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.

Pre-clinical evaluation and efficacy studies of a melanin-binding IgM antibody labeled with 188Re against experimental human metastatic melanoma in nude mice.

PURPOSE: Currently there is no satisfactory treatment for metastatic melanoma. Radioimmunotherapy (RIT) uses the antigen-antibody interaction to deliver lethal radiation to target cells. Recently we established the feasibility of targeting melanin in tumors with 188-Rhenium ((188)Re)-labeled 6D2 mAb to melanin. Here we carried out pre-clinical development of (188)Re-6D2 to accrue information necessary for a Phase I trial in patients with metastatic melanoma. RESULTS: TCEP proved to be effective in generating a sufficient number of -SH groups on 6D2 to ensure high radiolabeling yields with (188)Re and preserved its structural integrity. (188)Re-6D2 was quickly cleared from the blood with the half-life of approximately 5 hrs and from the body--with the half-life of 10 hr. The doses of 0.5, 1.0 and 1.5 mCi significantly (p < 0.05) slowed down A2058 tumor growth in nude mice, also causing release of melanin into the extracellular space which could provide additional target for repeated treatments. Transient effects of RIT on WBC and platelet counts resolved by Day 14 post-treatment. EXPERIMENTAL DESIGN: Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) was evaluated as potential agent for generation of -SH groups on 6D2 mAb. TCEP-treated 6D2 mAb was radiolabeled with (188)Re and its radiochemical purity and stability was measured by ITLC and HPLC and its immunoreactivity--by melanin-binding ELISA. The pharmacokinetics, therapeutic efficacy and acute hematologic toxicity studies were performed in nude mice bearing lightly pigmented A2058 human metastatic melanoma tumors. CONCLUSIONS: We have developed radiolabeling and quality control procedures for melanin-binding (188)Re-6D2 mAb which made possible currently an on-going Phase I clinical trial in patients with metastatic melanoma.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

Metabolite production in patients with lymphoma after radiometal-labeled antibody administration.

UNLABELLED: Radiometal-labeled monoclonal antibodies are retained longer in tumors than iodinated antibodies, leading to their increased use for radioimmunotherapy. Dissociation of radioiodine from the antibody during metabolism has been documented. We now report metabolites in the plasma of lymphoma patients given 111In- and 90Y-2-iminothiolane-2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-Lym-1 (111In/90Y-2IT-BAD-Lym-1). METHODS: Nineteen patients with non-Hodgkin's lymphoma (NHL) received 111In- and 90Y-2IT-BAD-Lym-1; 111In was used as a surrogate tracer for 90Y, which emits no gamma-photon. Plasma was obtained up to 7 d and analyzed by high-performance liquid chromatography to determine the fraction of radiolabel associated with monomeric antibody, metabolites, and complexed antibody. Planar images of conjugate views were acquired up to 7 d and used to quantitate 111In in organs and tumors. RESULTS: Metabolites and complexes were observed in the plasma of every patient who received 111In-2IT-BAD-Lym-1. At 3 d, the mean percentages of 111In in the patients' plasma in monomeric, metabolite, and complexed forms were 54%, 36%, and 10%, respectively. Metabolites of 90Y-2IT-BAD-Lym-1 were formed to a similar extent. In comparison, in groups of breast and prostate cancer patients who received the radioimmunoconjugate 111In-2IT-BAD-m170, 91% and 94% of 111In in the patients' plasma were in monomeric form, respectively. Metabolites and complexes of 111In-2IT-BAD-Lym-1 contributed a mean 10% of the total area under the time-activity curve (AUC) for blood. Little formation of metabolites and complexes occurred in vitro in NHL patient or volunteer plasma or in Raji cell culture. The clinical and in vitro data supported the processing of 111In/90Y-2IT-BAD-Lym-1 in the hepatocytes as the dominant mechanism for the production of metabolites. CONCLUSION: Metabolites of 111In/90Y-2IT-BAD-Lym-1 accounted for 10% of blood AUC in patients. The therapeutic index was adversely affected by metabolism of 111In/90Y-2IT-BAD-Lym-1 to the extent that the tumor specificity of the radioactive metabolites was lost.

Radioimmunotherapy with (111)In/(90)Y-2IT-BAD-m170 for metastatic prostate cancer.

