Determining molecular mass distributions and compositions of functionalized dendrimer nanoparticles.

This study demonstrates that a combination of unconventional electron microscopy techniques provides a quantitative means of assessing the degree of monodispersity of gadolinium (Gd) diethylenetriamine pentaacetic acid-conjugated polyamidoamine (PAMAM) dendrimers, which are designed for diagnostic imaging and delivering chemotherapeutics. Specifically, analysis of images acquired in the scanning transmission electron microscopy mode yields the distribution of molecular weights of individual dendrimers, whereas analysis of images acquired in the energy-filtering transmission electron microscopy mode yields the distribution of Gd atoms bound to the dendrimer nanoparticles. Measured compositions of Gd-conjugated G7 and G8 PAMAM dendrimers were consistent with the known synthetic chemistry. The G7 dendrimers had a mass of 330 +/- 4 kDa and 266 +/- 4 Gd atoms (+/- standard error of the mean). The G8 dendrimers had a mass of 600 +/- 8 kDa and 350 +/- 5 Gd atoms (+/- standard error of the mean). This approach will be particularly attractive for assessing the mass, composition and homogeneity of metal-containing organic nanoparticles used in nanomedicine.

Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen.

The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a (99m)Tc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab' fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab')(2) fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG x Fab' < F(ab')(2) x Fab' < Fab' x Fab' conjugate). The IgG x Fab' bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab')(2) x Fab' conjugate allowed for superior tumor accretion of the (99m)Tc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab' x Fab' conjugate, albeit tumor uptake was lower than with the F(ab')(2) x Fab' conjugate. Because the IgG x Fab' bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG x Fab' bsMAb remaining in the blood to take advantage of its 3-4-fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalent-DTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG x Fab' conjugate (time of maximum tumor accretion for this bsMAb), (99m)Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the (99m)Tc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG x Fab'/blocking agent combination and the F(ab')(2) x Fab' (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab')(2) x Fab'. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab')(2) x Fab' by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.

Successful therapy of a human lung cancer xenograft using MAb RS7 labeled with residualizing radioiodine.

We have recently reported that a radioiodinated, DTPA-appended peptide, designated IMP-R1, is a residualizing iodine label that overcomes many of the limitations that have impeded the development of residualizing iodine for clinical use. In this study the potential of 131I-IMP-R1-RS7, an internalizing anti-EGP-1 monoclonal antibody, was evaluated by performing preclinical therapy studies in nude mice bearing Calu-3 human non-small cell carcinoma of the lung xenografis. Elimination of 6 of 9 established tumors (mean tumor volume=0.3 cm(3)) was observed using a single dose of 350 microCi/mouse of 131I-IMP-R1-RS7, with all animals tolerating the dose. At the same dose and specific activity of 131I-RS7, labeled using the conventional chloramine-T method, there were four deaths, and one complete remission in nine treated mice. At the maximum tolerated dose of conventionally 131I-labeled RS7, 275 microCi, mean stable disease for approximately 5 weeks was observed, with no complete responses. Specificity of the therapeutic effect was shown in an isotype-matched control experiment, where 131I-IMP-R1-RS7 was markedly more effective than the (131)I-IMP-R1-labeled control antibody. These studies demonstrate that (131)I-IMP-R1-RS7 provides a therapeutic advantage in comparison to conventional 131I-labeled RS7, as predicted by the increased tumor accretion observed previously in targeting studies. A direct comparison of the maximum tolerated doses of (131)I-IMP-R1-RS7 (350 microCi) and 90Y-DOTA-RS7 (105 microCi) was performed in this tumor model using large established tumors (mean tumor volume=0.85 cm(3)). Anti-tumor efficacy and toxicity of the two treatments were comparable.

Radioimmunotherapy of a human lung cancer xenograft with monoclonal antibody RS7: evaluation of (177)Lu and comparison of its efficacy with that of (90)Y and residualizing (131)I.

