Transgenic rabbits that overexpress the neonatal Fc receptor (FcRn) generate higher quantities and improved qualities of anti-thymocyte globulin (ATG)

Abstract

Immune suppression with rabbit anti-thymocyte globulin (rATG) is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn) and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α1-antitrypsin was performed, resulting in significantly greater numbers of antigen-specific B-cells in the FcRn Tg rabbits as compared with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies.

Figure 1. Kinetics of Jurkat specific immune response in Tg and wt rabbits.

A. Total IgG concentrations of serum samples collected from rabbit FcRn transgenic (Tg) and wild type (wt) rabbits immunized with Jurkat T cells (determined by ELISA assay). B. Development of immune response to Jurkat T cell surface antigens during immunization (determined by flow cytometry binding assay of 1∶250-fold diluted individual rabbit serum samples). Each dot represents one animal as an average of three measurements; (* P<0.05; ** P<0.01).

Figure 2. Binding of rATG to Jurkat cells from serum samples at different dilutions.

Flow cytometry binding assay was performed and the geometric means of fluorescence intensities obtained are presented for different immune sera dilutions. Antigen binding of individual samples at immune serum dilutions of 1∶500 (A), 1∶1000 (B), 1∶5000 (C) and 1∶10000 (D) are shown in small inserts. Data show that Tg sera bindings were significantly higher than its wt controls in all dilutions. Each dot represents one animal as an average of three measurements (* P<0.05; *** P<0.001).

Figure 3. Binding of Protein-G purified, pooled Tg and wt IgG antibodies to Jurkat cells.

The binding capacity of the Tg IgG analyzed by flow cytometry in a concentration range from 10 - 0.039 µg/ml was higher at all concentrations compared to their wt controls. The non-linear regression analysis indicated that binding activities of MFI 20 was achieved at concentration of 6.92 µg/ml or 4.23 µg/ml, in cases of wt or Tg preparations, respectively, indicating that Tg IgG binding was more than 60% higher than its wt control.

Figure 4. Complement activated cytotoxicity of rATG from Tg and wt rabbits.

A. Complement activated cytotoxicity of pooled immune serum samples collected from wt and Tg rabbits on day 21 of immunization. Tg-pool of immune sera was more efficient in killing Jurkat cells at all dilutions. B. Concentration dependent induction of complement activated cytotoxicity by protein-G purified IgG collected on day 21 of immunization from wt and Tg rabbits (ATG-Fresenius S IgG was used as reference). We observed more efficient cytotoxicity of the Tg samples at all concentrations, and when cytotoxicity curves were analyzed with non-linear regression analysis, data showed that 20% cytotoxic activity was achieved by adding 259 µg/ml, 273 µg/ml or 99 µg/ml IgG preparations from the wt IgG, ATG-Fresenius or Tg IgG preparations, respectively, indicating that Tg IgG mediated cytotoxicity was more than 260% more efficient than its wt control. Percent of lysed cells was determined by flow cytometry analyzing propidium iodide incorporation (samples were analyzed in triplicate and error bars indicate standard deviations of these measurements).

Figure 5. FcRn Tg rabbits have augmented antigen specific B-cell activation.

Immunization with ovalbumin (OVA) and human α1-anti-trypsin (hA1AT) resulted in generally higher level IgM and IgG titers measured by ELISA, more antigen specific IgM and IgG producing B cells determined by ELISPOT, and larger spleen in Tg rabbits as compared to their wt controls (* P<0.05; ** P<0.01).