Field of the Invention

The invention relates to a method for producing anti-thymocyte globulin (ATG) comprising the steps of contacting a first mammal with a thymocyte derived from a second mammal or a T-lymphoblastic cell line, and harvesting the immunoglobulin G (IgG) produced by said first mammal, wherein the first mammal overexpresses the alpha-chain of a mammalian FcRn protein. Furthermore, an anti-thymocyte globulin composition is provided, which is obtainable or obtained by the method according to the invention. In particular, a rabbit ATG composition is provided, wherein the ATG is produced by using a transgenic rabbit, which overexpresses the alpha-chain of a mammalian FcRn protein.

Background of the Invention

Polyclonal-anti-thymocyte globulin (ATG) - a composition of polyclonal antibodies against thymocytes, more specifically a composition contain a (preferably purified) immunoglobulin fraction of serum (e.g. a purified IgG fraction thereof) from animals, e.g. rabbits, horses, cattle or less commonly goats, immunized with human thymocytes or immunized with a T- lymphoblastic cell line (e.g. the Jurkat T cell line) - contains antibodies with a wide range of specificities against antigens expressed on various normal and malignant hematopoietic cells including T cells, B cells, NK cells, and dendritic cells (Mohty M (2007): Official journal of the Leukemia Society of America, Leukemia Research Fund, UK 21 : 1387-1394). Due to their immunosuppressive potency, ATG compositions are extensively used in clinical applications, mainly in the field of human transplantation. These applications include therapy of aplastic anemia (Marsh J, Schrezenmeier H, Marin P, llhan O, Ljungman P, et al. (1999) Blood 93: 21 91 -2195), conditioning of recipients of bone marrow transplantation (Aversa F, Tabilio A, Velardi A, Cunningham I, Terenzi A, et al. (1 998) N Engl J Med 339: 1 186-1 193; Ringden O, Remberger M, Carlens S, Hagglund H, Mattsson J, et al. (1 998) Transplantation 66: 620-625), treatment of graft-versus-host disease after bone marrow transplantation (Deeg HJ, Loughran TP, Jr., Storb R, Kennedy MS, Sullivan KM, et al. (1985) Transplantation 40: 1 62-1 66; Storb R, Gluckman E, Thomas ED, Buckner CD, Clift RA, et al. (1974) Blood 44: 56-75), and prevention and treatment of acute rejection of organ allografts, including steroid resistant rejection (Cosimi AB, editor (1988) Saunders, Philadelphia, PA. 343 p). There has been growing evidence that ATG compositions have potent cytotoxic effects particularly on lymphatic and to a lesser extent on myeloid malignancies (Bonnefoy-Berard N, Cenestier L, Flacher M, Rouault JP, Lizard G, et al. (1994) Blood 83: 1051 -1059). The mechanisms involved in the cytotoxic effects include complement-dependent cytolysis, cell-mediated antibody-dependent cytotoxicity, opsonisation and subsequent phagocytosis by macrophages, activation-induced cell death as well as apoptosis (Ayuk FA, Fang L, Fehse B, Zander AR, Kroger N (2005) Exp Hematol 33: 1531 -1 536; Bonnefoy-Berard N, Revillard JP (1996) J Heart Lung Transplant 1 5: 435- 442; Timm MM, Kimlinger TK, Haug JL, Kline MP, Greipp PR, et al. (2006) Leukemia 20: 1863-1869; Zand MS, Vo T, Pellegrin T, Felgar R, Liesveld JL, et al. (2006) Blood 107: 2895-2903). There is also evidence that ATG-mediated immunosuppression is delivered in part via immunologically specific actions involving the generation of regulatory T cells (Lopez M, Clarkson MR, Albin M, Sayegh MH, Najafian N (2006) J Am Soc Nephrol 1 7: 2844-2853; Theurich S, Reisberg A, Christopeit M, von Bergwelt-Baildon M, Weber T, et al. (2010) Transplantation 1 6: S220).

Currently, three commercially available preparations of ATG are provided on the market. ATG-Fresenius® (Fresenius-Biotech GmbH) is produced by immunization of New Zealand White (NZW) rabbits with the Jurkat human T-lymphoblastic cell line. Thymoglobulin® (Genzyme) is produced by immunizing NZW rabbits with human thymocytes, while ATGAM® (Pharmacia Upjohn) is produced by immunizing horses with human thymocytes. Due to differences in manufacturing processes, these different ATG products contain non- identical specificities and amounts of antibodies explaining the wide variability in doses used for each of these ATG products in clinical settings. For Thymoglobulin®, a total recommended dose is 4.5 to 8 mg/kg body weight for matched unrelated donor allogenic hematopoetic stem cell transplantation; for ATG-Fresenius® , however, the recommended dose for the same indication is about ten times as high (Ayuk F, Zander A, Kroger N (2009) Annals of hematology 88: 401 -404). As demand for ATG is increasing, producers have expanded their facilities to house hundreds of thousands of rabbits in costly high-standard facilities under specified pathogen-free (SPF) conditions. In addition, commercial issues arise, as it is a challenge to produce sufficient amounts of ATG by vaccination of larger animals, such as horses (ATGAM ^® ). Additionally, SPF environments cannot be established for horses and thus the percentage in antigen specific antibodies is relatively low (due to high baseline IgG levels).

Given the increased demand for ATG and the technical difficulties in providing large amounts of ATG, it is therefore highly desirable to increase the efficiency of ATG, e.g. rabbit (r)ATG, production per animal (e.g. rabbit), thereby reducing the number of animals involved in the production process. It is therefore an object of the present invention to provide an animal production system for producing ATG with greater efficiency, in particular a greater efficiency per animal, in a cost-effective manner while maintaining a high quality production standard. Hereby, improvement of the ATG composition's quality and yield is therefore desired.

The object underlying the present invention is solved by the claimed subject matter. Summary of the Invention

The present invention provides a method for producing anti-thymocyte globulin (ATG) comprising the steps of contacting a mammal with thymocytes, a composition containing thymocytes as a major fraction, or (e.g. lysed) thymocyte fractions or cells of a T cell line, e.g. a human T cell line (e.g. a (human) T-lymphoblastic cell line, such as the Jurkat T cell line) and harvesting the ATG produced by said mammal, wherein the mammal overexpresses the alpha-chain of mammalian Fc n protein.

By using the method according to the invention, the amount of ATG obtained from a mammal is significantly increased with respect to the amount obtained by using conventional methods, in particular in comparison to the amount obtained when using a reference animal, which is identical with the animals employed according to the present invention, but does not overexpress the alpha-chain of a mammalian FcRn protein. The neonatal Fc receptor (FcRn) is an Fc receptor which is similar in structure to MHC class I. It was first discovered in rodents as a unique receptor capable of transporting IgG from mother's milk across the epithelium of newborn rodent's gut into the newborn's bloodstream. Further studies revealed a similar receptor in humans. In humans, however, it is found in the placenta to help facilitate transport of mother's IgG to the growing fetus. This receptor also plays a role in salvage of IgG (and typically albumin) through its occurrence in the pathway of endocytosis in vascular endothelial cells and bone marrow derived cells (e.g. monocytes, macrophages and dendritic cells). Fc receptors in the acidic endosomes bind to monomeric IgG internalized through pinocytosis, recycling it to the cell surface, releasing it at the basic pH of blood, thereby preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the greater half-life of IgG in the blood compared to other isotypes.

In addition, FcRn plays major roles in antigen-lgG immune-complex phagocytosis by neutrophils (Vidarsson G, Stemerding AM, Stapleton NM, Spliethoff SE, Janssen H, et al. (2006) Blood 108: 3573-3579), and in antigen presentation of IgG immune complexes by professional antigen presenting cells (Qiao SW, Kobayashi K, Johansen FE, Sollid LM, Andersen JT, et al. (2008). Proc Natl Acad Sci U S A 105: 9337-9342; Mi W, Wanjie S, Lo ST, Gan Z, Pickl-Herk B, et al. (2008) J Immunol 181 : 7550-7561 .; Liu X, Lu L, Yang Z, Palaniyandi S, Zeng R, et al. (201 1 ) J Immunol 186: 4674-4686.; Baker K, Qiao SW, Kuo TT, Aveson VG, Platzer B, et al. (201 1 ) Proceedings of the National Academy of Sciences of the United States of America 108: 9927-9932.; and Vegh A, Farkas A, Kovesdi D, Papp K, Cervenak J, Schneider Z, et al. Antibodies. PLoS One 2012; 7:e36286.) and in generating antigen specific antibodies (Liu X, Lu L, Yang Z, Palaniyandi S, Zeng R, et al. (201 1 ) J Immunol 186: 4674-4686). Furthermore, genetically modified animals that overexpress the alpha-chain of the FcRn showed augmented antigen-specific humoral immune response with larger numbers of antigen specific B cells Cervenak J, Bender B, Schneider Z, Magna M, Carstea BV, et al. (201 1 ) J Immunol 186: 959-968. and Schneider Z, Cervenak J, Baranyi M, Papp K, Prechl J, et al. (201 1 ) Immunology Letters 137: 62-69.), more efficient hybridoma production (Schneider Z, Cervenak J, Baranyi M, Papp K, Prechl J, et al. (201 1 ) Immunology Letters 137: 62-69.), increased diversity of induced antibodies (Vegh A, Farkas A, Kovesdi D, Papp K, Cervenak J, et al. (2012) PLoS One 7: e36286.) and the generation of antibodies against weakly immunogenic antigens (Vegh A, Cervenak J, Jankovics I, Kacskovics I (201 1 ) mAbs 3: 1 73-180.).

The inventors of the present invention identified unexpected improvements of the quality and quantity in terms of binding and complement-mediated cytotoxicity characteristics of polyclonal antibodies raised against thymocytes or a T-lymphoblastic cell line, such as the Jurkat T cell line) for providing ATG compositions, if FcRn transgenic animals are employed for polyclonal antibody production (instead of wt reference animals as used in the art). These findings were even more surprising, as improved quality and increased quantity of antibody production against a multitude of antigens residing on the cell surface, here the thymocyte cell surface, has not been observed yet at all when using FcRn transgenic animals. It was - in view of the role and the underlying mechanism of FcRn - expected that overexpression of the FcRn receptor in a transgenic animal are exclusively suitable to provide a more robust immune response against soluble antigens.

The method according to the invention is employed in order to increase the ATG yield per individual animal following immunization with thymocytes or a T cell line, in particular a T-lymphoblastic cell line, such as the Jurkat T cell line. Thereby, the number of animals required for producing a certain amount of ATG is reduced. The method according to the invention is therefore useful in enhancing the cost-effectiveness of the ATG production process. A further advantage of the reduced number of animals required for obtaining the desired amount of ATG is a reduced quality control due to a lower number of serum batches from individual animals.

The increased ATG yield per animal obtained by using the method according the invention also has a positive influence on the quality of the ATG composition. In comparison to conventional methods for producing ATG, a wider spectrum of B cell clones producing a higher yield of antibodies against thymocytes or a T cell line, in particular a (human) T- lymphoblastic cell line, such as the Jurkat T cell line. is obtained in an individual FcRn transgenic animal. As a consequence, ATG obtained by the method according to the invention is characterized by a significantly increased yield of anti-thymocyte antibodies per FcRn transgenic animal. Finally, the inventors have found that ATG obtained by the method according to the invention is characterized by greater binding capacity and cytotoxic activity as compared to conventional ATG compositions obtainable by methods known in the art. IgGs purified from polyclonal serum obtained from an animal overexpressing the alpha-chain of a mammalian FcRn protein additionally contain a proportionally higher amount of e.g. antigen-specific IgG. Higher specific activity of IgG harvested from FcRn overexpressing animals is highly valuable, as it reduces doses of e.g. purified IgG as obtainable according to the method of the invention that is needed for therapy.

In summary, the method according to the invention allows harvesting polyclonal ATG samples according to the invention, which is characterized by a broader clonal spectrum, enhanced binding capacity and enhanced cytotoxic activity isolated from a reduced number of animals, which provide a higher antibody yield per immunized animal. Thereby, consistency problems and exposure to non-antigen specific IgG are reduced. Accordingly, the ATG product obtainable by the inventive method is characterized by the above advantages as well.

