Molecular specific and cell selective cytotoxicity induced by a novel synthetic HLA-DR antibody mimic for lymphoma and leukemia.

Like rituximab, monoclonal antibodies reactive with human leukocyte antigen have potent antilymphoma activity. However, size limits their vascular and tissue penetration. To mimic monoclonal antibody binding, nanomolecules have been synthesized, shown specific for the beta subunit of HLA-DR10, and selective for cells expressing this protein. Selective high affinity ligands (SHALs) containing the 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid (Ct) ligand residualized and had antilymphoma activity against expressing cells. Herein, we show the extraordinary potency in mice with human lymphoma xenografts of a tridentate SHAL containing this ligand. After titrating antilymphoma activity in cell culture, a randomized preclinical study of a tridentate SHAL containing the Ct ligand was conducted in mice with established and aggressive human lymphoma xenografts. Mice having HLA-DR10 expressing Raji B- or Jurkat's T-lymphoma xenografts were randomly assigned to receive either treatment with SHAL at a dose of 100 ng i.p. weekly for 3 consecutive weeks, or to be untreated. Primary end-points were cure, overall response rates and survival. Toxicity was also evaluated in these mice, and a USFDA general safety study was conducted in healthy Balb/c mice. In Raji cell culture, the threshold and IC50 concentrations for cytotoxic activity were 0.7 and 2.5 nmol (pm/ml media), respectively. When compared to treated Jurkat's xenografts or untreated xenografts, Raji xenografts treated with the SHAL showed an 85% reduction in hazard of death (P=0.014; 95% confidence interval 32-95% reduction). There was no evidence for toxicity even after i.p. doses 2000 times greater than the treatment dose associated with cure of a majority of the mice with Raji xenografts. When compared with control groups, treatment selectively improved response rates and survival in mice with HLA-DR10 expressing human lymphoma xenografts at doses not associated with adverse events and readily achievable in patients.

NanoFerrite particle based radioimmunonanoparticles: binding affinity and in vivo pharmacokinetics.

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 were >90% 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.

A new era for radiolabeled antibodies in cancer?

Radioimmunotherapy (RIT), a therapy targeted to tumor cells, is a modality that can currently deliver radiation to tumor cells at levels 3-50-times higher than to the normal tissue with the next highest dose. RIT appears promising for future cancer therapy. Clinical responses in patients with advanced cancer have frequently been achieved with RIT as a single agent. Extended complete remissions and even increased survival have been achieved in lymphoma. Similar results in other cancers seem likely with RIT in combination therapy.

Stable bifunctional chelates of metals used in radiotherapy.

Monoclonal antibody technology allows the specificity of an antibody for its antigen to be used in targeting cancer cells. The conjugation of metals, particularly radionuclides such as 90Y or 67Cu, to monoclonal antibodies results in agents for radioimmunotherapy and other medical applications. Chelators that can hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to nontarget cells. Derivatives of polyazamacrocycles (bearing a C-substituted functional group for antibody attachment) can exhibit remarkable kinetic inertness; for example, the copper complex of the 14-membered 6-(p-nitrobenzyl)-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''- tetraacetic acid is very stable in human serum under physiological conditions, and a conjugate of this complex with a monoclonal antibody has tested well in tumor-bearing mice. Desreux and coworkers [Loncin, M. F., Desreux, J. F., and Merciny, E. Inorg. Chem., 25: 2646-2648, 1986] have shown that complexes of lanthanides with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid have formation constants that are several orders of magnitude higher than that of 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid; thus the 12-membered macrocycle is the favored target for binding trivalent yttrium. We have developed a new synthetic route to these macrocycles via peptide synthesis and intramolecular tosylamide ring closure. Incubation of the 88Y-(III) complex of 2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''- tetraacetic acid for 18 days in serum results in loss of so little Y(III) from the complex (less than 0.5%) that the rate of loss cannot be measured under these conditions.

Radioimmunotherapy for breast cancer using escalating fractionated doses of 131I-labeled chimeric L6 antibody with peripheral blood progenitor cell transfusions.

