Internal antigens accessible in breast cancer: implications for tumor targeting

Proponents of monoclonal antibody (MAb)-mediated cancer therapy often assume that a major limitation in clinical application of MAbs is their lack of absolute specificity for malignant cells. In addition, the presence of surface target antigens is thought to be essential. These requirements may be more stringent than necessary for the clinical usefulness of MAbs. We have demonstrated selective localization of a MAb to keratin polypeptides in malignant breast epithelium under conditions of passive infusion of antibody in fresh surgical specimens of breast carcinoma. Although these proteins are normal intracellular constituents of epithelial cells throughout the body, localization of antikeratin antibodies only within the tumor population is most probably associated with the presence of cells permeable to macromolecules. This permeable tumor cell fraction could be recruited for targeting neighboring impermeable tumor cells with radioisotopes or other antitumor agents conjugated to antibodies directed against intracellular antigens.     

Targeting tomoregulin for radioimmunotherapy of prostate cancer

Radiotherapy is an effective approach for the treatment of local prostate cancer. However, once prostate cancer metastasizes, radiotherapy cannot be used due to the distribution of multiple metastases to lymph nodes and bones. In contrast, radioimmunotherapy should still be efficacious in metastatic prostate cancer as radioisotopes are brought to tumor cells by targeting antibodies. Here we identify and validate a prostate-expressed molecule, tomoregulin, as a target for radioimmunotherapy of prostate cancer. Tomoregulin is a transmembrane protein selectively expressed in the brain, prostate, and prostate cancer, but not expressed in other normal tissues. Immunohistochemical studies of tomoregulin protein in clinical samples show its location in the luminal epithelium of normal prostate, benign prostatic hyperplasia, and prostatic intraepithelial neoplasia. More importantly, the tomoregulin protein is expressed in primary prostate tumors and in their lymph node and bone metastases. The nature of tomoregulin as a transmembrane protein and its tissue-specific expression make tomoregulin an attractive target for radioimmunotherapy, in which tomoregulin-specific antibodies will deliver a radioisotope to prostate tumor cells and metastases. Indeed, biodistribution studies using a prostate tumor xenograft model showed that the (111)In-labeled anti-tomoregulin antibody 2H8 specifically recognizes tomoregulin protein in vivo, leading to a strong tumor-specific accumulation of the antibody. In efficacy studies, a single i.p. dose of 150 microCi (163 microg) (90)Y-labeled 2H8 substantially inhibits the growth rate of established LNCaP human prostate tumor xenograft in nude mice but produces no overt toxicity despite cross-reactivity of 2H8 with mouse tomoregulin. Our data clearly validate tomoregulin as a target for radioimmunotherapy of prostate cancer.     

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules

MUC1 glycoprotein that is overexpressed in aberrant forms in epithelial cancers has been used for diagnosis, staging and therapy. As normal prostate and prostate cancer tissues express MUC1, it represents a potential target, but MUC1 epitopes specific to prostate cancer have not been well characterized. In order to assess MUC1 epitopes in prostate cancer, and their correlation with Gleason grades, binding of 7 well-characterized anti-MUC1 monoclonal antibodies (MAbs) (BrE-3, SM3, BC2, EMA, B27.29, HMFG-1 and NCL MUC1 core), were studied on a prostate tissue microarray. This microarray contained 197 prostate tissue cores representing: i) normal/benign prostate; ii) prostatic intraepithelial neoplasia and Gleason grades 1 and 2; and iii) Gleason grades 3-5. These MAbs bind the MUC1 extracellular domain, but have variable sensitivity to MUC1 glycosylation. To further characterize the effect of glycosylation on their binding, MAb reactivities with unglycosylated MUC1 core peptide and breast and prostate cancer cell lysates were compared. These studies demonstrated strong binding of BrE-3, BC2 and EMA to the peptide core and recognition by BrE-3, SM3, BC2 and EMA of hypoglycosylated MUC1. The results for the microarray indicated that higher Gleason grades were associated with markedly increased cellular staining by MAbs that preferentially recognize less glycosylated MUC1 (BrE-3, p<0.001; SM3, p<0.004; EMA, p=0.009; and BC2, p<0.001). Staining by MAbs that bind preferentially to hyperglycosylated MUC1 (B27.29, p=0.33; HMFG-1, p=0.89; and NCL MUC1 core, p=0.96) did not correlate with Gleason grade. These results demonstrated that hypoglycosylated MUC1 expression increased with Gleason grade, thus supporting the targeting of hypoglycosylated MUC1 epitopes in prostate cancer for more specific imaging and therapy applications.     

