Classifications

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  • C07K16/289—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
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  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response

Definitions

• the invention is in the field of immunotherapy. More particularly, the invention relates to an isolated anti-CD45RC antibody for use in preventing or treating transplant rejection, autoimmune diseases, unwanted immune responses against proteins expressed in the course of gene therapy and/or therapeutic proteins, allergy as well as lymphoma or cancer which are associated with CD45RC + cells.
• the protein CD45 is a highly glycosylated transmembrane tyrosine phosphatase expressed by hematopoietic cells and described as an essential regulator of T and B cell antigen receptor signaling.
• the cytoplasmic domain of CD45 regulates the activity of Lck and JAKs, which are essential components of the signal transduction process of antigen recognition by T and B cells.
• Lck and JAKs are essential components of the signal transduction process of antigen recognition by T and B cells.
• There isoforms of the CD45 protein generated by alternative splicing of exons 4 (A), exon 5 (B) and exon 6 (C) (the inclusion of exons 4 and 6 in CD45 mRNA is tightly regulated whereas the inclusion of exon 5 appears stochastic). These different extracellular domains differ in size and charge (3, 4).
• CD45 isoforms Individuals express different levels of CD45 isoforms (5) and while the function of the different CD45 isoforms is not clear, differential expression of these isoforms have been associated with the level of activation of T cells and allows dissociation of naive vs. memory T cells (6). Importantly, this pattern of iso forms expression is highly conserved across species emphazing its functional role and importance (7).
• CD45RA, CD45RB and CD45RC isoforms are mainly expressed by naive cells while the shortest isoform CD45RO is express by activated/memory T cells.
• CD45RC + cells also express the A and B isoforms whereas CD45RC " do not (8).
• CD45RO activated/memory T cells can also revert and express again A, B or C isoforms (8).
• CD4 and CD8 + T cells had high levels of message for exon B with variable expression of double-exons AB and BC (8).
• CD45RB low a population of CD4 Treg can be defined as CD45RB low .
• Treatment of mice with anti-CD45RB antibodies can prevent and reverse kidney allograft rejection (9, 10).
• This antibody was also shown to prevent the onset of diabetes in a non-obese diabetic mouse model (11). Comparing two different CD45RB specific mAbs, it has been found that the antibody that was effective for tolerance induction also showed the most effective peripheral cell depletion capacity (9).
• the tolerogenic mAb was able to stimulate tyrosine phosphatase activity on splenic lymphocyte membranes, thereby suggesting a possible immunoregulatory capacity of this antibody.
• the mechanisms of CD45RB mAbs were either induction of apoptotic cell death or dysregulation the signaling apparatus causing lack of effector cell function followed by activation induced cell death.
• CD45RC has been described in rodents and human (12). In rodents, it has been shown that both CD4 + and CD8 + T cells CD45RC + are potent Thl effector cells capable of promoting transplant rejection and organ inflammation (13, 14), while T cells expressing undetectable or low levels of CD45RC are Th2 and regulatory T cells and inhibit allograft rejection, GVHD and cell-mediated autoimmune diseases (15-17).
• CD40Ig a fusion molecule capable of blocking CD40-CD40L interaction
• CD40Ig a fusion molecule capable of blocking CD40-CD40L interaction
• CD45RC + T cells exhibit different cytokine profiles after polyclonal stimulation and that the frequency of these cells is imbalanced in patients with vasculitis (5).
• the international patent application WO 98/1 1918 relates to the use of antibodies to CD45R leukocyte antigens for immunomodulation based on results obtained mainly with anti-CD45RB antibodies corresponding to the results described above (9, 11) and very succinctly with anti-CD45RO antibodies. Nevertheless, said application has also suggested that an anti-CD45RC antibody could be useful in the prevention and/or treatment of transplant rejection as well as treatment of autoimmune disease in generic terms. However no prophylactic or therapeutic effect has been concretely evaluated in this document. The proposed prevention and/or treatment of such disease is thus absolutely not substantiated.
• anti-porcine CD45RC IgM, MIL 15
• human PBMCs were incubated with porcine PBMCs in comparison with anti-CD45 and anti-CD45RA Abs (25).
• an anti-CD45RC antibody in particular an anti-CD45RC monoclonal antibody may be useful in therapy, especially for preventing allogeneic transplant rejection such as GVHD in a patient in need thereof.
• the invention relates to an isolated anti-CD45RC antibody for use as drug.
• the invention in a second aspect, relates to an isolated anti-CD45RC antibody for use in inducing immune tolerance in a patient in need thereof.
• the invention relates to an isolated anti-CD45RC antibody for use in preventing or reducing transplant rejection in a patient in need thereof.
• the invention relates to an isolated anti-CD45RC antibody for use in preventing or treating autoimmune diseases, unwanted immune response against therapeutic proteins, allergies lymphoma or cancer which are associated with CD45RC + cells in a patient in need thereof.
• the invention in a fifth aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising an isolated anti-CD45RC monoclonal antibody and a pharmaceutically acceptable excipient.
• the invention relates to pharmaceutical composition or a kit-of-part composition comprising an isolated anti-CD45RC monoclonal antibody and an immunosuppressive drug.
• the invention relates to an isolated anti-CD45RC antibody for use in expanding and/or potentiating regulatory T cells in a patient in need thereof.
• the inventors fulfill this need in providing antibodies that have the capacity to decrease aggressive effector T cells or B cells while increasing tolerogenic regulatory T cells.
