Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Generation of T cell clones binding F(ab′)2 fragments of the idiotypic immunoglobulin in patients with monoclonal gammopathy

Summary Lymphocytes from two patients with multiple myeloma stage I and one patient with monoclonal gammopathy of undetermined significance were found to proliferate specifically in response to low concentrations of F(ab)₂ fragments of the autologous M component. T cell clones isolated from repeatedly stimulated cultures bound specifically the autologous idiotype and proliferated after addition of soluble idiotype and exogenous interleukin-2. The majority of clones were CD8⁺ and showed negligible staining for CD4. Idiotype-binding clones could not be isolated from cultures of lymphocytes from a healthy control stimulated under the same conditions. The study provides support for the existence of idiotype-reactive T cells in monoclonal gammopathies. Such cells might have a regulatory role on the tumour cell clone and may be important for a future therapeutic approach.     

Differences in biodistribution of the anti-(carcinoembryonic antigen) murine monoclonal antibody CE-25, its F(ab′)2 fragment and its intact mainly human chimeric form CE 4-8-13

The effect of the size of the tumour and the amount of antibody injected on the biodistribution of a family of radioiodinated antibodies was studied. The intact mouse anti-(carcinoembryonic antigen) (anti-CEA) monoclonal antibody CE-25, its F(ab)₂ fragment and the intact human-mouse chimeric from CE 4-8-13 were evaluated in a model system using the human CEA-producing colon xenograft T 380 grown in nude mice. The relative retention (the percentage of the injected dose per gram of tissue), of mouse mAb and F(ab)₂ in tumour and most normal tissues 1 day after injection was independent of the antibody dose; after 4 days the mAb values increased with increasing antibody dose. The relative retention of chimeric mAb increased with increasing antibody dose 1 day after injection and also slightly after 4 days. The relative retention in tumour tissue was lower in bigger xenografts for all antibodies. The relative retention of mouse mAb in small tumours increased from day 1 to day 4; for chimeric mAb this value decreased. In normal tissues the relative retention of mouse mAb decreased from day 1 to day 4, but the relative retention of chimeric mAb in normal tissue dropped rapidly and changed little afterwards. Thus the biokinetics of antibodies is species-dependent: foreign, mainly human, chimeric antibody clears faster from normal mouse tissue than mouse antibody and reaches lower concentrations.     

Tumor targeting in a murine tumor xenograft model with the (sFv′)2 divalent form of anti-c-erbB-2 single-chain Fv

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific crosslinking of this sFv′ to make divalent (sFv′)₂. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv′ and (sFv′)₂ derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv′)₂ demonstrated by its complex formation with ECD. The tumor binding properties of¹²⁵I-labeled anti-c-erbB-2 741F8 sFv, sFv′, and (sFv′)₂ were compared with radiolabeled antidigoxin 26-10 sFv′ and (sFv′)₂ controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which overexpress c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv′, and (sFv′)₂ had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv′, and (sFv′)₂, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv′)₂ having Gly₄Cys tails than for monovalent 741F8 sFv′ or Fab. The superior properties of the 741F8 (sFv′)₂ in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.     

Improved immunoscintigraphy by subcutaneous injection of 99mTc or 111In labelled F(ab′)2 fragments of an anti-melanoma monoclonal antibody

Technetium-99m and/or ¹¹¹In labelled F(ab′)₂ fragments of a melanoma associated MoAb 225.28S were injected i.v. in 80 patients affected by stage I to IV malignant melanoma. Seventy five percent of metastatic lesions already documented by other methods were detected by immunoscintigraphy, which was also capable of detecting a certain number of unknown metastases. However, we observed a lower percentage of positive scans in liver, lung and skin because of the poor tumour to background ratio. In some patients, subcutaneous (s.c.) injection allowed us to visualize documented metastases undetected by i.v. administration. An equal amount of non-specific F(ab′)₂ fragments (MoAb 4C4) injected s.c. as a negative control showed no positive scans. Clinical studies and Chromatographic patterns of patient serum samples suggest that the s.c. route of administration offers, with respect to the i.v. route, the advantage of reducing vascular background and aspecific accumulation in liver, probably because of retention of possible contaminants by the lymphatic system.     

