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Because breast cancer cells often express either Her2/neu or carcinoembryonic antigen (CEA) or both, these tumor markers are good targets for radioimmunotherapy using Y-90-labeled antibodies. We performed studies on nude mice bearing xenografts from MCF7, a cell line that has low Her2 and CEA expression, to more accurately reflect the more usual situation in breast cancer. Although uptake of In-111 anti-CEA into tumors was lower than that for In-111-labeled anti-Her2, radioimmunotherapy (RIT) with Y-90 anti-CEA was equivalent to that of Y-90 anti-Her2. When either Y-90 antibody was combined with a split-dose treatment with Taxol, the antitumor effect was greater than with either agent alone. When Y-90 anti-CEA was combined with a single dose of Taxol, the results were equivalent to the split-dose regimen. RIT plus cold Herceptin had no additional effects on tumor size reduction over RIT alone. When animals were first treated with Y-90 anti-Her2 and imaged 1−2 weeks later with In-111 anti-CEA or anti-Her2, tumor uptake was higher for anti-CEA and improved over tumor uptake with no prior RIT. These results suggest that a split dose of RIT with anti-Her2 antibody followed by anti-CEA antibody would be more effective than a single dose of either. This prediction was partially confirmed in a controlled study comparing single- vs split-dose anti-Her2 RIT followed by either anti-Her2 or anti-CEA RIT. These studies suggest that combined RIT and Taxol therapy are suitable in breast cancers expressing either low amounts of Her2 or CEA, thus expanding the number of eligible patients for combined therapies. They further suggest that split-dose RIT using different combinations of Y-90-labeled antibodies is effective in antitumor therapy.

Arano and co-workers (Arano et al. (1999) Cancer Res. 59, 128−134) have synthesized peptides with an N-terminal radioiodinated hippuric acid and a C-terminal lysine linked to antibody fragments via the ε-amino group of lysine that show reduced kidney uptake compared to antibody fragments directly radioiodinated. This approach takes advantage of the lysine specific carboxypeptidase activity of the kidney brush border enzymes that cleave off the radiolabeled peptide linker from the antibody fragment prior to uptake by proximal tubule cells. On the basis of their approach, we have synthesized a tetrapeptide with an N-terminal DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and a C-terminal (N^ε-maleoyl)lysine that was site-specifically conjugated to an anti-CEA diabody (Yazaki et al. (2001) Bioconjugate Chem. 12, 220−228) that was engineered to contain a C-terminal cysteine (Cys-diabody). Biodistributions of the In-111-radiolabeled conjugate in nude mice show significantly reduced kidney uptake (a maximum of 82%ID/g at 6 h) compared to In-111 radiolabeled DOTA-diabody (184%ID/g at 6 h) in which DOTA was conjugated to endogenous lysine residues using DOTA-active ester chemistry. To further reduce kidney uptake, a homologous compound with a C-terminal (N^ε-amino-1,6-hexane-bis-vinyl sulfone)lysine was synthesized and site-specifically conjugated to the Cys-diabody. Biodistributions of this In-111-labeled conjugate reduced kidney uptake to 54%ID/g at 6 h. To explore the effect of the relative positions of the chelate vs the cys-diabody on kidney uptake, we also synthesized a tetrapeptide with an N-terminal bromoacetate for conjugation to Cys-diabody and a C-terminal (N^ε-amidino-propyl-3-thio-vinylsulfonyl-DO3A)lysine. This peptide essentially reverses the positions of the chelate and Cys-diabody attachment points on the peptide, while retaining the linker length on the ε-amino group of the lysine. In this case, biodistributions of the In-111-radiolabeled conjugate in nude mice showed high kidney uptake (189%ID/g at 6 h), comparable to that obtained with the In-111-radiolabeled active ester conjugated DOTA-diabody (184%ID/g at 6 h). We conclude that the peptide linker strategy of Arano and co-workers to reduce kidney uptake can be successfully applied to chelate/radiometal complexes and requires that the chelate/radiometal be located at the N-terminus of the peptide and the antibody fragment attachment site on the ε-amino group of the lysine. Furthermore, we demonstrated a role for the attachment chemistry to the ε-amino group of the lysine on the magnitude of kidney uptake.

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 ¹¹¹In to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 °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.

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.

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that ¹¹¹In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-ε-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s−5720s]. To determine the metabolic profile of another widely used metal-chelate, [¹¹¹In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [¹¹¹In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [¹¹¹In]DOTA-ε-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [¹¹¹In]DOTA-Fab. Since injection of [¹¹¹In]DOTA-ε-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [¹¹¹In]DOTA-Fab, followed by lysosomal degradation to [¹¹¹In]DOTA-ε-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [¹¹¹In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [¹¹¹In]DOTA-ε-amino-lysine.

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N‘,N‘‘,N‘‘‘-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3−7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66−DOTA conjugate was labeled to high specific activity with ¹¹¹In, ⁹⁰Y, ⁸⁸Y, ⁶⁴Cu, and ⁶⁷Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66−DOTA for clinical radioimmunotherapy trials.