Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine.

We have generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) designated 3H1, which mimics a specific epitope on the carcinoembryonic antigen (CEA). Patients with CEA positive tumors are immunologically "tolerant" to CEA. We used 3H1 as a surrogate for CEA for vaccine therapy of 12 patients with advanced colorectal cancer. Each of the patients received a minimum of four intracutaneous injections of aluminum hydroxide precipitated 3H1 at either 1, 2, or 4 mg dosage per injection. 9 of 12 patients demonstrated anti-anti-idiotypic (Ab3) response to 3H1. All nine patients generated specific anti-CEA antibody demonstrated by reactivity with radiolabeled purified CEA; some cases were confirmed by immunoprecipitation of purified CEA. We also demonstrated Ab3 stained both autologous and allogeneic colonic tumors. 7 of 12 patients demonstrated idiotype specific T cell proliferative responses and four also showed T cell proliferation to CEA. Toxicity was limited to local reaction with mild fever and chills. All 12 patients eventually progressed after finishing 4-13 dosages. This is the first report demonstrating that a vaccine therapy is capable of breaking "immune tolerance" to CEA in patients with CEA positive tumors. Future studies will focus on treating patients with minimal residual disease.

Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen expressed on most gastrointestinal adenocarcinomas and is a putative target for cancer immunotherapy. We developed a murine monoclonal anti-idiotype (anti-Id) antibody, 3H1, which mimics a specific epitope of CEA, for cancer immunotherapy. In this study, the efficacy of 3H1 as a tumor vaccine was evaluated in a murine tumor model. In this model, the murine colorectal cancer cell line MC-38 was transduced with the human CEA gene and injected into syngeneic C57BL/6 (H-2b) mice. Immunization of naive mice with 3H1 conjugated with keyhole limpet hemocyanin Freund's adjuvant induced humoral and cellular anti-3H1 as well as anti-CEA immunity. Mice immunized with 3H1 were protected against a challenge with lethal doses of MC-38-cea, whereas no protection was observed when 3H1 vaccinated mice were challenged with CEA negative MC-38 cells or when mice were vaccinated with an unrelated anti-Id antibody and challenged with MC-38-cea cells (P < 0.003). These data demonstrate that the 3H1 vaccine can induce protective CEA-specific antitumor immunity.

Induction of human breast cancer-specific antibody responses in cynomolgus monkeys by a murine monoclonal anti-idiotype antibody.

We have generated and characterized a murine monoclonal anti-idiotype (Id) antibody, designated 11D10, which biologically and antigenically mimics a distinct and specific epitope of the high molecular weight human milk fat globule primarily expressed by human breast and some other tumor cells at high density. This epitope is identified by mAb BrE1, which was used as the immunizing antibody or Ab1 to generate the anti-Id (Ab2) 11D10. 11D10 induced antitumor immune responses across species barriers, i.e., in mice and rabbits. In preclinical studies, cynomolgus monkeys were immunized with 2 mg of either 11D10 or the isotype- and allotype-matched control Ab2 3H1 after precipitation with aluminum hydroxide. All monkeys developed high titers of antibodies against the immunizing mouse immunoglobulin. Immunization with 11D10 induced anti-anti-idiotype antibodies (Ab3) which reacted with breast cancer cell lines but not with control T-cell and melanoma cell lines. The Ab3 shared idiotypes with BrE1 (Ab1), as demonstrated by their ability to inhibit 11D10 binding to BrE1. The Ab3 obtained with 11D10 bound specifically to human milk fat globule antigen and competed with BrE1 for binding to breast cancer cell lines, suggesting that Ab1 and Ab3 may bind to the same epitope. In addition, Id-specific cellular immune responses were demonstrated in monkeys immunized with 11D10 by T-cell proliferation assays. These results indicate that aluminum hydroxide-precipitated anti-Id 11D10 can induce breast cancer-specific antibodies in nonhuman primates and can serve as a potential network antigen for breast cancer patients.

