New lyophilized kit for rapid radiofluorination of peptides.

Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with (18)F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH(2)], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)(2+) in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al(3+) ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of (18)F(-) and ethanol (1:1), heated at 100-110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al(18)F-IMP485 was isolated as a single isomer complex, in high yield (45-97%) and high specific activity (up to 223 GBq/µmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al(18)F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al(18)F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43, and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h postinjection, with increased tumor/nontumor ratios. The NOTA-somatostatin-binding peptide also was fluorinated with good yield and high specific activity in the same kit formulation. However, yields were somewhat lower than those achieved with IMP485 containing the NODA-MPAA ligand, likely reflecting this ligand's superior binding properties over the simple NOTA. These studies indicate that (18)F-labeled peptides can be reproducibly prepared as stable Al-F complexes with good radiochemical yield and high specific activity using a simple, one-step, lyophilized kit followed by a rapid purification by SPE that provides the (18)F-peptide ready for patient injection within 30 min.

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody.

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.

Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex.

We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.

Pretargeted versus directly targeted radioimmunotherapy combined with anti-CD20 antibody consolidation therapy of non-Hodgkin lymphoma.

UNLABELLED: We determined whether therapeutic responses using a bispecific antibody that pretargeted (90)Y-hapten-peptide radioimmunotherapy or a directly radiolabeled, humanized, (90)Y-anti-CD20 IgG (veltuzumab) could be improved by combining these treatments with unlabeled humanized antibodies against CD22 (epratuzumab), CD74 (milatuzumab), or veltuzumab. METHODS: Nude mice bearing established subcutaneous Ramos human Burkitt lymphoma were treated with antibodies alone or in combination with pretargeted radioimmunotherapy (PT-RAIT) or radioimmunotherapy, and tumor growth was monitored. Biodistribution studies examined the effect that predosing with unlabeled veltuzumab had on radioimmunotherapy and PT-RAIT targeting. RESULTS: None of the unconjugated antibodies was effective against established and rapidly growing xenografts, but PT-RAIT, at approximately 30% of its maximum tolerated dose, and radioimmunotherapy alone, at its maximum tolerated dose, were able to arrest growth and even entirely ablate tumors in some animals. Only combinations with veltuzumab improved therapeutic responses, most significantly when a veltuzumab regimen (weekly, 1.0 mg followed by 3 x 0.5 mg) was initiated 1 wk after PT-RAIT or (90)Y-veltuzumab. Biodistribution data indicated that when unlabeled veltuzumab (1.0 or 0.25 mg) was administered in advance of the radiolabeled veltuzumab or bispecific antibody injection, tumor uptake was significantly reduced ((111)In-veltuzumab, 47% and 25%, respectively; (111)In-hapten-peptide, 74% and 49%, respectively). Despite an approximately 50% decrease in radioactivity uptake in the tumor, antitumor responses were not diminished significantly for (90)Y-veltuzumab, and in the case of PT-RAIT responses were improved. However, higher amounts of predosed veltuzumab reduced the effects of PT-RAIT. CONCLUSION: These studies suggest that administering unlabeled anti-CD20 IgG therapy after the radioactivity dose provides the best efficacy and that the amount of unlabeled anti-CD20 IgG administered as a predose to anti-CD20-targeted radionuclide therapy should be minimized.

A novel method of 18F radiolabeling for PET.

UNLABELLED: Small biomolecules are typically radiolabeled with (18)F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein (18)F is first attached to aluminum as Al(18)F, which is then bound to a chelate attached to a peptide, forming a stable Al(18)F-chelate-peptide complex in an efficient 1-pot process. METHODS: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl(3).6H(2)O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of (18)F to form the Al(18)F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH(2)) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100 degrees C for 15 min. In vitro and in vivo stability and targeting ability of the Al(18)F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). RESULTS: The radiolabeled peptide was produced in 5%-20% yield with an estimated specific activity of 18,500-48,100 GBq (500-1,300 Ci)/mmol. The Al(18)F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al(18)F had higher tissue uptake, particularly in the bones, than the chelated Al(18)F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al(18)F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. CONCLUSION: The ability to bind highly stable Al(18)F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

Therapy of advanced B-lymphoma xenografts with a combination of 90Y-anti-CD22 IgG (epratuzumab) and unlabeled anti-CD20 IgG (veltuzumab).

