Jose Corvalan

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (κ) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 Vκ genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Development of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody, for cancer therapy

Overexpression of epidermal growth factor receptor (EGFr) has been demonstrated on many human tumors, and the increase in receptor expression levels has been linked with a poor clinical prognosis. Blocking the interaction of EGFr and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. To this end, using XenoMouse technology, ABX-EGF, a human IgG2 monoclonal antibody (mAb) specific to human EGFr, has been generated. ABX-EGF binds EGFr with high affinity (5x10(-11) M), blocks the binding of both EGF and transforming growth factor-alpha (TGF-alpha) to various EGFr-expressing human carcinoma cell lines, and inhibits EGF-dependent tumor cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. In vivo ABX-EGF prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, administration of ABX-EGF without concomitant chemotherapy results in complete eradication of established tumors. No tumor recurrence was observed for more than 8 months following the last antibody injection, further indicating complete tumor cell elimination by the antibody. Inhibition of human pancreatic, renal, breast and prostate tumor xenografts which express different levels of EGFr by ABX-EGF was also achieved. Tumor expressing more than 17000 EGFr molecules per cell showed significant growth inhibition when treated with ABX-EGF. ABX-EGF had no effect on EGFr-negative tumors. The potency of ABX-EGF in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for treatment of multiple EGFr-expressing human solid tumors, including those where no effective chemotherapy is available. Utilization of mAbs directed to growth factor receptors as cancer therapeutics has been validated recently by the tumor responses obtained from clinical trials with Herceptin, the humanized anti-HER2 antibody, in patients with HER2 overexpressing metastatic breast cancer. Being a fully human antibody, ABX-EGF is anticipated to exhibit a long serum half-life and minimal immunogenicity with repeated administration, even in immunocompetent patients. These results demonstrate the potent anti-tumor activity of ABX-EGF and its therapeutic potential for the treatment of multiple human solid tumors that overexpress EGFr.

Fully human anti-interleukin-8 monoclonal antibodies: potential therapeutics for the treatment of inflammatory disease states.

Interleukin‐8 (IL‐8) is a potent chemotactic cytokine implicated in the pathogenesis of a number of inflammatory disease states. Agents that block the binding of IL‐8 to its receptor have been shown to block inflammation in animal models of disease. This suggests that drugs specifically targeting IL‐8 may prove efficacious in treating multiple human diseases. To this end, we developed a panel of fully human anti‐IL‐8 monoclonal antibodies (mAbs). These human antibodies were generated from XenoMouse™ strains, mice created by introducing megabase‐size unrearranged human immunoglobulin heavy and κ light chain loci into a mouse genome in which the corresponding endogenous loci have been inactivated. From the panel of more than 50 mAbs, two antibodies, K4.3 and K2.2, were further characterized and evaluated for their specificity, productivity, affinity, and biological activity. Both K4.3 and K2.2 bind human IL‐8 with high affinity (K d of K4.3 = 2.1 × 10−10 M; K d of K2.2 = 2.5 × 10−10 M). In vitro, in addition to blocking IL‐8 binding to human neutrophils, K4.3 and K2.2 blocked a number of IL‐8‐dependent cellular functions including neutrophil activation, up‐regulation of the cell adhesion receptor CD11b/ CD18, and neutrophil chemotaxis, suggesting that the fully human anti‐IL‐8 mAbs derived from XenoMouse strains are potent anti‐inflammatory agents. This was further supported by in vivo studies in which K4.3 and K2.2 significantly inhibited IL‐8‐induced skin inflammation in rabbits. A pharmacokinetic study in Cynomolgus monkeys demonstrated that the α phase half‐life is 9.4 h and the β phase 10.9 days, typical of human mAbs in monkeys. These data support advancing a fully human anti‐IL‐8 mAb into clinical trials to treat inflammatory diseases. J. Leukoc. Biol. 66: 401–410; 1999.

Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci

ABSTRACT Infections with Streptococcus pneumoniae remain a significant cause of morbidity and mortality. To gain insight into structure-function relationships for human antibodies to pneumococcal capsular polysaccharide (PPS), we studied the response of transgenic mice reconstituted with human immunoglobulin loci, XenoMouse, to PPS antigens in a pneumococcal vaccine. Enzyme-linked immunosorbent assays of sera from mice vaccinated with a 23-valent pneumococcal vaccine revealed that they produced serotype-specific human antibodies, with the greatest response being to the PPS of serotype 3 (PPS 3). Molecular sequence analysis of three monoclonal antibodies (MAbs) to PPS 3 generated from lymphoid cells from mice vaccinated with a 23-valent pneumococcal vaccine or a PPS 3-bovine serum albumin conjugate revealed that they all used heavy-chain immunoglobulin genes from the VH3 family, two expressed light chain genes from the human Vκ1 family, and one expressed a mouse λ light chain. The protective efficacy of the two MAbs was examined in mice. A 10-μg dose of both, and a 1-μg dose of one, significantly prolonged survival from a lethal serotype 3 infection in CBA/N mice. Our data show that XenoMouse mice produced protective, serotype-specific human antibodies to PPS 3, and they lend support to the proposal that these animals represent a useful model to study the human antibody response to PPS antigens.

Transgenic mice as a source of fully human antibodies for the treatment of cancer.

The last two years have seen a renaissance of monoclonal antibodies for the treatment of disease. Of the eight antibodies currently approved for human therapy, two are for the treatment of cancer. In large part, the revival of antibodies has been driven by technology developments geared toward making antibodies less likely to elicit an anti-antibody response in humans. The development of transgenic mice, XenoMouse™ animals, capable of making fully human antibodies offers new opportunities for generating antibodies of therapeutic quality. Recently, this technology has been applied to the generation of a fully human antibody to the epidermal growth factor receptor. A description of the development of this antibody serves to illustrate the power and ease of use of XenoMouse technology.