PURPOSE: Over 31,000 Americans die of androgen-independent metastatic prostate cancer each year. New strategies that do not involve hormonal manipulation but instead recognize the biochemical and molecular characteristics of prostate cancer are needed. Radioimmunotherapy (RIT) uses a tumor-specific monoclonal antibody to deliver systemic, targeted radiation to cancer. The objectives of this Phase I study of (111)In-2IT-BAD-m170 (for imaging) and (90)Y-2IT-BAD-m170 (for therapy) were to determine the toxicity and maximum tolerated dose (MTD), the specificity for targeting metastatic prostate cancer, and the efficacy for palliation of pain. EXPERIMENTAL DESIGN: M170 is a mouse monoclonal antibody that targets adenocarcinomas. Patients with adequate renal and liver function, rising prostate-specific antigen, and androgen-independent metastatic prostate cancer were eligible. After estimation of dosimetry and pharmacokinetics with (111)In-2IT-BAD-m170, a single dose of (90)Y-2IT-BAD-m170 (0.185, 0.370, 0.555, or 0.740 GBq/m(2)) was administered to cohorts of three patients. Pain was assessed objectively by questionnaires before and for 8 weeks after RIT; weekly prostate-specific antigen levels were obtained for 2 months after RIT. RESULTS: The MTD of (90)Y-2IT-BAD-m170 was 0.740 GBq/m(2) for patients that had up to 10% of the axial skeleton involved with prostate cancer. Toxicity was almost exclusively confined to reversible myelosuppression. Metastatic prostate cancer was targeted by (111)In-2IT-BAD-m170 in all 17 patients. The mean radiation dose delivered to 39 bone and 18 nodal metastases by (90)Y-2IT-BAD-m170 was 10.5 Gy/GBq (range 2.8-25.1). Thirteen of 17 patients reported pain before (90)Y-2IT-BAD-m170; 7 of these 13 had a partial or complete resolution of pain that lasted an average of 4.3 weeks. CONCLUSIONS: This study determined the MTD of (111)In/(90)Y-2IT-BAD-m170 in patients with metastatic prostate cancer. The drugs were well tolerated, targeted metastases, and temporarily palliated pain.

67Copper-2-iminothiolane-6-[p-(bromoacetamido)benzyl-TETA-Lym-1 for radioimmunotherapy of non-Hodgkin's lymphoma.

Copper-67 (67Cu) has ideal properties for radioimmunotherapy. The 62-h half-life is similar to the residence time of antibodies in tumor, and the therapeutic beta emission of 67Cu is comparable to that of 131I. 67Cu, however, has gamma emissions similar to 99mtechnetium that are favorable for imaging. The macrocyclic chelating agent 1,4,7,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA) binds 67Cu tightly and selectively, facilitating linkage to Lym-1, a mouse monoclonal antibody that preferentially targets malignant lymphocytes. The safety, efficacy, and practicality of 67Cu-2-iminothiolane (2IT)-6-[p-(bromoacetamido)benzyl]-TETA (BAT)-Lym-1 was assessed in this Phase I/II clinical trial for patients with non-Hodgkin's lymphoma (NHL) who had failed standard therapy. Up to four doses of 67Cu-2IT-BAT-Lym-1, 25 or 50-60 mCi/m2/dose (0.93 or 1.85-2.22 GBq/m2/dose, respectively) were administered; the lower dosage was used when NHL was detected in the bone marrow. 67Cu-2IT-BAT-Lym-1 provided good imaging of NHL, had favorable radiation dosimetry, and had a response rate of 58% (7 of 12). Hematological toxicity was dose-limiting, but no significant nonhematological toxicity was observed. The ability to image and treat NHL patients with a single radiopharmaceutical with useful physical properties makes 67Cu-labeled monoclonal antibody an option for future clinical trials, as this study showed that 67Cu-2IT-BAT-Lym-1 was safe, effective, and practical.

