Antigen-Specific Human Monoclonal Antibodies from Transgenic Mice

Abstract

Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.

Human immunology and immunotherapy: main achievements and challenges

“…The generation of fully human mAbs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or P1 vectors. The generation of knockout mice (in which mice lack Ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mAbs 229 , 230 . Other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies (Table 2 ).…”

A Review on The Development, Production Strategies, and Utilization of Monoclonal Antibodies

“…These mice were engineered in a way to prevent the expression of murine Ig chains by silencing the endogenous murine Ig loci by gene targeting techniques. Then, to these animals with the ability to express human antibody genes, the conventional immunization strategy is applied and the mAb technique (activated B cells being fused with mouse myeloma cells, testing/ screening, freezing) is followed (28). Panitumumab (Vectibix, approved in 2006 for the treatment of epidermal growth factorreceptor -EGFR-positive colorectal cancer) was the first fullyhuman therapeutic antibody derived from a transgenic mouse.…”

“…High grade mRNA from human PBMCs (peripheral blood mononuclear cells) is extracted and cDNA is reverse-transcribed. The synthesized cDNA carries the genetic codes for all antibodies specific to various antigens consisting of numerous lymphocyte clones with an approximation of 109 to 1011 clones (28). The VH and VL polymerase chain reaction products, making up the Ig-encoding repository, are bound into a phageamid, a phage display vector (46).…”

“…Using the specific antibody -antigen-binding property, the phage carrying the targeted antibody can be isolated because all surface antibody fragments are functional. This step involves the enrichment by biopanning of the targeted antibody present on the phage surface based on their affinity to bind target antigen (28). For in vitro selection, target antigens are immobilized on a solid surface.…”

Strategies for Generating Diverse Antibody Repertoires Using Transgenic Animals Expressing Human Antibodies

“…The demonstration that large portions of the intact human immunoglobulin loci could be introduced into the mouse genome was a significant achievement and is the subject of a series of excellent reviews ( 1 , 12 – 14 ). These efforts culminated in the world’s first fully human transgenic antibody generation platforms (XenoMouse ® and HuMab-Mouse ® ) and have been followed by a series of related, next-generation animals ( 15 – 20 ). These platforms largely recapitulate critical aspects of the human antibody repertoire including V-, D-, and J-segment usage patterns.…”