Molecular mimicry of carcinoembryonic antigen by peptides derived from the structure of an anti-idiotype antibody.

Our goal was to use carcinoembryonic antigen (CEA) as a target for immunotherapy in CEA-positive cancer patients who are all immune tolerant to the native antigen. We isolated and characterized an anti-idiotype monoclonal antibody 3H1, which mimics a distinct and specific epitope of the Mr 180,000 CEA and can be used as a surrogate for CEA. In Phase Ib clinical trials in a group of 23 advanced colorectal cancer patients, 3H1 induced both humoral and cellular anti-3H1 responses, as well as anti-CEA immunity. To study the cellular immunity invoked by 3H1 at the molecular level, we have cloned and sequenced the cDNAs encoding the variable heavy and light chains of 3H1 and deduced the amino acid sequences of the encoded proteins. To identify any cross-reactive peptides of 3H1 and CEA, we compared the amino acid sequences of 3H1 with those of CEA and found several regions of homology in 3H1 heavy and light chain variable domains, as well as in the framework regions. To search for potential cross-reactive T-cell epitopes, a number of peptides were synthesized based on 3H1/CEA homology and were used as stimulants in cell proliferation assays, using peripheral blood mononuclear cells from a group of 3H1-immunized CEA-positive cancer patients in the adjuvant setting. Two partially homologous peptides, designated LCD-2 (from 3H1) and CEA-B (from CEA), were identified in 10 of 21 adjuvant patients by strong proliferation responses (stimulation index, 3-50-fold), which were extensively studied in five of these individuals over an extended period of time (12-24 months). We saw no correlation with the MHC class I haplotype of the patients. Analysis of the subtype of the responding T cells demonstrated that primarily CD4+ T cells were stimulated by both 3H1 and 3H1-derived peptides. Interleukin 2, interleukin 4, and IFN-gamma were assayed in the culture medium of peripheral blood mononuclear cells stimulated with 3H1, CEA, and LCD-2 to determine the T-cell helper subset induced by these stimulants. The in vitro responses were mainly associated with secretion of IFN-gamma, which suggested that the induced T cells were most likely CD4+ Th1 type. Future studies will include the design of second-generation LCD-2 and CEA peptides to further enhance antigenicity, to characterize the responding T-cell populations more fully, and to test refined peptides for immunogenicity.

Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen.

PURPOSE: We generated an anti-idiotype antibody, designated CeaVac, that is an internal image of the carcinoembryonic antigen (CEA). We previously demonstrated that the majority of patients with advanced colorectal cancer generate specific anti-CEA responses. The purpose of the current study was to treat patients with surgically resected colon cancer with CeaVac to determine the immune response and clinical outcome to treatment with vaccine. We also compared the immune responses between patients treated with fluorouracil (5-FU) chemotherapy regimens plus vaccine versus vaccine alone. PATIENTS AND METHODS: Thirty-two patients with resected Dukes' B, C, and D, and incompletely resected Dukes' D disease were treated with 2 mg of CeaVac every other week for four injections and then monthly until tumor recurrence or progression. Fourteen patients were treated concurrently with 5-FU chemotherapy regimens. RESULTS: All 32 patients entered onto this trial generated high-titer immunoglobulin G and T-cell proliferative immune responses against CEA. The 5-FU regimens did not have a qualitative or quantitative effect on the immune response. Three of 15 patients with Dukes' B and C disease progressed at 19, 24, and 35 months. Seven of eight patients with completely resected Dukes' D disease remained on study from 12 to 33 months; one patient with resected Dukes' D disease relapsed at 9 months. One patient with incompletely resected Dukes' D disease remained on study at 14 months without evidence of progression; eight experienced disease progression at 6 to 31 months. CONCLUSION: CeaVac consistently generated a potent anti-CEA humoral and cellular immune response in all 32 patients entered onto this trial. A number of very high-risk patients continue on study. 5-FU regimens, which are the standard of care for patients with Dukes' C disease, did not affect the immune response. These data warrant a phase III trial for patients with resected colon cancer.