Radioimmunotherapy (RAIT) using a humanized murine monoclonal antibody, chimeric L6 (ChL6), has produced objective tumor reduction in 50% of chemotherapy-refractory patients with metastatic breast cancer in our prior studies. Because myelosuppression limited dose escalation, we evaluated the ability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cell (PBPC) transfusions to ameliorate this problem. 131I-labeled ChL6 was given at a starting dose of 150 mCi/m2 (2,5 times the maximum tolerated dose without PBPCs) for a planned three treatments. When blood radioactivity declined to less than 1 microCi/ml after treatment, PBPCs were transfused, and G-CSF was administered. Patient 1 had minimal myelosuppression, received two cycles of therapy, and then developed human antimonoclonal antibody (HAMA). Patient 2 had prolonged thrombocytopenia that resolved after additional PBPC transfusion. Progressive disease as well as HAMA prevented further treatment. Patient 3 received all three cycles of 150 mCi/m2 at 8-week intervals. Thrombocytopenia (< 25,000/microliter) occurred but was transient (0-7 days). Because HAMA developed in all prior patients who received G-CSF with ChL6 RAIT, including patients 1 and 2, who received PBPC, patient 3 was given cyclosporin for 14 days. She did not develop HAMA or significant toxicity following 3 cycles of RAIT. Cumulative radiation doses to her lungs and tumor were estimated at 3,100 and 11,200 cGy, respectively. For 9 months, she had a reduction in bone pain, a decline in serum tumor markers, and decreased tumor uptake of F-18-deoxyglucose on a positron emission scan. Her performance status improved, and she had no pulmonary toxicity. We conclude that: (a) PBPC transfusion can modify the myelotoxicity of RAIT and can permit repetitive dosing; (b) cyclosporin is a promising means to abrogate HAMA; and (c) fractionation of intensive-dose RAIT may increase the antitumor effect and reduce normal organ toxicity.

Effect of mass of 111In-benzyl-EDTA monoclonal antibody on hepatic uptake and processing in mice.

In patients, the pharmacokinetic behavior of murine monoclonal antibodies has been observed to vary with the amount of antibody administered. It has been suggested that this reflects human recognition of the foreign mouse protein. We have found that the amount of antibody administered also influenced pharmacokinetic behavior when murine monoclonal antibody was administered to mice. p-Isothiocyanatobenzyl-EDTA, a new chelator which forms complexes with 111In that are stable in vivo, was conjugated to Lym-1, a murine anti-Burkitt's lymphoma monoclonal antibody. The pharmacokinetics of two doses (20 and 0.2 micrograms) of the 111In labeled radiopharmaceutical were studied in non-tumor bearing BALB/c mice. About 20% (0.04 microgram) of the 0.2-microgram dose, compared with 8% (1.6 micrograms) of the 20-micrograms dose, was found in the liver at 48 h after injection. Both doses demonstrated a biological half-life of approximately 120 h. At least 75% of the 111In was excreted by the kidneys, and essentially all 111In in the urine remained chelated by the EDTA portion of p-isothiocyanatobenzyl-EDTA. From these observations of a dose dependent uptake of this radiopharmaceutical by the liver we conclude that there is a recognition phenomenon in mice for this murine monoclonal antibody.

Fractionated radioimmunotherapy of B-cell malignancies with 131I-Lym-1.

Eighteen patients with Stage 4 B-cell malignancies, which were primarily of intermediate or high grade and progressive despite multiple drug chemotherapy and external irradiation, were treated with fractionated doses of 131I-labeled Lym-1. Lym-1 is an IgG2a monoclonal antibody that was produced by immunizing mice with Raji cell nuclei that originated from a patient with African Burkitt's lymphoma. Despite advanced disease, 10 of the patients had objective evidence for a complete or partial remission. Toxicity was very modest except in one patient who developed hypotension. Dose-dependent hepatic uptake of Lym-1 was observed in the patients and in BALB/c mice suggesting receptor-mediated recognition of this murine antibody.

