Antibody biodistribution coefficients

Tissue vs. plasma concentration profiles have been generated from a physiologically-based pharmacokinetic model of monoclonal antibody (mAb). Based on the profiles, we hypothesized that a linear relationship between the plasma and tissue concentrations of non-binding mAbs could exist; and that the relationship may be generally constant irrespective of the absolute mAb concentration, time, and animal species being analyzed. The hypothesis was verified for various tissues in mice, rat, monkey, and human using mAb or antibody-drug conjugate tissue distribution data collected from diverse literature. The relationship between the plasma and various tissue concentrations was mathematically characterized using the antibody biodistribution coefficient (ABC). Estimated ABC values suggest that typically the concentration of mAb in lung is 14.9%, heart 10.2%, kidney 13.7%, muscle 3.97%, skin 15.7%, small intestine 5.22%, large intestine 5.03%, spleen 12.8%, liver 12.1%, bone 7.27%, stomach 4.98%, lymph node 8.46%, adipose 4.78%, brain 0.351%, pancreas 6.4%, testes 5.88%, thyroid 67.5% and thymus is 6.62% of the plasma concentration. The validity of using the ABC to predict mAb concentrations in different tissues of mouse, rat, monkey, and human species was evaluated by generating validation data sets, which demonstrated that predicted concentrations were within 2-fold of the observed concentrations. The use of ABC to infer tissue concentrations of mAbs and related molecules provides a valuable tool for investigating preclinical or clinical disposition of these molecules. It can also help eliminate or optimize biodistribution studies, and interpret efficacy or toxicity of the drug in a particular tissue.     

Immunohistochemical analysis of steroid sulfatase in human tissues

Steroid sulfatase (EC 3.1.6.2) is an enzyme that removes the sulfate group from 3β-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production via the fetal adrenal–placental pathway during pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. The objective of this study was to examine the distribution of steroid sulfatase in normal human tissues and in breast cancers using immunohistochemistry, employing a newly developed steroid sulfatase antibody. A rabbit polyclonal antiserum was generated against a peptide representing a conserved region of the steroid sulfatase protein. In Western blotting experiments using human placental microsomes, this antiserum crossreacted with a 65 kDa protein, the reported size of steroid sulfatase. The antiserum also crossreacted with single protein bands in Western blots of microsomes from two human breast cancer cell lines (MDA-MB-231 and MCF-7) and from rat liver; however, there were some size differences in the immunoreactive bands among tissues. The steroid sulfatase antibody was used in immunohistochemical analyses of individual human tissue slides as well as a human tissue microarray. For single tissues, human placenta and liver showed strong positive staining against the steroid sulfatase antibody. ER+/PR+ breast cancers also showed relatively strong levels of steroid sulfatase immunoreactivity. Normal human breast showed moderate levels of steroid sulfatase immunoreactivity, while ER−/PR− breast cancer showed weak immunoreactivity. This confirms previous reports that steroid sulfatase is higher in hormone-dependent breast cancers. For the tissue microarray, most tissues showed some detectable level of steroid sulfatase immunoreactivity, but there were considerable differences among tissues, with skin, liver and lymph nodes having the highest immunoreactivity and brain tissues having the lowest. These data reveal the utility of immunohistochemistry in evaluation of steroid sulfatase activity among tissues. The newly developed antibody should be useful in studies of both humans and rats.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Analysis of brain chromatin subunit composition using different endonucleases]

A comparison of the processes of chromatin digestion in brain and liver nuclei by Ca, Mg-dependent and staphylococcal endonucleases demonstrates a similarity of the subunit composition of chromatin from both tissues and reveals the same type of linked DNA regions. However, a formation of low molecular weight DNP fragments during hydrolysis and the DNA spectra of soluble and insoluble DNP fragments suggest that brain chromatin contains these fragments alongside with the regions, which are specific for this particular tissue, predominate in it and are resistant to staphylococcal and, particularly, to Ca, Mg-dependent endonucleases. This is paralleled with a non-histone protein enrichment of different brain chromatin fractions and an expansion of the electrophoretic monomer band towards the fragment with a greater molecular weight. It may be assumed that brain nucleosomes are characterized by a higher size heterogeneity of linked DNA, part of which are mostly covered by non-histone proteins, and/or are characterized by a greater set variety.     

HSP70-related proteins in bovine skeletal muscle

Constitutive expression of HSP70-related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co-migrated on two-dimensional gels with the stressed-induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two-dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70-related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one-dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.     