Clinical and immune responses in advanced melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD2.

PURPOSE: To determine immune responses and toxicity to the anti-idiotype vaccine, as well as clinical responses and survival, we initiated a clinical trial for patients with advanced melanoma treated with an anti-idiotype antibody (TriGem) that mimics the disialoganglioside GD2. PATIENTS AND METHODS: Forty-seven patients with advanced melanoma received either 1-, 2-, 4-, or 8-mg doses of TriGem (Titan Pharmaceuticals Inc, South San Francisco, CA) mixed with QS-21 adjuvant (Aquila Biopharmaceuticals, Inc, Worcester, MA) 100 microg subcutaneously weekly for 4 weeks and then monthly until disease progression. Median age was 57 years, there were 32 men and 15 women, 43% of patients had undergone prior therapy for metastatic disease, 55% had disease confined to soft tissue, and 45% had visceral metastasis. RESULTS: Hyperimmune sera from 40 of 47 patients showed an anti-anti-idiotype (Ab3) response. Patient Ab3 was truly Ab1' because it specifically bound purified disialoganglioside GD2. The isotypic specificity of the Ab3 antibody consisted of predominantly immunoglobulin (Ig)G, and all IgG subclasses were represented. One patient had a complete response that persisted at 24 months, and 12 patients were stable from 14+ to 37+ months (median, 18+ months). Disease progression occurred in 32 patients on study from 1 to 17 months (median, 5.5 months), and 21 have died at 1 to 16 months (median, 6 months). The Kaplan-Meier-derived overall median survival has not been reached. Median survival has not been reached for the 26 patients with soft tissue disease only and was 13 months for 21 patients with visceral metastasis. Toxicity consisted of local reaction at the site of injection and mild fever and chills. CONCLUSION: TriGem has minimal toxicity and generates robust and specific IgG immune responses against GD2. Objective responses were minimal, but there may be a favorable impact on disease progression and survival that will require prospective randomized trials.

Preclinical evaluation in nonhuman primates of murine monoclonal anti-idiotype antibody that mimics the disialoganglioside GD2.

The antiganglioside GD2 monoclonal antibody 14G2a (Ab1) served as an immunogen to generate the anti-idiotype (anti-Id) 1A7 (IgG1,kappa), which mimics GD2 both antigenically and biologically. Anti-Id 1A7 induced anti-GD2 antibodies in mice and rabbits. In this preclinical study, a pair of cynomolgus monkeys, immunized with 1A7 that had been mixed with QS-21 adjuvant, produced anti-anti-Id antibodies (Ab3), which reacted with the GD2-positive melanoma cell line M21/P6 cells but not with GD2-negative LS174-T cells. The Ab3 shared Ids with mAb 14G2a (Ab1), as demonstrated by their ability to inhibit binding of 1A7 to this Ab1. The Ab3 bound specifically to purified GD2 antigen and competed with the Ab1 14G2a in binding to a GD2-positive melanoma cell line or to purified GD2, suggesting that Ab1 and Ab3 may bind to the same epitope and may behave as an Ab1-like antibody (Ab1'). The isotype of the GD2-specific antibodies was mostly IgG in nature. The specificity of the antibodies for GD2 was further confirmed by dot blot analysis. These antisera also specifically lysed GD2-positive target cells in an antibody-dependent cellular cytotoxicity assay. The induction of anti-GD2 responses in monkeys did not cause any apparent side effects, despite the fact that GD2 antigen is expressed by many normal tissues of these animals. Taken together, these results suggest that anti-Id 1A7 can induce GD2-specific antibodies in nonhuman primates and can thus serve as a potential network antigen for triggering active anti-GD2 antibodies in patients with GD2-positive neuroectodermal tumors.

Antibody responses in melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD2.

