Sorafenib in patients with Child-Pugh class A and B advanced hepatocellular carcinoma: a prospective feasibility analysis.

BACKGROUND: Sorafenib has shown survival benefits in patients with advanced hepatocellular carcinoma (HCC) and Child-Pugh (CP) class A liver function. There are few prospective data on sorafenib in patients with HCC and CP class B. PATIENTS AND METHODS: A consecutive prospective series of 300 patients with CP class A or B HCC were enrolled in a dual-phase trial to determine survival and safety data according to liver function (class A or B) in patients receiving oral sorafenib 800 mg daily. [Results of this study were presented in part at the ASCO 2012 Gastrointestinal Cancers Symposium, 19-21 January 2012. J Clin Oncol 2012; 30 (Suppl 4): abstract 306.] RESULTS: Overall progression-free survival (PFS), time to progression (TTP) and overall survival (OS) were 3.9, 4.1 and 9.1 months, respectively. For patients with CP class A versus B status, PFS was 4.3 versus 2.1 months, TTP was 4.2 versus 3.8 months and OS was 10.0 versus 3. 8 months. Extrahepatic spread was associated with worse outcomes but taken together with CP class, liver function played a greater role in reducing survival. Adverse events for the two CP groups were similar. CONCLUSION: Although patients with HCC and CP class B liver function have poorer outcomes than those with CP class A function, data suggest that patients with CP class B liver function can tolerate treatment and may still benefit from sorafenib.

Low glomerular filtration rate is a risk factor for ribavirin-associated anaemia in old patients with chronic hepatitis C.

Elderly patients with chronic hepatitis C have a reduced responsiveness to antiviral therapy with Peg-interferon and ribavirin. The dose reduction or the discontinuation of ribavirin due to the occurrence of anaemia is one of the most important causes for the low sustained viral response observed in older patients. We aimed to evaluate the relationship between baseline renal function and the early onset of ribavirin-associated anaemia in older (≥60 years) patients. Using data from 348 patients with chronic hepatitis C consecutively treated with peg-interferon plus ribavirin, we investigated which factors were associated with the occurrence of anaemia in elderly patients (≥60 years). Ribavirin-induced anaemia occurred in 40.5% of patients. Older patients showed a rate of anaemia significantly higher than younger patients (51.5% vs 36.3%; P = 0.009). Consequently, the rate of ribavirin dose reduction or discontinuation due to anaemia was 35.1% in older patients and 23.5% in younger patients (P = 0.029). A significantly higher proportion of older patients had a low baseline glomerular filtration rate (GFR) compared with younger patients (56.7% vs 27.1%; P < 0.001). At the multivariate regression analysis, low baseline GFR (<70 mL/min) was associated with an increased risk of ribavirin-associated anaemia only in the older patients (OR: 3.526; 95% CI: 1.385-8.979; P = 0.008). In this subset, baseline GFR was significantly correlated with both absolute (r = -0.320; P < 0.001) and relative (r = -0.324; P < 0.001) haemoglobin decrease within the first 8 weeks of treatment. In patients aged >60 years, a low pre-treatment GFR was strongly associated with the risk to develop ribavirin-related anaemia with consequent reduction in ribavirin doses.

phoenixin(smim20), a gene coding for a novel reproductive ligand, is expressed in the brain of adult zebrafish.

Gonadotropin-releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. In addition to its role as hypothalamic releasing hormone, GnRH has multiple functions including modulator of neural activity within the nervous system and of resulting behaviors. These multiple functions are reflected by the existence of multiple isoforms. Despite its importance as a critical hypothalamic releasing hormone, the gnrh1 gene has been lost in zebrafish, and its reproductive function is not compensated for by other GnRH isoforms (GnRH2 and GnRH3), suggesting that, surprisingly, zebrafish do not use any of the GnRH peptides to control reproduction and fertility. Previously we proposed that Phoenixin/SMIM20, a novel peptide identified in mammals and the ligand for the orphan GPR173, is a potential candidate to control the initiation of sexual development and fertility in the zebrafish. Here we confirm the sequence of the zebrafish phoenixin/smim20 gene and by RT-PCR show that it is expressed early in development through adulthood. Subsequently we show that phoenixin/smim20 is expressed in the adult brain including the regions of the hypothalamus important in the control of fertility and reproduction.

