Antigen-specific human antibodies from mice comprising four distinct genetic modifications

Abstract

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

In VitroCharacterization of the Anti-PD-1 Antibody Nivolumab, BMS-936558, andIn VivoToxicology in Non-Human Primates

“…Transgenic mice comprising germline configuration human immunoglobulin miniloci in an endogenous IgH and Igk knockout background (28,29) were used to generate human anti-PD-1 monoclonal antibodies (mAb). The transgenic mice were immunized with recombinant human PD-1-Fc protein consisting of the extracellular domain of PD-1 (amino acids 1-167) and the Fc portion of human IgG1, and Chinese hamster ovary (CHO) cells expressing human PD-1 (CHO-PD-1 cells).…”

Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti–human DEC205 monoclonal antibody

“…Human Ig Tg mice 15 were immunized with 5-25 g of purified V5.hDEC205/ hIgG1Fc protein using either complete/incomplete Freund or RIBI monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) as adjuvants (Sigma-Aldrich). Mice with anti-hDEC205 titers were administered with fusion-protein intravenously 3-4 days before harvesting spleen cells for fusion with the murine myeloma cell line, P3 ϫ 63Ag8.653.…”

“…Human Ig Tg mice 15 were used to generate human anti-hDEC205 mAbs by standard immunization and hybridoma technology. Both complete/incomplete Freund and RIBI MPL plus TDM adjuvants resulted in good antibody titers to hDEC205.…”

“…14 To extend the concept of protein targeting to DCs to human therapeutics, a series of human immunoglobulin G (IgG) mAbs to human DEC205 (hDEC205) were produced from human Ig transgenic mice that lack mouse Ig genes, but carry the human Ig locus. 15 While these mAbs also react with DEC205 in rhesus macaques, the latter are expensive to explore the many variables that are needed to move this vaccine strategy into proof-of-concept studies in humans. Therefore, we generated transgenic (Tg) mice expressing the hDEC205 receptor on DCs.…”

Transgenèse animale et humanisation des anticorps

“…La première description de souris exprimant un répertoire restreint de gènes d'Ig humains date de 1989 [2]. L'utilisation de longs transgènes humains qui incluaient les séquences codant pour deux à cinq régions variables [capables de réarrangements V(D)J] et plusieurs gènes codant pour les régions constantes (capables de subir la commutation de classe) a ensuite été combinée à l'inactivation des gènes endogènes codant les immunoglobulines de souris [3][4][5]. Ces modèles ont permis d'obtenir des hybridomes producteurs d'anticorps humains spécifiques malgré un répertoire V assez restreint : en effet, une part notable de la diversité des anticorps provient de la diversité jonctionnelle puis de la maturation d'affinité par hypermutation somatique, part plus importante que celle que crée la diversité combinatoire des différents segments germinaux.…”