Prevention of graft rejection by prior use of modified grafts

The invention generally relates to the field of grafts and transplantations thereof. In particular, the invention relates to modified grafts and unmodified grafts for use in methods of treating diseases treatable by transplantation as well as related uses. The said methods generally comprise a step of introducing into a subject a modified graft, and a second step of introducing into said subject an unmodified graft. The invention makes use of inhibition of CD4 e.g. in said modified grafts.

In certain aspects, the invention relates to the field of solid organ grafts and transplantations thereof. The invention relates to modified grafts, where introduction thereof is e.g. followed by transplantation of a solid organ. According to the invention the need for conventionally immunosuppressive drugs may be avoided. The invention also describes methods of obtaining the modified grafts to be used in the invention. In certain aspects, the invention relates to modification of allogeneic grafts containing immune competent viable cells by anti human CD4 antibodies before transplantation of solid organs.

BACKGROUND OF THE INVENTION

In many cases, transplantation of solid organs is still the only curative treatment for many patients with organ dysfunction. Transplantations of solid organs are well established methods in medicine and were performed for circa 50 years [Sayegh, 2004]. The investigation of the blood types A, B, 0 was done by K. Landsteiner in 1901 [Landsteiner, 1901; Durand & Willis, 2010] followed by AB by Alfredo von Castello and Adriano Sturli [de Castello & Sturli, 1902]. The first transplantation of a kidney in animals was performed by Alexis Carrel (1873-1944) in 1906 [Carrel & Guthrie, 1906]. A milestone in the development of solid organ transplantation was the investigation of the tissue compatibility and immune reactions during skin transplantation by Sir Peter Medawar (1915-1987) in 1944 [Medawar, 1944]. The first syngeneic and allogeneic human kidney transplantations were achieved by Joseph E. Murray in 1954 and 1959, respectively [Murray et al, 1976], and the HLA antigens responsible for graft rejection were detected by Jean Dausset in 1958 [Dausset, 1958]. In 1962, the use of the immunosuppressive drug azathioprine permitted the kidney transplantation of a deceased person [Calne et al, 1962]. In Germany, the first (clinical) kidney transplantation between relatives was done by Wilhelm Brosig in 1963 [Eigler, 2002]. First transplantation of pancreas was done by Richard Lillehei in 1966 [Kelly et al, 1967], first successful transplantation of a liver by Tom Starzl in 1967 [Starzl et al., 1968; Starzl et al, 1982]. Also in 1967, the first heart transplantation was performed by Christiaan N. Barnard (1922-2001) in Cape Town, South Africa [Barnard et al, 1967], followed by the first transplantation of heart and lung by Denton A. Cooley in 1969 [Cooley et al, 1969]. For protection of graft rejection, cyclosporin A was first used in 1983 [Kapturczak, 2004]. From the first organ transplantation in 1963 until 2013, 116.650 solid organ transplantations were conducted in Germany [Eurotransplant, 2015a]. In January 2015, 12.500 solid organs were needed, which is more than available [Eurotransplant, 2015b]. Moreover, allogeneic solid organ transplantation can be associated with severe complications. Graft rejection due to genetic disparities, side effects, toxicity, infections and the development of secondary tumors due to life-long treatment with immunosuppressive drugs are serious side effects [Hsu & Katelaris, 2009]. Also the mental impact for the patients due to waiting for a (matching) solid organ graft should be considered [Schulz & Koch, 2005].

Strategies for prophylaxis of graft rejection by conventional immunosuppressive drugs have not been able to distinguish between different T cell clones and therefore not between T cell clones responsible for graft rejection and responsible for maintenance of healthy immunological reactions (e.g., destruction of bacteria, viruses, tumor cells). Generally, the use of conventionally immunosuppressive drugs can lead to suppression of the entire immune system, which enhances the possibility for infections and/or development of malignant tumors [Fricke et al. 2012, 2014].

The investigation of alternative or improved therapeutic approaches or procedures for prevention of graft rejection after solid organ transplantation without conventional immunosuppressive drugs by not ameliorating the desired healthy immunological reactions is still needed.

In a previous work depicted in patent application WO 2012/072268, which is incorporated by reference herein in its entirety, it was e.g. shown that an in vitro method of modifying a graft comprising the steps of a) incubating a graft with an anti-CD4 antibody wherein said incubating is carried out for from 1 min to 7 days and b) removing unbound antibody from said graft, allowed the prevention of Graft-versus-Host-Disease (GvHD) and not decreasing the Graft-versus-Tumor-effect in allogeneic hematopoietic stem cell transplantation at the same time due to induction of immune tolerance. This immune tolerance is considered to be due to modulation of CD4 ⁺ T cells by MAX.16H5 IgGi or CD4.16H5.chimIgG4. CD4 molecules directly bind to constant regions of HLA molecules of antigen presenting cells (APCs) to allow complete T cell activation [In: Murphy KM, Travers P, Walport M., English translation by Seidler L, HauBer-Siller I. (Hrsg): Immunologie, 7. Edition -Heidelberg, Berlin: Spektrum, Akad. Verl. 2008]. To interfere with this binding by non-depleting monoclonal antibodies may inhibit this activation by a total steric blockage, by shortening of cell-cell contact between APC and T cell [cf. Harding, 2002], by induction of negative signals through inhibition of protein tyrosine phosphorylation [June et al, 1990] or induction of T cell anergy [cf. Madrenas, 1996a; Madrenas, 1996b].

CD4 is a surface glycoprotein primarily expressed on cells of the T lymphocyte lineage including a majority of thymocytes and a subset of peripheral T cells [Harrison, 1993; Luckheeram et al, 2012]. Low levels of CD4 are also expressed by some non- lymphoid cells [Stewart et al, 1986]. On mature T cells, CD4 functions as a co-recognition signal through interaction with MHC Class II molecules expressed on antigen presenting cells [Harrison, 1993]. CD4 ⁺ T cells primarily constitute the helper subset which regulates T and B cell functions during T-dependent responses to e.g. viral, bacterial, fungal and parasitic infections [Jiang & Dong, 2013]. During the pathogenesis of autoimmune diseases, in particular when tolerance to self-antigens breaks down, CD4 ⁺ T cells contribute to inflammatory responses which inter alia result in tissue destruction [Bellone, 2005]. These processes are facilitated by the recruitment of inflammatory cells of the hematopoietic lineage, production of antibodies, inflammatory cytokines and mediators, and by the activation of killer cells [Bellone, 2005].

Previously, the murine anti human CD4 monoclonal antibody MAX.16H5 IgGi was used in patients with autoimmune diseases or as a protective therapy against transplant rejection [Emmrich, 1991a; Emmrich, 1991b; Reinke, 1991]. In human kidney transplantation, MAX.16H5 IgGi had the potential to effectively reduce graft rejection [Reinke, 1991; Reinke, 1994; Reinke, 1995]. The use of MAX.16H5 IgGi not only resulted in suppression of immune activity but also in the induction of tolerance against tetanus toxoid in a triple transgenic mouse model [Laub, 2000; Laub, 2001; Laub, 2002; Strauss, 1994; Fricke, 2014]. The development of these transgenic mice was performed by scientists mentioned in the literature [Laub, 2000; Laub, 2001; Laub, 2002; Strauss, 1994]. CD4/DR3 mice expressing human CD4 and HLA-DR17, a split antigen of HLA-DR3, on a murine CD4- deficient background, were initially bred at the Institute for Laboratory Animal Science, Medical School Hanover (Germany) [Fricke, 2014]. Using these mice, anti human CD4 antibodies can be used directly, and host and donor hematopoiesis could be distinguished by means of human and murine CD4 molecules [Fricke, 2014; Cooke, 1998; O'Connell, 2010; Liu, 2006]. In a T cell activating environment, MAX.16H5 IgGi preincubated CD4 ⁺ T cells were shown to express lower IL-2 mRNA levels and not to activate Lck dependent signal transduction in contrast to controls without prior MAX.16H5 IgGi incubation [Fricke, 2014].

Direct application of antibodies to human beings can be associated with an unwanted immune reaction to the therapeutic protein [de Groot & Scott, 2007]. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response [Becker, 2012]. The direct application of antibodies to patients may results in severe anaphylactic reactions [de Groot & Scott, 2007]. In previous work, a murine GvHD and tumor transplantation model was shown that allows the direct testing of anti human CD4 antibodies for long-term prevention of GvHD [Fricke, 2014]. However, advantageously, the GvL ("Graft-versus-Leukemia") effect was not decreased [Fricke, 2014]. Without intending to be bound by theory, it may be considered that this effect is CD4 epitope specific [Fricke, 2014]. Advantageously, GvHD was prevented when the graft was short-term pre-incubated (2 hours) with MAX.16H5 IgGi and unbound antibodies were removed [Fricke, 2014]. A direct application of the antibodies was not necessary [Fricke, 2014].

However, there still remains a need for methods for introduction of grafts, particularly of non-modified grafts. Also, there still remains a need for advantageous ways of treating diseases treatable by transplantation of grafts. Generally, there still remains a need for promising alternative or improved therapeutic approaches that lack disadvantages of the prior art methodologies.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an unmodified graft for use in a method of treating one or more disease(s) treatable by transplantation in a subject, wherein said method comprises a first step of introducing into said subject a modified graft, and a second step of introducing into said subject said unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4.

In a second aspect, the present invention relates to a modified graft for use in a method of treating one or more disease(s) treatable by transplantation, wherein said method comprises a first step of introducing said modified graft into said subject, and a second step of introducing an unmodified graft into said subject, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4. In a third aspect, the present invention relates to a CD4 antagonist for use in a method of treating one or more disease(s) treatable by transplantation; wherein said method comprises a first step of introducing into said subject a modified graft, and a second step of introducing into said subject an unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4.

In a fourth aspect, the present invention relates to an unmodified graft, a modified graft and/or a CD4 antagonist for use in i) a chirurgical transplantation method, ii) a method of inducing transplantation tolerance, wherein a modified graft is used for inducing transplantation tolerance towards an unmodified graft, iii) a method of transferring one or more cell suspension(s), particularly stem cell containing cell suspension(s) from a donor to a subject, iv) a method of transferring one or more tissue(s) from a donor to a subject, v) a method of transferring one or more partial organ(s) from a donor to a subject, vi) a method of transferring one or more organ(s) from a donor to a subject, vii) a method of transferring hematopoietic system reconstituting cells from a donor source into a subject, optionally followed by transferring one or more organ(s) from said donor source into said subject, and/or viii) a method for enhancing immune reconstitution in a subject, optionally followed by transferring one or more organ(s) into said subject; wherein said method comprises a first step of introducing into said subject a modified graft, and a second step of introducing into said subject an unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4.

Generally herein, in preferred embodiments, the CD4 antagonist is a CD4 antibody. Generally herein, in preferred embodiments, the modified graft and the unmodified graft are from HLA-related donors.

SHORT DESCRIPTION OF THE FIGURES Fig. 1 shows Balb/c (TTG) mice and wildtype Balb/c™ ¹ (control) mice 24h and 50 days (d) after transplantation of skin from TTG donor mice. No graft rejection could be observed in Balb/c (TTG) mice.

Fig. 2 contains an explanation of C57Bl/6-triple transgenic mice (TTG) as donors. TTG mice express human CD4 and HLA-DR while murine CD4 molecules are knocked out. That allows the determination of specific human surface molecules after transplantation and the direct testing of anti-human CD4 antibodies in mice.

Fig. 3 shows the explanation of an experimental design herein. Bone marrow cells and splenocytes were taken from TTG mice, pooled, incubated in medium containing either anti-human CD4 antibodies or no antibodies for 2 hours, and transplanted in lethally irradiated Balb/c mice. The experiment results were compared with results obtained by using donor cells from C57B1/6 wild-type mice. Therapeutic effects after transplantation were investigated.

Moreover, SEQ ID NOs 1-10 depict the DNA and amino acid sequences of exemplary modified heavy chain variable regions of a particular anti-CD4 antibody for use in the invention.

SEQ ID NOs 11-20 depict the DNA and amino acid sequences of exemplary modified light chain variable regions of a particular anti-CD4 antibody for use in the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention solves above described objects and overcomes above described deficiencies of prior art methods. It includes the first report showing that ex vivo modulation of an allogeneic hematopoietic stem cell graft by anti-human CD4 antibodies MAX.16H5 IgGi followed by third party skin transplantation prevents skin graft rejection in a full MHC-mismatch transplantation model. Without intending to be bound by theory, the present inventors consider that differentiation of or distinction between T cell clones responsible for graft rejection and those responsible for maintenance of healthy immunological reactions could be achieved by modulation of CD4 T-helper cells.