UNLABELLED: Tumor targeting and therapeutic efficacy of (177)Lu-labeled monoclonal antibody (mAb) RS7 (antiepithelial glycoprotein-1) was evaluated in a human nonsmall cell lung carcinoma xenograft model. The potential of (177)Lu-labeled RS7 was compared with that of RS7 labeled with (90)Y and a residualizing form of (131)I. METHODS: A 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) conjugate of RS7 was used for radiolabeling with (177)Lu-acetate or (88/90)Y-acetate. Biodistribution and therapy studies were conducted in nude mice with subcutaneous Calu-3 xenografts. Therapy studies were performed using the maximal tolerated doses (MTDs) of (90)Y-DOTA-RS7 (3.9 MBq [105 microCi]) and (177)Lu-DOTA-RS7 (10.2 MBq [275 microCi]) and compared with the data obtained using the MTD (13.0 MBq [350 microCi]) of a residualizing form of (131)I-RS7. RESULTS: Radiolabeling of RS7-DOTA conjugate with (177)Lu-acetate was facile. (177)Lu-DOTA-RS7 displayed biodistribution results that were nearly identical to that of the (88)Y analog in a paired-label study. The mean percentage injected doses per gram (%ID/g) for (177)Lu-RS7 and (88)Y-RS7 (in parentheses) in tumor were 38.3 %ID/g (39.1 %ID/g), 63.0 %ID/g (66.0 %ID/g), 63.0 %ID/g (65.8 %ID/g), and 34.0 %ID/g (34.9 %ID/g) on days 1, 3, 7, and 14, respectively. Elimination of established tumors, with an initial mean tumor volume of 0.24 cm(3), was shown using doses of (177)Lu-DOTA-RS7 ranging from 5.6 to 9.3 MBq (150--250 microCi) per nude mouse, with no significant difference in response rate noted between the doses in this range. Specificity of the therapeutic effect was shown in an isotype-matched control experiment, in which (177)Lu-DOTA-RS7 was markedly more effective than the (177)Lu-DOTA control antibody. A comparison of the therapeutic efficacies of (177)Lu-DOTA-RS7 and (90)Y-DOTA-RS7, using mice with established tumors with an initial mean tumor volume of 0.85 cm(3), indicated similar tumor growth inhibition and similar tumor regrowth profiles. The therapy data were similar to those obtained with residualizing (131)I-RS7 obtained at the same time. CONCLUSION: (177)Lu-RS7 is an effective radioimmunoconjugate for radioimmunotherapy. With its radiophysical properties similar to those of (131)I, coupled with its facile and stable attachment to mAb, (177)Lu promises to be an alternative to (131)I, and a complement to (90)Y, in radioimmunotherapy.

Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide.

The aim of this study was to localize (99m)Tc and (188)Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 x 734 (Fab' x Fab'), anti-carcinoembryonic antigen [CEA] x anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N'-o-Phenylenedimaleimide was used to cross-link the Fab' fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH(2) ¿TscG = 3-thiosemicarbazonylglyoxyl¿], with two indium-DTPAs and a chelate for selectively binding (99m)Tc or (188)Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with (99m)Tc (94-99%) at up to 1836 Ci/mmol and with (188)Re (97%) at 459-945 Ci/mmol of peptide. [(99m)Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 x murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) x 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 x m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 x m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 microg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 x 734, respectively), [(188)Re]IMP-192 (7 microCi) or [(99m)Tc]IMP-192 (10 microCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [(99m)Tc] or [(188)Re]IMP-192 was 12.6 +/- 5.2 and 16.9 +/- 5.5% ID/g at 3 h postinjection, respectively. Tumor/nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with (99m)Tc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when (188)Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.

Radionuclides linked to a CD74 antibody as therapeutic agents for B-cell lymphoma: comparison of Auger electron emitters with beta-particle emitters.

