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Supplementary Figures 1-4, Methods from Halogenated Benzimidazole Carboxamides Target Integrin α4β1 on T-Cell and B-Cell Lymphomas

Integrin α4β1 is an attractive but poorly understood target for selective diagnosis and treatment of T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships, and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin α4β1 antagonists. We documented tumor uptake of derivatives labeled with 125I in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high-affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. Cancer Res; 70(13); 5448–56. ©2010 AACR.

Immunoscintigraphic studies for breast cancer localization have been performed in several centers worldwide but clinical studies of radioimmunotherapy (RAIT) for metastatic breast cancer have been rare and usually unrewarding. Oncofetal antigens such as carcinoembryonic antigen represent a major group of possible RAIT targets for breast cancer. Tumor imaging requires a high target to nontarget ratio, but tumor therapy with radiolabeled antibodies is related to the cumulative radiation energy deposited per volume of cancer tissue. The development of systemic tumor-targeted radiotherapy as part of the multimodality approach to metastatic breast cancer requires a focused multidisciplinary team. Metastatic breast cancer poses a unique challenge by its variation in cell biology which is most easily summarized by the descriptor "heterogeneous". Theoretically this ideal target for tumor therapy would be tumor specific and generously expressed on all the breast cancer cells from all breast cancer patients.

There is a need for effective “broad spectrum” therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of188Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi188Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of188Re-6D2 was escalated to 54 mCi. SPECT/CT revealed188Re-6D2 uptake in melanoma metastases. The mean effective half-life of188Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that188Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Although the term has been coined recently, the concepts underlying theranosis have been applied in patient care for more than one-half century. However, advanced technologies are used now. Theranosis describes processes used to tailor therapy for a patient. It is the use of diagnostic tests to identify those patients better-suited for a drug (or drugs) or to determine how well a drug is working. (131)I-iodide for imaging and for therapy of hyperthyroidism and thyroid cancer is an excellent example of personalized theranosis and has withstood challenge for more than 50 years. Radioimmunotherapy for non-Hodgkin lymphoma is a more recent example of theranosis. Either of 2 anti-CD20 monoclonal antibodies, one labeled with indium for imaging or (90)Y for radiotherapy or a second labeled with (131)I for both imaging and radiotherapy, is used for salvage and first-line therapy of multifocal non-Hodgkin lymphoma. The efficacy of these drugs is greater than that of alternative therapies. To mimic the molecular specificity and cell selectivity of a monoclonal antibody, smaller molecules that also bind to proteins upregulated by malignant cells can be used to transport cytotoxic agents to the malignant cells. Smaller carrier molecules like peptides, aptamers, affibodies, and selective, high-affinity ligands facilitate intensification of therapy because of their size. Personalized genomics, proteomics, and molecular imaging are among technologies currently used for theranosis. Molecular emission tomographic imaging with radiolabeled drugs has been used to examine the pharmacology of anticancer therapies and their effectiveness. Increased glycolysis, a molecular phenotype of many malignancies, can be imaged using (18)F-fluoro-2-deoxyglucose (FDG). Tomographic imaging using FDG allows stratification of patients into those responding and likely to respond to the therapy and those better treated in another manner. Prediction of therapeutic response avoids useless therapy so that FDG imaging is included in official response evaluation criteria. Although a fixed approach to therapy may be more practical, an individualized approach is more likely to ensure that each patient receives an effective drug and drug dose that has acceptable and definable tissue effects. Drugs that work in one individual may be ineffective or cause adverse events in others.

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-α-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric α/δ-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en α/δ-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with 111In, incubated in human blood plasma at 37 °C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural α-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

Although most patients with locoregional cancer are cured by surgery, radiotherapy, chemotherapy, and combinations thereof, those with distant metastases are not despite systemic chemotherapy. These patients respond to local radiotherapy but generally need systemic therapy. Non-Hodgkin's lymphoma (NHL) provides a paradigm for the role of molecular targeted radiotherapy (MTRT) because these patients have multifocal disease in most cases. Although patients with NHL achieve remissions after multiple cycles of chemotherapy, less than one half of those with aggressive NHL are cured and almost none of those with low grade NHL. Furthermore, NHL, like other cancers, becomes chemoresistant, yet remains responsive to radiotherapy. MTRT, radiation targeted by molecules, is a good strategy for the treatment of multifocal and radiosensitive cancers. Radioimmunotherapy (RIT) is an MTRT approach using MAbs, or parts thereof, to target the radionuclide that delivers radiation. Two anti-CD20 monoclonal antibodies (MAbs), one labeled with (111)In for imaging or (90)Y for therapy and a second labeled with (131)I for imaging and therapy, have proven effective and safe for MTRT for NHL patients. The importance of the radiation is demonstrated in the data from the randomized pivotal trial of (90)Y-ibritumomab; response rates were distinctly better in the (90)Y-ibritumomab arm than in the rituximab arm. Furthermore, the efficacy of (131)I-tositumomab was greater than that of the same MAb alone in another pivotal trial. Although hematologic toxicity is dose limiting for MTRT, febrile neutropenia is uncommon. MTRT is also not associated with mucositis, hair loss, or persistent nausea or vomiting, unlike chemotherapy. Randomized trials of MTRT in different strategies have not been conducted, but there is evidence of better outcomes, particularly for strategies that provide dose intensification, such as pretargeted MTRT, multiple dosing ("fractionation"), and MTRT with stem cell transplantation (SCT). Pretargeted RIT separates delivery of the targeting molecule from radionuclide delivery, provides dose escalation, and is more effective than direct one-step RIT, although more complicated to implement. Improved drugs and strategies for MTRT have documented potential for better patient outcomes. Smaller radionuclide carriers, such as those used for pretargeted MTRT, should be incorporated into the management of patients with NHL and other cancers soon after the patients have proven incurable. Expected improvements using better drugs, strategies, and combinations with other drugs seem likely to make MTRT integral in the management of many patients with cancer and likely to lead to cures of NHL.

Multivalency results from the simultaneous binding of multiple ligands with multiple receptors. Understanding the effect of multivalency on binding kinetics in molecular and cellular systems may aid the development of new types of therapeutics or countermeasures to pathogen infection. Here, we describe a method using single-molecule dynamic force spectroscopy to determine the binding strength of antibody-protein complexes as a function of binding valency in a direct and simple measurement. We used the atomic force microscope to measure the force required to rupture a single complex formed by the MUC1 protein, a cancer indicator, and therapeutic antibodies that target MUC 1. We will show that nanomechanical polymer tethers can be used in a new manner to count the number of biological bonds formed. Mechanical work will disrupt these bonds and can be used to quantify the overall kinetics. This ability to form, count, and dissociate biological bonds with nanomechanical forces provides a powerful method to study the physical laws governing the interactions of biological molecules.

