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Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8- T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-x(L) was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-x(L), provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.

We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity., (Copyright 2004 Elsevier B.V.)

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.

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 kappa 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 (Kd of K4.3 = 2.1x10(10) M; Kd of K2.2 = 2.5x10(-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 alpha phase half-life is 9.4 h and the beta 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.

A fully human IgG2kappa monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and kappa light chain loci. The E7.6.3 MAb exhibits high affinity (KD = 5 x 10(-11) M) to the receptor, blocks completely the binding of both EGF and transforming growth factor alpha (TGF-a) to various EGFr-expressing human carcinoma cell lines, and abolishes EGF-dependent cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. The antibody (0.2 mg i.p. twice a week for 3 weeks) prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, the administration of E7.6.3 without concomitant chemotherapy results in complete eradication of established tumors as large as 1.2 cm3. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total E7.6.3 doses as low as 3 mg, administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice. No tumor recurrence was observed for more than 8 months after the last antibody injection, which further indicated complete tumor cell elimination by the antibody. The potency of E7.6.3 in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for the treatment of multiple EGFr-expressing human solid tumors, including those for which no effective chemotherapy is available. Being a fully human antibody, E7.6.3 is expected to exhibit minimal immunogenicity and a longer half-life as compared with mouse or mouse-derivatized MAbs, thus allowing repeated antibody administration, including in immunocompetent patients. These results suggest E7.6.3 as a good candidate for assessing the full therapeutic potential of anti-EGFr antibody in the therapy of multiple patient populations with EGFr-expressing solid tumors.

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.