Regulatory t cells and uses thereof

Abstract

To provide methods for treating disease mediated by immune response in a subject.SOLUTION: The invention relates to generation of regulatory T cells, in particular, those generated in the presence of anti-CD80 and anti-86 antibodies. The invention also relates to the use of the regulatory T cells in the treatment of a subject undergoing to organ transplantation. The invention also relates to the use of the regulatory T cells in the treatment of a subject receiving an implant. The regulatory T cells may be administered to the subject with one or more antibodies.SELECTED DRAWING: None

Description

Technical Field

[0001] <Cross - Reference to Related Applications> This application claims the benefit of U.S. Provisional Patent Application No. 61 / 824,590, filed on May 17, 2013, which is incorporated herein by reference in its entirety.

Background Art

[0002] Liver transplantation has been widely used as a final treatment for patients with end - stage liver failure. There are 20,000 cases overseas and more than 500 cases in Japan every year.

[0003] Transplantation is one of the main treatment options for end - stage kidney, heart, liver, and pancreas organ failure. Despite significant progress in treating transplant rejection in recent years, most transplants are ultimately rejected. The current immunosuppressive regimens that rely on continuous drug therapy make patients susceptible to infections and cancers because the drugs themselves cannot inhibit the reaction clearly directed against the transplant.

Summary of the Invention

Means for Solving the Problems

[0004] Provided herein is a method of treating a subject's disease mediated by an immune response, the method comprising administering to the subject a composition comprising an antibody or an antigen - binding fragment thereof that specifically binds to CD80 and CD86, thereby generating a population of regulatory T lymphocytes.

[0005] In one embodiment, the composition comprises an antibody that specifically binds to CD80 and an antibody that specifically binds to CD86.

[0006] In another embodiment, the antibody binds to one or more epitopes on CD80 and one or more epitopes on CD86.

[0007] In yet another embodiment, the antibody blocks and / or neutralizes CD80 and CD86.

[0008] Also provided herein is an ex vivo method of generating a population of regulatory T lymphocytes, the method comprising culturing T cells in the presence of cells presenting an alloantigen or a non-cellular protein antigen using a composition comprising an antibody that specifically binds to CD80 and CD86.

[0009] In one embodiment, the non-cellular protein antigen is human gamma globulin, equine gamma globulin, or ovalbumin.

[0010] In another embodiment, the T cells are obtained from a recipient animal and the cells presenting the alloantigen are either cells obtained from a donor animal or cells pulsed with an antigen obtained from a donor animal.

[0011] Also provided herein is a cell culture prepared by the above method, the cell culture comprising cultured cells obtained ex vivo in a medium. In one embodiment, the antibody that specifically binds to CD80 and CD86 is removed from the medium. The antibody may be removed from the culture medium, for example, by washing the cells and reconstituting them in the medium. The cells obtained from these methods may be further administered to a recipient subject in need thereof.

[0012] Also provided herein is a method of suppressing rejection of an organ or tissue transplant in a recipient subject, the method comprising the following steps: (a) obtaining a sample of T cells from the recipient subject; (b) obtaining a sample of an alloantigen from a donor subject who is the source of the transplanted organ or tissue; (c) exposing the sample of T cells to the sample of alloantigen in the presence of a composition comprising an antibody that specifically binds to CD80 and CD86, thereby producing a composition comprising a population of regulatory T lymphocytes; and (d) administering the composition comprising the population of regulatory T lymphocytes to the recipient subject. Step (c) may further comprise removing the antibody from the composition prior to step (d).

[0013] In one embodiment, about 1×10 9 to about 1×10 15 cells may be administered to the recipient subject. The population of regulatory T lymphocytes is administered to the recipient subject before, simultaneously with, or after transplantation of the organ or tissue. In one embodiment, the subject is human.

[0014] In another embodiment, the method may further comprise administering to the recipient subject one or more immunosuppressive agents. Non-limiting examples of immunosuppressive agents include, for example, calcineurin inhibitors (e.g., tacrolimus (FK-506), cyclosporin A (CsA), etc.), adriamycin, azathioprine (AZ), busulfan, cyclophosphamide, deoxyspergualin (DSG); FTY720 (fingolimod, also known as 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), fludarabine, 5-fluorouracil, leflunomide (LEF); methotrexate, mizoribine (MZ), mycophenolate mofetil (MMF), non-steroidal anti-inflammatory agents, sirolimus (rapamycin), corticosteroids (e.g., prednisone and methylprednisolone), agents that block CTLA-4 and / or CD28, antibodies (e.g., muromonab-CD3, alemtuzumab, basiliximab, daclizumab, rituximab, antithymocyte globulin, etc.), and combinations thereof. The one or more immunosuppressive agents may be administered to the recipient subject before, simultaneously with, or after transplantation of an organ or tissue. The present invention provides, for example, the following items. (Item 1) A method for treating a subject's disease mediated by an immune response, the method comprising administering to the subject a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD80 and CD86, wherein administration of the antibody or the antigen-binding fragment thereof induces the generation of a population of regulatory T lymphocytes. (Item 2) The composition comprises an antibody or an antigen-binding fragment thereof that specifically binds to CD80 and an antibody that specifically binds to CD86, according to the method of item 1. (Item 3) The antibody or the antigen-binding fragment thereof binds to one or more epitopes on CD80 and to one or more epitopes on CD86, according to the method of item 1 or 2. (Item 4) The method according to any one of items 1 to 3, wherein the antibody or its antigen-binding fragment blocks and / or neutralizes CD80 and CD86. (Item 5) An ex vivo method for generating a population of regulatory T lymphocytes, the method comprising culturing T cells in the presence of cells presenting an alloantigen or a non-cellular protein antigen using a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD80 and CD86. (Item 6) The method according to item 5, wherein the non-cellular protein antigen is selected from the group consisting of human gamma globulin, equine gamma globulin, and ovalbumin. (Item 7) The method according to item 5 or 6, wherein the T cells are obtained from a recipient animal, and the cells presenting the alloantigen are either cells obtained from a donor animal or cells pulsed with an antigen obtained from a donor animal. (Item 8) The method according to any one of items 5 to 7, wherein the regulatory T lymphocytes produced by the method are further administered to a subject in need thereof. (Item 9) A cell culture prepared by the method according to any one of items 5 to 7, comprising cells and a medium. (Item 10) The cell culture according to item 9, wherein the antibody is removed from the medium. (Item 11) The cell culture according to item 10, wherein the antibody is removed by washing. (Item 12) A method for suppressing rejection of an organ or tissue transplant in a recipient subject, the method comprising: (a) obtaining a sample of T cells from the recipient subject; (b) obtaining a sample of an alloantigen from a donor subject who is the source of the transplanted organ or tissue; (c) A step of exposing a sample of T cells to a sample of an alloantigen in the presence of a composition comprising an antibody that specifically binds to CD80 and CD86, thereby generating a composition comprising a population of regulatory T lymphocytes; and (d) A step of administering a composition comprising a population of regulatory T lymphocytes to a recipient subject A method comprising the above steps. (Item 13) The method according to item 12, wherein step (c) further comprises a step of removing the antibody from the composition. (Item 14) About 1×10 9 to about 1×10 15 The cells are administered to the recipient subject, and the method according to item 12 is characterized by this. (Item 15) The population of regulatory T lymphocytes is administered to the recipient subject before, simultaneously with, or after transplantation of an organ or tissue, and the method according to item 12 is characterized by this. (Item 16) The subject is a human, and the method according to item 12 is characterized by this. (Item 17) The method according to any one of items 12 to 16, further comprising a step of administering one or more immunosuppressive drugs to the recipient subject. (Item 18) The one or more immunosuppressive drugs are calcineurin inhibitors, adriamycin, azathioprine (AZ), busulfan, cyclophosphamide, deoxyspergualin (DSG); FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), fludarabine, 5-fluorouracil (5-FU), leflunomide (LEF), methotrexate, mizoribine (MZ), mycophenolate mofetil (MMF), non-steroidal anti-inflammatory agents, sirolimus (rapamycin), corticosteroids, agents that block CTLA-4, agents that block CD28, antibodies, or combinations thereof, and the method according to item 17 is characterized by this. (Item 19) The method according to item 17, wherein the immunosuppressive agent is administered to the recipient subject before, simultaneously with, or after transplantation of an organ or tissue. (Item 20) The method according to item 18, wherein the calcineurin inhibitor is tacrolimus (FK-506) or cyclosporin A (CsA). (Item 21) The method according to item 18, wherein the corticosteroid is prednisolone or methylprednisolone. (Item 22) The method according to item 18, wherein the antibody is muromonab-CD3, alemtuzumab, basiliximab, daclizumab, rituximab, or antithymocyte globulin.