Microfluidic preparation of drug-loaded PEGylated liposomes,and the impact of liposome size on tumour retention and penetration

Understanding the effect of liposome size on tendency for accumulation in tumour tissue requires preparation of defined populations of different sized particles. However, controlling the size distributions without changing the lipid composition is difficult, and differences in compositions itself modify distribution behaviour. Here, a commercial microfluidic format as well as traditional methods was used to prepare doxorubicin-loaded liposomes of different size distributions but with the same lipid composition, and drug retention, biodistribution and localization in tumour tissues were evaluated. The small (～50?nm diameter) liposomes prepared by microfluidics and large (～75?nm diameter) liposomes displayed similar drug retention in in vitro release studies, and similar biodistribution patterns in tumour-bearing mice. However, the extent of extravasation was clearly dependent on size of the liposomes, with the small liposomes showing tissue distribution beyond the vascular area compared to the large liposomes. The use of microfluidics to prepare smaller size distribution liposomes compared to sonication methods is demonstrated, and allowed preparation of different size distribution drug carriers from the same lipid composition to enable new understanding of tissue distribution in compositionally consistent materials is demonstrated.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Summary Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.In honour of Prof. P. van Duijn     

Qualitative and quantitative changes in nitrogenous compounds in senescing leaf and bark tissues of the apple

Quantitative and qualitative changes in proteins and ethanol-soluble nitrogen were followed in senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). While senescing leaves lost 46% of their proteins, total bark protein increased 240% during senescence. However, the protein nitrogen gain in bark tissue was about the same as the protein nitrogen loss in leaf tissue per unit fresh weight of tissues. The pattern of bark protein accumulation appears to be gradual from early August to November and sequential from lower to higher molecular weight species of proteins. The final electrophoretic profile of total bark proteins was established at the later stages of senescence. By late November, 89% of the nitrogen in the bark tissue was found in proteins with 11% in the ethanol-soluble fractions. The total protein content of dormant bark tissue was 3.5% per gram dry tissue. Fractionation of the total bark proteins by DEAE-cellulose chromatography indicated that the final upsurge of bark proteins observed in November was associated primarily with one group of proteins (Peak III).     

Detection of disordered regions in globular proteins using 13C‐detected NMR

Characterization of disordered regions in globular proteins constitutes a significant challenge. Here, we report an approach based on ¹³C‐detected nuclear magnetic resonance experiments for the identification and assignment of disordered regions in large proteins. Using this method, we demonstrate that disordered fragments can be accurately identified in two homologs of menin, a globular protein with a molecular weight over 50 kDa. Our work provides an efficient way to characterize disordered fragments in globular proteins for structural biology applications.     

Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions

Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein–protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.     

In situ phage screening. A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.     

Amino Acid Residues 226–240 of τ Which Encompass the First Lys-Ser-Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament-τ

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.     

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Modular cloning and protein expression of long, repetitive resilin-based proteins

Resilin has emerged as a promising new biomaterial possessing attractive properties for tissue engineering applications. To date, proteins with repeating resilin motifs have been expressed with molecular weights less than 30 kDa. This work describes the development of resilin-based proteins (repeating motif derived from Anopheles gambiae) 50 kDa in size. A modular cloning scheme was utilized and features a recursive cloning technique that can seamlessly and precisely tune the number of resilin repeats. Previously-established resilin expression protocols (based on the Studier auto-induction method) were employed to express the proteins in Escherichia coli BL21(DE3)pLysS. Western blot and densitometry results demonstrated that only ~50% of expressed proteins were the desired molecular weight. This finding suggested that either protein truncation or degradation occurred during protein expression. Preventing leaky expression, lowering the culture temperature, and harvesting during exponential phase resulted in up to 94% of the expressed proteins having the desired molecular weight. These expression conditions differ from previously-published resilin expression methods and are recommended when expressing proteins with a larger number of repetitive resilin sequences.     

Comparison of Amino-acid Sequences of Hypothalamic Peptide,Brain-specific Histone and Myelin Basic Protein

SEVERAL studies have described the basic proteins of porcine central nervous tissue. Shome and Saffran¹ have isolated and purified a large peptide (or small protein) of molecular weight about 14,000 from acetone-dried hog hypothalamic powder. Sixteen of the approximately twenty-six tryptic digest fragments of this protein have been sequenced. The protein has no pressor or ACTH-releasing activity. Tomasi-and Kornguth² purified and partially characterized a basic protein from pig brain and, on the basis of fluorescent antibody studies, they concluded that this protein is a brain-specific histone, found in neurone nuclei (or nucleoli) of several animals^3–5.     

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array

To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.