PURPOSE: Antibodies are effective therapeutic agents in cancer, but cures are rarely if ever obtained. Combination therapies are likely to be more effective than a single agent. In this study, the combination of a new unconjugated humanized anti-CD20 IgG, veltuzumab, with a (90)Y-conjugated humanized antibody to CD22 (epratuzumab) was evaluated for the treatment of B-cell lymphoma in a nude mouse model system. EXPERIMENTAL DESIGN: Nude mice were grafted with the Ramos human B-lymphoma and treatment initiated when tumors were >0.1 cm(3). In most experiments, mice were injected first with unconjugated anti-CD20, then with (90)Y-anti-CD22 1 day later. Additional weekly injections of the unconjugated veltuzumab were administered for 3 weeks. Controls included a single agent only and a nonreactive control radiolabeled antibody. RESULTS: Unconjugated anti-CD20 veltuzumab alone did not have a significant therapeutic effect, even at a total dose of 2.5 mg per mouse. The (90)Y-anti-CD22 epratuzumab alone induced marked regressions of all tumors, but they regrew in a few weeks. The combination of these agents cured approximately 80% of the mice. A nonreactive control antibody labeled with (90)Y, used without veltuzumab, had no therapeutic effect. The therapeutic effect of (90)Y-epratuzumab required the maximum tolerated dose of radioactivity, which was 160 muCi per mouse. CONCLUSIONS: These studies illustrate how combinations of unconjugated and radioconjugated antibodies against different B-cell markers can improve therapeutic outcome, and offer a new therapeutic paradigm for the treatment of B-cell lymphomas.

Metastatic human colonic carcinoma: molecular imaging with pretargeted SPECT and PET in a mouse model.

PURPOSE: To prospectively determine if a bispecific monoclonal antibody (MoAb) pretargeting method with a radiolabeled hapten peptide can depict small (<0.3 mm in diameter) microdisseminated human colon cancer colonies in the lungs of nude mice. MATERIALS AND METHODS: Animal studies were approved in advance by animal care and use committees. Animals injected intravenously with a human colon cancer cell line to establish microdisseminated colonies in the lungs were pretargeted with TF2--a recombinant, humanized, anti-carcinoembryonic antigen (CEA) and anti-histamine-succinyl-glycine (HSG) bispecific MoAb; 21 hours later, a radiolabeled HSG peptide was given. Imaging and necropsy data for tumor-bearing animals given the anti-CEA bispecific MoAb (n = 38, all studies) were compared with those of animals given fluorine 18 ((18)F) fluorodeoxyglucose (FDG) (n = 15, all studies), peptide alone (n = 20, all studies), or an irrelevant anti-CD22 bispecific MoAb (n = 12, all studies). Uptake of these agents in the lungs of non-tumor-bearing animals enabled assessment of specificity (n = 15, 4, and 6 for TF2 pretarget, hapten peptide alone, and (18)F-FDG, respectively). RESULTS: TF2-pretargeting helped localize tumors in the lungs within 1.5 hours of the radiolabeled HSG peptide injection, while the peptide alone, irrelevant bispecific MoAb pretargeted peptide, and (18)F-FDG failed. Necropsy data indicated that the signal in tumor-bearing lungs was five times higher than in blood within 1.5 hours, increasing to 50 times higher by 24 hours. Peptide uptake in tumor-bearing lungs pretargeted with TF2 was nine times higher than in non-tumor-bearing lungs, while it was only 1.5-fold higher with (18)F-FDG or the peptide alone. Micro-positron emission tomographic (PET) images showed discrete uptake in individual metastatic tumor colonies; autoradiographic data demonstrated selective targeting within the lungs, including metastases less than 0.3 mm in diameter. CONCLUSION: Bispecific antibody pretargeting is highly specific for imaging micrometastatic disease and may thus provide a complementary method to (18)F-FDG at clinical examination.

