Positively charged templates for labeling internalizing antibodies: comparison of N-succinimidyl 5-iodo-3-pyridinecarboxylate and the D-amino acid peptide KRYRR.

Receptor-mediated internalization of monoclonal antibodies (mAbs), such as those specific for the epidermal growth factor receptor variant III (EGFRvIII), can lead to rapid loss of radioactivity from the target cell. In the current study, the anti-EGFRvIII mAb L8A4 was radioiodinated using two methods -N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) and via a D-amino acid peptide LysArgTyrArgArg (D-KRYRR). Paired-label internalization assays performed on EGFRvIII-expressing U87DeltaEGFR cells in vitro demonstrated that labeling L8A4 using D-KRYRR resulted in significantly higher retention of radioiodine in the intracellular compartment. In athymic mice with D256 human glioma xenografts, tumor uptake was similar for both labeling methods through 24 hr. However, an up to fourfold higher tumor retention was observed for mAb labeled with the D-amino acid peptide at later time points. Radiation absorbed dose calculations based on these biodistribution data indicated that L8A4 labeled using D-KRYRR exhibited better tumor-to-normal-organ radiation dose ratios, suggesting that this labeling method may be of particular value for labeling internalizing mAbs.

Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody.

The mutant epidermal growth factor receptor variant III (EGFRvIII) has been found on gliomas and other tumors but not on normal tissues, including those that express the wild-type receptor. Monoclonal antibodies (mAbs) specific for EGFRvIII are rapidly internalized and degraded after binding to EGFRvIII-expressing cells. If anti-EGFRvIII mAbs are to be useful for radioimmunotherapy, then methods for trapping radionuclides in target cells after mAb processing are required. Because lysosomes are known to retain positively charged molecules, we have evaluated a new reagent for this purpose that uses a polycationinc peptide composed of D-amino acids (D-Lys-D-Arg-D-Tyr-D-Arg-D-Arg; D-KRYRR). D-KRYRR was first labeled using lodogen and then coupled to the murine anti-EGFRvIII mAb L8A4 via maleimido bond formation in 60% yield. In vitro assays with the U87deltaEGFR cell line indicated that internalized and total cell-associated activity for the 125I-labeled D-KRYRR-L8A4 conjugate were up to 4 and 5 times higher, respectively, than for L8A4 labeled with 131I using Iodogen. Paired-label comparisons in athymic mice with s.c. U87deltaEGFR xenografts demonstrated up to 5-fold higher tumor uptake for mAb labeled using D-KRYRR. Higher levels of radioiodine activity also were observed in kidney when L8A4 was labeled using D-KRYRR. Another paired-label study directly compared L8A4 labeled using radioiodinated D-KRYRR and L-KRYRR, and confirmed the role of D-amino acids in enhancing tumor uptake. These results suggest that D-KRYRR is a promising reagent for the radioiodination of internalizing mAbs, such as the anti-EGFRvIII mAb L8A4.

Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications.

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate.

Monoclonal antibodies (MAbs) such as the anti-epidermal growth factor variant III (EGFRvIII) MAb L8A4 are rapidly internalized, which can lead to rapid loss of radioactivity from the tumor cell. The aim of this study was to evaluate the potential utility of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate ([211At]SAPC) for labeling murine L8A4 with 211At. SAPC was synthesized by astatodestannylation of N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate and then coupled to L8A4 in approximately 50% yield. The affinity and immunoreactive fraction for 211At-labeled L8A4 were comparable to those obtained when the MAb was labeled with 131I via N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). Paired-label comparisons of the 211At- and 131I-labeled MAbs demonstrated similar internalization and catabolism by EGFRvIII-positive cells in vitro, and with the exception of the stomach, similar tissue distribution in athymic mice with EGFRvIII-expressing U87MGdeltaEGFR xenografts. These results suggest that SAPC may be a useful reagent for labeling L8A4, and possibly other internalizing proteins, with 211At.

Radioiodinated antibody targeting of the HER-2/neu oncoprotein: effects of labeling method on cellular processing and tissue distribution.

Monoclonal antibody (MAb) internalization can have a major effect on tumor retention of radiolabel. Two anti-HER-2/neu MAbs (TA1 and 520C9) were radioiodinated using the iodogen, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), and tyramine-cellobiose (TCB) methods. Paired-label studies compared internalization and cellular processing of the labeled MAbs by SKOv3 9002-18 ovarian cancer cells in vitro. Intracellular radioiodine activity for 520C9 was up to 2.6 and 3.0 times higher for SIPC and TCB labeling, respectively, compared with iodogen. Likewise, intracellular activity for TA1 was up to 2.3 and 2.9 times higher with the SIPC and TCB methods compared with iodogen labeling. Unfortunately, similar advantages in tumor accumulation were not achieved in athymic mice bearing SKOv3 9008-18 ovarian cancer xenografts.

Astatine-211-labeled biotin conjugates resistant to biotinidase for use in pretargeted radioimmunotherapy.

We report herein the preparation and biological evaluation of two radioastatinated biotin conjugates, (3-[211At]astatobenzoyl)norbiotinamide and ((5-[211At]astato-3-pyridinyl)carbonyl)norbiotinamide. Both conjugates were stable in the presence of human serum and cerebrospinal fluid as well as murine serum, indicating a resistance to degradation to biotinidase. The normal tissue clearance of (3-[211At]astatobenzoyl)norbiotinamide and ((5-[211At]astato-3-pyridinyl)carbonyl)norbiotinamide was rapid, as observed previously with their iodo analogues. Also reported are the first syntheses of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate and 3-[211At]astatoaniline, two reagents of potential utility for labeling proteins and peptides with 211At.