• the invention is indeed based on the discovery that induction of transplantation tolerance is obtained with a treatment with an anti-CD45RC monoclonal antibody. Accordingly, the inventors targeted the cell populations expressing CD45RC using an anti- CD45RC monoclonal antibody (0X22 clone) administered for 10 to 20 days every 2.5 days and demonstrated the potency and advantages of such strategy for allograft survival in a rat cardiac transplantation model. They showed that short-term anti-CD45RC antibody treatment resulted in permanent allograft survival with no signs of chronic rejection and associated to inhibition of effector T cells, as well as induction of regulatory T cells of increased activity and that this tolerance could be transferred to secondary irradiated recipients. Both CD4 + and CD8 + Tregs transferred tolerance.
• CD45 also known as CD45R or PTPRC
• CD45R also known as CD45R or PTPRC
• CD45R transmembrane glycoprotein existing in different isoforms.
• CD45R also known as CD45R or PTPRC
• the various isoforms of CD45 have different extracellular domains, but have the same transmembrane and cytoplasmic segments having two homologous, highly conserved phosphatase domains of approximately 300 residues.
• CD45RC refers to a 200-220 kD single chain type I membrane glycoprotein well known from the skilled man in the art (3). It is an exon 6 splice variant (exon C) of the tyrosine phosphatase CD45.
• the CD45RC isoform is expressed on B cells, majority of CD8 + T cells and a subset of CD4 + T cells, but not on myeloid cells.
• CD45 and its isoforms non-covalently associate with lymphocyte phosphatase-associated phosphoprotein (LPAP) on T and B lymphocytes.
• LPAP lymphocyte phosphatase-associated phosphoprotein
• CD45 has been reported to be associated with several other cell surface antigens, including CD1, CD2, CD3, and CD4. CD45 is involved in signalling lymphocytes activation.
• mAbs monoclonal antibodies
• anti-CD45R anti-CD45 receptor
• mAbs recognizing the product of exon A are consequently designated anti- CD45RA
• those recognizing the product of exon B are consequently designated anti-CD45RB
• those recognizing the product of exon C are consequently designated anti-CD45RC.
• an antibody that specifically binds to CD45RC binds to a polypeptide sequence defined by SEQ ID NO: 2 and encoded by the nucleotide sequence SEQ ID NO: 1 (exon 6, isoform C) as listed in Table A:
• an "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a CD45RC protein is substantially free of antibodies that specifically bind antigens other than CD45RC proteins).
• An isolated antibody that specifically binds a human CD45RC protein may, however, have cross-reactivity to other antigens, such as CD45RC proteins from other species.
• an isolated antibody can be substantially free of other cellular material and/or chemicals.
• antibody or “immunoglobulin” have the same meaning, and will be used equally in the present invention.
• the term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
• two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
• the heavy chain includes two domains, a variable domain (VL) and a constant domain (CL).
• the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
• VL variable domain
• VH variable domain
• CH constant domain
• the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
• the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
• the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
• Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site.
• Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
• the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
• An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
• Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
• the anti-CD45RC monoclonal antibody has preferably an IgG isotype (such as IgGl, IgG2, IgG3 or IgG4).
• Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
• F(ab')2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
• Fab refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
• a single chain Fv (“scFv”) polypeptide is a covalently linked VH:: VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
• dsFv is a VH:: VL heterodimer stabilised by a disulfide bond.
• Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
• a peptide linker such as divalent sc(Fv)2.
• the term "diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL).
• Antibodies directed against the CD45RC surface marker can be raised according to known methods by administering the appropriate antigen or epitope (such as the peptide of SEQ ID NO: 2) to a host animal selected, e.g., from rats, pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• a host animal selected, e.g., from rats, pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• a host animal selected, e.g., from rats, pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• Various adjuvants known in the art can be used to enhance antibody production.
• antibodies useful in practicing the invention can be polyclo
• Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
• techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against the CD45RC surface marker.
• Useful antibodies according to the invention also include antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments as well as scFv and dsFv as above-mentioned.
• Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the CD45RC surface marker.
• Useful antibodies according to the invention also include different antibody formats created from these fragments, in particular formats of chimerized or humanized, multispecific and/or multivalent antibodies.
• the "antibody formats” as referred herein correspond to different combinations of domains and regions such as variable domains of heavy single chain antibodies (VHH) from Camelidae (camel, dromedary, llama), specifically recognizing a type of antigen.
• VHH variable domains of heavy single chain antibodies
• Examples of antibodies which bind the CD45RC antigen that are contemplated by the invention include the mouse monoclonal antibodies OX-22 (anti-rat CD45RC), OX-32 (anti- rat CD45RC), MT2 (anti-human CD45RC), and RP1/12 (anti-rat CD45RC).
• Monoclonal antibodies directed against CD45RC are well known from the skilled man in the art such as the antibodies commercialized by Santa Cruz Biotech.
• antibodies which bind the CD45RC antigen that are contemplated by the invention include antibodies such as the monoclonal antibody OX-22 or a derivative thereof.
• Such monoclonal antibody is well known from the skilled man in the art and has been initially described in Spickett et al. 1983 and is commercialized by several companies.
• the anti-CD45RC monoclonal antibody is an anti-human CD45RC monoclonal antibody.
• antibodies which bind the human CD45RC antigen that are contemplated by the invention include antibodies such as the monoclonal antibody MT2 or a derivative thereof and the monoclonal antibody RP1/12 or a derivative thereof.