Effect of the extent of chelate substitution on the immunoreactivity and biodistribution of 2IT-BAT-Lym-1 immunoconjugates.

Trial therapy for lymphoma with the radiolabeled chelate-antibody conjugate 67Cu-2IT-BAT-Lym-1 has been promising. It is desirable to deliver therapeutic doses of radiometal using a minimum amount of 2IT-BAT-Lym-1 to minimize the risks of adverse patient reaction and antigenic response to antibody. This is readily accomplished by increasing the number of metal-binding sites (i.e., chelating agents) conjugated per antibody, but the ability of the antibody to bind antigen and target tumor cells in vivo must not be impaired by the conjugation reaction. To determine the maximum chelator:antibody ratio (c/a) of 2IT-BAT-Lym-1 at which functional integrity is preserved, immunoconjugates with a c/a of 1.3-23 were prepared and examined by radioimmunoassay and competitive antigen binding versus lightly iodinated Lym-1. The biodistribution in tumored mice of conjugates with c/a of 2.1, 4.3, 8.4, and 11.4 also was examined. Conjugates with c/a up to 5 exhibited no loss of immunoreactivity, and conjugates with c/a up to 11 retained 75% or greater immunoreactivity relative to unmodified Lym-1. All conjugates examined competed less effectively than did unmodified Lym-1 for antigen binding, but the effect at c/a 5 was slight. Tumor uptake declined with increasing c/a, but the effect was insignificant at c/a 2.1 and 4.3. Conjugates of c/a 4-5 were found to be optimal for the preparation of radioimmunoconjugate of high specific activity with minimal, if any, loss of functional integrity.

Prelabeling of chimeric monoclonal antibody L6 with 90yttrium- and 111indium-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) chelates for radioimmunodiagnosis and therapy.

90Y and 111In have been attached to chimeric monoclonal antibody L6 with a bifunctional chelating agent (DOTA-peptide isothiocyanate). The bifunctional chelating agent was prelabeled with either radiometal and then conjugated to the antibody. Studies in human patients showed excellent 111In single-photon emission computed tomography images of breast cancer lesions 24 h after injection.

Prognostic factors for radioimmunotherapy in patients with B-lymphocytic malignancies.

The Ann Arbor staging classification has proven less useful in nonHodgkin's lymphoma, because this malignancy is inherently a multifocal disorder. Since 1985, 57 adult patients with advanced B-lymphocytic malignancies that progressed despite standard therapy entered into one of three different therapy trials using radiolabeled Lym-1 antibody. Tumor regression in 31 (54%) of these patients fulfilled conventional requirements for an oncological response to the therapy. To define the role of radioimmunotherapy in B-lymphocytic malignancies better and to find opportunities for improving its therapeutic efficacy, the records of these patients were reviewed to assess the significance of various parameters as prognostic indicators. Twenty-one pretherapy characteristics were evaluated, including age at diagnosis, age at study entry, sex, Karnofsky performance status, prior chemotherapy and radiation therapy, interval since diagnosis, histology, constitutional B symptoms, extranodal malignancy (excluding marrow), bone marrow malignancy, tumor bulk, and circulating malignant cells; blood tests included lymphocyte, granulocyte, platelet, hematocrit, serum lactate dehydrogenase (LDH), interleukin 2 receptor, and human antimouse antibody levels. In the multivariate analysis, LDH and Karnofsky performance status were the parameters that best predicted survival, complete and partial remission, and time to progression; interleukin 2 receptor and LDH best predicted complete remission. These prognostic factors for radioimmunotherapy outcome are consistent with the pretherapy characteristics observed to be significant for chemotherapy.

Yttrium-90 chimeric L6 therapy of human breast cancer in nude mice and apoptosis-related messenger RNA expression.

