Radioisotopic localization of (90)Yttrium-ibritumomab tiuxetan in patients with CD20+ non-Hodgkin's lymphoma.

PURPOSE: (90)Yttrium-ibritumomab-tiuxetan (Zevalin) is an effective treatment for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. The purpose of this study is to assess whether tissue and cellular localization of (90)Y-ibritumomab-tiuxetan determined by autoradiography and radioactivity localized to tumor tissue might enhance our understanding of the mechanism of action of radioimmunotherapy. METHODS: Eight eligible patients had CD20+ NHL, a bulky peripheral lymph node, and were scheduled for (90)Y-ibritumomab-tiuxetan treatment. 2-Deoxy-2-[F-18]fluoro-D: -glucose-positron emission tomography/computed tomography (FDG-PET/CT) was performed prior to treatment and at 12 weeks after therapy for assessment of response. Bone marrow, lymph node, and blood samples were collected 114 +/- 3 h after 14.8 MBq/kg (90)Y-ibritumomab-tiuxetan and processed for histology, scintillation counting, and microscopic autoradiography. RESULTS: Pericellular membrane localization of (90)Y-ibritumomab-tiuxetan to lymphoma cells was observed by autoradiography in the involved areas of lymph node with absence of significant localization in histologically normal sections of bone marrow. Pericellular radioactivity and the highest quantitative radioactivity were observed in lymph node samples of responding patients. CONCLUSIONS: (90)Y-ibritumomab-tiuxetan localizes to the surface membrane of CD20+ lymphoma cells in affected lymph nodes. The patients with the highest quantitative concentration of radioactivity to the lymph node as determined by scintillation counting were observed to have a clinical and FDG-PET/CT response.

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.

Cyclophosphamide followed by fludarabine for untreated chronic lymphocytic leukemia: a phase II SWOG TRIAL 9706.

B-cell chronic lymphocytic leukemia (CLL) accounts for 95% of chronic leukemia cases and 25% of all leukemia. Despite the prevalence of CLL, progress in its treatment has been only modest over the past three decades. Based upon the ability of fludarabine to produce high-grade remissions especially among patients with low initial tumor mass, and the ability of alkylators to reduce tumor mass, we hypothesized that sequential administration of a limited number of cycles of intermediate-dose cyclophosphamide followed by fludarabine could result in a larger percentage of patients with complete remissions (CRs). In all, 27 of the 49 eligible patients achieved overall responses of CR, unconfirmed complete remission (UCR), or PR, for a total response rate of 55% (95% confidence interval (CI) 40-69%). Considering the confounding medical issues of this patient population with advanced aggressive disease, the regimen was generally well tolerated. This study demonstrates that high-dose cyclophosphamide followed by fludarabine was relatively well tolerated in this group of advanced CLL patients. The study's criterion for testing whether the regimen is sufficiently effective to warrant further investigation was met: 14 (32%) of the first 44 eligible patients achieved CR or UCR.

Intratumor localization of monoclonal antibody in patients with melanoma treated with antibody to a 250,000-dalton melanoma-associated antigen.

Antibody localization at the tumor site was assessed in melanoma patients who received the murine monoclonal antibody 9.2.27. Antibody was administered twice weekly in escalating doses from 1 to 500 mg. Localization was assessed by biopsies of cutaneous and lymph node lesions obtained 24-96 hours following therapy. The percentage of tumor cells that bound the antibody in vivo was dose dependent, with similar findings obtained by either flow cytometry or immunoperoxidase staining techniques. Little or no in vivo binding of the 9.2.27 antibody to tumor cells was found following 1- and 10-mg doses, whereas all specimens demonstrated in vivo binding of the antibody following 200- and 500-mg doses. Fluorescence staining intensity, as quantitated by flow cytometry, was employed to determine the degree of in vivo saturation of antibody binding sites following therapy. The degree of saturation was found to vary substantially among patients: Some patients demonstrated nearly 100% saturation after 200-mg doses of 9.2.27 antibody, whereas others demonstrated only half maximal saturation after doses of 500 mg. Although immunoperoxidase staining provided important qualitative information regarding the distribution of antigen and antibody within the tumor, these studies demonstrated the usefulness of immunofluorescent flow cytometry for quantitative assessment of antibody localization in solid tumors and provided information necessary for the design of further trials of monoclonal antibodies and immunoconjugates.

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.

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.

Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen.

The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression.

Antibody-dependent, cell-mediated cytotoxicity by an anti-class II murine monoclonal antibody: effects of recombinant interleukin 2 on human effector cell lysis of human B-cell tumors.

