However, although gangliosides are important cell type-specific markers, they are poor immunogens in eliciting humoral or cellular immune responses. A small portion of gangliosides are present in tumor cells and tissues in the form of a lactone thereof. For example, less than 0. Ganglioside lactones are defined as the inner ester between the carboxyl group of the sialic acid and the primary or secondary hydroxyl group of the sugar residues within the same molecule.
While galactoside lactones have been detected and are believed to be naturally occurring plasma membrane components, their quantity is extremely low and thus their natural occurrence has been disputed Nores, G. Despite the question about their natural occurrence, it has been demonstrated that ganglioside lactones are strong immunogens, which can cause a much greater immune response than native gangliosides Nores, G.
Nores et al found that the antibodies produced using ganglioside lactones as immunogens are of the IgG 3 isotype. In the present invention, it has been found that the use of tumor cells and tumor cell membranes followed by a booster with purified ganglioside lactones in the immunization step of producing hybridomas provides new hybridomas that produce monoclonal antibodies directed to a variety of tumor-associated gangliosides, including human cancer-associated fucogangliosides.
Further, while some of the new antibodies show similar binding specificity to known antibodies, their isotypes are different. Therefore their pharmacodynamic activity is also expected to be different. Also many of the new antibodies have unique cross-reactivity. Accordingly, an object of the present invention is to provide an improved method for the production of antibodies to tumor-associated gangliosides, and especially tumor-associated fucogangliosides.
It is also an object of the present invention to provide novel hybridomas that produce monoclonal antibodies to particular tumor-associated antigens. It is further an object of the present invention to provide novel monoclonal antibodies to particular tumor-associated antigens. Another object of the present invention is to provide a passive immunization method for treating tumors containing gangliosides.
Methods of Hybridoma Formation | Arie H. Bartal | Springer
Still another object of the present invention is to provide a method for detecting tumors containing gangliosides. These and other objects of the present invention, which will be apparent from the detailed description of the invention provided hereinafter, have been met by the following embodiments. In one embodiment, the present invention relates to a method of producing monoclonal antibodies that bind to tumor-associated gangliosides, the method comprising:.
The present invention also provides a hybridoma cell line that produces a monoclonal antibody having the following identifying characteristics:. A hybridoma cell line that secretes a monoclonal antibody having the following identifying characteristics:. A hybridoma cell line that produces a monoclonal antibody having the following identifying characteristics:. In a further embodiment, the present invention provides the monoclonal antibodies produced by the above-identified hybridomas.
In an even further embodiment, the present invention provides a passive immunization method for treatment of tumors containing gangliosides comprising administering to a subject: a pharmaceutically effective amount of an antibody produced by a method comprising:. In an even further embodiment, the present invention provides a method for detecting tumors containing gangliosides comprising:.
The ordinate represents the activity of protein A binding to primary and secondary antibody, in counts per minute. The abscissa represents the antigen dilution wherein the first well denoted as 1 contained ng antigen, the second well denoted as 2 contained 50 ng antigen. The ordinate represents the activity of protein A binding to secondary antibody which reacts with primary antibody bound to the antigen. The abscissa represents the antigen dilution as described for FIG.
The open, upside down triangles represent the same antigen, but isolated from meconium also contains disialyl Le a. The closed squares represent sialylparagloboside SPG type 1 chain. The closed circles represent bivalent Le a on lactoisooctaosylceramide iso-Le a structure 8, Table I. According to the present invention there is provided a novel method of producing monoclonal antibodies directed to tumor-associated gangliosides. An important aspect of the method is that the immunization of the host includes a booster step using a lactone of a tumor associated ganglioside.
The particular tumor cells and tumor cell membrane used in steps 1 and 2 , respectively, of the method are not particularly limited and any of those conventionally used in the production of hybridomas that produce antibodies to tumor cell antigens can be used Fukushi et al. Specific examples include colonic cancer cells e.
SW and colo and human lung carcinoma cells e. The membrane fraction used in step 2 is prepared in a conventional manner Fukushi, et al J. A mixture of a tumor cell membrane and at least one purified lactonized tumor-associated ganglioside is used for immunization in step 2. The mixture can be prepared by adding purified lactonized tumor-associated ganglioside to a membrane fraction and incubating at pH 6.
