Antibody with Infinite Affinity for In Vivo Tracking of Genetically Engineered Lymphocytes.

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. Methods: DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection of 86Y/177Lu-AABD was studied and radiation doses determined. Results: A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake of 86Y-AABD in vitro when compared with nontransduced cells. Imaging studies showed 86Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo with 177Lu-AABD seems feasible for clustered T-cell populations. Conclusion: We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene.

The pharmacokinetics of Zr-89 labeled liposomes over extended periods in a murine tumor model.

(89)Zr (t1/2=78.4h), a positron-emitting metal, has been exploited for PET studies of antibodies because of its relatively long decay time and facile labeling procedures. Here, we used (89)Zr to evaluate the pharmacokinetics of long-circulating liposomes over 168h (1week). We first developed a liposomal-labeling method using p-isothiocyanatobenzyl-desferrioxamine (df-Bz-NCS) and df-PEG1k-DSPE. Df-Bz-NCS was conjugated to 1mol% amino- and amino-PEG2k-DSPE, where the 1mol% df-PEG1k-DSPE was incorporated when the liposomes were formulated. Incubation of (89)Zr with df, df-PEG1k, and df-PEG2k liposomes for one hour resulted in greater than 68% decay-corrected yield. The loss of the (89)Zr label from liposomes after incubation in 50% human serum for 48h ranged from ~1 to 3% across the three formulations. Tail vein administration of the three liposomal formulations in NDL tumor-bearing mice showed that the (89)Zr label at the end of the PEG2k brush was retained in the tumor, liver, spleen and whole body for a longer time interval than (89)Zr labels located under the PEG2k brush. The blood clearance rate of all three liposomal formulations was similar. Overall, the results indicate that the location of the (89)Zr label altered the clearance rate of intracellularly-trapped radioactivity and that df-PEG1k-DSPE provides a stable chelation site for liposomal or lipid-based particle studies over extended periods of time.

Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor.

UNLABELLED: Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand's affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a (64)Cu-labeled affinity peptide tag, (64)Cu-L19K-(5-fluoro-2,4-dinitrobenzene) ((64)Cu-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of (64)Cu-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, (64)Cu-L19K-(2,4-dinitrophenyl) ((64)Cu-L19K-DNP) and (64)Cu-L19K. METHODS: The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with (64)Cu with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. (64)Cu-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of (64)Cu-L19K-FDNB, (64)Cu-L19K-DNP, and the native (64)Cu-L19K were compared in HCT-116 xenografts. Blocking studies of (64)Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. RESULTS: In vitro binding studies confirmed the covalent and irreversible binding of (64)Cu-L19K-FDNB to VEGF, whereas (64)Cu-L19K-DNP and (64)Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with (64)Cu-L19K-FDNB than with (64)Cu-L19K-DNP and (64)Cu-L19K, with mean standardized uptake values of 0.62 ± 0.05, 0.18 ± 0.06, and 0.34 ± 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 ± 0.05, 0.32 ± 0.14, and 0.30 ± 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with (64)Cu-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection. CONCLUSION: In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.

Enhancing peptide ligand binding to vascular endothelial growth factor by covalent bond formation.

Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide.

Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation.

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a ß(-) emitter such as (90)Y with an optical imager.

Imaging cancer using PET--the effect of the bifunctional chelator on the biodistribution of a (64)Cu-labeled antibody.