[0015] <Incorporation by reference> All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference.

Brief Description of the Drawings

[0016] The novel features of the embodiments are, in particular, specified within the scope of the appended claims. A better understanding of the features and advantages of the present embodiments can be obtained by referring to the following detailed description that specifies specific examples in which the principles of the embodiments are utilized, and the following appended drawings.

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Mode for Carrying Out the Invention

[0017] When the transplanted tissue is rejected by the recipient's immune system, a transplant rejection reaction occurs and destroys the transplanted tissue. The transplant rejection reaction can be alleviated by the determination of molecular similarity between the donor and the recipient and by the use of immunosuppressive drugs after transplantation. The present inventors have identified a new subgroup of regulatory T cells useful for suppressing organ transplant rejection.

[0018] <Problems of Conventional Immunosuppressive Therapy in Liver Transplantation> Liver transplantation has been widely used as the ultimate treatment for patients with end-stage liver failure. This development of liver transplantation depends on progress in surgical techniques, organ preservation, and pre- and post-operative management, among which improvements in immunosuppressive agents have contributed greatly. The 1-year and 5-year survival rates have increased dramatically to 35% and 20%, 70% and 60%, and 80% and 70%, respectively, by the azathioprine (1960s - 70s), cyclosporine (1980s), and tacrolimus (since the 1990s) used (Citation 1, 2). The world's first clinical liver transplant was performed in 1963. To date, more than 300,000 cases have been performed, with more than 20,000 cases overseas and more than 500 cases in Japan every year. These patients have to take immunosuppressive drugs for life to control rejection reactions and are constantly exposed to the risks of drug-induced side effects such as infections and carcinogenesis, so there are important unsolved problems that affect both medicine and the medical economy. To eliminate these problems, so-called induction of immune tolerance is required, whereby the graft functions properly even if immunosuppressive drugs are discontinued.

[0019] CD4+ T-helper (T H ) lymphocytes play an essential role in the immune response that protects the subject from pathogens such as bacteria and viruses in healthy individuals. However, when a subject receives a transplanted organ, these cells mainly cause organ transplant rejection. It has been previously determined that administration of immunosuppressive agents such as anti-CD4 antibodies targeting CD4+ T cells can reduce organ transplant rejection. In recent years, antibody therapy has been shown to lead to the generation of a subpopulation of T cells that can control or regulate harmful rejection responses in several instances. Since the presence of anti-CD4 antibodies prevents complete T cell activation, regulatory cells can be generated in such instances, and the cells are initialized to a regulatory or inhibitory phenotype (default).

[0020] <Immune Tolerance and Regulatory T Cells> Since the early 1970s, suppressor T cells have been found in recipients with a state of immune tolerance in models of autoimmune diseases and organ transplantation using small animals, and these lymphocytes can transfer immune tolerance (infectious tolerance) to naive hosts by adoptive transfer.

[0021] These findings advanced research on suppressor T cells. Co-investigator Okumura (Reference 3) is a pioneer in the field of transplantation immunology who first reported these suppressor T cells worldwide in the early 1970s. Research on suppressor T cells has been negated because it was difficult to establish a cytological identification method, but in recent years, phenotypes / markers such as + CD25 + and Foxp3 + have been discovered, and regulatory T cells: Tregs have attracted attention again (Reference 4), and the effects of using the same cells concentrated in in vitro and small animal transplantation models have been reported in many studies.

[0022] <Ex Vivo Induction and Proliferation of Regulatory T Cells> In allogeneic organ transplantation, graft rejection of the graft is mainly caused by the recipient's cell-mediated immunity. Since this cell-mediated immunity is a donor antigen-specific reaction, donor antigens presented by antigen-presenting cells such as dendritic cells recognize helper CD4 T cells, activate effector CD8 T cells, and ultimately cause rejection. Costimulation is required for the activation of helper T cells and was first known in the early 1990s. However, the group of Okumura et al. found that costimulation is transmitted by the binding of CD28 on T cells to CD80 / CD86 on antigen-presenting cells, and recipient T cells do not elicit an immune response against donor antigen-presenting cells by adding anti-CD80 and anti-CD86 antibodies in cell culture, resulting in a state of donor antigen-specific anergy. In recent studies, anergic T cells have been found to act as donor antigen-specific regulatory T cells (Tregs). In addition, co-investigators Okumura, Bashuta, Seino et al. successfully induced antigen-specific Treg-like cells ex vivo by adding anti-CD80 and anti-CD86 antibodies in lymphocyte culture medium and confirmed long-term graft survival in a mouse heart transplantation model by injecting the collected cells (citation 6). Furthermore, in a study attempting the same protocol for a preclinical trial using a monkey kidney transplantation model, injection of recipient Treg-like cells collected from co-culture and induction of peripheral blood mononuclear cells (PBMCs) under donor splenocytes and anti-CD80 / CD86 antibodies two weeks before and two weeks after transplantation enabled early withdrawal of the immunosuppressant cyclosporine, the transplanted kidney achieved long-term (>600 days) survival, and donor antigen-specific immune tolerance was successfully induced even in the immunosuppression-free state (citation 7).

[0023] <Antibodies, Generation of Regulatory T Cells, Cell Culture, and Uses Thereof> This specification provides a method for treating a disease of a subject mediated by an immune response, the method comprising administering to the subject a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD80 and CD86, thereby generating a population of regulatory T lymphocytes.

[0024] In one embodiment, the composition comprises an antibody that specifically binds to CD80 and an antibody that specifically binds to CD86.

[0025] In another embodiment, the antibody binds to one or more epitopes on CD80 and one or more epitopes on CD86.

[0026] In yet another embodiment, the antibody blocks and / or neutralizes CD80 and CD86.

[0027] In yet another embodiment, the antigen-binding fragment may be, for example, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, an Fv fragment, a scFv fragment, a single-chain binding polypeptide, an Fd fragment, a variable heavy chain, a variable light chain, a dAb fragment, an AVIMER, a bispecific antibody, or a heavy chain dimer. The heavy chain dimer may be, for example, a camelid or shark heavy chain construct.