Bispecific antibody pretargeting of radionuclides for immuno single-photon emission computed tomography and immuno positron emission tomography molecular imaging: an update.

Molecular imaging is intended to localize disease based on distinct molecular/functional characteristics. Much of today's interest in molecular imaging is attributed to the increased acceptance and role of 18F-flurodeoxyglucose (18F-FDG) imaging in a variety of tumors. The clinical acceptance of 18F-FDG has stimulated research for other positron emission tomography (PET) agents with improved specificity to aid in tumor detection and assessment. In this regard, a number of highly specific antibodies have been described for different cancers. Although scintigraphic imaging with antibodies in the past was helpful in patient management, most antibody-based imaging products have not been able to compete successfully with the sensitivity afforded by 18F-FDG-PET, especially when used in combination with computed tomography. Recently, however, significant advances have been made in reengineering antibodies to improve their targeting properties. Herein, we describe progress being made in using a bispecific antibody pretargeting method for immuno-single-photon emission computed tomography and immunoPET applications, as contrasted to directly radiolabeled antibodies. This approach not only significantly enhances tumor/nontumor ratios but also provides high signal intensity in the tumor, making it possible to visualize micrometastases of colonic cancer as small as 0.1 to 0.2 mm in diameter using an anti-carcinoembryonic antigen bispecific antibody, whereas FDG failed to localize these lesions in a nude mouse model. Early detection of micrometastatic non-Hodgkin's lymphoma is also possible using an anti-CD20-based bispecific antibody pretargeting procedure. Thus, this bispecific antibody pretargeting procedure may contribute to tumor detection and could also contribute to the detection of other diseases having distinct antigen targets and suitably specific antibodies.

A divalent hapten-peptide induces apoptosis in human non hodgkin lymphoma cell lines targeted by anti-CD20xanti-hapten bispecific antibodies.

PURPOSE: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20. METHODS: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')2, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody. RESULTS: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab'xFab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')2xFab', IgGxFab', and the recombinant bsMAbs by approximately 1.5-fold. CONCLUSIONS: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti-hapten-binding arm of the bsMAb was well defined, such as in the Fab'xFab' and recombinant forms of bsMAb.

Bispecific antibody pretargeting PET (immunoPET) with an 124I-labeled hapten-peptide.

UNLABELLED: We previously described a highly flexible bispecific antibody (bs-mAb) pretargeting procedure using a multivalent, recombinant anti-CEA (carcinoembryonic antigen) x anti-HSG (histamine-succinyl-glycine) fusion protein with peptides radiolabeled with 111In, 90Y, 177Lu, and 99mTc. The objective of this study was to develop a radioiodination procedure primarily to assess PET imaging with 124I. METHODS: A new peptide, DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), was synthesized and conditions were established for radioiodination with yields of approximately 70% for 131I and 60% for 124I. Pretargeting with the 131I- and 124I-labeled peptide was tested in nude mice bearing LS174T human colonic tumors that were first given the anti-CEA x anti-HSG bs-mAb. Imaging (including small-animal PET) and necropsy data were collected at several intervals over 24 h. Comparisons were made between animals given 124I-anti-CEA Fab', 18F-FDG, the same peptide radiolabeled with 111In and pretargeted with the bs-mAb, and the radioiodinated peptide alone. RESULTS: The radioiodinated peptide alone cleared quickly from the blood with no evidence of tumor targeting, but when pretargeted with the bs-mAb, tumor uptake increased 70-fold, with efficient and rapid clearance from normal tissues, allowing clear visualization of tumor within 1-2 h. Tumor uptake measured at necropsy was 3- to 15-fold higher and tumor-to-blood ratios were 10- to 20-fold higher than those for 124I-Fab' at 1 and 24 h, respectively. Thyroid and stomach uptake was observed with the radioiodinated peptide several hours after injection (animals were not premedicated to reduce uptake in these tissues), but gastric uptake was much more pronounced with 124I-Fab'. Tumor visualization with 18F-FDG at approximately 1.5 h was also good but showed substantially more uptake in several normal tissues, making image interpretation in the pretargeted animals less ambiguous than with 18F-FDG. CONCLUSION: Bispecific antibody pretargeting has a significant advantage for tumor imaging over directly radiolabeled antibodies and could provide additional enhancements for oncologic imaging, particularly for improving targeting specificity as compared with 18F-FDG.

Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods.

The article reviews the background and current status of pretargeting for cancer imaging and therapy with radionuclides. Pretargeting procedures were introduced approximately 20 years ago as an alternative to directly radiolabeled antibodies. Because they were multistep processes, they were met with resistance but have since progressed to simple and improved procedures that could become the next generation of imaging and therapy with radionuclides. The separation of the radiolabeled compound from the antibody-targeting agent affords pretargeting procedures considerable flexibility in the radiolabeling process, providing opportunities for molecular imaging using gamma- or positron-emitting radionuclides and a variety of beta- and alpha-emitting radionuclides of therapeutic applications. Pretargeting methods improve tumor/nontumor ratios, exceeding that achieved with directly radiolabeled Fab' fragments, particularly within just a few hours of the radionuclide injection. In addition, tumor uptake exceeds that of a Fab' fragment by as much as 10-fold, giving pretargeting a greatly enhanced sensitivity for imaging. Advances in molecular biology have led to the development of novel binding proteins that have further improved radionuclide delivery in these systems. Studies in a variety of hematologic and solid tumor models have shown advantages of pretargeting compared with directly radiolabeled IgG for therapy, and there are several clinical studies under way that are also showing promising results. Thus, the next generation of targeting agents will likely employ pretargeting approaches to optimize radionuclide delivery for a wide range of applications.

Therapeutic advantage of pretargeted radioimmunotherapy using a recombinant bispecific antibody in a human colon cancer xenograft.

PURPOSE: To assess if pretargeting, using a combination of a recombinant bispecific antibody (bsMAb) that binds divalently to carcinoembryonic antigen (CEA) and monovalently to the hapten histamine-succinyl-glycine and a (90)Y-peptide, improves therapeutic efficacy in a human colon cancer-nude mouse xenograft compared with control animals given (90)Y-humanized anti-CEA immunoglobulin G (IgG). EXPERIMENTAL DESIGN: Clearance and biodistribution were monitored by whole-body readings and necropsy. Animals were monitored for 34 weeks with a determination of residual disease and renal pathology in survivors. Hematologic toxicity was assessed separately in non-tumor-bearing NIH Swiss mice. RESULTS: Hematologic toxicity was severe at doses of 100 to 200 microCi of (90)Y-IgG, yet mild in the pretargeted animals given 500 or 700 microCi of the (90)Y-peptide. Evidence of end-stage renal disease was found at 900 microCi of the pretargeted (90)Y-peptide whereas animals given 700 microCi showed only mild renal pathology, similar to that seen in control animals given (90)Y-IgG. Biodistribution data indicated that the average amount of tumor radioactivity by a 700-microCi dose of the pretargeted peptide over a 96-hour period was increased 2.5-fold (48 microCi/g) compared with 150 microCi of (90)Y-IgG (18.9 microCi/g). At these doses, survival (i.e., time to progression to 2.5 cm(3)) was significantly improved (P < 0.04) compared with (90)Y-IgG, with ablation of about one third of the tumors, whereas viable tumor was present in all of the (90)Y-IgG-treated animals. CONCLUSION: Pretargeting increases the amount of radioactivity delivered to colorectal tumors sufficiently to improve the therapeutic index and responses as compared with conventional radioimmunotherapy.

Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.

PURPOSE: To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. EXPERIMENTAL DESIGN: hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In. RESULTS: Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection. CONCLUSIONS: The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

A universal pretargeting system for cancer detection and therapy using bispecific antibody.