Enzymatic scarification of Anacamptis morio (Orchidaceae) seed facilitates lignin degradation, water uptake and germination.

The seed coat of many species contains hydrophobic lignins, and in soil the action of microbial ligninases may contribute to release from dormancy. Laboratory use of ligninases to stimulate germination is promising because of the specific action on the seed coat, whereas chemical scarification agents may also corrode the embryo. We hypothesised that exposure of Anacamptis morio (Orchidaceae) seeds to fungal laccase would stimulate germination, and that the mechanism involves lignin degradation and increased imbibition. Germination capacity in vitro was quantified with 1 U filter-sterilised laccase added to agar medium following autoclaving, compared to a 10% bleach solution (standard bleach surface sterilisation/scarification method used in orchid seed sowing). Lignin degradation was quantified using an optical method (phloroglucinol-HCl staining) combined with image analysis, following experimental pre-treatments involving immersion in laccase solution, distilled water (negative control) or bleach (positive control). Water uptake after experimental treatments was quantified as the proportion of seeds exhibiting visible uptake of an aqueous fluorochrome under UV excitation. Laccase stimulated a doubling of germination in vitro with respect to bleach surface sterilisation/scarification alone, from 23.7 to 49.8% (P = 0.007). Laccase and bleach methods both significantly decreased the optical signal of phloroglucinol (for laccase, to 79.9 ± 1.3% of controls; anova: F = 10.333, P = 0.002). Laccase resulted in a modest but highly significant (P < 0.0001) increase in water uptake with respect to the control (11.7%; cf 99.4% for bleach). Laccase scarification can stimulate germination of A. morio through a mechanism of targeted seed coat degradation. The results demonstrate the potential of this relatively non-invasive enzymatic scarification technique.

Comparison of simple tests for the non-invasive diagnosis of clinically silent cirrhosis in chronic hepatitis C.

BACKGROUND: Biopsy is the gold standard for assessing cirrhosis in patients with chronic hepatitis C virus infection, but it is expensive and at risk of complications. Alternative non-invasive methods have been developed but their usefulness remains uncertain. AIM: To compare the accuracy of five non-invasive scores in detecting cirrhosis. METHODS: We reviewed the charts and liver biopsies of 228 consecutive, treatment-naïve, hepatitis C virus-positive patients, 13.2% of whom with histological diagnosis of cirrhosis. The five alternative scores were age-platelet index, cirrhosis discriminant score, aspartate transaminases to platelet ratio index, Pohl's index, and aspartate transaminases/alanine transaminases ratio. RESULTS: The specificities of the scores were good (87-100%), but not so their sensitivities (17-67%). Accordingly positive likelihood ratios were generally good but negative likelihood ratios were suboptimal. Combinations of the scores independently related to cirrhosis only slightly change this diagnostic accuracy. Using double cut-offs to exclude/diagnoses cirrhosis, cirrhosis discriminant score classified 21% of patients without misdiagnoses and aspartate transaminases to platelet ratio index classified 85% of case with 9% of misdiagnoses. CONCLUSIONS: The five scores showed variable sensitivities and specificities in detecting liver cirrhosis, both individually and in combination. The use of double cut-off points may make the cirrhosis discriminant score and aspartate transaminases to platelet ratio index useful to reduce the number of patients submitted to liver biopsy.

Immunologic methods for the identification of cell types. II. Expression of normal mouse mammary epithelial cell antigens in mammary neoplasia.

Mouse mammary epithelial (MME) cell antigens were cell type-specific and were retained, to a large extent, by MME cells after neoplastic transformation. These MME cell antigens were expressed in mammary tumors of BALB/c, C3H, GRS/A, RIII, and Is/Bi mice tested and were not expressed in tumors whose normal counterpart was other than mammary tissue. They were not dependent on cell culture conditions or on the presence of murine mammary tumor virus; therefore, they can be used in an ubiquitous cell type marker for MME cells in both normal and neoplastic tissues.

Immunologic methods for the identification of cell types. I. Specific antibodies that distinguish between mammary gland epithelial cells and fibroblasts.