Radioimmunotherapy (RIT) in breast cancer patients using I-131-chimeric L6 (ChL6) and in human breast cancer xenografts in nude mice using Y-90-1,4,7,10-tetraazacylododecant N,N',N",N"'-tetraacetic acid-peptide ChL6 (Y-90-ChL6) has shown promise. Tumor cell response to low-dose rate (5-25 rads/h) irradiation from Y-90-ChL6 RIT, therefore, was correlated with levels of tumor cell mRNA for selected genes linked to programmed cell death (apoptosis). Three groups of 10-16 mice with 1-2 HBT 3477 xenograft tumors were treated with 100, 150, or 250 microCi Y-90-ChL6. Three tumors were taken before and two tumors each were taken 3, 6, and 24 h after injection of 150 microCi Y-90-ChL6. Tumor expression of mRNA was amplified by PCR for p53, PIC1, c-myc, and transforming growth factor-beta 1; quantitated; and standardized to N-ras. Tumors received radiation doses of 2000, 3000, and 5000 rads, respectively, for the groups of mice that received 100, 150, and 250 microCi Y-90-ChL6, and tumor regression occurred in each group, with mean tumor volumes decreased by 10, 50, and 95% at nadir after Y-90-ChL6 injection. At the highest dose level, 30% of mice had complete remissions, and no treatment deaths occurred, although tumors subsequently recurred. Continuous up-regulation of transforming growth factor-beta 1 and c-myc mRNA expression was observed from 3 to 24 h after treatment. Expression of p53 and PIC1 increased at 3 h and subsequently decreased to the untreated control levels. These observations are consistent with previous observations of early responses of p53 and PIC1 to cellular DNA damage and subsequent G1 cell cycle arrest or apoptosis. Apoptosis-associated gene expression patterns observed in this tumor model provide evidence that changes are initiated in the first 24 h of RIT associated with radiation doses of 100-700 rads. These preliminary data suggest that insight into the molecular basis of RIT-induced tumor regression may be gained by further studies using different radiation doses.

Radioimmunotherapy: recent results and future directions.

PURPOSE: To review antibody structure, function, and production; suitable radioisotopes for radioimmunotherapy; challenges facing the field; recent clinical results; toxicity; and future directions. DESIGN: The radioimmunotherapy literature was reviewed, with an emphasis on clinical results and future directions. RESULTS: The highest complete response rates (overall, approximately 50%) have been achieved in patients with B-cell non-Hodgkin's lymphoma. Challenges that currently face radioimmunotherapy include circulating free antigen, binding of antibodies to nonspecific Fc receptors, insufficient tumor penetration, antigenic heterogeneity and insufficient antigen expression, antigenic modulation, and development of human antimouse antibodies. Possible approaches to these challenges, including high-dose radioimmunotherapy and chemotherapy followed by autologous bone marrow transplantation, the use of radionuclides such as yttrium 90 (90Y) and copper 67 (67Cu), and the development of humanized and bifunctional antibodies, are under investigation. CONCLUSION: Although radioimmunotherapy is a relatively new field, substantial progress has been made. Additional research will ultimately resolve many of the challenges that currently face radioimmunotherapy and hopefully lead to the cure of some currently incurable malignancies.

Maximum-tolerated dose, toxicity, and efficacy of (131)I-Lym-1 antibody for fractionated radioimmunotherapy of non-Hodgkin's lymphoma.

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).

Accuracy of Doppler catheter measurements: effect of inhomogeneous beam power distribution on mean and peak velocity.

