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Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation.

ABSTRACT: A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine h?NGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of h?NGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.

SUBMITTER: Bowers PM

PROVIDER: S-EPMC3597809 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation.

Bowers Peter M PM Neben Tamlyn Y TY Tomlinson Geoffery L GL Dalton Jennifer L JL Altobell Larry L Zhang Xue X Macomber John L JL Wu Betty F BF Toobian Rachelle M RM McConnell Audrey D AD Verdino Petra P Chau Betty B Horlick Robert A RA King David J DJ

The Journal of biological chemistry 20130125 11

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with ...[more]

PMID: 23355464

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Project description:BackgroundGraft failure resulting from rejection or any other adverse event usually originates from an aberrant and/or exaggerated immune response and is often catastrophic in renal transplantation. So, it is essential to monitor patients' immune status for detecting a rejection/graft failure early on.MethodsWe monitored the sequence change of complementary determining region 3 (CDR3) in B-cell receptor (BCR) immunoglobulin heavy-chain (IGH) immune repertoire (iR) in 14 renal transplant patients using next-generation sequencing (NGS), correlating its diversity to various clinical events occurring after transplantation. BCR-IGH-CDR3 in peripheral blood mononuclear cells was sequenced along the post-transplantation course by NGS using the iRweb server.ResultsDatasets covering VDJ regions of BCR-IGH-CDR3 indicated clonal diversity (D50) variations along the post-transplant course. Furthermore, principal component analysis showed the clustering of these sequence variations. A total of 544 shared sequences were identified before transplantation. D50 remained low in three patients receiving rituximab. Among them, one's D50 resumed after 3 m, indicating graft tolerance. The D50 rapidly increased after grafting and decreased thereafter in four patients without rejection, decreased in two patients with T-cell-mediated rejection (TCMR) and exhibited a sharp down-sliding after 3 m in two patients receiving donations after cardiac death (DCD). In another two patients with TCMR, D50 was low just before individual episodes, but either became persistently low or returned to a plateau, depending on the failure or success of the immunosuppressive treatments. Shared CDR3 clonal expansions correlated to D50 changes. Agglomerative hierarchical clustering showed a commonly shared CDR3 sequence and at least two different clusters in five patients.ConclusionsClonal diversity in BCR-IGH-CDR3 varied depending on clinical courses of 14 renal transplant patients, including B-cell suppression therapy, TCMR, DCD, and graft tolerance. Adverse events on renal graft failure might lead to different clustering of BCR iR. However, these preliminary data need further verification in further studies for the possible applications of iR changes as genetic expression biomarkers or laboratory parameters to detect renal graft failure/rejection earlier.

Dataset Information

Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation.

Publications

Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation.

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