Antibodies were produced against intact mouse mammary epithelial cells and cleared fat pad fibroblasts; after appropriate absorption, two specific antibody preparations were obtained. The antimammary epithelial cell preparation did not appear to be strain- or species-specific. The antimammary fibroblast preparation recognized both mammary and fetal fibroblasts. These findings suggested that each cell type possessed distinct immunogenic components. These components were characteristic of each cell type and could be used to identify the respective cells by an immunofluorescence technique. Characterization of the antigenic component of mouse mammary epithelial cells demonstrated that this antigen was released during enzymatic cell dissociation and was regenerated underin vitro culture.

Primary human breast carcinomas transplantable in the nude mouse.

Of 19 primary human breast carcinomas implanted into noninbred female nude mice, 3 produced transplantable tumors. Membrane components specific for human mammary epithelial cells were demonstrated in the cells from heterotransplants even after four or five passages in nude mice.

Experimental therapy of human breast tumors with 131I-labeled monoclonal antibodies prepared against the human milk fat globule.

Breast tumors are susceptible to attack by unconjugated anti-human milk fat globule monoclonal antibodies (MoAbs) and most particularly by their mixture (cocktail) (Cancer Res., 47: 532-540, 1987). In the present study the same MoAbs (Mc1, Mc3, Mc5, and Mc8) labeled with 131I, either singly or in cocktails, were used for a similar purpose. Biodistribution studies showed that a transplantable human breast tumor line (MX-1) implanted in BALB/c nude mice (nu/nu) had the maximum incorporation of injected 131I-MoAbs at day 4 while levels in circulation and in normal tissue declined steadily from day 1. Also, these studies showed that the amount of radiolabeled Mc3 MoAb incorporated by MX-1 tumors was greater than that for cocktail of MoAbs and MoAb Mc5. Tumor destruction by injected 131I-MoAb cocktail was shown in therapy experiments to be dose dependent. A single injection (1500 [corrected] microCi/mouse) of 131I-MoAb Mc3, or of cocktail, produced large breast tumor volume diminution and inhibition of growth for up to 30 days while a similar dose of 131I-labeled control IgG had no effect. A second dose of 1500 [corrected] microCi 131I-MoAb of Mc3 or of cocktail, injected at an appropriate interval, again diminished tumor mass significantly and inhibited its growth for another 20 days. In control experiments, non-breast tumors (colon) were marginally affected by the 131I-MoAbs. These results show that the systemic injection of radioiodinated MoAbs against human milk fat globule destroy the epithelial cells of human breast tumors and control their growth for an appreciable length of time. Radioiodoconjugated MoAbs proved to be more effective than unconjugated MoAbs in reducing breast tumor mass and also in inhibiting growth for longer periods of time at immunoglobulin doses 100 to 200 times lower. Further exploration of their role in breast cancer treatment seems warranted by these results.

Significance of breast carcinoma-associated antigens as a monitor of tumor burden: characterization by monoclonal antibodies.

Use of monoclonal antibodies (Mc 3 and Mc 8) prepared against human mammary-epithelial antigens of human mild fat globule membranes has enabled characterization of breast carcinoma (BC) associated antigens (BCAA), antibodies, and circulating immune complexes (CIC). For this study, BC patients were grouped on the basis of measurable tumor burden: Group I patients with no evidence of disease at sampling time; Group II patients with tumor burden less than or equal to 5 g; and Group III patients with known regional or distal metastases. In an in vitro simulation of tumor burden change, selected BC patients' sera were admixed with Mc 3 and Mc 8 at optimal concentration. CIC reduction (dissociation) for Groups I and III and increment (formation) for Group II were noted. Unlike Group I sera, Groups II and III sera required 4- to 16-fold dilution of Mc 3 and 4-fold more concentrated Mc 8 to achieve maximal CIC changes. Serum BCAA isolated by use of both Mc 3 and Mc 8 immunobead procedures showed apparent Mr 33,000 monomer, 66,000 dimer, and 95,000 trimer. When BCAA were added to BC patients' sera, autologous combinations resulted in small (7.7S) CIC for Groups I and III, and medium (9 to 12S) CIC for Group II. Conversely, allogenic combinations resulted in mainly small CIC for Group I, and intermediate CIC for Group II and Group III. Evaluation of circulating BCAA concentration by use of a three-step radioligand technique demonstrated significant discrimination between BC patients' sera (mean = 105 ng/ml) and normal control sera (less than or equal to 20 ng/ml). BCAA were found to be elevated in 31 of 46 (67%) Group I (mean = 70 ng/ml), 41 of 43 (95%) Group II (mean = 197 ng/ml), and 30 of 46 (65%) Group III (mean = 50 ng/ml) patients' sera, as compared to "background" levels in malignant melanoma and normal controls. Benign breast disease sera showed moderate BCAA increases (mean = 48 ng/ml) in 20 of 35 (57%) patients. Furthermore, serial sample determination of BCAA in 36 selected BC patients confirmed the above pattern, indicating that this assay can be used with some restriction to monitor tumor burden. Whereas in early breast carcinoma increase in BCAA concentration was concurrent with or antedated clinical objective evidence of tumor burden increase, significant decreased BCAA concentration was observed with tumor burden reduction. Overall, increased BCAA levels were associated with limited tumor burden (Group II) while decreased BCAA levels were observed with no evidence of disease (Group I) and known regional or distal metastatic advanced disease (Group III) during patients' follow-up.