[0028] Any antibody that specifically binds to CD80 or CD86 may be used in the compositions described herein, such as those found in the following examples. Commercially available antibodies and hybridomas may be obtained, for example, from ATCC; the sequences of variable heavy and light chains may be found in public databases such as NCBI PubMed; and companies such as Bay Bioscience Co., Ltd., Thermo Scientific Pierce Antibodies, Lifespan Biosciences, Inc., and BD Biosciences also produce anti-CD80 and anti-CD86 antibodies commercially.

[0029] As used herein, the term "antibody" refers to an immunoglobulin (Ig), whether natural or produced by partial or total synthesis. This term also encompasses any polypeptide or protein having an antigen-binding domain or a binding domain homologous thereto. This term further includes "antigen-binding fragments" and other terms interchangeable with similar binding fragments as described below.

[0030] Native antibodies and native immunoglobulins are usually about 150,000 Dalton heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is typically bound to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. The heavy and light chains each have regularly spaced intrachain disulfide bridges. Each heavy chain has, at one end, a variable domain ("V H " or "VH") followed by a number of constant domains ("C H " or "CH"). Each light chain has, at one end, a variable domain ("V L " or "VL") and, at the other end, a constant domain ("C L " or "CL"); the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the variable domains of the light and heavy chains.

[0031] As used herein, the terms "synthetic polynucleotide", "synthetic gene", or "synthetic polypeptide" mean that the corresponding polynucleotide sequence or a part thereof, or the amino acid sequence or a part thereof, is derived from a sequence that has been designed, newly synthesized, modified, or compared to an equivalent naturally occurring sequence. Synthetic polynucleotides (antibodies or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including but not limited to chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes typically differ from naturally occurring genes at either the amino acid or polynucleotide level (or both), and are typically positioned within the context of synthetic expression control sequences. The synthetic gene polynucleotide sequences do not necessarily encode proteins with different amino acids compared to natural genes; for example, they can also include synthetic polynucleotide sequences that incorporate different codons but encode the same amino acids (i.e., the nucleotide changes represent silent mutations at the amino acid level).

[0032] With respect to an antibody, the term "variable domain" refers to the variable domain of the antibody that is used in the binding and specificity of an individual particular antibody with respect to its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. Rather, it is concentrated in three segments, called hypervariable regions, in both the variable domains of the light and heavy chains (also known as CDRs). The more highly conserved portions of the variable domain are called the "framework regions" or "FRs". The variable domains of the unmodified heavy and light chains each contain four FRs (FR1, FR2, FR3, FR4), and mostly adopt a β-sheet structure with three CDRs interspersed that form loop junctions, and in some cases, part of the β-sheet structure. The CDRs in each chain are held together in proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669).

[0033] As used herein, the terms "hypervariable region" and "CDR" refer to the amino acid residues of an antibody that are responsible for antigen binding. CDRs contain amino acid residues from three sequence regions that bind in a complementary fashion to an antigen and are known as CDR1, CDR2, and CDR3 for each of the VH and VL chains. In the light chain variable domain, the CDRs typically correspond to approximately residues 24 - 34 (CDRL1), 50 - 56 (CDRL2), and 89 - 97 (CDRL3), and in the heavy chain variable domain, the CDRs typically correspond to approximately residues 31 - 35 (CDRH1), 50 - 65 (CDRH2), and 95 - 102 (CDRH3) according to "Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)". It should be understood that the CDRs of different antibodies may contain inserts and thus the amino acid numbering may vary. The Kabat numbering system accounts for such inserts by using letters assigned to specific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain) in order to reflect any inserts in the numbering between different antibodies. Alternatively, in the light chain variable domain, the CDRs typically correspond to approximately residues 26 - 32 (CDRL1), 50 - 52 (CDRL2), and 91 - 96 (CDRL3), and in the heavy chain variable domain, the CDRs typically correspond to approximately residues 26 - 32 (CDRH1), 53 - 55 (CDRH2), and 96 - 101 (CDRH3) according to "Chothia and Lesk, J. Mol. Biol., 196: 901 - 917 (1987)".

[0034] As used herein, "framework region" or "FR" refers to framework amino acid residues that form part of an antigen-binding pocket or groove. In some embodiments, framework residues form loops that are part of the antigen-binding pocket or groove, and the amino acid residues in the loops may or may not contact the antigen. The framework region typically includes regions between the CDRs. In the light chain variable domain, the FR typically corresponds to approximately residues 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109, and in the heavy chain variable domain, the FR typically corresponds to approximately residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133 according to "Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)". As discussed above with respect to Kabat numbering for the light chain, the heavy chain also occupies insertions in a similar fashion (e.g., 35A, 35B of CDRH1 in the heavy chain). Alternatively, in the light chain variable domain, the FR typically corresponds to approximately residues 0-25 (FRL1), 33-49 (FRL2), 53-90 (FRL3), and 97-109 (FRL4), and in the heavy chain variable domain, the FR typically corresponds to approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4) according to "Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)".

[0035] The constant domain (Fc) of an antibody is not directly involved in the binding of the antibody to the antigen, but rather exhibits various effector functions, such as the involvement of the antibody in antibody-dependent cytotoxicity through interaction with, for example, Fc receptors (FcR). The Fc domain can also increase the bioavailability of the antibody in circulation after administration to a patient. Substitution of the mouse Fc domain with the human Fc domain can also reduce the side HAMA reaction.

[0036] Depending on the amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classifications. There are five major classifications of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2). The heavy chain constant domains (Fc) corresponding to different classifications of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional arrangement of different classifications of immunoglobulins are well known.

[0037] The "light chain" of an antibody from any vertebrate species can be assigned to one of two clearly different types, called kappa or (κ) and lambda or (λ), based on the amino acid sequence of the constant domain.

[0038] The terms "antigen-binding portion of an antibody", "antigen-binding fragment", "antigen-binding domain", "antibody fragment", or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Non-limiting examples of antibody fragments included in such terms are, without limitation, (i) Fab fragment (a monovalent fragment consisting of the domains of V L , V H , C L , and C H1 ); (ii) F(ab’)2 fragment (a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); (iii) V H and CH1 an Fd fragment consisting of the domains of; (iv) the V of a single arm of an antibody L and V H an Fv fragment containing the domains of; (v) V H a dAb fragment containing the domain (Ward et al., (1989) Nature 341:544 546); and (vi) isolated CDRs. In addition, in this definition, "half" antibodies containing a single heavy chain and a single light chain are included. Other forms of single-chain antibodies such as bispecific antibodies are also encompassed herein.

[0039] The "F(ab’)2" and "Fab" portions can be created by treating Ig with proteases such as pepsin and papain, and include antibody fragments generated by digesting the immunoglobulin near the disulfide bonds present between the hinge regions in each of the two heavy chains. For example, papain cleaves the IgG upstream of the disulfide bonds present between the hinge regions in each of the two heavy chains to generate two homologous antibody fragments, where L and C L a light chain composed of (light chain constant region), and, in the constant region of the heavy difference, V H and C Hγ1 (γ1) region, and the heavy chain fragments are connected at their C-terminal regions via disulfide bonds. Each of these two homologous antibody fragments is called Fab’. Pepsin also cleaves the IgG downstream of the disulfide bonds present between the hinge regions in each of the two heavy differences to generate an antibody fragment slightly larger than the fragment in which the two aforementioned Fab’ are connected by the hinge region. This antibody fragment is called F(ab’)2.