Surprisingly, and further to the previous work described herein above, the present inventors e.g. found that GvHD was not re-induced after single incubation of an allogeneic graft by MAX.16H5 IgGi even when cells were transferred in a third party recipient without re-incubation of MAX.16H5 IgGi. Also, GvL was not prevented. To the inventor's knowledge, such a therapy for induction of tolerance with regard to GvHD by preserving the GvL effect was surprising. Without intending to be bound by theory, the present inventors consider that, advantageously, the maintenance of the immunological effect (e.g. freedom from GvHD) was induced at the day of transplantation and kept up by regulatory T cells.

Among others, the present inventors provide the first report showing that ex vivo modulation of an allogeneic hematopoietic stem cell graft by anti human CD4 antibodies MAX.16H5 IgGi followed by third party skin transplantation prevents skin graft rejection in a full MHC-mismatch transplantation model.

Currently used drugs for prevention of graft rejection are associated with side effects and do not distinguish between different T cell clones and therefore not between T cell clones responsible for graft rejection and responsible for maintenance of healthy immunological reactions (e.g., destruction of bacteria, viruses, tumor cells) [Fricke, 2014].

Inter alia, anti- human CD4 antibodies MAX.16H5 IgGi were tested in murine stem cell transplantation and in a skin transplantation model. Ex vivo modified grafts from human CD4 ⁺ C57B1/6 mice (cf. Fig. 2) were transplanted into Balb/c™ ¹ mice. After 119 days, third party skins from the human CD4 ⁺ C57B1/6 mice were transplanted to these recipient mice. The survival rate of these skin grafts was significantly increased in recipients receiving a MAX.16H5 IgGi short-term (2 hours) pre-incubated graft (cf. Fig. 1). A direct application of the antibodies to the recipients was not necessary. The anti-human CD4 antibodies MAX.16H5 IgGi were long-term effective with regard to survival of solid skin grafts. The nature of the observed induction of immune tolerance by short-term pre-incubation of an antibody with regard to solid organs without the need of conventionally immunosuppressive drugs was advantageous and surprising.

These findings are considered to e.g. make allogeneic organ transplantation safer, and prevent graft rejection due to genetic disparities, reduce side effects, toxicity, and infections as well as the development of secondary tumors due to life-long treatment with immunosuppressive drugs.

Within this patent application, the present inventors could e.g. show that a prior use of a modified graft is suitable to achieve tolerance to an unmodified graft, e.g. in solid organ transplantation (cf. Fig. 3).

Accordingly, the methods of the present invention are e.g. useful i) for reducing the likelihood of donor graft rejection, and organ rejection of a third or higher party organ; ii) for achieving tolerance within the transplanted immunocompetent cells against the third or higher party organ; iii) for achieving tolerance or partial tolerance within the recipient's tissue against the third or higher party organ; and/or iv) for silencing cell activation against a third or higher party organ.

The observed induction of immune tolerance by short-term pre-incubation of an antibody with regard to solid organs without the need for conventional immunosuppressive drugs is advantageous. The present findings are considered to make e.g. allogeneic organ transplantation safer, prevent graft rejection due to genetic disparities, and reduce side effects, toxicity, and infections as well as the development of secondary tumors because life-long treatment with immunosuppressive drugs may be unnecessary.

In certain embodiments, the present invention uses the in vitro treatment of cell grafts containing immune cells with antibodies, thereby avoiding their direct application in vivo. In detail, the present invention may e.g. use a short term incubation of a (stem cell) graft such as cell suspensions containing T cells, in particular CD4 ⁺ cells, with the aim of tolerance induction or immunosuppression. Without intending to be bound by theory, it is considered that e.g. the anti-CD4 antibody incubation of (stem cell) grafts comprising CD4 positive (immune) cells and subsequent removing of unbound antibodies, preferably removing of only unbound antibodies, results in a modified graft, wherein the antibody labeled cells are are not activated, in particular not activated as soon as they encounter specific antigen. Preferably, the antibody labeled cells in the modified graft are anergic cells. Accordingly, e.g. GvHD is not initiated. Without intending to be bound by theory, it is envisaged that the CD4 antagonist such as anti-CD4 antibody may for example inhibit immune cells (such as lymphocytes) bearing CD4 (e.g. by binding to them) and thereby exerts its beneficial effect. Also, as will be readily apparent to the skilled person, substantially reducing or avoiding the administration of free CD4 antagonists (such as anti-CD4 antibodies, i.e. anti-CD4 antibodies that are not bound to an antigen located on the graft) is advantageous.

In any case, it is considered feasible that CD4 is antagonized by any other way. Such alternative ways of inhibiting CD4 are well-known and easily appliable by the skilled person. CD4 antagonists are well studied and numerous options are known and available to the skilled person - and are e.g. further described herein below. In general, practicing the present invention is considered to involve one or more of the following advantages: i) no direct application of the CD4 antagonist (such as anti-CD4 antibodies) to the graft recipients is required; ii) suitable use of a short-term incubation of the graft, such as cell suspensions, tissues, and organs containing T cells, in particular CD4 ⁺ cells; iii) GvHD prevention; iv) prevention of other immunological complications after transplantation of the graft of the invention (e.g. cytokine-release syndrome); v) reduction of costs due to the avoided treatment with conventional immunosuppressive drugs or reduction of their dosage and the significantly reduced amount of antibodies as compared to systemic application; vi) improvement of survival of patients receiving (a) graft(s) in accordance with the invention; vii) facilitation of transplantation of grafts also into patients, into whom regular grafts cannot be transplanted due to expected immunological complications, such as older patients; viii) transplantation of grafts from HLA mismatch donors or grafts from less good HLA matched donors than without the invention; ix) maintenance of the GvL effect; and/or x) facilitation of repeated transplantation of grafts into a subject. Generally, the present invention relates to subject matter as defined in the claims and herein below. That is, in a first aspect, the present invention relates to an unmodified graft for use in a method of treating one or more disease(s) treatable by transplantation in a subject, wherein said method comprises a first step of introducing into said subject a modified graft, and a second step of introducing into said subject said unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4 (or involves CD4 inhibition, respectively).

In a second aspect, the present invention relates to a modified graft for use in a method of treating one or more disease(s) treatable by transplantation, wherein said method comprises a first step of introducing into said subject said modified graft, and a second step of introducing into said subject an unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4 (or involves CD4 inhibition, respectively).

In a third aspect, the present invention relates to an CD4 antagonist, preferably an anti- CD4 antibody, for use in a method of treating one or more disease(s) treatable by transplantation; wherein said method comprises a first step of introducing into said subject a modified graft, and a second step of introducing into said subject an unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4 (or involves CD4 inhibition, respectively).

Preferably, the said modification involves use of the said CD4 antagonist. Preferably, the said modification is effected by the said CD4 antagonist.

Generally herein, the subject may also be referred to as recipient. Preferably herein, the subject is an animal, especially a mammal, particularly a human.

Likewise, preferably herein, the donor(s) (from which the graft(s) is/are isolated and/or derived) is/are an animal, especially a mammal, particularly a human. Generally herein, the modified and unmodified graft(s) may be from the same donor or from different donors. Preferred embodiments with respect to the donor(s) are defined elsewhere herein.

Preferably, said one or more diseases treatable by transplantation is/are selected from the group consisting of acute myeloid leukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia (CML); myelodysplastic syndrome (MDS) / myeloproliferative syndrome; malign lymphomas, particularly selected from Morbus Hodgkin, high grade Non-Hodgkin Lymphoma (NHL), mantle cell lymphoma (MCL), low malign NHL, chronic lymphatic leukemia (CLL), multiple myeloma; severe aplastic anemia; thalassemia; sickle cell anemia; immunological defects particularly selected from severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and hemophagocytic lymphohistiocytosis (HLH); inborn errors of metabolism particularly selected from lysosomal storage disorders and disorders of peroxisomal function; autoimmune diseases; rheumatologic diseases; and recidivisms of any of the above.

Even more preferably, said one or more diseases are one or more hematological malignancies especially selected from acute myeloid leukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia (CML); myelodysplasia syndrome (MDS) / myeloproliferative syndrome; malign lymphomas, particularly selected from Morbus Hodgkin, high grade Non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), low malign Non-Hodgkin lymphoma (NHL), chronic lymphatic leukemia (CLL), multiple myeloma; severe aplastic anemia; thalassemia; and sickle cell anemia.

In certain embodiments herein, the disease treatable by transplantation is any condition requiring transplantation.

In certain preferred embodiments herein, the disease treatable by transplantation is a disease involving organ malfunction. Accordingly, said disease may e.g. require transplantation to replace an organ, which does not function as desired.

In certain embodiments herein, the disease treatable by transplantation is transplant rejection, such as organ rejection. Further examples are GvHD and donor graft rejection. That is to say, in the latter cases, a previously transplanted transplant, such as an organ, may be replaced by another transplant, such as an organ, in line with the present invention.

Generally herein, said one or more diseases treatable by transplantation also include recidivisms of any of the above as well as any combination of diseases mentioned herein.

As used herein, a "cell graft containing immune cells" essentially refers to a graft comprising immune cells. Generally, the cell graft containing immune cells is not particularly limited, with particular embodiments corresponding to those described herein below, e.g. in context with the modified graft.

Generally, grafts, and also cell grafts containing immune cells, are very well known to the person skilled in the art. Also the use of grafts including cell grafts containing immune cells in transplantation is well known in the art. According to the present invention, the graft may comprise a cell suspension, a tissue and/or an organ. Preferably, the graft is a cell suspension, a tissue and/or an organ. More preferably, the grafts are selected from the group consisting of a cell suspension, a tissue and an organ. Generally, herein, a graft may also be a combination of grafts, such as a combination of one or more of the grafts referred to above, e.g. a combination of an organ and a cell suspension. In another preferred embodiment, the graft is not an artificial graft. In a preferred embodiment, it is understood that grafts, and also cell grafts containing immune cells, are intended for and useful for therapeutic purposes in vivo, in particular intended for and useful for treating a disease involving organ malfunction. Said disease may e.g. require transplantation to replace an organ, which does not function as desired. Accordingly, in another preferred embodiment, grafts, and also cell grafts containing immune cells, exclude cells and compositions comprising cells, which are intended for and/or useful only for mechanistic studies in animal models, which do not aim at a therapy of a disease.

In preferred embodiments herein, each of the said grafts is selected from the group consisting of a cell suspension, a tissue and an organ. Accordingly, in the present invention, the modified graft may be selected from the group consisting of a cell suspension, a tissue and an organ. Accordingly, in the present invention, the unmodified graft may be selected from the group consisting of a cell suspension, a tissue and an organ. In particular embodiments, said modified graft is a cell suspension and said graft is a cell suspension. In particular embodiments, said modified graft is a cell suspension and said graft is a tissue. In particular embodiments, said modified graft is a cell suspension and said graft is an organ.

In some particular embodiments the graft does not include embryonic stem cells that are not derived and/or reprogrammed from adult stem cells. Hence, in some particular embodiments any embryonic stem cells included in the graft are embryonic stem cells that are derived and/or reprogrammed from adult stem cells. In some particular embodiments, the grafts do not consist of or do not comprise totipotent embryonic stem cells. In certain embodiments herein, the grafts do not consist of or do not comprise embryonic stem cells. In particular embodiments herein, the grafts do not comprise embryonic stem cells derived from a human embryo. As used herein, an unmodified graft is a regular graft as it is well-known to a skilled person. In particular, it will be readily understood by the skilled person that such unmodified graft is not modified as the modified graft described herein, i.e. does not comprise the modifications of the modified graft herein. Accordingly, the unmodified graft may be described as not having been modified by use of a CD4 antagonist. Accordingly, the unmodified graft may be described as not comprising CD4 antagonists (such as anti- CD4 antibodies). Accordingly, the unmodified graft may be described as not comprising a modification that inhibits CD4. Preferred particular embodiments of an unmodified graft are described herein below.

The present invention e.g. advantageously uses a modified graft. Generally, modified grafts used in the invention may be used as it is known in the art for unmodified grafts, e.g. as regards their introduction (e.g. transplantation) into a subject. As used herein, a modified graft is a graft that has been modified (preferably that has artificially been modified), e.g. via a method described herein, preferably by an in vitro method described herein, preferably by use of a CD4 antagonist. Hence, said modification may be said to involve use of a CD4 antagonist. Likewise, said modification may be said to be effected by a CD4 antagonist. The modified graft herein may have been modified prior to the uses of the invention or during the uses of the invention.