In conclusion, the object of the present invention is solved by the provision of an improved method for producing ATG as described herein and by the provision of a polyclonal ATG product of clearly superior properties, which is obtainable or obtained by a reduced number of immunized FcRn transgenic animals, whose anti-serum is pooled and opionally purified to provide the ATG composition of the invention..

Brief Description of the Figures

The figures shown in the following are merely illustrative and shall describe the present invention in more detail. These figures shall not be construed to limit the present invention thereto.

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

A. Total IgG concentrations of serum samples collected from rabbit FcRn transgenic (Tg) and wild type (wt) rabbits immunized with Jurkat T cells was determined by ELISA assays. Samples were measured at day 0, day 7, day 14 and day 21 .

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) Samples were taken out at day 7, day 14 and day 21 .

Each dot in Figure 1 A and 1 B represents one animal as an average of three measurements; (* P < 0.05; ** P < 0.01 ). Clearly, an increased immune response (expressed by the measured IgG levels) is observed, which increases over time.

Figure 2: Binding of rATG to Jurkat cells from serum samples at different dilutions was plotted as mean fluorescent intensity (MFI). 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. The dilution is provided in Fig. 2 on a logarithmic scale. Serial dilutions of each test serum sample were applied and antibody cell binding (mean fluorescence intensity (MFI)) was used to determine the MFI 50 value (50%) by cubic spline curve fit algorithm. Data show that binding activities reflecting the MFI 50 value, were achieved by diluting the wt or Tg sera by 1 :1318-fold or 1 :3012-fold, respectively, indicating that Tg sera binding was more than 200% higher than for its wt controls. 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 was studied. The binding capacity of the Tg IgG analyzed by flow cytometry in a concentration range from 10 - 0.039 pg/ml was higher at all concentrations compared to wt control samples. The non-linear regression analysis indicated that binding activities at the MFI 20 data point (20%) was achieved at concentration of 6.92 g/ml or 4.23 pg/ml, for wt or Tg preparations, respectively, indicating that Tg IgG binding was more than 60% higher compared to wt control.

Figure 4: Complement activated cytotoxicity of rATG from Tg and wt rabbits was studied. A. Complement activated cytotoxicity was determined for pooled immune serum samples collected from wt and Tg rabbits on day 21 of immunization. Tg-pool of immune sera was more efficient in ki lling Jurkat cells at all dilutions; cytotoxicity curves were analyzed with the cubic spline curve fit algorithm. The results show that lysis of 20% of the cells was observed for the wt or Tg sera at dilutions of 1 :72 or 1 :204, respectively. The serum of Tg rabbits had 280% more efficient cytotoxic activity compared to wt control.

B. Concentration dependent induction of complement activated cytotoxicity was studied by protein-G purified IgG collected on day 21 of immunization from wt and Tg rabbits (ATG-Fresenius S IgG was used as reference). More efficient cytotoxicity of the Tg samples was observed at all concentrations. Cytotoxicity curves were analyzed with the cubic spline curve fit algorithm. The results show that 20% cytotoxic activity was achieved by adding 1 54 μg/ml, 1 37 μg/ml or 68 μg/ml IgG preparations from wt IgG, ATG-Fresenius or Tg IgG preparations, respectively. Tg IgG mediated cytotoxicity was more than 200% more efficient compared to wt control and about 200% more efficient than ATG-Fresenius, which is one of the currently commercialized ATG products being available on the market. The percentage 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 show augmented antigen specific B-cell activation. Immunization with ovalbumin (OVA) and human alphal -anti-trypsin (hA1 AT) results in generally higher level IgM and IgG titers measured by ELISA, more antigen specific IgM and IgG producing B cells determined by ELISPOT, and a larger spleen, whenever Tg rabbits were immunized as compared to wt controls (* P < 0.05; ** P < 0.01 ).

Detailed Description of the Invention

In a first aspect, the invention relates to a method for producing ATG (i.e. a composition derived from anti-thymocyte or anti-T cell line (such as Jurkat cells) polyclonal serum of the immunized first animal or mammal). This method comprises the steps of (i) contacting a first mammal with a thymocyte or a composition comprising thymocytes as a major cell fraction (typically derived from a second mammal) or T cells of a (human) T cell line, in particular a (human) T-lymphoblastic cell line, such as a Jurkat T cell line, and (ii) harvesting the immunoglobulines, e.g. the IgG fraction, produced by said first mammal, wherein the first mammal overexpresses the alpha-chain of a mammalian FcRn protein. Preferably, the first animal, e.g. rabbit, overexpresses the alpha-chain of an FcRn protein derived from mouse, rat, rabbit, human, sheep, cattle, possum, swine, donkey, goat, dog, horse or camel. In one embodiment, the overexpressed FcRn protein corresponds to the species specific sequence of the overexpressing first animal. If e.g. the first animal is a rabbit or a horse, the overexpressed alpha chain FcRn protein is derived from the corresponding species, i.e. rabbit or horse, respectively. In another embodiment, the overexpressed FcRn protein is not drived from the species of the first mammal, e.g. a bovine alpha-chain of bovine FcRn may be overexpressed in a rabbit or a mammal other than cattle. It is preferred that the FcRn protein, which is overexpressed in the first mammal, specifically and advantageously selectively binds to the IgG that is produced by the first said mammal. The IgG fraction produced by the first mammal upon immunization is typically either the species-specific IgG of the first mammaPs species or may not be species-specific, if the first mammal produces IgG specific for another species, e.g. due to a genetic modification in the antibody encoding region of the first mammal. E.g. the non-human first mammal may additionally be transgenic for expressing human IgG as described below.

As a second aspect, the invention relates to a polyclonal ATG composition obtainable or obtained according to the methods of the present invention, e.g. obtained or obtainable from a transgenic rabbit overexpressing the alpha-chain of a mammalian FcRn protein. Such an ATG composition according to the invention is preferably an anti-human thymocyte or an anti-T-lymphoblastic cell line, e.g. of human origin, e.g. an anti-Jurkat cell line, polyclonal antibody composition. Such an ATG composition is obtainable by contacting the first mammal with e.g. human thymocytes and by harvesting or collecting the immunoglobulin fraction from e.g. blood or serum samples of the first animal or rather a larger number of first animals. As a further aspect, the invention provides prophylactic and/or therapeutic methods or uses of treating a mammal by administering ATG or an ATG-like composition to a mammal in need of the treatment, preferably a human, e.g. at a dose of less than 1 mg/kg per day. The present invention is based on the finding that ATG compositions obtainable by an inventive method reflect diverse spectrum of antibodies having a greater binding capacity and cytotoxic activity, thus, being superior to ATG compositions obtained from methods known in the art. Hereby, the spectrum of therapeutic and/or prophylactic uses/methods for in vivo applications is improved. It also allows for more specifically addressing experimental issues for in vitro applications. Accordingly, both therapeutic or prophylactic in /Vo use and, e.g. experimental, in vitro use of the ATG composition obtainable according to the inventive method is enabled by the present invention.

As used herein, the term "ATG" ot "ATG composition" refers to the whole anti-serum harvested from the first mammal according to the inventive method (e.g. by immunization of a first mammal, e.g. rabbits or horses, with human thymocytes or compositions containing thymocytes or cells of a (Jurkat) T-lymphoblastic cell line) or to purified fractions thereof.

Purified fractions may e.g. correspond to a globulin fraction of the harvested anti-serum containing essentially immunoglobulins. Under such circumstances, e.g. other serum proteins have been removed by the purification step. In case of further purification, a subfraction of the globulin fraction that essentially contains polyclonal anti-thymocytic antibodies may be provided. Alternatively, the globulin fraction may be purified to essentially contain exclusively the IgG antibody subfraction of the harvested anti-serum (against whatever antigen). Finally, a subfraction may be provided, which is purified such that it contains essentially a (sub)subfraction of anti-thymocytic antibodies of the IgG type only. Whatever purification step is applied to the harvested anti-serum of the first mammal, which results in fractions or subfractions containing anti-thymotic antibodies, such a fraction or subfraction is understood to be an ATG composition within the meaning of the present invention.

It is noted that - by virtue of their mode of production - ATGs contain a diverse spectrum of antibodies targeting a wide range of antigens expressed on various normal and malignant hematopoetic cells including T-, B-, NK, dendritic, and plasma cells (Ayuk F, Zander A, Kroger N. Ann Hematol 2009; 88:401 -4.). That is due to the initial thymocyte composition used for immunization, which may contain other hematopoietic cells than T cells, e.g. if a thymocyte composition is administered which is derived from the thymus, e.g. after surgery, or due to surface structures which are expressed not only on T cells but on other hematopoietic cells as well.

The polyclonal ATG composition of the invention may contain an ensemble of antibodies against any (T cell) surface structure (e.g. any T cell surface protein), such as CD1 , CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD1 1 , CD1 6, CD1 8, CD20, CD28, CD38, CD40, CD44, CD45, CD56, CD58, MHC class 1 , and the T cell receptor.

Preferably, a mammal to be contacted with thymocytes or cells of T cell lines, e.g. T- lymphoblastic cells, is a non-human mammal selected from the group consisting of mouse, rat, rabbit, sheep, cattle, possum, swine, donkey, goat, dog, -horse or camel. More preferably, the mammal is a rodent, a rabbit, or a horse or goat. Most preferably, the mammal to be contacted with thymocytes or (Jurkat) T-lymphoblastic cells is a rabbit. Rabbits generate one isotype of IgG immunoglobuline, that binds efficiently to e.g. human Fc gamma receptors, allowing the development of powerful human T cell opsonization and subsequent cell-mediated antibody-dependent cytotoxicity by NK cells or phagocytosis by macrophages..

According to the method of the invention, such a first mammal is contacted with thymocytes. Typically, thymocytes are administered as a composition containing thymocytes and optionally other cells, in particular other cells of the hematopoietic system. The thymocytes may also be derived from an established T lymblastic cell line, like the Jurkat cell line. Specifically, the term "thymocyte" refers to a mammalian T cell, be it a normal, an immortalized or a malignant T cell. Preferably, a thymocyte is derived from mammal selected from the group consisting of human, mouse, rat, rabbit, sheep, cattle, possum, swine, donkey, goat, dog, horse or camel, in particular a human. In a preferred embodiment, live thymocytes are used to contact a mammal. Live thymocytes may be derived from a donor subject or from cell culture. As used herein, the term also refers to inactivated or lysed thymocytes, which are no longer capable of dividing (e.g. as a consequence of chemical treatment). In particular, the term refers to thymocytes, which have an intact cytoplasmic membrane, but may also refer to lysed thymocytes. Accordingly, the thymocytes may be intact or lysed or otherwise modified.

The cellular preparation of e.g. human thymuses can be obtained either from cells of lines in culture, or from fresh thymocytes which are purified preferentially from human thymus fragments or optionally from, for example, suspensions of spleen, tonsils, lymph nodes, thoracic trachea or peripheral blood. E.g. human thymus fragments can in particular be easily removed during surgical acts, in particular subsequent to cardiac surgery on children. Virological tests are carried out on the donor's blood in order to avoid any contamination and to eliminate any contaminated thymic fragment showing a positive serological result.

Thymocytes may also be derived from a cultured cell or cultured cell line, in particular a T lymphoblastic cell line. Alternatively, thymocytes are used that are derived from a donor subject (e.g. from a blood sample or from the fraction of peripheral blood monocytic cells (PBMC's) therein or from lymph nodes or e.g. from the thymus, in particular the children's thymus). Preferably, a thymocyte according to the invention is a thymocyte derived from a cultured cell line, e.g. a T lymphoblastic cell line, in particular a human T lymphoblastic cell line. As an example of such a cell line, the Jurkat cell line of human origin may be employed. In a particularly preferred embodiment of the invention, a Jurkat E6.1 T cell is used for the first mammal's immunization.

