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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

BACKGROUND Patients with B-cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) frequently have splenomegaly, which has been reported to cause poor tumor targeting of radiolabeled antibodies. Consequently, patients with splenomegaly have been ineligible for some trials of radioimmunotherapy because of the assumption that they would not benefit. METHODS Forty-nine patients with NHL and five with CLL received an initial dose of 131I-Lym-1 ranging from 740-8140 MBq. Six patients had prior splenectomy. The remaining 48 patients had spleen volumes ranging from 140-2830 ml determined using x-ray computed tomography. Medical Internal Radiation Dose Committee formalism was used to determine dosimetry, and spleen volume was used to adjust the S value for the spleen of each patient. RESULTS Spleen radiation dose decreased as spleen volume increased, although there was a positive correlation (r = 0.75) between spleen volume and spleen cumulated activity. There was no clear relationship between spleen volume and tumor radiation dose, although tumor radiation doses were low in five patients whose spleen volumes were greater than or equal to 970 ml. There was no apparent relationship between spleen volume and therapeutic response to 131I-Lym-1. Two of five patients whose spleen volumes were greater than or equal to 970 ml responded despite low tumor radiation doses, whereas two of six patients with prior splenectomy did not respond. CONCLUSIONS The results of this study provide no clear evidence that patients with splenomegaly should be excluded from radioimmunotherapy trials because of the assumption that they will not benefit. Splenomegaly was associated with decreased radiation dose to the spleen, and to tumors only for extraordinarily large spleens. Cancer 1997; 80:2553-7. © 1997 American Cancer Society.

BACKGROUND Because most patients with advanced non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) respond to radioimmunotherapy (RIT), the study was designed to evaluate the relationship between response and survival in patients treated with radiolabeled Lym-1. METHODS Fifty-seven patients with NHL (52) or CLL (5) were treated with radiolabeled Lym-1 antibody between 1985 and 1994. Influence of response to treatment on survival was examined directly and was also adjusted for other factors previously found to be significant predictors of survival. A multivariate model that was based on baseline Karnofsky performance status (KPS) and serum lactic dehydrogenase (LDH) predicted response and survival and was used to define risk groups. Proportional-hazards, Kaplan-Meier, and Landmark models were used to evaluate parameters for their ability to predict outcome. RESULTS By using a proportional-hazards model with response as a time-dependent variable, overall response (P < 0.001) and complete response (P = 0.006) were predictive of increased survival in univariate analyses. Overall response continued to be significant (P = 0.02) in multivariate analyses, even when risk groups that also predicted survival were included. Median survival of responders was 84 weeks, whereas that of nonresponders was 22 weeks when the Landmark method, based on response status at 16 weeks from start of therapy, was used to generate survival curves. CONCLUSIONS Response to treatment with radiolabeled Lym-1 was associated with increased survival, even when adjusted for baseline clinical parameters that also predicted for survival. The results provide the first unbiased evidence for survival effects of RIT in patients. Cancer 1997; 80:2706-11. © 1997 American Cancer Society.

