Efficient conjugation of DTPA to an IgM monoclonal antibody in ascites fluid

We have developed a simple, rapid, and reproducible method for conjugating the bifunctional metal chelator diethylenetriaminepentaacetic acid (DTPA) to an IgM monoclonal antibody (MoAb) without first isolating the MoAb from the ascites fluid. Treatment of the protein mixture in the ascites fluid with cyclic DTPA anhydride (cDTPAA) followed by HPLC purification on a size exclusion column allowed isolation of the DTPA-IgM conjugate which could then be labeled with ¹¹¹ In ⩾80% yield. Over the range of total protein concentrations used (11–44 mg/mL), the number of DTPA molecules per molecule of IgM was approximately one-half the molar ratio of cDTPAA to total protein. We have used this method to prepare an ¹¹¹In labeled anti-Thy 1.2 IgM, a MoAb with specificity for a murine cell-surface antigen found on normal and malignant T cells and neuroectodertmal tissues. Analysis of the DTPA-IgM conjugate prior to and after ¹¹¹In labeling using indirect immunofluorescence flow cytometry and a target-cell binding assay showed that the antigen specificity of this anti-Thy 1.2 MoAb is not substantially altered by the presence of up to 8 DTPA molecules per IgM molecule.     

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity ¹³¹I may damage the proteins. We found that 1000 Gy of external ¹³⁷Cs γ radiation was sufficient to destroy 80–90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [¹³¹I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of ¹³¹I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.     

鼠单克隆抗体人源化

鼠源性单克隆抗体应用于临床由于HAMA反应的存在而受到阻碍。以CDR移植为核心,以同源分析及分子模建为辅助设计的人源化方案已成为克服HAMA反应的主要手段。本文结合单抗人源化的最新进展对该领域的工作作一综述。     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

Lack of detectable nuclease activity in highly purified preparations of murine fibroblast interferon

The size distribution of radiolabeled mouse C-243 cell poly-A containing RNA, and SV40 DNA was analyzed electrophoretically before and after incubation with high concentrations of electrophoretically pure murine fibroblast interferon. Using this sensitive assay, no evidence for any nuclease activity directly associated with interferon could befound, under a wide variety of incubation conditions.     

Monoclonal antibody production in murine ascites. II. Production characteristics

OBJECTIVE: To characterize monoclonal antibody production parameters of five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample. RESULTS: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Antibody production was greatest at tap 1 for Groups 2B11 and 2C6D9; tap 2 for Group 3C9; and tap 3 for Groups RMK and 3D6. Total antibody production ranged from 422.90 to 996.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1, 2, and 3 ranged from 2.50 to 15.03 mg/ml among cell lines. CONCLUSION: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites.     

Treatment of murine lupus with monoclonal anti-T cell antibody

Three strains of autoimmune mice (MRL/lpr, NZB/NZW, and BXSB) were treated with repeated injections of rat monoclonal anti-T cell antibody (anti-Thy-1.2) in order to determine 1) the extent and duration of target cell depletion, 2) the effect of T cell depletion on the course of autoimmunity, and 3) the magnitude and consequences of the host immune response to the monoclonal antibody. Mice were treated with 6 mg of anti-Thy-1.2 every 2 wk beginning early in their disease. Treatment produced a substantial reduction in circulating T cells in all three strains. Therapy was beneficial in MRL/lpr mice. It reduced lymphadenopathy, lowered autoantibody concentrations, retarded renal disease, and prolonged life. In contrast, treatment did not improve autoimmunity in NZB/NZW mice, and it caused fatal anaphylaxis in BXSB mice. These findings demonstrate that monoclonal antilymphocyte antibodies can serve as specific probes to examine the cells that contribute to autoimmunity. Moreover, they illustrate the potential therapeutic value of monoclonal antilymphocyte antibodies when a pathogeneic cell subset can be identified. However, the same antibody may have a broad range of effects, from efficacy to severe toxicity, even in diseases that share clinical features.     