In case thymus fragments are isolated from donors (e.g. human donors), they are - after removal - optionally purified. If isolated T cells are purified prior to their use for immunization, "purified T cell subpopulation" are provided. Such a T cell subpopulation is preferably a substantially pure T cell subpopulation that is isolated from a sample that natural source (e.g. after thymus surgery) which contains other cells as well. The purified T cell subpopulation is e.g. enriched for at least one or all of the of the following cell types: NK T cells, CDld-reactive T cells, or JaQ+ T cells and does preferably essentially not contain cells other than immune cells or, more preferably, does essentially not contain other immune cells than those belonging to the T cell subpopulation. The purified T cell subpopulation is more pure than the purity of the T cell subpopulation found in nature. Desirably, the purified T cell subpopulation is at least 1 , 5, 15, 30, 50, 75, 90, or 99%, by number, free from cells with which it is naturally associated. Typically, these other cells that are associated with the T cell subpopulation differ from the T cells belonging to the subpopulation by not expressing a cell-surface molecule, not binding a ligand, or not having an activity of the T cell subpopulation.

Further thymocytes, preferably purified are additionally additionally preferably grinded, filtered and placed in suspension, e.g. suspended in an isotonic solution, e.g. a solution containing 0.9% saline. Other well known components, which are used to establish the desired osmolality may be used as well, e.g. dextran 40, dextrose, etc. The cell suspension is then preferably filtered, e.g. through a nylon cloth and then, if required, subjected to a centrifugation step. Such a suspension is thereafter applied (in the process step of "contacting") for eliciting an immunization reaction in the first animal.

Typically, thymocytes are suspended, e.g. in buffer solution (which is preferably isotonic by osmolar components, e.g. salts or sugar components, e.g. dextran, dextrose) and injected, e.g. intravenously. Typically, thymocytes or suspensions of thymocytes are administered to the first mammal more than once to booster the first mammal's immune response. For instance, the administration may be repeated once or twice or more (e.g. up to 4 times) after a certain period of time, e.g. within 1 to 14 days or 1 to 10 days or within 1 to 5 days. The dose and immunization regimen will depend on the species of the first mammal immunized, its immune status, body weight, and/or calculated surface area, etc.

As used herein, the phrase "contacting an animal (with a thymocyte or thymocytes)" refers to any appropriate way of bringing the animal in physical contact with thymocytes of whatever orginin, e.g. from a natural source of of a cell line, e.g. a lymphoblastic cell line, such as Jurkat T-lymphoblastic cells. It is desired to ensure that the physical contact is sufficient to elicit an immune response in said animal, in particular "contacting" is meant to elicit a humoral immune response in the first mammal. Typically, "contacting" refers to a process that allows direct contact between immune cells of the first mammal overexpressing the alpha-chain of a mammalian FcRn protein and a thymocyte, which is used for contacting said mammal, in vivo. Preferably, a mammal is contacted with thymocytes, e.g. a suspension of thymocytes of whatever origin or any suitable preparation of thymocytes, by administering said thymocytes (of e.g. another (second) species than the first mammal) to the first mammal intravenously, intramuscularly, intranodally, subcutaneously, intradermally or intraperitoneal ly. The parenteral administration route is preferred. Administration may be carried out by injection or infusion, e.g. via a control led- rate pump. For injection purposes, it may be a needle injection or a needle free injection. Thymocytes, e.g. isolated from a natural source, e.g. thymus or thymus of a human donor, or derived from T cell lines of the second mammal's species (e.g. human) are thereby brought into contact with another (the first transgenic mammal's) species (e.g., a transgenic rabbit or horse).

When contacting the first mammal with thymocytes of whatever nature, also adjuvants may be administered to the first mammal that are typically used to augment the immune response during immunization. Adjuvants vary according to the first mammal's species used for immunization with thymocytes. Examples of adjuvants include, but are not limited to: Freund's complete adjuvant ("FCA"), Freund's incomplete adjuvant ("FIA"), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions), peptides, oil emulsions, keyhole limpet hemocyanin ("KLH"), dinitrophenol ("DNP"), and potentially useful human adjuvants such as Bacille Calmette-Guerin ("BCG") and corynebacterium parvum. Other adjuvants may be TLR agonists, e.g. TLR-7/8, TLR-9 or TLR-10 agonists. Such adjuvants are typically administered separately from the thymocytes.

Preferably, the administration, e.g. injection, of a cellular preparation of (e.g. human) thymocytes is carried out into a specific-pathogen-free (SPF) first mammal, preferably a pathogen-free rabbit, as the recipient. The SPF animals are breeding animals whose environment and food are strictly controlled according to health standards. Reference is also made to animals "with controlled health status". The SPF animals are bred in closed, sterilized compartments, the air environment being filtered, for example through a HEPA filter (sterilizing filtration), and the water and foodstuff being decontaminated before introduction, which makes it possible to eliminate any pathogenic element from their direct environment (Yanabe et al. Exp. Animal 48(2), 101 -106 (1 999)). The term "pathogen" here denotes an infectious agent capable of causing a clinical disease and/or of modifying the biological response of the animal with regard to the desired use. The SPF status refers to a list (which evolves) of microorganisms and of methods of control (clinical, serological, histological, or using culturing) for detecting the targeted microorganisms or, conversely, demonstrating their absence. Documents that contain guidelines and recommendations for managing SPF animals are available and may be considered, preferably the regularly updated guideline or protocols as provided by the Federation of European Laboratory Animal Science Associations (FELASA) and American Association for Laboratory Animal Science (AALAS) may be followed.

In the method according to the invention, thymocytes are preferably used, which are derived from a mammal, to which the ATG composition obtainable or obtained by that method is e.g. to be administered thereafter for therapeutic or prophylactic purposes. For instance, if ATG is desired for use in a human subject, human thymocytes of e.g. human donors or a human T cell line, such as Jurkat cells, are typically used for contacting a mammal overexpressing the alpha-chain of a mammalian FcRn protein. If the resulting ATG composition is to be provided for e.g. veterinary use in a non-human mammal (e.g. swine), thymocytes are preferably used that are derived from the species of said non-human mammal (e.g. swine). Accordingly, the second animal belongs preferably to the same mammalian species as the mammalian to be treated with the resulting ATG composition.

In the method according to the invention, a (first) mammal that is contacted with thymocytes of a natural source or a cell line, e.g. the Jurkat T-lymphoblastic cell line, overexpresses the alpha-chain of a mammalian FcRn protein. In a preferred embodiment, the alpha-chain of an FcRn protein is derived from the same mammalian species which is used as the first mammal for overexpression (homologous overexpression). For example, in the case of a rabbit as the first mammal, which is contacted with thymocytes for immunization purposes, the alpha-chain of rabbit FcRn is overexpressed in said rabbit.

In an alternative embodiment, the alpha-chain of an FcRn protein that is overexpressed in a first mammal, which is contacted with thymocytes of a natural source or a cell line, e.g. the Jurkat T-lymphoblastic cell line, is derived from the native sequence of another mammalian species that is distinct from the native FcRn sequence of the species of the first mammal (heterologous overexpression). For instance, when a rabbit is contacted with thymocytes, the alpha-chain of e.g. the bovine, murine, horse, swine FcRn protein may be overexpressed by the FcRn transgenic rabbit.

In the meaning of the present invention, the term "overexpression" refers to a situation, where the expression of the alpha-chain of a mammalian FcRn protein in a mammal to be contacted with a thymocyte or thymocytes, typically a suspension of thymocytes, is elevated with respect to the expression level in a reference mammal having two genomic wildtype alleles.

Overexpression can be assessed at several levels. For instance, it is possible - by methods known in the art - to measure the amount of transgenic RNA as well as the amount of expressed protein. Preferably, the overexpression of the alpha-chain of a mammalian FcRn protein and RNA is measured using a method selected from the group consisting of Western Blot, flow-cytofluorimetry, confocal microscopy, quantitative RT-PCR and/or Northern Blot analysis.

The overexpression is typically due to an increased amount of gene transcript of said alpha- chain of a mammalian FcRn protein in the first mammal. The increased amount of that protein is typically achieved a manipulation of the fist mammal's genom, i.e. by providing a transgenic first mammal. That may be due to a change in the regulation of an endogenous allele (i.e. of a native allele in its natural location in the genome of the first mammal) or by the incorporation of one or more additional alleles for the alpha chain of FcRn. The regulation of a sequence encoding the alpha-chain of a mammalian FcRn protein can be altered, for instance, by mutation, insertion or deletion of regulatory elements (acting in cis or in trans) or by changes in the concentration of factors regulating gene expression (in trans). Alternatively (or in addition) to the altered regulation of a sequence encoding the alpha-chain of a mammalian FcRn protein, the increase in transcript amount may preferably be due to a change in copy number, such that at least one additional allele (of the FCGRT gene encoding the alpha-chain of a mammalian FcRn protein, preferably together with its regulatory sequences), preferably at least two additional alleles, is/are present in the mammal to be contacted with the second animal's thymocyte or T cells derived from a T cell line. In one embodiment, the at least one additional allele of the FCGRT gene encoding the alpha-chain of a mammalian FcRn protein is derived from an endogenous allele by gene duplication. In another embodiment, the at least one additional allele has been introduced into a cell of the first mammal, e.g. by transfection, microinjection, infection, transduction or fusion of the (acceptor) cell with a donor cell. Preferably, the at least one additional allele is a homolog or an ortholog with respect to an endogenous allele of the mammalian FCGRT gene encoding the alpha-chain of a mammalian FcRn protein. In a further preferred embodiment, the upregulation of the expression of an endogenous allele or of an additional allele of FCGRT gene encoding the alpha-chain of a mammalian FcRn protein is induced by the activation of a transcription factor, wherein the term "activation" refers to a process resulting in the increase of the nuclear concentration of a transcription factor in its active form, i.e. in the form that is actually capable of activating gene transcription. Preferably, the activation comprises de novo synthesis of a transcription factor. Alternatively, the activation comprises a transition of a transcription factor (which is already present) from an inactive to an active state. Preferably, that transition consists in a post-translational modification and/or in a translocation from one cellular compartment to another, preferably translocation into the nucleus. In a more preferred embodiment, the transcription factor regulating the expression of the alpha-chain of a mammalian FcRn protein is NF-κΒ. According to an embodiment of the invention, NF-kB is activated in a first mammal, which overexpresses the alpha-chain of a mammalian FcRn protein and which is contacted with thymocytes, thus leading to an expression increase of the alpha-chain of a mammalian FcRn protein, the transcription of which is controlled by a promoter sequence that has at least one NF-kB binding site. Preferably, the activation of NF-kB is achieved by administration of an immunostimulatory substance to that mammal or a tissue of that mammal. More preferably, the immunostimulatory substance acts through a Toll-like receptor (TLR), preferably through TLR4. Most preferably, the substance used for activating NF-kB in a mammal overexpressing the alpha-chain of a mammalian FcRn protein is selected from the group consisting of pro-inflammatory cytokines (e.g. IL-1 , tumor necrosis factor a (TNFa)), bacterial toxins (e.g. lipopolysaccharide (LPS), exotoxin B), viruses/viral products (e.g. HIV-1 , HTLV-I, HBV, EBV, Herpes simplex) and pro-apoptotic or necrotic stimuli (oxygen free radicals, UV light, gamma-irradiation)

In a preferred embodiment, the at least one additional allele encoding the alpha-chain of a mammalian FcRn protein is identical to the respective endogenous alleles of the first mammal. For example, an additional wildtype allele derived from the same species of the first mammal is introduced into an animal endogenously carrying at least one such wildtype allele encoding the alpha-chain of a FcRn protein.

In another preferred embodiment, the at least one additional allele encoding the alpha- chain of a mammalian FcRn protein differs from at least one of the endogenous alleles encoding the alpha-chain of a mammalian FcRn protein of the first mammal. For instance, the at least one additional allele encoding the alpha-chain of a mammalian FcRn protein may be a wildtype allele of a different species or another allele of the same species, which is modified (e.g. through mutation, insertion or truncation) with respect to the endogenous allele encoding the alpha-chain of a mammalian FcRn protein. Whenever the at least one additional copy of the FCGRT gene is heterologous to the corresponding wt allele of the first animal, one or both of the first animal's wt alleles may be or remain functional or not (e.g. by disruption of one or both of the homologous i alleles). In the context of the present invention, the phrase "overexpression of the alpha-chain of a mammalian FcRn protein" is typically meant to reflect the total amount of mammalian FcRn protein (i.e. the sum of the expression product of any (functional) allele present) in the first animal, which is increased as compared to the total amount of expression in the corresponding wt animal. For example, a rabbit expressing the alpha-chain of rabbit FcRn protein by its two endogenous wildtype alleles and, additionally, by expressing one or more additional (homologous or heterologous) alleles (as a result of the first mammal's trangenic nature), is referred to as "overexpressing the alpha-chain of a mammalian FcRn protein".

