Compositions and methods for generating antibodies
View Patent ↗The compositions and methods of the present invention comprise the efficient and effective presentation of antigens to the appropriate components of the immune system resulting in the production of species-specific antibodies in vitro. In general, these compositions comprise one or more antigenic components together with a colloidal metal, optionally combined with derivatized PEG (polyethylene glycol) or other agents. The invention also comprises methods and compositions for making such colloidal metal compositions.
1. A method for producing species-specific antibodies comprising:
co-culturing B cells and antigen presenting cells (APCs) in the presence of a denatured antigen,
generating activated B cells by adding a stimulation media comprising CD40 ligand (CD40L), or an anti-CD40 antibody and one or more of the following cytokine stimuli: a) interleukin-2 (IL-2) and interleukin-21 (IL-21); b) interleukin-5 (IL-5), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-β); c) interleukin-4 (IL-4), interleukin-13 (IL-13), or both IL-4 and IL-13; or d) Interleukin-6 (IL-6);
detecting the production of a species-specific antibody; and
generating a hybridoma from the activated B-cells, wherein the hybridoma produces the species-specific antibodies.
2. The method of claim 1 , wherein the B cells and APCs are co-cultured at a ratio of 1:5.
3. The method of claim 2 , wherein the denatured antigen is denatured by application of external stress, by application of heat, by the addition of an acid, by the addition of a base, by exposure to a chaotropic agent, by physical stress, or by binding a colloidal metal nanoparticle.
4. The method of claim 3 , wherein the denatured antigen is heat denatured by application of heat.
5. The method of claim 4 , wherein the denatured antigen is bound to a colloidal metal.
6. The method of claim 5 , wherein the colloidal metal is pegylated with a derivatized PEG.
7. The method of claim 6 , wherein the derivatized PEG is a thiol PEG derivative.
8. The method of claim 3 , wherein the denatured antigen is denatured by binding to a colloidal metal.
9. The method of claim 8 , wherein the colloidal metal is pegylated with a derivatized polyethylene glycol (PEG).
10. The method of claim 9 , wherein the derivatized PEG is a thiolated PEG derivative.
11. The method of claim 1 , wherein the APCs are isolated peripheral blood APCs.
12. The method of claim 1 , wherein the antigen is a human self-antigen or a non-human non-self antigen.
13. The method of claim 12 , wherein the human self-antigen is derived from nucleic acids, tumor antigens, foreign blood cells, or the cells of transplanted organs.
14. The method of claim 12 , wherein the non-human non-self antigen is derived from toxins, bacteria, viruses, protozoa, nucleic acids, tumor antigens, foreign blood cells, or the cells of transplanted organs.
15. The method of claim 1 , wherein the antigen is a cytokine.
16. The method of claim 15 , wherein the cytokine is a lymphokine, monokine, chemokine, interleukin.
17. The method of claim 15 , wherein the cytokine is selected from the group consisting of; interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, the interferon class including, interferon alpha, beta and gamma, the B7 class of molecules, Type I interferon, Type II interferon, tumor necrosis factor-alpha, or lymphotoxin.
18. The method of claim 1 , wherein the antigen is tumor necrosis factor-alpha.
19. The method of claim 1 , wherein the antigen is a growth factor.
20. The method of claim 19 , wherein the growth factor is selected from the group consisting of; fibroblast growth factor, kerotinocyte growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, epidermal growth factor, vascular endothelial growth factor, nerve growth factor, transforming growth factor-alpha (TGF-a), transforming growth factor-beta (TGF-b), Schwann-cell derived growth factor, nerve growth factor, platelet-derived growth factor, insulin like growth factor 1, insulin like growth factor 2, and glial growth factor.
21. The method of claim 19 , wherein the growth factor is epidermal growth factor.
22. The method of claim 1 , wherein the antigen is an alpha or beta subunit of an interleukin-2 receptor.
23. The method of claim 1 , wherein the stimulation media comprises 11-2, CD40L, and IL-21.
24. The method of claim 23 , wherein the species-specific antibody is an IgG species-specific antibody.
25. The method of claim 1 , wherein the stimulation media comprises anti-CD40, I1-5, IL-10, and TGF-β.
26. The method of claim 25 , wherein the species-specific antibody is an IgA species-specific antibody.
27. The method of claim 1 , wherein the stimulation media comprises CD40L, and IL4, or IL-13, or both.
28. The method of claim 27 , wherein the species-specific antibody is an IgE species-specific antibody.
29. The method of claim 1 , wherein the species-specific antibody is a human species-specific antibody.