Alternatively the mixture can be prepared by treating the mixture by known methods to lactonize the tumor cell membrane gangliosides and the purified ganglioside. This procedure induces lactonization and enrichment of membrane gangliosides. The lyophilized material is suspended in PBS and injected intravenously, according to well known methods. The particular tumor-associated ganglioside employed in steps 2 and 3 of the present invention is not critical thereto. Examples of such tumor-associated gangliosides include GD 3 found in melanomas Pukel.
USA, : sialyl Le a found in gastrointestinal and pancreatic cancers Magnani. Cancer Res. Lactones of the gangliosides for use in step 3 of the method can be prepared by known methods Nores, G. For example, the lactones can be prepared by dissolving any ganglioside in glacial acetic acid and allowing the solution to stand for at least 48 hours, followed by lyophilization of the acetic acid. Formation of the ganglioside lactones can be monitored by thin layer chromatography, using high performance thin layer chromatography plates obtained from J.
Baker Chemical Co. The above solvent composition is not critical and any well known solvent which can separate gangliosides from the lactones thereof can be employed, for example, as described in Nores, G. Alternatively, and more efficiently, ganglioside lactones can be prepared by dissolving any ganglioside in chloroform:methanolN HCl Two main components and several minor components, the structures of the latter remain to be elucidated, are resolvable in this system.
Lipid Res. The structure of the purified ganglioside lactones can be verified by direct probe fast atom bombardment mass spectrometry as described in Riboni, L. Ganglioside lactones can also be prepared by treatment with carbodiimide Sonnino. The purified lactonized ganglioside is administered to the host intravenously along with a suitable carrier. The particular carrier to be used along with the lactone of the tumor-associated ganglioside is not critical to the present invention.
Examples of such acceptable carriers include acid-treated Salmonella minnesotae, reconstituted viral membranes such as Newcastle's disease virus membranes and any reconstituted cell membranes incubated with octylglucose followed by dialysis. The particular host being immunized for eventual production of hybridomas is not critical to the present invention. Examples of suitable hosts include mice, rabbits, rats and goats. As used herein "immunized cells" refers to the sensitized spleen cells of the immunized host, e.
The particular myeloma cells employed in the present invention are not critical thereto and can be any well known myeloma cell useful for preparing hybridomas of mouse, rat, rabbit, goat and human origin. By way of example, one suitable immunogenic effective amount of the lactone is about 2. Immunizing the animals, e.
Monoclonal antibodies secreted by hybridomas thus isolated according to the method of the present invention can be produced in quantity by growing large batches of hybridoma cell cultures and purifying the antibody from the supernatant or by injecting mice with the hybridoma line to stimulate the production of ascites fluid. Both methods are well known in the art. The hybridomas isolated according to the present invention can be grown in large batches in suspension culture, or more conveniently in a fiberglass container in which cells are packed and grown in high density, wherein antibodies can diffuse into the culture medium.
Methods of producing the monoclonal antibody in quantity according to the present invention are described in Young et al supra. The monoclonal antibodies can be purified by known methods, for example, by affinity separation using protein A or high pressure liquid chromatography on reverse phase alkylated silica gel or with a synthetic polystyrene gel filtration column. According to the above described method, new hybridomas that produce new monoclonal antibodies directed to a variety of tumor-associated gangliosides, including human cancer-associated fucogangliosides can be made.
Hybridoma Development: The Path to Identifying and Isolating a Specific Monoclonal Antibody
These new antibodies have isotypes different from known antibodies showing similar binding specificity and many of the new antibodies have unique cross-reactivity. Further, this method differs over the previous method for immunizing with fucogangliosides in that, unexpectedly monoclonal antibodies having isotypes other than IgG 3 are obtained These include IgM and IgG 1.
