HK1168605A - Methods for inducing transplantation tolerance - Google Patents

Abstract

A method is provided for preventing rejection by an immune system of a recipient subject of a tissue transplanted from a donor subject into the recipient subject without the need for long-term administration of non-specific immunosuppressive drugs.

Description

Method for inducing transplantation tolerance

The disclosure herein was made with government support under grant number U19AI46132 from the national institutes of health. Accordingly, the U.S. government also has certain rights in this invention.

The various publications cited in this application are indicated by numbers in parentheses. The provenance of these references can be found before the claims at the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background

The normal function against infectious agents is based on how the immune system achieves self/non-self discrimination, and this has been the core problem of immunology (1). What the immune system sees as "self" and what as "foreign" determines how the immune system distinguishes self from non-self. In this regard, early work by Burnet and Medawar suggested that self versus non-self definitions are arbitrary for the immune system, since foreign antigens presented during fetal life are subsequently considered self (2, 3). In addition, all T cells are known to be self-directed (self-presenting) (8-10) in the sense that T cells survive positive selection during thymic positive selection (4-7) prior to thymic negative selection (where thymocytes expressing TCRs with high affinity for most self-antigens are removed) relying on self-peptides bound to MHC molecules.

Thymic negative selection is generally thought to eliminate the "emergency risk" of pathogenic autoimmunity in the periphery, and is the primary mechanism of self-tolerance. However, under normal circumstances, thymic negative selection also allows the release of a large fraction of autoreactive T cells with "higher" or "medium" affinity into the periphery when releasing "harmless" autoreactive T cells with low affinity (11-13). The presence of "moderate affinity" autoreactive T cells in the periphery represents a "potential risk" for pathogenic autoimmunity inherited in individuals, since these T cells are often activated when encountering self-peptides presented at sufficient levels, and some may differentiate into potentially pathogenic effector cells to initiate autoimmune disease (13-16). Self/non-self discrimination must continue in the periphery following thymic negative selection and one of the major functions of the peripheral regulatory mechanisms is to selectively down-regulate the immune response to self-antigens without compromising the normal response to foreign pathogens to maintain self-tolerance (17).

How does the immune system distinguish self from non-self in the periphery? Since the self/non-self differentiation initiated during thymic negative selection is based on the affinity of thymocyte activation (8-10) and most autoreactive T cells escape thymic negative selection (11-13), understanding of how the resulting pool of peripheral T cells is regulated to accomplish the self and non-self differentiation initiated by thymic negative selection to maintain self tolerance is crucial. To this end, the inventors propose and tested a "peripheral T cell regulated affinity model" capable of self-non-self differentiation in the periphery by selective down-regulation of T cells with intermediate affinity to both self and foreign antigens by Qa-1/HLA-E restricted CD8+ T cells (18, 19). Since potentially pathogenic autoreactive T cells are contained in the intermediate affinity T cell pool, selective down-regulation of intermediate affinity T cells can directly control autoimmune diseases. On the other hand, the unitary mechanism of selectively downregulating moderate affinity T cells does not largely inhibit the immunity mediated by high affinity T cells to foreign infectious agents or alloantigens, since only high affinity T cells are unaffected by this downregulation. Thus, through a unified simple mechanism, the immune system is able to achieve self-non-self differentiation in the periphery to specifically maintain self-tolerance without paying the cost of subduing anti-infection and anti-tumor immunity.

The concept that the perception of affinity for T cell activation can be translated into peripheral T cell regulation is at the heart of the "affinity model". Defining the cellular mechanisms of how the affinity of perceived T cell activation is translated into peripheral T cell regulation and the molecular structures recognized by regulatory T cells that enable them to distinguish between self and non-self in the periphery is a major problem in regulatory T cell biology. To this end, an alternative target structure (Qa-1/Hsp60sp) (1) was recently identified that was specifically recognized by Qa-1 restricted CD8+ T cells. As a biological consequence of T cell activation, the common alternative target structure (Qa-1/Hsp60sp complex) is preferably expressed at higher levels only on medium affinity (rather than high and low affinity) T cells (1). Thus, at the level of biological systems, through specific recognition of common target structures expressed on moderate affinity T cells, the immune system is able to achieve the goal of distinguishing self-non-self in the periphery by sensing the affinity of T cell activation.

The biggest challenge in transplant medicine is the responsiveness of the immune system to foreign organs, i.e. rejection. Often, recipients of transplanted organs must receive immunosuppressive drugs indefinitely to combat rejection which is prone to failure and puts the patient at high risk of infection. The invention disclosed herein provides a novel therapeutic strategy for inducing persistent tolerance to transplanted organs without the need for long-term use of immunosuppressive drugs, which introduces an affinity model.