Lym-1 is an IgG2a murine monoclonal antibody that reacts with variant Class II molecules expressed on B-cell malignancies. Lym-1 was shown to mediate antibody-dependent cellular cytotoxicity (ADCC) of human effector cells against a variety of malignant B-cell lines. Tumor cell lysis was Lym-1 specific because (a) the reaction was dose dependent with significant ADCC detectable at Lym-1 concentrations as low as 1 microgram/ml; (b) tumor targets not expressing the Lym-1 antigen were unaffected; (c) an isotype-matched irrelevant monoclonal antibody and an IgG1 anti-Class II monoclonal antibody failed to mediate ADCC; and (d) addition of Protein A (which binds avidly to Lym-1) blocked ADCC by 90 to 100%. Peripheral blood mononuclear cells obtained from normal donors as well as from cancer patients were able to interact with Lym-1 to elicit ADCC. Recombinant interleukin 2 (rIL-2) enhanced non-antibody-mediated tumor lysis and Lym-1 ADCC with an optimal concentration of 100 units/ml. Pulse treatment of normal peripheral blood mononuclear cells with rIL-2 was able to augment Lym-1 ADCC but was less effective than having the rIL-2 present through the assay. Peripheral blood mononuclear cells obtained from patients being treated with high doses of rIL-2 administered by continuous i.v. infusion demonstrated Lym-1 ADCC levels which were higher than normal individuals and which were further augmented by in vitro incubation with rIL-2.

Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy.

Human anti-murine immunoglobulin responses were assessed in serum from three groups of patients receiving murine monoclonal antibody therapy. Each of the three patient groups responded differently. Chronic lymphocytic leukemia patients demonstrated little or no preexisting murine immunoglobulin G-reactive antiglobulin prior to treatment, while the cutaneous T-cell lymphoma and melanoma patients demonstrated preexisting antiglobulin levels in the same range as those demonstrated in healthy controls. None of 11 chronic lymphocytic leukemia patients receiving the T101 monoclonal antibody demonstrated an antiglobulin response, whereas all four of the cutaneous T-cell lymphoma patients receiving the same antibody developed increased levels of antiglobulins. Three of nine malignant melanoma patients receiving the 9.2.27 monoclonal antibody showed an increase in antiglobulin titers. In patients developing antiglobulin responses, the response was rapid, typically being detectable within 2 weeks. The antiglobulins were primarily immunoglobulin G and, with the exception of a single melanoma patient in whom the response appeared to have a substantial 9.2.27-specific component (i.e., antiidiotype), were cross-reactive with most murine immunoglobulin G preparations tested. This pattern of results suggested that the antiglobulin was a secondary immune reaction with elevation of the levels of preexisting antiglobulin which was cross-reactive with the mouse antibody administered. While the presence of serum antiglobulin would be expected to present major complications to monoclonal antibody therapy, no clinical toxicity related to antiglobulin responses was observed in these patients, and no inhibition of antibody localization on tumor cells was seen.

Fate of gamma-interferon-activated killer blood monocytes adoptively transferred into the abdominal cavity of patients with peritoneal carcinomatosis.

Five patients with colorectal cancer widely metastatic to peritoneal surfaces have been treated i.p. with infusions of autologous blood monocytes made cytotoxic by in vitro incubation with human gamma-interferon. The monocytes were purified by a combination of cytapheresis and counter-current centrifugal elutriation procedures; each week approximately 350 million activated monocytes were given to patients as adoptive immunotherapy by a single i.p. instillation. On the eighth cycle of treatment the trafficking of i.p. infused blood monocytes was studied in two patients by prelabeling the cells with 111In. These activated cells became distributed widely within the peritoneal cavity. Two and 5 days after infusion their position within the peritoneum had not changed. When peritoneal specimens were obtained 36 h after 111In-labeled monocyte infusion, labeled monocytes were demonstrated to be associated with the serosal surfaces by autoradiographic analysis. Scintiscanning structures outside the abdominal cavity revealed that 111In-labeled monocytes infused i.p. did not traffic to other organs during the 5 days of the study. We conclude that i.p. adoptive transfer of autologous killer blood monocytes is an effective way of delivering these cytotoxic cells to sites of tumor burden on peritoneal surfaces in these 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.

Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice.