• Such monoclonal antibodies are well known from the skilled man in the art and are commercialized by several companies.
• a derivative of MT2 refers to an anti-CD45RC antibody which specifically binds to human CD45RC and which comprises the 6 CDRs of MT2.
• the derivative of MT2 is an antibody which comprises the VL chain and the VH chain of MT2.
• the derivative of MT2 is a chimeric antibody or humanized antibody, which comprises the variable domains of MT2.
• a derivative of RP1/12 refers to an anti-CD45RC antibody which specifically binds to human CD45RC and which comprises the 6 CDRs of RP1/12.
• the derivative of RP 1/12 is an antibody which comprises the VL chain and the VH chain of RP 1/12.
• the derivative of RP 1/12 is a chimeric antibody or humanized antibody, which comprises the variable domains of RP 1/12.
• chimeric antibody refers to a genetically engineered fusion of parts of an animal antibody, typically a mouse antibody, with parts of a human antibody. Generally, chimeric antibodies contain approximately 33% mouse protein and 67% human protein. Developed to reduce the Human anti-animal antibodies response elicited by animal antibodies, they combine the specificity of the animal antibody with the efficient human immune system interaction of a human antibody.
• Humanized antibodies and antibody fragments therefrom can also be prepared according to known techniques.
• “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• the human chimeric antibody of the invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell.
• the CH domain of a human chimeric antibody it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used.
• the CL of a human chimeric antibody may be any region which belongs to Ig, and those of kappa class or lambda class can be used.
• Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See patent documents US5,202,238; and US5,204,244).
• the humanized antibody of the invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell.
• the humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type).
• humanized antibody expression vector of the tandem type In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred.
• tandem type humanized antibody expression vector include pKANTEX93 (WO 97/10354), pEE18 and the like.
• amino acid sequence modification(s) of the MT2 or RP1/12 antibody are also contemplated.
• a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non-human animal in FRs of the VH and VL of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal.
• several amino acid residues of the VH and VL of the non- human antibody are directly or indirectly associated with the antigen binding activity.
• substitution of these amino acid residues with different amino acid residues derived from FRs of the VH and VL of the human antibody would reduce of the binding activity.
• attempts have to be made to identify, among amino acid sequences of the FR of the VH and VL of human antibodies, an amino acid residue which is directly associated with binding to the antibody, or which interacts with an amino acid residue of CDR, or which maintains the three-dimensional structure of the antibody and which is directly associated with binding to the antigen.
• the reduced antigen binding activity could be increased by replacing the identified amino acids with amino acid residues of the original antibody derived from a non- human animal.
• hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
• each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: iso leucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
• the antibodies are fully human monoclonal antibodies.
• transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
• transgenic and transchromosomic mice include mice referred to herein as the HuMAb Mouse® and KM Mouse®, respectively, and are collectively referred to herein as "human Ig mice.”
• the HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (see e.g., Lonberg et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg et al.
• Fully human antibodies of the invention can also be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
• This mouse is referred to herein as a "KM mouse” and is described in detail in PCT Publication WO 02/43478.
• a modified form of this mouse, which further comprises a homozygous disruption of the endogenous FcyRIIB receptor gene, is also described in PCT Publication WO 02/43478 and referred to herein as a "KM/FCGR2D mouse®.”
• mice with either the HCo7 or HCol2 heavy chain transgenes or both can be used.
• Additional transgenic animal embodiments include the Xenomouse (Abgenix, Inc.,
• Still additional transgenic animal embodiments include the OmniRatTM (OMT, Inc., PCT Publication Nos. WO2008/151081 ; Geurts et al. (2009) Science. Jul 24;325(5939):433 and Menoret at al. (2010) Eur J Immunol. Oct;40(10):2932-41.
• OmniRatTM OMT, Inc., PCT Publication Nos. WO2008/151081 ; Geurts et al. (2009) Science. Jul 24;325(5939):433 and Menoret at al. (2010) Eur J Immunol. Oct;40(10):2932-41.
• the contents of these patents and publications are specifically incorporated herein by reference in their entirety.
• the anti-CD45RC antibody is an antibody modulating the immune response by competing with CD45RC for its binding site in vivo.
• Such anti-CD45RC modulating antibody includes antibody fragments and different antibody formats created from these fragments, in particular formats of chimerized or humanized, multispecific and/or multivalent antibodies.
• the anti-CD45RC antibody is a CD45RC + cell depleting antibody.
• CD45RC + cell depleting antibodies are defined as those antibodies which bind to a CD45RC + cell surface marker on the surface of CD45RC + cells (preferably CD45RC) and mediate their destruction or depletion (i.e. reduce circulating CD45RC + T cell levels in a patient treated therewith) when they bind to said cell surface marker.
• depletion may be achieved via various mechanisms such as antibody-dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of CD45RC + cell proliferation and/or induction of CD45RC + cell death (e.g. via apoptosis).
• ADCC antibody-dependent cell mediated cytotoxicity
• CDC complement dependent cytotoxicity
• the CD45RC + cell depleting antibodies more preferably an anti-CD45RC antibody, optionally conjugated with or fused to a cytotoxic agent.
• the term includes antibody fragments and different antibody formats created from these fragments, in particular formats of chimerized or humanized, multispecific and/or multivalent antibodies.