Rockville, Md. Monoclonal antibody SNH3 according to the present invention is produced by hybridoma SNH3 and has the following important identifying characteristics:. Monoclonal antibody NKHl according to the present invention is produced by hybridoma NKHl and has the following important identifying characteristics:. Monoclonal antibody NKH2 according to the present invention is produced by hybridoma NKH2 and has the following important identifying characteristics:. Monoclonal antibody NKH2 is unique in its cross-reactivity with Le a -containing structures. Thus the pharmacodynamic activities of monoclonal antibodies NKH3 and NKH4 are expected to be different from the pharmacodynamic activity of monoclonal antibody CA The present invention also provides a passive immunization method for treatment of tumors containing gangliosides.
The method comprises administering to a subject:. A pharmaceutically acceptable diluent can be employed in the immunization method of the present invention. The particular pharmaceutically acceptable diluent employed is not critical thereto. Examples of such diluents include physiological saline, Ringer's solution, vitamin cocktail and amino acid vitamin cocktail. For administration of antibody to produce an anti-tumor effect in vivo, no carrier should be used. Purified antibodies are given intravenously without carrier material.
The pharmaceutically effective amount of the antibodies of the present invention to be administered will vary depending upon the age, weight, sex and species of the animal to be administered. Generally, the pharmaceutically effective amount is about 1. Generally, from 5 to 10 injections of the antibodies are employed but the present invention is not limited thereto.
The particular antibody which will be administered will depend upon the particular ganglioside present in the tumor which is intended to be treated. Information as to the particular ganglioside present in the tumor can be obtained by a serum assay or biopsy assay for the various gangliosides. As used herein, "treatment" means both prevention of tumor formation and treatment of existing tumors. The present invention also provides a method for detecting tumors containing gangliosides comprising:.
In the method for detecting tumors containing gangliosides of the present invention, "test sample" means, for example, tissue biopsies, serum, ascites fluid and spinal fluid. Detection can occur either in vitro or in vivo. In vitro detection can be carried out using any of the well known in vitro immunological assays, such as those described by Young. Further, in vivo detection can be carried out using any of the well known in vivo immunological assays, such as those described in Burchell, J.
Cancer, ; Epenetos, A. Nuclear Med. The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present invention. SNH3 antibody was established after immunization of mice with tumor cells and lactonized sialyl difucosyl Le x as follows.
Next, 10 6 human lung carcinoma PC9 cells were injected intraperitoneally at weeks 3 and 4. Finally, at week 5, lactonized sialyl difucosyl Le x adsorbed on Salmonella minnesotae was injected intravenously.
Lactonization of sialyl difucosyl Le x ganglioside and adsorption on S. Lactonization of sialyl difucosyl Le x was performed in glacial acetic acid Nores, G. Glycoconjugate J. Three days after the lactone injection, spleen cells were harvested and fused with mouse myeloma SP2 or NS1. Clones were screened by positive reactivity with the sialyl difucosyl Le x antigen structure 2, Table I , and specificity of the antibody secreted by established hybridoma SNH3 was examined by TLC immunostaining and solid-phase radioimmunoassay according to known methods. Open circles, sialyl difucosyl Le x structure 2, Table I isolated from tumor.
Closed, upside down triangles, sialyl Le x hexasaccharide ceramide isolated from tumor structure 1, Table I. Solid-phase radioimmunoassay was performed as previously described Kannagi. The ordinate indicates the activity of protein A binding to primary and secondary antibody. The abscissa indicates antigen dilution, i. The data in FIG. SNH3 is non-reactive with other related structures. This reactivity is distinctive from known reactivities of similar antibodies. This antibody is particularly useful as a diagnostic for serum assay of a large variety of human carcinomas.
The material from lyophilization was suspended in PBS and injected intravenously at week 4. The idea of " magic bullets " was first proposed by Paul Ehrlich , who, at the beginning of the 20th century, postulated that, if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. In the s, the B-cell cancer multiple myeloma was known. It was understood that these cancerous B-cells all produce a single type of antibody a paraprotein. This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.
In , Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,  eliminating the reactions that many monoclonal antibodies caused in some patients. In , James P. Allison and Tasuku Honjo received the Nobel Prize in Physiology or Medicine for their discovery of cancer therapy by inhibition of negative immune regulation, using monoclonal antibodies that prevent inhibitory linkages. Polyethylene glycol is used to fuse adjacent plasma membranes,  but the success rate is low, so a selective medium in which only fused cells can grow is used.