Multistep targeting systems represent highly selective alternatives to targeting systems using directly radiolabeled antibodies for diagnostic and therapeutic applications. A flexible bispecific antibody (bsMAb) multistep, pretargeting system that potentially can be developed for use with a variety of different imaging or therapeutic agents is described herein. The flexibility of this system is based on use of an antibody directed against histamine-succinyl-glycine (HSG) and the development of peptides containing the HSG residue. HSG-containing peptides were synthesized with either 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid for the chelation of 111In, 90Y, or 177Lu, or a technetium/rhenium chelate. The peptides can be radiolabeled to a high specific activity in a facile manner that avoids the need for purification. In vivo studies in nude mice bearing human colon tumor xenografts showed that the radiolabeled peptides cleared rapidly from the body with minimal retention in tumor or normal tissues. For pretargeting, these peptides were used in combination with a bsMAb composed of the anti-HSG Fab' that was covalently coupled with the Fab' of either an anticarcinoembryonic antigen or an anticolon-specific antigen-p antibody to provide tumor targeting capability. When the radiolabeled peptides were administered 1-2 days after a pretargeting dose of the bsMAbs, tumor uptake of the radiolabeled peptides increased as much as 28-175-fold over that seen with the peptides alone with tumor:nontumor ratios exceeding 2:1 to 8:1 within just 3 h of the peptide injection, which was a marked improvement over the tumor:nontumor ratios seen with a directly radiolabeled 99mTc-anti-anticarcinoembryonic antigen Fab' at this same time. The anticolon-specific antigen-p x anti-HSG F(ab')2 bsMAb had the highest and longest retention in the tumor, and when used in combination with the 111In-labeled peptide, radiation dose estimates for therapeutic radionuclides, such as 90Y and 177Lu, suggested that antitumor effects would be expected with tolerable radiation exposure to the normal tissues. These results suggest that this multistep, pretargeting system has diagnostic imaging and therapeutic potential.

Identification of a Mu-9 (anti-colon-specific antigen-p)-reactive peptide having homology to CA125 (MUC16).

Mu-9, an anti-colon specific antigen-p (CSAp) monoclonal antibody (MAb), has shown excellent gastrointestinal cancer targeting in both pre-clinical and clinical trials. With the recent development of the humanized version of this MAb, Mu-9 will receive further attention as a potential therapeutic agent for colorectal cancer. Hence, we have undertaken studies to examine the nature of the CSAp antigen and the structure of the Mu-9 epitope. M13 phage displaying random 12-mer peptides were used to isolate a peptide that binds both murine and humanized Mu-9 MAbs. The peptide, Mu-9-p16, was synthesized and found to inhibit binding of Mu-9 to CSAp with a Ka for the humanized antibody of 4.28 x 10(-7) +/- 0.91 x 10(-7) M. Control peptides did not bind Mu-9, nor did they inhibit the Mu-9-CSAp interaction. Three overlapping peptides were synthesized and used to demonstrate that the last six residues were sufficient to inhibit the Mu-9-CSAp interaction. A search of GenBank revealed that the peptide sequence IHPRP, was also present within CA125, a very high molecular weight ovarian cancer-associated antigen. The sequence is present outside of the known antigenic regions identified by the Oc125, M-11 and Ov197 anti-CA125 antibodies. To demonstrate that CSAp and CA125 may be the same protein, a heterologous sandwich enzyme immunoassay was developed with anti-CA125 MAbs used as capture reagents and Mu-9 as probe. By use of this ELISA system, we were able to specifically identify CSAp. In conclusion, our results indicate that the Mu-9-p16 peptide isolated through our screen identifies a peptide epitope shared by CSAp and CA125 and suggest that these proteins are related.

Improved targeting of pancreatic cancer: experimental studies of a new bispecific antibody, pretargeting enhancement system for immunoscintigraphy.