Factors affecting 131I-Lym-1 pharmacokinetics and radiation dosimetry in patients with non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

UNLABELLED: Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced therapeutic responses in patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) when labeled with 131I. Responders had statistically significant prolongation of survival compared with nonresponders. The nonmyeloablative, maximum tolerated dose for each of two doses of 131I-Lym-1 was 3.7 GBq/m2 (total 7.4 GBq/m2 [100 mCi/m2, total 200 mCi/m2]) of body surface area. The purpose of this study was to determine the pharmacokinetics and radiation dosimetry for the initial 131I-Lym-1 therapy dose in patients with NHL and CLL and to compare tumor dosimetry with 131I-Lym-1 dosing and other patient parameters. METHODS: Fifty-one patients with stage 3 or 4 lymphoma were treated with 131I-Lym-1 (0.74-8.04 GBq [20-217 mCi]) in either a maximum tolerated dose (MTD) or low-dose (LD) trial. Total Lym-1 given to each patient was sufficient in all instances to exceed the threshold required for stable pharmacokinetics. Quantitative imaging and physical examination, including caliper and CT measurement of tumor size and analysis of blood, urine and feces, were performed for a period of 7 to 10 d after infusion to assess pharmacokinetics and radiation dosimetry. Clinical records were reviewed to obtain data required for comparative assessments. RESULTS: The concentration (%ID/g) and biologic half-time of 131-Lym-1 in tumor were about twice those in normal tissues, although tumor half-time was similar to that of the thyroid. Pharmacokinetics were similar for patients in the MTD and LD trials, and for NHL and CLL patients in the LD trial, except that the latter group had less tumor concentration of 131I. Mean tumor radiation dose per unit of administered 131I was 1.0 Gy/GBq (3.7 rad/mCi) for patients with NHL whether in MTD or LD trials, about nine times greater than that for body or marrow. Tumor radiation dose was less and liver radiation dose was more in patients with CLL. Otherwise, radiation dosimetry was, on average, remarkably similar among groups of patients and among individual patients. Pharmacokinetics and dosimetry did not appear to be influenced by the amount of 131I or Lym-1 within the ranges administered. Tumor concentration of 131I and radiation dose per gigabecquerel were inversely related to tumor size but did not seem to be related to histologic grade or type, tumor burden or therapeutic response. CONCLUSION: The therapeutic index of 131I-Lym-1 was favorable, although the index for patients with CLL was less than that for patients with NHL. Pharmacokinetics and radiation dosimetry were, on average, remarkably similar among patients and groups of patients in different trials.

Milestones in the development of Lym-1 therapy.

Lym-1, a monoclonal antibody (MAb) that preferentially targets malignant lymphocytes, has induced therapeutic remissions in patients with advanced non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) when labeled with iodine-131 (131I). Based on the strategy of fractionating the total radiation dose, trials were designed to define the safety, toxicity, and efficacy of a series of doses of 131I-Lym-1 given 2-6 weeks apart. All patients had disease resistant to standard therapy. 131I-Lym-1 was given after unconjugated Lym-1 and the 131I dose was escalated in Phase I-II trials. Therapy proved safe. The dose-limiting toxicity was thrombocytopenia. Nonhematological toxicities did not exceed grade 2 except for infrequent instances of grade 3 hypotension. In a low-dose (LD) trial of 131I-Lym-1, tumor regression occurred in 25 (83%) of 30 patients and 17 (57 %) had durable remissions; 3 of the remissions were complete. In a maximum tolerated dose (MTD) trial of 131I-Lym-1, 10 (71%) of 14 entries that received at least two doses of 131I-Lym-1 therapy and 11 (52%) of 21 total entries had remissions; 7 of the remissions were complete. All 3 entries in the MTD cohort of 100 mCi/m2 [3.7 MBq/m2] of body surface area had durable complete remissions. Therapeutic remission and human anti-mouse antibody (HAMA) after Lym-1 therapy were associated with increased survival that was significant in multivariate analyses. Evidence for an Ab3 idiotypic network with an antibody cytotoxic for Raji human lymphoma was found in the only patient examined in detail thus far; this patient was studied because she had a high titer, HAMA and prolonged survival. In conclusion, 131I-Lym-1 induced durable remissions in patients with chemotherapy-resistant NHL or CLL and was associated with acceptable toxicity. In a subset of the patients, survival was quite prolonged perhaps related to development of Ab3.

67Cu-2IT-BAT-Lym-1 pharmacokinetics, radiation dosimetry, toxicity and tumor regression in patients with lymphoma.