In a preferred embodiment, the mammal to be contacted with thymocytes is transgenic for the alpha-chain of a mammalian FcRn protein, i.e. the (first) mammal to be contacted with a thymocyte comprises at least one additional allele encoding the alpha-chain of a mammalian FcRn protein as a transgene. As used herewithin, the term "transgenic", refers to an animal that would not have been obtained through normal breeding or mating processes. Accordingly, the term "transgene" refers to a gene (i.e. a coding sequence comprising a regulatory sequence), which has been introduced into the genome of a cell or an animal by transformation and which is stably maintained.

Preferably, the at least one additional allele encoding the alpha-chain of a mammalian FcRn protein is introduced as transgene into a (first) mammal to be contacted with a thymocyte through a genetic construct. According to the invention, the term "genetic construct" includes artificially recombinant DNA comprising a nucleic acid sequence that, upon introduction into the recipient cell, provides the expression of the alpha-chain of a mammalian FcRn protein. A genetic construct may comprise coding sequences as well as regulatory sequences. The term "coding sequence" refers to a DNA or RNA sequence encoding a specific peptide or protein. The term "regulatory sequences" refers to nucleotide sequences upstream (i.e. 5'), within, or downstream (i.e. 3') of a coding sequence, which influence the transcription, RNA processing/stability and/or translation of the coding sequence. Regulatory sequences comprise promoters, enhancers, 5' or 3' untranslated regions, intron, polyadenylation signals, internal ribosomal entry sites (IRES) and the like.

In a preferred embodiment, a transgenic (first) animal, which is contacted with a thymocyte, overexpresses the alpha-chain of a mammalian FcRn protein. The transgenic animal is generated using a technology known in the art, without any limitation regarding the applied methods. Stable integration of a transgene into a genomic site may be achieved employing several strategies and using various constructs containing a sequence encoding the alpha- chain of a mammalian FcRn protein, such as plasmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or PACs (P1 phage artificial chromosomes).

For instance, a transgenic construct carrying the alpha-chain of a mammalian FcRn protein can be introduced into a recipient cell (e.g. by injection into the pronucleus of a fertilized oocyte) and allowed to integrate in a random manner into the genome of the recipient cell. Alternatively, a transgenic construct may also be introduced by lenti viral transgenesis. As another option for introducing a transgenic construct, embryonic stem cells can be transfected with the transgenic construct, which are subsequently injected into developing embryos with the object to obtain a transgenic (non-human) first mammal..

According to the invention, a linearized BAC clone comprising a sequence encoding the alpha-chain of a mammalian FcRn protein is preferably microinjected into fertilized zygotes, which are transferred into pseudopregnant females. Offspring is screened for the presence of the transgene (for example, using BAC backbone specific PCR primers for amplification). The BAC copy number may be determined by, for instance, quantitative PCR. In a preferred embodiment, the transgenic animal contains one extra copy (in a hemizygous animal) or two extra copies (in a homozygous animal) of a sequence encoding the alpha-chain of a mammalian FcRn protein. Depending on - amongst other factors - the genomic insertion site, the expression of the additional copy of the alpha-chain of a mammalian FcRn protein may vary as determined by quantitative methods known in the art. The amount of alpha-chain of a mammalian FcRn protein expressed from the additional copy introduced into the transgenic animal may be higher or lower than the amount expressed from an endogenous allele coding for the FcRn alpha chain. In the context of the invention, the transgenic animal is regarded as "overexpressing the alpha-chain of a mammalian FcRn protein", if the total amount of alpha-chain expressed from an additional allele and the endogenous alleles is larger than the total amount of alpha-chain expressed in a homozygous animal having two genomic copies of the wildtype allele.

In a preferred embodiment, the construct used for the generation of a transgenic animal comprises a BAC clone harboring a sequence encoding the alpha-chain of an FcRn protein from a non-human mammal selected from the group consisting of mouse, rat, rabbit, sheep, cattle, possum, swine, donkey, goat, dog, horse or camel. More preferably, the construct comprises a BAC clone containing a sequence encoding the alpha-chain of an FcRn protein from a rodent, cattle (bovine) or rabbit, preferably rabbit or cattle. Most preferably, the construct comprises a rabbit BAC clone encoding the rabbit FCGRT gene. In a preferred embodiment, the construct used for transgenesis comprises rabbit BAC clone 262E02.

In a further preferred embodiment, a linearized BAC clone encoding the FcRn alpha chain, preferably a linearized rabbit BAC clone, in particular rabbit BAC clone 262E02, is microinjected into a first mammal, preferably rabbit, zygote and transferred into a pseudopregnant first mammal, e.g. rabbit, in order to generate a transgenic animal, e.g. rabbit (see also Catunda Lemos AP, Cervenak J, Bender B, Hoffmann Ol, Baranyi M, et al. (2012) PLoS One 7: e28869). If rabbits are used for transgenesis, the genetic background of the rabbits may e.g. be chosen from "New Zealand White" or "California" or hybrid strains thereof may be employed. The transgenic animal, e.g. rabbit, preferably comprises two additional copies of the alpha-chain of the rabbit FcRn protein (in the homozygous state). More than two copies may be provided as well.

For therapeutic or prophylactic administration to mammals, e.g. to humans, in particular in case of long-term administration, fully or partially human or humanized forms of antibodies as components of the ATG composition may be preferred. By "humanized" is preferably meant an alteration of the amino acid sequence of an antibody so that fewer antibodies and/or immune responses are elicited against the humanized antibody when it is administered to a human. For the use of the antibody in a mammal other than a human, an antibody of the invention may be converted to that species format. Such human or humanized antibodies, which are harvested from the first animal to provide the ATG composition may be obtained from transgenic non-human (first) animals that have been genetically engineered to express fully or partially human immunoglobulins. For example, polyclonal antibodies of such a modified type against thymocytes can be produced in the first mammal, e.g., horse, goats or, preferably, rabbits, which are transgenic not only in terms of FcRn alpha chain overexpression but also transgenic, as e.g. described in WO 2003/081993 and U.S. 2005/246782, in terms of antibody expression. Such animals have disrupted endogenous immunoglobulin production and, when challenged with e.g. a thymocyte suspension or any other thymocyte preparation, produce human or humanized immunoglobulins encoded by engineered human DNA incorporated in the first animal's DNA. In e.g. (in terms of antibody expression) transgenic rabbits, e.g. human or humanized immunoglobulins are produced and can be recovered from the first animal's blood. As additional examples, methods for producing (partially) human antibodies in transgenic non- human animals are described in, e.g., U.S. 2006/026696, WO 2005/007696, WO 01/19394, WO 2003/081992, WO 2003/097812 and WO 2004/044156.

Another method for producing human(ized) antibodies is described in U.S. 5,789,650 which describes transgenic mammals that produce antibodies of another species (e.g., humans) with their own endogenous immunoglobulin genes being inactivated. The genes for the heterologous antibodies are encoded by human immunoglobulin genes. The transgenes containing the unrearranged immunoglobulin encoding regions are introduced into a non-human animal. The resulting transgenic animals are capable of functionally rearranging the transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes.

Immunoglobulin fractions may be obtained by extraction of the anti-thymocyte immunoglobulins from the first mammal's immune body fluid, e.g. serum or blood. Typically, the body fluid containing anti-thymotic polyclonal antibodies of a larger number of immunized first transgenic animals is harvested and pooled or sampled prior to its further use and, optionally, assayed for anti-mesothelin antibodies using appropriate screening assays. To ensure a sufficient level of purity of these preparations, which may contain e.g. anti-basal membrane immunoglobulins produced by the first mammal during the immunization, several methods may be employed. Such techniques may include a step of hemadsorption, e.g. adsorption on human red blood cells to eliminate e.g. anti-human erythrocyte immunoglobulines, which may occur, if required. A step of removal of the anti- tissue antibodies, may also be envisaged, in order to e.g. adsorb the anti-basal membrane antibodies. Such steps are specifically preferred, if anti-human thymocyte immunoglobulins shall be prepared according to the present invention, but hemoadsorption may be envisaged for any other anti thymocyte immunoglobulines which may be adsorbed on the erythrocytes of the tissue of the respective mammal. Red blood cells or adsorption on (human) tissues (such as placenta in particular), stroma or crude extracts of these tissues may be used for the hemadsorption step.

Alternatively, the ATG raw material, in particular if production of ATG based on anti-human thymocyte immunoglobulins is envisaged, may be purfied without a step of (hem)adsorption. Upon the harvesting step of the immune serum produced by the first mammal, the anti-thymocyte immunoglobulins, e.g. the anti-human thymocytes from the serum, are preferably isolated such that the total fraction of gammaglobulins from the serum after immunization is obtained. Avoidance of the (hem)adsorption steps does not allow for viral contamination or contamination with unconventional agents of the prion type. In addition, any contamination associated with (hem)adsorption, namely contamination with the hemoglobin released by the (e.g. human) red blood cells during the hemolysis is avoided. Thereby, the safety is improved and the cost of the preparation are reduced. That holds in particular, as (hem)adsorption conventionally uses whole (e.g. human) red blood cells which are fresh and formalin-treated, which requires large quantities of cells to be treated, making the production of antithymocyte immunoglobulins intricate.

Isolation of the anti-thymocyte immunoglobulins, in particular anti-human thymocytes, from the body fluid of the first mammal, in particular blood or serum, of the first mammal may be carried out and makes it possible to eliminate other undesired proteins of whatever nature, in particular (serum) proteins other than immunoglobulins. Such proteins for elimination do not necessarily, but preferably include immunoglobulins against non-thymotic targets. Such an elimination may be achieved by using adequate protein purification methods, e.g. chromatographic methods, e.g. ion exchange chromatography, preferably on a column, and/or one or more precipitations. Advantageously, such a precipitation step can be carried out in two or more successive steps using an immunoglobulin precipitating reagent. The reagent preferentially used is sodium or ammonium sulfate or other well-known precipitation reagents, e.g. polymers, like dextran or polyethylene glycols. The antibodies harvested can also be purified by one skilled in the art using standard techniques such as those described by Ausubel et al. (Current Protocols in Molecular Biology, volume 2, p. 1 1 .13.1 -1 1 .13.3, John Wiley & Sons, 1995). The antibody is desirably at least 2,5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or western analysis to detect a reduction in the amount of contaminating proteins or ELISA to detect an increase in specific activity for binding to markers for the T cell subpopulation in general or specific types of T cell specifically..

The chromatography step e.g. may be based on an ion exchange resin, such as anions (DEAF). The IgG fraction is not retained by the column and rapidly removed from the column, which makes it possible to harvest them selectively. As an alternative or additional step, specific affinity column chromatography may be employed, which removes undesirable antibodies against other target structures. Additionally, preferably prior to the chromatography step, various steps of filtration, concentration, diafiltration and/or precipitation of the immunoglobulin fraction (for example alcoholic fractionation of the COHN type or ammonium sulfate fractionation) can be carried out.

The ATG composition is preferably purified by separating the relevant immunoglobulines, in particular the anti-thymotic immunoglobulines, more particularly the IgG fraction of anti- thymotic immunoglobulines, from other components that accompany such immunoglobulines in the harvested blood or serum sample. Typically, the fraction to be purified, e.g. as mentioned above, is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated.