Criteria for the selection of second-generation platinum compounds.

Our selection of a potential second-generation platinum compound began with an initial short list of 8 compounds selected on the basis of antitumour and toxicity studies in mice. We now report further, more detailed investigations of the renal toxicity and antitumour activity of one of these compounds, cis-dichloro trans-dihydroxy bis isopropylamine platinum (IV) (CHIP), in comparison with cis-dichloro diammine platinum (II) (Neoplatin). CHIP was a more effective anti-tumour agent against both alkylating-agent sensitive and resistant strains of the Yoshida sarcoma (YSS and YSR respectively) than was Neoplatin. In addition CHIP produced negligible kidney toxicity as measured by blood urea levels. We have also compared the effects of these two drugs on nuclear-protein phosphorylation, in an attempt to gain insight into their molecular mode of action. Both Neoplatin and CHIP induced increased nuclear-protein phosphorylation in the YSS tumour cells, and loss of condensed chromatin. However, CHIP also induced increased nuclear-protein phosphorylation and loss of condensed chromatin in the YSR tumour cells. These changes correlated well with cell death. In addition Neoplatin, but not CHIP, treatment caused increased nuclear-protein phosphorylation in kidney tissues. This correlated with kidney damage as measured by blood urea levels. These selection criteria suggested that CHIP would be a more selective antitumour agent than Neoplatin, and will provide a basis for its comparison with the other 7 compounds.     

Beta dosimetry for radioimmunotherapy of cancer using labeled antibodies

Starting with the tabulation of scaled monoenergetic electron dose point kernels F in water, according to the calculations of Berger, we have developed analytic approximations that facilitate the task for convolving F with beta spectra of specific nuclides to derive their dose point kernels J. J is well fitted by a single log-normal distribution, involving three independent parameters. For large atomic numbers, the addition of an exponential function near the origin is required. The radiation dose distribution D in a homogeneous medium of soft tissue with an activity distribution C is given by the convolution of J with C. Computer programs using fast Fourier transform techniques have been developed to evaluate the convolution efficiently. To study the perturbation of tissue inhomogeneity for D, we have used a point source of 32P and measured the increase in backscattered dose from a polystyrene/aluminum interface simulating soft tissue/bone. We have also measured the decrease in dose at a polystyrene/air interface.     

Combined gemcitabine and radioimmunotherapy for the treatment of pancreatic cancer.

MAb-PAM4 is an anti-MUC1 antibody that has been shown to be reactive with 85% of pancreatic adenocarcinomas with no reactivity with normal pancreas or other tissues. Initial clinical studies have shown excellent targeting with high tumor/nontumor ratios. Gemcitabine, an analog of deoxycytidine, is currently a frontline treatment for pancreatic cancer. Acting via a number of metabolic pathways, gemcitabine is also a powerful radiosensitizer. Combined-modality, chemo/radiosensitization with gemcitabine and low dose (131)I-PAM4 radioimmunotherapy was performed to determine if a more effective treatment procedure could be developed. Athymic nude mice bearing large (1 cm(3)) CaPan1 human pancreatic tumors were given a single treatment cycle consisting of gemcitabine, 333 mg/m(2) administered on Days 0, 3, 6, 9 and 12 by intraperitoneal injection, along with either 100 or 200 microCi, (131)I-PAM4 administered on Day 0 by intravenous injection. Gemcitabine did not interfere with the biodistribution of radiolabeled antibody. Specific tumor targeting was observed for (131)I-PAM4, with a tumor/blood radiation dose ratio of 2.6 over the first 14 days. Gemcitabine alone and low dose radioimmunotherapy alone, each had no affect upon tumor growth; no statistical differences were noted in comparison to the untreated group. When combined, however, a statistically significant (p = 0.0324), synergistic anti-tumor effect was observed. Median survival time doubled for the combined treatment regimen compared to single modality treatment groups. The combined treatment modality was well tolerated by the mice. Our data show that combined gemcitabine with radioimmunotherapy may provide an improved alternative for the treatment of pancreatic cancer, achieving successful anti-tumor effects with low toxicity.     

肿瘤放射免疫治疗的问题及对策

综述放射免疫治疗中肿瘤对抗体的摄取、靶/非靶的比、免疫原性、放射性核素的选择等问题.     

Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer

Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer-testis antigens that are upregulated in MHC class I-deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus, SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study, we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines, but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably, SSX expression in patient tumor samples was restricted to metastatic lesions (5/22; 23%) and no expression was detected in primary prostate tumors examined (0/73; P < 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2-specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting.     

Predictors of long-term outcome from intraperitoneal radioimmunotherapy for ovarian cancer

Data was analyzed from 92 patients > 5 years after intraperitoneal (IP) radionuclide therapy (RIT) with (90)Y- or (177)Lu-CC49 to determine prognostic factors. Patients had CC49 antibody-reactive ovarian cancer confined to the abdominal cavity after primary debulking and chemotherapy. The first 27 patients received IP (177)Lu-CC49 alone; the remainder received Interferon (IFN), to increase the expression of the tumor-associated glycoprotein-72 (TAG-72) antigen, +/- IP paclitaxel (25-100?mg/m(2)) 2 days before RIT. Factors assessed by univariate (and some multivariate) analysis included age, race, body size, interval between initial diagnosis and RIT, interval between 2nd look surgery and RIT, (90)Y versus (177)Lu, MBq dose, paclitaxel dose, grade of tumor, extent of initial surgery, size of disease deposits prior to RIT, intensity of TAG reactivity, the addition of unlabeled antibody, and the development of human anti-mouse antibody and/or serum sickness after murine antibody. A statistically significant improvement in progression-free survival (p ≤ 0.05) was noted for less bulky disease and younger age. Administration of paclitaxel plus IFN, an immune response, and use of (90)Y showed a favorable nonsignificant trend. Dose escalation of radionuclide did not change risk of progression; thus, this therapy may have therapeutic efficacy at modest dose levels.     

FACTORS INFLUENCING THE EFFECT OF RADIOIMMUNOTHERAPY ON LIVER CANCER

Factors influencing the therapeutic effect of radiolmmunotherapy with 131I labeled anti- human hepa-tocellular carcinoma (HCC) ferritin antibody (131I -FtAb) on thirty three patients with surgically proven unresectable HCC were studied. Multi- variable analysis with Cox' s regression model revealed that the statistically sig-nifieant factors include tumor size, activity of 131I administered each time and the second-look resection. Survival of patients with tumor diameter less than 10 cm was higher than that of patients with tumor diameter more than 10 cm (1-year survival; 84% versus 50%) 3-year survival; 63% versus 9% ). Patients administered with 5. 55×108 Bq to 9. 25× 10(?) of 131I-FtAb each time yielded better effect than those administered with more than 9. 25×108 Bq of 131I -FtAb (1-year, survival: 86% ver- sus 55%; 3-year survival: 50% versus 18%). When tumor shrank, patients underwent second-look resection had a higher survival than those without receiving second- look resection (1- year survi     

Radiobiology of radioimmunotherapy: targeting CD20 B-cell antigen in non-Hodgkin's lymphoma

The radiobiology of radioimmunotherapy is an important determinant of both the toxicity and the efficacy associated with the treatment of B-cell non-Hodgkin's lymphoma with radiolabeled anti-CD20 monoclonal antibodies. The properties of the target, CD20, and the mechanisms of action of both the monoclonal antibodies and the associated exponentially decreasing low-dose-rate radiotherapy are described. The radiation dose and dose-rate effects are discussed and related to both the tumor responses and normal organ toxicity. Finally, the use of either unlabeled or radiolabeled anti-CD20 monoclonal antibodies as a component of combined modality therapy (including the sequential or concurrent use of sensitizers) and future directions of the field are discussed.     

Evaluation of CD20, CD22, and HLA-DR targeting for radioimmunotherapy of B-cell lymphomas