UNLABELLED: Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced therapeutic responses and prolonged survival in patients with non-Hodgkin's lymphoma when labeled with 1311. Radiometal-labeled antibodies provide higher tumor radiation doses than corresponding 1311 antibodies. 67Cu has an exceptional combination of properties desirable for radioimmunotherapy, including gamma and beta emissions for imaging and therapy, respectively, a biocompatible half-time and absence of pathways contributing to myelotoxicity. The radioimmunoconjugate, 67Cu-21T-BAT-Lym-1, has been shown to be efficacious in nude mice bearing human Burkitt's lymphoma (Raji) xenografts. Based on these results, a clinical study of the pharmacokinetics and dosimetry of 67Cu-21T-BAT-Lym-1 in patients with lymphoma was initiated. METHODS: Eleven patients with advanced stage 3 or 4 lymphoma were given a preload dose of unmodified Lym-1, then an imaging dose of 126-533 MBq (3.4-14.4 mCi) 67Cu-21T-BAT-Lym-1. Total Lym-1 ranged from 25 to 70 mg dependent on the specific activity of the radioimmunoconjugate and was infused at a rate of 0.5-1 mg/min. Imaging, physical examination, including caliper measurement of superficial tumors, and analysis of blood, urine and fecal samples were performed for a period of 6-13 d after infusion to assess pharmacokinetics, radiation dosimetry, toxicity and tumor regression. RESULTS: In 7 patients, in whom superficial tumors had been accurately measured, tumors regressed from 18% to 75% (mean 48%) within several days of 67Cu-21T-BAT-Lym-1 infusion. The uptake and biological half-time of 67Cu-21T-BAT-Lym-1 in tumors were greater than those of normal tissues, except the mean liver half-time exceeded the mean tumor half-time. The mean tumor-to-marrow radiation ratio was 32:1, tumor-to-total body was 24:1 and tumor-to-liver was 1.5:1. Images were of very good quality; tumors and normal organs were readily identified. Mild and transient Lym-1 toxicity occurred in 6 patients; 1 patient developed a human antimouse antibody. There were no significant changes in blood counts or serum chemistries indicative of radiation toxicity. CONCLUSION: Because of the long residence time of 67Cu-21T-BAT-Lym-1 in tumors, high therapeutic ratios were achieved and, remarkably, numerous tumor regressions were observed after imaging doses. The results indicate considerable therapeutic potential for 67Cu-21T-BAT-Lym-1.

Transfer of copper from a chelated 67Cu-antibody conjugate to ceruloplasmin in lymphoma patients.

The Lym-1 monoclonal antibody was conjugated with the bifunctional chelating agent 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N'',N'''-tetraacetic acid (BAT), using 2IT as a linker, and radiolabeled with 67Cu to make the radiopharmaceutical, 67Cu-2IT-BAT-Lym-1. Ten patients received a total of 18 doses of 67Cu-2IT-BAT-Lym-1 as targeted, systemic radiotherapy. The beta phase of blood clearance, when corrected for 67Cu decay, was positive or flat, a phenomenon not observed in similar patients treated with 131I-Lym-1. The flat beta phase of blood clearance suggested recycling of 67Cu from 67Cu-2IT-BAT-Lym-1 to another plasma protein. Therefore, the amount of 67Cu transferred from the radiopharmaceutical to CP, Alb, and TF was measured using affinity-purified polyclonal antibodies. The fraction of plasma 67Cu precipitated by anti-human CP increased daily; most blood radioactivity was 67Cu-CP after a median of 4 days (range 2-7 days). The transfer of 67Cu to CP was observed in all patients and was consistent from dose to dose within the same patient. An average of 2.8 +/- 1.5% (range 0.8-7.8%) of the 67Cu dose (%ID) was transferred to CP. The release rate of 67Cu-CP from the liver into the blood was 0.9 +/- 0.4 %ID/day for the first 3 days. The 67Cu-CP effective clearance half-life was 3.7 +/- 0.7 days. Subtraction of the 67Cu-CP activity from the total blood radioactivity yielded a biphasic blood clearance similar to that obtained for patients given 131I-Lym-1. Cu-67-CP increased the AUC for whole blood by 24 +/- 10%. The %ID of 67Cu recycled correlated with GGT, ALT, and alkaline phosphatase levels; r = 0.958 (p < 0.001), 0.857 (p < 0.01), and 0.822 (p < 0.01), respectively. Albumin levels correlated negatively with recycled copper (r = -0.745, p < 0.05). The data suggest that the liver metabolizes 67Cu-2IT-BAT-Lym-1 and recycles a small fraction of the 67Cu, transferring it to CP.