Another aspect of the present invention encompasses the isolated and preferably purified ATG composition as obtainable or obtained according to the present invention. Preferably, the (preferably purified) ATG composition according to the invention contains immunoglobulines, whereby at least 80%, preferably at least 90 %, more preferably at least 95% and even more preferably at least 99% of the immunoglobulines of such a (purified) fraction of the harvested body fluid (typically containing a larger variety of immunoglobulins prior to the purification step) are anti-thymocyte immunoglobulins, preferably anti-human thymocytes (if human thymocytes were used for immunization) with only a low fraction of remaining immunoglobulines recognizing non-thymocytic targets. Preferably, such a (purified) immunoglobuline fraction is essentially composed of the IgG- type immunoglobuline sub-fraction, which means that at least 80%, preferably a least 90%, more preferably at least 95% of the anti-thymocyte immunoglobulines of such a purified fraction are of the IgG type, the remainder, if any, being of other immunoglobuline types, e.g. IgM or IgA. The IgG purity of the subfraction may be tested by e.g. agarose gel electrophoresis. Alternatively, however, the composition obtained according to the present invention is a mixture of various immunoglobuline subtypes, as no purification step to isolate specifically the IgG subtype is applied. Such compositions may contain IgG and IgM, if just these two immunoglobuline subtypes have been isolated or it may contain all subtypes of immunoglobulins, preferably (as a result of purification) all subtypes of anti- thymocytic immunoglobulines, e.g. anti-human thymocytic immunoglobulines.

It may also be prefered to harvest ATG compositions from the first mammal which contain IgG polyclonal antibodies which do not contain the full-length sequence, but rather a fragment therof, e.g. a fragment without an Fc portion. Hereby, the polyclonal antibodies are harvested from the first animal, which correspond to full length polyclonal antibodies. Thereafter, an additional step, is typically carried after harvesting and purification of the full- length antibody fraction from the first ammal, if ATG compositions (containing partial sequences of the full-length antibodies) are envisaged. E.g. the Fc portion of the ensemble of full-length polyclonal antibodies is removed such that the final polyclonal ATG composition essentially contains e.g. F(ab') ₂ and/or Fab fragments of the polyclonal antibodies produced by the first mammal. Hereby, the harvested polyclonal antibody fraction may be subjected to known techniques for obtaining purified Fab and F(ab') ₂ fragments therefrom. Specifically, harvested ATG polyclonal antibody preparations may be subjected to treatment with effective amounts of papain or pepsin and then subjected to chromatographic methods, e.g. column chromatography, for the isolation of Fab or F(ab') ₂ fragments, respectively. Techniques for obtaining both Fab and F(ab') ₂ fragments from polyclonal antibody preparations are well known in the art (see, e. g., Abbas et al., Cellular and Molecular Immunology, 2nd ed., W. B. Saunders Company (1994) and the Examples below) and can be readily employed for the preparation of both the Fab and F(ab') ₂ anti-thymotic polyclonal antibodies. By administering ATG compositions only containing such fragments without an Fc portion, massive lymphocytopenia in the patient, who receives the ATG composition for prophylactic or therapeutic purposes, may be avoided. Prior to its use, the ATG composition of the invention as obtainable by the present invention may be tested by assays. E.g. the optionally purified ATG composition may be tested to determine its pH, its bacterial or fungal sterility or it may be tested for pyrogens. Also hemolysin titres and hemagglutinin titres may be determined.

The host, once an ATG composition of the invention is used for whatever purpose, as recipient may be domestic animals, pets, laboratory animal and, in particular humans. Therefore, it is another aspect of the present invention to provide a method of therapeutic or prophylactic treatment in which a therapeutically effective quantity of immunoglobulins, in particular anti-thymotic immunoglobulins, thus obtained is administered to a patient or subject (for human or veterinary treatment), e.g. requiring his lymphocyte load to be decreased. Such methods may be applied in transplant surgery prior, in the course and/or after organ transplantation. Other therapeutic methods directed to the treatment of other disease or disorders are described in the following in the context of the use of an ATG composition according to the present invention.

The present invention is directed to the use of the ATG compositions obtainable or obtained according to the present invention for producing a medicament for decreasing the quantity of circulating lymphocytes of the blood and of the lymphoid tissues in a human or non- human mammal, in particular in a human patient. The use may be therapeutic and/or prophylactic. The inventive anti-thymocyte polyclonal antibodies (in particular anti-human thymocytic), in particular the IgG fraction thereof, may e.g. be used as selective immunosuppressants. They act on the immune response by decreasing, by depletion, according to various mechanisms, the quantity of circulating lymphocytes of the blood and of the various lymphoid tissues of the patient/subject to be treated. The depletion (via e.g. ADCC and/or activation induced apoptotic cell death) may be due to receptor mediated complement dependent lysis, opsonization, and subsequent phagocytosis by macrophages and/or immunomodulation. Such therapeutic/prophylactic methods of depleting thymocytes are within the scope of the present invention.

Accordingly, "ATG compositions" according to the invention and as obtainable as descirbed above may be used for the treatment of various clinical conditions including prevention or rescue treatment of acute rejection in e.g. solid organ transplantation, for conditioning for hematopoietic stem cell transplantation, for the treatment of severe aplastic anemia, or for the treatment of various autoimmune diseases, or for the treatment or prevention of acute or chronic graft-versus-host disease (GVHD), in particular for the treatment or prevention of steroid-resistant acute or chronic graft-versus-host disease (GVHD), and for the treatment of B cell malignancies and multiple myeloma .The immunosuppressive activity of ATG is expected to result from the depletion of peripheral lymphocytes from the circulating pool through complement-dependent lysis or activation- associated apoptosis (Beiras-Fernandez et al., Exp. Clin. Transplant. 1 :79-84 (2003); Genes- tier et al., Blood 91 :2360-2368 (1998); Michallet et al., Transplantation 75:657-662 (2003); Zand et al., Transplantation 79:1 507-1515 (2005)). Other potential mechanisms of action include modulation of surface adhesion molecules or chemokine receptor expression (Brennan, Transplantation 75:577-578 (2003)).

If used for transplantation associated disorders or conditions, the use of ATG compositions of the invention is specifically directed to administration before; during and/or after transplantation of organ or tissue transplants (e.g. kidney, heart, lung, eye lid, gut, vascular vessel or liver transplants), e.g. for the prevention and treatment of transplant or tissue graft rejection, and also for the treatment of acute graft versus host reaction (e.g. for the treatment of steroid-resistant acute rejection) The ATG composition according to the invention enables host or donor to accept the transplanted organ, tissue or cell. Accordingly, the ATG composition according to the invention may also be used for the treatment of tissue or cell transplantation, e.g. beta-islet cells or bone marrow cells. In any case, the transplant, be it cells, tissue or organ, may be either allogeneic or xenogeneic.

The inventive ATG composition may also be used in the treatment of medullary aplasia or aplastic anemia, in particular for the treatment of patients which are unsuitable or suitable for bone marrow transplantation, and bone marrow aplasia. It may also be used for the treatment of aplastic anemia which is secondary to a neoplastic disease, storage disease, myelofibrosis or Fanconi's syndrome.

The use of the inventive ATG composition also refers to the treatment of (blood) cancer diseases, e.g. B-cell malignancies and multiple myeloma. Since ATG compositions may be provided according to the invention which contain antibodies against antigens expressed on various hematopoetic cells (and not only T cells), they may induce cell death not only in healthy T-, B-, NK, and dendritic cells but also in malignant cells of lymphatic and potentially of myeloid lineage. Thus, the inventive ATG composition may also be used in the treatment of such hematological malignancies.

Other fields of use include the area of autoimmune diseases to counteract the auto- aggressive immune reaction in the subject to be treated. Examples of autoimmune diseases, which may be addressed by the treatment with the inventive ATG composition, are rheumatoid arthritis, Crohn's disease, asthma, Alzheimer, Insulin-dependent diabetes mellitus, multiple sclerosis, Acute disseminated encephalomyelitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyotrophic lateral sclerosis, Motor Neuron Disease, Ankylosing Spondylitis, Antiphospholipid syndrome, Antisynthetase syndrome, Antisynthetase syndrome, Atopic allergy, Atopic dermatitis, Atopic dermatitis, Aplastic anemia, Autoimmune cardiomyopathy, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Churg-Strauss syndrome, Autoimmune urticaria, Autoimmune uveitis, Balo concentric sclerosis, Beh et's disease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, Bullous pemphigoid, Cancer, Castleman's disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal osteomyelitis, Chronic obstructive pulmonary disease, Churg-Strauss syndrome , Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome, Crohn's disease (one of two types of idiopathic inflammatory bowel disease "IBD"), Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformi, Dermatomyositis, Diabetes mellitus type 1 , Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Drug-induced lupus erythematosus, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Eosinophilic gastroenteritis, Eosinophilic pneumonia, Epidermolysis bullosa acquisita, Erythema nodosum, Erythroblastosis, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibrosing alveolitis, Idiopathic pulmonary fibrosis, Gastritis, Gastrointestinal pemphigoid, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schonlein purpura, Herpes gestationis, Gestational Pemphigoid, Hidradenitis suppurativa, Hughes-Stovin syndrome, Hypogammaglobulinemia, Idiopathic inflammatory demyelinating diseases, Idiopathic pulmonary fibrosis, Autoimmune thrombocytopenic purpura, Idiopathic thrombocytopenic purpura, nephropathy, Inclusion body myositis, Chronic inflammatory demyelinating polyneuropathy, Interstitial cystitis, Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lupoid hepatitis, Autoimmune hepatitis, Lupus erythematosus, Majeed syndrome, Meniere's disease, Microscopic polyangiitis, Miller-Fisher syndrome, Guillain-Barre Syndrome, Mixed connective tissue disease, Morphea, Mucha-Habermann disease, Pityriasis lichenoides et varioliformis acuta, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (also Devic's disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Opsoclonus myoclonus syndrome, Ord's thyroiditis, Palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonage-Turner syndrome, Pars planitis, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary, sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatic fever, Sarcoidosis, Schizophrenia, Schmidt syndrome (another form of APS), Schnitzler syndrome, Scleritis, Scleroderma, Serum Sickness, Sjogren's Syndrome, Spondyloarthropathy, Juvenile idiopathic arthritis, Juvenile Rheumatoid Arthritis, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham chorea, PANDAS, Sympathetic ophthalmia, Systemic lupus erythematosus, Takayasu's arteritis, Temporal arteritis (also known as „giant cell arteritis", Thrombocytopenia, Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis (one of two types of idiopathic inflammatory bowel disease "IBD"), Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Urticarial vasculitis, Vasculitis, Vitiligo, and Wegener's granulomatosis. The ATG composition obtainable according to the invention may be provided as a medicament and may preferably be prepared in formulations comprising pharmaceutically acceptable media, in particular liquid media, e.g. saline, PBS, aqueous ethanol etc. or liquid polyethylene glycol. A (preferably sterile) aqueous solution may preferably contain the polyclonal anti-thymotic antibodies and, if required further components. Preferably, the liquid solution is stabilized. Sterility may be established by filtration through a sterile membrane. The ATG composition may also be provided in solid, e.g. lyophylized, form (preferably in powder form) and diluted prior to its administration, preferably in water for injection (e.g. 5 mg/ml). Preferably, complete dissolution is envisaged and recorded. The freeze-dried form preferably contains or is composed of one or more of the following components: a salt (in particular a chloride salt, e.g. NaCl), a sugar component (e.g. mannitol) and/or an amino acid (e.g. glycine) in addition to the anti-thymocyte antibody. In a preferred embodiment, the liquid formulation, e.g. the sterile aqueous solution (water for injection, WFI), contains purified polyclonal antibodies, e.g. (monomeric) IgG polyclonal antibodies, against thymocytes, preferably directed against human thymocytes. The liquid formulation may be buffered, e.g by a phosphate, acetate or citrate buffer. The pH of that solution is preferably from pH 6.5 to 7.5, more preferably from 6.7 and 7.2, even more preferably from 6.8 to 7.0. It may contain stabilizers, e.g. glycine, dextrose etc., and/or may contain bactericidial agents, e.g. benzyl alcohol. More specifically, further components of a liquid formulation may be selected from one or more of Tween 80, formaldehyde, isopropyl alcohol, disodium phosphate, hydrochloric acid, sodium hydroxide, mannitol, NaCl, and glycine. Typically, the (concentrated) solution will contain between 10 mg and 100 mg anti- thymocyte immunoglobulin, in particular anti-thymocyte IgG, more preferably between 20 and 80 mg and more preferably between 40 and 60 mg per ml solution.