Experimental immunotherapy of human breast carcinomas implanted in nude mice with a mixture of monoclonal antibodies against human milk fat globule components.

Immunological therapy of BALB/c nude mice (nu/nu) implanted with human breast tumors, estrogen receptor negative MX-1 and estrogen receptor-positive MCF-7, was carried out with four monoclonal antibodies (MoAbs) raised against human milk fat globule membrane glycoproteins also present on normal breast epithelial cells. MoAbs injected singly or as a partial mixture arrested growth of the tumors but to a lesser extent than a mixture ("cocktail") of all four MoAbs. Two model systems were developed in order to examine the capabilities of the four MoAbs to arrest human mammary tumor growth. In the first model the ability of these MoAbs to arrest tumor growth during a 6- to 8-week period was tested by injection of the MoAbs immediately before and after implantation (passive immunization) and thereafter every other day. In the second model the effect of these MoAbs on established and growing tumors was tested. Using the cocktail in the passive immunization protocol, human mammary tumor growth in nu/nu mice was arrested either completely or averaging to one-tenth the size of the controls for those mice in which the tumors had taken. Other human carcinomas, colon and lung, under the same protocol, were not affected. Injection of cocktail every 2 days into nu/nu mice with established and growing human breast tumors (both estrogen receptor positive and negative) produced arrests of tumor growth of 44.1, 45.2, 49.8% of their controls after 7 to 8 days of treatment. Previously, it has been established that human mammary tumors are heterogeneous in expression of the human milk fat globule antigens recognized by our antibodies to the extent that some cells may have large amounts and others no detectable amount of a particular antigen. Those MX-1 tumors treated for a prolonged time with the cocktail of MoAbs that survived and continued to grow could be the result of the preferential multiplication of those cells in the heterogeneous population which had low or no antigen content. The breast tumors that did grow in the nu/nu mice after 8 weeks of injection of the cocktail revealed by immunoperoxidase staining a 90% reduction in the antigen content as recognized by these MoAbs when compared with untreated tumors. These results attest to the effectiveness of unconjugated anti-human milk fat globule MoAbs to arrest human breast tumor growth in nu/nu mice, and they also suggest that to best arrest tumor growth the use of a mixture of MoAbs should be considered.

Variability in surface antigen expression of human breast epithelial cells cultured from normal breast, normal tissue peripheral to breast carcinomas, and breast carcinomas.