Generally herein, the modification of the modified graft is said to inhibit CD4. Hence, the modified graft is preferably described as comprising a modification that inhibits CD4. Accordingly, it may be said that the modified graft herein has been / is modified to inhibit CD4.

Generally, inhibition as referred to herein may not mean absolute inhibition but also includes partial inhibition. Sufficient degrees of inhibition may readily be determined by the skilled person. Particular embodiments of degrees of inhibition e.g. correspond to embodiments (e.g. including the percentage values recited in context with antibody binding to a graft) of the modified graft described elsewhere herein. A particular preferable degree of preferred inhibition achieves at least one of the advantageous effects disclosed herein.

As used herein, a "modified graft wherein the modification inhibits CD4" ("... involves CD4 inhibition", or other suchlike and/or equivalent terms) preferably refers to a modified graft, where the expression and/or function, preferably the function, of CD4 is inhibited (or impaired). Ways to inhibit CD4 are known to the skilled person and are not particularly limited. They may involve use of (or be effected by, respectively) any CD4 antagonist(s), and particularly of any antagonist(s) of CD4 described herein. Accordingly, in any of the aspects herein, a CD4 antagonist (or e.g. also a mixture of CD4 antagonists) may be used.

In certain embodiments herein, as the skilled reader will readily appreciate, the term "wherein the modification inhibits CD4" and suchlike terms is/are replaced by the term "wherein the modification inhibits cellular functions via CD4". In a preferred embodiment, the term "wherein the modification inhibits CD4" and suchlike terms is/are replaced by the term "wherein the modification inhibits or impairs cellular functions downstream of CD4, in particular binding and/or recruitment". Cellular functions that may be inhibited via CD4 are readily known to the skilled person, particularly in view of his knowledge on CD4 and its mode of action in the cell. In certain embodiments herein, at least one such cellular function is inhibited.

Likewise, in certain embodiments herein, the term "wherein the modification involves CD4 inhibition" and suchlike terms is/are replaced by the term "wherein the modification involves CD4-mediated inhibition of function". In one preferred embodiment, the term "wherein the modification involves CD4 inhibition" and suchlike terms is/are replaced by the term "wherein the modification involves inhibition or impairment of function". In another preferred embodiment, the term "wherein the modification involves CD4 inhibition" and suchlike terms is/are replaced by the term "wherein the modification involves inhibition or impairment of CD4-mediated function"

Generally, non-limiting embodiments of CD4 antagonists to be used in context with the present invention include CD4 antagonists, which modulate CD4 expression and/or function. Accordingly, in certain embodiments a CD4 antagonist herein may modulate, particularly inhibit, expression of CD4. Accordingly, in certain embodiments a CD4 antagonist herein may modulate, particularly inhibit, the function of CD4 - especially by binding to it. Accordingly, the CD4 antagonists herein may be CD4 ligands. Generally, CD4 antagonists such as CD4 inhibitors or CD4 ligands, respectively, are known in the art. Numerous CD4 antagonists, and particularly CD4 ligands that bind to CD4, are commercially available. CD4 antagonists are generally well known in the art. Non- limiting examples include the ones described herein. Generally herein, said CD4 antagonists, e.g. said CD4 inhibitors or CD4 ligands, respectively, may be extra- or intracellular agents (such as ligands). Generally herein, and particularly also in the third aspect, said CD4 inhibition may directly or indirectly be caused by said CD4 antagonist. In preferred embodiments herein, the CD4 antagonist binds to CD4.

In particularly preferred embodiments herein, the CD4 antagonist is an anti-CD4 antibody. Anti-CD4 antibodies are generally well-known in the art. Numerous anti-CD4 antibodies are commercially available.

Antibodies and also anti-CD4 antibodies are well known in the art. As used herein, by "antibody" is meant inter alia a protein of the immunoglobulin family that is capable of specifically combining, interacting or otherwise associating with an antigen, wherein said combining, interacting or otherwise associating (such as binding) of the antibody to the antigen is mediated by complementarity-determining regions (CDRs). Similarly, term "antigen" is used herein to refer to a substance that is capable of specifically combining, interacting or otherwise associating with said antibody. In the context of the anti-CD4 antibody of the present invention the antigen is meant to be CD4, particularly human CD4. As used herein, the term "CDR" refers to the "complementarity-determining region" of an antibody, i.e. to one of the hypervariable regions within an immunoglobulin variable domain contributing to the determination of antibody specificity. CDRs are well known to a person skilled in the art. Typically, both the heavy chain immunoglobulin variable domain and the light chain immunoglobulin variable domain contain three CDRs. In the context of the present invention, the term "antibody" is considered to also relate to antibody fragments including for example Fv, Fab, Fab' and F(ab') ₂ fragments. Such fragments may be prepared by standard methods [for example; Coligan et al, 1991-1997, incorporated herein by reference]. The present invention also contemplates the various recombinant forms of antibody derived molecular species well known in the art. Such species include stabilized Fv fragments including single chain Fv forms (e.g., scFv) comprising a peptide linker joining the VH and VL domains, or an Fv stabilized by interchain disulphide linkage (dsFv) and which contain additional cysteine residues engineered to facilitate the conjoining of the VH and VL domains. Equally, other compositions are familiar in the art and could include species referred to as "minibodies"; and single variable domain "dAbs". Other species still may incorporate means for increasing the valency of the modified antibody V-region domain, i.e. species having multiple antigen binding sites for example by engineering dimerization domains (e.g., "leucine zippers") or also chemical modification strategies. Moreover, the term "antibody" also relates to multimers of scFv such as diabodies, triabodies or tetrabodies, tandabs, flexibodies, bispecific antibodies, and chimeric antibodies, all known in the art. As used herein, antibodies are considered to also include any bivalent or multivalent antibodies. They also include any antibody derivatives and any other derivatives known to the skilled person. In some embodiments, the antibody is a polyclonal antibody. In preferred embodiments, the antibody is a monoclonal antibody. Further embodiments of "antibody" may be taken from WO 2012/072268.

According to the invention, the term "anti-CD4 antibody" refers to an antibody, which has the ability to bind to CD4. Preferably herein, the anti-CD4 antibody is an anti human CD4 antibody. "CD4" or "cluster of differentiation 4" refers to a protein, more precisely a surface glycoprotein, well known to the person skilled in the art [cf. above, cf. also Bowers et al, 1997]. In the present context CD4 may also refer to a fragment of full-length CD4, or an otherwise modified form of CD4, provided that the fragment or otherwise modified form still functions as an antigen in the context of the antibody of the present invention. Particularly preferred examples of anti-CD4 antibodies that may be used in accordance with the present invention are described elsewhere herein.

Non-limiting (further) examples of CD4 antagonists are peptide ligands (including naturally occurring peptide ligands and peptide constructs).

Certain further embodiments include aptamers. In a further embodiment, the CD4 antagonist is cyclo(CNSNQIC). In a further embodiment, the CD4 antagonist is 4,4'- diisothiocyano-2,2'-dihydrostilbenedisulfonic acid. Generally, in preferred embodiments of the invention, the modified graft comprises a CD4 antagonist.

In further aspects herein, which are related to the first, second and third aspects, said method(s) comprise(s) a step of introducing into said subject an unmodified graft, wherein said subject has previously been introduced with a modified graft, wherein the modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4. In a preferred embodiment, said method(s) comprise(s) a step of introducing into said subject an unmodified graft, wherein a modified graft has previously been introduced into said subject, wherein the modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4. Generally, preferred embodiments of those related aspect correspond to those of the respective aspects as described herein.

In a fourth aspect herein, the present invention relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in any one or more method(s) of the group consisting of i) a chirurgical transplantation method, ii) a method of inducing transplantation tolerance, wherein a modified graft is used for inducing transplantation tolerance to an unmodified graft, iii) a method of transferring one or more cell suspension(s), particularly stem cell containing cell suspension(s) from a donor to a subject, iv) a method of transferring one or more tissue(s) from a donor to a subject, v) a method of transferring one or more partial organ(s) from a donor to a subject, vi) a method of transferring one or more organ(s) from a donor to a subject, vii) a method of transferring hematopoietic system reconstituting cells from a donor source into a subject, optionally followed by transferring one or more organ(s), from said donor source into said subject, and viii) a method for enhancing immune reconstitution in a subject, optionally followed by transferring one or more organ(s) into said subject. Generally, the above method(s) preferably comprise(s) a first step of introducing into said subject a modified graft, and a second step of introducing into said subject an unmodified graft, wherein said modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4 (or involves CD4 inhibition, respectively).

Alternatively, in a further variant related to the said fourth aspect, said method(s) comprise(s) a step of introducing into said subject an unmodified graft, wherein said subject has previously been introduced with a modified graft, wherein the modified graft is a cell graft containing immune cells, wherein said modification inhibits CD4 (or involves CD4 inhibition, respectively). Preferred embodiments of the latter aspect correspond to those of the fourth aspect. In detail, in one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a chirurgical transplantation method.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of inducing transplantation tolerance, wherein a modified graft is used for inducing transplantation tolerance to an unmodified graft.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of transferring one or more cell suspension(s), particularly stem cell containing cell suspension(s) from a donor to a subject.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of transferring one or more tissue(s) from a donor to a subject.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of transferring one or more partial organ(s) from a donor to a subject.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of transferring one or more organ(s) from a donor to a subject. In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method of transferring hematopoietic system reconstituting cells from a donor source into a subject, optionally followed by transferring one or more organ(s), from said donor source into said subject.

In one particular embodiment the fourth aspect relates to an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in a method for enhancing immune reconstitution in a subject, optionally followed by transferring one or more organ(s) into said subject.

In certain embodiments, the fourth aspect relates to an unmodified graft for use in any of said method(s). In certain embodiments, the fourth aspect relates to a modified graft for use in any of said method(s). In certain embodiments, the fourth aspect relates to an anti-CD4 antibody for use in any of said method(s). Generally herein, embodiments of the fourth aspect (and the said variant related thereto) correspond to embodiments of the other aspects described herein.

Generally herein, in embodiments of the first, second, third and fourth aspects herein, the said first step recited in context with the respective method(s) is carried out before said second step recited in context with the said method(s).

In particular embodiments of the first, second, third and fourth aspects herein, said first step is carried out from 30 min to 50 years, particularly from one hour to 10 years, such as to 9, 8, 7, 6, 5, 4, 3, or two years, such as from one day to 1 year, such as to 11, 10, 9, 8, 7, 6, 5, 4, 3, or two months before said second step. In certain embodiments, said first step is carried out from one hour to two months before said second step. In certain embodiments, said first step is carried out at least one hour, particularly at least three weeks, before said second step.

In particular embodiments, said first step is carried out at least one hour, particularly at least one day, particularly at least one month, particularly at least one year before said second step. In particular embodiments herein, said first step is carried out at least 12 hours, particularly at least one day, particularly at least one week, particularly at least three weeks, particularly at least six months before said second step.

In particular embodiments, said first step is carried out up to 30 years, particularly up to 10 years, particularly up to 1 year, particularly up to 1 month before said second step. In particular embodiments, said first step is carried out up to 20 years, particularly up to 5 years, particularly up to 2 years, particularly up to 6 months, particularly up to 12 weeks before said second step.

Similarly, in the related further aspects herein, where the said method(s) herein are defined as comprising a step of introducing into said subject an unmodified graft, wherein said subject has previously been introduced with a modified graft, the timing of the previous introduction is preferably further defined as detailed above. Preferably, said method(s) herein are defined as comprising a step of introducing into said subject an unmodified graft, wherein a modified graft has previously been introduced into said subject, the timing of the previous introduction is preferably further defined as detailed above. Generally, in preferred embodiments of the first, second, third and fourth aspects herein, said modified graft comprises stem cells.

In preferred embodiments of the invention, the modified graft is a cell graft containing immune cells that has been modified by a CD4 antagonist, particularly wherein said modification inhibits CD4. Generally, in preferred embodiments of the invention, the modified graft, particularly a cell graft containing immune cells, comprises a CD4 antagonist.

In particularly preferred embodiments of the invention, the modified graft is a cell graft containing immune cells that comprises an anti-CD4 antibody bound to CD4 epitopes thereof.