PURPOSE: The early detection and diagnosis of pancreatic cancer remains a major clinical challenge in which imaging procedures have a central role. The purpose of this study was to evaluate a pretargeting method with a bispecific PAM4 (bsPAM4; anti-MUC1) antibody for radioimmunoscintigraphy of experimental human pancreatic cancer. EXPERIMENTAL DESIGN: A bispecific F(ab')(2) antibody was generated from chimeric PAM4 Fab' and murine 734 (anti-indium-diethylenetriaminepentaacetic acid) Fab' fragments and then used in conjunction with 2 peptide haptens ((111)In-IMP-156 and (99m)Tc-IMP-192). Biodistribution studies and radioimmunoscintigraphic imaging properties of the radiolabeled bsPAM4, and pretargeted, radiolabeled peptides were examined in the CaPan1 human pancreatic tumor grown as s.c. xenografts in athymic nude mice. Tumor uptake and tumor:nontumor ratios were compared with a nontargeting irrelevant anti-CD20, bispecific rituximab, radiolabeled peptides alone, and with directly labeled PAM4. RESULTS: Biodistribution results indicated significantly greater tumor uptake of radiolabeled peptides at 3 h after injection when pretargeting was performed with bsPAM4 as compared with the bispecific rituximab [20.2 +/- 5.5 percentage of injected dose per gram of tissue (%ID/g) versus 0.9 +/- 0.1%ID/g, respectively, for (111)In-IMP-156, and 16.8 +/- 4.8%ID/g versus 1.1 +/- 0.2%ID/g, respectively, for (99m)Tc-IMP-192]. Similar results were obtained at the 24-h time point. Tumor:nontumor ratios were >30 for all of the tissues except the kidneys, where a ratio of 7.8 +/- 2.8 was observed. By immunoscintigraphy, tumors could be visualized as early as 30 min after injection of the radiolabeled peptide. CONCLUSIONS: These studies demonstrate the feasibility of using the pretargeted, bispecific antibody technology for nuclear imaging of pancreatic cancer. The advantage of pretargeted bsPAM4 antibody as an imaging platform is the high specificity for pancreatic cancer as compared with the physicochemical parameters identified by current imaging technologies.

Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy.

PURPOSE: Two bispecific diabodies (BS1.5 and BS1.5H) and two bispecific trivalent proteins (BS6 and BS8) were produced and tested as potential agents for pretargeted delivery of radiolabeled bivalent haptens to tumors expressing carcinoembryonic antigen. EXPERIMENTAL DESIGN: Each of the four proteins was expressed in Escherichia coli and purified from the soluble fraction. BS1.5 and BS1.5H (a humanized version of BS1.5) were evaluated in the GW-39 human colonic tumor-nude mouse model using a di-HSG-1,4,7,10-tetra-azacyclododecane-N,N',N" N"'-tetraacetic acid peptide (IMP-241) radiolabeled with (111)In. The biodistribution and T/NT ratios were compared with those of hMN-14 x m679 (Fab' x Fab') prepared chemically. RESULTS: In animals, both BS1.5 and BS1.5H cleared more rapidly than hMN-14 x m679 and showed tumor to nontumor ratios far superior to those of hMN-14 x m679. For example, with BS1.5 injected 8 h before (111)In-IMP-241, the tumor uptake of (111)In was 10.3 +/- 2.7 and 6.3 +/- 2.2% ID/g at 3 and 24 h, respectively, with the tumor to blood ratios being 167 +/- 35 at 3 h and 631 +/- 231 at 24 h. In comparison, the tumor to blood ratios of (111)In observed for hMN-14 x m679 given 24 h earlier were 8 +/- 2 at 3 h and 16 +/- 3 at 24 h. CONCLUSIONS: These results indicate that BS1.5 and BS1.5H are promising candidates for use in a variety of pretargeting applications, including tumor therapy with radionuclides and drugs. BS6 and BS8 may be even more attractive because of their potential to achieve higher levels of tumor uptake because of divalent carcinoembryonic antigen binding.

Optimizing bispecific antibody pretargeting for use in radioimmunotherapy.

