Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.     

抗肿瘤免疫毒素稳定性的研究进展

免疫毒素 (immunotoxins)是由某些细菌和植物产生的毒素蛋白与抗体或生长因子等靶向分子连接而成 ,用于杀伤表面带有特定抗原或受体的细胞。在试验中 ,免疫毒素在肿瘤周围环境中的稳定性是影响其抗肿瘤效果的主要因素之一。本文主要对几种由不同形式的抗体Fv段构建的免疫毒素的稳定性进行了比较 ,并对定点突变和PEG化学修饰提高免疫毒素稳定性的方法做一简要介绍。     

Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins

Seven different monoclonal antibodies of the IgG class that are reactive with four different antigens on human lymphoid cells were utilized to form immunotoxins with the ribosome-inactivating proteins gelonin and the three known pokeweed antiviral proteins. Thirteen different immunotoxin combinations were prepared. The ribosome-inactivating proteins were modified with 2-iminothiolane. The sulfhydryl groups so introduced were reacted with maleimido groups or with dithiopyridyl groups that had been introduced into the antibodies. The toxin-antibody conjugates so formed were purified by affinity chromatography on protein A-Sepharose CL-4B, ion exchange chromatography, and by gel filtration and were characterized by polyacrylamide-dodecyl sulfate gel electrophoresis. The purified immunotoxins were free of nonconjugated monomeric proteins and aggregates of very high molecular weight. All the immunotoxins showed the specific binding of the component antibody as measured by indirect immunofluorescence binding assays. The activities of the ribosome-inactivating proteins were unaffected by conjugation where the cross-link to the antibody contained a disulfide bond and when assayed after reductive cleavage of the linker. Disulfide-linked immunotoxins with six of the antibodies were highly cytotoxic for the target cells. However, immunotoxins containing an anti-B1 antibody showed no cytotoxicity.     

The effect of antibody valency and lysosomotropic amines on the synergy between ricin A chain- and ricin B chain-containing immunotoxins

The cytotoxicity of A chain immunotoxins containing IgG or Fab fragments specific for the surface immunoglobulin of the Daudi cell line was assessed in the presence of B chain immunotoxins (IgG or Fab) or lysosomotropic amines, or both. The concentration required for 50% inhibition of protein synthesis (IC50) in Daudi cells was 1.3 X 10(-8) M for IgG-A and 5 X 10(-8) M for Fab-A. The toxicity of both A chain immunotoxins was enhanced twofold by ammonium chloride. In the presence of A chain immunotoxins and ammonium chloride, a maximum of 99 and 90% reduction of clonal precursors was obtained with IgG and Fab-A chain immunotoxins respectively. Immunotoxins containing ricin B chain and IgG or Fab fragments specific for the antibody portion of A chain immunotoxins were used as secondary "piggyback" immunotoxins to treat cells that were pretreated with A chain immunotoxins. Both B chain immunotoxins were nontoxic at 1 X 10(-6) M. When added to target cells pretreated with specific A chain immunotoxins, the IC50 of the A chain immunotoxins was decreased up to 16-fold in the absence of ammonium chloride. In contrast to the results obtained with A chain immunotoxins alone, ammonium chloride significantly increased the toxicity of the complete piggyback system, resulting in the killing of 99.999% or five logs of target cells in the clonal assay. This decreased the IC50 of A chain immunotoxins up to 116-fold when compared with A chain immunotoxin alone. This enhanced toxicity was independent of the valency of either immunotoxin.     

Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Conjugates of the single-chain ribosome-inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000-10,000 molecules/cells to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of immunotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.     

Preclinical evaluation of anti-CD86 immunotoxin in rhesus monkeys: analysis of systemic toxicity,pharmacokinetics, and effect on primary T-cell responses

CD86 and CD80 costimulatory antigens are highly overexpressed on Hodgkin/Reed-Sternberg cells in patients with Hodgkin's disease (HD) and are candidate target antigens for immunotoxins in order to eliminate minimal residual disease. In this study we have evaluated the pharmacokinetics (and immunological) toxicity in rhesus monkeys of immunotoxins consisting of gelonin conjugated to anti-CD86 (alphaCD86-IT). Both alphaCD86-IT and alphaCD80-IT inhibited protein synthesis in B cell lines from rhesus monkeys and inhibited the mixed leukocyte reaction. Reactivity of the alphaCD86 antibody with rhesus monkey CD86 (RhCD86) was shown by cloning and transfecting RhCD86, which conferred reactivity of the alphaCD86 antibody used in this study. alphaCD86-IT was administered as single intravenous bolus injection in four rhesus monkeys, and achieved plasma concentration in 50-fold excess able to eliminate cultured Hodgkin/Reed-Sternberg cells up to 6 h. The animals were capable of generating primary immune responses to both gelonin and murine IgG within 9 days after infusion with alphaCD86-IT. No evidence could be found of significant (immunological) toxicity. The results of this study show that alphaCD86-IT can be applied safely in an effective dose.     

Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.     

抗癌胚抗原免疫毒素生物学功能的研究

为了研究免疫毒素发挥生物学作用的全过程,构建了两种不同形式的基于假单胞菌外毒素(Pseudomonas exotoxin A,PE)的抗癌胚抗原（carcinoembryonic antigen, CEA）免疫毒素CEA(Fv)/PE38/KDEL和PE35/CEA(Fv)/KDEL.用流式细胞术经间接免疫荧光法测定免疫毒素的抗原结合活性.免疫毒素的内化(internalization)通过间接免疫荧光标记的方法在激光共聚焦显微镜下进行检测,采用流式细胞术进行免疫毒素内化的定量分析.细胞凋亡采用FITC-annexin V/PI双荧光染色方法进行分析.用噻唑蓝(MTT)法测定免疫毒素的靶细胞杀伤功能.对两种形式的免疫毒素在各个功能阶段的性质进行研究和比较,全面展现了免疫毒素发挥生物学功能的过程及各个生物作用过程之间的相互影响,为详尽的机制研究和免疫毒素的进一步优化改造提供了重要的依据.     

Effect of Antigen Shedding on Targeted Delivery of Immunotoxins in Solid Tumors from a Mathematical Model

Most cancer-specific antigens used as targets of antibody-drug conjugates and immunotoxins are shed from the cell surface (Zhang & Pastan (2008) Clin. Cancer Res. 14: 7981-7986), although at widely varying rates and by different mechanisms (Dello Sbarba & Rovida (2002) Biol. Chem. 383: 69–83). Why many cancer-specific antigens are shed and how the shedding affects delivery efficiency of antibody-based protein drugs are poorly understood questions at present. Before a detailed numerical study, it was assumed that antigen shedding would reduce the efficacy of antibody-drug conjugates and immunotoxins. However, our previous study using a comprehensive mathematical model showed that antigen shedding can significantly improve the efficacy of the mesothelin-binding immunotoxin, SS1P (anti-mesothelin-Fv-PE38), and suggested that receptor shedding can be a general mechanism for enhancing the effect of inter-cellular signaling molecules. Here, we improved this model and applied it to both SS1P and another recombinant immunotoxin, LMB-2, which targets CD25. We show that the effect of antigen shedding is influenced by a number of factors including the number of antigen molecules on the cell surface and the endocytosis rate. The high shedding rate of mesothelin is beneficial for SS1P, for which the antigen is large in number and endocytosed rapidly. On the other hand, the slow shedding of CD25 is beneficial for LMB-2, for which the antigen is small in number and endocytosed slowly.     

Expression cloning of cDNAs that render cancer cells resistant to Pseudomonas and diphtheria toxin and immunotoxins.

BACKGROUND: Several immunotoxins in which antibodies are coupled to plant or bacterial toxins are now in clinical trials for the treatment of cancer. One of these is B3-LysPE38 in which MAb B3 which reacts with many human cancers, is coupled with a genetically modified form of Pseudomonas exotoxin (PE). MATERIALS AND METHODS: To investigate how cells can become resistant to PE-derived immunotoxins, we constructed an immunotoxin-sensitive MCF-7 breast cancer cell line that contains SV40 T antigen and allows episomal replication of SV40 origin containing plasmids. We transfected a pCDM8/HeLa cDNA expression library into these cells, thereby causing over-expression of the plasmid-encoded genes. The transfected cells were treated with immunotoxin to select for resistance-mediating plasmids, which were reisolated from these cells and amplified in Escherichia coli. The resulting plasmid pool was transfected into cells for two further rounds of selection and plasmid reisolation. RESULTS: Several plasmids that caused immunotoxin resistance were enriched by this selection procedure. Four plasmids were stably transfected into MCF-7 cells and found to increase their resistance to PE-derived immunotoxins by 5- to 20-fold. These plasmids also confer resistance to native PE and to diphtheria toxin but not to ricin or cycloheximide. Thus, they appear to specifically interfere with the action of ADP-ribosylating toxins. CONCLUSION: Cancer cells can become resistant to immunotoxins by deregulated expression of normal genes. The clinical significance of this type of resistance will be evaluated in clinical trials.     

Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ～ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ～ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.     

Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified “HV4” and “LV4” in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.     

Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis

Immunotoxins were prepared by conjugating saporin, a ribosome-inactivating protein from Saponaria officinalis, to a monoclonal antibody against the Thy1.1 antigen, or to its F(ab')2 fragment. The immunotoxins were eight- to 16-fold more toxic to mice than free saporin. Injection of the immunotoxins induced necrosis of the liver and spleen, whereas free saporin caused necrosis of the epithelium of the kidney tubules. The cytoplasm of the hepatic parenchymal cells was affected by the immunotoxins, lesions being apparent in the rough endoplasmic reticulum and, later, in the mitochondria. These changes were associated with a reduced capacity to synthesise proteins both in the intact liver and by isolated liver microsomes. Studies of the in vivo distribution showed that 90% of the free saporin was removed from the bloodstream, mainly by the kidneys, within 10 min of injection. By contrast, the immunotoxins persisted in the blood for several hours and the only organ in which they consistently accumulated was the liver. The hepatotoxic effect of the immunotoxins was not due to their binding to liver cells via the antigen-binding sites or the Fc-piece of the antibody moiety, nor was it due to hepatic recognition of carbohydrate in the immunotoxin. It is concluded that free saporin, although capable of entering liver cells, is filtered so rapidly by the kidney that liver damage does not occur to a significant extent. Filtered saporin, however, is reabsorbed by renal tubules, whose epithelial cells are damaged. The antibody-saporin conjugate is too large to filter at the glomerulus and so has greater opportunity to penetrate into and to damage the hepatic parenchymal cell.     

抗CEA免疫毒素的表达、纯化、复性与生物学活性的研究

以抗癌胚抗原（Carcinoembryonic antigen, CEA）单链抗体与假单胞菌外毒素（Pseudomonas exotoxin A, PEA）的截短和修饰形式PE38/KDEL构建重组免疫毒素CEA/PE38/KDEL,并在大肠杆菌菌株BL21(DE3)-star中表达。采用镍离子螯合层析法纯化变性的包涵体样品,并用连续梯度透析的方法对纯化后的包涵体进行复性。采用流式细胞术鉴定复性产物与靶细胞的结合活性,结果表明免疫毒素CEA/PE38/KDEL具有与靶细胞特异性结合的活性。以MTT法检测免疫毒素对肿瘤细胞的体外杀伤活性,结果表明该免疫毒素对SW1116和CNE_2细胞具有特异性杀伤活性。证明了经包涵体复性的抗CEA免疫毒素CEA/PE38/KDEL对表达CEA抗原的肿瘤细胞具有良好的结合和杀伤活性。     

Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin.

We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.     

Surface marker antigens in the characterization of human embryonic stem cells

The use of cell surface antigens to characterise embryonic stem (ES) cells, and to monitor their differentiation, has had a long history, stretching back to the early studies of differentiation antigens in the haematopoietic system, and their application to teratocarcinomas and embryonal carcinoma (EC) cells in the laboratory mouse. A wide series of such antigens, which include both glycolipids and glycoproteins are now extensively used in studies of human ES cells. Many of these were first identified using both mouse and human EC cells, although the cell surface antigen phenotype of human EC and ES cells has proved to be significantly different from that of murine EC and ES cells.     

Endocytosis and Presentation of Liposome-Associated Antigens by B Cells

B cells have limited endocytic capacity and are reported to endocytose and present liposome-encapsulated antigens poorly. B cells also endocytose soluble antigens poorly, except those for which their surface immunoglobulin is specific, which are taken up and presented efficiently. We present results indicating that, in vitro, B cells endocytose small liposomes bearing antigen with affinity for their surface immunoglobulin. Antigen encapsulated in liposomes targeted by antibody specific for surface immunoglobulin is presented to T cells as efficiently as specific antigen in soluble form. These studies provide a rational basis for the design of liposomes optimized to stimulate T-dependent B-cell responses.     

Immunotoxins to the HER-2 oncogene product: Functional and ultrastructural analysis of their cytotoxic activity

Two immunotoxins were prepared using monoclonal antibodies (mAb) directed towards two distinct epitopes of the gp185^HER-2 extracellular domain, and the type I ribosome inactivating protein (RIP) plant toxin saporin 6. Cell protein synthesis inhibition assay reveals that the immunotoxins display a potent and specific cytotoxicity that is characterized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxins by immunoelectron microscopy. The results of this study have demonstrated that the distribution pattern of the immunotoxins and of the unconjugated mAb over the cell surface overlaps. Similarly the mAb and immunotoxins are internalized into the cell by two different pathways: via clathrin-coated pits or via smaller uncoated pits and vesicles. A higher degree of internalization is achieved when the two immunotoxins are used in combination. Unlike the slow kinetics of cell intoxication the process of immunotoxin endocytosis is characterized by a rapid rate of internalization (above 40% at 5 min in the SK-BR-3 cell line). Although these findings provide no clue to explain the mechanisms of the slow rate of cytotoxicity of the two immunotoxins their rapid internalization indicates that these reagents can be exploited in immunotherapeutic approches to gp185^HER-2-expressing malignancies.     

Chemical construction of immunotoxins

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as “in vitro” and “in vivo” testing of the immunotoxins efficacy.