BACKGROUND In clinical trials of radiolabeled monoclonal antibodies targeting metastatic breast carcinoma, myelosuppression has been the initial dose-limiting toxicity. We previously have shown that mobilized peripheral blood stem cell transfusions ameliorate this toxicity of radioimmunotherapy (RIT). Because of the difficulty we experienced harvesting adequate numbers of precursor cells from some of these heavily pretreated patients, we have compared the mobilization results and clinical histories of the metastatic breast carcinoma patients referred for RIT with those of metastatic breast carcinoma patients referred for high dose chemotherapy (HDCT) with transplantation. METHODS Mobilization of stem cells into the peripheral blood was accomplished using granulocyte-colony stimulating factor with or without chemotherapy. Granulocyte macrophage colony-forming assays (CFU-GM) and flow cytometric analysis for CD34 positive cells were used to evaluate the number of hematopoietic precursors mobilized and collected with each apheresis procedure. Clinical characteristics, including prior chemotherapy and external beam radiotherapy, tumor bulk, and performance status, were determined by chart review. RESULTS Significantly fewer hematopoietic precursors (both CD34 positive cells and CFU-GM) were harvested per procedure from the six RIT patients compared with a group of six patients with metastatic breast carcinoma who had stem cells harvested prior to HDCT (P = 0.002). There was no significant difference between the groups with regard to age or prior radiotherapy. All the RIT patients had received more chemotherapy, had a less favorable performance status (1 vs. 0), and had measurable tumor, whereas all the HDCT patients had minimal residual disease. CONCLUSIONS Patients enrolled in RIT studies had lower stem cell yields than metastatic breast carcinoma patients scheduled to receive HDCT with stem cell transplantation. Poor mobilization is unlikely to be due to the mobilizing regimen alone and may be related to the intensity of prior therapy and/or tumor bulk. Mobilizing adequate stem cells for multiple treatment cycles from patients on Phase I/II RIT trials may require new, more effective mobilizing regimens, but referral of patients earlier in their disease course, prior to chronic bone marrow damage and disease progression, is recommended strongly. Cancer 1997; 80:2728-32. © 1997 American Cancer Society.

BACKGROUND The severity of myelotoxicity after radioimmunotherapy has been predicted from body and blood radiation doses to marrow. However, marrow radiation can be increased substantially if the marrow or skeleton contains the malignancy targeted by the radiolabeled monoclonal antibodies. A study of 29 patients treated with iodine-131 (131I)-Lym-1 showed that radiation doses to marrow from body and blood had little correlation with myelotoxicity. The purpose of the present study was to assess the significance of marrow targeting and other factors for prediction of myelotoxicity. METHODS Injected radioactivity and nontargeted radiation doses to marrow were compared with peripheral blood cell counts after the first therapy dose of 131I-Lym-1 in 16 heavily pretreated patients with non-Hodgkin's lymphoma (NHL). Bone marrow biopsy, targeted marrow radiation doses, marrow image uptake scores, age, Karnofsky performance score (KPS), previous chemotherapy, and tumor burden were also compared with blood counts. RESULTS Myelotoxicity was not predicted well by injected radioactivity, total body radiation, or body and blood radiation doses contributed to marrow (P > 0.1). Biopsy-proven bone marrow lymphoma also failed to predict myelotoxicity (P > 0.1). Thrombocytopenia and leukopenia were predicted well by targeted radiation dose to marrow (P < 0.05) obtained by 131I imaging. Similarly, marrow image scores predicted decreases in platelets and white blood cells (WBCs; P < 0.05). Prediction of myelotoxicity using marrow radiation dose methods was slightly improved when serum lactic dehydrogenase (LDH), age, KPS, and prior chemotherapy were included in the analysis (P ≤ 0.01). CONCLUSIONS Prediction of myelotoxicity was improved in this group of patients by assessment of the targeting component of marrow radiation and was better predicted and obtained more easily by semiquantitative marrow image scores. Further improvement in prediction was slight when other factors were considered. Cancer 1997; 80:2558-66. © 1997 American Cancer Society.

