Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library.

We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Ablation of colorectal xenografts with combined radioimmunotherapy and tumor blood flow-modifying agents.

Radioimmunotherapy (RIT) does not readily eradicate common solid tumors and therefore requires augmentation by complementary therapies that do not increase normal tissue damage. We have examined the efficacy of RIT combined with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a drug which induces immunomodulation and cytokine production and preferentially reduces tumor blood flow, using a colorectal xenograft model in nude mice. Although an optimal i.p. dose (27.5 mg/kg) of drug alone induced massive hemorrhagic necrosis of all but a thin peripheral rim of viable tumor cells, survival was unaffected. However, when combined with i.v. 18.5 MBq 131I-labeled anti-carcinoembryonic antigen IgG, DMXAA significantly potentiated the RIT without increased toxicity, with five of six mice showing complete cures. Scheduling was critical because the antibody must be allowed to reach maximum tumor accumulation before initiation of drug-induced blood flow inhibition. Subsequently, the antibody was retained preferentially in the tumor, reaching approximately twice control levels by 5 days after drug delivery. In combined studies, the drug had a narrow therapeutic window, 30 mg/kg being toxic to two of six mice, whereas 20 mg/kg were ineffective. However, the addition of a second vasoactive agent, serotonin, to RIT plus 20 mg/kg DMXAA enhanced therapy without increasing systemic toxicity. Tumor histology and phosphor image plate analysis reflected these results. When given without RIT, the two drugs combined, although not alone, also significantly inhibited tumor growth. Drug-induced tumor necrosis and tumor retention of radioantibody may both contribute to the enhanced RIT produced by this combined complementary therapy.

Improved radioimmunodetection of tumours using liposome-entrapped antibody.

The discrimination of radioimmunodetection of tumours is reduced by the presence of circulating radiolabelled antibody (primary antibody). We have prepared liposomes containing an antibody to the primary antibody (secondary antibody), with the intention of complexing and delivering to the liver primary antibody which is not associated with the tumour. In mice bearing xenografts of human tumours which secrete the marker carcinoembryonic antigen (CEA), liposomally entrapped secondary antibody was able to reduce the blood levels of 125I-labelled anti-CEA within 2 h, without reducing the amount of anti-CEA bound to the tumour. We therefore suggest that the use of liposomally entrapped secondary antibody would improve the diagnostic potential of radioimmunodetection of tumours and their metastases.

Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: implications for cellular adhesion.

Carcinoembryonic antigen (CEA) is one of the most widely used cell-surface tumour markers for tumour monitoring and for targeting by antibodies. It is heavily glycosylated (50% carbohydrate) and a monomer is constructed from one V-type and six C2-type fold domains of the immunoglobulin superfamily. The solution arrangement at low resolution of the seven domains in CEA cleaved from its membrane anchor was determined by X-ray and neutron scattering. Guinier analyses showed that the X-ray radius of gyration RG of CEA was 8.0 nm. The length of CEA was 27 to 33 nm, and is consistent with an extended arrangement of seven domains. The X-ray cross-sectional radius of gyration RXS was 2.1 nm, and is consistent with extended carbohydrate structures in CEA. The neutron data gave CEA a relative molecular mass of 150,000, in agreement with a value of 152,500 from composition data, and validated the X-ray analyses. The CEA scattering curves were analysed using an automated computer modelling procedure based on the crystal structure of CD2. The V-type and C2-type domains in CD2 were separated, and the C2-type domain was duplicated five times to create a linear seven-domain starting model for CEA. A total of 28 complex-type oligosaccharide chains in extended conformations were added to this model. By fixing the six interdomain orientations to be the same, three-parameter searches of the rotational orientations between the seven domains gave 4056 possible CEA models. The best curve fits from these corresponded to a family of zig-zag models. The long axis of each domain was set at 160(+/-25) degrees relative to its neighbour, and the two perpendicular axes were orientated at 10(+/-30) degrees and -5(+/-35) degrees. Interestingly, the curve fit from this model is within error of that calculated from a CEA model generated directly from the CD2 crystal structure by the superposition of adjacent domains. Zig-zag models of this type imply that the protein face of the GFCC' beta-sheet in neighbouring CEA domains lie on alternate sides of the CEA structure. Such a model has implications for the adhesion interactions between CEA molecules on adjacent cells or for the antibody targeting of CEA.

The use of cellulose carbonate-based immunoadsorbents in the isolation of minor allotypic components of rabbit immunoglobulin populations.

Cellulose trans-2,3-carbonate has been used as a new insoluble matrix for the simple coupling of a1- and b4-positive rabbit immunoglobulin to make immunoadsorbents capable of purifying from serum, with great efficiency, alloantibodies to these allotypic determinants. The antibodies have themselves been conjugated to prepare specific antibody immunoadsorbents of high binding activity for their allotypic target molecules. With these anti-allotypic solid-phase reagents it has been possible to affinity purify a1- and b4-positive immunoglobulin molecules and to deplete serum immunoglobulin of these molecules to leave in the eluates only the allotypically uncontaminated minor immunoglobulin components which are a-negative or b-negative (lambda chain-bearing) molecules. lambda chain molecules were also purified in very small quantities by affinity chromatography on a sheep anti-rabbit lambda chain column. This method of purifying minor populations of rabbit immunoglobulin from normal serum by special immunoadsorbent applications offers new opportunities to study the products of rarely expressed immunoglobulin genes in normal rabbits.