• the "antibody formats” as referred to in the invention correspond to different combinations of domains and regions such as variable domains of heavy single chain antibodies (VHH) from Camelidae (camel, dromedary, llama), specifically recognizing a type of antigen.
• VHH variable domains of heavy single chain antibodies
• CD45RC + cell surface marker or “CD45RC + cell antigen” refer to an antigen expressed on the surface of a CD45RC + cells (including T and B cells) which can be targeted with an anti-CD45RC agent which binds thereto (such as an antibody or an aptamer).
• exemplary CD45RC + T cell surface markers include but are not limited to the CD45RC as previously described or other antigens that characterize said population of T cells.
• the CD45RC + T cells surface marker of particular interest is preferentially expressed on CD45RC + T cells compared to other non-CD45RC + T cells of a mammal.
• CD45RC + cells for example those that deplete CD45RC + cells via antibody-dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), inhibition of CD45RC + cell proliferation or induction of CD45RC + cell death (e.g. via apoptosis).
• ADCC antibody-dependent cell mediated cytotoxicity
• CDC complement dependent cytotoxicity
• inhibition of CD45RC + cell proliferation or induction of CD45RC + cell death e.g. via apoptosis.
• ADCC antibody-dependent cell-mediated cytotoxicity
• FcRs Fc receptors
• cytotoxic cells e.g. NK cells, neutrophils, monocytes and macrophages
• an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
• complement dependent cytotoxicity refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies which are bound to their cognate antigen.
• a CDC assay e.g. as described in Gazzano-Santoro et al. (1997) may be performed.
• the anti-CD45RC antibody may consist in an antibody directed against the CD45RC surface marker which is conjugated to a cytotoxic agent or a growth inhibitory agent.
• the invention contemplates the use of immuno conjugates comprising an antibody against the CD45RC surface marker conjugated to a cytotoxic agent or a growth inhibitory agent.
• a "growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially CD45RC + cells, either in vitro or in vivo.
• growth inhibitory agents include agents that block cell cycle progression, such as agents that induce Gl arrest and M-phase arrest.
• Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes (e.g.
• chemo therapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleo lytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, e.g., gelonin, ricin, saporin, and the various antitumor or anticancer agents disclosed below.
• chemo therapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin
• Conjugation of the antibodies of the invention with cytotoxic agents or growth inhibitory agents may be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6diisocyanate), and bis-active fluorine compounds (such
• a ricin immunotoxin can be prepared as described in Vitetta et al (1987).
• Carbon labeled 1- isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radio nucleotide to the antibody (WO 94/11026).
• the invention provides methods and compositions (such as pharmaceutical compositions) for use in inducing immune tolerance in a patient in need thereof.
• the invention also provides methods and compositions for use in preventing or reducing transplant rejection in a patient in need thereof.
• the invention further provides methods and compositions for use in preventing or treating autoimmune diseases, unwanted immune response against therapeutic proteins, allergies and lymphoma or cancer, which are associated with CD45RC + cells in a patient thereof.
• the invention relates to an isolated anti-CD45RC antibody for use as drug.
• the anti-CD45RC antibody is an anti-CD45RC monoclonal antibody, more particularly an anti-human CD45RC monoclonal antibody as described above.
• the invention in a second aspect, relates to an isolated anti-CD45RC antibody for use in inducing immune tolerance in a patient in need thereof.
• the anti-CD45RC antibody is an anti-CD45RC monoclonal antibody, more particularly an anti-human CD45RC monoclonal antibody as described above.
• immune tolerance refers to a state of unresponsiveness of the immune system to specific substances or tissues that have the capacity to elicit an immune response while preserving immune responses against other substances or tissues.
• immune response includes T cell mediated and/or B cell mediated immune responses.
• immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity
• immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
• Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4 + , CD8 + , Thl and Th2 cells); antigen presenting cells (e.g. professional antigen presenting cells such as dendritic cells); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.
• the invention relates to an isolated anti-CD45RC antibody for use in expanding and/or potentiating regulatory T cells in a patient in need thereof.
• regulatory T cells refers to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance.
• the regulatory T cells are typically forkhead box P3 (Foxp3 + ) regulatory T cells" and "CD45RC low cells".
• forkhead box P3 (Foxp3 + ) regulatory T cells or “Foxp3 + Treg cells” refer to 2-10% of CD4 + and CD8 + T cells in humans and rodents (rats or mice) whose the characteristic marker is the transcription factor Foxp3.
• expanding refers to the process of converting and/or amplifying a given population of cells (e.g. immune cells such as Treg cells).
• the term “potentiating” refers to the process of increasing the function of a given population of cells (e.g. increasing the suppressive capacity of Treg cells as detailed in the Section Examples below).
• said Treg cells are Foxp3 + and/or CD45RC low Treg cells.
• the invention relates to an isolated anti-CD45RC antibody for use in preventing or reducing transplant rejection in a patient in need thereof.
• the anti-CD45RC antibody is an anti-CD45RC monoclonal antibody, more particularly an anti-human CD45RC monoclonal antibody as described above.
• the term "preventing or reducing transplant rejection” is meant to encompass prevention or inhibition of immune transplant rejection, as well as delaying the onset or the progression of immune transplant rejection.
• the term is also meant to encompass prolonging survival of a transplant in a patient, or reversing failure of a transplant in a patient.
• the term is meant to encompass ameliorating a symptom of an immune transplant rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as for example, interstitial fibrosis, chronic graft arteriosclerosis, or vasculitis.