[0040] The Fab fragment also includes the constant domain of the light chain and the first constant domain of the heavy chain (C H 1). The Fab’ fragment is a heavy chain C containing one or more cysteines from the antibody hinge region HAt the carboxyl terminus of the domain, by adding a few residues, it is different from the Fab fragment. Fab’-SH is the designation herein for a Fab’ in which the cysteine residue of the constant domain has a free thiol group. The F(ab’)2 antibody fragment is originally produced as a pair of Fab’s having a hinge cysteine in between. Other chemical linkages of antibody fragments are also known.

[0041] “Fv” refers to an antibody fragment that contains the complete antigen recognition and antigen-binding site. This region consists of a dimer of the variable domains of one heavy chain and one light chain in a rigid, non-covalent, or covalent association (disulfide-linked Fv’s are described in the art, Reiter et al. (1996) Nature Biotechnology 14:1239-1245). In this configuration, V H -V L To define the antigen-binding site on the surface of the dimer, the three CDRs of the individual variable domains interact with each other. Generally, one or more combinations of CDRs from each of the V H and V L chains confer antigen-binding specificity to the antibody. For example, CDRH3 and CDRL3 are sufficient to confer antigen-binding specificity to an antibody when transferred to the V H and V L chains of a recipient antibody or its antigen-binding fragment, and it should be understood that this combination of CDRs can be tested for binding, affinity, etc. using the techniques described herein. Even a single variable domain (or half of an Fv containing only the three CDRs specific for an antigen) has the ability to recognize and bind the antigen, but the affinity is probably lower than when combined with a second variable domain. Furthermore, the two domains of the Fv fragment (V L and V H ) are encoded by separate genes, but they pair as V L and V HUsing recombinant methods with synthetic linkers that allow regions to be made as single protein chains that form monovalent molecules, which can be linked (known as single-chain Fv (scFv); Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85:5879-5883; and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such scFv are also intended to be included within the antibody term "antigen-binding portion". The sequences of any specific scFv V H and V L can be joined to an Fc region cDNA or genomic sequence to generate an expression vector encoding a complete Ig (e.g., IgG) molecule or other isotype. V H and V L can also be used in the generation of Fab, Fv, or other fragments of Ig using either protein chemistry or recombinant DNA technology.

[0042] An antibody fragment of "single-chain Fv" or "sFv" can contain the V H and V L domains of an antibody, where these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the V H and V L domains that allows the sFv to form the structure desired for antigen binding. For an evaluation of sFv, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0043] The term "AVIMER™" refers to a class of human-derived therapeutic proteins, which are unrelated to antibodies and antibody fragments and are composed of various modular and reusable binding domains called A-domains (also referred to as Class A modules, complement-type repeats, or the Class A domains of the LDL-receptor). They were developed from human extracellular receptor domains by in vitro exon shuffling and phage display (Silverman et al., 2005, Nat. Biotechnol. 23:1493-1494; Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteins can contain multiple independent binding domains and can exhibit improved affinity (in some cases, sub-nanomolar) and specificity compared to single epitope-binding proteins. See, for example, U.S. Patent Application Publication Nos. 2005 / 0221384, 2005 / 0164301, 2005 / 0053973, 2005 / 0089932, 2005 / 0048512, and 2004 / 0175756, each of which is incorporated herein by reference in its entirety.

[0044] Each of the known 217 human A-domains contains ~35 amino acids (~4 kDa). These domains are separated by linkers that are on average 5 amino acids in length. Native A-domains rapidly and efficiently fold into a uniform and stable structure, mainly mediated by calcium binding and disulfide formation. A conserved scaffold motif of only 12 amino acids is required for this common structure. The end result is a single protein chain containing multiple domains, each representing a distinct function. The domains of the protein bind independently, and the energetic contributions of each domain are additive. These proteins were called "AVIMER™" from the binding-active multimers.

[0045] The term "bispecific antibody" refers to a small antibody fragment that has two antigen-binding sites, the fragment containing a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and to generate two antigen-binding sites. Bispecific antibodies are described more fully, for example, in European Patent No. 404,097; WO93 / 11161; and Hollinger et.al., Proc. Natl. Acad. Sci. USA, 90:6444 6448 (1993).

[0046] Antigen-binding polypeptides also include, for example, heavy-chain dimers such as camel and shark antibodies. Camel and shark antibodies contain a pair of homodimeric chains of V-like and C-like domains (both lacking light chains). Since the V H region of the heavy-chain dimer IgG in camels does not need to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts the light chain is changed to hydrophilic amino acid residues in camels. The V H domain of the heavy-chain dimer IgG is called the V HH domain. Shark Ig-NAR contains a homodimer of one variable domain (referred to as the V-NAR domain) and five C-like constant domains (C-NAR domains). In camels, the repertoire of antibody diversity is determined by CDR1, 2, and 3 in the V H or V HH region. The CDR3 in the V HH region of camels is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to the CDR3 regions of antibodies of many other species. For example, mouse V HThe CDR3 has an average of 9 amino acids. A library of camelid-derived antibody variable regions (maintaining the in vivo diversity of camelid variable regions) can be made, for example, by the method disclosed in U.S. Patent Application No. 20050037421.

[0047] The "chimeric" form of a non-human (e.g., mouse) antibody includes chimeric antibodies containing a minimal sequence derived from non-human Ig. Most often, the chimeric antibody is a mouse antibody, where at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin, is inserted in place of the mouse Fc. See, e.g., Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0048] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a population in which each antibody is identical except for possible naturally occurring variants that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Further, in contrast to conventional (polyclonal) antibody preparations that may include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by the hybridoma method first described in "Kohler et al., Nature 256:495 (1975)" or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). In certain embodiments, monoclonal antibodies can be isolated from phage antibody libraries using, for example, the techniques described in "Clackson et al., Nature 352:624-628 (1991)" and "Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0049] Antibodies can be isolated and purified from the above-described culture supernatants or ascites by saturated ammonium sulfate precipitation, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), or affinity chromatography using, for example, anti-Ig columns or columns of protein A, G, or L as detailed below.

[0050] When constructing immunoglobulin molecules, the variable region or a portion thereof can be fused, joined, or otherwise linked to one or more constant regions or portions thereof to create any of the antibodies described herein. This may be accomplished by various methods known in the art, including, but not limited to, molecular cloning techniques of nucleic acids encoding the molecule or direct synthesis.

[0051] As used herein, "immunoreactive" refers to a binding agent, antibody, or fragment thereof that is specific for the sequence of an amino acid residue ("binding site" or "epitope"), provided that when they cross-react with other peptides / proteins, they are not toxic at the levels prescribed for administration for human use. The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions under physiological conditions, including interactions such as salt bridges and water bridges, and other conventional binding means. The term "binds preferentially" means that the binding agent binds to the binding site with a greater affinity than it binds to unrelated amino acid sequences. The affinity may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the binding agent for unrelated amino acid sequences. The terms "immunoreactive" and "binds preferentially" are used interchangeably herein.

[0052] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and is expressed as Kd. The affinity of a binding protein for a ligand, such as the affinity of an antibody for an epitope, can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). As used herein, the term "binding activity" refers to the resistance of a complex of two or more agents to separation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA), or any other technique familiar to those skilled in the art. Binding activity can be determined by methods such as Scatchard analysis, or any other technique familiar to those skilled in the art.