Biodistribution, pharmacokinetic, and radioimaging studies were performed with 186Re-labeled NR-LU-10 whole antibody in athymic nude mice bearing the LS174T tumor growing either s.c. or in an experimental hepatic metastasis model. NR-LU-10 is an IgG2b murine monoclonal antibody (MAb) that reacts with virtually all human tumors of epithelial origin. NR-BC-1, a IgG2b murine MAb that reacts with normal human B-cell and B malignancies, was used as an isotype-matched control. These MAbs were radiolabeled with 186Re (3.7-day physical half-life; 1.07-MeV beta particle and 137-keV gamma, 9% abundance) by a preformed chelate approach by using the triamide thiolate ligand system. 186Re-labeled NR-LU-10 (50 microCi) was injected into nude mice bearing LS174T tumors growing s.c. Biodistribution studies revealed that the LS174T tumor retained the highest concentration of 186Re-labeled NR-LU-10 (5.3% injected dose/g) at day 6. The tumor:blood ratio ranged from 0.1:1 to 10.8:1 by day 6, the last day of analysis. In contrast the tumor:blood ratio of 186Re-labeled NR-BC-1, the isotype-matched MAb control, was 1:1 on day 6. Pharmacokinetic analysis indicated that the t1/2 beta of NR-LU-10 for blood and other tissues ranged from 21 to 25 h, while the t1/2 beta for the LS174T tumor averaged 52 h. The area under the curve for tumor compared to blood was 2.8- to 5.7-fold higher than the area under the curve for all other tissues and organs. The mean residence time for NR-LU-10 in blood and all other organs ranged from 23 to 26 h, while the mean residence time for NR-LU-10 in the LS174T tumor was 72 h. Scintigraphic images revealed selective uptake of the 186Re-labeled NR-LU-10, but not of the 186Re-labeled NR-BC-1, at the LS174T tumor site. Studies in an experimental model of hepatic metastasis revealed a similar selective pattern of 186Re-labeled NR-LU-10 accumulation. Scintigraphic images of the LS174T tumor growing within the athymic nude mouse liver were obtained. The biodistribution, pharmacokinetic, and scintigraphic image results suggest that 186Re-labeled NR-LU-10 shows promise as a therapeutic agent for gastrointestinal cancer.

Kinetic model for the biodistribution of an 111In-labeled monoclonal antibody in humans.

Using data from 12 patients, we have analyzed the pharmacokinetics of 111In-9.2.27, an antimelanoma monoclonal antibody, following i.v. infusion. Plasma data and scintillation camera images obtained from patients receiving either 1, 50, or 100 mg of monoclonal antibody indicated dose-dependent (i.e., saturable) kinetics. Based on these observations and known immunoglobulin kinetics, we developed a nonlinear compartmental model to describe the biodistribution of 111In-9.2.27 and the other coinjected 111In-associated compounds. The model included (a) three compartments representing intact 111In-9.2.27 ("plasma," "nonsaturable," and "saturable binding" compartments), (b) four compartments representing 111In-diethylenetriaminepentaacetic acid, and (c) one compartment representing 111In in an undetermined chemical form ("extravascular delay" compartment). Analysis of the rate of urinary excretion relative to plasma concentration indicated that the saturable binding compartment was a site for catabolism of monoclonal antibody. Further examination of the urinary data, together with previous studies of the site(s) of immunoglobulin catabolism, suggested that additional elimination took place from either the plasma or the nonsaturable compartment. The model indicated that to fill the saturable sites would require a dose of approximately 0.5 mg and suggested that greater than 3.5 mg would maintain saturation for 200 h. Computer integration of gamma camera counts over the spleen revealed a clear saturable component of uptake, whereas integration over the liver showed no such pattern. The proposed model was fitted to the liver and spleen imaging data by summing fractions of model simulations of each compartment. That analysis confirmed the suspected saturable uptake by the spleen (21% of the saturable binding compartment) and revealed a quantitatively important component of saturation in the liver (35% of the saturable binding compartment) that was not obvious from initial examination of the images. When the results were expressed on a concentration basis, the spleen accounted for 247% of the saturable compartment per kg, whereas the liver accounted for 25%/kg. The bone marrow also showed saturable uptake; hence, the saturable uptake may relate to the sinusoidal blood supply characteristic of liver, spleen, and marrow. The model predicts the dose levels required to overcome saturable background, suggests appropriate doses and schedules for cold loading strategies, and provides a format for explicit inclusion of tumor antigen.

Localization and imaging with radioiodine-labeled monoclonal antibodies in a xenogeneic tumor model for human B-cell lymphoma.

