Tumor Targeting of Radiometal Labeled Anti-CEA Recombinant T84.66 Diabody and T84.66 Minibody:  Comparison to Radioiodinated Fragments

Abstract

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.

Biodistribution and tumor imaging of an anti-CEA single-chain antibody–albumin fusion protein

“…To provide a perspective on the immunobumin imaging potential, [ 125 I]-and [ 111 In]-DOTA immunobumin blood and tumor uptake data were compared with data for the T84.66 diabody [22], minibody [10] and humanized antibody [23] previously obtained in the same mouse tumor xenograft model (Figure 4). Although similar in molecular size, the immunobumin (90 kDa) had significantly slower blood clearance than the minibody (80 kDa), but cleared faster than the intact humanized antibody.…”

“…The radiometal labeled immunobumin achieved a tumor uptake of 37 %ID/g, which was substantially higher than the [ 111 In]-minibody's 26.2 %ID/g. The key difference between the radiometal labeled immunobumin and the minibody is that the [ 111 In]-labeled minibody had much higher hepatic retention that approached 19 %ID/g [10], resulting in tumor to liver ratios of 1.4 compared to 3.4 for the immunobumin. Previous reports suggested the albumin fusion protein's extended blood circulation time was due only to the increased molecular mass.…”

“…We and others have shown that the monovalent scFv does not provide sufficient accumulation of activity in tumors for in vivo imaging, due to its small molecular size, valency and very rapid blood clearance [8]. While the multivalent constructs, T84.66 minibody and diabody, have entered pilot human imaging trials [9], currently their use has been restricted to radioiodinated agents because the radiometal labeled versions have resulted in increased retention in normal liver and kidney [10].…”

Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments forIn vivoImaging

“…Our radiolabeled anticarcinoembryonic antigen (CEA) T84.66 minibody (scFv-C H 3 dimers; Fig. 1A) has shown excellent tumor uptake (21.4-32.9% ID/g at 6 hours) in vivo (2,5,9). In addition, high-resolution microPET images of xenografts in nude mice were achieved when this fragment was radiolabeled with the positron emitters 64 Cu (T 1/2 = 12.7 hours; ref.…”

“…In addition, high-resolution microPET images are also obtained when this antibody fragment is labeled with 124 I (15) and 64 Cu positron emitters. 9 Recently, we described the construction and characterization of a minibody specific for p185 HER2 , a transmembrane glycoprotein of 185 kDa encoded by the HER2/neu proto-oncogene (17). The overexpression of the tyrosine kinase receptor HER2/ neu (c-erbB-2) in 20% to 30% of breast cancers and in a variety of other tumors of epithelial origin is often associated with poor prognosis (18).…”