of the most important tumour markers in the management of human carcinoma, including lung cancer. So far, however, because of the nonspecificity of anti-CEA antibodies, it remains unclear whether the experimental measurements of CEA expression really reflect genuine CEA. In normal lung, nonspecific cross reacting antigen (NCA) has been described as a major component of CEA-related antigens. Recently isolated CEA and NCA cDNA clones enabled us to analyse CEA and NCA expression of in vivo tumour specimens and tumour cell lines at mRNA levels. NCA-specific mRNA (but not CEA-specific mRNA) was detected in all normal lung tissues examined. Of 21 lung cancer tissue specimens, nine expressed both NCA and CEA and five expressed only NCA. Of 16 tumour cell lines, two expressed only NCA and one expressed both NCA and CEA, although its level of CEA mRNA was weaker than that of NCA mRNA. Therefore, CEA-related mRNA expression was always accompanied by NCA mRNA expression; there were no cases of CEA mRNA expression alone. These findings suggest that NCA is a major member of the CEA-related gene family expressed in lung cancer.     

Vaccine-based therapy directed against carcinoembryonic antigen demonstrates antitumor activity on spontaneous intestinal tumors in the absence of autoimmunity

By virtue of its tissue-specific expression, carcinoembryonic antigen (CEA) is an important self, tumor-associated antigen, which is expressed by different human adenocarcinomas and also serves as a target for active-specific immunotherapy. Similar to humans, CEA expression in mice transgenic for the human CEA gene (CEA.Tg) occurs predominantly along the gastrointestinal tract. CEA.Tg mice were crossed with mice bearing a mutation in the Apc gene (MIN mice), and the CEA.Tg/MIN progeny developed multiple intestinal neoplasms, which overexpress CEA to levels that are reminiscent of those reported for tubulovillous intestinal adenomas from patients. CEA.Tg/MIN mice were vaccinated with an aggressive diversified prime/boost vaccine regimen: (a) a primary vaccine consisting of recombinant vaccinia virus-expressing CEA and a triad of costimulatory molecules (TRICOM): B7.1, ICAM-1, and LFA-3 (rV-CEA-TRICOM); and (b) a booster vaccine using CEA-TRICOM in a recombinant avipox (fowlpox) virus (rF-CEA-TRICOM). Granulocyte/macrophage colony-stimulating factor was administered as a biological adjuvant with all vaccinations, either as a recombinant protein (with rV-CEA-TRICOM) or as a recombinant avipox virus (with rF-CEA-TRICOM). That vaccine regimen generated strong CEA-specific host immune responses in CEA.Tg/MIN mice, which resulted in (a) a delayed onset of adult anemia and weight loss, (b) a significant reduction in the number of intestinal tumors, and (c) improved overall survival. No evidence of autoimmunity directed against normal tissues expressing CEA was observed in mice in which the CEA-based vaccine significantly reduced intestinal tumor load. The CEA.Tg/MIN mice present a clinically relevant model in which different CEA-based vaccine strategies can be tested on the spontaneous onset of intestinal tumorigenesis.     

A monkey antigen crossreacting with carcinoembryonic antigen, CEA.

Normal monkey tissues were found to contain an antigen which crossreacts immunologically with the carcinoembryonic antigen (CEA) of the human digestive tract. The monkey antigen reacted with complete or partial identity to the normal crossreacting antigen (NCA) in humans when tested in immunodiffusion against anti-CEA or anti-NCA. Extracts of monkey tissues inhibited in radioimmunoassays measuring human NCA. It is possible that monkey foetuses and colonic tumours contain CEA.     

Cell mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) recognizing the carcinoembryonic antigen (CEA) and CEA-related molecules

We report a novel anti-CEA monoclonal antibody (MAb) designated R4, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC) of human colon carcinoma cells and displays differential reactivity for human carcinomas versus the normal counterparts. R4 (IgG1) reacted with the cell surface of 6 colon carcinoma cell lines expressing CEA. Western blot analysis and epitope mapping using native and baculovirus recombinant CEA and non specific cross-reacting antigen (NCA) demonstrated that MAb R4 recognizes a proteinic epitope located on the 3' end of the domain I shared by CEA and NCA molecules. Immunohistochemical analysis demonstrated an intense staining of MAb R4 with the majority of the neoplastic tissues tested, including colon (13/13), stomach (2/2), breast (9/10), lung (7/10) and endometrial (2/4) carcinomas, whereas no reactivity with the correspondent normal tissues was observed. Using human PBLs from healthy donors as effector cells, we have shown that MAb R4 mediated antibody dependent-cell mediated cytotoxicity (ADCC) of human carcinoma cells LS-174T, CBS and WiDr. This activity was enhanced after PBLs activation with interleukin-2 (IL-2). The specificity of MAb R4 for an epitope shared by two tumor overexpressed antigens, CEA and NCA, resulting in an intense reactivity with neoplastic cells and the peculiar property to mediate ADCC, indicate that MAb R4 might be a novel powerful reagent for diagnostic and immunotherapy of carcinoma patients.     

Mimicry of a carcinoembryonic antigen epitope by a rat monoclonal anti-idiotype antibody

Anti-idiotype antibodies (Ab2) that immunologically mimic tumor antigens are auspicious agents for the active immunization of cancer patients. We have developed W12, a rat monoclonal IgG₁ Ab2 to MN -14, a murine anti-carcinoembryonic antigen (CEA) monoclonal antibody. W12 is specific for MN-14 and does not react with other isotype-matched anti-CEA monoclonal antibodies. Moreover, W12 inhibits the binding between MN-14 and CEA. Anti-CEA antibodies can be induced by immunization with W12 (but not with control rat IgG) in xenogenic animals (mice or rabbits). Immunoblotting studies indicate that the internal image determinant borne by W12 is conformational and requires the association of the heavy and light chains of the Ab2 molecule. This study indicates that W12 is a potential idiotype vaccine in patients with CEA-producing cancers.     

Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates.

PURPOSE: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. EXPERIMENTAL DESIGN: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. RESULTS: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). CONCLUSION: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.