A strategy to produce monoclonal antibodies against gp96 by prime-boost regimen using endogenous protein and E. coli heterologously-expressed fragment

Abstract

Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).

Keywords

monoclonal antibody / priming-boost / gp96 / low immunogenic protein

References

[1] ShiuR. P., PouyssegurJ., PastanI.. Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts [J]. Proc Natl Acad Sci USA, 1977, 74: 3840-3844

[2] GuptaR. S.. Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species [J]. Mol Biol Evol, 1995, 12: 1063-1073

[3] ChavanyC., MimnaughE., MillerP., BittonR., NguyenP., TrepelJ., WhitesellL., SchnurR., MoyerJ., NeckersL.. p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2 [J]. J Biol Chem, 1996, 271: 4974-4977

[4] FerreiraL. R., NorrisK., SmithT., HebertC., SaukJ. J.. Hsp47 and other ER-resident molecular chaperones form heterocomplexes with each other and with collagen type IV chains [J]. Connect Tissue Res, 1996, 33: 265-273

[5] MelnickJ., AvielS., ArgonY.. The endoplasmic reticulum stress protein GRP94, in addition to BiP, associates with unassembled immunoglobulin chains [J]. J Biol Chem, 1992, 267: 21303-21306

[6] MelnickJ., DulJ. L., ArgonY.. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum [J]. Nature, 1994, 370: 373-375

[7] SchaiffW. T., HruskaK. A., MccourtD. W.Jr, GreenM., SchwartzB. D.. HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells [J]. J Exp Med, 1992, 176: 657-666

[8] WassenbergJ. J., ReedR. C., NicchittaC. V.. Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94: II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity [J]. J Biol Chem, 2000, 275: 22806-22814

[9] LeeA. S.. Mammalian stress response: Induction of the glucoseregulated protein family [J]. Curr Opin Cell Biol, 1992, 4: 267-273

[10] SrivastavaP. K., UdonoH., BlachereN. E., LiZ.. Heat shock proteins transfer peptides during antigen processing and CTL priming [J]. Immunogenetics, 1994, 39: 93-98

[11] StrboN., PodackE. R.. Secreted heat shock protein gp96-Ig: An innovative vaccine approach [J]. Am J Reprod Immunol, 2008, 59: 407-416

[12] CsermelyP., SchnaiderT., SotiC., ProhaszkaZ., NardaiG.. The 90-kDa molecular chaperone family: Structure, function, and clinical applications. A comprehensive review [J]. Pharmacol Ther, 1998, 79: 129-168

[13] ArnoldD., FaathS., RammenseeH., SchildH.. Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunization with the heat shock protein gp96 [J]. J Exp Med, 1995, 182: 885-889

[14] SrivastavaP. K., UdonoH.. Heat shock protein-peptide complexes in cancer immunotherapy [J]. Curr Opin Immunol, 1994, 6: 728-732

[15] SchildH., RammenseeH. G.. gp96—The immune system’s Swiss army knife [J]. Nat Immunol, 2000, 1: 100-101

[16] Singh-jasujaH., HilfN., SchererH. U., Arnold-schildD., RammenseeH. G., ToesR. E., SchildH.. The heat shock protein gp96: A receptor-targeted cross-priming carrier and activator of dendritic cells [J]. Cell Stress Chaperones, 2000, 5: 462-470

[17] MengS. D., GaoT., GaoG. F., TienP.. HBV-specific peptide associated with heat-shock protein gp96 [J]. Lancet, 2001, 357: 528-529

[18] MengS. D., SongJ., RaoZ., TienP., GaoG. F.. Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides [J]. J Immunol Methods, 2002, 264: 29-35

[19] SrivastavaP. K., DeleoA. B., OldL. J.. Tumor rejection antigens of chemically induced sarcomas of inbred mice [J]. Proc Natl Acad Sci USA, 1986, 83: 3407-3411

[20] LiH., ZhouM., HanJ., ZhuX., DongT., GaoG. F., TienP.. Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments [J]. J Immunol, 2005, 174: 195-204

[21] WargerT., HilfN., RechtsteinerG., HaselmayerP., CarrickD. M., JonuleitH., von LandenbergP., RammenseeH. G., NicchittaC. V., RadsakM. P., SchildH.. Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses [J]. J Bio Chem, 2006, 281: 22545-22553

[22] LiuS., TobiasR., McclureS., StybaG., ShiQ., JackowskiG.. Removal of endotoxin from recombinant protein preparations [J]. Clin Biochem, 1997, 30: 455-463

[23] FerrariniM., HeltaiS., ZocchiM. R., RugarliC.. Unusual expression and localization of heat-shock proteins in human tumor cells [J]. Int J Cancer, 1992, 51: 613-619

[24] ZhuX. D., LiC. L., LangZ. W., GaoG. F., TienP.. Significant correlation between expression level of HSP gp96 and progression of hepatitis B virus induced diseases [J]. World J Gastroenterol, 2004, 10: 1141-1145