Disclosure of Invention

A method of preventing rejection of a transplanted tissue from a donor subject into a recipient subject, comprising:

a) administering to the recipient subject prior to the tissue transplant the following agents so as to inhibit activation of alloreactive T cells in the recipient subject: (i) a plurality of Peripheral Blood Mononuclear Cells (PBMCs) from the donor subject, wherein the PBMCs have been irradiated such that they cannot proliferate in vivo, and (ii) a monoclonal antibody that specifically binds to a CD40 ligand, thereby inhibiting activation of alloreactive T cells in the recipient subject; and

b) administering to the recipient subject after the tissue transplantation a CD8 restricted by HLA-E⁺HLA-E with T cells enhancing donor tissue activation⁺An agent for down-regulation of T cells, thereby enhancing activated HLA-E of said donor tissue⁺The down-regulation of T-cells is achieved,

thereby preventing rejection of the tissue transplanted into the recipient subject.

Drawings

FIG. 1: the Qa-1 (or HLA-E) restricted CD8+ T cell mediates cellular events in the pathway initiated by the activation of naive T cells during a primary immune response in which the TCR on the T cell interacts with the MHC/antigenic peptide complex presented by conventional APC. One of the results of initial T cell activation is the differential expression of a specific "target antigen" (in this case including the "Qa-1/self-peptide complex" on the surface of the target T cell). Importantly, the expression of the "target antigen" recognized by the TCR on the regulatory T cell is determined by the affinity interaction of T cell activation, regardless of which antigen the target T cell is triggered by. Thus, since T cells are not professional APCs, professional APCs such as dendritic cells may be recruited and act to provide a costimulatory signal during the induction phase of regulatory T cells. A "target antigen" expressed on certain activated T cells triggers the regulatory T cells to differentiate into effector cells, which in turn down-regulate any activated T cells expressing the same target antigen during a secondary immune response.

Detailed Description

A method of preventing rejection of a transplanted tissue from a donor subject into a recipient subject, comprising:

a) administering to the recipient subject prior to the tissue transplant the following agents so as to inhibit activation of alloreactive T cells in the recipient subject: (i) a plurality of Peripheral Blood Mononuclear Cells (PBMCs) from the donor subject, wherein the PBMCs have been irradiated such that they cannot proliferate in vivo, and (ii) a monoclonal antibody that specifically binds to a CD40 ligand, thereby inhibiting activation of alloreactive T cells in the recipient subject; and

b) administering to the recipient subject after the tissue transplantation a CD8 restricted by HLA-E⁺HLA-E with T cells enhancing donor tissue activation⁺An agent for down-regulation of T cells, thereby enhancing activated HLA-E of said donor tissue⁺The down-regulation of T-cells is achieved,

thereby preventing rejection of the tissue transplanted into the recipient subject.

A method of preventing rejection of a transplanted tissue from a donor subject into a recipient subject, comprising:

a) administering to the recipient subject prior to the tissue transplant the following agents so as to inhibit activation of alloreactive T cells in the recipient subject: (i) a plurality of Peripheral Blood Mononuclear Cells (PBMCs) from the donor subject, wherein the PBMCs have been irradiated such that they cannot proliferate in vivo, and (ii) a monoclonal antibody that specifically binds to a CD40 ligand, thereby inhibiting activation of alloreactive T cells in the recipient subject; and

b) administering to the recipient subject after the tissue transplantation a CD8 restricted by HLA-E⁺HLA-E with T cells enhancing donor tissue activation⁺An agent for down-regulation of T cells, thereby enhancing activated HLA-E of said donor tissue⁺The down-regulation of T-cells is achieved,

thereby preventing rejection of the tissue transplanted into the recipient subject.

In one embodiment, the method further comprises administering an immunosuppressive drug to the recipient subject during step a), during step b), or during both step a) and step b).

In one embodiment, the agent is an HLA-E/IgG fusion protein or an HLA-E/Hsp60sp tetramer.

In one embodiment, the HLA-E⁺The T cell is CD4⁺/HLA-E⁺T cells or CD8⁺/HLA-E⁺T cells.

In one embodiment, the agent is a class B self peptide-loaded HLA-E bearing exosome derived from dendritic cells.

In one embodiment, the type B self peptide has the amino acid sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, the agent is an HLA-E bearing exosome loaded with Hsp60sp peptide derived from dendritic cells.

In one embodiment, the agent is an autologous dendritic T cell loaded with Hsp60sp peptide.

In one embodiment, the Hsp60sp peptide comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In one embodiment, prior to administering the autologous dendritic T cells to the recipient subject as the agent, the autologous dendritic T cells have been removed from the recipient subject, cultured ex vivo, and contacted with an Hsp60sp peptide, thereby loading the cells with an Hsp60sp peptide.

In one embodiment, the agent is administered intravenously, intramuscularly, or orally.

In one embodiment, in step a), the monoclonal antibody directed against CD40 ligand is administered to the recipient subject for up to 10 weeks.

