Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

• This invention relates to antibodies and to methods of making them.
• Antibodies raised by conventional techniques to a relatively potent antigen such as dinitrophenyl (DNP) will bind to relatively few specific sites on the antigen.
• Kipps et al. (1978) Proc. Nat'l. Acad. Sci. _7_5: 2914-2917 report that they can increase the number of DNP sites recognized and the amount of DNP antibody produced by diminishing suppressor T-cell activity in mice before they immunize the mice with DNP.
• Spleens were removed from the treated animals and spleen cells sensitized i ⁇ vitro with preparations containing normal NPF. This procedure yielded antibodies which recognized and inhibited the neurite outgrowth activity of normal NPF.
• Certain molecules tend not to cause an immunogenic response; such molecules include, for example, functional sites of enzymes and other proteins with highly conserved structures, including hormones, other regulatory molecules, cell surface receptors, certain viral coat proteins, and ubiquitous molecules such as glycosaminoglycans and proteoglycans. It would be extremely useful to produce antibodies which bind such non-immunogenic molecules in order to perform various procedures such as immuno-purifications, immunoassays, or selective immuno-delivery of active molecules.
• the invention features, in general, producing antibodies which are specific for (i.e., undergo a specific binding reaction with) molecules that are not normally immunogenic.
• these molecules are not normally immunogenic or non-immunogenic.
• the method comprises first administering to the mammal a sensitizing agent (either the non-immunogenic molecule or a molecule which has a specific binding site for the non-immunogenic molecule) in order to elicit in the mammal an immune-attenuating response specific for the sensitizing agent.
• specific binding site includes binding or recognition sites of enzyme/substrate pairs, hormone/receptor pairs, or antigen/antibody pairs.
• mice are immunized with the non-immunogenic agent, i.e., glycosaminoglycans or proteoglycans such as heparin and hyaluronate, or a synthetic molecule which is non-immunogenic such poly-fucose.
• the mammals are then immunosuppressed by administering an agent such as cyclophosphamide that is toxic to suppressor T-lymphocytes, and the same sensitizing agent is readministered.
• the antibodies can be anti-idiotypic antibodies, which can be produced when the non-immunogenic molecule is a first member of a specific binding pair, and the sensitizing agent includes the second member of that pair (or an analog of it) .
• a hybridoma is formed from an antibody-producing cell of the mammal, and the hybridoma is cultured to produce the antibody in monoclonal form.
• the invention enables production of antibodies which are able to bind molecules that do not normally elicit an immune response sufficient to make antibody production possible.
• Specific types of such non-immunogenic molecules are highly conserved enzymes, hormones or other regulatory molecules, cell surface receptors, ubiquitous molecules such as glycosaminoglycans and proteoglycans, or certain viral coat proteins.
• Figures 1-3 are flow charts of antibody production processes. Immunization and Cyclophosphamide Treatment
• Fig. 1 is a flow chart of the steps in the process for producing antibodies.
• monoclonal antibodies which recognize two non-immunogenic glycosaminoglycans (GAG) , chondroitin sulfate and heparin, are made by the following procedure.
• GAG glycosaminoglycans
• Six week old female BALB/c mice are immunized intraperitoneally with 1.0 mg of chondroitin sulfate (whale and shark cartilage, Sigma Chemical Co., St. Louis) , and 1.0 mg of porcine heparin (Sigma) attached to positively charged control pore glass (treated with aminopropyltriethoxysilane) , suspended in Freund's complete adjuvant.
• mice After 24 hours, the mice are injected intraperitoneally with 100-150 mg/kg of the immunosuppressant drug, cyclophosphamide (Cytoxan, Mead Johnson) in saline. After an additional 13 days, 0.5 mg of each GAG in saline is injected intravenously.
• the spleens of the sensitized-immunosuppressed-sensitized animals are removed and hybridomas are made by fusing antibody-producing cells to NS1 myeloma cells using standard techniques, such as those described by Koehler and Milstein Nature (London) 256:495 (1975) .
• Hybridomas producing the desired antibodies are screened by solid phase radioimmunoassay and immunohistochemistry.
• Fig. 2 illustrates generally a method for making an anti-idiotypic antibody which recognizes a non-immunogenic enzyme or a non-immunogenic ligand which is part of a binding pair (e.g. a hormone/receptor pair or other specific binding pairs) .
• the method is specifically illustrated by making anti-idiotypic antibodies which recognize bacterial and mammalian hyaluronidase using 1.0 mg hyaluronate attached to the positively charged control pore glass for the first immunization, and using 1.0 mg of hyaluronate in saline in the second immunization.
• the monoclonal antibodies obtained are able to recognize hyaluronidase as evidenced by the fact that hyaluronate inhibits antibody binding to hyaluronidase; the antibodies are able to inhibit the enzymatic activity of hyaluronidase by at least 25%, and the antibodies are able to recognize both mammalian and bacterial hyaluronidases.
• Fig. 3 illustrates the use of poly-fucose (specifically fucoidan, a sulfated poly-fucose from algae) as a sensitizing agent to recover primary antibodies (Ab,) that bind to fucose as well as secondary or idiotypic antibodies (Ab 2 ) that recognize the enzyme fucosidase or fucose-specific plant lectins.
• poly-fucose specifically fucoidan, a sulfated poly-fucose from algae
• anti-idiotypic antibodies which bind to fucose-specific plant lectins and fucosidase are made by the above method using 1.0 mg fucoidan (Sigma) attached to the positively charged control pore glass as a sensitizing agent in the first immunization and 1.0 mg of fucoidan in saline as a sensitizing agent in the second immunization.
• the fucose-binding monoclonal antibodies are obtained by screening hybridomas with polyfucose.
• the monoclonal antibodies (Ab 2 ) that bind to fucosidase and fucose-binding plant lectin are obtained by screening with plant lectins.
• the anti-idiotypic antibodies are able to bind fucose-specific plant lectins as well as mammalian fucosidase. In competitive binding assays, fucose inhibits the anti-idiotypic antibody from binding to lectins.
• a hybridoma cell line has been prepared by the above technique using hyaluronate as the sensitizing agent. The antibodies produced by that cell line recognize hyaluronidase.
• a hybridoma cell line has also been prepared by the above techniques using polyfucose as a sensitizing agent. The antibodies produced by that cell line recognize fucosidase as well as fucose-specific plant lectins. A specimen of each cell line will be deposited with a public depository and
• the method described can be used to raise antibodies to other non-immunogenic molecules, particularly to other glycosoaminoglycans.
• the method can also be used to raise anti-idiotypic antibodies that
• the sensitizing agent can be a specific binding partner (or an analog of a specific binding partner) to the non-immunogenic molecule.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=25219163

Family Applications (1)

Country Status (2)

Families Citing this family (1)

• 1986
  • 1986-12-30 WO PCT/US1986/002820 patent/WO1987004186A1/en unknown
  • 1986-12-30 EP EP19870900611 patent/EP0250569A1/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events