• transplant rejection encompasses both acute and chronic transplant rejection.
• Acute rejection is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporin, anti-CD40L monoclonal antibody and the like. "Chronic rejection” generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants.
• transplantation refers to the insertion of a transplant (also called graft) into a recipient, whether the transplantation is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species).
• the host is human and the graft is an isograft, derived from a human of the same or different genetic origins.
• the graft is derived from a species different from that into which it is transplanted, including animals from phylogenically widely separated species, for example, a baboon heart being transplanted into a human host.
• the transplant rejection is an allogeneic transplant rejection.
• the donor of the transplant is a human.
• the donor of the transplant can be a living donor or a deceased donor, namely a cadaveric donor.
• the transplant is an organ, a tissue or cells.
• organ refers to a solid vascularized organ that performs a specific function or group of functions within an organism.
• the term organ includes, but is not limited to, heart, lung, kidney, liver, pancreas, skin, uterus, bone, cartilage, small or large bowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limb including upper and lower, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, uterus.
• tissue refers to any type of tissue in human or animals, and includes, but is not limited to, vascular tissue, skin tissue, hepatic tissue, pancreatic tissue, neural tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura, pericardia, muscle tissue, glandular tissue, facial tissue, ophthalmic tissue.
• the transplant is a cardiac allotransplant.
• the term "cells" refers to a composition enriched for cells of interest, preferably a composition comprising at least 30%, preferably at least 50%, even more preferably at least 65 % of said cells.
• the cells are selected from the group consisting of multipotent hematopoietic stem cells derived from bone marrow, peripheral blood, or umbilical cord blood; or pluripotent (i.e. embryonic stem cells (ES) or induced pluripotent stem cells (iPS)) or multipotent stem cell-derived differentiated cells of different cell lineages such as cardiomyocytes, beta-pancreatic cells, hepatocytes, neurons, etc...
• the cell composition is used for allogeneic hematopoietic stem cell transplantation (HSCT) and thus comprises multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood.
• HSCT allogeneic hematopoietic stem cell transplantation
• HSCT can be curative for patients with leukemia and lymphomas.
• an important limitation of allogeneic HCT is the development of graft versus host disease (GVHD), which occurs in a severe form in about 30-50% of humans who receive this therapy.
• GVHD graft versus host disease
• Antibodies of the invention are useful in preventing or reducing Graft-versus-Host- Disease (GvHD).
• GvHD Graft-versus-Host- Disease
• the patient in need thereof is affected with a disease selected from the group consisting of acute myeloid leukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia (CML); myelodysplasia syndrome (MDS) / myeloproliferative syndrome; lymphomas such as Hodgkin and non-Hodgkin lymphomas, chronic lymphatic leukemia (CLL) and multiple myeloma.
• AML acute myeloid leukemia
• ALL acute lymphoid leukemia
• CML chronic myeloid leukemia
• MDS myelodysplasia syndrome
• lymphomas such as Hodgkin and non-Hodgkin lymphomas, chronic lymphatic leukemia (CLL) and multiple myeloma.
• the invention relates to an isolated anti-CD45RC antibody for use in preventing or treating autoimmune diseases, unwanted immune responses against proteins expressed in the course of gene therapy or therapeutic proteins, allergies and lymphoma or cancer which are associated with CD45RC + cells in a patient thereof.
• the anti-CD45RC antibody is an anti-CD45RC monoclonal antibody as described above.
• the terms “prevent”, “preventing” and “prevention” refer to the administration of therapy to an individual who may ultimately manifest at least one symptom of a disease, disorder, or condition, but who has not yet done so, to reduce the chance that the individual will develop the symptom of the disease, disorder, or condition over a given period of time. Such a reduction may be reflected, for example, in a delayed onset of the at least one symptom of the disease, disorder, or condition in the patient.
• the terms “treat”, “treating” or “treatment” refers to the administration of therapy to an individual in an attempt to reduce the frequency and/or severity of symptoms of a disease, defect, disorder, or adverse condition of a patient.
• autoimmune disease refers to a disease in which the immune system produces an immune response (for example, a B-cell or a T-cell response) against an antigen that is part of the normal host (that is an auto-antigen), with consequent injury to tissues.
• an autoimmune disease the immune system of the host fails to recognize a particular antigen as "self and an immune reaction is mounted against the host's tissues expressing the antigen.
• Exemplary autoimmune diseases affecting humans include rheumatoid arthritis, juvenile oligoarthritis, collagen- induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia, thrombotic thrombocytopenic purpura and the like.
• the term "unwanted immune response against a therapeutic protein” refers to any unwanted immune reaction directed to proteins expressed in the course of gene therapy, and/or therapeutic proteins, such as factor VIII (hemophilia A) and other coagulation factors, enzyme replacement therapies, monoclonal antibodies (e.g. natalizumab, rituximab, infliximab), polyclonal antibodies, enzymes or cytokines (e.g. IFN ⁇ ).
• factor VIII hemophilia A
• enzyme replacement therapies e.g. natalizumab, rituximab, infliximab
• monoclonal antibodies e.g. natalizumab, rituximab, infliximab
• polyclonal antibodies e.g. cytokines (e.g. IFN ⁇ ).
• an isolated anti- CD45RC antibody according to the invention may be used to prevent immune reactivity towards proteins normally absent in the patient due to mutations, while their reconstitution is achieved by gene therapy.
