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Monoclonal antibodies (MAbs) have been prepared with specificity for diethylenetriaminopentaacetic acid (DTPA) used to chelate metal radioisotopes to immunoglobulins for radioimmunoimaging and radioimmunotherapy. The use of fusion partners of lymph node-derived B cells resulted more frequently in the isolation of IgG secreting hybridomas than with splenocytes. All MAbs have been selected for simultaneous recognition of chelated and unchelated DTPA, and have been characterized in their biochemical, physico-chemical and immunochemical features. In view of the potential use in development of bifunctional MAb, these novel MAbs were also proven to lack detectable cross-reactivity with normal human tissues.

Four different proteases were screened for their capability of selectively digesting murine monoclonal IgGl to obtain active F(ab)2. For the screening, a series of five different mouse monoclonal antibodies (IgGl, k) was used, recognizing different tumor-associated antigens and currently used for radioimmunoimaging studies. The enzymes (pepsin, bromelain, ficin and elastase) showed different fragmentation capability and the fragments obtained showed different stability and immunoreactivity. No digestion was noticed using elastase. Pepsin gave discontinuous results, in that its activity ranged from reduction of IgG to small inactive fragments to an inability to digest the immunoglobulin. Pepsin activity was strongly pH-dependent and immunoreactivity of the obtained fragments was not always conserved. Bromelain and, in particular, ficin gave excellent results. Digestion was always rapid and stable, all five MAbs were reduced to F(ab)2 in a comparable time range and with high yields. Moreover, ficin-obtained F(ab)2 showed a highly conserved immunoreactivity. Therefore, ficin was selected as the murine monoclonal IgGl digestion enzyme to obtain active bivalent antibody fragments. The digestion procedure gave a uniform result for all five different MAbs and was easily scaled up to produce hundreds of milligrams of F(ab)2.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.

This report deals with the use of gene transfer by retrovirus-derived shuttle vectors in a novel model aimed at the generation of hybrid hybridomas secreting bispecific monoclonal antibodies (biMAbs). Following this approach, two genes conferring dominant resistance trait to the neomycine analogue geneticin (G418) and to methotrexate (MTX) respectively, were infected in two established hybridoma lines, each producing a well characterized MAb. The vectors used here were replication-deficient, being dependent on the complementation of helper virus provided by packaging lines. The infection procedure involved co-cultivation of the hybridomas with irradiated packaging cell lines, previously transfected with the vectors and producing the recombinant retroviruses, not inclusive of helper virus in their genome. The packaging lines used were psi2 ecotropic cells made able to produce high titers of virus. Further, the vector pMV7 was carrying G418 resistance while the pSDHT render the cells able to survive MTX. Easy and fast transfer of the dominant selection markers yielded lines of hybridomas to be fused according to the conventional somatic fusions. The resulting double hybridomas were tested for the production of hybrid molecules retaining parental specificity and successively underwent extensive cloning. The purification system featuring the most efficiently between the true biMAbs and the parental immunoglobulins (or other combination products) proved to be HPLC on hydroxylapatite column. The method described above was successful in producing two biMAbs targeting simultaneously molecules expressed on cytotoxic cells (such as CD3 on T-lymphocytes and CD16 on NK cells) and the melanoma-associated antigen Ep2.

This pharmacokinetic study was performed in order to assess the potential usefulness of the murine monoclonal antibody (MoAb) AR-3-IgG1 as an immunoscintigraphy agent for pancreatic cancer. This MoAb, which defines a mucin-like antigen (CAR-3) expressed by a large fraction of pancreatic cancers, shows in fact favourable in vivo localizing properties in the experimental animal model of human tumor xenograft. 131I-AR-3-IgG1 was injected i.v. into 5 patients with suspected pancreatic cancer. Whole-body maps and spot views of the abdominal area were recorded with a computerized gamma-camera, and specific regions of interest drawn over the liver and spleen helped to define the kinetics of activity in these organs. Blood samples taken from 0.1-144 hours post-injection and daily urine collections over the same interval served to define the kinetics of plama distribution and removal of activity from the body. Different multicompartmental models were tested to fit the experimental data, assuming as the starting hypothesis that there was to be significant nonspecific tracer accumulation in the liver, spleen and bone marrow, as already observed for the majority of radioiodinated murine MoAbs injected into humans. Surgery confirmed pancreatic cancer in 3 out of the 5 patients (chronic pancreatitis and periampullary cancer in one each); in all these 3 patients immunostaining with the MoAb AR-3 demonstrated the presence of the CAR-3 antigen (with a cytoplasmic and endoluminal/secretory pattern of distribution). Nonspecific radioactivity accumulation in the liver, spleen and bone marrow was extremely low, linked essentially to the blood pool effect of circulating activity in these organs.(ABSTRACT TRUNCATED AT 250 WORDS)