This is possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase HGPRT , an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. Exposing cells to aminopterin a folic acid analogue, which inhibits dihydrofolate reductase , DHFR , makes them unable to use the de novo pathway and become fully auxotrophic for nucleic acids , thus requiring supplementation to survive.
The selective culture medium is called HAT medium because it contains hypoxanthine , aminopterin and thymidine. This medium is selective for fused hybridoma cells. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the medium because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal similar to a cancer cell.
This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen with a test such as ELISA or Antigen Microarray Assay or immuno- dot blot.
The most productive and stable clone is then selected for future use. The hybridomas can be grown indefinitely in a suitable cell culture medium.
They can also be injected into mice in the peritoneal cavity , surrounding the gut. There, they produce tumors secreting an antibody-rich fluid called ascites fluid. The medium must be enriched during in vitro selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-media conditioned by macrophages can be used.
Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, ascites is considered unethical. Several monoclonal antibody technologies had been developed recently, such as phage display , single B cell culture,  single cell amplification from various B cell populations      and single plasma cell interrogation technologies. Different from traditional hybridoma technology, the newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by PCR and produce in either bacterial or mammalian systems with recombinant technology.
One of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals in the laboratory. After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted.
Cell culture sample contaminants consist primarily of media components such as growth factors, hormones and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases , nucleases , nucleic acids and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture and thus the contaminants, one or the other method in vivo or in vitro may be preferable.
The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids and clotted material are first removed, typically by centrifugation followed by filtration with a 0. These large particles can cause a phenomenon called membrane fouling in later purification steps.
In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is produced by a low-secreting cell line. The sample is therefore concentrated by ultrafiltration or dialysis. Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These can be separated by ion exchange chromatography.
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Various proteins can also be separated along with the anions based on their isoelectric point pI. In proteins, the isoelectric point pI is defined as the pH at which a protein has no net charge. For example, albumin has a pI of 4. Thus, at a pH between 4. Transferrin, on the other hand, has a pI of 5. A difference in pI of at least 1 is necessary for a good separation.
Transferrin can instead be removed by size exclusion chromatography.
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This method is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times.
However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used.
A low pH can break the bonds to remove the antibody from the column. Gentle elution buffer systems that employ high salt concentrations are available to avoid exposing sensitive antibodies to low pH. To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support.
If the antigen is a peptide , it is commonly synthesized with a terminal cysteine , which allows selective attachment to a carrier protein, such as KLH during development and to support purification. The antibody-containing medium is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support. Product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing.
These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. The generally accepted purification method of process streams for monoclonal antibodies includes capture of the product target with protein A , elution, acidification to inactivate potential mammalian viruses, followed by ion chromatography , first with anion beads and then with cation beads. Displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pharmacokinetic studies.
Recombinant antibody engineering involves antibody production by the use of viruses or yeast , rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. While mouse and human antibodies are structurally similar, the differences between them were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production of human anti-mouse antibodies HAMA.
Recombinant DNA has been explored since the late s to increase residence times. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this " chimeric " or "humanised" DNA through cell culture yielded part-mouse, part-human antibodies.
Is hybridoma production about to take a quantum leap forward?
Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of fully human products to reduce the side effects of humanised or chimeric antibodies. Two successful approaches have been identified: transgenic mice  and phage display. As of November , thirteen of the nineteen fully human monoclonal antibody therapeutics on the market were derived from transgenic mice technology. Phage display can be used to express variable antibody domains on filamentous phage coat proteins Phage major coat protein.
Monoclonal antibodies have been approved to treat cancer , cardiovascular disease , inflammatory diseases , macular degeneration , transplant rejection , multiple sclerosis and viral infection. Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. Proteins can be detected using the Western blot and immuno dot blot tests. In immunohistochemistry , monoclonal antibodies can be used to detect antigens in fixed tissue sections, and similarly, immunofluorescence can be used to detect a substance in either frozen tissue section or live cells.
Antibodies can also be used to purify their target compounds from mixtures, using the method of immunoprecipitation.
Related Methods of Hybridoma Formation
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