BACKGROUND Radiation-induced DNA damage resulting in p53 protein attachment and downstream gene activation has been considered a major mechanism for tumor response to low dose rate radiation therapy. In this study, the mechanism of tumor response, and p53 gene status as well as levels of expression of p53 pathway genes were investigated in a human breast tumor (HBT 3477) before and after yttrium-90-DOTA-peptide-ChL6 (Y-90-ChL6) treatment of these xenografts. METHODS Mice with HBT 3477 xenografts were treated with 260 μCi Y-90-ChL6 and sacrificed 3, 24 and 48 hours after injection. Reverse transcriptase-polymerase chain reaction and/or Western blotting were used to measure the tumor levels of p53, p21CDKN1/WAF1 (p21), GADD45, and bcl-2. Single strand conformation polymorphism and direct sequencing were used to determine the mutational status of p53. Evidence of apoptosis was determined by cleavage of poly(ADP-ribose) polymerase (PARP). RESULTS Tumors regressed 4-7 days after treatment with 260 μCi Y-90-ChL6, resulting in a 79% tumor response. The p53 gene mutation found at codon 342 in HBT 3477 resulted in truncation of the p53 protein, and correlated with undetectable basal p21 protein levels. GADD45 and p53 mRNA decreased after therapy. bcl-2 mRNA was abundant, but decreased. Retinoblastoma phosphorylation showed no changes. Cleavage of PARP was detected at 3 hours and levels were increased greatly at 6 hours after therapy. CONCLUSIONS Response in the Y-90-ChL6 treated HBT 3477 xenograft tumors was independent of p53 and occurred by apoptosis. The down-regulation of bcl-2 may be the key in this apoptotic response to low dose rate radioimmunotherapy. Cancer 1997; 80:2529-37. © 1997 American Cancer Society.

BACKGROUND. The beneficial effects of radioimmunotherapy (RIT) may result from activation of molecular pathways that lead to programmed cell death (apoptosis). The influences of sequence and timing of 99 Y-DOTA-peptide-ChL6 antibody ( 90 Y-ChL6) and anti-epidermal growth factor receptor antibody (ch225) or paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) on efficacy and toxicity were examined. METHODS. Groups of human breast carcinoma (HBT 3477) tumored mice received paclitaxel (300 or 600 μg) or ch225 (70, 150, or 350 μg) at various intervals before or after 90 Y-ChL6. Mortality, hematologic toxicity, weight loss, and therapeutic efficacy were evaluated. RESULTS. Mice receiving paclitaxel within 24 hours of 90 Y-ChL6 had a 100% response rate; 48% were cured when paclitaxel was given 6 or 24 hours after 90 Y-ChL6. When 150 μg ch225 was given 24 hours before 90 Y-ChL6, the response and cure rates surpassed those of 90 Y-ChL6 alone. Timing of administration was critical, with mortality rates as high as 80% in some groups receiving 350 μg ch225 and 90 Y-ChL6. CONCLUSIONS. In this aggressive human breast carcinoma model, combined 90 Y-ChL6 and paclitaxel had a high therapeutic index with many cures. Sequence of administration was critical in order for ch225 or paclitaxel, when combined with 90 Y-ChL6, to enhance the response rate.

Large hospitals and biomedical research centers utilize decay-in-storage programs to minimize the volume of their low level radioactive waste. However, some medically useful radionuclides often contain small amounts of long-lived radionuclidic impurities which may complicate simple waste management procedures. We have evaluated the extent of this problem in low level radioactive waste involving 67Cu and (111)In over a 6-mo cycle of decay-in-storage by identifying the residual radionuclides in our dry waste using a multichannel analyzer. The multichannel analyzer was also used to quantify the radionuclide constituents of our liquid waste at the beginning of a decay-in-storage cycle. Radionuclides were identified by the presence of characteristic photopeaks of each isotope in the gamma spectrum and quantified by region of interest analysis. After a decay-in-storage cycle, long-lived 58Co, 57Co, and 56Co isotopes were observed in dry 67Cu waste and (114m)In identified in dry (111)In waste. The (114m)In was detected in dry (111)In waste containing initial (114m)In activity of 740 kBq (20 microCi), while the cobalt radionuclides were detected in dry 67Cu waste containing initial 58Co, 57Co, and 56Co activities of 444, 148, and 148 kBq (12, 4, and 4 microCi), respectively. Such dry low level radioactive waste was thus disqualified from short-term radioactive waste storage programs. The radionuclide constituents in the liquid waste were quantified in microCi mL(-1) and confirmed to be within the Nuclear Regulatory Commission set limits of 2 x 10(-4), 6 x 10(-4) and 6 x 10(-5) microCi mL(-1) for 58Co, 57Co, and 56Co, respectively, before disposal. The highest levels of long-lived isotopes that have been found in our liquid low level radioactive waste at the beginning of decay-in-storage were 5.5 x 10(-4), 4.8 x 10(-4), and 1.4 x 10(-4) microCi mL(-1) for 58Co, 57Co, and 56Co, respectively. Gamma spectrometry can be used to aid waste segregation and final management decisions on low level radioactive waste.