[0053] "Epitope" refers to a part of an antigen or other macromolecule that can form a binding interaction with the variable region binding pocket of an antibody. Such binding interactions can be characterized as intermolecular contacts with one or more amino acid residues of one or more CDRs. Antigen binding can involve, for example, CDR3 or a pair of CDR3s, or, in some cases, interactions up to a total of 6 CDRs from the V H and V L chains. An epitope can be a linear peptide sequence (i.e., "continuous") or can be composed of non-adjacent amino acid sequences (i.e., "conformational" or "discontinuous"). An antibody can recognize one or more amino acid sequences; thus, an epitope can define more than one different amino acid sequence. The epitope recognized by an antibody can be determined by techniques of peptide mapping and sequence analysis well known to those skilled in the art. The binding interaction can be characterized as intermolecular contacts with one or more amino acid residues of the CDR.

[0054] The term "specific" refers to a situation where the antibody does not show any significant binding to molecules other than the antigen containing the epitope recognized by the antibody. This term is also applicable, for example, when the antigen-binding domain is specific for a particular epitope carried by many antigens, in which case the antibody can bind to the diverse antigens carrying the epitope. The terms "binds preferentially" or "binds specifically" mean that the antibody binds to the epitope with a greater affinity than it binds to amino acid sequences to which it is not related, and is not toxic at the levels prescribed for administration to humans if it cross-reacts with other polypeptides containing the epitope. In one embodiment, such affinity is at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the antibody for the unrelated amino acid sequence. The terms "immunoreactive", "binds", "binds preferentially", and "binds specifically" are used interchangeably herein. The term "binds" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions under physiological conditions, including interactions such as salt bridges and water bridges, as well as other conventional means of binding.

[0055] "Isolated" (used interchangeably with "substantially pure"), when applied to a polypeptide, means the polypeptide or a portion thereof that by virtue of its origin or manipulation: (i) is present in a host cell as a product of expression of a portion of an expression vector; or (ii) is bound to a protein or other chemical moiety other than one to which it is naturally bound; or (iii) does not occur naturally and is a protein that has been chemically engineered, for example, by supplementing or adding at least one hydrophobic moiety to the protein, whereby the protein is in a form not found in nature. By "isolated," it further means a protein that is: (i) chemically synthesized; or (ii) expressed in a host cell and purified away from associated and contaminating proteins. This term generally means a polypeptide separated from other naturally-occurring proteins and nucleic acids. Typically, the polypeptide is also separated from substances such as antibodies or a gel matrix (polyacrylamide) used to purify it.

[0056] Also provided herein is an ex vivo method of generating a population of regulatory T lymphocytes, the method comprising culturing T cells in the presence of cells presenting an allogeneic antigen or a non-cellular protein antigen using a composition comprising an antibody that specifically binds to CD80 and CD86. In one embodiment, the non-cellular protein antigen is human gamma globulin, equine gamma globulin, or ovalbumin.

[0057] The cells may be obtained from a subject who is to receive an organ or graft transplant. For example, the T cells are obtained from a recipient animal and the cells presenting the allogeneic antigen are either cells obtained from a donor subject or cells pulsed with an antigen obtained from a donor subject.

[0058] Also provided herein is a cell culture prepared by the above method, which cell culture comprises cultured cells obtained by an ex vivo method in a medium. In one embodiment, an antibody that specifically binds to CD80 and CD86 is removed from the medium. Removal of the antibody may be by any conventionally accepted laboratory method such as those described in the Examples. The antibody may be removed from the culture medium, for example, by washing the cells and reconstituting the cells in the medium. The cells obtained from these methods may be further administered to a recipient subject in need thereof. Any commercially acceptable medium (e.g., DMEM, RPMI, etc.) may be used to culture the cells under conditions according to conventional laboratory methods.

[0059] After culturing, the regulatory T cells used in the treatment methods described herein are, for example, CD4 + , CD25 + , and Foxp3 + and show cell surface markers such as.

[0060] Also provided herein is a method of suppressing rejection of an organ or tissue transplant in a recipient subject, the method comprising the following steps: (a) obtaining a sample of T cells from the recipient subject; (b) obtaining a sample of an alloantigen from a donor subject who is the source of the organ or tissue being transplanted; (c) exposing the sample of T cells to the sample of alloantigen in the presence of a composition comprising an antibody that specifically binds to CD80 and CD86, thereby generating a composition comprising a population of regulatory T lymphocytes; and (d) administering the composition comprising the population of regulatory T lymphocytes to the recipient subject. Step (c) may further comprise removing the antibody from the composition prior to step (d).

[0061] In one embodiment, about 1×10 9 to about 1×10 15The cells may be administered to the recipient subject. The number of cells to be administered to the subject may be determined based on the experience of the physician based on the age, weight, height, and health status of the recipient subject. The population of regulatory T lymphocytes is administered to the recipient subject before, simultaneously with, or after transplantation of an organ or tissue (graft). In one embodiment, the subject is human. Administration of the cells or antibodies to the subject may be by any means, such as injection or infusion.

[0062] Provided herein are methods for inhibiting or delaying rejection of a graft or organ to prevent or inhibit various modes of attack (e.g., inhibition of T cell attack, inhibition of antibody response, and inhibition of the effects of cytokines and complement). Donor pre-screening to match the recipient is performed to assist in preventing rejection, particularly in preventing hyperacute rejection.

[0063] For the purposes of this embodiment, a "therapeutically effective amount" means, in the conventional sense, i.e., an amount sufficient to provide a health benefit to the subject being treated, such that in one embodiment the transplanted organ is not rejected. In another embodiment, a therapeutically effective amount is an amount sufficient to provide a health benefit to the subject being treated, such that in one embodiment the time to rejection is delayed by about 1 month, about 6 months, about 12 months, about 1.5 years, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15 years, about 20 years or more compared to a subject not receiving treatment.

[0064] The recipients described herein may be, for example, recipients of solid organ transplants selected from the group consisting of hematopoietic cell or bone marrow transplants, islet cell allografts, or heart transplants, kidney-pancreas transplants, kidney transplants, liver transplants, lung transplants, and pancreas transplants. Additional examples of grafts or transplants include, but are not limited to, allogeneic cells, tissues, or organs such as vascular tissue, eyes, corneas, lenses, skin, bone marrow, muscle, connective tissue, gastrointestinal tissue, nerve tissue, bone, stem cells, cartilage, hepatocytes, or hematopoietic cells. In some embodiments, the graft rejection reaction is an acute humoral rejection reaction of the transplanted cells, tissues, or organs. In other embodiments, the graft rejection reaction is a chronic humoral rejection reaction of the transplanted cells, tissues, or organs.

[0065] In some embodiments, the cell populations described herein are administered prior to transplantation. In other embodiments, the cell populations described herein are administered at the time of transplantation. In other embodiments, the cell populations described herein are administered after transplantation.

[0066] Non-limiting examples of specific protocols that may be used in the methods described herein are provided in more detail in the Examples and the Drawings.