UNLABELLED: We demonstrated previously that human B-cell lymphomas were effectively and specifically killed in vitro by an antibody to CD74 (LL1) linked to (111)In or other Auger electron emitters. This study was intended to more accurately compare the potency and specificity of 3Auger electron emitters, (111)In, 67Ga, and 125I, and to evaluate beta-particle emitters, 131I and 90Y. The unique property of LL1 is its high level of intracellular uptake. METHODS: Raji B-lymphoma cells were incubated with serial dilutions of the radiolabeled Abs for 2 d and then monitored for cell growth by 2 assays: a cell counting assay and a clonogenic assay. The uptake of radioactivity per cell was monitored at various time points, and the radiation dose was calculated using published S values for radioactivity located in the cytoplasm. Both specific and nonspecific toxicity were evaluated. RESULTS: The beta-particle emitters had considerably higher levels of nonspecific toxicity than the Auger electron emitters, but both 131I and 90Y, and particularly 131I, still had high levels of specificity. Both of these results were consistent with dosimetry calculations. Relative to the delivered disintegrations per cell, 131I and 67Ga were the most potent of the radionuclides tested, with 125I and (111)In being significantly weaker and 90Y being intermediate. The high potency of 67Ga, together with its low nonspecific toxicity, caused this radionuclide to have the highest specificity index. CONCLUSION: When delivered by Ab LL1, both Auger electron and beta-particle emitters can produce specific and effective toxicity. The choice of the optimal radionuclide for therapy may depend on the ease and efficiency of labeling, the specific activity obtained, the nature of the tumor being targeted, and other factors, but the high specificity indices of the Auger electron emitters may be an advantage.

Radiolabeling of an anti-carcinoembryonic antigen antibody Fab' fragment (CEA-Scan) with the positron-emitting radionuclide Tc-94m.

The goal of this work was to test whether an antibody-based agent approved for use as a single-photon-emitting imaging agent when radiolabeled with technetium-99m could be labeled comparably with a positron-emitting nuclide, technetium-94m. "Instant kits" containing lyophilized NP-4 antibody Fab' fragment of an anticarcinoembryonic antigen IgG (CEA-Scan) from the same manufactured lot were reconstituted with either Tc-99m or Tc-94m, as solutions of sodium pertechnetate in isotonic saline solution. Radioanalyses of the labeled Fab' fragments by size-exclusion high-performance chromatography and TLC were carried out. Equivalent results were obtained for radioimmunoconjugates when each was analyzed with both methods. Facile incorporation of Tc-94m into tumor-targeting Fab' antibody fragments will enable investigation of such agents for tumor-specific imaging using positron emission tomography.

Targeting human cancer xenografts with monoclonal antibodies labeled using radioiodinated, diethylenetriaminepentaacetic acid-appended peptides.

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacetic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1- and 125I-IMP-R2-labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, (111)In, and 131I-dilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 125I-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 +/- 4.1% and 18.1 +/- 7.9% (P < 0.05) for the conventional 131I- and 125I-IMP-R1-RS7, respectively, and tumor:nontumor ratios were 2.6-4.5-fold higher with the 125I-IMP-R1-RS7. It is estimated that 131I-IMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 90Y-labeled RS7, and 0.7 times that of 131I-DLT-labeled RS7. Tumor accretion of 125I-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor:nontumor ratios were observed when 125I-IMP-R1-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way.

Pretargeting of renal cell carcinoma: improved tumor targeting with a bivalent chelate.

