A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance.

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

Non-invasive monitoring of immunization progress in mice via IgG from feces.

A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity.

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.

Environmental effects on the structure of metal ion-DOTA complexes: an ab initio study of radiopharmaceutical metals.

Quantum chemical calculations were performed to study the differences between the important radiopharmaceutical metals yttrium (Y) and indium (In) bound by DOTA and modified DOTA molecules. Energies were calculated at the MP2/6-31+G(d)//HF/6-31G(d) levels, using effective core potentials on the Y and In ions. Although the minimum energy structures obtained are similar for both metal ion-DOTA complexes, changes in coordination and local environment significantly affect the geometries and energies of these complexes. Coordination by a single water molecule causes a change in the coordination number and a change in the position of the metal ion in In-DOTA, but Y-DOTA is hardly affected by water coordination. When one of the DOTA carboxylates is replaced by an amide, the resulting structures show a large variation between the Y and In ions. A six-residue model of the active site containing metal ion-DOTA showed that the Y-DOTA structure optimized to a structure similar to the crystal structure but that the water molecule in In-DOTA disrupts the salt bridge between Arg98B and a carboxylate side chain of DOTA. These observed differences could in part explain the differential binding constants for Y-DOTA and In-DOTA to the antibody 2D12.5.

Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions.

We have synthesized a bifunctional vinyl sulfone-cysteineamido derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that can be conjugated to the sulfhydryls of mildly reduced recombinant antibody (chimeric anti-CEA antibody cT84.66) at pH 7 or to the amino groups of lysine residues at pH 9. The conjugation is sulfhydryl specific at pH 7 (case 1), and amino specific at pH 9 (case 2) as long as the antibody has no free sulhydryl groups. At a molar ratio of 50 BCA (bifunctional chelating agent) to mAb, the number of chelates conjugated is 0.8 for case 1, and 4.6 for case 2. The resulting conjugates can be radiolabeled with (111)In to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 degrees C in 60 min. The radiolabeled conjugates retained >95% immunoreactivity and are stable in serum containing 1mM DTPA over 3 d. When the radiolabeled conjugates were injected into nude mice bearing LS174T human colon tumor xenografts, over 40% ID/g accumulated in tumors during the period 24-72h. Tumor-to-blood ratios were 4.5, 3.5, and 2.5 for the sulfhydryl coupled conjugate at 24, 48, and 72 h, respectively, and 2.7, 2.5, and 2.3 for the amino-coupled conjugate at the same time points. For other organs the biodistributions were nearly identical whether the conjugates were attached via sulfhydryl or amino groups. These novel BCAs are easy to synthesize, offer versatile conjugation options, and give equivalent biodistributions that result in high tumor uptake and good tumor-to-blood ratios.

Anti-HLA-DR/anti-DOTA diabody construction in a modular gene design platform: bispecific antibodies for pretargeted radioimmunotherapy.

Recombinant immunoglobulin libraries of single chain molecules (sc) from the variable domains of antibody light and heavy chains (Fv), have great promise for new approaches to radioimmunotherapy (RIT). However, creating and evaluating scFv from diverse sources is time consuming and differences in molecular format can influence in vitro and in vivo characteristics. Furthermore, scFv do not have optimal characteristics for targeting therapy to tumor because of their small size and univalent binding. Diabody molecules at least twice the size of scFv are better for RIT because bivalent and bispecific molecules can be constructed. A polymerase chain reaction (PCR) based primer system was created to easily convert scFv genes into a diabody gene format, once they have been placed into pCANTAB 5E, a readily available vector. The primer system for this diabody gene platform was developed and tested by constructing an anti-lymphoma/anti-chelate, bispecific diabody (anti-HLA-DR/anti-DOTA). Two mouse scFv libraries were screened for reactive clones using recombinant phage display techniques. Selected mouse anti-HLA-DR and anti-DOTA scFv genes were combined, ligated into the pCANTAB 5E vector that co-expressed these self-assembling scFv in E. coli as two mismatched nonlinked pairs (VHA-link-VLB; VHB-link-VLA). The diabody protein that was purified from periplasm had the expected molecular characteristics when analyzed by sequencing, chromatography, electrophoresis and Western blot. This modular gene design platform provides methodology for easy and rapid creation of diabody molecules from diverse scFv libraries. Diabodies from various scFv can easily be produced, thereby facilitating comparative preclinical studies en route to development of new tumor targeting molecules.