The solution will typically be isotonic, e.g. by the addition of an osmolar component, which may be a salt (e.g. a saline solution of typically less than 1 %) or e.g. a sugar or sugar polymer (dextran etc.). Dextrose may be incorporated into the solution (e.g. from 3 to 8%). If provided as solution, the solution should be stored at 2 to 8°C. The ATG composition for medical use may also be delivered in non-liquid form, in e.g. lyophylized form. Dilution should - under such circumstances - be carried out shortly before administration. The solution for dilution of the non-liquid form is typically as described above, e.g. an isotonic solution, which may be buffered and may contain further pharmaceutically acceptable components. By another embodiment, the polyclonal ATG antibodies (e.g. as isolated from the first animal's body fluid, optionally purified and/or furthermodified) as obtainable by the present invention may be encapsulated for providing a formulation thereof, e.g. in the lumen of liposomes, to extend their life time in vivo. Other modifications of the ATG polyclonal antibodies may be envisaged as well either to extend their half-life in vivo or to influence their distribution in the body of the patient or to diminish or enhance binding to blood components upon administration.

Modifications of polyclonal ATG antibodies (either for extending the half-life in vivo or for any other purpose) result in antibody-derived molecules ("derivative") which may be covalently attached to the polyclonal antibodies either via a linker, which may be cleavable or not, or without a linker, or may be provided by gene fusion technology. The attachment will typically be realized via functional amino acid side chain groups (e.g. hydroxy, thiol, amino, carboxyl) or via the N- and/or C-termini of the polypeptide chains of the antibodies/antibody fragments. Such modifications may be based on covalent attachment of e.g. toxic compounds, labels or (affinity) tags or carbohydrates (glycosylation, e.g. fucosylation), lipids, PEGs (pegylation), phosphate (phosphorylation) or other synthetic or non-synthetic polymers. "Affinity tag" is preferably meant to refer to a peptide, protein, or compound that binds another peptide, protein, or compound. In a desirable embodiment, the affinity tag is used for purification or immobilization of the derivative. In another desirable embodiment, the affinity tag or toxin is used to target the antibody or fragment to a specific cell, tissue, or organ system in vivo. In still another desirable embodiment, the fluorescent or radiolabel is used for imaging of the derivative. In yet another desirable embodiment, the therapeutically active compound or radiolabel is used for the treatment or prevention of a disease or disorder. In another embodiment, the derivative or fragment of an antibody of the invention has increased stability or increased solubility compared to the antibody. It is also contemplated that the antibody or its fragment, or derivative of the invention may be bound non-covalently to another antibody covalently linked to a toxin, therapeutically active compound, enzyme, cytokine, radiolabel, fluorescent label, magnetic label, or affinity tag.

Slow release composition may be provided as well to achieve an enhanced half life in vivo, e.g. by appropriate matrix formulations. The ATG composition according to the invention is typically administered to the mammal to be treated, in particular a human, by injection or infusion, e.g. needle injection or needle-free injection methods. Preferably, intradermal, intramuscular or subcutaneous or intravenous or intranodal injection is preferred, but any other route of injection is possible as well. Parenteral administration is preferred.

The dosage regimen of the ATG composition obtainable according to the invention depends on various factors, e.g. which ATG composition is administered, e.g. a purified IgG subfraction or a whole immunoglobuline fraction (without an isolation step for anti- thymotic antibodies), on which antibody concentration is provided by the inventive ATG composition and on which disorder is treated and on ATG's efficacy to deplete the patient's T cells in the peripheral blood or other lymphoid organs and further depend on which subject is treated (human or vererinary use). Moreover, the dosage regimen depends on age, sex and other factors that influence the therapy regimen. For immunosuppression prior, in the course and/or after transplantation, e.g. for adult renal allograft patients, 0.5 mg/kg to 50 mg/kg body weight per day, e.g. for less than 28 days, in particular for 2-14 days). While usually administration for no more than 28 days after transplantation surgery is preferred, it is quite possible that under specific circumstances the treatment may be administered chronically, e.g. as long as the implant is present in the host. For the treatment of aplastic anemia a dosage regimen of e.g. 1 .0 mg kg to 30 mg/kg body weight per day for e.g. less than 1 5 days, in particular 3 to 10 days, may be appropriate depending on the particularities of each individual patient. The administration may be carried out on a daily basis or may be carried out according to an interval scheme of administration (e.g. once or twice every two, three or more days). It is preferred, if the dosage is chosen such that the patient's or subject's total serum levels of the anti-thymocytic immunoglobulines administered are between 10-500 pg/ml, more preferably between 10-100 pg/ml, e.g. 10 to 1 50 Yg/ml may be preferred. It may be the object to establish a steady-state of the ATG serum concentration for the patient/subject treated over a period of time, e.g. two to six weeks. Under such circumstances, continuous infusion of the ATG composition (instead of repetitive dosages) may be envisaged.

The use or the method of treatment of the ATG composition obtainable according to the present invention may be accompanied by a co-therapy, e.g. with other immunosuppressants, e.g. anti-metabolites or corticosteroids or immunophilin binding agents (e.g. calcineurin inhibitors or mTOR inhibitors) or other antibodies (e.g. IVIG, anti- CD25 and/or anti-CD3 monoclonal antibodies, e.g. basiliximab) or inhibitors of de novo nucleotide synthesis (e.g. mycophenolic acid and/or leflunomide). Under certain circumstances, e.g. for allograft patients or for any condition, which requires further immunosuppression, concomitant administration of another immunosuppressant, e.g. azathioprine, cyclosporin A, FK506, tacrolimus, sirolimus, everolimus, dactinomycin (in particular for kidney transplantation) or of a corticosteroid, e.g. triamcinolone, dexamethason, hydrocortison, fluocortolone, and/or prednisolone, may be envisaged, in particular to reduce e.g. the to suppress the patient's immune system to avoid an immune reaction against the ATG composition. Double or triple co-therapy by co-administration of more than one additional immunosuppressants (e.g. two or three immunosuppressants of the same or different classes (e.g. a triple therapy by co-administering an antibody (other than the polyclonal ATG composition of the invention), a corticosteroid and an immunophilin binding agent) may be envisaged, e.g. administration of ATG compositions in co-therapy with both, a corticosteroid (e.g. prednisolone) and cyclosporine or e.g. with tacrolimus and cyclosporine or e.g. with tacrolimus and a corticosteroid (as options of a double co-therapy). For patients suffering from aplastic anemia (prophylactic) coadministration (in particular transfusion) of platelets may be envisaged as co-therapy. Such co-administration is preferably based on concomitant administration, whereby the ATG composition of the invention may be administered together, prior or after the co-therapeutic medicament by the same or a different administration route.

The ATG composition obtainable according to the invention may also be used for nonmedical application, i.e. for in vitro or ex vivo use or for in vitro or ex vivo methods. Anti- thymocytes antibodies of the ATG composition may e.g. be used in vitro to select specific thymocytes of cell samples. Also in vitro studies for analyzing mechanisms underlying antibody-induced immunosuppression may be carried out by using the inventive ATG compositions. In vitro cell lysis inhibition of target antigen presenting cells by effector cells may be studied. Accordingly, in e.g. basic research it may be envisaged to inhibit lysis so that the cellular population may be maintained while under investigation. Hereby, one may wish to maintain mixtures of cells, where cytotoxic lymphocytes would be activated and kill antigen presenting cells, such as macrophages or B-lymphocytes, or other cells, which might serve as target cells, e.g., neoplastic cells, viral infected cells, or the like.. Alternatively, such a composition according to the invention may be cultured in vitro together with T-lymphocytes, e.g. in order to generate or to promote the generation of functional regulatory T cells. Hereby, the regulatory T cell population is expanded by either converting non-regulatory T cells into regulatory T cells or by maturing pre-mature regulatory T cells.

An ex vivo application may be directed to preparing organs, tissue, cells for implantation by bathing the xenogeneic or allogeneic donor material in a medium comprising the ATG composition according to the invention. In this way, cytotoxic lymphocytes present within the implant will be prevented from participating in graft-versus-host disease. Also, during the period when the subject antibodies remain bound to the implant, the recipient's lymphocytes will be inhibited from being activated. Generally, the concentration of the antibodies will vary in the medium, depending upon the activity of the antibodiess, the level of inhibition desired, the presence of other compounds affecting lymphocyte activation, and the like. Other immunosuppressants which may additionally be present in the bathing medium include cyclosporin A, FK506, and the like. Subtherapeutic dosages will be employed, generally when present, not less than about 1 % of the normal dosage, and not more than about 75%, usually in the range of about 5 to 50%. Other components of the bathing medium will generally be constituents normally used in an organ preservation solution, e.g. HBSS. The time for the organ to be maintained in the bathing medium will generally be in the range of about 2 to 72 hours.

Advantages of the present invention

By using the method according to the invention, the amount of ATG obtained per mammal is increased in comparison to the amount obtained in a reference animal, which does not overexpress the alpha-chain of a mammalian FcRn protein.

Furthermore, in comparison to conventional methods for producing ATG, a wider spectrum of B cell clones producing antibodies against thymocytes or T-lymphoblastic cells, such as Jurkat cells, is obtained in an individual animal. As a consequence, ATG obtained by the method according to the invention is characterized by a greater amount and by a greater diversity of antibodies targeting a wider group of antigens expressed on T cells and other hematopoetic cells including T-, B-, NK, dendritic, and plasma cells as compared to ATG produced by conventional methods.

The ATG composition obtainable or obtained according to the present invention is characterized by significant advantages over ATG compositions known from the art or currently commercialized. These advantages are outlined in the following and refer to pooled ATG compositions, e.g. composition which are obtained from e.g.pooled samples of blood or serum of a larger number of first mammals immunized, e.g. pooled from at least 5 mammals, preferably at least 10 mammals or further preferably more than 50 mammals:

Employing the method according to the invention, the pooled ATG yield (i.e. the harvestable amount of anti-thymotic immunoglobulines, in particular anti-thymotic IgG, is preferably enhanced by a factor of at least 1 .5, more preferably at least by a factor of 2.0, even more preferably at least by a factor of 2.5 and further preferably at least by a factor of 3.0 and further preferably by a factor of at least 3.9. as compared to anti-thymotic samples derived from the same number of wt reference animals of the same species (not overexpressing FcRn), whereby the ATG composiions are treated in the very same way. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits by a method according to the invention as compared to identical methods by using wt reference rabbits.

Specifically, the total IgG concentration in a pooled anti-thymocyte polyclonal serum produced according to the method of the invention is elevated as compared to the total IgG concentration in anti-thymocyte polyclonal ATG composition obtained according to prior art methods using a wt reference animal of the same species. Preferably, the total IgG concentration in the serum obtained or obtainable according to the invention is elevated at least by a factor of 1 .2, more preferably at least by a factor of 1 .3, even more preferably at least by a factor of 1 .4 and most preferably at least by a factor of 1 .5. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits by a method according to the invention as compared to the same method using wt reference rabbits.

Furthermore, the IgG mediated cytotoxicity of a pooled ATG composition obtainable or obtained by using the method of the invention is increased in comparison to the IgG mediated cytotoxicity of ATG composition obtained by using a reference mammal of the same species not overexpressing the alpha-chain of a mammalian FcRn protein. Preferably, the IgG mediated cytotoxicity is increased at least by 100 percent, more preferably at least by 150 percent, even more preferably at least by 200 percent and most preferably at least by 250 percent. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits by a method according to the invention as compared to identical methods using wt reference rabbits.

The thymocyte binding capacity of pooled anti-thymocyte polyclonal ATG composition obtained or obtainable by the method according to the invention is also increased as compared to the binding capacity of a reference ATG composition obtained by using wt reference mammals of the same species not overexpressing the alpha-chain of a mammalian FcRn protein. Preferably, the thymocyte binding capacity of anti-thymocyte polyclonal ATG composition obtainable by the method according to the invention is augmented at least by 100 percent, more preferably at least by 150 percent, even more preferably by at least 1 75 percent and most preferably by at least 200 percent. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits obtainable by a method according to the invention as compared to identical methods using wt reference rabbits.

Accordingly, the number of mammals required for producing a certain amount of ATG is significantly reduced by using the method according to the invention, preferably by at least 20 percent, more preferably by at least 30 percent, even more preferably by at least 40 percent, even more preferably by at least 50 percent, even more preferably by at least 60 percent and most preferably by at least 70 percent. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits obtainable by a method according to the invention as compared to identical methods using wt reference rabbits.