The selectivity of Lym-1 for malignant B lymphocytes makes this monoclonal antibody a promising candidate for the delivery of toxic agents to malignant B cells. The original immunogen used for the development of Lym-1 was Raji Burkitt's lymphoma cell nuclei [Epstein A. L., Marder R. J., Winter J. N., Stathopoulos E., Chen F. M., Parker J. W., Taylor C. R. (1987) Cancer Res 47: 830]. The Lym-1 antigen was characterized at that time as a polymorphic HLA-DR variant. We prepared an affinity column using immobilized Lym-1 to isolate the Lym-1 antigen from Raji cell lysate. Immunological characterization of the immunoaffinity-purified Lym-1 antigen on Western blots led to the conclusion that the antigen is the beta chain of HLA-DR10. This was confirmed by Edman sequencing of the isolated polypeptide chain. Western blots further show that the Lym-1 epitope is only recognized if the beta chain disulfide bonds are intact. These results imply that Lym-1 binds a discontinuous epitope on the beta chain of HLA-DR10.

Yttrium-90 (90Y), a pure beta emitter, is an attractive radionuclide for radioimmunotherapy of cancer. Therapeutic management requires quantitative imaging to measure the pharmacokinetics of the radionuclide in the patient for radiation dosimetric calculations. The bremsstrahlung emissions can be utilized to acquire an image of beta sources using a gamma camera. Quantitation of 90Y by bremsstrahlung imaging is difficult because of poor image quality that results from septal penetration and scatter secondary to the broad bremsstrahlung energies. In this work, quantitative methods for bremsstrahlung imaging of 90Y sources that involved the use of (a) a Wiener filter to deconvolve the septal penetration and scatter while suppressing image noise, and (b) the geometric mean of the conjugate view (GM) and effective point source (EPS) methods to quantify activities were investigated. An abdominal phantom was prepared with 90Y activities in the liver, spleen, tumors, and background volumes that were similar to those observed in patient studies. A twofold improvement in resolution recovery for full width at tenth maximum of the line spread function at 11 cm depth in water was achieved using Wiener restoration. Definition of the organ and tumor edges was greatly enhanced and cross talk between adjacent sources was suppressed after Wiener restoration. These improvements in image quality led to more accurate estimation of organ and tumor activities. Using the optimum attenuation correction method for GM and EPS quantitation of filtered bremsstrahlung images, estimates of individual activities (or = 17% error) and cumulated activities (or = 8% error) in all of the sources were accurate except for a tumor of 2 cm diameter. The results of this study provide the basis for a method to quantify beta source distribution and demonstrate the potential use of bremsstrahlung imaging in clinical settings.

Background. The development of new chelating agents and radiolabeling protocols is essential to progress in radioimmunotherapy with antibody-chelate conjugates. Methods. Immunoconjugates of four polyazamacrocycles with N-bonded acetate groups were prepared by conjugation via 2-iminothiolane to Lym-1, a murine anti-lymphoma immunoglobulin G 2a MoAb. To optimize 67 Cu radiolabeling, complexation conditions were explored. The kinetic stabilities in vitro in human serum of four 67 Cu labeled immunoconjugates were investigated