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

Analysis of radioimmunodetection of tumors by the subtraction technique.

The subtraction method for radioimmunodetection (RID) of cancer is showing potential as a diagnostic tool. An objective assessment of this technique is needed in order to determine its clinical usefulness. A numerical analysis of the RID scintigrams was developed and applied to 21 RID studies on 20 patients for whom a final diagnosis had been established. The subtraction technique was shown to give rise to artifactual positive regions, which could be avoided by experienced clinicians. When artifact-producing regions were excluded, 97 regions were examined (80 nontumor, 17 tumor-containing). The "background" scintigrams were shown to be good models for nontumor regions of the antibody scintigrams, and tumor and nontumor regions were shown to form statistically distinct groups.

Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine.

UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.

The history and rationale of hormone replacement therapy.

Robert Wilson rendered women aged over 45 a great service. He provoked a line of research which has in 25 years resulted in a sensible approach towards the use of medication in treating the climacteric and post-menopausal oestrogen deficiency. The history of hormone replacement therapy (HRT) is described, as is the current approach to rational HRT. Some ideas are outlined for future development.

Contraceptive choice in the completed family.

PIP: In Belgium, the contraceptive choices and views on completed family size were studied. The study involved a 2-page questionnaire; the 1st part was self-administered while the 2nd half of the questionnaire allowed for the physician to interview the respondents. The survey studied the demographic characteristics of the respondents and the relationship of these characteristics to contraceptive choice, family size and possible future choice in contraception. Demographic information assayed included age and occupation and religion of the respondent and the number of unplanned children. 235 of the 359 respondents (2/3) considered their family complete. 70% of those surveyed used some form of contraception with oral contraceptives and female sterilization being the most popular currently used methods and oral contraceptives and condoms being the most popular ever used method. Motivations for contraceptive choice were also evaluated (family health, age, socioeconomic conditions, etc). Although a large number of unplanned pregnancies were reported, they were not necessarily unwanted. The failure a contraceptive method may account for this. Over 80% of those surveyed had used oral contraceptives at some time. Although the couples agreed on family size, the reasoning behind their decision was obtuse and not well planned. Sterilization created mixed emotions among both partners. Its popularity stems from the standard recommendation of discontinued use of oral contraceptives after age 35. However, doctors need to be considerate of the ambivalent feelings of their patients and recommend choices that leave options open without presenting health risks.^ieng

Hysterectomy in six European countries.

Gynaecologists have been criticized in recent years because of their attitude towards hysterectomy; it is often stated that they are too ready to consider hysterectomy to be indicated and that hysterectomies are carried out too frequently. In order to obtain insight into the incidence of hysterectomy, 2066 women between 40 and 70 yr of age, composing a sample covering 6 European countries, were asked whether they had undergone this operation. Of the total sample, 11.4% had been hysterectomized, the highest percentage being found in Italy (15.5%) and the lowest in France (8.5%). The most important factor that influenced the relative frequency of hysterectomy was age: the percentage of women who had undergone hysterectomy increased with age up to 55-59 yr, but fell thereafter. Another significant factor was civil status; there were fewer divorcees or widows at the moment of operation than married or never-married women. The relationship between the incidence of hysterectomy and age is a parabolic one. To explain this curve the authors postulate that two trends must have played a rôle: firstly; a greater need for hysterectomy with increasing age and, secondly a decrease in the reluctance of the gynaecological profession to perform a hysterectomy and/or of women to undergo this operation. Statistical analysis confirms the existence of this second trend over the period 1960-1975. The chances of a young woman losing her uterus before her 70th year went up linearly to 19.8% in 1975. By extrapolation a figure of 21% is obtained for 1980. Satisfaction with the result of the operation was lowest in Italy and Germany; satisfaction was also lowest in rural areas.

Vaginal cytology in the postmenopause. A study into some correlates.

A considerable number of women continue to have proliferative vaginal smears indicating a certain level of endogenous estrogen production. Some correlates of this were studied. No significant correlation was found with age since menopause or with overweight. No correlation was found between the proliferation of the vaginal smear and climacteric complaints, circulatory or nervous. The implications of a proliferative smear for the postmenopausal woman and of the postmenopausal estrone production are briefly discussed. They appear at least to be ambiguous, because, although they protect against some facets of the estrogen deficiency syndrome, the factors which are correlated to a high level of endogenous estrone production are known as risk factors, predisposing for a higher incidence of uterine bleedings and perhaps malignant degeneration of some target cell tissues.