A clinical trial of radioimmunotherapy with 67Cu-2IT-BAT-Lym-1 for non-Hodgkin's lymphoma.

UNLABELLED: Encouraged by the results of 131I-Lym-1 therapy trials for patients with B-cell non-Hodgkin's lymphoma (NHL), this phase I/II clinical trial of 67Cu-2IT-BAT-Lym-1 was conducted in an effort to further improve the therapeutic index of Lym-1-based radioimmunotherapy. Lym-1 is a mouse monoclonal antibody that preferentially targets malignant lymphocytes. 67Cu has beta emissions comparable to those of 131I but has gamma emissions more favorable for imaging. The macrocyclic chelating agent 1,4,7,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid binds 67Cu tightly to form a stable radioimmunoconjugate in vivo. METHODS: All 12 patients had stage III or IV NHL that had not responded to standard therapy; 11 had intermediate- or high-grade NHL. At 4-wk intervals, patients received up to four doses of 67Cu-2IT-BAT-Lym-1, 0.93 or 1.85-2.22 GBq/m2 (25 or 50-60 mCi/m2), with the lower dose used when NHL was detected in the bone marrow. RESULTS: 67Cu-2IT-BAT-Lym-1 provided good imaging of NHL and favorable radiation dosimetry. The mean radiation ratios of tumor to body and tumor to marrow were 28:1 and 15:1, respectively. Tumor-to-lung, -kidney and -liver radiation dose ratios were 7.4:1, 5.3:1 and 2.6: 1, respectively. This 67Cu-2IT-BAT-Lym-1 trial for patients with chemotherapy-resistant NHL had a response rate of 58% (7/12). No significant nonhematologic toxicity was observed. Hematologic toxicity, especially thrombocytopenia, was dose limiting. CONCLUSION: 67Cu remains an option for future clinical trials. This study established 67Cu-2IT-BAT-Lym-1 as a safe, effective treatment for patients with NHL.

L6 monoclonal antibody binds prostate cancer.

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.

Maximum tolerated dose of 67Cu-2IT-BAT-LYM-1 for fractionated radioimmunotherapy of non-Hodgkin's lymphoma: a pilot study.

PURPOSE: Lym-1, a monoclonal antibody (MoAb) that preferentially targets malignant lymphocytes, has induced therapeutic responses in patients with non-Hodgkin's lymphoma (NHL) when labeled with iodine-131 (131I). Radiometal labeled antibodies provide a higher tumor radiation dose than the corresponding 131I labeled antibodies. Based on the strategy of fractionating the total radiation dose, this study was designed to define the maximum tolerated dose (MTD) of the first 2, of a maximum of 4, doses of 67Cu-2IT-BAT-Lym-1 given 4 weeks apart. Additionally, toxicity, radiation dosimetry and efficacy were assessed. MATERIALS AND METHODS: Patients had Ann Arbor stage IVB NHL, resistant to standard therapy, including multiple chemotherapy regimens. Each dose of 67Cu-2IT-BAT-Lym-1 was given after a preload of unmodified Lym-1. A 10 mCi imaging dose of 67Cu-2IT-BAT-Lym-1 was given in order to assess pharmacokinetics and radiation dosimetry prior to therapy. Based on the MTD for 131I-Lym-1 and comparative dosimetry for 131I-Lym-1 and 67Cu-2IT-BAT-Lym-1, the trial was initiated at 60 millicuries per square meter of body surface area (mCi/m2) in cohorts of 3 patients. RESULTS: A single cohort of patients proved sufficient to define the MTD as 60 mCi/m2 for each of the first 2 doses of 67Cu-2IT-BAT-Lym-1. The dose-limiting toxicities were grade 3-4 thrombocytopenia and neutropenia. Neutropenic sepsis and bleeding did not occur. Mean radiation dose contributed to the bone marrow by 67Cu in the body and blood was 0.2 (range, 0.2 to 0.3) rads/mCi. Copper-67 incorporated into ceruloplasmin contributed 25% of the dose to marrow from blood. Non-hematologic toxicities did not exceed grade 2. The three patients had substantial tumor regression even after imaging doses of 67Cu-2IT-BAT-Lym-1. After therapy, one response was complete with a duration of 12 months. Radiation doses to tumors in this patient varied from 7.0-21.9 rads/mCi or 5420-7000 total rads from the course of therapy. CONCLUSION: 67Cu-2IT-BAT-Lym-1 provided good imaging, favorable radiation dosimetry and a remarkably high therapeutic index (ratio of tumor to marrow radiation doses). The non-myeloablative MTD for each of 2 doses was 60 mCi/m2.