N-[[4-[[[(2-ethylphenyl)amino]carbonyl]amino]phenyl]acetyl]-N(epsilon)-6-[(2E)-1-oxo-3-(3-pyridinyl-2-propenyl)]-l-lysyl-l-2-aminohexanedioyl-(1-amino-1-cyclohexane)carboxamide (LLP2A) is a high-affinity, high-specificity peptidomimetic ligand (inhibitory concentration of 50% = 2 pM) that binds the activated alpha4beta1 integrin found on a variety of malignant lymphoid cell lines. To better determine whether this ligand holds promise for imaging and therapy in lymphoid malignancies, 6 LLP2A derivatives, as LLP2A-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (LLP2A-DOTA) and LLP2A-DOTA-polyethylene glycol (LLP2A-DOTA-PEG), were designed, synthesized, and radiolabeled with (111)In. Comparative pharmacokinetic studies in mice with Raji B-cell lymphoma xenografts were then complemented by small-animal PET of the lead molecular LLP2A format using (64)Cu-LLP2A-11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane ((64)Cu-LLP2A-CB-TE2A).LLP2A-DOTA and LLP2A-CB-TE2A were prepared using solid-phase synthesis; LLP2A-DOTA-PEG(2,000), LLP2A-DOTA-PEG(5,000), LLP2A-DOTA-PEG(10,000), (LLP2A-DOTA)(2)PEG(10,000), and (LLP2A-DOTA)(4)PEG(10,000) were prepared by PEGylation. (111)In radiolabeling of DOTA and (64)Cu radiolabeling of CB-TE2A conjugates yielded 370-1,850 and 3,700-7,400 kBq/microg (10-50 and 100-200 microCi/microg), respectively. The pharmacokinetics of the six (111)In radioconjugates were studied in vivo using biodistribution data (4 and 24 h) and whole-body autoradiography (24 h) in mice with Raji tumor xenografts. (64)Cu-LLP2A-CB-TE2A was imaged (4 and 24 h) on a small-animal PET scanner in the same mouse model.The highest tumor uptake in pharmacokinetic studies was obtained with LLP2A-DOTA and (LLP2A-DOTA)(4)-PEG(10,000). For (111)In-LLP2A-DOTA (1 nM) at 4 and 24 h after injection, ratios of tumor to blood and tumor to nontumor (normal) organ (T/NT) were 8 to 35:1 for all organs or tissue except the spleen, marrow, and kidney, which were between 2:1 and 1:1. Tetravalent (LLP2A-DOTA)(4)-PEG(10,000) (1.1 nM) had tumor uptake similar to the univalent LLP2A-DOTA but higher liver, marrow, and kidney uptake. The excellent T/NT of LLP2A was also demonstrated by small-animal PET with (64)Cu-LLP2A-CB-TE2A at both 4 and 24 h after injection; obvious spleen targeting was apparent, but little kidney or liver activity was observed.Of the conjugates investigated, the univalent, non-PEGylated ligand (111)In-LLP2A-DOTA exhibited the best T/NT ratios and showed the greatest potential for imaging of alpha4beta1 in human lymphoma. Furthermore, this univalent non-PEGylated LLP2A format, as (64)Cu-LLP2A-CB-TE2A, demonstrated excellent tumor targeting by small-animal PET and warrants further investigation as an agent for the study of alpha4beta1 expression in human lymphoid malignancies.

Radioimmunotherapy (RIT) is a method for selectively delivering radionuclides to cancer cells while reducing the radiation dose to normal tissues. However, because of slow clearance of MAbs, normal tissues also received radiotoxicity. One of the promising strategies is linking on-demand cleavable (ODC) peptides between radiometal chelates and the tumor targeting agents. We have tested this proof-of-concept by using ODC peptides that are designed to be cleaved only by TNKase and are resistant to cleavage by enzymes present in the plasma and the tumor. TNKase-specific peptide linkers using l- and d-amino acids were screened by OBOC combinatorial peptide libraries. One of the best peptides was linked to radiometal chelate and ChL6-MAb to prepare radioimmunoconjugate (RIC). Optimization and characterization of the linker conjugation to MAb show (a) 1-2 peptides linked to each MAb; (b) immunoreactivity >80%; (c) specific activity of the RIC 0.7-1 microCi/microg; (d) RIC stable over 7 days in human plasma; and (e) radiometal-chelated ODC peptide cleaved from the RIC in plasma by TNKase at clinical dose levels of 10 microg/ml. The percent release of radiochelate from RIC was 50% at 24h and 85% over 7 2h in vitro. This novel ODC-linked RIC could be a potential molecule for RIT.

Dextran and PEG-coated iron oxide nanoparticles (NP), when suitably modified to enable conjugation with molecular targeting agents, provide opportunities to target cancer cells. Monoclonal antibodies, scFv, and peptides conjugated to 20 nm NP have been reported to target cancer for imaging and alternating magnetic field (AMF) therapy. The physical characteristics of NPs can affect their in vivo performance. Surface morphology, surface charge density, and particle size are considered important factors that determine pharmacokinetics, toxicity, and biodistribution. New NanoFerrite (NF) particles having improved specific AMF absorption rates and diameters of 30 and 100 nm were studied to evaluate the variation in their in vitro and in vivo characteristics in comparison to the previously studied 20 nm superparamagnetic iron oxide (SPIO) NP. SPIO NP 20 nm and NF NP 30 and 100 nm were conjugated to (111)In-DOTA-ChL6, a radioimmunoconjugate. Radioimmunoconjugates were conjugated to NPs using 25 microg of RIC/mg of NP by carbodiimide chemistry. The radioimmunonanoparticles (RINP) were purified and characterized by PAGE, cellulose acetate electrophoresis (CAE), live cell binding assays, and pharmacokinetics in athymic mice bearing human breast cancer (HBT 3477) xenografts. RINP (2.2 mg) were injected iv and whole body; blood and tissue data were collected at 4, 24, and 48 h. The preparations used for animal study were90% monomeric by PAGE and CAE. The immunoreactivity of the RINP was 40-60% compared to (111)In-ChL6. Specific activities of the doses were 20-25 microCi/2.2 mg and 6-11 microg of mAb/2.2 mg of NP. Mean tumor uptakes (% ID/g +/- SD) of each SPIO 20 nm, NF 30 nm, and 100 nm RINP at 48 h were 9.00 +/- 0.8 (20 nm), 3.0 +/- 0.3 (30 nm), and 4.5 +/- 0.8 (100 nm), respectively; the ranges of tissue uptakes were liver (16-32 +/- 1-8), kidney (7.0-15 +/- 1), spleen (8-17 +/- 3-8), lymph nodes 5-6 +/- 1-2), and lung (2.0-4 +/- 0.1-2). In conclusion, this study demonstrated that 100 nm NF NP could be conjugated to (111)In-mAb so that the resulting RINP had characteristics suitable for AMF therapy. Although 100 nm RINP targeted tumor less than 20 nm SPIO RINP, their heating capacity is typically 6 times greater, suggesting the 100 nm NF RINP could still deliver better therapy with AMF.