Rapid and potent induction of cell death and loss of NK cell cytotoxicity against oral tumors by F(ab′)2 fragment of anti-CD16 antibody

Freshly isolated untreated NK cells undergo rapid apoptosis and lose their cytotoxic function upon the addition of F(ab′)₂ fragment of anti-CD16 antibodies. Loss of NK cell cytotoxic function after treatment with F(ab′)₂ fragment of anti-CD16 antibody can be seen against K562 and UCLA-2 oral tumor cells when either added immediately in the co-cultures of NK cells with the tumor cells or after pre-treatment of NK cells with the antibody before their addition to the tumor cells. Addition of Interleukin-2 (IL-2) in combination with anti-CD16 antibody to NK cells delayed the induction of DNA fragmentation in NK cells, and even though decreased cytotoxicity could still be observed against K562 and UCLA-2 oral tumors when compared to IL-2 alone treated NK cells, the cytotoxicity levels remained relatively higher and approached those obtained by untreated NK cells in the absence of antibody treatment. No increases in IFN-γ, Granzymes A and B, Perforin and TRAIL genes could be seen in NK cells treated with anti-CD16 antibody. Neither secretion of IFN-γ nor increased expression of CD69 activation antigen could be observed after the treatment of NK cells with anti-CD16 antibody. Furthermore, IL-2 mediated increase in CD69 surface antigens was down-modulated by anti-CD16 antibody. Finally, the addition of anti-CD16 antibody to co-cultures of NK cells with tumor target cells was not inhibitory for the secretion of VEGF by oral tumor cells, unlike those co-cultured with untreated or IL-2 treated NK cells. Thus, binding and triggering of CD16 receptor on NK cells may enhance oral tumor survival and growth by decreased ability of NK cells to suppress VEGF secretion or induce tumor cell death during the interaction of NK cells with oral tumor cells. This work was supported by RO1-DE18830 from NIH.     

Immunoscintigraphic localization of renal tumours in an extracorporeal perfusion model with a monoclonal antibody against γ-glutamyltransferase

Summary Monoclonal antibody 138H11 against human -glutamyltransferase has been shown to react immunohistochemically with 98% of all tested clear-cell type and chromophilic renal cell carcinomas, but not with renal chromophobic carcinomas, Duct-Bellini carcinomas or oncocytomas. In normal kidney the target epitopes of mAb 138H11 are located in the luminal brush-border membrane of proximal tubule cells, whereas in renal carcinomas the epitopes are found surrounding the whole tumour cells. These results form the basis of the present immunoscintigraphic study designed to evaluate mAb 138H11 in an extracorporeal perfusion model. Immediately after nephrectomy, human tumour-bearing kidneys were perfused with^99mTc-labelled mAb 138H11 in Euro-Collins solution. High specific uptake in 4/4 renal clear cell carcinomas could be demonstrated by planar immunoscintigraphy and single-photon-emission computed tomography, regions of interest investigation and immunohistochemistry. In contrast, a perfused oncocytoma showed up as an unlabelled lesion. The results indicate a possible use for mAb 138H11 in immunoscintigraphy or even therapy, provided high tumour uptake can be confirmed in patients.     

Anticooperativity in diffusion-controlled reactions with pairs of anisotropic domains: a model for the antigen–antibody encounter

The encounter between anisotropic agents in diffusion-controlled reactions is a topic of very general relevance in chemistry and biology. Here we introduce a simplified model of encounter of an isotropic molecule with a pair of partially reacting agents and apply it to the encounter reaction between an antibody and its antigen. We reduce the problem to the solution of dual series relations, which can be solved iteratively, yielding the exact solution for the encounter rate constant at any desired order of accuracy. We quantify the encounter effectiveness by means of a simple indicator and show that the two binding centers systematically behave in an anti-cooperative fashion. However, we demonstrate that a reduction of the binding active sites allows the composite molecule to recover binding effectiveness, in spite of the overall reduction of the rate constant. In addition, we provide a simple formula that enables one to calculate the anti-cooperativity as a function of the size of the binding site for any values of the separation between the two active lobes and of the antigen size. Finally, some biological implications of our results are discussed.     