PURPOSE: With increasing interest in pretargeting procedures for improving the delivery of radionuclides for cancer imaging and therapy, this investigation was undertaken to examine how to optimize a bispecific monoclonal antibody (bsMAb) pretargeting procedure for therapeutic applications. EXPERIMENTAL DESIGN: The model system examined was a bsMAb composed of two Fab' fragments, one from a humanized anti-carcinoembryonic antigen antibody (hMN-14), and the other a murine antibody (679) against histamine-succinyl-glycine. These Fab' fragments were chemically conjugated to form a F(ab')(2) that is joined by a stable thioether bond. The peptide used for these studies (IMP-241) contained two histamine-succinyl-glycine moieties for binding to the 679 portion of the bsMAb and a single 1,4,7,10-tetra-azacyclododecane N,N',N",N"'-tetraacetic acid chelate for radiolabeling with (111)In. RESULTS: The bsMAb cleared rapidly in nude mice bearing the GW-39 human colonic cancer xenograft. Administration of a radiolabeled peptide 1 day after the bsMAb, using a bsMAb/peptide mole injection ratio of 10:1, allowed for higher tumor accretion than if delayed by 2 days. Tumor uptake measured 3 h after the peptide injection given 1 day after the bsMAb was 11.3 +/- 2.2% percentage of injected dose/gram (%ID/g), with just 2.9 +/- 0.4% ID/g of the bsMAb in the tumor at this time. Tumor/blood ratios were 8.1 +/- 2.1. Peptide uptake was highest in the kidneys, but even so, the tumor/kidney ratio was 2.5 +/- 1.9 just 3 h after the peptide injection. Although low bsMAb/peptide mole injection ratios allow for greater concentrations of the peptide in the tumor, kidney uptake is increased at a proportionally higher amount than in the tumor. Therefore, a bsMAb/peptide injection ratio of 10:1 with a 24-h interval was preferred for pretargeting. Increasing the bsMAb dose, and thereby increasing the bsMAb/peptide injection ratio, further enhanced the delivery of the radiolabeled peptide to the tumor, but the interval spacing between the bsMAb and peptide had to be increased. Despite having a lower %ID/g of the bsMAb in the tumor, with a bsMAb/peptide injection ratio of 50:1 and a 48-h interval, tumor uptake of the (111)In-peptide was nearly 30% ID/g, a 1.6-fold improvement over that seen with the 10:1/24-h interval pretargeting group, and tumor/blood was 35:1, and tumor/kidney ratio was 8:1. Two fractionation strategies were also examined. Giving two equal fractions of peptide after a single injection of bsMAb loaded more moles of peptide into the tumor but would not permit higher radioactivity delivery than what could be achieved with a single injection. However, area under the curve analysis indicated that giving repeated cycles of the bsMAb followed by the peptide would enable improvements in the amount of radioactivity delivered to the tumor without increasing the amount delivered to normal tissues, but the timing of the bsMAb/peptide cycles was important to optimize this process. Finally, it was noted that larger tumors (e.g. those > 0.3 g) were more likely to have higher peptide uptake in a pretargeting procedure than smaller tumors (e.g., those of approximately 0.1 g), perhaps due to the greater mass of the bsMAb localized in the larger tumors, but also possibly because of better blood supply in these tumors. CONCLUSIONS: These studies reveal principles that might be applied generally to other pretargeting procedures and demonstrate how a bsMAb pretargeting method could potentially exceed a directly radiolabeled antibody in its ability to deliver radionuclides for cancer therapy.

Molecular advances in pretargeting radioimunotherapy with bispecific antibodies.

The use of antibodies against tumor-associated cell surface antigens for the targeted delivery of radionuclides was introduced >20 years ago. Although encouraging results have been achieved with radiolabeled antibodies in the management of hematopoietic malignancies, there remains a need for successfully treating solid tumors with this modality. One promising approach involving pretargeted delivery of radionuclides has been shown to be capable of significantly increasing the radioactive uptake in tumor relative to normal organs, thereby potentially improving the efficacy of both detection and therapy of cancer. Uncoupling of the radionuclide from the tumor-targeting antibody allows the relatively slow process of antibody localization and clearance to occur before a very rapid and highly specific delivery of the radioactive payload carried on a small molecule, such as a peptide. This minireview discusses the various strategies and advancements made since the concept of pretargeting was proposed in the mid-1980s, with emphasis on those comprising bispecific antibodies for cancer therapy. Critical aspects of these pretargeting systems for achieving higher tumor:nontumor ratios are considered. In addition, both preclinical and clinical results obtained from a pretargeting method known as the Affinity Enhancement System are presented. Future directions of pretargeting technology are also suggested.

Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates.

UNLABELLED: The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol. METHODS: We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates. The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit. The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies. A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer. RESULTS: A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work. We synthesized the bivalent peptide termed IMP 241 [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)] and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody. For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide. The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins. High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively. CONCLUSION: The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application.