In certain preferred embodiments of the invention the modified graft, the modified graft is a cell graft containing immune cells that comprises an anti-CD4 antibody bound to from 40% to 100% of the CD4 epitopes thereof. Such graft is preferably obtainable by the in vitro method disclosed herein. Preferably, the modified cell graft containing immune cells, comprises anti-CD4 antibodies bound to from 50% to 100%, particularly 60% to 100%, particularly 70% to 100%, more particularly 80% to 100%, more particularly 90% to 100%, more particularly 95% to 100%), more particularly 99% to 100%), of the accessible CD4 epitopes of said graft. Most preferably, essentially all of the accessible CD4 epitopes of the cell graft containing immune cells are bound to anti-CD4 antibodies.

In particular embodiments herein, said modified graft is obtained by or obtainable by an in vitro method comprising the step a) incubating a cell graft containing immune cells with an anti-CD4 antibody, and optionally comprising the step b) removing unbound antibody from said graft. In one preferred embodiment, said modified graft is obtained by or obtainable by an in vitro method comprising the step a) incubating a cell graft containing immune cells with an anti-CD4 antibody, and optionally comprising the step b) removing unbound antibody from said graft, thereby reducing the amount of unbound antibody in said graft. In a more preferred embodiment, the amount of unbound antibody in said graft is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or is reduced by 100%.

In one preferred embodiment, said modified graft is obtained by or obtainable by an in vitro method comprising the step a) incubating a cell graft containing immune cells with an anti-CD4 antibody, and optionally comprising the step b) removing unbound antibody from said graft by washing the modified graft at least one time or at least 2 times, such as 2, 3, 4, 5 or 6 times. By washing the modified graft, the amount of unbound antibody in said graft is reduced. In a more preferred embodiment, the amount of unbound antibody in said graft is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or is reduced by 100%. Suitable washing liquids are known to a skilled person and include buffered saline solutions. In a preferred embodiment, it is understood that a certain amount of CD4-positive immune cells may be lost or removed unspecifically during the washing step(s). In a yet further preferred embodiment, the modified graft contains immune cells, in particular CD4- positive immune cells, such as CD4 ⁺ -T cells, to which anti-CD4 antibody is bound. In a more preferred embodiment, the modified graft contains at least 10%, 20%, 30%>, 40%>, 50%, 60%, 70%, 80%, 90% or 95% of the CD4-positive immune cells of the cell graft containing immune cells prior to incubation.

In another preferred embodiment, said modified graft is obtained by or obtainable by, respectively, an in vitro method comprising the step a) incubating a cell graft containing immune cells with an anti-CD4 antibody, and optionally comprising the step b) removing only unbound antibody from said graft. Accordingly, the modified cell graft containing immune cells may be obtainable in accordance with an in vitro method described herein.

As used herein, an "m vitro method" refers to a method that is performed outside a living subject. It particularly also includes an "ex vivo method", such as in case of the graft comprising or being a tissue or an organ, but particularly excludes an "in vivo method" performed inside a living subject.

As used herein, "unbound antibody" refers to an antibody which, following the step of incubating, is not bound to the graft. In other words, it refers to an antibody which is not essentially associated with its ligands on the graft. Said in vitro method to be used in the invention may be a method of modifying a cell graft containing immune cells that comprises the steps of a) incubating a cell graft containing immune cells with an anti-CD4 antibody, especially wherein said incubating is carried out for from 1 min to 7 days, b) removing unbound antibody from said graft. Preferably, said incubating in step a) of said in vitro method is carried out for from 1 min to 1 day. In one preferred embodiment, said in vitro method to be used in the invention may be a method of modifying a cell graft containing immune cells that comprises the steps of a) incubating a cell graft containing immune cells with an anti-CD4 antibody, especially wherein said incubating is carried out for from 1 min to 7 days, b) removing unbound antibody from said graft, thereby reducing the amount of unbound antibody in said graft. In a more preferred embodiment, the amount of unbound antibody in said graft is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or is reduced by 100%. Preferably, said incubating in step a) of said in vitro method is carried out for from 1 min to 1 day. In another preferred embodiment, said in vitro method to be used in the invention may be a method of modifying a cell graft containing immune cells that comprises the steps of a) incubating a cell graft containing immune cells with an anti-CD4 antibody, especially wherein said incubating is carried out for from 1 min to 7 days, b) removing unbound antibody from said graft, by washing the modified graft at least one time or at least 2 times, such as 2, 3, 4, 5 or 6 times. By washing the modified graft, the amount of unbound antibody in said graft is reduced. In a more preferred embodiment, the amount of unbound antibody in said graft is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%), or is reduced by 100%. Preferably, said incubating in step a) of said in vitro method is carried out for from 1 min to 1 day.

In a preferred embodiment, it is understood that a certain amount of CD4-positive immune cells may be lost or removed unspecifically during the washing step(s). In a yet further preferred embodiment, the modified graft obtained by the method contains immune cells, in particular CD4-positive immune cells, such as CD4 -T cells, to which anti-CD4 antibody is bound. In a more preferred embodiment, the modified graft contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the CD4-positive immune cells of the cell graft containing immune cells prior to incubation.

In another preferred embodiment, said in vitro method to be used in the invention may be a method of modifying a cell graft containing immune cells that comprises the steps of a) incubating a cell graft containing immune cells with an anti-CD4 antibody, especially wherein said incubating is carried out for from 1 min to 7 days, b) removing only unbound antibody from said graft. Preferably, said incubating in step a) of said in vitro method is carried out for from 1 min to 1 day.

Generally, preferred embodiments for said in vitro methods may be taken from WO 2012/072268, and are incorporated by reference in their entirety herein.

In step a) of the in vitro method used herein, the (step of) incubating is preferably carried out for a time sufficient to allow binding of said antibody to said graft. Preferably, said incubating is carried out for a time sufficient to allow the binding of anti-CD4 antibodies to from 40% to 100%, particularly 50% to 100%, particularly 60% to 100%, particularly 70% to 100%, more particularly 80% to 100%, more particularly 90% to 100%, more particularly 95% to 100%), more particularly 99% to 100%), of the accessible CD4 epitopes of said graft. Most preferably, following said incubating, anti-CD4 antibodies bind to essentially all of the accessible CD4 epitopes of said graft. In a preferred embodiment, it is understood that the accessible CD4 epitopes or CD4-positive immune cells (or immune cells bearing the CD4 antigen) of said graft are not removed or substantially not removed. Accordingly, in a further preferred embodiment, immune cells bearing the CD4 antigen are not depleted, or not substantially depleted from the unmodified graft, and/or the modified graft obtained by or obtainable by the in vitro method comprises or substantially comprises immune cells bearing the CD4 antigen of the unmodified graft.

An appropriate incubation period will easily be determined by the person skilled in the art. Usually, an appropriate incubation period will depend on the type of graft used. A preferred incubation period may also dependent on the amount of antibody used. Generally, where the graft is e.g. a cell suspension, shorter incubation periods will be required than where the graft is e.g. an organ. Generally, where the graft comprises or is a tissue or an organ, longer incubation periods are preferred to allow the antibody to be transported - e.g. via diffusion - into the respective compartments.

Moreover, in any case, the skilled person may easily test the (status of the) binding of the anti-CD4 antibodies according to methods well known within the art that may, for example, involve flow cytometry.

In certain embodiments, said incubating in step a) of said in vitro method is carried out for from 1 min to 7 days. Preferably, said incubating in step a) of said in vitro method is carried out for from 1 min to 1 day.

Generally, short incubation periods are preferred herein over long incubation periods in order to minimize any possible damage to the graft due to in vitro processing.

Particularly, said incubating may be carried out for from 1 to 150 min, particularly for from 5 min to 150 min, more particularly for from 10 min to 150 min, more particularly for from 30 min to 150 min, more particularly for from 40 minutes to 120 min, more particularly for from 45 min to 90 min, especially for from 50 min to 70 min.

In alternative embodiments, said incubating may be carried out for from 150 min to 7 days, particularly for from 150 min to 5 days, more particularly from 150 min to 3 days, more particularly from 150 min to 1 day, especially for from 150 min to 8 hours. In a further preferred embodiment, incubating may be carried out for from 1 min to 1 day.

As to the "removing" of unbound (anti-CD4) antibody in accordance with e.g. step b) of the in vitro method used herein, various ways of performing said step are known to the skilled person. One exemplary way of removing unbound antibody from the graft is by washing the graft. Washing may e.g. occur by employing centrifugation where the graft comprises or is a cell suspension. In one preferred embodiment, the amount of unbound antibody in said graft is reduced by washing the graft. In a more preferred embodiment, the amount of unbound antibody in said graft is reduced by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or is reduced by 100%. Preferably, only unbound (anti- CD4) antibody is removed in accordance with e.g. step b) of the in vitro method used herein. A few CD4-positive immune cells may be lost unspecifically during removal of unbound antibodies, in particular be washing. In a preferred embodiment, the modified graft contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the CD4-positive immune cells of the cell graft containing immune cells prior to incubation.

In said step, preferably at least 40%, more particularly at least 50%, more particularly at least 60%, more particularly at least 70%, more particularly at least 80%, more particularly at least 90%, of unbound (anti-CD4) antibody are removed from the graft. Preferably, up to 100% of unbound (anti-CD4) antibody are removed from the graft.

The amount of antibody employed in the above step of incubating is not particularly limited. Appropriate amounts may easily be determined by the person skilled in the art and may depend on e.g. the type of graft used. Preferably according to the invention, said incubating is carried out with an antibody amount of from O. ^g to lOOmg. In a preferred amount, Preferably, in accordance with the invention, particularly in case of the graft being a cell suspension, an amount of from 2 x 10 ⁶ cells to 2 x 10 ¹⁰ nucleated cells, particularly of from 4 x 10 ⁶ to 1 x 10 ⁹ nucleated cells, more particularly of from 1 x 10 ⁷ to 1 x 10 ⁸ nucleated cells are administered to said subject, preferably to the human subject, accordingly, in a yet further embodiment, said incubating is carried out with an antibody amount of from 0. ^g/l x 10 ⁹ nucleated cells to 100mg/2 x 10 ⁶ nucleated cells, more preferably 0. ^g/l x 10 ⁹ nucleated cells to 100mg/2 x 10 ⁷ nucleated cells.

In particular embodiments said incubating in step a) of said in vitro method is carried out with an antibody amount of from 0.1 μg/ml to 1 Omg/ml. In certain embodiments, particularly where the graft is a cell suspension, said incubating is carried out with an antibody concentration of from 0. ^g/ml cell suspension to 150μg/ml cell suspension, particularly from 7μg/ml cell suspension to 100μg/ml cell suspension, more particularly from 30μg/ml cell suspension to 100μg/ml cell suspension, especially from 40μg to 60μg/ml cell suspension. In some embodiments, particularly where the graft is a tissue or where the graft is an organ, said incubating is carried out with an antibody amount of from 0.1 mg to lOmg, particularly from lmg to lOmg, more particularly from 2mg to 9mg, more particularly from 3mg to 8mg, especially from 4mg to 6mg.

In some embodiments, particularly where the graft is a tissue or where the graft is an organ, said incubating is carried out with an antibody concentration in the incubation solution of from O.lmg/ml to lOmg/ml, particularly from lmg/ml to lOmg/ml, more particularly from 2mg/ml to 9mg/ml, more particularly from 3mg/ml to 8mg/ml, especially from 4mg/ml to 6mg/ml. Preferably, the specified volume includes the volume of said tissue or organ as well as the volume of the (antibody-containing) solution, in which said tissue or organ is incubated.

In some embodiments, particularly where the graft is a tissue or where the graft is an organ, said incubating is carried out by incubating said tissue or organ in a solution having an antibody concentration of from l(^g/ml to 15(^g/ml, particularly from 2(^g/ml to 10(^g/ml, more particularly from 3(^g/ml to 10(^g/ml, especially from 4(^g/ml to 6(^g/ml. Preferably, the specified volume includes the volume of said tissue or organ as well as the volume of the (antibody-containing) solution, in which said tissue or organ is incubated.

When incubating tissues and/or organs with a CD4 antagonist (e.g. an anti-CD4 antibody- containing solution), the skilled person will preferably readily perform such incubation e.g. by means of a suitable container.

In any case, the selection of suitable amounts of CD4 antagonist, such as of anti-CD4 antibody, is well within the expertise of the skilled person. Generally, higher amounts or concentrations, respectively, of antagonist (e.g. antibody) are preferred where the graft comprises or is a tissue or an organ. Moreover, the selection of an exact amount or a concentration, respectively, of antagonist (e.g. antibody) used will also depend on the size of such tissue or organ.

