Apoptosis-related gene and protein expression in human lymphoma xenografts (Raji) after low dose rate radiation using 67Cu-2IT-BAT-Lym-1 radioimmunotherapy.

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to 67Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, 67Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 micrograms Lym-1, or 335-500 microCi 67Cu on less than 400 micrograms Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-beta 1 and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. 67Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after 67Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with 67Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.

Antibody phage display applications for nuclear medicine imaging and therapy.

Antibody-based constructs genetically engineered from genes of diverse origin provide a remarkable opportunity to develop functional molecular imaging techniques and specific molecular targeted radionuclide therapies. Phage display libraries of antibody fragment genes can be used to select antibody-based constructs that bind any chosen epitope. A large naive human antibody-based library was used to illustrate binding of antibody constructs to a variety of common and unique antigens. Antibody-based libraries from hybridoma cells, lymphocytes from immunized humans or from mice and human antibody repertoires produced in transgenic mice have also been described. Several orders of magnitude of affinity enhancement can be achieved by random or site specific mutations of the selected binding peptide domains of the scFv. Affinities (Kd) as high as 10(-11) M (10 pM) for affinity-matured scFv have been documented. Such gene libraries thus offer an almost limitless variety of antibody-based molecular binding peptide modules that can be used in creative ways for the construction of new targeting agents for functional or molecular imaging and therapy.

Apoptotic and proliferation indexes in canine lymphoma.

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.

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.

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.

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.

Yttrium-90-DOTA-peptide-chimeric L6 radioimmunoconjugate: efficacy and toxicity in mice bearing p53 mutant human breast cancer xenografts.

UNLABELLED: The novel radioimmunoconjugate, 90Y-DOTA-peptide-chimeric L6 (ChL6), was designed to reduce radiation to critical normal tissues with an exceptionally stable 90Y chelate moiety and a biodegradable linker. Human breast cancer tumors (HBT 3477) in mice were treated with 90Y-DOTA-peptide-ChL6 to examine the effects of increasing dose on the therapeutic efficacy and toxicity of this new agent. METHODS: Groups of athymic mice bearing HBT 3477 xenografts received 4.1- to 14.1-MBq doses of 90Y-DOTA-peptide-ChL6 intravenously. The lethal dose (LD)(50/30), general well-being (weight loss), hematotoxicity and therapeutic efficacy were studied. RESULTS: The LD(50/30) was 12.8 MBq, which corresponded to doses of 17.9 and 50.9 Gy to the total body and tumor (200 mm3), respectively. Deaths were associated with hematotoxicity; no deaths occurred at doses of 9.6 MBq or less. At sublethal doses, the rate of tumor response (cures +/- complete responses + partial responses) increased with increasing dose: 4.1 MBq, 27%; 5.9 MBq, 41%; 8.5 MBq, 69%; and 9.6 MBq, 79% (maximum tolerated dose, MTD). In mice receiving doses of 4.1-9.6 MBq, 6 of 74 (8%) of tumors were cured. Increasing the 90Y dose led to smaller tumor size at nadir and longer tumor regrowth delay but no increase in cure. Although the HBT 3477 p53 gene was found to be mutant resulting in p53 protein not binding DNA breaks, tumors at MTD demonstrated evidence of apoptosis. CONCLUSION: In the human breast cancer athymic mouse model, 90Y-DOTA-peptide-ChL6 had a high therapeutic index and LD(50/30) leading to a 79% response rate at the MTD. The evidence of apoptosis as a mechanism for this tumor response in p53 mutant breast cancer warrants further studies because these observations are relevant to the treatment of lethal breast cancer.

p53-independent response of a human breast carcinoma xenograft to radioimmunotherapy.

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 microCi 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, p21(CDKN1/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 microCi 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.

Decreased C-MYC and BCL2 expression correlates with methylprednisolone-mediated inhibition of Raji lymphoma growth.

Methylprednisolone (MP) and related corticosteroids are a fundamental part of regimens used to treat lymphoma and leukemia. In many of these malignancies, oncogenic activation of C-MYC and BCL2 is seen. Abnormalities of the tumor suppressor p53, which exerts growth-suppressing and apoptosis-enhancing functions through the transcriptional regulation of downstream genes including CDKN1, GADD45, and BCL2, are also often found. The goal was to determine the modulation of expression of the oncogenes (C-MYC and BCL2), the p53 pathway described above, and the apoptosis marker TGF-beta 1 in the human Raji lymphoma following MP treatment. Raji xenografts were grown in nude mice and growth curves characterized by sequential measurement. Mice were treated daily for 8 days with MP. Tumors were harvested untreated, or at 1 or 8 days after cessation of MP treatment, and the RNA was extracted. RT-PCR was used to determine the level of mRNA expression of the genes. Tumor growth was greatly reduced in the MP-treated mice. Gene expression levels for C-MYC and BCL2 were reduced at 1 day following MP and approached control levels 8 days after MP treatment. Expression levels of p53, CDKN1, and GADD45 were moderately and coordinately decreased at 1 day after cessation of MP treatment and remained repressed a week later. TGF-beta 1 exhibited no change in expression levels. These results suggest that decreased expression of C-MYC and BCL2 may play a role in the molecular events that initiate and are responsible for the growth inhibition of Raji lymphoma xenografts by MP.

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.