Prolonged survival associated with immune response in a patient treated with Lym-1 mouse monoclonal antibody.

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.

Combined modality radioimmunotherapy with Taxol and 90Y-Lym-1 for Raji lymphoma xenografts.

Despite effective therapies for non-Hodgkin's lymphoma (NHL), the majority of patients are not cured. Radioimmunotherapy (RIT) has shown good results in preclinical and clinical trials even in patients that are non-responsive to standard chemotherapy. To make RIT more effective, agents such as paclitaxel (Taxol), that can enhance radiation effects, are being tested. Nude mice bearing human Burkitt's lymphoma (Raji) xenografts were treated with: 1) 150 or 200 microCi (5.5 or 7.3 MBq) of 90Y-2IT-BAD-Lym-1 alone, 2) 600 micrograms of Taxol alone, 3) 150 or 200 microCi of 90Y-2IT-BAD-Lym-1 plus 600 micrograms of Taxol given 24 hours after RIT, or 4) no treatment. Tumor size, survival, mouse weight and blood counts were monitored to assess efficacy and toxicity. Survival for mice treated in this 84 day trial was: 71% for 90Y-2IT-BAD-Lym-1 (200 microCi) plus Taxol, 29% for Taxol alone, 6% for 90Y-2IT-BAD-Lym-1 (200 microCi) alone and 14% in the untreated group. Average tumor volume in the 90Y-2IT-BAD-Lym-1 (200 microCi) plus Taxol group was reduced by 89 and 99% compared to the RIT alone and Taxol alone groups, respectively. Mice treated with 150 microCi had less toxicity than those treated with 200 microCi of 90Y-2IT-BAD-Lym-1, however, the higher radiation dose, and Taxol, were required for improved survival. Mouse weights and myelotoxicity in the combined modality (RIT plus Taxol) groups were similar to those receiving the same dose of RIT alone. In the Raji tumored nude mouse model, addition of Taxol to 90Y-2IT-BAD-Lym-1, in doses clinically achievable in humans, provided therapeutic synergy without increased or excessive toxicity.

Antibody responses to macrocycles in lymphoma.

UNLABELLED: Metallic radioimmunoconjugates have promise for radioimmunoimaging and therapy. Macrocyclic chelating agents allow formation of stable metallic radioimmunoconjugates but have been reported to be immunogenic. This study assesses human antibody responses in patients that were imaged or treated with radiolabeled Lym-1 containing the macrocyclic chelators 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA) or 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). METHODS: One to six doses (median 1) and 6 to 285 mg (median 33) 67Cu-2IT-BAT (2-iminothiolane bromoacetamidobenzyl TETA)- or 111In-2IT-BAD (2-iminothiolane bromoacetamidobenzyl DOTA)-Lym-1 were administered to each of 18 patients with lymphocytic malignancies. Solid-phase ELISA, utilizing unchelated Lym-1 or human serum albumin conjugated to DOTA, TETA or 2IT-bromoacetamidobenzyl-ethylenediaminetetraacetic acid (BABE) as coating antigens, was used to characterize antibody responses against 67Cu-2IT-BAT-Lym-1 and 111In-2IT-BAD-Lym-1 by quantitating antibodies against the Lym-1, DOTA, TETA or 2IT moieties, respectively. RESULTS: None of the patients had evidence for serum sickness. No patient that received 111In-2IT-BAD-Lym-1 developed antibodies to Lym-1 or DOTA. Two (15%) of the 13 patients that received 67Cu-2IT-BAT-Lym-1 developed antibodies against both TETA and Lym-1, and an additional patient developed antibodies against Lym-1 only. No patient developed an antibody response solely against the macrocycle, nor did any of the patients generate antibodies against the 2IT molecule. HAMA levels were many times greater in amount than HATA levels even when their relative molecular masses were considered. CONCLUSION: Although macrocycles such as DOTA and TETA, and other chelates, can be haptens and thus potentially immunogenic, our findings do not support the view that macrocycles are more immunogenic than other radiometal chelating agents.