Background. BrE-3 is a monoclonal antibody that has promise for imaging and therapy of human adenocarcinoma. Because of observations in therapeutic trials of yttrium-90 ( 90 Y) escape from radioimmunoconjugates and uptake by the skeleton with resultant bone marrow toxicity, the authors attempted to evaluate the importance of this factor by a comparison of the LD 50 in healthy mice treated with 90 Y that had been chelated with either of two high affinity chelators, methylbenzyldiethylenetriaminepentaacetic acid (MX-DTPA) or bromoacetamidobenzyl-1,4,7,10-tetraazocyclododecane-N,N',N'',N'''-tetra-acetic acid (BAD)

Background. There has been little success in using radioimmunotherapy in patients with adenocarcinoma, partly because of the low tumor uptake of the administered monoclonal antibody (MoAb). The authors recently reported therapeutic response in advanced cancer patients who received 131 I chimeric-L6 MoAb. The L6 MoAb identifies abundant, nonshed antigen that is expressed in many human carcinomas, including carcinomas of the lung, breast, colon, and ovary. In vitro both mouse L6 (L6) and chimeric L6 (ChL6) mediate complement-dependent tumor cytolysis with human serum, and antibody-dependent tumor cell cytolysis with normal human peripheral blood mononuclear cells

Nanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death.Using carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here.NPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher.In human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.

Selective high-affinity ligands (SHALs) were synthesized as molecular targeting agents for HLA-DR10, a cell surface receptor upregulated on malignant B-cell lymphocytes in non-Hodgkin's lymphoma and leukemia. SHALs are designed to mimic the affinity and selectivity of Lym-1, an antibody that binds to the beta-subunit of HLA-DR10. To bind selectively to HLA-DR10, SHALs were constructed to bind to two adjacent pockets on the surface of the beta-subunit of HLA-DR10 located within an epitope recognized by the Lym-1 antibody. A series of multivalent SHALs with molecular masses of 1500-3000 Da were synthesized using solid/polymer-supported synthesis on chlorotrityl chloride resin in 50-80% yield. To enable their use as radionuclide carriers in mouse studies, SHALs were conjugated to DOTA in a solution-phase reaction with 70-100% yield. 57Co/CoCl2 titrations revealed that 50-60% of the DOTA in the DOTA-conjugated SHALs was available for radiometal chelation. These DOTA-SHALs were labeled with 111In and used to carry out pharmacokinetic studies in mice. Radiolabeling reactions of DOTA-SHALs, with exactly one DOTA entity per targeting SHAL molecule, yielded products with greater than 90% radiochemical purity and specific activities ranging from 97 to 150 muCi/mug.

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.

Although radioimmunotherapy (RIT) has been effective in non-Hodgkin's lymphoma (NHL) as a single agent, solid tumors have shown less clinically significant therapeutic response to RIT alone. The clinical impact of RIT or other forms of targeted radionuclide therapy for solid tumors depends on the development of a high therapeutic index (TI) for the tumor vs. normal tissue effect, and the implementation of RIT as part of synergistic combined modality therapy (CMRIT). Preclinical and clinical studies have provided a wealth of information, and new prototypes or paradigms have shed light on future possibilities in many instances. Evidence suggests that combination and sequencing of RIT in CMRIT appropriately can provide effective treatment for many solid tumors. Vascular targets provide RIT enhancement opportunities and nanoparticles may prove to be effective carriers for RIT combined with intracellular drug delivery or alternating magnetic frequency (AMF) induced thermal tumor necrosis. The sequence and timing of combined modality treatments will be of critical importance to achieve synergy for therapy while minimizing toxicity. Fortunately, the radionuclide used for RIT also provides a signal useful for nondestructive quantitation of the influence of sequence and timing of CMRIT on events in animals and patients. This can be readily accomplished clinically using quantitative high-resolution imaging (e.g., positron emission tomography [PET]).