Synthesis and preliminary biological evaluation of (3-iodobenzoyl)norbiotinamide and ((5-iodo-3-pyridinyl)carbonyl)norbiotinamide: two radioiodinated biotin conjugates with improved stability.

A new class of radioiodinated biotin conjugated is described in which the amido bond between biotin and the labeled prosthetic group is reversed. One conjugate, (3-[125I]iodobenzoyl)norbiotinamide (4c, [125I]IBB) was labeled with Na125I in one step from (3-(tributylstannyl)benzoyl)norbiotinamide (4b, TBB) via a demetalation reaction. However, the analogous reaction with ((5-(tributylstannyl)-3-pyridinyl)carbonyl)norbiotinamide (6b, TPB) failed to yield ((5-[131I]iodo-3-pyridinyl)carbonyl)norbiotinamide (6c, [131I]IPB, necessitating a two-step approach for synthesizing [131I]IPB. The binding of [125I]IBB and [131I]IPB to streptavidin in vitro was identical to that of biotinyl-3-[125I]iodoanilide, a conjugate with an amido bond with normal configuration. Both [125I]IBB and [131I]IPB were stable in serum while the first-generation compound was rapidly degraded. The biodistribution patterns of [125I]IBB and [131I]IPB in mice are consistent with limited degradation of these conjugates by biotinidase and deiodinases.

Intrathecal 5-[125I]iodo-2'-deoxyuridine in a rat model of leptomeningeal metastases.

UNLABELLED: The antitumor effect of 5-[125I]iodo-2'-deoxyuridine (125IUdR) was examined in a rat model of leptomeningeal metastases. In this model, 50% of rats develop paralysis of hind limbs. In 9.20 +/- 0.02 days and die in 12.1 +/- 2.1 days after intrathecal (i.t.) implantation of 5 x 10(5) 9L rat gliosarcoma cells. METHODS: Three days after implantation of 9L gliosarcoma cells, 125IUdR was administered intrathecally to rats as: (a) a single injection (500 microCi/rat), (b) five daily injections (100 microCi/day) or (c) a continuous 5-day infusion (0.5 microliter/hr, total of 500 microCi), and the animals were monitored for the onset of paralysis. Control groups received physiologic saline. For biodistribution studies, rats received a bolus injection of 125IUdR (10 microCi) 5 days after tumor-cell implantation and were killed 1, 8, 24, and 48 hr later. Tissues and organs, including the spinal cord, were isolated and their radioactive content determined. The results were expressed as percent injected dose per gram of wet tissue. Histological sections of the spinal cord were also prepared and used for autoradiographic detection of DNA-incorporated 125IUdR. RESULTS: Treatment with i.t. administered 125IUdR (500 microCi/rat) significantly (p < or = 0.005) prolonged the median time of paralysis to 11.2 +/- 0.1, 12.3 +/- 0.1 and 15.2 +/- 0.4 days for the single-dose, five daily injections and continuous infusion groups, respectively. Radioactivity cleared rapidly from all tissues except the thyroid and tumor cells growing within the spinal cord. Autoradiography demonstrated that normal cells in the tumor-bearing spinal cord were void of radioactivity. CONCLUSION: The results suggest that a selective antitumor effect could be achieved in treating leptomeningeal metastases with i.t. administered 125IUdR.

Preparation and biological evaluation of an astatine-211 labeled biotin conjugate: biotinyl-3-[211 At]astatoanilide.

Biotinyl-3-[211 At]astatoanilide ([211 At]AtBA) was prepared in more than 80% yield by destannylation. In vitro, [211 At]AtBA exhibited a high affinity for streptavidin, and was stable after incubation in human serum, cerebrospinal fluid and distilled water, whereas it was rapidly degraded in mouse serum. HPLC analysis showed that the main degradation pathway in mouse serum was the cleavage of [211 At]astatoaniline. In mice, [211 At]AtBA and its 125I-labeled analogue cleared rapidly from most tissues; however, there was some evidence for dehalogenation of both tracers.

Kit formulation for the preparation of radiolabeled iododeoxyuridine by demetallation.

UNLABELLED: The fastest and most reliable preparation of radiolabeled 5-iodo-2'- deoxyuridine (*IUdR) is accomplished by iododemetallation. METHODS: We describe a kit formulation for the preparation of *IUdR by demercuration whereby [123I/125I/131I]IUdR is synthesized virtually instantaneously following the incubation of an aqueous solution of the chloromercuric precursor with Na123I/125I/131I in the presence of lodogen. We also report the conditions for the radiosynthesis of IUdR by destannylation of the tributylstannyl precursor using hydrogen peroxide as the oxidant. RESULTS: In each case, the total procedure is completed in 5 min. HPLC indicates the total transformation of iodide into IUdR with no detectable UV-absorbing by-products. The metal content of the sample is low. The product, therefore, does not require purification. CONCLUSION: *IUdR can be prepared instantly, by either demercuration of ClHgUdR or destannylation of Bu3SnUdR. The use of a mercuric precursor favors a kit formulation since the metallic derivative is stable when kept in aqueous solution, aliquoted in vials coated with the oxidant, for up to 3 mo.

Instantaneous preparation of radiolabeled 5-iodo-2'-deoxyuridine.

The fastest and most reliable preparation of radiolabeled 5-iodo-2'-deoxyuridine ([123/125/131I]IUdR) is accomplished by iododemetallation. We describe a series of improvements to a previously published synthesis of radiolabeled IUdR by demercuration whereby [123/125/131I]IUdR is synthesized instantly following the incubation of an aqueous solution of the chloromercury precursor with Na123/125/131I in the presence of Iodogen. The total procedure is completed in 5 min and does not require purification. Mercury content is low and HPLC indicates the total transformation of iodide into IUdR with no detectable UV-absorbing by-products.