[0067] Additional drugs may be utilized to delay graft rejection (i.e., to extend graft engraftment or the survival of organ transplant recipients), as required in some instances. Any of the methods described herein may be performed in conjunction with another treatment. For example, a patient may be administered one or more immunosuppressive drugs during the treatment. The immunosuppressive drugs may assist in preventing the immune system from rejecting the organ transplant. Non-limiting examples of immunosuppressive drugs include, but are not limited to, calcineurin inhibitors (e.g., tacrolimus (FK-506), cyclosporine A (CsA), etc.), adriamycin, azathioprine (AZ), busulfan, cyclophosphamide, deoxyspergualin (DSG); FTY720 (fingolimod, also known as 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), fludarabine, 5-fluorouracil, leflunomide (LEF); methotrexate, mizoribine (MZ); mycophenolate mofetil (MMF), non-steroidal anti-inflammatory agents, sirolimus (rapamycin), corticosteroids (e.g., prednisone and methylprednisolone), agents that block CTLA-4 and / or CD28, antibodies (e.g., muromonab-CD3, alemtuzumab, basiliximab, daclizumab, rituximab, antithymocyte globulin, etc.), or combinations thereof.

[0068] Cyclosporin A is one of the most widely used immunosuppressive drugs for inhibiting graft rejection by inhibiting interleukin-2 (IL-2) (it prevents the mRNA transcription of interleukin-2). More directly, cyclosporin inhibits calcineurin activation that normally occurs after T cell receptor stimulation. Calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), thereby enabling NFAT to enter the nucleus and bind to the interleukin-2 promoter. By blocking this process, cyclosporin A inhibits the activation of CD4+ T cells and the cascade resulting from other subsequent events. Tacrolimus is another immunosuppressive agent that acts by inhibiting the production of interleukin-2 via calcineurin inhibition. Rapamycin (sirolimus), SDZ RAD, and interleukin-2 receptor blockers are drugs that inhibit the action of interleukin-2 and thus prevent the cascade of the above-described events. Inhibitors of purine or pyrimidine biosynthesis are also used to inhibit graft rejection. These inhibitors prevent DNA synthesis and thereby inhibit cell division, including the proliferation of T cells. The result is the inhibition of T cell activity by preventing the formation of new T cells. Inhibitors of purine synthesis include azathioprine, methotrexate, mycophenolate mofetil (MMF), and mizoribine (bredinin). Inhibitors of pyrimidine synthesis include brequinar sodium and leflunomide. Cyclophosphamide is an inhibitor of both purine and pyrimidine synthesis. Another way to inhibit T cell activation is to treat the recipient with an antibody that specifically binds to T cells. For example, OKT3 is a mouse monoclonal antibody against CD3. This antibody first activates T cells via the T cell receptor and then induces cell death of the activated T cells.

[0069] Other drugs and methods for delaying allograft rejection are available. One method is, for example, to deplete T cells by irradiation. T cell depletion has been frequently used in bone marrow transplantation, especially when there is a partial mismatch of the major HLA. The recipient may be administered an inhibitor (blocking agent) of the CD40 ligand-CD40 interaction.

[0070] In some embodiments, the population of cells and immunosuppressive agent described herein are administered prior to transplantation. In other embodiments, the population of cells and immunosuppressive agent described herein are administered at the time of transplantation. In other embodiments, the population of cells and immunosuppressive agent described herein are administered after transplantation.

Example

[0071] This application will be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented to more fully illustrate the embodiments, but should not be construed as limiting the broad scope of the application.

[0072] <Preparation of regulatory T cells> <Composition> Donor lymphocytes and patient lymphocytes are obtained by suspending cells collected by culturing for 2 weeks in 100 ml of saline in the presence of anti-CD80 antibody (2D10.4) and anti-CD86 (IT2.1).

[0073] <Raw materials> The main raw materials are as follows.

[0074] 1. Donor lymphocytes: Peripheral blood mononuclear cells collected in a blood collection device for components (4×10 9 or more).

[0075] 2. Patient (recipient) lymphocytes: Peripheral blood mononuclear cells collected in a blood collection device for components (5×10 9 or more).

[0076] If the amount of lymphocytes is inappropriate, add lymphocytes derived from the patient's spleen.

[0077] 3. Anti-CD80 antibody (2D10.4) of Bay Bioscience Co., Ltd.; Quality specification: non-GMP manufacturing, endotoxin-free.

[0078] 4. Anti-CD86 antibody (IT2.2) of Bay Bioscience Co., Ltd.; Quality specification: non-GMP manufacturing, endotoxin-free.

[0079] <Mechanism of action> Predict the induction of immune tolerance by cultured regulatory T cells.

[0080] <Effect> According to the mechanism of action described in Section 2.4, it is predicted that the dosage of the immunosuppressant can be reduced and discontinued at the early stage after liver transplantation.

[0081] <Manufacturing method> Describe the manufacturing method in the standard procedure of the test substance. The main manufacturing process is as follows.

[0082] 1. Collect more than 4×10 9 mononuclear cells from the peripheral blood of the donor using a blood collection device for components, and cryopreserve them.

[0083] 2. Collect more than 5×10 9 mononuclear cells from the peripheral blood of the patient using a blood collection device for components. If the number of cells is insufficient, it is possible to collect lymphocytes from the spleen removed at the time of liver transplantation and add them.

[0084] 3. Thaw 2×10 9 donor mononuclear cells and co-culture them with the patient's mononuclear cells in the presence of the patient's plasma, anti-CD86 antibody, and anti-CD80 antibody.

[0085] 4. After one week, the cultured cells were collected, and again, 2×10 of the donor's mononuclear cells 9 were thawed and co-cultured with the patient's mononuclear cells in the presence of the patient's plasma, anti-CD86 antibody, and anti-CD80 antibody.

[0086] 5. After one week, the cultured cells were collected, washed, and suspended in 100 ml of saline.

[0087] <Quality specifications> The quality specifications are described in the standard procedures of the test substance. The quality specifications of the intermediate test substance and the final test substance are as follows. Samples of the intermediate test substance are taken from the culture medium four days before the preparation of the final test substance. Samples of the final test substance are taken from the cell suspension immediately after the collection of the final test substance.

[0088]

Table 1

[0089] <Results of non-clinical studies> Small-scale culture test of healthy human peripheral blood samples

[0090] <Test method> Peripheral blood was collected from two healthy adults, one similar to the donor and the other similar to the patient (recipient), and a small number of cells were used. The experiment for inducing regulatory T cells was performed with the same conditions as the production method and the antibodies used. Regarding the cells before and after culture, the cell number and surface antigens were analyzed, and the effect of immunosuppression was examined by MLR.

[0091] <Results of induction of regulatory T cells> The experiment was performed four times according to the above method. A summary of the results is shown in Table 1.

[0092] The total number of lymphocytes was 23.89±11.39×10 before culture 6 to 6.80±7.46×10 after two weeks of culture 6is reached. The specific fraction of lymphocytes that cannot remain in the current culture system, and the death of other white blood cells, are considered for the decrease in the number of these cells.

[0093] According to the analysis of surface antigens, the phenotypes of these lymphocytes were analyzed; CD3 + CD4 + Following the two-week culture of the cells, an increase of approximately 15% from 40.83 ± 3.52% to 55.01 ± 5.39% was recognized. On the other hand, regulatory T cells, CD4 + CD25 + Foxp3 + cells increased more than 10-fold at a ratio from 0.21 ± 0.04% to 2.73 ± 1.27%. Therefore, regulatory T cells are considered to be selectively induced by the current culture system. Furthermore, regarding the ratio among CD4 + cells, it increased from 1.29 ± 0.60% to 6.16 ± 2.01% for CD4 + CD25 + Foxp3 + cells, from 1.90 ± 1.27% to 4.68 ± 1.49% for CD4 + CD25 + CTLA4 + cells, and from 1.04 ± 0.79% to 2.40 ± 2.24% for CD4 + CD127 lo Foxp3 + cells, respectively.