Moreover, the thymocyte binding capacity of ATG (e.g. IgG purified from anti-thymocyte polyclonal serum) produced according to the method of the invention is higher than the thymocyte binding capacity of e.g. IgG purified from anti-thymocyte polyclonal serum produced according to a conventional method, in particular higher than the thymocyte binding capacity of e.g. IgG purified from anti-thymocyte polyclonal serum produced using a mammal not overexpressing the alpha-chain of a mammalian FcRn protein. Preferably, the thymocyte binding capacity of e.g. IgG purified from anti-thymocyte polyclonal serum obtained by the method according to the invention is increased at least by 15 percent, more preferably at least by 30 percent, even more preferably by at least 45 percent and most preferably by at least 60 percent. Such advantageous properties hold in particular for ATG compositions derived from FcRn transgenic rabbits obtainable by a method according to the invention as compared to identical methods using wt reference rabbits.

Finally, in comparison to conventional methods for producing ATG, a wider spectrum of B cell clones producing antibodies against thymocytes is obtained in an individual animal. As a consequence, ATG obtained by the method according to the invention is characterized by a greater diversity of antibodies specific for thymocytes with respect to ATG produced by conventional methods. This can be verified by using model antigens (such as ovalbumin or α,-antitrypsin) and measuring the number of B cell clones specific for each of those antigens.

Examples

The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.

1 . Animals and housing

Tg rabbits that have enhanced FcRn activity because they carry and express one extra copy of the rabbit FcRn alpha-chain encoding gene (rabbit FCGRT) in addition to the endogenous rabbit FCGRT gene on New Zealand White (NZW) rabbit genetic background are coded as NZW Tg1 rabbit FCGRT (78) wherein 78 refers to the founder line (Catunda Lemos AP, Cervenak J, Bender B, Hoffmann Ol, Baranyi M, et al. (2012) Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn. PLoS One 7: e28869). These Tg rabbits and their wild type (wt) littermates were housed in specified pathogen free facility. 262E02, a rabbit BAC clone containing the FcRn alpha-chain coding sequence (FCGRT) located on a 1 10 kb genomic insert was identified using PCR screening method A rabbit BAC library which was constructed from white blood cells of a New Zealand rabbit was used to identify a BAC clone that contains the rabbit FCGRT gene, with rabbit FcRn specific PCR screening method (primers: OCU_FCGRTf: 5'-GGG ACT CCC TCC TTC TTT GT-3' and OCU_FCGRTr: 5' AGC ACT TCG AGA GCT TCC AG-3').

BAC transgenic rabbits were generated with the linearized 262E02 BAC clone harboring the rabbit FCGRT gene and its regulatory region which was microinjected into fertilized rabbit zygotes. BAC clone 262E02 harboring the rabbit FCGRT gene was purified using Qiagen Large-Construct kit (Qiagen GmbH, Germany) and was linearized by Pmel restriction enzyme digestion. Subsequently, the linearized DNA was run on a pulsed-field gel and recovered from the gel by GEIase digestion (Epicentre Biotechnologies, Madison, Wl, USA) [72]. The linearized BAC DNA was injected into pronuclei of New Zealand White rabbit zygotes. Injected eggs were transferred into pseudopregnant females. Genomic DNA samples of founder rabbits were collected from ear biopsies. The founder rabbits were genotyped by a pair of primers: 5'-CGA AAC AGT CGG GAA AAT CT-3' and 5'-GGC ATC GTG TGT AAG CAG AA-3' which are specific for the BAC backbone. Transgenic rabbit lines were maintained by sibling mating. 1 724 injected embryos were transferred into 95 pseudopregnant females. Altogether 125 rabbits were born from 29 does. Five transgenic founders were identified by BAC backbone specific PCR primers, one of which was stillborn. Three transgenic lines were originally established carrying the transgene. One transgenic line - #78 - was selected to further characterize its immunophenotype. BAC copy number was determined by absolute quantification of the transgene by real time PCR and found that one and two extra copies of the transgene had been integrated in hemizygous and homozygous Tg rabbits, respectively.

To determine the copy number of the BAC construct carrying the rabbit FCGRT transgene integrated into the genome, a quantitative PCR was carried out using primers designed to amplify a 250 bp fragment of the BAC sequence (5'-CGA AAC AGT CGG GAA AAT CT-3' and 5'-GGC ATC GTG TGT AAG CAG AA-3'). Two-fold dilutions of the purified BAC DNA samples were spiked into 1 .5 ng/μΙ rabbit genomic DNA (final concentration). This created a series of standard samples such that the ratio of BAC molecules ranged from 1 to 1 6 BAC copies per diploid rabbit genome. These samples were used to generate calibration curve with equation for estimating the copy number from sample rabbit line #78. Amplification was analyzed using the Power SYBR Green PCR Master Mix (Life Technologies, USA) run on Rotorgene RG-3000.

Homozygous transgenic animals were selected using genomic qPCR. qPCR was conducted with 40 ng DNA in a 20 μΙ reaction mix using the TaqMan PCR Universal Master Mix (Applied Biosystems, USA). Custom TaqMan chemistry assays were used for assaying rabbit β-actin (endogenous control) and rabbit FcRn (Life Technologies, USA and Integrated DNA Technologies, Germany, respectively) run on Rotorgene RG-3000.

Integrated extra copies of the rabbit FcRn gene in homozygous Tg rabbits by quantitative genomic PCR were detected by using ΔΔΟ method (RotorGene software, Corbett Research, Sidney. To test if rabbit FcRn expression increases in Tg rabbits, real time quantitative RT- PCR was setup, in which RNA was isolated from rabbit leukocytes with the RNeasy Plus Mini kit (that includes a DNase digestion step, Qiagen GmbH, Germany) and first strand of cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, USA). Quantitative PCR was performed with RotorGene RG-3000 and analyzed.

To distinguish between the rabbit FcRn endogene and transgene expression, a quantitative real time RT-PCR assay was established. Rabbit FcRn levels were normalized to rabbit beta- actin. RNA was isolated and pooled from the leukocytes of 5-5-4 homozygous, hemizygous and control animals, respectively. Elevated rabbit FcRn levels were detected in the leucocytes of the transgenic animals (1 .83-fold and 2.49-fold higher expression of the rabbit FcRn mRNA in hemizygous and homozygous animals, respectively, than those of the wild- type rabbits).

In conclusion, the data show that the integrated transgene is expressed as the rabbit FcRn alpha-chain mRNA level is higher in both the hemizygous and homozygous rabbits as compared to their wt controls.

In order to analyze if FcRn overexpression results in reduction of IgG catabolism in rabbits, the pharmacokinetic behavior of rabbit IgG in Tg (+/+) animals that carry two extra copies of the rabbit FcRn was analyzed and compared these results with wt rabbits. The clearance curves of the measured OVA-specific IgG were biphasic, with phase 1 (alpha phase) representing equilibration between the intravascular and extravascular compartments, and phase 2 (beta-phase) representing a slow elimination. Beta phase half-lives of rabbit IgG from day 2 to 13 were analyzed. It was found that the Tg rabbits demonstrated increased serum persistence of rabbit igG because the beta phase half-lives were 7.1 + 0.46 days (mean ± SEM) as compared to their controls which showed 5.3 ± 0.3 days. Following a pre- bleed, five, age and weight matched Tg (homozygous #78 animals that carry two extra copies of the rabbit FcRn; Tg +/+) and wt siblings as controls, respectively, were injected intravenously (ear vein) with a single injection of 1 mg anti-OVA rabbit IgG in 1 ml of sterile PBS, and blood samples were collected from ear vein during the next 13 days. OVA specific IgG levels in the serum samples were measured by an ELISA assay (as it is described below) and expressed as OD values. The serum concentrations of OVA specific IgGs were presented as percent remaining in the circulation at different time points after injection compared with day 1 values (100%). IgG clearance data was analyzed by fitting the data of days 2-13 to the one-compartmental model using WinNonLin professional, version 5.1 (Pharsight, Mountain View, CA).

As a result, it was concluded that the transgenic FcRn was functionally expressed and elongated the half-life of rabbit IgG in these animals.

2. Immunizations

Preparation of anti-thymocyte serum:

Jurkat E6.1 T cells obtained from the European Collection of Cell Cultures (Salisbury, UK) were used as antigen. Before immunization, Jurkat cells were harvested, washed twice with phosphate buffered saline (Sigma-Aldrich, Budapest), and used as a suspension at a concentration of 1 x10 ⁸ cells per ml. 10 Tg and 10 littermate wt rabbits received two successive intravenous injections 14 days apart of 5x10 ⁸ living Jurkat T cells, as was suggested in earlier reports, indicating that longer courses of injection usually yields less effective antisera (Levey RH, Medawar PB (1 966) Nature and mode of action of anti lymphocytic antiserum. Proc Natl Acad Sci U S A 56: 1 130-1 137; Jooste SV, Lance EM, Levey RH, Medawar PB, Ruszkiewicz M, et al. (1968) Notes on the preparation and assay of anti-lymphocytic serum for use in mice. Immunology 1 5: 697-705). Blood samples were collected before the first immunization and on days 7, 14 and 21 and Jurkat-specific antibodies were analyzed by binding studies using flow cytometry based on previous report (Preville X, Nicolas L, Flacher M, Revillard JP (2000) A quantitative flow cytometry assay for the preclinical testing and pharmacological monitoring of rabbit antilymphocyte globulins (rATG). Journal of Immunological Methods 245: 45-54). Sera were prepared by centrifugation, heated to 56°C for 30 minutes, and stored at -20°C. Normal rabbit serum as complement source for cytotoxicity assays was obtained from non-immunized NZW rabbits and processed in an identical manner but without heating.

Immunization with soluble antigens:

Ovalbumin (OVA) immunization: 7 Tg and 7 wt rabbits were immunized subcutaneously with 300 pg OVA (Sigma-Aldrich, Budapest) in complete Freund's Adjuvant (CFA) and challenged 21 days later with 150 g OVA in incomplete Freund's Adjuvant (IFA). Human <x1 -antitrypsin (hA1 AT) immunization: 10 Tg and 4 wt rabbits were immunized subcutaneously with 300 g hA1 AT (Sigma-Aldrich, Budapest) in CFA and challenged 21 , 42 and 63 days later with 1 50 pg antigen in IFA.

3. Protein G-agarose chromatography for IgG purification

Serum samples of Tg and wt rabbits were pooled, diluted with binding buffer (0.2 M Na- phosphate buffer, pH 6.0) and bound to Protein G agarose beads (Pierce Protein G Plus Agarose, Thermo Scientific) by incubation for 30 minutes at room temperature with slow agitation. IgG was eluted using 0.1 M Na-citrate buffer (pH 2.9), dialyzed against PBS, aliquoted and stored at -20°C until use.

4. Flow cytometry

Flow cytometry measurements were carried out using fluorescence-activated cell sorting (FACS) on a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA). Results were analyzed using FCS Express Version 3 software (De Novo Software, Los Angeles, CA, USA). Binding assay. Dilutions of the individual sera and Protein G purified pooled IgG from the Jurkat-immunized Tg and wt rabbits were prepared in FACS buffer (phosphate-buffered saline complemented with 1 % fetal bovine serum and 0.04 % azide). 5x10 ^s Jurkat cells per sample were washed with FACS buffer, then 100 μΙ diluted immune serum was added and incubated on ice for 30 minutes. After washing with FACS buffer, cell bound antibodies were detected by incubation with 30 pi of 1000-times diluted DyLightTM 649-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 30 minutes on ice. Results were visualized by plotting the mean fluorescence intensities (MFI), which were measured at the same instrumental setting for all samples. Serial dilutions of each test samples were applied and antibody cell binding was determined by GraphPad Prism version 5 for Windows (GraphPad Software, La Jolla California USA) using nonlinear regression - one site binding (hyperbola) or curve fit the cubic spline curve fit algorithm.