Two MoAbs directed towards human B-cell malignancies have been studied in a preclinical animal model to evaluate their potential for in vivo imaging and therapy of B-cell lymphomas. Anti-B1 reacts with virtually all immunoglobulin-bearing malignancies and non-T acute lymphoblastic leukemia. Anti-J5 reacts with the common acute lymphoblastic leukemia antigen found on non-T acute lymphoblastic leukemia and follicular lymphomas. Anti-T1 which recognizes the CD5 antigen on most T-cell leukemias and lymphomas was used as a control antibody. These monoclonal antibodies were radiolabeled with 125I or 131I by the ICl method. Namalwa (B-cell) and MOLT-4 (T-cell) tumors were grown s.c. in irradiated nude mice. The highest tissue concentration of 125I-labeled anti-J5 in Namalwa-bearing mice was in blood and tumor. The tumor/blood ratio ranged from 0.7-1.2, with the highest ratio 4 days after injection. Pharmacokinetic analysis indicated that the t1/2 beta of anti-J5 from blood and other tissues ranged from 40-50 h, while the t1/2 beta for tumor averaged 65 h. The area under the curve of tumor was 2- to 5-fold higher than the area under the curve of liver, kidney, skin, and muscle. The peak tissue levels of 125I-labeled anti-B1 in Namalwa-bearing mice were again in blood and tumor and 6 days following injection more than 5-fold greater activity was found in tumor compared to normal tissues other than blood. The tumor/blood ratio was 1.2 and 0.7 at 4 and 6 days after injection. 125I-labeled anti-B1 showed minimal uptake in antigen-negative MOLT-4 tumors and 125I-labeled anti-T1 showed little uptake in Namalwa tumors. Scintigraphic images were obtained following the injection of 131I-labeled anti-J5 and anti-B1 in nude mice bearing Namalwa tumors. These results indicate that radiolabeled anti-J5 and anti-B1 show promise as diagnostic and possibly therapeutic agents for human B-cell lymphoma, although there may be a limitation to clinical utility due to cross-reactivity with some normal cells.

Immunolymphoscintigraphy and the dose dependence of 111In-labeled T101 monoclonal antibody in patients with cutaneous T-cell lymphoma.

Serial gamma camera imaging was performed in 11 patients with cutaneous T-cell lymphoma after s.c. injection between the toes of 111In-labeled T101, a pan T-cell monoclonal antibody. Two of the patients also received 111In-labeled 9.2.27, an isotype-matched control monoclonal antibody. Three doses of T101 (0.1, 0.5, and 1.0 mg), coupled with 0.5 mCi 111In, were used to measure lymph node uptake and dose response. A single 0.5-mg (0.5 mCi) dose of 111In-9.2.27 was used as control antibody to assess nonspecific uptake. Low-dose (0.1 mg) T101 administration produced rapid, intense uptake in inguinal-femoral and iliac nodes with minimal uptake in the paraaortic nodes and liver. The 0.5-mg dose and higher produced consistent uptake in all subdiaphragmatic nodes with greatest accumulation of liver activity at the 1.0-mg dose. In the two control studies, the ratio of specific:nonspecific antibody uptake in the inguinal-femoral, iliac, and paraaortic nodes averaged 7.7,5.9, and 1.9 at 48 h, respectively. In another patient, inguinal-femoral node biopsy contained over 2% of the injected dose of 111In-T101 per gram at 7 days. These findings suggest efficient and specific antibody binding in nodes nearest to the injection site, with progressive uptake of remaining antibody in more distant nodes as proximal sites approach saturation. Higher doses increase overflow of unbound antibody into the systemic circulation. There appears to be an optimal s.c. dose of approximately 0.5 mg (0.25 mg/foot) for T101 immunolymphoscintigraphy of subdiaphragmatic lymph nodes in patients with cutaneous T-cell lymphoma.

Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

Localization of 111In- and 125I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors.

A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either 125I or 111In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

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.

Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration.

The murine antimelanoma monoclonal antibody, 9.2.27, was administered intravenously to eight patients with metastatic malignant melanoma. Biopsies of metastatic nodules clearly demonstrate the selective localization of this antibody on the melanoma cell surface with a dose-response relationship to the quantity of administered antibody. The antibody infusions were clinically well tolerated and the pharmacokinetics of the antibody and the antiglobulin responses are described. This study indicates that murine monoclonal antibodies have potential as selective targeting agents in the design of future therapeutic trials using monoclonal antibodies or conjugates thereof in the treatment of cancer.