In the following, further particular embodiments of the modified graft to be used in the invention are described:

In some preferred embodiments of the invention, the modified graft comprises stem cells. A graft comprising stem cells may also be referred to herein as a stem cell graft.

According to the present invention, the modified graft may comprise cells bearing the CD4 antigen. Preferably, the modified graft comprises immune cells, particularly immune cells bearing the CD4 antigen. Such cells are well known to the person skilled in the art. In certain preferred embodiments, these immune cells are CD4 positive T lymphocytes or precursor cells thereof. In certain preferred embodiments, these immune cells include, but are not limited to T helper cells and cells belonging to the monocyte and macrophage lineage, such as monocytes and macrophages. Another example for such cells are microglia. In some embodiments, the modified graft comprises, preferably is, a tissue, preferably a stem-cell-containing tissue. According to the present invention, suitable tissues include, but are not limited to blood, muscle, adipose tissue, connective tissue, epithelium, embryonic, and cellular tissue. In some embodiments, the modified graft comprises, preferably is, an organ, preferably a stem-cell-containing organ. Suitable organs include, but are not limited to skin, intestine, kidney, and liver. Preferably, said organ is an intestine.

In preferred embodiments, the modified graft comprises, preferably is, a cell suspension, preferably a stem-cell-containing cell suspension. Suitable cell suspensions and methods for obtaining them are well known to the skilled person. For example, a cell suspension graft may be obtained by puncture of bones comprising bone marrow, e.g. puncture of the iliac crests or sterna or taken from stem cell niches throughout the whole body, e.g. fat tissue, tooth root, root of a hair and any other source mentioned above.

In preferred embodiments, the modified graft, particularly the cell suspension, particularly the stem-cell-containing cell suspension, comprises T cells, monocytes and macrophages. In another preferred embodiment, the modified graft, particularly the cell suspension, comprises T cells and non-immune cells, such as stem cells. In another preferred embodiment, the stem-cell-containing tissue, the stem-cell-containing organ, or the stem- cell-containing cell suspension, comprises immune cells, more preferably immune cells bearing the CD4 antigen, in particular CD4 positive T lymphocytes or precursor cells thereof.

In another preferred embodiment, the graft or cell graft, such as the stem-cell-containing tissue, the stem-cell-containing organ, or the stem-cell-containing cell suspension, comprises immune cells bearing the CD4 antigen, in particular CD4 positive T lymphocytes or precursor cells thereof.

In certain preferred embodiments the modified graft, particularly the cell suspension, comprises any of bone marrow stem cells, peripheral blood stem cells, umbilical cord blood stem cells, adult stem cells of the bone marrow such as NA-BMCs, embryonic stem cells (particularly derived and/or reprogrammed from adult stem cells), and any pluripotent stem cells, wherein the latter includes induced pluripotent cells, such as cells derived and/or reprogrammed from adult stem cells (i.e. including pluripotent embryonic stem cells).

In preferred embodiments, the modified graft is a bone marrow suspension, particularly comprising bone marrow stem cells. Generally, the modified graft, particularly the bone marrow suspension, may additionally comprise any of stem cells comprised in blood cells, cord blood cells, donor lymphocytes, peripheral blood stem cells, adult stem cells of the bone marrow, embryonic stem cells (particularly derived and/or reprogrammed from adult stem cells), and any pluripotent stem cells, wherein the latter includes induced pluripotent cells, such as cells derived and/or reprogrammed from adult stem cells (i.e. including pluripotent embryonic stem cells).

The modified graft, particularly the bone marrow suspension, may additionally comprise any of stem cells comprised in blood cells, cord blood cells, donor lymphocytes, peripheral blood stem cells, and/or adult stem cells of the bone marrow. Generally, it is intended that the cell suspension also includes any cell suspension that comprises (any combination of) stem cells, optionally along with any (combination of) other cells.

In preferred embodiments herein, said modified graft is selected from the group consisting of a cell suspension containing T cells and monocytes/macrophages the cell suspension comprising bone marrow cells, non-adherent bone marrow cells, peripheral blood cells, and/or cord blood cells; a cell suspension comprising lymphocytes, monocytes and/or macrophages; a stem-cell-containing tissue; a stem-cell-containing organ; an immune cell containing tissue; and an immune cell containing organ.

Further embodiments of the unmodified graft herein correspond to the further particular embodiments described above for the modified graft.

Generally herein, the amount of cells contained in the graft (e.g. in the modified graft) is not particularly limited. Any person skilled in the art will easily be able to choose appropriate amounts of a graft and of cells of the graft for transplantation. Furthermore, suitable guidance is also available e.g. from the specific guidelines for transplantation developed by the "Deutsche Bundesarztekammer", e.g. for human hematopoietic stem cells into patients.

In preferred embodiments herein, said modified graft is a cell suspension and the respective said method (or use, respectively) comprises administration of an amount of from 2xl0 ⁶ cells to 2x1ο ¹⁵ nucleated cells to said subject, wherein preferable amounts will readily be selected by the skilled person. Exemplary preferred ranges include from lxlO ⁷ to lxlO ¹⁴ nucleated cells, from lxlO ⁷ to lxlO ¹⁴ nucleated cells, from lxlO ⁸ to lx 10 ¹³ nucleated cells, from lxlO ⁹ to lxlO ¹² nucleated cells, from lxlO ¹⁰ to lxlO ¹¹ nucleated cells, from lxl 0 ⁶ to lxlO ⁸ nucleated cells, from lxl 0 ⁷ to lxl 0 ⁹ nucleated cells, from lxl 0 ⁸ to lxlO ¹⁰ nucleated cells, from lxlO ⁹ to lxlO ¹¹ nucleated cells, from lxlO ¹⁰ to lxlO ¹² nucleated cells, from lxlO ¹¹ to lxlO ¹³ nucleated cells, from lxlO ¹² to lxlO ¹⁴ nucleated cells, and from lxlO ¹³ to lxlO ¹⁵ nucleated cells.

Further embodiments of the unmodified graft herein correspond to the said preferred embodiments described above for the modified graft. Where the graft to be used herein comprises or is a tissue or an organ, any suitable amounts of said tissue or organ may be administered to said subject. As will be understood by the skilled person, cell numbers in tissues or organs are difficult to determine. Particularly for this reason, the amount of cells contained in the grafts is not particularly limited. Appropriate amounts will easily be determined or selected by the skilled person, e.g. taking into consideration the particular type of subject, graft and/or disease to be treated. In case of organs, the administration of whole organs is preferred.

In preferred embodiments of first, second, third and fourth aspects herein, the respective said method i) implies tolerance or partial tolerance within said unmodified graft against the recipient's tissue; and/or ii) implies tolerance or partial tolerance within the recipient's tissue against said unmodified graft; and/or iii) implies a reduced likelihood of developing any one of the group consisting of GvHD, donor graft rejection, and organ rejection, upon translation of the unmodified graft.

Accordingly, in some embodiments herein, the respective said method implies tolerance or partial tolerance within said unmodified graft against the recipient's tissue. Accordingly, in some embodiments herein, the respective said method implies tolerance or partial tolerance within the recipient's tissue against said unmodified graft. Accordingly, in some embodiments herein, the respective said method implies a reduced likelihood of developing any one of the group consisting of GvHD, donor graft rejection, and organ rejection, upon transplantation of the unmodified graft. In some embodiments herein, the use of the modified graft implies a reduced likelihood of developing any one of the group consisting of GvHD, donor graft rejection, and organ rejection; particularly of GvHD, upon transplantation of said graft. In other embodiments, the use implies tolerance within the transplanted immunocompetent cells against the recipient's tissue upon transplantation of said modified graft. In other embodiments, the use implies tolerance against the modified graft upon transplantation of said modified graft. In other embodiments, the use implies tolerance or partial tolerance within the recipient's tissue against the modified graft upon transplantation of said modified graft. In other embodiments, the use is for silencing cell activation within said graft. In a preferred embodiment, the use is for reducing the CD4-positive immune cells' ability to be activated within said graft. In a preferred embodiment, the use is for obtaining anergy of the CD4- positive immune cells within said graft. In preferred embodiments, the use implicates / is for any combination of the above.

In some preferred embodiments herein, the use of the modified graft results in a reduced likelihood of developing any one of the group consisting of GvHD, donor graft rejection, and organ rejection; particularly of GvHD, upon transplantation of said graft. In other preferred embodiments, the use results in tolerance of the transplanted immunocompetent cells against the recipient's tissue upon transplantation of said modified graft. In other preferred embodiments, the use results in tolerance against the modified graft upon transplantation of said modified graft. In other preferred embodiments, the use results in tolerance or partial tolerance of the recipient's tissue against the modified graft upon transplantation of said modified graft. In other preferred embodiments, the use is for preventing cell activation or reducing the cells' ability to be activated within said graft. In a preferred embodiment, the use is for reducing the CD4-positive immune cells' ability to be activated within said graft. In a preferred embodiment, the use is for obtaining anergy of the CD4-positive immune cells within said graft. In preferred embodiments, the use implicates / is for any combination of the above.

In the present invention, generally, the said subject (or recipient, respectively) may be allogeneic or xenogeneic with respect to the donor(s).

As will readily be understood by the skilled person, the recipient and the respective donor(s) are referred to as being allogeneic when they are separate individuals of the same species, whereas the recipient and the respective donor(s) are referred to as being xenogeneic when they are derived from different species.

Accordingly, in certain preferred embodiments herein, the said subject is allogeneic with respect to the donor of the said unmodified graft and to the donor of the said modified graft. In other embodiments herein, the said subject is xenogeneic with respect to the donor of the said unmodified graft and to the donor of the said modified graft.

Similarly, in the present invention, generally, the donors may be allogeneic or xenogeneic with respect to each other.

As will readily be understood by the skilled person, the donor(s) are referred to as being allogeneic when they are separate individuals of the same species, whereas the donor(s) are referred to as being xenogeneic when they are derived from different species.

Accordingly, in certain embodiments herein, the donor of the modified graft is xenogeneic with respect to the donor of the said unmodified graft. In certain preferred embodiments herein, the donor of the modified graft is allogeneic with respect to the donor of the unmodified graft.

In the present invention, the nature of and relationship between the donors of the modified graft and the unmodified graft and the recipient are not particularly limited. Hence, in certain non-limiting examples, and e.g. where the donor(s) and recipient are mice, the donor(s) may comprise full HLA mismatches with respect to the recipient. Hence, in certain preferred non-limiting examples, and e.g. where the donor(s) and recipient are mice, the donor(s) may have full HLA mismatches with respect to the recipient.

In other non-limiting examples, the donor(s) may e.g. be haploidentical (such as transplantation between parents and infants) to the recipient and may e.g. be HLA-partially matched family members, parents, siblings or children of the recipient.

In any case, it is preferred in the present invention that the donor(s) and the recipient (or subject, respectively) are HLA-related.

Likewise, in the present invention, the nature of and relationship between the donors of the modified graft and the unmodified graft are not particularly limited. Hence, in certain non- limiting examples, the donor(s) may comprise, preferably have, full HLA mismatches with respect to each other. In other non-limiting examples, one donor may e.g. be haploidentical to the other donor, and may e.g. be HLA-partially matched family members, parents, siblings or children of the other donor.

In any case, it is preferred in the present invention that the modified graft and the unmodified graft are from HLA-related donors. Accordingly, the respective donors in context with the present invention are preferably HLA-related (i.e. HLA-related to each other).

As used herein, the term "HLA-related donors" is preferably understood as referring to donors comprising, preferably having, not more than 50% HLA mismatches. In preferred embodiments, the donors comprise, preferably have, not more than 40% HLA mismatches, preferably not more than 30% HLA mismatches, preferably not more than 20% HLA mismatches, preferably not more than 10% HLA mismatches, preferably no HLA mismatch.

Similarly, a given donor and recipient are referred to as being "HLA-related" when they comprise, preferably have, not more than 50% HLA mismatches. In preferred embodiments, a given donor and recipient comprise, preferably have, not more than 40% HLA mismatches, preferably not more than 30% HLA mismatches, preferably not more than 20% HLA mismatches, preferably not more than 10% HLA mismatches, preferably no HLA mismatch.

Accordingly, and particularly in case of humans, the term "HLA-related" is preferably understood as referring to the presence of not more than 50% HLA mismatches, preferably not more than 40% HLA mismatches, preferably not more than 30% HLA mismatches, preferably not more than 20% HLA mismatches, preferably not more than 10% HLA mismatches, preferably no HLA mismatch.

