John E. Shively

Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers

BACKGROUND Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo. OBJECTIVES To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model. STUDY DESIGN ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts. RESULTS 125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h. CONCLUSION Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.

A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36)

A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supernatant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two peptides were consistent with the structures: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(1-36)] and--IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(1-36) differs from porcine PYY only at position 3, with Ile instead of Ala, and position 18, with Asn instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(1-36).

CEACAM1-4S, a cell–cell adhesion molecule, mediates apoptosis and reverts mammary carcinoma cells to a normal morphogenic phenotype in a 3D culture

In a 3D model of breast morphogenesis, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) plays an essential role in lumen formation in a subline of the nonmalignant human breast cell line (MCF10A). We show that mammary carcinoma cells (MCF7), which do not express CEACAM1 or form lumena when grown in Matrigel, are restored to a normal morphogenic program when transfected with CEACAM1-4S, the short cytoplasmic isoform of CEACAM1 that predominates in breast epithelia. During the time course of lumen formation, CEACAM1-4S was found initially between the cells, and in mature acini, it was found exclusively in an apical location, identical to its expression pattern in normal breast. Lumena were formed by apoptosis as opposed to necrosis of the central cells within the alveolar structures, and apoptotic cells within the lumena expressed CEACAM1-4S. Dying cells exhibited classical hallmarks of apoptosis, including nuclear condensation, membrane blebbing, caspase activation, and DNA laddering. Apoptosis was mediated by Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm, and was partially inhibited by culturing cells with caspase inhibitors. The dynamic changes in CEACAM1 expression during morphogenesis, together with studies implicating extracellular matrix and integrin signaling, suggest that a morphogenic program integrates cell–cell and cell–extracellular matrix signaling to produce the lumena in mammary glands. This report reveals a function of CEACAM1-4S relevant to cellular physiology that distinguishes it from its related long cytoplasmic domain isoform.

Microsequence analysis of peptides and proteins. II. Separation of amino acid phenylthiohydantoin derivatives by high-performance liquid chromatography on octadecylsilane supports.

A comparison of the separation of the common phenylthiohydantoin derivatives of amino acids on DuPont octadecylsilane with that obtained with Ultrasphere octadecylsilane supports is given together with the effect of acetate, phosphate, and trifluoroacetate buffers in the elution solvents. An important change in performance for two different batches of DuPont Zorbax octadecylsilane was noted. The use of combined trifluoroacetate/acetate buffer with Ultrasphere octadecylsilane gives optimal separations and peak sharpness. Practical examples of the performance of this system in low-nanomole NH2-terminal sequence analysis are discussed with emphasis on identification of unusual amino acid derivatives and interfering background peaks.

Partial structure of a large canine cholecystokinin (CCK58): Amino acid sequence

A cholecystokinin molecule larger than any previously chemically characterized was purified from canine proximal small intestine mucosa. The purification procedure consisted of sequential steps of affinity chromatography, gel filtration, and high pressure liquid chromatography. Activity was detected and quantitated by radioimmunoassay with an antibody that recognized the carboxyl terminal sequence of porcine cholecystokinin. Microsequencing of the purified peptide revealed an amino terminal nonadecapeptide sequence (AQKVNSGEPRAHLGALLAR) not present in known cholecystokinin molecules followed by a nonadecapeptide sequence (YIQQARKAPSGRMSVIKNL) that corresponds exactly to the amino terminal sequence of porcine cholecystokinin 39 except for reversed positions of a Met and a Val residue. Based on the sequence analysis, immunoreactivity, and presence of biological activity in two bioassay systems, this peptide, tentatively named cholecystokinin 58, may be a biosynthetic precursor of the smaller forms previously characterized in gastrointestinal and brain tissues.

Microsequence analysis of peptides and proteins. I. Preparation of samples by reverse-phase liquid chromatography.

Abstract Reverse-phase supports for the separation of peptides and proteins are compared in two high-performance liquid chromatographic systems. One uses a trifluoroacetic acid-acetonitrile solvent system with a 206-nm detector, and the other uses pyridine-formate or pyridine-acetate and 1-propanol with a postcolumn fluorescence detector. Each system was examined with RP8, RP18, and alkylphenyl supports. In most applications, the trifluoroacetic acid-acetonitrile solvent system used in conjunction with an alkylphenyl column performed best. The use of this system for the preparation of low-microgram amounts of samples for microsequence analysis is illustrated.

Radioiodinated versus Radiometal-Labeled Anti–Carcinoembryonic Antigen Single-Chain Fv-Fc Antibody Fragments: Optimal Pharmacokinetics for Therapy

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti-carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2beta, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2beta, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy.

The Role of Peroxiredoxin II in Radiation-Resistant MCF-7 Breast Cancer Cells

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.

Purification and molecular characterization of human fibroblast interferon.

Abstract Human fibroblast interferon was purified from serum-containing culture medium by a combination of concanavalin A or Blue Dextran Sepharose affinity chromatography with high-performance liquid chromatography to material exhibiting a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interferon could be chromatographed and purified at acidic pH in volatile buffers on RP-8, RP-18, cyclohexyl, phenylalkyl, diphenyl, cyanopropyl, and diol supports. A specific activity averaging around 4 × 108 units/mg was found for the pure material with a molecular weight of 20,000–21,000 after 20,000- to 50,000-fold purifications. In some preparations, low activity levels were also found at positions corresponding to 10,000, 17,000–18,000, 35,000, and 40,000 daltons. Amino acid and amino sugar analysis, partial NH2- and COOH-terminal sequences, and tryptic peptide patterns determined at the picomole level are reported for the purified interferon.

Pilot Trial Evaluating an 123I-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (cT84.66 Minibody) in Patients with Colorectal Cancer

Purpose: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (VL-linker-VH-CH3; 80 kDa) that demonstrates bivalent and high affinity (4 × 1010 m−1) binding to carcinoembryonic antigen (CEA). The variable regions (VL and VH) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the CH3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab′)2 fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether 123I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer. Experimental Design: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5–10 mCi (1 mg) of 123I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48–72 h. Surgery was performed immediately after the last nuclear scan. Results: Tumor imaging was observed with 123I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. 123I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9–65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed. Conclusion: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as 111In.