Single-cell heterogeneity and variability in expression of several surface antigens on human mammary epithelial cells in short-term culture were studied with immunofluorescence techniques, using polyclonal and monoclonal antibodies. The cultures, derived from normal breast, a fibroadenoma, a gynecomastia, normal breast tissue peripheral to breast carcinomas, and breast carcinomas and their metastases, were studied after one passage in vitro. The percentage of positive cells varied considerably from one tissue sample to another in all categories from normal to malignant, although there was an overall trend toward a decreasing percentage of positive cells of malignant tissues. The relative antigen content varied 3- to 8-fold among individual samples of cells from normal, peripheral, and carcinoma tissue, while the mean values in the three categories were similar. The single-cell variability in relative antigen content was considerable in all individual samples of normal, peripheral, and carcinoma tissues, as reflected in the high coefficients of variation. However, the coefficients of variation were significantly higher for cells from carcinoma and peripheral tissues [69 +/- 10% (S.E.) and 75 +/- 9%, respectively] than for cells from normal breast (48 +/- 5%). By analyzing in clonal colonies the appearance of quantitative variants in expression of a specific surface antigen, detected with a monoclonal antibody, the carcinoma cells were found to have a 10-fold higher rate of phenotypic variability (mean, 1.21 X 10(-2)/cell/generation) than did cells from normal breast (mean, 0.119 X 10(-2)) and one gynecomastia (0.045 X 10(-2)). Mammary epithelial cells from apparently "normal" tissue peripheral to a carcinoma had an intermediate rate of phenotypic variability (mean, 0.310 X 10(-2)) that was significantly higher than that of the normal tissue.

Effect of multiple, repeated doses of radioimmunotherapy on target antigen expression (breast MUC-1 mucin) in breast carcinomas.

The effect of radioimmunotherapy (RIT) on target antigen expression was studied in breast carcinomas transplanted in immunodeficient mice. In nine separate experiments, a single dose of 1500 microCi of 131I-labeled monoclonal antibody (MAb) Mc5 was given to groups of mice carrying well-established, vascularized, transplantable breast tumors (MX-1). Mc5 recognizes an epitope on the tandem repeat of the breast epithelial MUC-1 mucin. This dose suppressed tumor growth for at least 20 days, after which the tumors began to regrow. At various times thereafter, tumors were removed and analyzed for target antigen expression by flow cytometry and immunohistochemistry. In no case was there any significant decrease in antigen content/cell in the tumors of treated mice compared to tumors in control untreated mice. Similar results were obtained with four other breast carcinomas (MCF-7, MDA-MB-331, MDA-MB-435, and MX-2A). To assess the effect of repeated RIT doses on target antigen expression, groups of mice with MX-1 tumors were given 2, 3, and 4 consecutive doses of 1200 microCi of 131I-labeled Mc5. One mouse each at 2, 3, and 4 doses (3 of 18) was cured of its tumor. Control mice were sacrificed after 50 days due to the excessive size of their tumors. Tumors from four mice from each group (2, 3, and 4 doses), after they began to regrow, were excised and analyzed for mucin content and compared to tumors from untreated mice with similar-size tumors transplanted at later dates. In none of the treated groups was there any decrease in mucin content. These results demonstrate that RIT with an anti-breast mucin MAb does not result in the appearance of antigen-negative tumor cells, thus indicating that repeated fractionated doses, which will most likely be necessary for an eventual cure of breast cancer with MAb therapy, are possible.

Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization.

Mc3 is a murine mAb that is highly effective in treating breast tumors in experimental radioimmunotherapy. Mc3 binds to BA46, a 46-kDa glycoprotein of the human milk fat globule membrane that is also expressed in breast carcinoma cells. We cloned and sequenced cDNAs encoding the variable regions of Mc3 and constructed an IgG1, kappa human/mouse chimeric antibody. We then humanized the variable regions of Mc3 using a positional consensus method and retaining residues that might either contact the complementarity-determining regions or the opposite chain. This positional consensus is novel in that it does not include residues with buried side chains. Humanized Mc3 retained full binding affinity, and fully competes with murine Mc3 for antigen binding. Humanized and murine 131I-labeled Mc3 behaved identically in athymic nu/nu mice biodistribution studies. The tumor uptake levels for both antibodies increased over a period of 4 days within a range of 13-20% of the injected dose per g with extremely favorable tumor:normal ratios. Also, a single therapeutic dose of 131I-labeled humanized Mc3 in the same animal model reduced the average tumor size and produced one of five cures while in the uninjected control tumor growth continued unabated. We believe that these results justify the implementation of Phase I human clinical trials for imaging and radioimmunotherapy of breast cancer.

Comparison of rates of phenotypic variability in surface antigen expression in normal and cancerous human breast epithelial cells.