Accordingly, and particularly in case of humans, the term "HLA-related" is preferably understood as referring to the presence of not more than six HLA mismatches, preferably not more than five HLA mismatches, preferably not more than four HLA mismatches, preferably not more than three HLA mismatches, preferably not more than two HLA mismatches, preferably not more than one HLA mismatch, preferably to no HLA mismatch.

In certain preferred embodiments, in case of humans, the said percentages and or numbers of mismatch(es) are determined with respect to the HLA loci HLA- A, -B, C-, -DR, -DQ, and -DP (corresponding to a total of 12 HLA loci). In other preferred embodiments, the said mismatch(es) are determined with respect to the HLA loci HLA- A, -B, C-, -DR, and - DQ (corresponding to a total of 10 HLA loci). In other preferred embodiments, the said mismatch(es) are determined with respect to the HLA loci HLA-A, -B, C-, and -DR (corresponding to a total of 8 HLA loci).

Generally herein, methods for determining HLA mismatches are not particularly limited, but are readily available and apparent to the person skilled in the art.

Furthermore, as will be readily understood by the skilled person, in the above definitions of the term "HLA related", HLA loci may be replaced by corresponding appropriate loci where non-human donors and recipients are compared to each other. Said loci are readily known to the skilled person.

Moreover, further envisaged herein are cases where the modified graft (e.g. without or after genetic manipulation) is HLA-related to the unmodified graft and vice versa. Hence, in certain preferred embodiments of the present invention, the modified graft and the unmodified graft are "HLA-related", wherein particular embodiments for "HLA-related" correspond to any of those above. Moreover, further envisaged herein are cases where the modified graft and/or the unmodified graft (e.g. without or after genetic manipulation) is HLA-related to the recipient. Hence, in certain preferred embodiments of the present invention, the modified graft is HLA-related to the recipient. Hence, in certain preferred embodiments of the present invention, the unmodified graft is HLA-related to the recipient. Again, particular embodiments for "HLA-related" correspond to any of those above.

Accordingly, as will also be understood by the skilled person, the modified and unmodified grafts may be HLA-related in case the respective donors are not HLA-related - and a given modified graft and/or unmodified may be HLA-related to a recipient in case the respective donor(s) and recipient are not HLA-related. Furthermore, as will also be understood by the skilled person, (grafts or) donors may also be HLA-related in case the donors are xenogeneic - and (modified and unmodified grafts or) a given donor and recipient may also be HLA-related in case the donor and recipient are xenogeneic. To the latter end, e.g. (one of) the donor(s) (or grafts, respectively) may be genetically manipulated to achieve one or more HLA matches or further HLA matches. Accordingly, in certain preferred embodiments, donor(s) with respect to the recipient (or the donors of the modified graft and the unmodified graft, respectively) preferably comprise, preferably have, not more than six HLA mismatches after genetic manipulation e.g. of donor cells, preferably comprise, more preferably have, not more than three HLA mismatches after genetic manipulation e.g. of donor cells, preferably comprise, more preferably have, not more than two HLA mismatches after genetic manipulation e.g. of donor cells, preferably comprise, more preferably have, not more than one HLA mismatch after genetic manipulation e.g. of donor cells, preferably comprise, more preferably have, no HLA mismatch after genetic manipulation e.g. of donor cells. Further embodiments in the latter context correspond to those as described above.

As also described above, in the present invention, the modified graft and the unmodified graft are preferably from HLA-related donors or, in other words, the donor of the modified graft and the donor of the unmodified graft are preferably HLA-related. Non-limiting particular examples of HLA-related donors include tissue-type matched donors and related twins. Hence, in certain embodiments, said HLA related donors are tissue-type matched donors (such as a tissue HLA type matched donor). Accordingly, in certain embodiments, the modified graft and the unmodified graft are from tissue-type matched donors. In certain embodiments, said HLA related donors are related twins, especially monozygotic twins. Accordingly, in certain embodiments, the modified graft and the unmodified graft are from related twins, especially from monozygotic twins.

Generally, in particularly preferred embodiments, the modified and unmodified grafts are from the same donor. Accordingly, in preferred embodiments, the said HLA-related donors may be one and the same donor. Hence, the term HLA-related donors as used herein may actually refer to one (particular) donor. Hence, in particular preferred embodiments, said HLA related donors are one particular donor. Hence, in certain preferred embodiments of the present invention, both the modified graft and the unmodified graft are from the same donor.

In preferred embodiments of the invention, the anti-CD4 antibody i) is selected from the group consisting of Maxl6H5, OKT4A, OKTcdr4a, cMT-412, YHB.46, particularly wherein said anti-CD4 antibody is Maxl6H5; or ii) is antibody 30F16H5; or iii) is obtainable from a cell line deposited with accession number ECACC 88050502; or iv) is obtainable from a cell line MAX.16H5/30F16H5 deposited with the DSMZ on December 2, 2011; or v) is antibody 16H5.chimIgG4; or vi) is obtainable from a cell line CD4.16H5.chimIgG4 deposited with the DSMZ on December 2, 2011; or vii) is an antibody comprising the VH and the VK of antibody 16H5.chimIgG4; or viii) is an antibody comprising a VH and a VK of an antibody obtainable from a cell line CD4.16H5.chimIgG4 deposited with the DSMZ on December 2, 2011; or ix) is an antibody comprising any combination of a VH selected from SEQ ID NOs: 1-10 and of a VK selected from SEQ ID NOs: 11-20, particularly wherein said combination is selected from VH1/VK1, VH2/VK2, VH4/VK2 and VH4/VK4, especially wherein said combination is VH2/VK2, or x) is a mixture of antibodies selected from the antibodies according to i) to ix) above.

Accordingly, referred anti-CD4 antibodies for use in accordance with the present invention are selected from the group consisting of Maxl6H5, OKT4A, OKTcdr4a, cMT-412, YHB.46. A particularly preferred anti-CD4 antibody is Maxl6H5. Cells for the production of Maxl6H5 have been deposited with the ECACC (European Collection of Cell Cultures) with accession number ECACC 88050502. Said antibody is also disclosed in DE 3919294, which is incorporated by reference herein. As used herein, the antibody "Maxl6H5" may also be referred to as "Max. l6H5", "MAX16H5" or "MAX.16H5", or also "30F16H5" (wherein the latter name is also the name of deposited cells producing said antibody). Max. l6H5 may also be obtained from the cell line MAX.16H5/30F16H5 (cf. deposit DSM ACC3148). Another particularly preferred anti-CD4 antibody for use in the invention is 16H5.chimIgG4. As used herein, said antibody may also be referred to as "16H5.chim" or as "CD4.16H5.chimIgG4" (wherein the latter name is also the name of deposited cells producing said antibody). 16H5.chimIgG4 may be obtained from the cell line CD4.16H5.chimIgG4 (cf. deposit DSM ACC3147).

In detail, certain preferred anti-CD4 for use in the present invention are e.g. obtainable from any of the following deposits of biological material: i) deposit with the European Collection of Cell Cultures having the accession number ECACC 88050502 (which is e.g. also described in application DE 3919294); ii) deposit "MAX.16H5/30F16H5", accession number DSM ACC3148, deposited with the DSMZ on December 2, 2011; iii) deposit "CD4.16H5.chimIgG4", accession number DSM ACC3147, deposited with the DSMZ on December 2, 2011. All of these deposits involve cells or cell lines, respectively, from which particular anti-CD4 antibodies in the context with the present invention may be obtained.

The present invention further relates to alternative embodiments of embodiments disclosed herein, where the term "ECACC 88050502" is replaced by "MAX.16H5/30F16H5". Likewise, the present invention further relates to embodiments, where the term "cell line ECACC 88050502" as used herein, or an equivalent term, is replaced by the term "cell line MAX.16H5/30F16H5" or an equivalent term. The present invention further relates to alternative embodiments of embodiments disclosed herein, where the term "ECACC 88050502" is replaced by "CD4.16H5.chimIgG4". Likewise, the present invention further relates to embodiments, where the term "cell line ECACC 88050502" as used herein, or an equivalent term, is replaced by the term "cell line CD4.16H5.chimIgG4" or an equivalent term.

In further embodiments of the present invention, particular anti-CD4 antibodies (including modified antibodies, etc.) that may be used correspond to the anti-CD4 antibodies described in patent application W02012/072268 (cf. particularly the "additional aspect" described therein), which document is incorporated by reference in its entirety herein. This particularly also applies to the embodiments according to items 1-33 disclosed on pages 42 et seqq. of W02012/072268.

Likewise, particular (embodiments for) methods to obtain / prepare further anti-CD4 antibodies that may be used in embodiments of the present invention correspond to those described in W02012/072268 as well as the references disclosed therein. Likewise, particular (embodiments for) methods to evaluate anti-CD4 antibodies (including e.g. method for the measurement of the affinity of antibodies) that may be used in embodiments of the present invention correspond to those described in W02012/072268 as well as the references disclosed therein.

Herein, the term "VH" generally refers to the heavy chain variable region of the heavy chain of an antibody. The "heavy chain variable region" is also referred to as "heavy chain immunoglobulin variable domain". Also these terms are well-known in the art. The term "VL" generally refers to the light chain variable region of the light chain of an antibody. The "light chain variable region" is also referred to as "light chain immunoglobulin variable domain". These terms are well-known in the art, too. VH preferably means a polypeptide that is about 110 to 125 amino acid residues in length. Similarly, VL preferably means a polypeptide that is about 95-130 amino acid residues in length.

In preferred embodiments herein, prior to introduction into said subject, said unmodified graft and/or modified graft is/are additionally incubated with soluble bioactive molecules. Accordingly, in preferred embodiments herein, prior to introduction into said subject, said unmodified graft is additionally incubated with soluble bioactive molecules. Accordingly, in preferred embodiments herein, prior to introduction into said subject, said modified graft is additionally incubated with soluble bioactive molecules.

In each case, in particular such bioactive molecules are agents promoting immunosuppression, immunotolerance and/or formation of regulatory T cells. Preferably, each case, such bioactive molecules are agents promoting immunosuppression, immunotolerance and/or (favored) formation and/or (favored) activation of regulatory T cells.

Preferred exemplary agents are selected from the group consisting of IL-2, TGF-β, rapamycin, retinoic acid, 4- IBB ligand, and anti-CD28 antibodies; or any combination thereof.

Generally, the grafts for use in the present invention may optionally be administered to the subject together with any medicament or combination of medicaments. This applies to both the modified and unmodified grafts. Said medicament(s) may be administered prior to, together with and/or following transplantation. Suitable administration modes and routes are not particularly limited and will easily be chosen by the skilled person. Preferably, such medicament(s) support the features or advantages, respectively, of the present methods, uses, modified grafts, or modified grafts for use described hereinabove, such as reducing the likelihood of any one of the group consisting of GvHD, donor graft rejection, and organ rejection. Non-limiting examples for such medicaments include rapamycin and retinoic acid.

In preferred embodiments herein, the modified graft is used i) for achieving tolerance or partial tolerance of the modified graft against the recipient's tissue; and/or ii) for achieving tolerance or partial tolerance of the recipient's tissue against the modified graft; and/or iii) for reducing the likelihood of any one of the group consisting of GvHD, donor graft rejection, and organ rejection upon transplantation of said modified graft.

In preferred embodiments herein, the modified graft is used i) for achieving tolerance or partial tolerance of the unmodified graft against the recipient's tissue; and/or ii) for achieving tolerance or partial tolerance of the recipient's tissue against the unmodified graft; and/or iii) for reducing the likelihood of any one of the group consisting of GvHD, donor graft rejection, and organ rejection upon transplantation of said unmodified graft.

In a further aspect, the invention features methods of treating a subject in need of such treatment in accordance with the used described herein. In preferred embodiments, said grafts, subjects, methods, and/or diseases are as described hereinabove.

In a further aspect, the invention features an unmodified graft, a modified graft and/or an anti-CD4 antibody for use in the manufacture of a medicament for the treatment of one or more diseases treatable by transplantation in a subject and/or for any of the (medical) uses described herein. In preferred embodiments, said use is further defined as described herein above. Accordingly, in preferred embodiments, said grafts, subjects, methods, and/or diseases are as described hereinabove.