Radiolabeled monoclonal antibodies (mAbs) can target tumors selectively. Sustained activity levels in nontarget tissues limit their application. Pretargeting approaches using bispecific mAbs (bsmAbs) or the biotinavidin interaction have been proposed to improve tumor:nontumor ratios. Pretargeting a tumor and subsequently administering the radioactivity as a low molecular weight ligand fundamentally changes the pharmacokinetics of the radiolabel. In previous studies, we have shown successful radioimmunotargeting of diethylenetriaminepentaacetic acid (DTPA) labeled with indium-111 to renal cell carcinoma (RCC) after pretargeting in nude mice. In this study, we aimed to optimize further a pretargeting strategy in nude mice with RCC xenografts based on a bispecific anti-RCC x anti-DTPA mAb. Using this two-step approach, we studied whether the use of a bivalent chelate ((111)In-diDTPA) could improve radioimmunotargeting. The (111)In-diDTPA dose greatly affected the uptake of the radiolabeled chelate in the tumor. At a low (111)In-diDTPA dose (< or = 7 pmol), tumor uptake of (111)In-diDTPA was very high [>50% injected dose (ID)/g, 1 h postinjection (p.i.)], whereas at higher doses (> or = 20 pmol), tumor uptake of (111)In-diDTPA decreased (<30% ID/g). With monovalent (111)In-DTPA uptake of the radiolabel in the tumor was much lower (<10% ID/g, 1 h p.i.). Furthermore, the bivalent chelate accreted rapidly in the tumor (78% ID/g, 4 h p.i.) and was virtually completely retained in the tumor during several days p.i. (92% ID/g, 72 h p.i.). Clearance of the (111)In-diDTPA from the blood and kidneys was rapid and complete without the need to clear the bsmAb from the blood, probably due to the relative lability of the univalent bsmAb-diDTPA complexes in the blood. As a result, with this two-step pretargeting approach tumor:blood ratios increased up to values as high as 3500 at 72 h p.i. High doses of diDTPA could be targeted preferentially to the tumor, indicating that this approach could also be used for radioimmunotherapy. Tumors could be imaged up to 1 week p.i. of 50 microCi of (111)In-diDTPA. Quantitative analysis of the images confirmed the biodistribution data and indicated that, at 20 h p.i., 50 +/- 15% of the whole-body activity was localized in the tumor. In conclusion, these studies indicate that the use of bivalent chelates can very effectively optimize two-step targeting of tumors with bsmAbs. Our data indicate that this approach could optimize radioimmunotherapy.

Cytotoxicity with Auger electron-emitting radionuclides delivered by antibodies.

We investigated the in vitro cytotoxic potential of Auger electron-emitting radionuclides delivered to the cytoplasm or, more specifically, to lysosomes, via antibodies. The antibody (Ab) used was LL1, which is specific for CD74, an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface. It is taken up in large amounts, approximately 10(7) Ab molecules per cell per day, and delivered to lysosomes. The radioisotopes tested included (111)In, 99mTc and 125I. With sufficient specific activity, approximately 10 mCi/mg Ab, all of these isotopes were potent cytotoxic agents. 125I was active only if a "residualizing" form was used, meaning a form that is trapped within cells after catabolism of the Ab to which it was conjugated (conventional oxidative iodination produces a non-residualizing label). The conjugates of (111)In and 99mTc used are known to be residualizing. One hundred percent cell kill in vitro was obtained with (111)In and 125I, under conditions in which a non-reactive control Ab, conjugated in the same way, produced no significant toxicity. 99mTc was also potent and specific, but appeared somewhat less active than the other isotopes under the conditions evaluated. Although few Abs are accreted by cells at the same rate as LL1, it may be possible to use other Abs to deliver similar amounts of radioactivity, if Abs with higher specific activity can be produced. Such conjugated radioisotopes may be useful for attacking tumor cells in vivo, particularly for single cells or micrometastases.

Labeling of monoclonal antibodies with diethylenetriaminepentaacetic acid-appended radioiodinated peptides containing D-amino acids.

The optimal use of radioiodinated internalizing monoclonal antibodies (mAbs) for radioimmunotherapy necessitates the development of practical methods for increasing the level of retention of 131I in the tumor. Lysosomally trapped ("residualizing") iodine radiolabels that have been previously designed are based mostly on carbohydrate-tyramine adducts, but these methods have drawbacks of low overall yields and/or high levels of mAb aggregation. We have developed a method using thiol-reactive diethylenetriaminepentaacetic acid (DTPA)-peptide adducts wherein the peptides are assembled with one or more D-amino acids, including D-tyrosine. Two such substrates, R-Gly-D-Tyr-D-Lys[1-(p-thiocarbonylaminobenzyl)DTPA], referred to as IMP-R1, and [R-D-Ala-D-Tyr-D-Tyr-D-Lys]2(CA-DTPA), referred to as IMP-R2, wherein R is 4-(N-maleimidomethyl)cyclohexane-1-carbonyl, were synthesized by preparing functional group-protected peptides on a solid phase, selectively derivatizing the lysine side chain with 1-(p-isothiocyanatobenzyl)DTPA or DTPA dianhydride (CA-DTPA), deprotecting other functional groups, and finally derivatizing the peptide's N-terminus so it contained a maleimide group. Radioiodinations of the peptides followed by conjugations to disulfide-reduced mAbs, carried out as a one-vial procedure, resulted in 32-89% overall yields, at specific activities of 1.8-11. 1 mCi/mg, with less than 2% aggregation. Two internalizing mAbs, LL2 (anti-CD 22 B-cell lymphoma mAb) and RS7 (an anti-adenocarcinoma mAb which targets EGP-1 antigen), labeled with this procedure exhibited a 2-3-fold better cellular retention in Ramos and Calu-3 tumor cell lines, in vitro, respectively, compared to the same mAbs radioiodinated with the chloramine-T method. The rationale for the new approach, syntheses, radiochemistry and in vitro data are presented.