A method is described for measuring the rate of phenotypic variability in normal and neoplastic breast epithelial cells. Three groups of normal human mammary epithelial cells were studied, two derived from reduction mammoplasties and one derived from the normal breast tissue of a patient with fibroadenoma. The breast carcinoma cells were all cell lines, four (MCF-7, SKBR-3, MDA-MB-157, and T47D) derived from pleural effusions of patients with breast cancer, and one (BT-20) derived from a primary breast tumor. The heterogeneity and variability in expression of a cell surface glycoprotein with apparent molecular weight of 400,000 were studied at the single-cell level with immunoperoxidase techniques using a specific monoclonal antibody, BLMRL-HMFG-Mc5, to a nonpenetrating glycoprotein. The rate of appearance of quantitative variants in expression of this specific surface antigen (rate of phenotypic variability) was determined in clonal colonies and was found to be severalfold higher in all five breast carcinoma cell lines (mean, 2.23 X 10(-2)/cell/generation) than in the normal breast epithelial cells (mean, 0.36 X 10(-2)/cell/generation). In addition, a considerable quantitative variation in expression of this surface antigen was demonstrated among the cells of each population in both normal and neoplastic breast cells which spread over an 8- to 10-fold range. Furthermore, the quantitative distribution among single cells was not random, for the cells tended to cluster around values that fit a geometric series.

Junctional intercellular communication pattern of cultured human breast cancer cells.

Junctional intercellular communication between several established human breast cancer cell lines and a variety of mammalian cells has been examined. All the cancer cell lines were found to be either noncommunicators or nonselective communicators. This contrasts with normal human mammary epithelium which shows selectivity in junctional communication. Loss of selectivity in junctional communication appears to be a general feature of cultured human breast cancer cells.

Cloning and sequencing of a complementary DNA encoding a Mr 70,000 human breast epithelial mucin-associated antigen.

The human milk fat globule (HMFG) membrane contains several glycoproteins that have been referred to as breast differentiation antigens and that are expressed in normal breast, breast tumors, breast tumor-derived cell lines, and are found in breast cancer patient serum. These antigens include a high molecular weight mucin and several smaller components including Mr 150,000; 70,000; and 46,000 glycoproteins. We have used 2 monoclonal antibodies (McR2 and Mc13) that bind the Mr 70,000 component of HMFG to immunoscreen a lambda gt11 expression library prepared from human lactating breast tissue. We report here the sequence of a complementary DNA clone (BA70-1) that codes for a peptide that binds both McR2 and Mc13 but not monoclonal antibodies to the breast mucin or other components of HMFG.A 1.8-kilobase RNA was detected in 9 of 9 breast tumor cell lines using 32P-labeled BA70-1 as probe. The BA70-1 RNA was highly expressed in 6 of 9 cells lines of breast and several other carcinomas lines compared with a lymphoblastoid cell line (Raji). The BA70-1 complementary DNA sequence has no extensive homology with previously reported sequences including the high-molecular weight mucin complementary DNA. Since the Mr 70,000 molecule appears to be associated with the breast mucin by disulfide bonds, its study could help elucidate the structure of this latter complex and how it is organized in the cell membrane, and prove useful in diagnosis and therapy of breast cancer.

Selection of tumor-specific epitopes on target antigens for radioimmunotherapy of breast cancer.