[0094]

Table 2

[0095] Figure 1 shows the results of flow cytometry regarding the surface antigen analysis before and after typical culture. This figure shows CD4 + cells to CD25 + Foxp3 + cells and CD25 + CTLA4 + cells.

[0096] <Characteristics of cultured cells> After culturing, regulatory T cells, CD4 + CD25 + Foxp3 + The ratio of cells increased significantly, but the frequency remained at a few percent. To understand the characteristics of the test substance, the cell fraction contained in the test substance was examined by analyzing surface antigens according to the cultured cells. Using the cells collected from the above experiment, the experiment was conducted 4 times. The results are shown in Table 2.

[0097] Among the cultured cells, CD4 + T cells contained 55.01 ± 5.39%, CD8 + T cells contained 26.5 ± 4.68%, and T cells contained more than 80%. In contrast, B and NK cells contained 5.98 ± 0.85% and 2.81 ± 1.45% respectively. In addition, monocytes contained 4.83 ± 3.41%. Furthermore, dendritic cells contained 2.3%, and granulocytes contained approximately 0.2%.

[0098]

Table 3

[0099] <Immunosuppressive effect of cultured cells> Next, to show the immunosuppressive effect of cells containing induced regulatory T cells, an experiment was conducted using the MLR method. An overview of the results of three experiments is shown in Figure 2.

[0100] The graph above the figure shows the results of MLR using the donor antigen (irradiated lymphocytes) used in the culture of regulatory T cells, and the graph below shows the results of MLR of the antigen from the third group of donors (irradiated lymphocytes). Columns 1 to 3 on each graph show the cell proliferation when the recipient lymphocytes, irradiated lymphocytes, and lymphocytes cultured for 2 weeks are cultured individually. Column 4 is the cell proliferation of the recipient lymphocytes after the addition of co-culture and stimulation by the donor light source (irradiated lymphocytes) (control). The results of adding 1 / 1, 1 / 2, and 1 / 4 amounts of the cultured cells in this system are shown in Columns 5 to 7. By adding the cultured cells, the proliferation of lymphocytes is strongly inhibited, and the addition of cell numbers shows a strong immunosuppressive effect even at 1 / 4 (0.25×10 5 ).

[0101] <Study on the remaining antibodies after washing> It is desirable that the antibody used in the culture does not remain in the final test substance administered to the patient. Therefore, regarding the manufacturing process of the test substance in this study, in order to study the number of detergents, the number of detergents and the remaining amount of the antibody were examined in a small-scale test (n = 4). Regarding the anti-human CD80 and CD86 antibodies used in this study, the isotype is mouse IgG; the remaining amount of these antibodies was studied by measuring mouse IgG using ELISA. Washing was performed a total of 4 times, and the remaining antibody concentration per hour was studied in 4 tests. The results are shown in Figure 3. The remaining antibody was found in all cases after one wash, the antibody was not detectable in 3 out of 4 cases after 2 washes, and the remaining antibody was not seen in any case after more than 3 washes. Therefore, 4 washes in the manufacturing process of the test substance in this study can avoid the risk of the antibody remaining in the test substance and entering the patient's body.

[0102] <Large-scale culture test of cells collected from healthy humans by apheresis> <Test method> Using the method of component collection (apheresis), peripheral blood mononuclear cells were collected from two healthy adults, one similar to the donor and the other similar to the patient (recipient), and a large number of cells similar to the cells for actual cell therapy were used. The induction experiment of regulatory T cells was carried out with the same conditions as the manufacturing method and the antibodies used. In addition, the cell number and surface antigens were analyzed for the cells before and after culture.

[0103] <Results of the induction of regulatory T cells> The experiment was carried out according to the above method. A summary of the results is shown in Table 3.

[0104] The total number of lymphocytes decreased from 10.95×10 9 before culture to 3.14×10 9 after 2 weeks of culture. The decrease in the number of these cells is considered due to the specific fraction of lymphocytes that cannot remain in the current culture system and the death of other white blood cells, which is similar to that in small-scale culture tests. In addition, this cell number is shown in the "four previously conducted clinical studies". In the regulatory T cell therapy for kidney transplantation at Tokyo Women's Medical College, it is comparable to the cell number in the actual treatment and is considered as one of the evidences for conducting this test appropriately.

[0105] According to the analysis of surface antigens, the phenotypes of these lymphocytes were analyzed; for CD3 + CD4 + cells, an increase of about 11% from 43.3% to 54.4% was found following 2 weeks of culture. On the other hand, for regulatory T cells, CD4 + CD25 + Foxp3 + cells, they increased more than 12-fold at a ratio from 0.36% to 4.41%. Therefore, regulatory T cells are considered to be selectively induced by the current culture system, which is similar to the results of small-scale tests.

[0106] Furthermore, regarding the ratio among CD4+ cells, it is for CD4 + CD25 + Foxp3 +For cells, from 1.2% to 9.2%, CD4 + CD25 + CTLA4 + For cells, from 0.7% to 14.1%, and CD4 + CD127 lo Foxp3 + For cells, from 1.6% to 3.3%, respectively increased (Table 3).

[0107] Figure 4 shows the results of flow cytometry for surface antigen analysis before and after typical culturing. This figure shows CD4 + cells to CD25 + Foxp3 + cells and CD25 + CTLA4 + cells.

[0108] These results are shown in "four previously conducted clinical studies". In the regulatory T cell therapy for kidney transplantation at Tokyo Women's Medical College, the cells are almost the same as those in the actual treatment. It is predicted that using the cells obtained from this study will yield an effect equivalent to that of the clinical study previously conducted at Tokyo Women's Medical College.

[0109]

Table 4

[0110] <Characteristics of Cultured Cells> After culturing, the ratio of regulatory T cells, CD4 + CD25 + Foxp3 + cells increased significantly, but their frequency remained at a few percent. To understand the characteristics of the test substance, the cell fraction contained in the test substance was examined by analyzing the surface antigen according to the cultured cells. The results are shown in Table 4.

[0111] Among the cultured cells, CD4 + T cells contain 54.4%, CD8 +T cells contain 30.0% and more than 84%. In contrast, B and NK cells contain 6.5% and 7.4% respectively. In addition, monocytes contain 1%. Furthermore, dendritic cells contain 0.2% and granulocytes contain approximately 0.1%.

[0112]

Table 5

[0113] <Research on Regulatory T Cell Therapy Using Animals> <Research Using Mice> The inventor successfully induced antigen-specific Treg-like cells ex vivo by adding anti-CD80 and anti-CD86 antibodies to lymphocyte culture medium and confirmed long-term graft survival in a mouse heart transplantation model by injecting the collected cells (Citation 6).

[0114] <Research Using Monkeys> In a study attempting the same protocol for a non-clinical trial using a monkey kidney transplantation model, immunosuppressive drug cyclosporine was withdrawn approximately 60 days after surgery by injecting regulatory T cells collected from co-culture and induction of peripheral blood mononuclear cells (PBMC) under donor spleen cells and anti-CD80 / CD86 antibodies 2 weeks before and 2 weeks after transplantation. Subsequently, the transplanted kidney achieved long-term survival even without immunosuppression (Figure 5). The following table provides data on the treatment and results of the transplanted monkeys.

[0115]

Table 6

[0116] Values are expressed as mean ± SD. Spleens were removed from all recipients. The numbers in the survival column represent values for each animal in the group. A Died due to acute rejection; B 4×10 6 Received cell inoculation; CDeath due to bleeding after renal biopsy; D Death due to hydronephrosis caused by urethral stricture.