Protease degradable linkers have been proposed to improve the therapeutic index (TI) (i.e., tumor to normal tissue) of molecular targeted radioisotope therapy by reducing unbound radiotargeting agent in the blood and other normal tissues. If the radioisotope is detached from the circulating targeting agent once the radioisotope level in the tumors has been maximized, the success of this system depends on the ability to anticipate a preferred intervention time that will lead to significantly improved TIs. This paper presents a method to predict preferred intervention times and TIs by using pharmacokinetic tracer studies carried out without intervention.Pharmacokinetic data for the blood and tumors from tracer doses of 111In-labeled chimeric and mouse monoclonal antibodies in patients and in mice were used as surrogates for corresponding 90Y radioimmunoconjugates. Data were fit with simple pharmacokinetic functions. A set of formulas was then developed to estimate the improvement in therapeutic index and the preferred intervention time, using simple modeling assumptions.A modeled introduction of enzymatic cleavable linkers resulted in an increase in the tumor-to-blood TI by a factor of 3.2-1.6 for the systems analyzed. As expected, the preferred intervention times varied depending on the pharmacokinetic data, but could be predicted based on a priori knowledge of the actual or anticipated pharmacokinetics in the absence of intervention.These results highlight the potential value of cleavable linkers in substantially increasing the TI, and provide an approach for estimating a preferred intervention time, using actual or predicted pharmacokinetic data obtained without intervention.

A first-generation series of novel small molecules, collectively known as selective high-affinity ligands (SHALs), were designed and synthesized to mimic the binding of Lym-1, a monoclonal antibody (mAb) shown to be an effective cytotoxic and radionuclide carrier molecule for targeting non-Hodgkin's lymphoma (NHL). Created as radionuclide targeting molecules, these SHALs were intended to have the human leukocyte antigen-DR (HLA-DR) selectivity of Lym-1 mAb and the pharmacokinetics of a small molecule. Because of the remarkable bioactivity of Lym-1 in vitro, the direct antilymphoma activity of three of these SHALs was tested. Two of these SHALs were bidentate and consisted of two ligands connected to the carboxyl and amino groups of lysine and polyethylene glycol (PEG); the third SHAL was a dimeric version of one of the former two SHALs linked with PEG. The three SHALs tested were: LeLPLDB, that contained one deoxycholate and one 5-leu-enkephalin as ligands; (LeacPLD)2LPB, a bis version of LeLPLDB intended to improve "functional affinity"; and ItPLDB, that contained the ligands, deoxycholate and triiodothyronine. Micromolar concentrations of all three SHALs showed binding to Raji, an HLA-DR10-positive human malignant B-cell line but no binding to CEM or Jurkat's, HLA-DR10-negative malignant T-cell lines. Additionally, the Raji cell membrane distributions of all three SHALs and of Lym-1 were remarkably similar. Unlike Lym-1, which causes substantial growth inhibition and cell death in NHL cell lines, these SHALs had no direct antilymphoma activity. In summary, three first-generation SHALs lacked direct antilymphoma activity, although they had selective NHL B-cell binding like Lym-1 mAb. Because of their small size, these SHALs have potential as radionuclide carrier substitutes for Lym-1 mAb to target the HLA-DR10 NHL-related cell-surface protein.

Purpose: Radioactivity deposition in normal tissues limits the dose deliverable by radiopharmaceuticals (RP) in radioimmunotherapy (RIT). This study investigated the absorbed radiation dose in normal tissues for prostate cancer patients in comparison to breast cancer patients for 2 RPs using the monoclonal antibody (MAb) m170. Methods and Materials: 111 In-DOTA-glycylglycylglycyl-l-p-isothiocyanatophenylalanine amide (GGGF)-m170 and 111 In-1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) 2-iminothiolane (2IT)-m170, representing the same MAb and chelate with and without a cleavable linkage, were studied in 13 breast cancer and 26 prostate cancer patients. Dosimetry for 90 Y was calculated using 111 In MAb pharmacokinetics from the initial imaging study for each patient, using reference man– and patient-specific masses. Results: The reference man–specific radiation doses (cGy/MBq) were not significantly different for the breast and the prostate cancer patients for both RPs in all but one tissue-RP combination (liver, DOTA-2IT). The patient-specific doses had differences between the groups most of which can be related to weight differences. Conclusions: Similar normal tissue doses were calculated for two groups of patients having different cancers and genders. This similarity combined with continued careful analysis of the imaging data might allow the use of higher starting doses in early phase RIT studies.

The most potent method for augmenting the cytocidal power of monoclonal antibody (MAb) treatment is to conjugate radionuclides to the MAb to deliver systemic radiotherapy (radioimmunotherapy; RIT). The antigen, MAb, and its epitope can make a difference in the performance of the drug. Additionally, the radionuclide, radiochemistry, chelator for radiometals and the linker between the MAb and chelator can have a major influence on the performance of drugs (radiopharmaceuticals) for RIT. Smaller radionuclide carriers, such as antibody fragments and mimics, and those used for pretargeting strategies, have been described and evaluated. All of these changes in the drugs and strategies for RIT have documented potential for improved performance and patient outcomes. RIT is a promising new therapy that should be incorporated into the management of patients with B-cell non-Hodgkin's lymphoma (NHL) soon after these patients have proven incurable. Predictable improvements using better drugs, strategies, and combinations with other drugs seem certain to make RIT integral to the management of patients with NHL, and likely lead to cure of currently incurable NHL.