CD20F(ab′)2抗体的发酵与纯化

转化了质粒PYZcpp3的 16C9大肠杆菌可高水平地分泌表达可溶性CD2 0F(ab′) 2 抗体 ;采用经优化的培养条件 ,在发酵罐上进行高密度培养OD550 值达 14 0 ;每升湿菌菌重 2 0 0g ;抗体的产量每升为 2 4 1mg ,其中F(ab′) 2 片段达 5 0 % ,F(ab′) 2 可以特异性的识别CD2 0 细胞并与CD2 0相结合 ;F(ab′) 2 对Raji细胞的IC50 值为 2 2 8μg mL ;而Fab′对Raji细胞的IC50 值为 4 5 9μg mL。     

Localization of the antigen for the monoclonal antibody ②9F11-B-E4 interspecific cross-reaction in the freshwater triclads

The localization of the antigen for monoclonal antibody 9F11-B-E₄ was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed.     

Reversible interference of Fe³(+) with monoclonal antibody analysis in cation exchange columns

The effect of Fe3(+) treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe3(+)-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance.     

Radioimmunolocalization of pelvic recurrences from rectosigmoid cancer employing 111In anti-CEA F(ab′)2

Thirty-two immunoscintigraphic studies have been performed in 26 patients previously operated upon for rectosigmoid cancer, for an early detection of pelvic recurrence, employing ¹¹¹In anti-CEA, F(ab′)₂. The sensitivity was 1, specificity 0.76 and accuracy 0.84, leading to a positive predictive value of 0.69 and a negative value of 1. The combined use of a non-imaging surgical probe in 6 of the 10 patients who underwent second look surgery, detecting the ¹¹¹In radioactivity used for RIS, improved diagnostic accuracy, ruling out the presence of tumors in 3 patients with false positive RIS.     

Immunolocalization of ecto-5′-nucleotidase in the kidney by a monoclonal antibody

Summary A monoclonal antibody IgG, has been raised against ecto-5-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 m thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative.The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.     

Flow cytometric analysis of sialyl Lewis A antigen on human cancer cells by using F(ab')2μ fragments prepared from a mouse IgM monoclonal antibody

F(ab')2 fragments, herein designated as F(ab')2μ fragments, were prepared from a mouse IgM monoclonal antibody specific to sialyl Lewis A antigen. The fragments were applied to flow cytometry to analyze the antigen on human cancer cells. The binding of the fragments to the antigen-positive cells was stronger than that of the original IgM. The non-specific binding of the IgM antibody to the antigen-negative cells was much decreased by using the F(ab')2μ fragments. These results indicate that the F(ab')2μ fragments are more suitable than the original IgM monoclonal antibody in flow cytometric analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Abnormal expression of NRF-2α in hepatocellular carcinoma identified with a newly prepared monoclonal antibody against human NRF-2α protein

Human nuclear respiratory factor 2 alpha subunit (NRF-2α) is fundamentally important to cell function and the development. We aimed to establish the monoclonal antibody (MAb) against the human NRF-2α protein and to investigate its distribution in human hepatocellular carcinoma (HCC) and tumor-adjacent tissues. The 6× His-NRF-2α fusion protein was successfully induced and purified. One monoclonal antibody (MAb) against human NRF-2α, 1-D10-E1-B11-G3 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. Using immunohistochemical analysis, the expression of NRF-2α was investigated in 38 cases of HCC specimens and 14 cases of tumor-adjacent specimens. Staining was found positive in 9 cases of HCC tissues (23.7%) and 8 cases of normal hepatic tissues (57.1%). The higher-grade frequency of expression of NRF-2α in tumor-adjacent tissues was significantly higher (P < 0.01) than that in tumor tissues, suggesting that NRF-2α may play important roles in carcinogenesis of HCC.     

Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

α₁-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α₁-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α₁-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α₁-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α₁-antitrypsin augmentation therapy contains latent α₁-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10⁻¹⁴). We conclude that latent α₁-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α₁-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α₁-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.     

Quantitative detection of 4-hydroxyequilenin–DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody

Estrogen–DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN–DNA adducts. Although the formation of 4-OHEN–DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN–DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN–dA adducts and of 4-OHEN–dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose–response between known amounts of 4-OHEN–DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10⁸ bases in 1 µg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN–DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN–DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN–DNA adducts in mammalian cells.     

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase α revealed the intranuclear localization of DNA polymerase α in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the α-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase a appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase a by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.