Despite the promise of radioimmunotherapy using anti-CD20 antibodies (Ab) for the treatment of relapsed patients with indolent non-Hodgkin lymphoma (NHL), most patients treated with conventional doses of (131)I-tositumomab or (90)Y-ibritumomab eventually relapse. We did comparative assessments using conventional radioimmunotherapy targeting CD20, CD22, and HLA-DR on human Ramos, Raji, and FL-18 lymphoma xenografts in athymic mice to assess the potential for improving the efficacy of radioimmunotherapy by targeting other NHL cell surface antigens. Results of biodistribution studies showed significant differences in tumor localization consistent with variable antigenic expression on the different lymphoma cell lines. Interestingly, the radioimmunoconjugate that yielded the best tumor-to-normal organ ratios differed in each tumor model. We also explored administering all three (111)In-1,4,7,10-tetra-azacylododecane N,N',N',N'-tetraacetic acid antibodies in combination, but discovered, surprisingly, that this approach did not augment the localization of radioactivity to tumors compared with the administration of the best single radiolabeled Ab alone. These data suggest that conventional radioimmunotherapy using anti-CD20, anti-HLA-DR, or anti-CD22 Abs is effective when used singly and provides targeted uptake of radiolabel into the tumor that is dependent on the levels of antigen expression. Improvements in tumor-to-normal organ ratios of radioactivity cannot be achieved using directly labeled Abs in combination but may be afforded by novel pretargeting methods.     

Intraperitoneal radioimmunotherapy in treating peritoneal carcinomatosis of colon cancer in mice compared with systemic radioimmunotherapy

Peritoneal spread is one of major causes of mortality in colorectal cancer patients. In the current investigation, the efficacy of radioimmunotherapy (RIT) with i.p. administration of an anti-colorectal cancer IgG1, 131I-A7, was compared to that with i.v. administration in BALB/c female mice bearing peritoneal nodules of LS180 human colon cancer cells, at the same toxicity level. Distribution of either i.p. or i.v. administered 131I-A7 and i.p. administered irrelevant 131I-HPMS-1 was assessed. Based on the results of toxicity determination at increments of 2 MBq and estimated dosimetry, an i.p. dose of 11 MBq and an i.v. dose of 9 MBq were chosen for treatment. Mice were monitored for long-term survival: untreated mice (n = 11), mice undergoing i.p. RIT with 131I-A7 (n = 11), mice undergoing i.v. RIT with 131I-A7 (n = 11) and mice undergoing non-specific i.p. RIT with 131I-HPMS-1 (n = 5). Intraperitoneal injection of 131I-A7 produced faster and greater tumor accumulation than i.v. injection: 34.2 +/- 16.5% of the injected dose per g (% ID/g) and 11.1 +/- 3.6% ID/g at 2 h, respectively (P < 0.0001). Consequently, cumulative radioactivity in tumors was 1.73-fold higher with i.p. injection. 131I-HPMS-1 did not show specific accumulation. Non-specific RIT with 131I-HPMS-1 (mean survival, 26.0 +/- 2.5 days) did not affect the survival as compared to no treatment (26.7 +/- 1.9 days). Intravenous RIT with 131I-A7 prolonged the survival of mice to 32.8 +/- 1.8 days (P < 0.01). Intraperitoneal RIT with 131I-A7 improved the survival more significantly and attained cure in 2 of 11 mice (P < 0.05 vs. i.v. RIT). In conclusion, i.p. RIT is more beneficial in treating peritoneal carcinomatosis of colon cancer than i.v. RIT in a murine model.     

Radioimmunodetection and radioimmunotherapy of head and neck cancer

Radiolabeled monoclonal antibodies (MAbs) can add a dimension to diagnostic imaging and staging of metastatic head and neck cancer, as well as in eradication of this disease. The vast majority of malignancies arising in the oral cavity, pharynx and larynx are squamous cell carcinomas. This common cellular origin makes it attractive to search for appropriate tumor-associated antigens, which are preferentially expressed in these neoplasms. Radiolabeled MAbs directed against these antigens can be used for tumor detection and selective therapy, known as radioimmunoscintigraphy and radioimmunotherapy, respectively. The combination of MAbs with positron emission tomography (PET) is an attractive novel option to improve tumor detection and to facilitate MAb quantification in a therapeutic setting. Basic aspects of tumor targeting with MAbs, as well as a review of the clinical trials reported in the literature, including own results, are presented.     

Targeting osseous metastases: Rationale and development of radioimmunotherapy for prostate cancer

For patients with metastatic prostate cancer, bone is the primary site of tumor localization and the major cause of disease-related morbidity and mortality. Hormonal therapy and chemotherapy alone cannot eradicate disease harbored in bone. The delivery of radiotherapy to the reservoir of disease is an approach previously only achievable using bone-seeking radiopharmaceuticals. Now, however, with the identification of tumor-specific targets, antibodies are being used to deliver radiotherapy to these sites. In this article, we review the rationale behind this approach, the targets being explored, the radiation sources available, and the antibodies currently under clinical development.     

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy

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