INTRODUCTION: Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the (64)Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with (64)Cu using these chelators in tumor-bearing mice. METHODS: The chelators [S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH(2)-Bn-NOTA): 6-[p-(bromoacetamido)benzyl]-1, 4, 8, 11-tetraazacyclotetradecane-N, N', N'', N'''-tetraacetic acid (BAT-6): S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododocane tetraacetic acid (p-NH(2)-Bn-DOTA): 1,4,7,10-tetraazacyclododocane-N, N', N", N"'-tetraacetic acid (DOTA): and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr)] were conjugated to the anti-GD2 antibody ch14.18, and the modified antibody was labeled with (64)Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from positron emission tomography images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. RESULTS: The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p < 0.05) were between blood, liver, spleen and kidney. For example, liver uptake of [(64)Cu]ch14.18-p-NH(2)-Bn-NOTA was 4.74 ± 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [(64)Cu]ch14.18-SarAr was 8.06 ± 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. CONCLUSIONS: The results of this study indicate that differences in the thermodynamic stability of these chelator-Cu(II) complexes were not associated with significant differences in uptake of the tracer by the tumor. However, there were significant differences in tracer concentration in other tissues, including those involved in clearance of the radioimmunoconjugate (e.g., liver and spleen).

Chemically modified antibodies as diagnostic imaging agents.

Notable new applications of antibodies for imaging involve genetically extracting the essential molecular recognition properties of an antibody, and in some cases enhancing them by mutation, before protein expression. The classic paradigm of intravenous administration of a labeled antibody to image not only its target but also its metabolism can be improved on. Protocols involving molecular targeting with an engineered unlabeled protein derived from an antibody, followed by capture of a small probe molecule that provides a signal, are being developed to a high level of utility. This is accompanied by new strategies for probe capture such as irreversible binding, incorporation of engineered enzyme active sites, and antibody-ligand systems that generate a signal only upon binding or uptake.

Liposomal Cu-64 labeling method using bifunctional chelators: poly(ethylene glycol) spacer and chelator effects.

Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A-PDEA), were synthesized and conjugated to long-circulating liposomes (LCLs) via attachment to a maleimide lipid. An in vitro stability assay of (64)Cu-TETA, (64)Cu-TETA-PEG2k, and (64)Cu-CB-TE2A-PEG2k liposomes showed that more than 86% of the radioactivity remains associated with the liposomal fraction after 48 h of incubation with mouse serum. The in vivo time activity curves (TAC) for the three liposomal formulations showed that approximately 50% of the radioactivity cleared from the blood pool in 16-18 h. As expected, the in vivo biodistribution and TAC data obtained at 48 h demonstrate that the clearance of radioactivity from the liver slows with the incorporation of a poly(ethylene glycol)-2k (PEG2k) brush. Our data suggest that (64)Cu-TETA and (64)Cu-CB-TE2A are similarly stable in the blood pool and accumulation of radioactivity in the liver and spleen is not related to the stability of Cu-64 chelator complex; however, clearance of Cu-64 from the liver and spleen are faster when injected as (64)Cu-TETA-chelated liposomes rather than (64)Cu-CB-TE2A-chelated liposomes.

Rates and equilibria for probe capture by an antibody with infinite affinity.

Probe-capture systems based on proteins and synthetic ligands have become important for new analytical and imaging applications. We have used kinetic measurements of luminescence and measurements of binding by isothermal calorimetry to determine essential rate and equilibrium constants for a system that permanently captures modified DOTA chelates for positron imaging. We used that information along with previous results to quantitatively characterize the behavior of this system in vitro and in vivo. Under physiological conditions at 37 degrees C, the equilibrium dissociation constant for yttrium S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate from antibody 2D12.5 is 2.0 (+/- 0.4) x 10(-9) M and the dissociation rate constant is 7.0 (+/- 0.7) x 10(-3) s(-1), leading to an inferred association rate constant of 3.5 x 10(6) M(-1) s(-1). Using these values to interpret data from earlier experiments leads to the rate constant 2.5 x 10(-2) s(-1) for covalent attachment of bound yttrium S-2-(4-acrylamidobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate to the G54C mutant of antibody 2D12.5. These values lead to a model for the detailed behavior of the latter system for tumor imaging in vivo that is consistent with experimental observations.

Mapping protein-protein interactions by localized oxidation: consequences of the reach of hydroxyl radical.