90Yttrium-labeled complementarity-determining-region-grafted monoclonal antibodies for radioimmunotherapy: radiolabeling and animal biodistribution studies.

90Yttrium-labeled monoclonal antibodies (mAbs) are likely to be important to radioimmunotherapy (RAIT) of a variety of cancers. The goal of this study was to select and evaluate a form of [90Y]mAb suitable for RAIT and determine conditions for high-yield, reproducible radiolabelings. 90Y-Labelings, at 2-40 mCi levels, of cdr-grafted versions of anti-B-cell lymphoma (hLL2) and anti-CEA (hIMMU-14) mAbs were optimized to >90% incorporations using the macrocyclic chelator DOTA as the metal carrier. In in vitro challenge assays, the stability of mAbs labeled with [90Y]DOTA was better than that of the corresponding [90Y]benzyl-DTPA conjugates. The retention of [90Y]DOTA-hLL2 on Raji tumor cells in vitro was similar to that of the same mAb labeled with [90Y]benzyl-DTPA and was about twice as much as with [125I]hLL2, indicating residualization of metalated mAb. Both [90Y]hLL2 conjugates, prepared using DOTA and Bz-DTPA, had similar maximum tolerated doses of 125 muCi in BALB/c mice and showed no discernible chelator-induced immune responses. Animal biodistribution studies in nude mice bearing Ramos human B-cell lymphoma xenografts revealed similar tumor and tissue uptake over a 10 day period, with the exception of bone uptake which was up to 50% lower for [88Y]DOTA-hLL2 compared to [88Y]Bz-DTPA-hLL2 at time points beyond 24 h. With [90Y]DOTA-hLL2 fragments, in vivo animal tumor dosimetries were inferior to those for the IgG, and kidney uptake was relatively high even with D-lysine administration. The ability of [111In]DOTA-hLL2 to accurately predict [90Y]DOTA-hLL2 biodistribution was established. These preclinical findings demonstrate that [90Y]DOTA-(CDR-grafted) mAbs are suitable for examination in clinical RAIT.

Pharmacokinetics, dosimetry and toxicity of rhenium-188-labeled anti-carcinoembryonic antigen monoclonal antibody, MN-14, in gastrointestinal cancer.

UNLABELLED: The biodistribution, pharmacokinetics and dosimetry of 188Re-labeled MN-14, an IgG anti-carcinoembryonic antigen monoclonal antibody (MAb), were assessed in patients in advanced gastrointestinal cancer. In addition, the dose-limiting toxicity (DLT) and maximum tolerated dose of fractionated doses of this agent were determined. METHODS: Eleven patients were administered radioactive doses of directly labeled 188Re-MN-14 IgG, ranging from 20.5 mCi to 161.0 mCi (2.0 mg-4.9 mg). Ten of these patients received two or three MAb infusions, given 3-4 days apart, delivering total doses of 30 mCi/m2-80 mCi/m2. External scintigraphy was used to evaluate the MAb biodistribution, and quantitative external scintigraphic methods were used to determine the organ and tumor radiation doses. RESULTS: The biodistribution studies showed enhanced 188Re-MN-14 uptake in the liver, spleen and kidneys, compared to that of 131I-MN-14. The biological T(1/2) values for 188Re-MN-14 in the blood and whole body (in hours) were 8.2 +/- 4.1 (n = 7) and 107.8 +/- 104.2 (n = 9), respectively (mean +/- s.d.). The radiation absorbed doses (cGy/mCi) delivered to the total body, red marrow, lungs, liver, spleen and kidneys were 0.5 +/- 0.05, 3.6 +/- 1.6, 2.0 +/- 0.8, 5.9 +/- 2.5, 7.1 +/- 1.9 and 8.5 +/- 2.8, respectively. Red marrow suppression was the only DLT observed. The maximum tolerated dose of fractionated doses of 188Re-MN-14 was estimated to be 60 mCi/m2. CONCLUSION: Despite its relatively increased renal and hepatic uptake, red marrow suppression is the only DLT of 188Re-MN-14. The feasibility of administering relatively high doses of 188Re on a completely outpatient basis may make this agent a preferred candidate for radioimmunotherapy.