The development of recombinant antibodies has facilitated the exploitation of the Ab-Ag interaction specificity for targeted therapies. A fully human antibody, with custom integrated designs, can be obtained in one-third the time, compared to development of antibodies by hybridoma technology. Recombinant antibodies can be tailored for specific applications, "armed" with cytotoxic agents in a controllable fashion, and used for extracellular and intracellular targeting. Multitargeted and combination therapies are rapidly evolving for the treatment of cancer. Antibody therapeutics, costly to develop and produce, have proven beneficial in the clinic.

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.

Human xenografts in athymic mice are frequently used as preclinical models of cancer to investigate the targeting of drugs. In order to distinguish specific from nonspecific targeting of the xenograft, the mice can be implanted with different malignant cell lines. We studied in xenograft success and growth rates after implantation of human lymphoma and breast cancer cells to begin an assessment of the validity of this approach for distinguishing specific from nonspecific targeting. Investigations were undertaken to determine the effect of two different cell-line xenografts, and prior radiation needed for one of the xenografts, on implantation success and growth rates.Female athymic mice were given 4 Gy of external beam radiation 4 days prior to subcutaneous (s.c.) abdominal implantation of 6 x 10(6) Raji human lymphoma cells. One week later, 3 x 10(6) hamster blood transfusion (HBT) 3477 human breast cancer cells were implanted s.c. in a contralateral abdominal site. Xenografts were evaluated frequently thereafter. Xenograft success and growth rates were compared to those observed in "historical" control groups, wherein only a single xenograft of each type was implanted.Raji xenografts developed from 73.7% of the implantations, and 100% of the HBT 3477 xenografts were successful in the experimental group. The "historical" Raji xenograft success rate was 74.1% (+/-9.3%), and the "historical" HBT 3477 xenografts success rate was 99.0% (+/-1.1%). HBT 3477 xenografts did not affect the growth rate of the Raji xenografts, and the mean doubling time for the experimental Raji xenografts was 6.3 days (+/-4.5 days), compared to the "historical" control group mean of 5.1 days (+/-3.9 days; p = 0.2). Similarly, the growth rates for the HBT 3477 xenografts were not affected by the Raji xenografts and the pre-radiation needed for this model. Mean doubling time for HBT 3477 xenografts in the presence of Raji xenografts was 9.2 days (+/-17.6 days), compared to a doubling time of 1.4 days (+/-15.2 days; p = 0.55 and 0.94 studies 1 and 2, respectively). Mean HBT 3477 xenograft doubling time for the "historical" control group was 4.4 days (+/-6.0 days).Implantation of Raji xenografts and HBT 3477 breast cancer xenografts in the same mouse did not affect xenograft success or growth rates, even when whole-body radiation of 4 Gy was used to promote Raji xenografts. These observations are not intended to imply an absence of differences in other biological parameters in this sytem or to encourage extrapolation of the conclusions indiscriminately to other preclinical models. Contrarily, our aim was to encourage other investigators to further validate these frequently used approaches.

Purpose: Monoclonal antibodies (mAb) in combination and mAbs combined with a radionuclide (radioimmunotherapy) have both been more effective in patients than mAb monotherapy.Experimental Design: Using assays of cell growth and viability, the dose response and temporal characteristics of CD20 (rituximab) and HLA-DR (Lym-1) mAbs, singly and in combination, and of 90Y-conjugated Lym-1 mAb have been characterized in five human lymphoma cell lines (B35M, Raji, SU-DHL-4, SU-DHL-6, and Ramos) spanning Burkitt's to diffuse large cell lymphoma. Although Ramos had a lower HLA-DR density, these cell lines were otherwise selected because of high cell surface CD20 and HLA-DR abundance. Assays of cell growth and death were done using microscopy and trypan blue dye.Results: Lym-1 and rituximab, used singly, showed direct antilymphoma effects; those of Lym-1 were often more potent than those of rituximab. Combinations of these mAbs were more effective, sometimes synergistic, than either mAb singly, even in more resistant SU-DHL-4 cells. Conjugation of 90Y to Lym-1 also augmented potency in all cell lines and overcame resistance to both Lym-1 and rituximab in Ramos cells.Conclusions: Lym-1 exhibited substantially greater direct antilymphoma effects than rituximab in lymphoma cells in culture. Combination of Lym-1 with rituximab or 90Y increased potency and overcame treatment resistance in lymphoma cells. Greater use of combination therapies of this type to increase potency and range of effectiveness seems likely to improve patient outcome.

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.

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.

Anti-HLA-DR and anti-CD20 monoclonal antibodies (MAbs) have been effective for immunotherapy and radioimmunotherapy in non-Hodgkin's lymphoma (NHL). The aim of our study was to compare the antilymphoma effects of Lym-1 and rituximab in human lymphoma cell lines, using assays of viability, apoptosis, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC), under conditions relevant to the clinic.To characterize response relationships at varied concentrations of Lym-1 and rituximab, growth inhibition and cell death were assayed over 96 hours in four NHL cell lines derived from Burkitt's or large-cell lymphoma patients. Untreated cells and cells treated with an mLym-1 isotype-matched MAb were used as negative controls for direct assays. Western blot was used to detect apoptosis through the activation of caspase-3 and cleavage of poly (ADP-ribase) polymerase (PARP). The indirect cytotoxicity of Lym-1 and rituximab was assayed at varied concentrations, using ADCC activity in the presence of purified peripheral blood leukocytes and CDC activity in the presence of human donor serum.Lym-1 and rituximab showed significant direct and indirect antilymphoma effects. Lym-1 had a substantial, and statistically greater, effect than rituximab over longer intervals of time. In Raji and B35M cells, Lym-1 induced potent growth inhibition reflected by 90% and 94% reductions in viable cells, respectively, whereas rituximab induced 63% and 56% reductions. Concurrently, Lym-1 increased nonviable cells by 372% and 153% in these cells, respectively, whereas rituximab induced 139% and 43% increases. Lym-1-induced apoptosis was greater than that of rituximab in all cell lines tested. Lym-1, both the chimeric form and the mouse parent, mediate ADCC more effectively, in the presence of a total peripheral blood leukocyte (PBL) population, than does rituximab, although the results for CDC activity were mixed.In conclusion, Lym-1 had more potent direct and indirect cytotoxic effects than rituximab in lymphoma cells under conditions achievable in patients. Because the HLA-DR target antigen of Lym-1 is enriched on most B-cell lymphomas, these results support its complementary use in patients as an alternative to CD20 for monoimmunotherapy and for combination immunotherapy with rituximab, because the HLA-DR and CD20 antigens are physically and functionally coupled on human B cells.