In an even further aspect, the graft (particularly the modified graft) for use in the present invention, may or may not comprise stem cells. That is, according to the latter aspect, the cell graft containing immune cells may be replaced by any graft and includes a graft comprising stem cells as well as a graft not comprising stem cells. In other words, the graft of the invention or used in accordance with the invention may be any graft or may be a cell graft containing immune cells. In even other words, in certain embodiments a graft used in accordance with the invention comprises stem cells, whereas in other embodiments, a graft used in accordance with the invention does not comprise stem cells. In certain embodiments, the graft does not comprise isolated CD4 ⁺ cells. In certain embodiments, the graft does not comprise purified DO.11.10 CD4 ⁺ T cells.

Generally, further embodiments of the various aspects herein correspond to those described in W02012/072268.

Generally, as to further mechanistic, experimental and theoretical characteristics related to (side) aspects of the present invention, reference is also made to W02012/072268.

Generally, the invention also relates to embodiments, where the term "comprises" or an equivalent term is replaced by "has" or an equivalent term. For example, the invention generally also relates to embodiments, where the term "comprising" or an equivalent term is replaced by "having" or an equivalent term.

In the following, the present invention is further illustrated by examples which are not intended to limit the scope of the present invention.

EXAMPLES Example 1 :

Animals and experimental design

Donor triple transgenic mice (human CD4 ^+/+ , murine CD4 ⁷ , HLA-DR3 ^+/+ ) TTG-C57B1/6 were bred at the Animal Facility at the University Leipzig [Fricke, 2014; Schmidt, 2015]. Recipient Balb/c™ ¹ mice were purchased from Charles River (Sulzfeld, Germany; http://jaxmice.jax.org) [Fricke, 2014; Schmidt, 2015]. All mice were maintained under standardized conditions [Fricke, 2014; Schmidt, 2015]. In transgenic mice, the CD4 transgene includes its own promoter ligated to a murine CD4 enhancer element thus leading to T cell subset-specific expression [Fricke, 2014; Schmidt, 2015]. Murine CD8 ⁺ cells are not affected [Fricke, 2014; Schmidt, 2015]. Transgenic mice express the HLA- DR3 molecule in addition to the murine MHC II complex [Fricke, 2014; Schmidt, 2015]. The TTG-C57B1/6 mice have complete functional murine immune systems [Fricke, 2014; Schmidt, 2015]. The mice were fed ad libitum [Fricke, 2014; Schmidt, 2015]. All mice were housed, treated, or handled in accordance with the guidelines of the University Leipzig Animal Care Committee and were approved by the Regional Board of Animal Care for Leipzig [Fricke, 2014; Schmidt, 2015].

Allogeneic hematopoietic stem cell grafts from donor TTG-C57BI/6 were incubated with MAX.16H5 IgGi or control antibodies for 2 hours before transplantation into Balb/c^ [Fricke, 2014; Schmidt, 2015]. Survival and GvHD occurrence were measured daily, engraftment, hematological (WBC subsets) and immunological reconstitution (murine CD4, human CD4, murine CD8, HLA-DR3, and H2K ^b ) were measured weekly [Fricke, 2014; Schmidt, 2015].

Irradiation protocol

For the irradiation of mice, the X-Ray apparatus (D3225, Orthovoltage, Gulmay Medical, Camberley, UK) was adjusted for animal irradiation as we described previously [Fricke, 2009]. Irradiation was performed before transplantation of recipient mice.

Preparation of bone marrow cells, splenocytes, and antibody incubation

As isotype control, the antibody from LEAF™ Purified Mouse IgGi (κ, Isotype Ctrl Antibody, San Diego, CA 92121) was used [Fricke, 2014]. MAX.16H5 IgGi antibodies were used as described previously [Fricke, 2014]. Bone marrow cells (BM) and splenocytes were prepared as described elsewhere [Fricke, 2009; Fricke, 2010; Fricke, 2011; Fricke, 2012, Fricke, 2014, Schmidt, 2015]. For antibody incubation, the calculated amount of antibodies was diluted before use to a final concentration of lmg/ml in DMEM without FCS [Fricke, 2014]. Subsequently, 1.4x10 ^s of BM and 1.4x10 ^s of splenocytes from donors were incubated with 800μ MAX.16H5 Igd in 15ml DMEM without FCS at room temperature in the dark for 2 hours [Fricke, 2014]. As control, BM and splenocytes from the donors without antibody pre-incubation were prepared under the same conditions [Fricke, 2014]. After 2 hours of incubation, cells were centrifuged at 300g for 10 min to pellet them and washed once in PBS (lx) at 300g for 10 min to remove unbound antibodies [Fricke, 2014].

Cell Transplantation

For co-transplantation experiments, 2xl0 ⁷ pre-incubated bone marrow cells of donor TTG- C57B1/6 mice were added to 2xl0 ⁷ MAX.16H5 IgGi pre-incubated splenocytes [Fricke, 2014]. The cell concentration was adjusted to a final volume of 150μ1 sterile 0.9% NaCl [Fricke, 2014]. Subsequently, the grafts were allogeneically transplanted by intravenous injection into the lateral tail vein of lethally irradiated recipient Balb/c™ ¹ mice [Fricke, 2014]. Survival, GvHD symptoms, and weight were assessed every day after transplantation [Fricke, 2014].

Flow cytometry

Before and after transplantation, donor TTG-C57B1/6 and recipient Balb/c^ mice were analyzed by flow cytometry. For cytometric analysis, cells were prepared and incubated as previously described [Fricke, 2009; Fricke, 2010; Fricke, 2011; Fricke, 2012, Fricke, 2014]. Additionally, the viability of splenocytes and bone marrow cells was tested before transplantation by staining with 7-Amino-Actinomycin D (7-AAD). lxlO ⁶ cells were incubated with 5μ1 (0.25μg/test) of 7-AAD in 300μ1 PBS at room temperature for 30 min and measured immediately. Before and after transplantation, recipient Balb/c™ ¹ mice were analyzed by flow cytometry and the full blood cell counts were determined. The following antibodies were used: murine CD4-PECy7, MHC-I (H-2D[b]-PE, and murine CD8-PerCP [BD Biosciences, Heidelberg, Germany]; murine CD3-FITC and human CD4-APC [Beckman Coulter, Krefeld, Germany]; and human HLA-DR3-FITC [Immunotools, Friesoythe, Germany]. For cytometric analysis of murine FoxP3, the murine T _reg Detection Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was used according to the manufacturer's protocol. The data were acquired on a BD FACSCanto™ II Flow Cytometer and analyzed using BD FACSDiva™ software (both BD Biosciences, Heidelberg, Germany) [Fricke, 2014].

Skin transplantation For allogeneic skin transplantation Balb/c (TTG) mice (showing C57B1/6 blood chimerism) and Balb/c™ ¹ mice were used as recipient and CD4/CD3 mice (TTG) as donors. Recipient mice were anesthetized by a standard protocol approved by the authorities. Skin of the donor mice from the tails was immediately prepared. The fur on the shoulder of recipient mice was shaved carefully before transplantation. Full-thickness grafts of 5 x5mm donor tail skin (TTG) were transplanted on Balb/c (TTG) or Balb/c™ ¹ recipients. Transplanted skin graft was secured by surgical sterile thread and additionally dressed with adhesive sterile bandage. Recipient mice were awakened by a standard protocol approved by the authorities. After surgery, mice were individually housed to avoid additional injuries. All procedures of skin transplantation took place under sterile conditions in an operating room and were approved by the authorities. Mice were monitored daily. Fifty days (50 days) after skin transplantation, the tissue was collected for histological analysis.

Histology of recipient mice

Histology was done as described previously [Fricke, 2011]. Statistical Analysis

All data are presented as means ± standard deviation [Fricke, 2012]. Statistical analysis and graphic presentations were made using SigmaPlot 10.0/SigmaStat 3.5 software (SYSTAT, Erkrath, Germany) [Fricke, 2012] and GraphPad Prism 5 (v5.03, GraphPad Software Inc, Dan Diego, California). P-values calculated with SigmaPlot 11.0/SigmaStat 3.5 software. Test variables were the GvHD score, weight, survival, and cell numbers. Analysis of survival curves was done using the log-rank test, analysis of other parameters with t-tests, the Mann- Whitney U test or the Holm-Sidak test [Fricke, 2009].

Results: (cf. also Fig. 1)

It was hypothesized that solid organs (e.g. skin) from third party TTG donor mice on Balb/c (TTG) recipient mice, which showed immune tolerance, would not be rejected after transplantation. For this, 5 Balb/c (TTG) mice (showing C57B1/6 blood chimerism) and 4 Balb/c™ ¹ (control) were transplanted using tail skin of TTG mice. It had to be mentioned that Balb/c (TTG) recipients were about 6 months older as control mice because of hematopoietic stem cell transplantation before. Sixty percent (60%) of skin transplanted Balb/c (TTG) mice survived and 100% of Balb/c™ ¹ mice. Nevertheless, all surviving Balb/c (TTG) mice (showing C57B1/6 blood chimerism, N=3), which received a third party skin graft from TTG mice did not show graft rejection unlike the controls (Fig. 1). Graft failure of TTG skin was observed in the Balb/c™* control group (N=4).

Example 2: As to further antibodies for use in the present invention, chimeric anti-CD4 antibodies may be made, and modified anti-CD4 antibodies may be designed and generated in accordance with Example 2 of W02012/072268, which is also incorporated in its entirety by reference herein. Example 3 :

Furthermore, as to an animal model for the human immune system that may supplementarily be used by the skilled person in context with certain issues of this invention, reference is made to WO 2006/122545 including all family members of the respective patent family such as EP 1887859 and US 2008216182, all of which are explicitly incorporated by reference herein.

LIST OF REFERENCES (all of which are explicitly incorporated by reference herein) DE 3919294

WO 2006/122545

WO 2012/072268

Barnard, C.N. The operation. A human cardiac transplant: an interim report of a successful operation performed at Groote Schuur Hospital, Cape Town. S Afr Med J. 41, 1271-1274 (1967).

Becker, C, Bopp, T. & Jonuleit,H. Boosting regulatory T cell function by CD4 stimulation enters the clinic. Front Immunol. 3. 164 (2012).

Bellone, M. Autoimmune Disease: Pathogenesis. Encyclopedia of Life Sciences John Wiley & Sons, 1-8 (2005).

Bowers, K., Pitcher, C. & Marsh, J.M. CD4:A co-receptor in the immune response and HIV infection.. Int. J. Biochem. Cell Biol. 29, 871-875 (1997).

Calne, R.Y. et al. A study of the effects of drugs in prolonging survival of homologous renal transplants in dogs. Ann N Acad Sci. 99, 743-761 (1962).

Carrel, A. & Guthrie, C.C. Successful Transplantation of Both Kidneys from a Dog into a Bitch with Removal of Both Normal Kidney from the Latter. Science 23, 394-395 (1906).

Chatenoud, L., Waldmann, H., & Emmrich,F. Tolerance induction in the adult: 'danger at Le Bischenberg. Immunol. Today 16, 121-123 (1995).

Coligan et al, Current Protocols in Immunology, John Wiley & Sons 1991-1997

Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J, Crawford JM et al. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation .1. The roles of minor H antigens and endotoxin. Blood 88(8):3230-3239 (1996)

Cooke, K.R. et al. Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J. C/in. Invest 102, 1882-1891 (1998).

Cooley, D.A. et al. Organ transplantation for advanced cardiopulmonary disease. Ann Thorac Surg. 8, 30-46 (1969).

Dausset, J. Iso-leuco-anticorps. Acta Haematol. 20, 1956-1966 (1958).

De Castello, A. & Sturli, A. Ueber die Isoagglutinine im Serum gesunder und kranker Menschen. Mfinch med Wschr. 49, 1090-1095 (1902).

De Groot, A.S. & Scott, D.W. Immunogenicity of protein therapeutics. Trend Immunol. 28, 482-490 (2007).

Durand, J.K. & Willis, M.S. Karl Landsteiner, MD: Transfusion Medicine. Laboratory Medicine 41, 53-55 (2009).

Eigler, F.W. Zur Geschichte der Nierentransplantation in Deutschland. Zentralbl Chir. Ill, 1001-1008 (2002).

Emmrich, F. et al. An anti-CD4 antibody for treatment of chronic inflammatory arthritis. Agents Actions Suppl 32, 165-170 (1991a).

Emmrich, J., Seyfarth, M., Fleig, W.E., & Emmrich, F. Treatment of inflammatory bowel disease with anti-CD4 monoclonal antibody. Lancet 338, 570-571 (1991b). Eurotransplant. Total numbers of organ transplantations in Germany in the period from 1963 to 2013 according to organs. 2015a [cited on June 19, 2015, URL: http ://de. statista. com/ statistik/daten/ studie/202037/umfrage/ anzahl-der- organtransplantationen-in-deutschland/ ]

Eurotransplant. 2015b [cited on June 19, 2015, URL: https://www. eurotransplant. org/cms/index.php?page=pat_germany]

Fricke, S. et al. Allogeneic non-adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment. PLoS. One. 4. e6157 (2009).