Intracellular processing of 99Tcm-antibody conjugates.

The catabolism of 99Tcm-antibody conjugates after internalization by B-cell lymphomas was investigated, using antibody LL1, an antibody to the MHC class II invariant chain which is internalized and catabolized very rapidly. Intact IgG antibodies were labelled with 99Tcm after mild reduction. The 99Tcm label was strongly retained within cells, similar to 'residualizing' labels such as 111In-diethylenetriamine pentaacetate (111In-DTPA), but different from a conventional iodine label. Unlike 111In-DTPA, 99Tcm was not retained in a low molecular weight form, but instead was found to be bound to a large number of different cellular proteins, and was retained in the cytoplasm rather than in lysosomes. Therefore, this form of 99Tcm represents a new paradigm of intracellular retention of a radiolabel.

Overcoming the nephrotoxicity of radiometal-labeled immunoconjugates: improved cancer therapy administered to a nude mouse model in relation to the internal radiation dosimetry.

BACKGROUND: Elevated renal uptake and extended retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. However, cationic amino acids have been shown to reduce renal accretion. The aims of the current study were to evaluate whether this methodology would benefit therapy with yttrium 90 (90Y)-labeled antibody fragments (Fab, F(ab)2), to establish the relationship between radiation dosimetry and observed biologic effects, and to compare the antitumor efficacy of antibody fragments with that of whole immunoglobulin (Ig)G. METHODS: The maximum tolerated dose (MTD) and the dose-limiting organ toxicity of 90Y-labeled anti-carcinoembryonic antigen (CEA) MN-14 monoclonal antibodies (Fab, F(ab)2, and IgG) were determined in nude mice bearing GW-39 human colon carcinoma xenografts. The mice were treated with or without kidney protection by administration of D-lysine, with or without bone marrow transplantation (BMT), or with combinations of each. Toxicity and tumor growth were monitored at weekly intervals after radioimmunotherapy. Dosimetry was calculated from biodistribution studies using 88Y-labeled antibody. Three different dosimetric models were examined: 1) taking solely self-to-self doses into account, using S factors for 90Y in spheroids from 0.1 to 1 g; 2) correcting for cross-organ radiation; and 3) using actual mouse anatomy as represented by nuclear magnetic resonance imaging with a three-dimensional internal dosimetry package (3D-ID). RESULTS: The kidney was the first dose-limiting organ with the use of Fab fragments. Acute radiation nephritis occurred at injected activities > or = 325 microCi, and chronic nephrosis at doses > or = 250 microCi. Activities of 200 microCi were tolerated by 100% of the animals (i.e., the MTD). Application of lysine decreased the renal dose by approximately fivefold, facilitating a 25% increase in the MTD (to 250 microCi), because myelotoxicity became dose-limiting despite red marrow doses of less than 5 gray (Gy). By using BMT and lysine, the MTD could be doubled from 200 to 400 microCi, where no biochemical or histologic evidence of renal damage was observed (kidney dose, < or = 40 Gy). With injected activities of > or = 325 microCi without kidney protection, and with a hepatic self-to-self dose of only 4 Gy, rising liver enzymes were observed, which could be explained only by cross-organ radiation from radioactivity in the kidneys (in the immediate neighborhood of the right kidney up to > or = 150 Gy). The MTD of F(ab)2 fragments could be elevated only by a combination of BMT and lysine. With IgG, the bone marrow alone was dose-limiting. Tumor dosimetry correlated well with antitumor effects; Fab was more effective than F(ab)2, which was consistent with its more favorable dosimetry, and it may also be more effective than IgG due to its higher dose rate and more homogenous distribution. Dosimetry Model 1 was insufficient for predicting biologic effects. Model 2 seemed to be more accurate, accounting for interorgan crossfire. However, Model 3 showed an additional substantial contribution to the red bone marrow dose due to crossfire from the abdominal organs. CONCLUSIONS: These data show that radiation nephrotoxicity is an important effect of cancer therapy with radiometal-conjugated antibody fragments or peptides. However, this effect can be overcome successfully with the application of cationic amino acids, which substantially increase the anti-tumor efficacy of radiometal-labeled immunoconjugates. For understanding the biologic effects (e.g., liver toxicity) of 90Y in a mouse model, accounting for cross-organ radiation is essential. Further studies with radiometal-conjugated monoclonal antibody fragments and peptides are necessary to determine the MTD, dose-limiting organs, antitumor effectiveness, and nephroprotective effects of cationic amino acids in humans.