Because of their remarkable effectiveness in radioimmunotherapy (RIT), 2 anti-CD20 monoclonal antibody (MAb) drugs, one labeled with indium 111 for imaging or yttrium 90 for therapy, and another labeled with iodine I 131 for imaging and therapy, have been approved for use in patients with non-Hodgkin's lymphoma (NHL). Successful RIT for lymphomas is due in large part to the rapid and efficient binding of the targeted MAb to lymphoma cells. Carcinomas are more difficult to access, necessitating novel strategies matched with radionuclides with specific physical properties. Because there are many radionuclides from which to choose, a systematic approach is required to select those preferred for a specific application. Thus far, radionuclides with γ emissions for imaging and particulate emissions for therapy have been investigated. Radionuclides of iodine were the first to be used for RIT. Many conventionally radioiodinated MAbs are degraded after endocytosis by target cells, releasing radioiodinated peptides and amino acids. In contrast, radiometals have been shown to have residualizing properties, advantageous when the MAb is localized in malignant tissue. β-emitting lanthanides like those of 90 Y, lutetium 177, etc. have attractive combinations of biologic, physical, radiochemical, production, economic, and radiation safety characteristics. Other radiometals, such as copper-67 and copper-64, are also of interest. α-emitters, including actinium-225 and bismuth- 213, have been used for therapy in selected applications. Evidence for the impact of the radionuclide is provided by data from the randomized pivotal phase III trial of 90 Y ibritumomab tiuxetan (Zevalin®) in patients with NHL; responses were about 2 times greater in the 90 Y ibritumomab tiuxetan arm than in the rituximab arm. It is clear that RIT has emerged as a safe and efficient method for treatment of NHL, especially in specific settings.

Radioimmunotherapy (RIT) delivered by radiometal immunoconjugates (RICs) is dose limited by deposition and retention of radioactivity in normal tissues. In order to increase elimination of radioactivity from the liver and body, a peptide having a specific cathepsin B cleavage site was placed between the radiometal chelate, 111In-DOTA, and the panadenocarcinoma monoclonal antibody (MAb), m170.Indium-111 (111In)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-2-iminothiolane (2IT)-m170 and 111In-DOTA-peptide-m170, representing the same MAb and chelate without and with a cleavable linkage, were studied in athymic mice and patients with breast or prostate cancer. Pharmacokinetics, cumulated activities and therapeutic indices (TI), were evaluated. Cumulated activities in the liver and tumors were calculated and used as a surrogate for radiation dose.Except for liver, the pharmacokinetics of 111In-DOTA-peptide-m170 were similar to those of the 111In-2IT-2-[p(bromoacetamido)benzyl]-1,4,7,10-tetraazocyclododecane-N,N',N",N"'-tetraacetic acid-m170 (111In-2IT-BAD-m170) in mice and patients. Liver cumulated activities for 111In-DOTA-peptide-m170 were consistently decreased when compared to those for 111In-2IT-BAD-m170, reductions varying between 22-30%. Cumulated activities for 111In-DOTA-peptide-m170 in the malignant tumors of the patients were as great as those for 111In-2IT-BAD-m170, so that the tumor-to-liver cumulated activity ratios (therapeutic indices) were better for 111In-DOTA-peptide-m170.A cathepsin-B-cleavable peptide used to link chelated 111In to MAb, m170, reduced liver cumulated activity (radiation dose) and improved the TI. This novel linker illustrates the importance of linker technology in the development of safer RICs for cancer therapy.

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.

Immunotherapies for cancer offer attractive alternatives to conventional therapies although human anti-globulin antibody (HAGA) against the antibody (Ab) administered to the patient can be an obstacle to repeated treatment. Monoclonal antibodies (mAb), whether foreign or human in origin, have been used safely in patients for two decades. Adverse events have not proven to be significant clinical obstacles, although alterations of pharmacokinetic behavior of subsequently administered Ab can lead to less effective therapy. Not only is HAGA safe, but it can be associated with beneficial immunity in patients. Studies have shown that some patients have unexpectedly prolonged survival associated with HAGA. In our own non-Hodgkin's lymphoma (NHL) patients treated with mouse Lym-1 anti-lymphoma mAb, a high human anti-mouse Ab (HAMA) titer was associated with increased survival. The possible mechanisms linking HAMA responses to survival are likely related to Ab generated against the idiotopes of the administered Ab. An induced immune cascade in these patients, including anti-idiotypic Ab (Ab2) and cytotoxic Ab1' or Ab3 probably contributed to survival. In summary, HAGA should not a priori preclude the therapeutic use of Ab for cancer.

Radionuclides provide biologically-distributed vehicles for radiotherapy of multifocal cancer. Two algorithms, fixed vs individualized, have been used to prescribe the therapeutic dose of radionuclide (GBq) for the patient. The individualized method for prescribing radionuclide dose takes variations in drug pharmacokinetics into consideration, whereas the fixed method depends, in part, on documentation that there is little interpatient pharmacokinetic variability for the radiolabeled drug. Two data bases, selected to compare iodine-131((131)I) and indium-111((111)In) labeled MAbs, were used to assess interpatient pharmacokinetic variability and its impact on radionuclide dose prescription. Pharmacokinetic data obtained over 7 days for non-Hodgkins lymphoma (NHL) patients given (131)I-Lym-1 (n = 46) or (111)In-Lym-1 (n = 13) were used to obtain cumulated activities. Although (131)I-Lym-1 often showed greater interpatient variability, (111)In-Lym-1 showed several-fold variability for many tissues. Both (131)I- and (111)In-Lym-1 had sufficient interpatient variability to be significant for radionuclide dose prescription, depending on the dose-limiting critical tissue. Interpatient variability exceeded intra- and interoperator variability and intrapatient variability over time for a single institution. In summary, the magnitude of interpatient pharmacokinetic variability for (131)I- and (111)In-Lym-1 suggested that an optimally safe and effective therapy can be best achieved when radionuclide dose is influenced by estimated radiation dose, if the latter is reproducible from institution to institution.