[0117] After performing donor and third-group skin grafts on these animals, the donor skin adheres (arrives), but the third-group skin is rejected, indicating the induction of immune tolerance specific to the donor antigen (citation 7).

[0118] <Results of previous clinical studies> <Regulatory T cell therapy in kidney transplantation (phase I trial)> Based on the results of preclinical studies of cell therapy using induced regulatory T cells, co-investigators Teraoka et al. from the Kidney Center of Tokyo Women’s Medical College attempted the same procedure for 9 cases of living donor kidney transplantation from August 2008 to October 2009 (citation 8).

[0119] <Patient background> The patient background tested in the phase I clinical study is shown in Table 5. The patients’ ages ranged between 26 and 53 years old, and other information such as underlying diseases is shown in Table 5.

[0120]

Table 7

[0121] <Typical clinical course> As seen in Case 2 (Figure 6), 1.5×10 9The regulatory T cells were infused 2 weeks after surgery, and the immunosuppressive agents (cyclosporine (CYA): 300 mg / day, mycophenolate mofetil (MMF): 2000 mg / day, methylprednisolone (MP): 500 mg / day) were gradually reduced. Here, the doses on day 225 were CYA 500 mg / day and MMF 50 mg / day, and MP was completely discontinued. During the same period, no acute rejection or obvious side effects were observed in renal function and renal biopsy. The doses of immunosuppressive drugs were also successfully reduced to 1 / 2 to 1 / 5 in other cases. For these cases, the same acute rejection or obvious side effects as those in case 2 were not observed in renal function and renal biopsy. The results of flow cytometry of the induced regulatory T cells are shown in Fig. 7. These cells are present in vitro and in kidney transplant patients (case 5) and inhibit donor antigen-specific lymphocyte proliferation (Figs. 8A, 8B).

[0122] <Results of cell culture> The results of cell culture in 9 cases performed in the phase I clinical study are shown in Table 6. The total lymphocyte count was 6.50 ± 1.17×10 9 before culture and reached 1.08 ± 0.51×10 9 after 2 weeks of culture. A decrease in the number of these cells is considered due to a specific fraction of lymphocytes that cannot remain in the current culture system and the death of other white blood cells, similar to the results of non-clinical trials.

[0123] According to the analysis of surface antigens, the phenotypes of these lymphocytes were analyzed; CD3 + CD4 + cells showed an approximately 5% increase from 36.69 ± 9.83% to 41.15 ± 11.18% following 2 weeks of culture. On the other hand, regulatory T cells, CD4 + CD25 + Foxp3 + cells increased more than 1.5-fold in the ratio from 3.04 ± 1.53% to 1.84 ± 0.57%. Therefore, regulatory T cells are considered to be selectively induced by the current culture system, which is similar to the results of non-clinical trials.

[0124] Furthermore, CD4+ Regarding the ratio in cells, it is CD4 + CD25 + Foxp3 + In cells, it increased from 4.63±0.03% to 8.87±3.41, and for CD4 + CD25 + CTLA4 + In cells, it increased from 4.09±0.13% to 8.54±3.29% respectively.

[0125]

Table 8

[0126] <Adverse event> In the Phase I clinical study, no adverse events have been reported that are determined to be caused by or have an accidental association with regulatory T cells.

[0127] <Cited references> 1. Todo S, Fung JJ, Starzl TE, Tzakis A, Doyle H, Abu-Elmagd K et al. Single-center experience with primary orthotopic liver transplantation with FK 506 immunosuppression. Ann Surg 1994; 220 (3): 297-308; discussion 308-299.

[0128] 2. Furukawa H, Todo S. Evolution of immunosuppression in liver transplantation: contribution of cyclosporine. Transplant Proc 2004; 36 (2 Suppl): 274S-284S.

[0129] 3. Okumura K, Herzenberg LA, Murphy DB, McDevitt HO. Selective expression of H-2 (i-region) loci controlling determinants on helper and suppressor T lymphocytes. J Exp Med 1976; 144 (3): 685-698.

[0130] 4. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155 (3): 1151-1164.

[0131] 5. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003; 3(3):199-210.

[0132] 6. Bashuda H, Seino K, Kano M, Sato K, Azuma M, Yagita H et al. Specific acceptance of cardiac allografts after treatment with antibodies to CD80 and CD86 in mice。Transplant Proc 1996; 28 (2): 1039-1041.

[0133] 7. Bashuda H, Kimikawa M, Seino K, Kato Y, Ono F, Shimizu A et al. Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman primates. J Clin Invest 2005; 115 (7): 1896-1902.

[0134] 8. Ichiro Koyama. Introduction of peripheral immune tolerance in SS3-8 kidney transplantation, the 45th General Meeting of the Japan Society for Transplantation, 2009.

[0135] <Ex vivo tolerance induction> This experiment considers the reduction and discontinuation of favorable outcomes of immunosuppressive agents by cell therapy based on regulatory T cells in living donor liver transplantation.

[0136] Purpose: The long-term use of immunosuppressive agents (IS) is associated with significant immunological and non-immunological adverse effects. Therefore, the minimization and subsequent complete discontinuation of IS have been the ultimate goal in organ transplantation.

[0137] The injection of donor antigen-specific regulatory T cells (Tregs) generated ex vivo enables the early withdrawal of IS and the induction of tolerance after kidney transplantation in non-human primates.

[0138] This study was conducted to determine the safety and efficacy of Treg-based cell therapy in living donor liver transplantation (LDLT).

[0139] Methods: The study was conducted on 10 consecutive adult LDLTs. Two weeks before LDLT, Tregs began to be generated ex vivo by co-culturing recipient-PBMC (+ splenocytes) with irradiated donor-PMBC, anti-CD80 mAb, and anti-CD86 mAb. Immunosuppressants were administered immediately after transplantation. The immunosuppressants were steroid + MMF + tacrolimus (TAC) or cyclosporine (CYA), while the first two (steroid + MMF) were discontinued within one month. Cyclophosphamide (40 mg / kg) was given on the 5th postoperative day (POD), and Tregs were injected on POD13. TAC (or CYA) was maintained for 6 months, and then reduced every 2 - 3 months, once a day and then three times a week, twice a week, and once a week, and finally discontinued.

[0140] Results: Cases 1 to 9, except for case 5, maintained excellent liver function during and after the reduction and discontinuation of Treg injection. Case 5 was replaced with conventional IS due to inappropriate generation of Tregs and excluded from the study. No adverse events were observed in all patients.

[0141] Two-week co-culture increased CD4 + CD25 + Foxp3 + (6.7 ± 3.8% to 28.1 ± 7.7%) and CD4 + CD127 lo Foxp3 + T cells (8.2 ± 6.0% to 26.2 ± 7.7%). Cultured cells inhibited the mixed lymphocyte reaction (MLR) in a cell number-dependent manner.

[0142]

Table 9

[0143] Conclusions: Based on cell therapy with donor antigen-specific Tregs generated ex vivo, an initial reduction of IS was achieved in 9 out of 10 cases, and finally discontinuation was possible in 3 out of 10 cases after LDLT.

[0144] While specific embodiments of this application are shown and described herein, it will be apparent that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the embodiments. It should be understood that various alternatives to the embodiments described herein may be utilized in practicing the methods described herein.

Claims

【Claim 1】 The composition etc. described in the specification.

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