Selection of radioimmunoconjugates for the therapy of well-established or micrometastatic colon carcinoma.

In order to optimize radioimmunotherapy (RAIT) as a cancer-treatment modality, it is necessary to select the appropriate radionuclide and antibody carrier. We evaluated the therapeutic potential of a single cycle of Mu-9 anti-CSAp monoclonal antibody (MAb) labeled with 3 different radionuclides, 131I, 90Y and 188Re. Intact antibodies and bivalent fragments with different blood clearance kinetics, normal organ distribution and varying tumor accretion and retention are also evaluated. Efficacy of treatment for large and small tumor burden was assessed in nude mice bearing s.c. GW-39 human colonic-carcinoma xenografts or intrapulmonary micrometastatic GW-39 colonies at the maximal tolerated dose of each agent. The magnitude and duration of myelosuppression associated with each radioantibody was considered by monitoring peripheral blood counts, marrow colony-forming unit activity and hematopoietic tissue weight. Radiation-dose estimates were calculated based on the kinetics of antibody accretion and elimination from tumor and normal tissues, and the results were correlated with tumoricidal activity and dose-limiting toxicity results. These studies, therefore, represent a detailed analysis, in a well-defined experimental tumor system, of several parameters (antibody form, radioisotope, tumor size) influencing the overall outcome of RAIT using equitoxic doses. It was found that myelosuppression is the primary dose-limiting toxicity for all radioantibodies except 90Y-F(ab')2, even though the different agents showed varied organ distribution. In a single-cycle treatment schedule of Mu-9 MAb, the 131I-labeled IgG is the radioimmunoconjugate of choice for the treatment of s.c. and intrapulmonary growth of the GW-39 human colonic-carcinoma xenograft in nude mice.

Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Reagent development.