• protein therapy is an area of medical innovation that is becoming more widespread, and involves the application of proteins, such as enzymes, antibodies or cytokines, directly to patients as therapeutic products.
• proteins such as enzymes, antibodies or cytokines
• Immuno suppressants are used in order to facilitate a longer lifetime of the protein and therefore increased uptake of the protein into the cells and tissues of the organism.
• General immune suppressants can however be disadvantageous due to the unspecific nature of the immune suppression that is carried out, resulting in unwanted side effects in the patient. Therefore, this approach can be applied to suppress an immune response against therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes or any other protein administered to a patient.
• allergy refers to a disorder (or improper reaction) of the immune system. Allergic reactions occur to normally harmless environmental substances known as allergens; these reactions are acquired, predictable, and rapid. Strictly, allergy is one of four forms of hypersensitivity and is called type I (or immediate) hypersensitivity. It is characterized by excessive activation of certain white blood cells called mast cells and basophils by a type of antibody known as IgE, resulting in an extreme inflammatory response. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees.
• compositions The invention also relates to pharmaceutical composition comprising an isolated anti-
• CD45RC antibody of the invention such as an anti-CD45RC monoclonal antibody.
• the pharmaceutical composition comprises an isolated anti- human CD45RC monoclonal antibody.
• the pharmaceutical composition comprises an isolated anti-CD45RC monoclonal antibody selected from the group consisting of MT2 or a derivative thereof, RP1/12 or a derivative thereof and a pharmaceutically acceptable excipient. Therefore, an antibody of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• pharmaceutically acceptable excipients such as biodegradable polymers
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
• the pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
• the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
• an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Dosages to be administered depend on individual needs, on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• the total dose required for each treatment may be administered by multiple doses or in a single dose.
• the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
• the daily dosage of the antibodies may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
• the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
• a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
• An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day.
• the pharmaceutical composition of the invention may further comprise an immunosuppressive drug.
• the immunosuppressive drug is selected from the group consisting of cytostatics such as mammalian target of rapamycin (mTOR) inhibitors and rapamycin (sirolimus); alkylating agents (cyclophosphamide) and antimetabolites (azathioprine, mercaptopurine and methotrexate); therapeutic antibodies (such as anti-CD40L monoclonal antibodies, anti-IL-2R monoclonal antibodies, anti-CD3 monoclonal antibodies, anti- lymphocyte globulin (ALG) and anti-thymocyte globulin (ATG)); calcineurin inhibitors (cyclosporine); glucocorticoids and mycophenolate mofetil.
• cytostatics such as mammalian target of rapamycin (mTOR) inhibitors and rapamycin (sirolimus); alkylating agents (cyclophosphamide) and antimetabolites (azathioprine, mercaptopurine and methotrexate); therapeutic
• the invention relates to methods of in vivo induction of tolerance.
• the methods include pre-treatment in vivo therapies to prevent rejection of transplanted organs, tissues or cells and post-transplant in vivo therapies to reverse an immune response.
• the invention relates to a method for inducing immune tolerance in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody).
• the invention relates to a method for expanding and/or potentiating regulatory T cells in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody).
• the invention also relates to a method for preventing or reducing transplant rejection in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody) previously and/or simultaneously and/or subsequently to the transplantation.
• an anti-CD45RC antibody such as an anti-CD45RC monoclonal antibody
• the transplant rejection is an allogeneic transplant rejection.
• the invention also relates to a method for preventing or reducing GVHD in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody) previously and/or simultaneously and/or subsequently to the allogeneic hematopoietic stem cell transplantation (HSCT).
• an anti-CD45RC antibody such as an anti-CD45RC monoclonal antibody
• HSCT allogeneic hematopoietic stem cell transplantation
• the term "simultaneously" means that the antibody of interest is administered to the recipient patient the same day that the transplantation.
• the term "subsequently" means that the antibody of interest is administered to the recipient patient after the transplantation, for instance 2, 3, 4, 5, 6 or 7 days following the transplantation.
• the invention also relates to a method for improving survival of the transplant in a transplanted patient (recipient), said method comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody).
• the invention also relates to a method for preventing or reducing GVHD in a patient in need thereof, comprising the steps of (a) pre-treating the allogeneic hematopoietic stem cells (or a hematopoietic stem cell graft) with a therapeutically effective amount of an anti- CD45RC antibody; and (b) administering to the patient in need thereof the pre-treated allogeneic hematopoietic stem cells obtained at step (a).
• the invention also relates to a population of allogeneic hematopoietic stem cells (or a hematopoietic stem cell graft) treated with an anti-CD45RC antibody.
• the invention also relates to a method for preventing or treating autoimmune diseases, unwanted immune response against therapeutic proteins, allergies and lymphoma or cancer which are associated with CD45RC + cells in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody).
• an anti-CD45RC antibody such as an anti-CD45RC monoclonal antibody
• terapéuticaally effective amount is meant an amount sufficient to achieve a concentration of antibodies which is capable of preventing, treating or slowing down the disease to be treated. Such concentrations can be routinely determined by those of skilled in the art.
• the amount of the polypeptide actually administered will typically be determined by a physician or a veterinarian, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the patient, the severity of the subject's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered polypeptide.
• the treatment with an anti-CD45RC antibody is administered in more than one cycle, i.e. the administration of an anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody) is repeated at least once.
• the intervals i.e. the time between the start of two subsequent cycles, are typically several days.