OBJECTIVES: We sought to determine the effect of inhomogeneous distribution of beam power produced by Doppler catheters on measurements of mean and peak velocity of coronary blood flow. BACKGROUND: Measurements of mean velocity of coronary blood flow by Doppler catheters have significant systematic errors that have not been completely characterized. We hypothesized that one error is the inhomogeneous distribution of the ultrasonic beam power and that this inhomogeneity makes measurements of mean, but not peak, velocity inaccurate. METHODS: We constructed a scaled-up model of a Doppler catheter to allow for accurate measurement of the distribution of beam power by miniature hydrophones. This catheter was placed in a model of coronary blood flow in which the fluid velocity was accurately measured by an external laser Doppler velocimeter. The laser Doppler measurements of mean velocity were compared with the measurements of mean velocity made by the catheter, using fast Fourier transform analysis, both without and with correction for inhomogeneous beam power distribution. Peak velocity measurements were also compared, as predicted from theory, without the need of correction for inhomogeneous beam power distribution. To investigate the clinical relevance of our results, we conducted studies using a clinical Doppler catheter both in a scaled model of coronary flow and in a series of eight patients. In the model and in each patient, we rotated the catheter without changing the axial position to systematically alter the relation of the beam power distribution to the local fluid dynamics. RESULTS: The measurement of beam power distribution revealed significant inhomogeneity. Comparison of the measured mean frequency shifts without correction for inhomogeneities in the distribution yielded a statistically significant difference. After correction for inhomogeneities, there was no statistically significant difference. Also, there was no significant difference for the peak frequency shifts. Rotation of the clinical catheter in the scaled model and in the patients changed the measured mean velocity (average change 18.8% and 20.6%, respectively), but not the measured peak velocity (average change 5.0% and 4.3%, respectively). CONCLUSIONS: For signal analysis using a fast Fourier transform, the inhomogeneous distribution of power of the ultrasonic beam produced by Doppler catheters makes measurements of mean, but not peak, velocity inaccurate. Measurements of peak velocity may therefore prove superior to measurements of mean velocity in estimating the response to pharmacologic intervention and in estimating stenosis severity.

Development of a hyperimmune anti-MUC-1 single chain antibody fragments phage display library for targeting breast cancer.

Radioimmunotherapy (RIT) has demonstrated potential for improving clinical cancer therapy. Optimizing the approach has proven difficult thus far. Antibody phage display libraries provide unique molecules that could improve RIT. A phage display library of single chain antibody fragments (scFv) against the MUC-1 mucin molecule, which is expressed on 90% of human breast cancers, was produced from the spleen cells of MUC-1 hyperimmunized BALB/c mice. Increased serum IgG levels, 15 times baseline, were detected following the third immunization. RNA from the spleen cells was isolated, cDNA was made, and variable heavy and variable light immunoglobulin chain gene regions were amplified using PCR technology. The variable heavy and variable light chain gene regions were combined with a flexible linker, ligated into the pCANTAB 5E phagemid vector, and electroporated into TG1 Escherichia coli cells. A library of 10(7) initial colonies was compiled. Forty-six of 288 colonies screened for reactivity demonstrated binding to MUC-1-expressing MCF-7 breast cancer cell membrane fragments. Anti-MUC-1 library diversity evaluated by BstNI digest demonstrated that 52% of the anti-MUC-1 scFv binding MCF-7 possessed individual banding patterns representative of approximately 5 x 10(5) colonies likely able to recognize distinct epitopes present on MUC-1 positive human breast cancers. In summary, the anti-MUC-1 scFv antibody phage library contains diverse scFv molecules, which should provide unique characteristics and epitope recognition. These molecules will be used in the development of pretargeting RIT strategies designed to improve the clinical outcome of patients with breast cancer.

Efficacy and toxicity of 67Cu-2IT-BAT-Lym-1 radioimmunoconjugate in mice implanted with human Burkitt's lymphoma (Raji).

Radioimmunotherapy has shown promising results for treatment of radiosensitive malignancies such as lymphoma. Positive responses have been reported in patients with non-Hodgkin's lymphoma treated with 131I-radiolabeled Lym-1, a mouse anti-lymphoma monoclonal antibody. In this study, the efficacy of 67Cu-radiolabeled Lym-1 was examined. Nude mice bearing human Burkitt's lymphoma (Raji) tumors (20-524 mm3) were treated with 12.4, 14.8, 18.5, and 23.3 MBq of 67Cu-2IT-BAT-Lym-1. Tumor size was measured to assess efficacy, and mouse weight, blood counts, and mortality were monitored to assess toxicity. In mice treated with 12.4, 14.8, and 18.5 MBq of 67Cu-2IT-BAT-Lym-1, 50% (9 of 18), 42% (5 of 12), and 50% (3 of 6) of tumors achieved remission or cure; 33% of tumors were cured overall; and significant regrowth delay was observed. The 23.3 MBq dose group did not yield meaningful efficacy data because of high mortality. In control groups receiving 14.8 and 18.5 MBq of the isotype-matched nonspecific monoclonal antibody radioimmunoconjugate, 67Cu-2IT-BAT-L6, 0% (0 of 15) and 17% (2 of 12) of tumors achieved a response; hence, targeted delivery of radiation was the dominant antitumor mechanism of 67Cu-2IT-BAT-Lym-1. LD50/30 for mice treated with 67Cu-2IT-BAT-Lym-1 and -L6 were 21.6 and 20.6 MBq, respectively. In conclusion, 67Cu-2IT-BAT-Lym-1 provided a therapeutic and frequently curative dose of radiation to tumored mice with modest toxicity.