Although fractionation can be used in a discrete radiobiologic sense, herein it is generally used in the broader context of administration of multiple, rather than single, doses of radionuclide for radioimmunotherapy (RIT) or other targeted radionuclide therapies. Fractionation is a strategy for overcoming heterogeneity of monoclonal antibody (MAb) distribution in the tumor and the consequent nonuniformity of tumor radiation doses. Additional advantages of fractionated RIT are the ability to 1) provide patient-specific radionuclide and radiation dosing, 2) control toxicity by titration of the individual patient, 3) reduce toxicity, 4) increase the maximum tolerated dose (MTD) for many patients, 5) increase tumor radiation dose and efficacy, and 6) prolong tumor response by permitting treatment over time. However, fractionated RIT has logistic and economic implications. Preclinical and clinical data substantiate the advantages of fractionated RIT, although the radiobiology for conventional external beam radiotherapy does not provide a straightforward rationale for RIT unless fractionation leads to more uniform distribution of radiation dose throughout the tumor. Preclinical data have shown that toxicity and mortality can be reduced while efficacy is increased, thereby providing inferential evidence of greater uniformity of radiation dose. Direct evidence of superior dosimetry and tumor activity distribution has also been found. Clinical data have shown that toxicity can be better controlled and reduced and the MTD extended for many patients. It is clear that fractionated RIT can only fulfill its potential if the effects of critical issues, such as the number and amount of radionuclide doses, the radionuclide physical and effective half-life, and the dose interval, are better characterized.

Radionuclide therapy extends the usefulness of radiation from localized disease of multifocal disease by combining radionuclides with disease-seeking drugs, such as antibodies or custom-designed synthetic agents. Like conventional radiotherapy, the effectiveness of targeted radionuclides is ultimately limited by the amount of undesired radiation given to a critical, dose-limiting normal tissue, most often the bone marrow. Because radionuclide therapy relies on biological delivery of radiation, its optimization and characterization are necessarily different than for conventional radiation therapy. However, the principals of radiobiology and of absorbed radiation dose remain important for predicting radiation effects. Fortunately, most radionuclides emit gamma rays that allow the measurement of isotope concentrations in both tumor and normal tissues in the body. By administering a small "test dose" of the intended therapeutic drug, the clinician can predict the radiation dose distribution in the patient. This can serve as a basis to predict therapy effectiveness, optimize drug selection, and select the appropriate drug dose, in order to provide the safest, most effective treatment for each patient. Although treatment planning for individual patients based upon tracer radiation dosimetry is an attractive concept and opportunity, practical considerations may dictate simpler solutions under some circumstances. There is agreement that radiation dosimetry (radiation absorbed dose distribution, cGy) should be utilized to establish the safety of a specific radionuclide drug during drug development, but it is less generally accepted that absorbed radiation dose should be used to determine the dose of radionuclide (radioactivity, GBq) to be administered to a specific patient (i.e., radiation dose-based therapy). However, radiation dosimetry can always be utilized as a tool for developing drugs, assessing clinical results, and establishing the safety of a specific radionuclide drug. Bone marrow dosimetry continues to be a "work in progress." Blood-derived and/or body-derived marrow dosimetry may be acceptable under specific conditions but clearly do not account for marrow and skeletal targeting of radionuclide. Marrow dosimetry can be expected to improve significantly but no method for marrow dosimetry seems likely to account for decreased bone marrow reserve.

Radioimmunotherapy of cancer utilizes anti-tumor antibodies or antibody fragments conjugated to radionuclides to deliver radiation selectively to tumors. However, radiolabeled proteins deposit radioactivity in normal organs that metabolize or conserve proteins and peptides, primarily liver and kidneys. To accelerate the clearance of radioactivity from normal tissues, linkers between the antibody or antibody fragment and the radioactive moiety have been designed for cleavage in the liver and kidneys, to liberate low molecular weight radioactive species for rapid excretion. Modest success in improving the tumor-to-liver and tumor-to-kidney radiation dose ratios have been achieved in preclinical studies. Such changes when taken to clinical studies have suggested useful impact on therapeutic work. Recent advances in the development of cleavable linkers are described.

One of the most promising strategies for treating cancer is the addition of antiangiogenic therapy to therapeutic regimens. Angiogenesis, or the growth of new blood vessels from preexisting vessels, is essential both for the growth of a primary tumor and for successful metastasis. As a result of intense research in this field, a number of antiangiogenic agents have been identified and have demonstrated varying degrees of success in inhibiting the growth of solid tumors and metastases in preclinical and clinical studies. The real potential of antiangiogenic agents for cancer therapy resides in strategic combinations with each other, with chemotherapy, with radiation, and with tumor-targeting agents, such as radioimmunotherapy. Along with this new opportunity to develop synergistic therapy comes the challenging complexities of the physiologic systems regulating angiogenesis. These multifaceted systems could intimidate investigators seeking to take advantage of the potential synergy in combined cancer therapy. To aid in these efforts, this overview of key antiangiogenic agent mechanisms, combination strategies and initial studies of the potential synergy with chemotherapy, radiation and radioimmunotherapy is presented.

Cancer-seeking antibodies (Abs) carrying radionuclides can be powerful drugs for delivering radiotherapy to cancer. As with all radiotherapy, undesired radiation dose to critical organs is the limiting factor. It has been proposed that optimization of radioimmunotherapy (RIT), that is, maximization of therapeutic efficacy and minimization of normal tissue toxicity, depends on a foreknowledge of the radiation dose distributions to be expected. The necessary data can be acquired by established tracer techniques, in individual patients, using quantitative radionuclide imaging. Object-oriented software systems for estimating internal emitter radiation doses to the tissues of individual patients (patient-specific radiation dosimetry), using computer modules, are available for RIT, as well as for other radionuclide therapies. There is general agreement that radiation dosimetry (radiation absorbed dose distribution, cGy) should be utilized to establish the safety of RIT with a specific radiolabeled Ab in the early stages (i.e. phase I or II) of drug evaluation. However, it is less well established that radiation dose should be used to determine the radionuclide dose (amount of radioactivity, GBq) to be administered to a specific patient (i.e. radiation dose-based therapy). Although treatment planning for individual patients based upon tracer radiation dosimetry is an attractive concept and opportunity, particularly for multimodality RIT with intent to cure, practical considerations may dictate simpler solutions under some circumstances.