• Kit-of-p rt compositions The anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody) and the immunusuppressive drug may be combined within one formulation and administered simultaneously. However, they may also be administered separately, using separate compositions. It is further noted that they may be administered at different times.
• the invention thus relates to a kit-of-part composition comprising an isolated anti-CD45RC antibody (such as an anti-CD45RC monoclonal antibody) and the immunusuppressive drug may be combined within one formulation and administered simultaneously. However, they may also be administered separately, using separate compositions. It is further noted that they may be administered at different times.
• the invention thus relates to a kit-of-part composition comprising an isolated anti-
• CD45RC antibody and an immunosuppressive drug.
• the invention also relates to a kit-of-part composition comprising an anti-CD45RC antibody and an immunosuppressive drug for use in preventing or reducing transplant rejection in a patient in need thereof.
• the transplant rejection is an allogeneic transplant rejection.
• the invention further relates to a kit-of-part composition comprising an anti-CD45RC antibody and an immunosuppressive drug for use in preventing or treating autoimmune diseases, alloimmune responses, allergies and lymphoma or cancer which are associated with CD45RC + cells in a patient in need thereof.
• kit means especially a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points.
• the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
• the ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied.
• the combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 7, days before the second partner.
• FIGURES are a diagrammatic representation of FIGURES.
• Figure 1 Tolerance induction after anti-CD45RC short-term treatment.
• Anti-CD45RC mAbs (0X22) was given i.v at 0.8 mg/kg/injection. **p ⁇ 0.01 versus untreated.
• FIG. 2 Lymphocyte phenotyping in the blood during and following 10 days of anti-CD45RC treatment. PBMCs were recovered at day 0, 4, and 10 from anti-CD45RC- treated recipients and analyzed by flow cytometry for absolute number of sub-populations.
• FIG. 3 Regulatory cells are increased in the spleen of long-surviving anti- CD45RC-treated recipients. Splenocytes were recovered at day 120 from anti-CD45RC- treated recipients compared to naive splenocytes and analyzed by flow cytometry for absolute numbers of sub-populations.
• Figure 4 Adoptive transfer of tolerance. LEW.1A recipients were sublethally irradiated (4.5 Gy) at day -1 and received heart allografts and i.v. injections at day 0 of splenocytes or sorted-subpopulations from long- surviving anti-CD45RC treated recipients. Graft survival was monitored by abdominal palpation.
• Figure 5 Increased suppressive capacity of CD8 + CD45RC
• CFSE-labeled LEW.1A dividing CD4 CD25 T cells stimulated with donor LEW.1W pDCs were analyzed after 6 days of culture in the absence or presence of dl20 LEW.1A naive or anti-CD45RC-treated CD8 + CD45RC /low Tregs at different effector/suppressor ratio.
• Anti-CD45RC mAb treatment reduces lethality and weight loss in a model of GVHD.
• Rats receiving 200.10 6 splenocytes were treated with anti-CD45RC mAb or isotype control to assess potency for preventing GVHD.
• Average weight loss (percentage of initial) for rats surviving on a given day was shown +/-SEM. p ⁇ 0.001.
• n 3.
• EXAMPLE 1 Short-term anti-CD45RC antibody treatment results in transplantation tolerance associated with induction of potent regulatory cells.
• Anti-CD45RC administration The 0X22 (IgGl anti-rat CD45RC) mouse hybridoma was used to produce the anti-CD45RC monoclonal antibody.
• Anti-CD45RC mAbs were given at a dose of 0.8 mg/kg/2.5days i.v. from day 0 to 5, 10 or 20 post-transplantation.
• Control IgGl (3G8, anti-human CD16, with no cross-reaction with the rat) was used at the same dose.
• tissue specimens were analyzed for chronic rejection at day 120 post-transplantation as previously described. Briefly, tissues were fixed in paraformaldhehyde, paraffin embedded, cut in 5 ⁇ - ⁇ 1 ⁇ 1 ⁇ sections and stained with hematoxylin-eosin-saffron.(24)
• Lymphocyte preparation preparation of subpopulations and adoptive transfert:
• Spleen were harvested at day 120 and splenocytes or sub-populations were purified as previously described (16). Cell transfers were performed by i.v. injection of total splenocytes or purified sub-populations on the day of transplantation of LEW.1W onto LEW.1A sublethally irradiated (4.5 Gy day -1) recipient.
• T cells and pDC were purified and sorted as previously described (20). Flow cytometry and cell sorting was performed using antibodies directed to T cells (TCRap, clone R7/3), CD4 + CD25 T cells (clones 0X35 and 0X39), CD8 + T cells (clone 0X8), CD8 + or CD4 + CD45RC low T cells (clones 0X8 and 0X22), and CD4 + CD45R + 85C7 + pDCs (clones His24, 0X35 and 85C7), CDl lb and CD45RA + B cells (0X33) obtained from the European Collection of Cell Culture (Salisbury, UK).
• T cells Naive Lewis 1A CD4 + T cells, naive Lewis 1W allogeneic pDC, and treated-syngeneic Lewis 1A CD8 + CD45RC low Tregs subsets were sorted as previously described (19). Proliferation of CFSE-labelled CD4 + CD25 " T cells was analyzed by flow cytometry 6 days later, by gating on TCR + CD4 + cells (R7/3-APC, Ox35-PECY7) among living cells (DAPI negative).