Cytotoxicity assay:

Complement-mediated cytotoxicity was performed as follows: 1 x106 Jurkat cells per sample were washed with FACS buffer and incubated for 30 minutes at 37°C with 100 μΙ of diluted heat inactivated individual immune sera or total IgG purified from pooled Tg and wt immune sera, and 10 μΙ normal rabbit serum as complement source. Cytotoxicity was determined by propidium iodide (PI) incorporation. The number of lysed cells from the cytotoxicity of the immune serum samples were determined by comparison with the negative control samples (with added complement source without immune serum). ATG- Fresenius S was used as reference and all samples were measured at the same instrumental setting. Serial dilutions of each test samples were applied and complement mediated cytotoxicity were determined by GraphPad Prism version 5 for Windows (GraphPad Software, La Jolla California USA) using curve fit the cubic spline curve fit algorithm.

Analysis of spleen cell populations:

Single-cell suspensions from OVA and hA1 AT immunized rabbit spleens were isolated and incubated with FITC-coupled mouse anti-rabbit T lymphocyte (AbD Serotec) and RPE- coupled goat anti-rabbit IgM (Southern Biotech) antibodies using standard protocol. Isotype controls were obtained from BD Pharmingen. Proportion of granulocytes was estimated based on forward scatter/side scatter dotplots.

5. ELISA measurements of antigen specific and total immunoglobulin levels

High binding ELISA plates (Corning Inc., NY, USA) were coated with 5 pg/ml OVA, 5 pg/ml hA1 AT or 1 g/ml unlabeled goat anti-rabbit IgG (H+L) (Southern Biotechnology Associates Inc., Birmingham, AL, USA) in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6) for 2 hours at room temperature (RT) or overnight a 4°C, washed with 0.1 M phosphate-buffered saline (PBS, pH 7.2) containing 0.05% Tween-20 (PBS-T) and blocked with PBS containing 1 % BSA for 1 hour at RT. Diluted serum samples were added to the wells (in independently diluted triplicates) and incubated for 1 hour at RT. In case of total IgC measurement each plate included standard controls of serially diluted, purified rabbit IgG (in duplicates). After washing, bound serum antibodies were detected by horseradish peroxidase labeled goat anti-rabbit IgG (Southern Biotechnology Associates Inc., Birmingham, AL, USA) using tetramethyl-benzidine (TMB, Sigma-Aldrich Co., St. Louis, MO, USA) as chromogen. Blank corrected optical density at 450 nm was measured with a Multiscan ELISA Plate Reader (Thermo Electron Corporation, USA) and interpreted using Scanlt Software 2.5.1 Research Edition for Multiscan FC (Thermo Fisher Scientific, Vantaa, Finland). For data analysis, GraphPad Prism Version 5 for Windows software (GraphPad Software, La Jolla, CA, USA) was used. Serum IgG concentrations were interpolated from the linear portion of the standard curve, based on the blank-corrected absorbance values using the one site binding hyperbola function of non-linear regression curve fit. IgG titers are given as half-maximal values (inflexion point titer, indicated as "titer") or as dilutions at an optical density of 0.05 (endpoint titer).

6. ELISPOT assays

MultiScreen HTS plates (Millipore, Bedford, MA) were coated with 100 g/ml OVA, or hA1 AT, respectively in PBS, at room temperature for 3 h. The plates were then washed with PBS and blocked with RPMI 1640 medium containing 5% FCS and mercaptoethanol (50 mM) for 30 min at room temperature. Serial dilutions (starting at 5 x 105 cells/well) of spleen lymphocytes harvested on day 26 of OVA and day 70 of hA1 AT immunizations were added to the wells. The plates were incubated at 37°C with 5% CO2 overnight and washed with PBS-T; HRP-conjugated goat anti-rabbit IgM and IgG (1 :4000-fold dilution; Southern Biotechnology Associates) was then added to each well. After 1 h incubation at room temperature, the plates were washed with PBS-T. The plates were then incubated in the presence of a chromogen, 3-amino-9-ethylcarbazole (Sigma-Aldrich), and H2O2 as substrate at room temperature, and the reaction was terminated by a water wash. The spots were counted in the ImmunoScan ELISPOT reader (Cellular Technology) and evaluated by ImmunoSpot software version 3.2 (Cellular Technology).

7. Statistical analysis Statistical differences were calculated by pair-wise comparisons of relevant groups using permutation tests. Briefly, values from the groups to be compared were randomly reassigned to two groups and the difference between the group means was determined. Distribution of 5000 randomizations was drawn and the two-tailed P-value corresponding to the real sample assignments was calculated. The arithmetic mean of 50 such P-values was accepted as the probability of a-error. Values of P<0.05 were considered significant and were indicated as follows: *, P<0.05; **, P<0.01 ; ***, P<0.001 .

8. Measurement of total IgG in FcRn Tg rabbits after live lurkat cell immunization

Antisera were raised by immunizing Tg rabbits and their wild type siblings with two successive intravenous injections 14 days apart of a single cell suspension of 5x108 living Jurkat cells and blood samples were collected before the first immunization and on days 7, 14 and 21 . Total IgG contents of individual rabbit immune sera were measured from all collected blood samples. During the first 14 days none of the rabbits showed significant increase in total IgG content compared to day 0 (wt = 5.20 ± 2.14 mg/ml, Tg = 6.32 + 1 .51 mg/ml). Tg rabbits had slightly, though not statistically significantly, higher total IgG levels even before immunization. By day 21 , Tg animals had significantly, 1 .5 times higher (P=0.001 6) total IgG as compared to their wt littermates (9.10 ± 1 .46 mg/ml and 5.95 + 2.1 1 mg/ml, respectively) (Figure 1 A).

9. Measurement of lurkat cell specific antibody amounts in FcRn Tg rabbits

Antibody levels of collected immune sera raised against Jurkat cell surface antigens were measured by flow cytometry using individual Tg and wt samples, initially at 1 :250-fold dilution. Prior to day 14 no significant antibody production could be detected. By day 21 a strong immune response was measured in both groups, with those of the Tg rabbits being significantly higher in Jurkat specific IgG titers (P=0.021 6) compared to the wt animals (Figure 1 B).

From the individual samples collected on day 21 , specific antibody binding was also examined using different immune serum dilutions. Data show that the antibody binding was significantly higher in Tg rabbits at every dilution analyzed, and when binding curves were analyzed with the cubic spline curve fit algorithm, data showed that mean fluorescence intensity (MFI) 50, was achieved by diluting the wt or Tg sera by 1 :131 8-fold or 1 :3012-fold, respectively, and thus the binding difference between the sera of these animals were more than twice (Figure 2). MFI 50 was chosen, as this value was plotted in the linear range of the fitted curves.

10. lurkat binding capacity of anti-thymocyte antibodies raised in FcRn Tg rabbits

The Jurkat binding capacity of the Protein G purified total IgG preparations in the range of 10-0.039 g/ml. At all concentrations the binding capacity of the Tg IgG was superior compared to their wt controls. The non-linear regression analysis indicated that MFI 20 was achieved at concentrations of 6.92 pg/ml or 4.23 pg/ml, in cases of wt or Tg preparations, respectively. The parameter MFI 20 was chosen as this value was plotted in the linear range of the fitted curves. Thus the IgG preparation of the Tg rabbits contains 60% more Jurkat specific IgGs compared to the IgG preparation from wt animals (Figure 3).

1 1 . Complement-mediated cytotoxicity of antibodies produced in FcRn Tg rabbits

The immunosuppressive activity of ATG has been thought to result primarily from the depletion of peripheral T lymphocytes from the circulating pool through complement- dependent lysis or activation associated apoptosis. In the experiments presented in this application, the efficiency of the pooled serum samples and purified IgG preparations have been analysed by complement-mediated cytolysis. The Tg-pool of immune sera was more efficient in killing Jurkat cells at all dilutions, and when cytotoxicity curves were analyzed, the data showed that 20% of cells lysed was achieved with wt or Tg sera in dilution of 1 :72 or 1 :204, respectively, indicating that the serum-pool of FcRn Tg rabbits had 2.8 times more efficient cytotoxic activity (Figure 4A).

The same tendency was evident when Protein G purified IgG preparations of wt and Tg rabbits were compared and also a commercially available ATG-Fresenius preparation, as internal control. More efficient cytotoxicity of the Tg IgG has been observed at all concentrations. 20% cytotoxic activity was achieved with 154 g/ml, 1 37 g/ml or 68 g/ml IgG preparations from the wt rabbits, ATG-Fresenius product or FcRn Tg rabbits, respectively, based on the cytotoxicity curve analysis. ATG-Fresenius IgG showed good correlation with the wt IgG preparation use in the experiments presented here, although immunization and purification protocols of ATG-Fresenius IgG differ from those used in this study and thus direct comparative conclusion cannot be made. It was therefore concluded that the purified Tg IgG was more than twice as efficient (2.2-fold) in mediating cytotoxicity as compared to the wt control (Figure 4B).

12. Titers of antigen-specific IgM and IgG antibodies in FcRn Tg rabbits and number of antigen specific B cells following immunization with soluble antigens

Binding and cytotoxicity analyses from Protein G purified IgGs indicated that FcRn Tg rabbits produced proportionally more Jurkat-specific antibody as compared to their wt controls. One explanation for this observation is that the FcRn Tg rabbits develop more antigen-specific B cells during their immune response, a feature that has been observed in FcRn Tg mice (Cervenak J, Bender B, Schneider Z, Magna M, Carstea BV, et al. (201 1 ) Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic Mice. J Immunol 1 86: 959-968). To test this hypothesis, two different soluble proteins, ovalbumin (OVA) and human 1 -anti-trypsin (hA1 AT), were used for immunizations and the antigen- specific B-cell numbers were analysed with ELISPOT assays. Soluble proteins have been chosen to analyze the number of antigen-specific B cells with ELISPOT assay, as analyzing living Jurkat cell specific B cell number is not feasible.

In both cases, FcRn Tg rabbits produced higher titers of antigen-specific IgM and IgG, showed larger spleen and produced many more antigen-specific B-cells than their wt controls (Figure 5). Analysis of the spleen cell composition showed a lower proportion of B and T lymphocytes in Tg animals compared to their wt controls, however calculating the total cell number, their absolute numbers were higher in Tg animals (data not shown). It has to be noticed that total spleen cells were used for ELISPOT assays, which means proportionally more antigen-specific B cells in Tg animals. These data indicate that FcRn Tg rabbits have augmented antigen specific B-cell activation as compared to their wt controls and thus the proportionally higher level of Jurkat cell specific antibodies in the purified Tg IgG preparation based on its binding capacity and complement mediated cytotoxic activity is the result of the many more activated Jurkat-specific B-cells in the Tg animals as compared to their wt controls. 1 3. Production of Immunoglobulins

1 3.1 Purification of Thymocytes:

Human thymus fragments are, after removal and grinding, filtered and placed in suspension. The cell suspension is then filtered through a nylon cloth and subjected to centrifugation at 1800 rpm at 5° C. 20 ml of Ficoll are added to 10 ml of cell solution containing 2 to 7*10<9 >cells and the mixture is centrifuged at 2000 rpm at 5° C. Two subsequent centrifugations are carried out at 1800 rpm. The cellular preparations are then stored at 5° C. overnight and are then diluted before being injected into SPF rabbits. The thymocytes can also be conserved by freezing.

1 3.2 Isolation of Antibodies

The rabbit immune serum is collected in the course of several bleeds and can be frozen for storage. During the preparation, batches of serum are formed which are gradually returned to ambient temperature. These batches of rabbit serum are decomplemented in order to remove the complement proteins, by bringing them to a temperature of 56-C+-2- C for a period of 30 to 45 min.

The rabbit serum is purified for the immunglobulin fraction by chromatography on an anion exchange resin (DEAE) at ambient temperature followed by two precipitations with sodium sulfate. After a further step of filtration, concentration and diafiltration, the anti-thymocyte immunoglobulins are pasteurized at a temperature of 60° C. for 10 hours in order to ensure the viral safety thereof.

The solution of immunoglobulins can, after formulation and sterilization by filtration, be conserved in the liquid state in a solution of 5 to 25 mg/ml or, according to one variant, in the lyophilized state. A series of quality controls is executed, including physicochemical tests, safety tests (presence of pyrogenic agents, of antiplatelet activity), sterility and purity tests and also lymphocytotoxic activity tests (inhibition of rosette formation in vitro and allogenic skin graft survival in monkeys).