Radioimmunotherapy (RIT) is a promising approach for treating metastatic breast cancer. Initial clinical trials using 131I radioimmunoconjugates, and more recent studies employing 90Y, have demonstrated objective, although transient, antitumor effects in heavily pretreated patients with minimal toxicity. Antibodies targeting unique epitopes of epithelial glycoprotein mucin (MUC-1) on breast cancer cell surfaces that have been studied in patients include BrE-3 (murine and humanized) and m170 (murine). Both antibodies react with at least 90% of breast cancers. In these and other RIT trials, myelosuppression has been the dose-limiting toxicity. However, this toxicity has been successfully circumvented with the use of autologous peripheral blood stem cell transplantation, and recent clinical trials have escalated 90Y doses up to 50 mCi/m(2). The therapeutic index indicates that using these agents with stem cell support should deliver 9000 to 18000 rads to metastatic tumors. Development of improved chelates that are readily metabolized in the liver may reduce doses to this organ, projected to be next in line as dose-limiting. Combination therapy will be required to produce durable benefits in metastatic breast cancer. Low dose taxanes are synergistic with RIT in preclinical studies and when administered in the optimal sequence could sensitize tumor cells to the continuous low dose radiation delivered by RIT, without increasing toxicity. The addition of systemically administered tumor targeting radiation therapy using RIT as part of combined modality therapy may enhance the rate of complete response and, in patients with minimal metastatic disease, could lead to curative therapy.

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.

BACKGROUND Radioimmunotherapy (RIT) is a new therapeutic modality capable of systemic delivery of radionuclides specifically to sites of metastatic cancer. The L6 monoclonal antibody has been shown to target prostate cancer in preclinical studies and, along with chimeric L6 (ChL6), has been used for RIT in breast cancer patients. METHODS Pharmacokinetics, blood counts, body weight, and antitumor activity of RIT with 90yttrium-(90Y)-DOTA-peptide-ChL6 (75–260 μCi) were determined in nude mice bearing human prostate cancer (PC3) xenografts. RESULTS RIT produced durable, dose-dependent antitumor effects with a 100% response rate using 112 μCi and 150 μCi (the maximum tolerated dose) of 90Y-DOTA-peptide-ChL6. Myelotoxicity was reversible, dose-limiting, and dose-related. RIT was associated with improved survival (P = 0.05). All 5 mice in the 150-μCi group survived the 84-day study period vs. 1/8 (13%) for untreated, tumored control mice. CONCLUSIONS 90Y-DOTA-peptide-ChL6 targets PC3 human prostate cancer xenografts in nude mice and has an antitumor effect. These results provide a basis for future RIT trials for patients with metastatic prostate cancer. Prostate 44:187–192, 2000. © 2000 Wiley-Liss, Inc.

Use of a single lot of monoclonal antibody (MoAb) or immunoconjugate for clinical studies provides efficiency of scale and consistent characteristics for MoAb-based pharmaceuticals. Lym-1, an anti-lymphoma mouse IgG2 alpha chimeric L6 (ChL6), an anti-adenocarcinoma mouse IgG2 alpha-human IgG1 chimera, and the immunoconjugate 2IT-BAD-Lym-1 were examined for stability following storage.Lym-1, ChL6, and 2IT-BAD-Lym-1 were aliquotted with filtration and stored at -70 degrees C for up to 8.5 years. 2IT-BAD-Lym-1 stored for 6.3 years (lot A) was compared to freshly prepared 2IT-BAD-Lym-1 (lot B). MoAbs were thawed and examined yearly by gel filtration high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), cellulose acetate electrophoresis (CAE), and Scatchard analysis of antigen binding. 2IT-BAD-Lym-1 was evaluated by HPLC, CAE, and radioimmunoassay. To assure the in vivo significance of the in vitro studies, 2IT-BAD-Lym-1 lots A and B were labeled with 111In and their pharmacokinetics in BALB/c mice were compared.Lym-1 demonstrated stability over 8.5 years, providing the following ranges of data over the interval: 98-100% chemical purity (HPLC), 96-100% monomeric fraction (CAE), 8.18-8.46 antigen binding pKa (Scatchard). Similar results were obtained for ChL6 for 7.4 years. HPLC and PAGE of Lym-1 and ChL6 have not changed from original manufacturer specifications, and both MoAbs remain sterile and apyrogenic. No significant differences between 2IT-BAD-Lym-1 lots A and B were observed by in vitro evaluation or pharmacokinetics in mice.Lym-1, ChL6, and 2IT-BAD-Lym-1 as manufactured and stored for 8.5, 7.4, and 6.3 years, respectively, demonstrated retention of structural and functional integrity.

Three planar, gamma camera methods for quantitating radiopharmaceuticals, such as radiolabeled antibodies, were investigated. Iodine-123 (123I), technetium-99m (99mTc) or indium-111 (111In) in the liver and spleen of an abdominal phantom were assessed in this study. In the first approach, the number of counts detected in a "single image" of the liver or spleen was used to measure radionuclide content using an attenuation correction factor (ACF) calculated from data obtained without radionuclide in the background volume of the phantom. In the other two methods, radionuclide content was derived from either the geometric mean (GM) of counts in conjugate, opposed images ("global conjugate") or in individual, opposed pixels of the conjugate, opposed images ("pixel conjugate") of the liver and spleen. Both of the conjugate image methods were corrected for attenuation with a first order ACF derived from a transmission image. The influence of background radionuclide on the accuracy of quantitation was studied by filling the background volume of the phantom with water containing 7 or 14 percent of the concentration of the radioactive water placed in the liver and spleen. The best estimates of radionuclide content were obtained by quantitation from the GM of counts in conjugate images of the liver and spleen. Radionuclide content of the liver and spleen could be determined from a single image if correction for attenuation was available. In all instances, measurements were less accurate for the spleen and for either organ when 111In was used. These results further validate and extend observations reported by others and provide a basis for radiation dosimetry for these and similar radionuclides and organs in patients.

In order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain antigen-binding (scFv) protein molecule was produced. Because the commonly used polymerase chain reaction (PCR) method frequently causes unexpected mutations, we developed a non-PCR method for scFv gene assembly. The method involved a stepwise linkage of doubly-restricted DNA fragments and re-digestion of the resultant concatamers. Using this strategy, the Lym-1 scFv expression gene was readily constructed without mutations. The recombinant gene was cloned into an expression vector and scFv protein was expressed. The method can be used for other genes or DNA recombination.

BACKGROUND Radioimmunotherapy (RIT) is a promising new modality for targeted, systemic delivery of radionuclides specifically to sites of androgen-independent metastatic prostate cancer. To be effective, RIT requires an antibody with specificity for malignant cells and appropriate pharmacokinetics in the body. METHODS Specific binding of the L6 monoclonal antibody to prostate cancer cell lines or cell lysates was determined by enzyme-linked immunoabsorbent assay (ELISA), solid-phase radioimmunoassay, and immunofluorescent staining. Biodistribution, tumor uptake, and whole body and blood clearances of 125I-L6 were determined in nude mice bearing human prostate cancer xenografts. RESULTS The L6 monoclonal antibody showed strong binding to the lysates of PC3 and DU145 prostate cancer cell lines, and 66% binding to live PC3 cells. The L6 antibody specifically targeted prostate cancer in PC3 and DU145-tumored nude mice, where approximately 10% of the injected dose of 125I-L6 bound to prostate cancer. Low-normal organ uptake was found, and the blood clearances were similar in each group of tumored mice. CONCLUSIONS The L6 monoclonal antibody targets human prostate cancer xenografts in nude mice and has low-normal organ uptake. Therefore, further study of the radiolabeled L6 monoclonal antibody for RIT of prostate cancer is warranted. Prostate 37:91–97, 1998. © 1998 Wiley-Liss, Inc.