We initiated a clinical trial for patients with advanced malignant melanoma treated with an anti-idiotype antibody that mimics the disialoganglioside GD2. We report the clinical and immune responses of the first 12 patients entered into this trial. Patients received 1-, 2-, 4-, or 8-mg doses of the anti-idiotype antibody mixed with 100 microg of QS-21 adjuvant every other week, four times, and then monthly. Twelve patients have been on trial for 2-23 months, and all of them have generated immune responses. Patients were removed from the study if they demonstrated disease progression. Hyperimmune sera from all 12 patients revealed an anti-anti-idiotypic Ab3 response, as demonstrated by the inhibition of Ab2 binding to Ab1 by patients' immune sera. To further test the anti-anti-idiotypic response, patients' Ab3 antibodies were affinity purified on Sepharose 4B columns containing adsorbed immunizing anti-idiotype immunoglobulin. Purified Ab3 of all patients studied inhibited binding of Ab1 to a GD2-positive cell line. Purified Ab3 also inhibited binding of Ab1 to purified GD2, in a manner comparable to equal quantities of purified Ab1. The patient Ab3 was truly an Ab1' because it specifically bound to purified disialoganglioside GD2. The isotypic specificity of the Ab3 antibody was predominantly IgG, with only minimal IgM. The predominant IgG subclass was IgG1, with approximately equal quantities of IgG2, IgG3, and IgG4. These Ab3 antibodies reacted specifically with tumor cells expressing GD2 by immune flow cytometry and immunoperoxidase assays. Five patients' Ab3 antibodies studied for antibody-dependent cellular cytotoxicity were positive. One patient had a complete clinical response, with resolution of soft tissue disease, and six patients had stable disease, ranging from 9 to 23 months, and are being continued on vaccine therapy. Toxicity consisted of local reaction at the site of the injection, including induration and pain that generally resolved within a few days. Mild fever and chills were observed in 75% of the patients but rarely required acetaminophen. There was no additional toxicity, including abdominal pain that was previously seen with infusion of murine monoclonal anti-GD2 antibody. Current trials include patients with stage III melanoma and small cell lung cancer. Future trials will attempt to enhance the antitumor response by the addition of interleukin 2, granulocyte macrophage colony-stimulating factor, and other cytokines, together with the 1A7 vaccine.

Clinical and immune responses in advanced colorectal cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is expressed in a wide variety of adenocarcinomas, and it is well recognized that cancer patients are immunologically "tolerant" to CEA. The purpose of this study was to determine whether we could break immune tolerance to CEA by vaccinating patients with a monoclonal anti-idiotype antibody that is the internal image of CEA and to determine what impact this might have on patient survival. Twenty-four patients with advanced CEA-positive colorectal cancer who failed standard therapies except for two were entered into this Phase Ib trial. One patient was considered not assessable, because on the day of entering into the study, she was diagnosed with acute myelogenous leukemia. Patients were treated with 1, 2, or 4 mg of aluminum hydroxide-precipitated 3H1 anti-idiotype antibody every other week for four injections and then monthly until tumor progression was observed. Immunological monitoring included humoral and cellular idiotypic and CEA responses, and all patients were evaluated for toxicity, response, and survival. Hyperimmune sera from 17 of 23 patients demonstrated an anti-anti-idiotypic Ab3 response, and 13 of these responses were demonstrated to be true anti-CEA responses (Ab1'). The antibody response was polyclonal, and 11 mediated antibody-dependent cellular cytotoxicity. Ten patients had idiotypic T-cell responses, and five had specific T-cell responses to CEA. None of the patients had objective clinical responses, but overall median survival for the 23 evaluable patients was 11.3 months, with 44% 1-year survival (95% confidence interval, 23-64%). Toxicity was limited to local swelling and minimal pain. Anti-idiotype monoclonal antibody 3H1 that mimics CEA was able to break immune tolerance in the majority of treated patients. Overall survival of 11.3 months was comparable to other phase II data with advanced colorectal cancer patients treated with a variety of chemotherapy agents, including irinotecan, with considerably less toxicity. Although it is not clear that the vaccine itself had an impact on survival, this should be determined in a Phase III randomized trial.