Treatment-related parameters predicting efficacy of Lym-1 radioimmunotherapy in patients with B-lymphocytic malignancies.

This study was designed to evaluate dosimetric, pharmacokinetic, and other treatment-related parameters as predictors of outcome in patients with advanced B-lymphocytic malignancies. Fifty-seven patients were treated with radiolabeled Lym-1 antibody in early phase trials between 1985 and 1994. Logistic regression and proportional hazards models were used to evaluate treatment parameters for their ability to predict outcome, taking into account patient risk group based on Karnofsky performance status and serum lactic dehydrogenase. The occurrence of a partial or complete response (31 of 57 patients) and development of human antimouse antibody (HAMA) predicted improved survival using a time-dependent proportional hazards model. The final multivariate model for survival with parameters significant at P

67Cu-versus 131I-labeled Lym-1 antibody: comparative pharmacokinetics and dosimetry in patients with non-Hodgkin's lymphoma.

Antilymphoma mouse monoclonal antibody (MoAb) Lym-1, labeled with 67Cu or 131I, has demonstrated promising results in radioimmunotherapy (RIT) for lymphoma. Although 131I has played a central role in RIT thus far, some properties of 67Cu are preferable. A subset of our patients received both 67Cu- and 131I-labeled Lym-1, allowing a comparative evaluation of the two radiopharmaceuticals administered to a matched population of patients. Four patients with B-lymphocytic non-Hodgkin's lymphoma that had progressed despite standard therapy entered trials of 67Cu- and 131I-labeled Lym-1, which were injected 3-26 days apart. Lym-1 was conjugated to 6-[p-(bromoacetamido)benzyl]-1,4,7,11-tetraazacyclotetradecane-N,N ',N",N'"-tetraacetic acid (BAT) via 2-iminothiolane (2IT) and radiolabeled with 67Cu to prepare 67Cu-2IT-BAT-Lym-1; 131I-Lym-1 was preparred by the chloramine-T reaction. Planar imaging was used to quantitate 67Cu-2IT-BAT-Lym-1 or 131I-Lym-1 in organs and tumors daily for 3 days or longer. 67Cu-2IT-BAT-Lym-1 exhibited higher peak concentration in 92% (12 of 13) of tumors and a longer biological half-time in every tumor than 131I-Lym-1. The mean tumor concentration (%ID/g) of 67Cu-2IT-BAT-Lym-1 was 1.7, 2.2, and 2.8 times that of 131I-Lym-1 at 0, 24, and 48 h after injection, respectively. The mean biological half-times of 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 in tumor were 8.8 and 2.3 days, respectively. Consequently, the mean tumor radiation dose delivered by 67Cu-2IT-BAT-Lym-1 was twice that of 131I-Lym-1, 2.8 (range 0.8-6.7), and 1.4 (range 0.4-35) Gy/GBq, respectively. 67Cu-2IT-BAT-Lym-1 delivered a lower marrow radiation dose than 131I-Lym-1; hence, the tumor:marrow therapeutic indices were 29 and 9.7, respectively. Radiation doses from 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 to normal tissues were similar except for liver, which received a higher dose from 67Cu-2IT-BAT-Lym-1. Images obtained with 67Cu-2IT-BAT-Lym-1 were superior. Radiation dosimetry data for 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 agreed with corresponding data from the larger populations of patients from which the matched population for the current study was drawn. In conclusion, 67Cu-2IT-BAT-Lym-1 given to non-Hodgkin's lymphoma patients in close temporal proximity to 131I-Lym-1 exhibited greater uptake and longer retention in tumor, resulting in higher radiation dose and therapeutic index than 131I-Lym-1. These as well as other factors suggest that 67Cu-2IT-BAT-Lym-1 may be superior to 131I-Lym-1 for RIT.