Analysis of antiglobulin (HAMA) response in a group of patients with B-lymphocytic malignancies treated with 131I-Lym-1.

Host development of human anti-mouse antibodies (HAMA) in response to administered antibodies has been reported as a problem for antibody imaging and therapy. However, radioimmunotherapy has been shown to be effective in patients with B-cell malignancies because their immunodeficient state precludes or delays development of a HAMA response to mouse antibodies. Baseline HAMA activity was assayed in 60 patients with B-lymphocytic non-Hodgkin's lymphoma or chronic lymphocytic leukemia and sequentially in 43 patients who were subsequently treated with radiolabeled Lym-1 antibody. Pre-existing "HAMA" activity was found in 3 (5%) of the 60 patients screened for treatment consideration. The incidence of development of HAMA in the 43 patients treated with multiple doses of radiolabeled Lym-1 antibody was 12 (28%). There was no evidence for an anaphylactoid or related response in the HAMA positive patients. HAMA activity interrupted therapy in 14% of the patients (6 of 43) but did not preclude therapeutic responses to radiolabeled Lym-1 therapy. Medial survival for the HAMA positive patients was longer (18 months) than for those who did not develop HAMA activity (9 months).

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.

The biologic window for chimeric L6 radioimmunotherapy.

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 131I 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. The authors have used L6 or ChL6 for radioimmunotherapy to exploit their biologic activity to create a "therapeutic window" of increased vascular permeability, allowing more 131I MoAb to reach the tumor. A reactive target is present in the vascular endothelium but can be covered by unlabeled L6 or ChL6. METHODS: Nine patients with metastatic breast cancer were treated on a therapy protocol and received imaging and therapy doses of 131I ChL6 on two sequential days at 4 week intervals. During each treatment cycle, serum cytokines, complement, albumin, and 131I ChL6 blood clearance were monitored, peripheral blood mononuclear cell activation was assessed, and tumor uptake and response were documented. RESULTS: After L6 or ChL6 was infused, patients demonstrated immediate serum-complement activation, manifested by rapidly decreasing levels of serum complements 3 and 4. Tumor uptake of the second 131I MoAb (therapeutic) injection, given after the second daily injections of 200 mg MoAb, was usually higher than the tumor uptake of the first 131I MoAb (imaging) dose given after a single 200 mg infusion of MoAb. Although serum complement frequently decreased after the first 50-100 mg dose of L6 or ChL6, elevation of soluble interleukin-2 receptor (IL-2R) in serum was seen only in patients who received 150 mg or more of L6 or ChL6. In the nine treated patients, with only one exception, the higher grade of therapeutic tumor response was seen in patients with a greater increase in IL-2R levels. CONCLUSIONS: The clinical importance of understanding these mechanisms is emphasized by the occurrence of measurable tumor regressions in five of the first nine advanced metastatic breast cancer patients treated in this manner. Absence of pulmonary edema and delayed release of dose-dependent IL-2R suggest that targeting of the pulmonary endothelium by L6 or ChL6 is not the major cause of the observed biologic effects. This unique response of a solid tumor to radioimmunoconjugate therapy may be secondary to both the increased delivery of the radioimmunoconjugate to tumor cells caused by enhanced vascular permeability, and to synergistic effects of radiation and activated effector cell mechanisms.

Body and blood clearance and marrow radiation dose of 131I-Lym-1 in patients with B-cell malignancies.

Fifty-eight per cent of patients with B-cell malignancies had durable responses to treatment with 131I-Lym-1. Myelosuppression manifested by peripheral blood cytopenia was the radiation dose-limiting toxicity. The mean biologic half-times were 3.3 and 31.2 h for the fast and slow phases, respectively, of the blood clearance and 33.5 h for the clearance from the total body. Nonpenetrating radiation from the blood contributed 0.18 rad and penetrating radiations from the total body contributed 0.18 rad per administered mCi to the bone marrow. The average total contribution from both of these sources was 0.36 +/- 0.14 rad mCi-1. Clearances and marrow radiation doses were remarkably constant among different patients and among different therapy doses for the same patient. These results are potentially useful as an initial approximation for other mouse monoclonal antibodies of the same isotype. While radiation to normal marrow from 'spill-over' incident to specific targeting of 131I-Lym-1 on malignant B-cells in the marrow is not addressed in this publication because it is unique for each patient, it should be considered in the case of individual patients.