Myelotoxicity can be ameliorated by peripheral blood stem cell (PBSC) infusion. Continuous irradiation by radioactivity retained in the body after high-dose radioimmunotherapy can damage PBSCs if they are transfused too early. Previously, infusion time was predetermined using the radioactivity concentration in the blood. This study proposes to plan PBSC infusion time based on noninvasive dosimetry that considers damage of PBSCs during PBSC circulation and residence in organs with high radioactivity.The method considers a time-varying distribution of PBSCs and radioactivity in tissues. Five breast cancer patients received (111)In-2IT-BAD-m170 for imaging, and 3 of the 5 received high doses of (90)Y-2IT-BAD-m170 therapy followed by PBSC infusion. (90)Y concentrations in tissues were extrapolated from quantitative imaging of (111)In, and (90)Y blood concentrations were determined from (90)Y in serial blood samples. The radiation dose to PBSCs was determined by time integration of the organ dose rate and PBSC distribution rate. The radiosensitivity of PBSCs was determined by measuring survival of granulocyte-macrophage colony-forming units with (90)Y in cell culture.The mean effective half-life of (90)Y within the imaging period (up to 6 d) was 3.7 d for liver, 2.4 d for spleen, 2.1 d for kidneys, 1.8 d for lungs, and 1.6 d for blood. The survival fractions of PBSCs in patients were determined as functions of the infusion time and the injected dose of (90)Y-2IT-BAD-m170. To achieve 90% PBSC survival rate for a 2.0-GBq injection dose, PBSC dosimetry suggested a time interval of 13 d after radioimmunotherapy for PBSC infusion. In contrast, the simple blood concentration method suggested an interval about 7 d for the same PBSC survival rate. In our clinical practice, an interval of 2 wk has been used and worked well.A noninvasive dosimetry method was developed for optimizing the time interval for PBSC infusion after high-dose radionuclide therapy. Our studies suggested that the PBSC dosimetry method was more effective than the blood concentration method in determining the optimal time to reinfuse PBSCs for radiopharmaceuticals that have much a higher activity concentration in organs than that in the blood.

Radioimmunotherapy for non-Hodgkin's lymphoma often results in surprisingly high response rates compared with those expected from estimated absorbed radiation doses. Several factors, including radiobiologic response, selective targeting, and heterogeneous absorbed radiation within the lymphoma, are likely to contribute to the lack of a dose-response relationship. This article investigates the impact of nodal regression on absorbed radiation dose and applies a correction factor to account for its effect.The radioactivity in and regression of 37 superficial lymph nodes were measured in 7 non-Hodgkin's lymphoma patients treated with 775-3,450 MBq/m(2) of (131)I-Lym-1 monoclonal antibody. Nodal dimensions were measured with calipers and radioactivity was quantitated using gamma-camera imaging on multiple days after (131)I-Lym-1 injection. Both nodal regression and radioactivity were fit with monoexponential functions. Formulas were developed to account for simultaneous change in nodal mass and radioactivity. All lymph nodes with size and radioactivity measurements, and exponential-fit coefficients of determination of0.8, were included in the analysis.A 3 orders-of-magnitude node-to-node variation in initial radiopharmaceutical concentration (MBq/g) was observed, with the highest concentrations in the smallest nodes. Reduction in radioactivity as a function of time (biologic half-life) varied by about a factor of 2. In contrast, the rate of nodal regression varied by orders of magnitude, from a 14-h half-time to no regression at all. Five nodes regressed with a half-time that was shorter than their observed effective radiopharmaceutical half-life. Accounting for the effect of nodal regression resulted in dose corrections ranging from 1 (no correction) to a factor of10, with 70% of nodes requiring a correction factor of at least 20% and50% of nodes requiring a correction factor of2. Corrected for nodal regression, 46% of nodes analyzed had absorbed radiation doses of10 Gy and 32% had doses of20 Gy.These results highlight the importance of accounting for change in mass, particularly tumor regression, when assessing absorbed radiation dose for tissues whose mass changes during the time the radiation dose is being absorbed. The increase in calculated absorbed dose when this change is considered provides better insight into the high nodal response rates observed in non-Hodgkin's lymphoma patients.