Hydroxyl radicals generated from a variety of methods, including not only synchrotron radiation but also Fenton reactions involving chelated iron, have become an accepted macromolecular footprinting tool. Hydroxyl radicals react with proteins via multiple mechanisms that lead to both polypeptide backbone cleavage events and side chain modifications (e.g., hydroxylation and carbonyl formation). The use of site-specifically tethered iron chelates can reveal protein-protein interactions, but the interpretation of such experiments will be strengthened by improving our understanding of how hydroxyl radicals produced at a point on a protein react with other protein sites. We have developed methods for monitoring carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. After activation of the chelated iron with ascorbate and peroxide produces new protein carbonyl groups, their positions can be identified using element-coded affinity tagging (ECAT), with carbonyl-specific tags {e.g., rare earth chelates of (S)-2-[4-(2-aminooxy)acetamidobenzyl]-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (AOD)} that allow for affinity purification, identification, and relative quantitation of oxidation sites using mass spectrometry. Intraprotein oxidation of single-cysteine mutants of Escherichia coli sigma(70) by tethered FeBABE was used to calibrate the reach of hydroxyl radical by comparison to the crystal structure; the application to protein-protein interactions was demonstrated using the same sigma(70) FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging.

Radiolabeling of liposomes with 64Cu (t(1/2)=12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel preloaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here, we report a 64Cu postlabeling method for liposomes. A 64Cu-specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during liposome formulation, liposomes were successfully labeled with 64Cu in 0.1 M NH4OAc pH 5 buffer at 35 degrees C for 30-40 min with an incorporation yield as high as 95%. After 48 h of incubation of 64Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64Cu label was still associated with liposomes. After injection of liposomal 64Cu in a mouse model, 44+/-6.9, 21+/-2.7, 15+/-2.5, and 7.4+/-1.1 (n=4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 h, respectively. The biodistribution at 48 h after injection verified that 7.0+/-0.47 (n=4) and 1.4+/-0.58 (n=3) % of the injected dose per gram of liposomal 64Cu and free 64Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.

The chemistry of irreversible capture.

The specific recognition and binding of biological molecules by antibodies is fundamentally important. Natural antibodies are multivalent, having at least two identical ligand-binding sites; this permits them to bind tightly at cell surfaces, which present multiple copies of their target ligands. Antibodies that bind to soluble monovalent ligands, such as most small molecules, do not share this multivalent advantage. Nor do engineered fragments of antibodies, such as single-chain Fv proteins or Fab fragments, which generally possess only a single ligand-binding site. Engineered monovalent antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate, are promising components of new delivery systems. These are based on a combination of genetic manipulation of the protein and chemical synthesis of appropriate ligands, examples of which are reviewed here.

Cysteinylated protein as reactive disulfide: an alternative route to affinity labeling.

Engineering the permanent formation of a receptor-ligand complex has a number of promising applications in chemistry, biology, and medicine. Antibodies and other proteins can be excellent receptors for synthetic ligands such as probes or drugs. Because proteins possess an array of nucleophilic sites, the placement of an electrophile on the synthetic ligand to react with a nucleophile on the macromolecule is a standard practice. Previously, we have used the site-directed incorporation of cysteine nucleophiles at the periphery of an antibody's binding site, paired with the chemical design of weakly electrophilic ligands, to produce receptor-ligand pairs that conjugate specifically and permanently (Corneillie et al. (2004) Bioconjugate Chem. 15, 1392-1402 and references therein). After protein expression in Drosophila S2 cells, we found, as is frequently observed, that the engineered cysteine was reversibly blocked by disulfide linkage to a cysteine monomer (cysteinylated). Removal of the cysteine monomer requires some care because of the need to preserve other disulfide linkages in the protein. Here, we report that cysteinylation can be used to advantage by treating the cysteine monomer as a leaving group and the protein disulfide as an electrophile with special affinity for thiols. Two ligands bearing thiol side chains were synthesized and incubated with the cysteinylated antibody Fab fragment 2D12.5 G54C, with the finding that both ligands become covalently attached within a few minutes under physiological conditions. The attachment is robust even in the presence of excess thiol reagents. This rapid, specific conjugation is particularly interesting for biomedical applications.