With pretargeting, radioisotope delivery to tumor is decoupled from the long antibody localization process, and this can increase tumor:blood ratios dramatically. Several reagents were prepared for each step of a "two-step" pretargeting method, and their properties were investigated. For pretargeting tumor, streptavidin-monoclonal antibody (StAv-mab) conjugates were prepared by cross-linking sulfo-SMCC-derivatized streptavidin to a free thiol (SH) group on MN-14 [a high-affinity anti-carcinoembryonic antigen (CEA) mab]. Thiolated mabs were generated either by reaction of 2-iminothiolane (2-IT) with mab lysine residues or by reduction of mab disulfide bonds with (2-mercaptoethyl)amine (MEA). Both procedures gave protein-protein conjugates isolated in relatively low yields (20-25%) after preparative size-exclusion (SE) chromatography purification with conservative peak collection. Both StAv-MN-14 conjugates retained their ability to bind to CEA, to an anti-idiotypic antibody to MN-14 (WI2), and to biotin, as demonstrated by SE-HPLC. Two clearing agents, WI2 mab and a biotin-human serum albumin (biotin-HSA) conjugate, were developed to remove excess circulating StAv-MN-14 conjugates in animals. Both clearing proteins were also modified with galactose residues, introduced using an activated thioimidate derivative, to produce clearing agents which would clear rapidly and clear primary mab rapidly. At least 14 galactose residues on WI2 were required to reduce blood levels to 5.9 +/- 0.7% ID/g in 1 h. Faster blood clearance (0.7 +/- 0.2% ID/g) was observed in 1 h using 44 galactose units per WI2. For the delivery of radioisotope to tumor, several biotinylated conjugates consisting of biotin, a linker, and a chelate were prepared. Conjugates showed good in vitro and in vivo stability when D-amino acid peptides were used as linkers, biotin-peptide-DOTA-indium-111 had a slightly longer blood circulation time (0.09 +/- 0.02% ID/g in 1 h) than biotin-peptide-DTPA-indium-111 (0.05 +/- 0.03% ID/g in 1 h) in nude mice. A longer circulation time with the neutral DOTA complex might allow higher tumor uptake.

Development of a streptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotin pretargeting method for radioimmunotherapy of colorectal cancer. Studies in a human colon cancer xenograft model.

Pretargeting methodologies can produce high tumor:blood ratios, but their role in cancer radioimmunotherapy (RAIT) is uncertain. A pretargeting method was developed using a streptavidin (StAv) conjugate of MN-14 IgG, an anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (mab) as the primary targeting agent, an anti-idiotype antibody (WI2 IgG) as a clearing agent, and DTPA- or DOTA-conjugated biotin as the radiolabeled targeting agent. A variety of reagents and conditions were examined to optimize this method. At 3 h, 111In-DTPA-peptide-biotin tumor uptake was 3.9 +/- 0.8% per gram and tumor:blood ratios were > 11:1. By 24 h, this ratio was 178:1, but tumor accretion declined in accordance with the gradual loss of StAv-MN-14 from the tumor. Tissue retention was highest in the liver and kidneys, but their tumor:organ ratios were > 2:1. Dosimetry predicted that radiolabeled MN-14 alone would deliver higher tumor doses than this pretargeting method. Increasing the specific activity and using DOTA-biotin in place of DTPA increased tumor uptake nearly 2-fold, but analysis of StAv-MN-14's biotin-binding capacity indicated over 90% of the initial biotin-binding sites were blocked within 24 h. Animals fed a biotin-deficient diet had 2-fold higher 111In-DOTA-biotin uptake in the tumor, but higher uptake also was observed in all normal tissues. Although exceptionally adept at achieving high tumor:blood ratios rapidly, the tumor uptake of radiolabeled biotin with this pretargeting method is significantly (p < 0.0001) lower than that with a radiolabeled antibody. Endogenous biotin and enhanced liver and kidney uptake may limit the application of this method to RAIT, especially when evaluating the method in animals, but with strategies to overcome these limitations, this pretargeting method could be an effective therapeutic alternative.

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22.

LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin's lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab')2, Fab'], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

Internalization and catabolism of radiolabelled antibodies to the MHC class-II invariant chain by B-cell lymphomas.

The fate of antibody (Ab) LL1, which reacts with the invariant chain (Ii) subunit of the immature MHC class-II antigen (CD74) after binding to the surface of B-cell lymphomas was investigated. This Ab was internalized and catabolized very rapidly, much faster than other Abs that are considered to be rapidly internalized, such as CD19, CD22 and anti-(transferrin receptor). Such internalization did not depend on Ab cross-linking. The capacity of this uptake process was determined in long-term experiments by increasing the Ab concentration: in 1 day, approx. 8 x 10(5) Ab molecules per cell were catabolized. This analysis was facilitated by the use of radiolabels that are trapped within cells after catabolism of the Abs to which they were conjugated. If the Ab is a reliable marker for the Ii antigen, which is likely, we can conclude that Ii directed to the cell surface appears to be sufficient, indeed more than sufficient, to account for the cell content of mature class-II molecules.