• Antibody detection Alloantibodies. Donor spleens were digested by collagenase D, stopped with 400 ⁇ 1 EDTA O. lmM, and red cells were lysed. Serum of recipients were added to donor splenocytes at a dilution 1/8, and incubated with either anti-rat IgG-FITC (Jackson ImmunoResearch Labs INC, Baltimore, USA), anti-rat IgGl (MCA 194, Serotec), anti-rat IgG2a (MCA 278, Serotec), or anti-rat IgG2b (STAR114F, Serotec) and anti-Ms Ig- FITC(115-095-164, Jackson ImmunoResearch).
• FACS Canto (BD Biosciences, Mountain View, CA) was used to measure fluorescence, and data were analyzed using the FLOWJO software (Tree Star, Inc. USA). Geometric mean of fluorescence (MFI) of tested sera was divided by mean of 5 naive Lewis 1 A sera MFI as control.
• Tolerance induction following short-term anti-CD45RC antibody treatment As we previously demonstrated the presence and potential of CD8 + T cells with absent or low expression of the CD45RC surface marker, we speculated that a targeting strategy using an anti-CD45RC MAb would promote tolerance in cardiac transplanted recipients. Thus, we have administered an anti-CD45RC antibody (0X22, 0.8 mg/kg/i.v.) every 2.5 days during 20, 10 or 5 days to transplanted rat recipients (cardiac allograft MHC mismatched, LEW.IW grafted onto LEW.1A), starting the day of the transplantation.
• an anti-CD45RC antibody (0X22, 0.8 mg/kg/i.v.) every 2.5 days during 20, 10 or 5 days to transplanted rat recipients (cardiac allograft MHC mismatched, LEW.IW grafted onto LEW.1A), starting the day of the transplantation.
• Cardiac histopathology analysis revealed a complete absence of fibrosis, infiltrate and vessel sclerosis at day 120 in 20-days anti-CD45RC -treated recipients, demonstrating that this short-term treatment efficiently inhibits both acute and chronic rejection.
• anti-CD45RC antibody treatment resulted in inhibition of both acute and chronic rejection, without compromising immunity and capacity of the recipients to mount antibody responses against cognate antigens.
• CD45RC-treatment we analyzed the proportion of regulatory T + CD4 + CD45RC "/low , T + CD8 + CD45RC /l0W or B + CD45RC /low cells in the spleen.
• analysis of the phenotype of the recipients in the spleen revealed a strong enrichment in absolute number of T cells and particularly of T CD4 + CD45RC int/" , T CD8 + CD45RC " , while other cell subsets were not modified (Figure 3).
• 0W Tregs and transferable tolerance To confirm in vivo that short-term treatment with anti-CD45RC antibodies resulted in induction of potent suppressive regulatory cells, we performed adoptive cell transfer of splenocytes or sub-populations into secondary irradiated cardiac grafted recipients (Figure 4). We observed that adoptive cell transfer of 150.10 e6 splenocytes resulted in indefinite allograft survival in all recipients compared to recipients transferred with naive splenocytes or non-transferred.
• CD8 + CD45RC /low CD4 + CD45RC /l0W and B + CD45RC /low cells and performed adoptive cell transfer. While we observed a slight prolongation of allograft survival in recipients adoptively transferred with B cells, we observed 100% allograft survival in recipients transferred with CD8 + CD45RC low Tregs and, CD4 + CD45RC low T cells, demonstrating that the depletion of CD45RC + cells from day 0 to 20 has induced and activated regulatory cells with a strong suppressive capacity.
• CD8 + CD45RC low Tregs were sorted at day 120 and added in a dose dependent manner in an assay were CFSE-labeled CD4 + CD25 ⁇ effector T cells were cocultured with donor-derived plasmacytoid dendritic cells (pDC) ( Figure 5). In absence of Tregs, we observed a strong proliferation of the CD4 + CD25 ⁇ Teff cells.
• EXAMPLE 2 Anti-CD45RC mAb treatment reduces lethality and weight loss in a model of GVHD.
• splenocytes were injected intravenously in syngeneic or allogeneic rats treated 24 hours earlier with whole-body sublethal irradiation of 7.8 Gy.
• Recipients received anti-CD45RC mAbs (0X22) or an irrelevant control mAbs starting day 0 and every 3 days at 2mg/kg/injection. Recipients were weighted every day and sacrifice when percentage of weight loss was >20% of their initial weight.
• Anti-CD45RC mAb treatment reduces lethality and weight loss in a model of
• GVHD To determine whether administration of the anti-CD45RC could prevent the development of acute graft versus host disease (GVHD), recipients received either syngeneic or allogeneic splenocytes following whole-body sublethal irradiation. Anti-CD45RC or irrelevant control mAbs were then administered i.p. every 3 days. We observed that recipients that received syngeneic splenocytes started to lose weight but then recovered from the whole- body irradiation and gained weight until the end of the experiment.
• CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients. PLoS One 4:e5287.
• CD45 a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol 21 : 107-137. 8. Hargreaves, M., and Bell, E.B. 1997. Identical expression of CD45R isoforms by CD45RC+ 'revertan memory and CD45RC+ naive CD4 T cells. Immunology 91 :323- 330.
• MRC OX-22 a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen. J
• AUoreactive CD4 T lymphocytes responsible for acute and chronic graft-versus-host disease are contained within the CD45RChigh but not the CD45RClow subset. Eur J Immunol 34:408-417.

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