Effect of 67Cu-2IT-BAT-Lym-1 therapy on BCL-2 gene and protein expression in a lymphoma mouse model.

Radioimmunotherapy using monoclonal antibodies against tumor-associated antigens has been particularly promising in the treatment of radiosensitive malignancies such as lymphoma. 67Cu has excellent physical and biochemical properties for radioimmunotherapy. 67Cu-2IT-BAT-Lym-1 has been used in preclinical and clinical trials, where an exceptionally long residence time of 67Cu on tumor was observed. BCL-2, a proto-oncogene that promotes cell survival by blocking apoptotic cell death, is overexpressed in most B-cell lymphomas including Raji human Burkitt's lymphoma cells. In this study, therapeutic efficacy and BCL-2 gene and protein expression levels were examined in Raji xenografts in mice after 67Cu-2IT-BAT-Lym-1 radioimmunotherapy. 67Cu-2IT-BAT-Lym-1 therapy induced a response rate (complete and partial responses) of approximately 50%. BCL-2 gene expression was decreased 3 h after radioimmunotherapy, followed by a decrease in Bcl-2 protein by 24 h. Decreases in BCL-2 gene and protein expression preceding observations of 67Cu-2IT-BAT-Lym-1 therapeutic effect suggest that down-regulation of BCL-2 leaves cells more likely to be killed by low dose-rate radiation from radioimmunotherapy.

Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics.

Pretargeting techniques have shown promise for enhancement of the therapeutic index of radioimmunotherapy for cancer. However, methods to vary and compare antibody configurations and select optimal combinations have proved rather formidable. New options for the construction of pretargeting molecules are provided by sophisticated use of the diversity and malleability of antibody genes. Diverse arrays of single-chain antibody fragments (scFvs) can now be obtained reactive with virtually any target antigen by selection from human naive phage antibody libraries. ScFvs can also be cloned directly from hybridoma for construction of phage libraries that facilitate subsequent manipulation: e.g., affinity maturation and modification of specificity. ScFvs affinity selected from these sources to their specific antigen targets have demonstrated a wide spectrum of binding characteristics. ScFvs selected from a large human naive phage antibody library by binding Cu-1,4,8,11-tetra-azacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA) or Y-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) have shown diversity by DNA fingerprints. DNA sequence information confirmed that the anti-TETA scFv represented diverse scFv gene families. ScFvs for Y-DOTA and those for lymphoma-associated HLA DR10 (Lym-1) were selected in a similar manner from mouse antibody gene libraries derived from hybridoma. ScFv clones for each of these antigens were chosen for further study based on the results of ELISA assays involving the respective cell membrane or metal chelate antigens. A PCR primer system built to pCANTAB 5E expression vector sequence was designed to facilitate cloning of antibody heavy (V(H)) and light (V(L)) genes from selected scFvs as cassettes into diabody modules. Thus, chosen scFvs could be expressed in the same diabody format for comparative study. Selected mouse anti-DOTA scFv and Lym-1 scFv genes were linked as V(HA) anti-DOTA-link-V(LB) Lym-1; V(HB) anti-DOTA-link-V(LA) Lym-1 and ligated into the pCANTAB 5E vector. Corresponding diabodies were expressed in Escherichia coli and purified by affinity chromatography. Here we provide a perspective on the power of antibody phage libraries and the possibilities of creating simple molecular formats that can be used en route to the development of new tumor targeting and pretargeting molecules.