A monoclonal antibody disrupting cell-cell adhesion of rat ascites hepatoma cells

A cell surface-associated adhesive factor (AF) separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) has been highly purified by chromatography. AF is assumed to mediate the cell-cell adhesion essential to island formation of the hepatoma cells. A substance, immunologically crossreactive with AF, is present in the ascites fluid or culture medium of the AH136B cells. Because the substance is almost identical to AF in molecular weight and aggregation-promoting activity, it has been concluded that AF is released into the ascites fluid where it is concentrated. Monoclonal antibodies have been raised against AF purified from ascites fluid of AH136B cells. We have obtained a monoclonal antibody, coded MoAF-6D6, that strongly abolishes the aggregation-promoting activity of AF. When AH136B cell islands are incubated in the presence of Fab fragments of MoAF-6D6, cell detachment from the islands is evident within 24 h. Cell islands following 36-h culture show a distinct dissociation and islands completely lose their organization 48 h after culture. The dissociating effect of MoAF-6D6 is neutralized by the addition of AF. These results suggest that AF plays a significant role in the maintenance of cell islands.     

Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody

A panel of hybridomas was constructed by fusion of P3X63Ag8 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with a C57BL/Ka x-ray-induced lymphoma, C6XL. One of forty-three hybridomas secreting antibodies reactive with the tumor cells was found to be unreactive with normal spleen cells in a radioimmunometric assay. This antibody, designated 124-40, was unreactive with normal adult thymus, spleen, lymph node, or bone marrow cells, or with fetal spleen or thymus cells in radioimmunometric or radioimmunoprecipitation assays. Flow microfluorometric analysis of these nonmalignant lymphoid cells failed to reveal subpopulations reactive with MAb 124-40. The antibody was highly specific for the lymphoma cells used for immunization and did not react with a panel of other spontaneous or x-ray-induced or chemically induced lymphomas. The antigen reactive with MAb 124-40 was isolated by radioimmunoprecipitation and found to be a glycoprotein composed of disulfide-bonded subunits of 39,000 m.w. and 41,000 m.w. A cell surface component of similar structure, but not reactive with MAb 124-40 could be detected by two-dimensional electrophoresis in extracts of purified T cells, but not B cells. These results suggest that the apparently individually specific lymphoma antigen reactive with MAb 124-40 might be a clonally expressed epitope carried by a T cell surface component.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Monoclonal antibody production in murine ascites. I. Clinical and pathologic features

BACKGROUND AND PURPOSE: Murine ascites production has been associated with appreciable morbidity and mortality, thus raising animal-welfare concerns. To address these concerns, the clinicopathologic changes associated with in vivo production of monoclonal antibodies in mice were characterized, and results were compared among cell lines. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally; 12 mice were sham treated (controls). Ascites fluid was collected a maximum of three times by abdominal paracentesis. Clinical observations and pre- and postabdominal tap body weights were recorded. Necropsies were performed on all mice. RESULTS: For all groups combined, overall survival to tap 1 was 98%, to tap 2 was 96%, and to tap 3 was 79%; survival among groups ranged from 90 to 100% for tap 1, 85 to 100% for tap 2, and 35 to 100% for tap 3. Disseminated intra-abdominal seeding with irregular soft tissue and/or solid tumor masses was observed at necropsy. CONCLUSIONS: Significant clinicopathologic changes were associated with monoclonal antibody production in mice, and differences between various hybridoma cell lines were apparent.     

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.     

Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

Separation of monoclonal antibodies from cell-culture supernatants and ascites fluid using thiophilic agarose

A one-step purification procedure will yield monoclonal antibodies from cell-culture supernatants and ascites fluids. The chromatographic adsorbant is thiophilic argose, i.e., beaded agarose gel coupled with ligands of thiophilic nature, often with a sulfone group and a sulfur atom. The chromatographic procedure is simply adsorption, wash, elution. The procedure is simple, efficient, and inexpensive.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

A phenyl-beta-galactoside-specific monoclonal antibody reactive with murine and rat NK cells

The phenyl-beta-galactoside (phi-beta-gal)-specific monoclonal antibody (mAb) 49H.8 cross-reacts with the terminal disaccharide structure of the asialo GM1 (AGM1) molecule. It was found to react with phi-beta-gal determinants on murine and rat splenic natural killer (NK) cells, as measured by complement depletion studies. Flow cytometric analysis identified the antigen on two IL 2-dependent cloned murine NK cell lines and the rat large granular lymphocyte leukemia RNK. We have compared the 49H.8 reactivity to that of anti-AGM1 antisera (alpha-AGM1) on NK cells and a panel of NK related killer cells, including bone marrow-derived killer cells, lymphokine-activated killer cells (LAK), and anomalous killer cells (AK). We found that the 49H.8 specificity closely paralleled that of alpha-AGM1. When tested against Con A-reactive T cells, the 49H.8 mAb was less reactive than the alpha-AGM1, indicating that it may be a more specific marker for splenic NK populations than the alpha-AGM1.