PURPOSE Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced remissions in patients with non-Hodgkin's lymphoma (NHL) when labeled with iodine 131 ((131)I). Based on the strategy of fractionating the total dose, this study was designed to define the maximum-tolerated dose (MTD) and efficacy of the first two, of a maximum of four, doses of (131)I-Lym-1 given 4 weeks apart. Additionally, toxicity and radiation dosimetry were assessed. MATERIALS AND METHODS Twenty patients with advanced NHL entered the study a total of 21 times. Thirteen (62%) of the 21 entries had diffuse large-cell histologies. All patients had disease resistant to standard therapy and had received a mean of four chemotherapy regimens. (131)I-Lym-1 was given after Lym-1 and (131)I was escalated in cohorts of patients from 40 to 100 mCi (1.5 to 3.7 GBq)/m2 body surface area. RESULTS Mean radiation dose to the bone marrow from body and blood (131)I was 0.34 (range, 0. 1 6 to 0.63) rad/mCi (0.09 mGy/MBq; range, 0.04 to 0.17 mGy/ MBq). Dose-limiting toxicity was grade 3 to 4 thrombocytopenia with an MTD of 100 mCi/m2 (3.7 GBq/m2) for each of the first two doses of (131)I-Lym-1 given 4 weeks apart. Nonhematologic toxicities did not exceed grade 2 except for one instance of grade 3 hypotension. Ten (71 %) of 14 entries who received at least two doses of (131)I-Lym-1 therapy and 11 (52%) of 21 total entries responded. Seven of the responses were complete, with a mean duration of 14 months. All three entries in the 100 mCi/m2 (3.7 MBq/m2) cohort had complete remissions (CRs). All responders had at least a partial remission (PR) after the first therapy dose of (131)I-Lym-1. CONCLUSION (131)I-Lym-1 induced durable remissions in patients with NHL resistant to chemotherapy and was associated with acceptable toxicity. The nonmyeloablative MTD for each of the first two doses of (131)I-Lym-1 was 100 mCi/m2 (total, 200 mCi/m2) (3.7 GBq/m2; total, 7.4 GBq/m2).

Accurate and reproducible quantification of tumor radioactivity by imaging requires definition of a region of interest (ROI) for the tumor. The use of a threshold for creating the tumor ROI based on tumor-to-background image contrast (image contrast) was examined. Quantification of iodine-131 in spheres in a phantom that simulated tumors in patients was investigated using planar imaging and geometric-mean and effective-point-source methods. Thresholds that provided the least quantitative error for spheres with different diameters (1-5 cm) and locations (0-11 cm deep in the body), 131I concentrations (0.037-3.2 MBq/ml), and sphere-to-background concentration ratios (1:0, 14:1 and 7:1) were investigated. The correlation between threshold and sphere image contrast was examined. The phantom study showed that an appropriate threshold value for quantification of tumor radioactivity could be determined using image contrast for a single view, provided that image contrast was/=1.5. The error of quantification was less than 10% for spheres with high image contrast (/=1.5) but was greater than 17% for spheres with low image contrast (1.5). When image contrast-dependent thresholds were applied to patient studies, 131I concentrations determined by imaging were in good agreement with the concentrations determined by counting biopsy samples. Additionally, reproducibility was improved when compared with a visual boundary method. It is concluded that accurate and reproducible quantification of radioactivity in tumors is achievable using thresholds based on image contrast if image contrast is greater than or equal to 1.5. Optimal thresholds for quantification of tumor radioactivity were similar if image contrast was similar despite differing tumor diameters, locations and 131I concentrations. Under certain circumstances, the effective-point-source method was preferable to the geometric-mean method.

Monoclonal antibodies were raised against yttrium(III)-1, 4, 7, 10-tetraazacyclododecane-N,N',N''N'''--tetraacetic acid (Y-DOTA) and copper(II)-1, 4, 8, 11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (Cu-TETA). Four hybridomas with high Y-DOTA binding activity and one hybridoma with Cu-TETA activity were selected. MAbs were purified from mouse ascites by Protein A affinity chromatography and characterized. Affinity constants were determined by equilibrium dialysis and the highest affinity Y-DOTA MAb (K(aff) = 1.9 x 10(8) M(-1)) was further characterized by competitive ELISA. Gd-DOTA competed as well as Y-DOTA, whereas In-DOTA required 740x higher concentrations for 50% inhibition of this Y-DOTA MAb binding to human serum albumin-Y-DOTA-coated microtiter plates. These anti-metal chelate MAbs have potential use as vehicles for the pretargeted delivery of radiometal chelates to tumors.

A patient with aggressive, chemotherapy-resistant non-Hodgkins lymphoma (NHL) was treated with 131I-Lym-1, a mouse antibody, on a protocol designed for serial therapy. Human anti-mouse antibody (HAMA) developed within 1 month of initial therapy. The patient also developed an antibody to the hypervariable region of the Lym-1 antibody (Lym-1 specific). Because the patient was responding to therapy, plasmaphoresis was used to reduce the level of HAMA followed by unlabeled Lym-1 calculated to be sufficient to block residual HAMA. This allowed additional therapy on three subsequent occasions over 5 months. Despite very high HAMA levels, no untoward effects from administrations of Lym-1 were observed. The HAMA response of the patient included anti-Lym-1 specific antibodies containing anti-idiotypic antibodies. The anti-Lym-1 antibody level has been sustained over the 9 year interval since 131I-Lym-1 therapy and has been associated with a uniquely long remission of the patient's disease. These observations demonstrate that, under certain circumstances, radioimmunotherapy (RIT) can be given safely and effectively despite HAMA. Anti-idiotypic antibodies could have induced an immune cascade that contributed to the prolonged disease-free survival of the patient.

The use of monoclonal antibodies (MoAbs) for immunotherapy and radioimmunotherapy has ushered in a new era in the treatment of non-Hodgkin's lymphoma.