Fricke, S. et al. Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis. Cell Mol. Life Sd. 67, 4095-4106 (2010).

Fricke, S. Measurement and illustration of immune interaction after stem cell

transplantation. Methods Mol. Biol. 690. 315-332 (2011).

Fricke, S. et al. Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure. Cytometry A 81, 476-488 (2012).

Fricke, S. et al. Prevention of graft-versus-host-disease with preserved graft-versus- leukemia-effect by ex vivo and in vivo modulation of CD4 ⁺ T-cells. Cell Mol Life Sci. 71, 2135-2148 (2014).

Harding, S., Lipp, P., & Alexander, D.R. A therapeutic CD4 monoclonal antibody inhibits TCR-zeta chain phosphorylation, zeta-associated protein of 70-kDa Tyr31 9

phosphorylation, and TCR internalization in primary human T cells. J. Immunol. 169, 230- 238 (2002).

Harrison, S.C. CD4: Structure and Interactions of an Immunoglobulin Superfamily Adhesion Molecule. Acc Chem. Res. 26, 449-453 (1993).

Hsu, D.C. & Katelaris, C.H. Long-term management of patients taking immunosuppressive drugs. Australian Prescriber 22, 68-71 (2009).

Jiang, S. & Dong, C. A Complex issue on CD4 ⁺ T-cell subsets. Immunol Rev. 252, 5-11 (2013).

June, C.H. et al. Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction. Proc.Natl.Acad.Sci. 87, 7722-7726 (1990).

Kapturczak, M.H. et al. Pharmacology of calcineurin antagonists. Transplant Proc. 36, 25- 32 (2004).

Kelly, W.D. et al. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery 61, 827-837 (1967).

Landsteiner, K. Ueber Agglutinationserscheinungen normalen menschlichen Blutes. Wiener Klinische Wochenschrift 49, 1132-1133 (1901).

Laub R. et al. A multiple transgenic mouse model with a partially humanized activation pathway for helper T cell responses. J. Immunol. Methods 246, 37-50 (2000).

Laub, R., Dorsch, M., Wenk, K., & Emmrich, F. Induction of immunologic tolerance to tetanus toxoid by anti-human CD4 in HL A-DR3 (+)/human CD4(+)/murine CD4(-) multiple transgenic mice. Transplant. Proc. 33, 2182-2183 (2001).

Laub, R. et al. Anti-human CD4 induces peripheral tolerance in a human CD4+, murine CD4-, HLA-DR+ advanced transgenic mouse model. J. Immunol. 169, 2947-2955 (2002). Liu,W. et al. CD 127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J. Exp. Med. 203, 1701-1711 (2006).

Luckheeram, R.V. et al. CD4 ⁺ T cells: differentiation and functions. Clin Dev Immunol. 2012, 1-12 (2012).

Madrenas, J., Schwartz, R.H., & Germain,R.N. Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists. Proc. Nat!. Acad. Sci. U. S. A 93, 9736- 9741 (1996).

Madrenas, J. & Germain, R.N. Variant TOR ligands: new insights into the molecular basis of antigen-dependent signal transduction and T-cell activation. Semin. Immunol. 8, 83- 101 (1996).

Medawar, P.B. The behaviour and fate of skin autografts and skin homografts in rabbits. J Anat. 78, 176-199 (1944).

Murray, J.E. et al. Renal transplantation: a twenty- five year experience. Ann Surg. 184, 565-573 (1976).

O'Connell, R.M. et al. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity. 33, 607-619 (2010).

Pfitzmann R, Neuhau P, Hetzer R (Hrsg.): Organtransplantation, ISBN 3-11-016849-9

Reinke, P. et al. Anti-CD4 therapy of acute rejection in long-term renal allograft recipients. Lancet 338. 702-703 (1991).

Reinke, P. et al. Late acute rejection in long-term renal allograft recipients. Diagnostic and predictive value of circulating activated T cells. Transplantation 58, 35-41 (1994).

Reinke, P. et al. Anti-CD4 monoclonal antibody therapy of late acute rejection in renal allograft recipients~CD4+ T cells play an essential role in the rejection process.

Transplant. Proc. 27, 859-862 (1995).

Robey, E. & Axel, R. CD4: Collaborator in immune recognition and HIV infection. Cell 60, 697-700 (1990).

Schmidt F. et al, Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice. Cytometry A. 87(4): 334-45 (2015).

Stewart, S.J. et al. Human T lymphocytes and monocytes bear the same Leu-3(T4) antigen. J Immunol. 136, 3773-3778 (1986).

Sayegh, M.H. et al. Transplantation 50 Years Later-Progress, Challenges, and Promises. N EnglJMed. 351, 2761-2766 (2004).

Schulz, K.-H. & Koch, U. Transplantationspsychiologie. In Balck, F. Anwendungsfelder der medizinischen Psychiologie. Springer, 101-114 (2005).

Starzl, T.E. et al. Orthotopic homotransplantation of the human liver. Ann Surg. 168, 392- 415 (1968).

Starzl, T.E. et al. Evolution of liver transplantation. Hepatology 2, 614-636 (1982).

Strauss, G., Vignali.D.A.. Schonrich,G.. & Hammerling,G.J. Negative and positive selection by HLA-DR3(DRwl7) molecules in transgenic mice. Immuno genetics 40. 104- 108 (1994). Applicant's or agent's International application No.

file reference F68779PC PCT/EP2017/053417

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule \ 3bis)

The indications made below relate to the deposited microorganism or other biological material referred to in the description on case 31 . line 7

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 1%

Name of depositary institution

ECACC European Collection of Cell Cultures (ECACC) Culture Collections Public Health England

Address of depositary institution (including postal code and country)

Porton Down

Salisbury, Wiltshire SP4 OJG

United Kingdom

Date of deposit Accession Number

05/05/1988 88050502

ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

I | This sheet was received with the international application I I This sheet was received by the International Bureau on:

Authorized officer Authorized officer

Ku i per-Cristi na, Nathal ie

Form PCT RO/134 (July 1998; reprint January' 2004)

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 31 27

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet \

Name of depositary institution

DSMZ Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zell-kulturen GmbH (DSMZ)

Address of depositary institution (including postal code and country)

Inhoffenstr. 7B

38124 Braunschweig

Germany

Date of deposit Accession Number

02/12/201 1 DSM ACC31 8

ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit ")

For receiving Office use only For International Bureau use only

I | This sheet was received with the international application □ This sheet was received by the International Bureau on:

Authorized officer Authorized officer

Ku iper-Cristi na, Nathal ie

Form PCT RO/134 (July 1998; reprint January 2004)

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 31 , line _32 .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Q

Name of depositary institution

DSMZ Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zell-kulturen GmbH (DSMZ)

Address of depositary institution (including postal code and country)

Inhoffenstr. 7B

38124 Braunschweig

Germany

Date of deposit Accession Number

02/12/2011 DSM ACC3147

ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated Slates)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit ")

For receiving Office use only For International Bureau use only

I | This sheet was received with the international application I I This sheet was received by the International Bureau on:

Authorized officer Authorized officer

uiper-Cristina, Nathalie

Form PCT/RO/134 (July 1998; reprint January 2004) ECACC

t'OslCj ia

Eu-opean Co!¾c -C ot C«' Cttlures

^ Pioseoion Agency

Cemie lor tmefg ncy ^e a-Mrfis jrd Keipc-M

Potosi Down, Sa!.sb.,iy Sf- OIG united Kingdom

Health Protection Agency

Porton Down

And

European Collection of Cell Cultures

This document certifies that:

Cell Line 30F16H5 (New stock received 2012 for a further 30 years)

Deposit Reference 88050502

Has been accepted as a patent deposit, in accordance with

The Budapest Treaty of 1977

With the European Collection of Cell Cultures on

05 Ma 1988 017 053417

46

FOR THE PURPOSES OF PATENT PROCEDURE

CKTERNATONAL FOR

Fraunhofer Gesellschaft 2ur FiSrderung

der angewandfen Forschang e.V,

Hansastr. 27c RECEIPT IN THE CASE OF AN ORIGINAL 01 POSIT

iuucd pursuant 10 Rule 7.1 byihi

S0686 MOnchen INTERNATIONAL DEPOSITARY ΛυΤΗΟ ^' ΚΠΎ

idcnlified -I dif boitom of this p¾c

L IDENTIFICATION' Of THE MICROORGANISM

!deciific3uOn refereact p'vin by Ac DEPOSITOR: Accession luuiibtT gi e by the

rjTftRNATIDXAL DEPOSITARY AUTHOR ΓΓΥ:

MAX. ) 6HSWT]6H5

DEM ACC3148

Π. SCIENTIFIC DESCRIPTION' AND/OR PROPOSED TAXONOM!C DESICNAT!ON

The microori-iii-ra identified unto Γ. o«oropiji>td b _j

( J o scientific decripsion

( I a ropoKd iiiDnotr.ic designation

{Mart with _ ivou epp Ikebiel.

I!!. RECEIPT AND ACCEPTANCE

This lmfrauuoriaJ Depositary Authority accepts she microorganism id-r.lifittl under I. ibove. ti h a-ss recti™) by ii oo 201 ] -12-02

(Dsr oi uw original deyan ^' l)'.

IV. RECEIP OF REQUEST FOR CONVERSION

The cnictoorgajiism identified under I ihovc was received by this Depositary Authority on (date of criiia-J deposit) and a request so convert the original deposit to - deposit under the Budipcs! Treaty received by is «n (dale of jweip! of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY

OS Z-DEUTSCHE SA ML ^' IFNG VON Sign-turcis) οΓ ίτωηΙί! hiring the power to ttprtscni the MI ROORGANISMEN U O ZELLKULTURE GmbH klem&tional Depositary Authority oi oiiuihoritcd ofEcieils):

Inhoffer-nr. 7 H

D-38124 Br-utis-huxig

Di«: 20.1 M 2- 16

' Where Rule 6.4 (dj applies, such date is the dait on which the stahis of iuttmotional d^»iuuy authority u¾s acquired.

Form DMZ-e (soli p<gt) 08/2006 T EP2017/053417

47

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Fraunhofcr-Gesellschaft zuT Fftriferung

tier angewandten Forschung e.V.

Hansasfr. 27c RECEIPT IN THE CAS E OF AN ORIGINAL DEPOSIT

iiiutd pursuant 1o Rule 7.1 by Lhe

80686 Manchen i TERNATIONAL DEPOSITARY AUTHORITY

iden tiTicd a! ihe botiom of this pace

I IDENTIFICATION OF THE M ICROORGANISM

Idcniific alien referent* givfti by the DEPOSITOR: Accession ituntber gkto by the

INTERNATIONAL DEPOSITARY AUTHORITY:

CD4.16H5.chim ^" lgG

DSM ACC3147

IL SCIENTIFIC DESCR IPTION AND.'OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under 1. above «s arcorop-nied by.

< χ > » ojeniific description

( ) a proposed [sxonomic dcsiirunion

(Mart wiifi a cross uiscre sppiicsbte),

III. RECEIPT AND ACCEPTANCE

This LotxmatSDnal Depositar Authority accepts thi microorganism identified under I. above, which u s rec i ed by it on 201 ] - 1 -02

{Dew of the original deposit/.

IV. RECEIPT OF REQUEST FOR CONVERSION

The ir.icroarg-niim identified under I er>o»c e¾j rrceiVed by this International Depositary Authority no (date Df original de osii) sod e request to convert the orii'inol deposit to deposii unit! the Budapest Treaty ·* ^■ « rtctivtd by it on [date of rcieipt ofrcqucst for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY

. ame: DSM2-DEUTSCHE SAMMLUNG VON Signatures) of perjoc{s ) r_ii½g Die pou-cr lo rcpr-jenl the MKROORGANISMEN UND Z£ LLt ULTUREN GmbH laicnutiarai Dcpwieuy Auihorii o of eut oriived offici»l(s): infioffensn-. 7 B

DO SI 24 Br-unschu 6/

Dale: 20] i - 12- 16

¹ Where R»le 6,4 (d) applies, ivth date is USt due en »'}ikh ihe iiitui of iDtemstional dcposiuiry «ui¾orii waj ac-juired.

Form DSMZ-BPM (sole page) OS/2006