Antibodies with infinite affinity: origins and applications.

Antibodies with infinite affinity were developed with the aim of improving targeted delivery of metal complexes to sites of disease. This is part of a series of chemical technology developments for biomedical imaging and therapy. Using a combination of genetics and chemical synthesis, it addresses challenges in developing proteins that specifically bind synthetic molecules and do not release them. The result is a set of reagents that promise to capture any of a large variety of metallic elements under physiological conditions and hold them for long periods of time.

Method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry.

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting.

Antibodies against metal chelates may potentially be used in biomedical applications such as targeted imaging and therapy of cancer. Highly specific monoclonal antibodies can be developed, but their binding strength needs to be maximized for them to be of practical use. In general, the half-life for dissociation of an antibody-ligand complex is more than an order of magnitude lower than the half-lifetimes for decay of medically useful radiometal ions. Practically speaking, the metal chelate-based ligand will not be bound to its receptor long enough for all of the bound radiometal to decay. A novel approach to this problem is a combination of synthetic chemistry and site-directed mutagenesis, to position a mildly reactive group on the metal chelate adjacent to a complementary reactive group on the antibody when the complex is formed. The partners are chosen to be sufficiently unreactive so that they coexist with other molecules in living systems without undergoing reaction. When the antibody-chelate complex is formed the effective local concentrations of the two groups can be non-physically large, so that a permanent link is formed in the complex even though no reaction occurs when the partners are free in solution.

High-dose radioimmunotherapy combined with fixed, low-dose paclitaxel in metastatic prostate and breast cancer by using a MUC-1 monoclonal antibody, m170, linked to indium-111/yttrium-90 via a cathepsin cleavable linker with cyclosporine to prevent human anti-mouse antibody.

PURPOSE: Although radioimmunotherapy alone is effective in lymphoma, its application to solid tumors will likely require a combined modality approach. In these phase I studies, paclitaxel was combined with radioimmunotherapy in patients with metastatic hormone-refractory prostate cancer or advanced breast cancer. EXPERIMENTAL DESIGN: Patients were imaged with indium-111 (111In)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-peptide-m170. One week later, yttrium-90 (90Y)-m170 was infused (12 mCi/m2 for prostate cancer and 22 mCi/m2 for breast cancer). Initial cohorts received radioimmunotherapy alone. Subsequent cohorts received radioimmunotherapy followed 48 hours later by paclitaxel (75 mg/m2). Cyclosporine was given to prevent development of human anti-mouse antibody. RESULTS: Bone and soft tissue metastases were targeted by 111In-m170 in 15 of the 16 patients imaged. Three prostate cancer patients treated with radioimmunotherapy alone had no grade 3 or 4 toxicity. With radioimmunotherapy and paclitaxel, two of three prostate cancer patients developed transient grade 4 neutropenia. Four breast cancer patients treated with radioimmunotherapy alone had grade 3 or 4 myelosuppression. With radioimmunotherapy and paclitaxel, both breast cancer patients developed grade 4 neutropenia. Three breast cancer patients required infusion of previously harvested peripheral blood stem cells because of neutropenic fever or bleeding. One patient in this trial developed human anti-mouse antibody in contrast to 12 of 17 patients in a prior trial using m170-radioimmunotherapy without cyclosporine. CONCLUSIONS: 111In/90Y-m170 targets prostate and breast cancer and can be combined with paclitaxel with toxicity limited to marrow suppression at the dose levels above. The maximum tolerated dose of radioimmunotherapy and fixed-dose paclitaxel with peripheral blood stem cell support has not been reached. Cyclosporine is effective in preventing human anti-mouse antibody, suggesting the feasibility of multidose, "fractionated" therapy that could enhance clinical response.