Evidence is presented for two different breast epithelial antigens that some epitopes have greater tumor specificity and are more effective targets for radioimmunotherapy than others. The two antigens, which are major components of the human milk fat globule membrane, are breast mucin and a M(r) 46,000 glycoprotein (BA46). Of five monoclonal antibodies (Mc5, Mc1, BrE-1, BrE-2, and BrE-3) against breast mucin, all recognize overlapping amino acid epitopes on the tandem repeat domain. However, each have unique and different tissue and tumor specificities and unique epitope structures on the fully glycosylated breast mucin. In preclinical studies, radioimmunoconjugates of all five monoclonal antibodies inhibit growth of transplantable breast tumors in immunodeficient mice. In human clinical trials, radioiodinated Mc5 was very poor in localizing breast tumor metastases. On the other hand, 111In-labeled BrE-3 imaged almost 90% of breast tumors and showed promise in radioimmunotherapy when labeled with 90Y. The failure of Mc5 in clinical trials may be partly attributed to the high levels of its epitope on circulating mucin compared to the epitope of BrE-3. The Mc5 binding affinity increased significantly with glycosylation, while the BrE-3 epitope was masked by glycosylation. The BA46 glycoprotein is a breast tumor-associated membrane antigen containing an NH2-terminal, epidermal growth factor-like domain into which a cell adhesion sequence (RGD) is inserted and a COOH-terminal domain with homology to the phospholipid binding C1/C2 domain of coagulation factors V and VIII. It promotes cell attachment in an RGD-dependent manner. Monoclonal antibody Mc8, which binds to the C2-like domain, is only moderately effective in experimental radioimmunotherapy, while Mc3, which binds an epitope in the EGF-like RGD domain, was highly effective in destroying breast tumors in nude mice. With 90Y-labeled Mc3, 6 of 7 mice are cured of the tumors. These results indicate that by selecting appropriate monoclonal antibodies, a normal antigen can be used as a target for radioimmunotherapy.

Biological activity of two humanized antibodies against two different breast cancer antigens and comparison to their original murine forms.

We have humanized two monoclonal antibodies (MoAbs), hu-BrE-3 and hu-Mc3, that are bound to two different antigens of the breast epithelial cell. They bind to the breast epithelial mucin (M(r) 400,000) and the BA46 antigen (M(r) 46,000). They could participate in a joint radioimmunotherapy strategy administering repeated or fractionated dosages, where increased irradiation could be delivered by their simultaneous administration. Both antibodies, hu-BrE-3 and hu-Mc3, had similar reactivity to their antigens and similar binding affinity as those of their original murine forms. However, because humanized MoAbs could have different pharmacokinetic and radioimmunotherapeutic characteristics than their original murine forms, the experimental biodistribution in vivo of both of these two humanized anti-breast tumor MoAbs was compared to their original murine forms. Biodistributions in immunodeficient mice grafted with transplantable human breast tumors, both after radioiodination and 111In labeling via 1-p-isothiocyanatobenzyl-methyl-diethylene-triaminepenta-ace tic acid (MXDTPA), demonstrated comparable tumor:normal tissue ratios for the humanized and murine forms. In radioimmunotherapy, the humanized forms for both MoAbs showed also similar tumoricidal activity as that of the original murine MoAbs. These results show that the new humanized forms are amenable to conjugation and radioisotope labeling without loss of biological activity. Furthermore, they demonstrate that these engineered molecules kept intact, both qualitatively and quantitatively, their binding ability, pharmacokinetics, and radioimmunotherapeutic characteristics after the humanization process.

Designing human consensus antibodies with minimal positional templates.

A humanized antibody retains from the original murine antibody the variable region amino acid residues that are required for antigen binding. These generally include the grafted complementarity determining regions, as well as a few key framework residues. Although the remainder of the framework sequences are imported from a human antibody, they nevertheless differ at a few positions from the human consensus sequences. These atypical residues, which arose by somatic mutation during the affinity maturation of the chosen human antibody, could elicit an immune response in some of the patients receiving the humanized antibody. Thus, ideally one should, instead, choose human consensus frameworks for humanizing murine antibodies. Because there is a different consensus sequence for each of the subclasses of variable light and heavy chains, a method is needed to choose the most appropriate one. We are developing a minimal positional template for such a purpose. A minimal positional template indicates which positions in the variable region frameworks are absolutely required for maintaining the integrity of the binding domains. Therefore, to choose a human framework for humanization, one screens the available human consensus sequences for those that are most similar to the original murine sequence, specifically at the positions indicated by the template. In the subsequent humanization protocol, one then retains all of the murine residues found in the positions indicated in the template while humanizing the residues found at all other positions. A conservative positional template has been applied to the humanizations of the antibreast epithelial mucin antibodies BrE-3 and KC4-G3 without loss of binding affinity. Now we are using progressive cycles of computer modeling and laboratory testing to develop a minimal template. The first of such cycles produced template B, which has been used successfully in the humanization of the antibreast epithelial antigen BA46 antibody Mc3. This prompted us to design template C, which further approaches the desired minimal template. Future constructs will test the validity of this template as well as the validity of this novel humanization approach.