In one embodiment, in step a), the plurality of cells from the donor subject is administered to the recipient subject in two separate fractions, first a first fraction and then a second fraction, wherein the second fraction is administered to the recipient subject about 7 days after administration of the first fraction.

In one embodiment, in step a) the monoclonal antibody directed to CD40 ligand is administered to the subject (1) on the first 1 day of the first part of the day of administration of the plurality of cells, and (2) on the day of administration of the first part of the day of administration of the plurality of cells, and (3) on the first 1 day of the first part of the day of administration of the plurality of cells, and (4) on the 3 days of the first part of the day of administration of the plurality of cells, and (5) on the 7 days of the first part of the day of administration of the plurality of cells, and (6) on the 14, 21, 28, 35, 42, 49, and 56 days of the first part of the day of administration of the plurality of cells.

In one embodiment, the immunosuppressive drug is administered daily for up to 10 weeks after administration of the plurality of cells or after administration of the first portion of the plurality of cells.

In one embodiment, the immunosuppressant drug is administered daily for 56 days.

In one embodiment, said tissue is transplanted into said recipient subject 2-3 weeks after administration of said plurality of cells or said first portion of said plurality of cells.

In one embodiment, the method further comprises administering an anti-thromboembolic agent to the recipient subject during the administration of the monoclonal antibody directed to CD40 ligand.

In one embodiment, the transplanted tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, genital tissue, ovarian tissue, bone tissue, tendon tissue, or vascular tissue.

In one embodiment, the transplanted tissue is transplanted as a whole organ.

In one embodiment, PBMCs have been irradiated with 2000-4000 rads (Rad) for a sufficient time to render them incapable of proliferation in vivo.

As used herein, a "recipient subject" is a subject who is to receive or has received a transplanted cell, tissue or organ from another subject.

As used herein, a "donor subject" is a subject from which cells, tissues or organs to be transplanted are removed prior to transplantation of the cells, tissues or organs to a recipient subject.

In one embodiment, the donor subject is a primate. In one embodiment, the donor subject is a human. In one embodiment, the recipient subject is a primate. In one embodiment, the recipient subject is a human. In one embodiment, the donor subject and the recipient subject are both human. Accordingly, the present invention includes embodiments of xenotransplantation.

As used herein, "rejection of the immune system" describes a hyperacute, acute, and/or chronic response event of the immune system of a recipient subject that recognizes transplanted cells, tissues, or organs from non-self donors.

As used herein, an "immunosuppressive drug" is a pharmaceutically acceptable drug for inhibiting an immune response in a recipient subject. Non-limiting examples include cyclosporine A, FK506 and rapamycin.

As used herein, a "prophylactically effective" amount is an amount of a substance that is effective to prevent or delay the onset of a given pathological condition in a subject to whom the substance is administered.

As used herein, a "therapeutically effective" amount is an amount of a substance that is effective to treat, ameliorate, or reduce the symptoms or causes of a given pathological condition suffered by a subject to whom the substance is administered.

In one embodiment, the therapeutically or prophylactically effective amount is about 1mg agent/kg subject to 1g agent/kg subject per dose. In another embodiment, the therapeutically or prophylactically effective amount is from about 10mg agent/kg subject to 500mg agent/kg subject. In yet another embodiment, the therapeutically or prophylactically effective amount is from about 50mg agent/kg subject to 200mg agent/kg subject. In yet another embodiment, the therapeutically or prophylactically effective amount is about 100mg agent/kg subject. In yet another embodiment, the therapeutically or prophylactically effective amount is selected from the group consisting of a 50mg agent/kg subject, a 100mg agent/kg subject, a 150mg agent/kg subject, a 200mg agent/kg subject, a 250mg agent/kg subject, a 300mg agent/kg subject, a 400mg agent/kg subject, and a 500mg agent/kg subject. In a preferred embodiment, the therapeutically or prophylactically effective amount is about 20mg agent/kg subject per dose.

As used herein, a "type B peptide" or "type B self peptide" is an HLA-E binding peptide that binds to HLA-E (i) does not inhibit NK cells by binding to CD94/NKG2A when bound to HLA-E, (ii) is recognized by regulatory CD8+ T cells when bound to HLA-E, and (iii) is capable of competing with a type a HLA-E binding peptide such as B7 sp. Preferably, the type B peptide is a monomer.

As used herein, "HLA-E" has the conventional meaning used in the art, i.e., the human leukocyte antigen system E.

As used herein, an "HLA-E-restricted CD8+ T cell" is a regulatory CD8+ T cell that recognizes peptides presented by HLA-E molecules on immune system Antigen Presenting Cells (APCs) or on HLA-E + dendritic cells. APCs for HLA-E-restricted CD8+ T cells encompassed herein are intermediate affinity T cells, which are also specific targets for these CD8+ T cells.

The "structurally related peptide" of Hsp60sp denotes a peptide corresponding to SEQ ID NO: 2, having a sequence similarity of from 70% to 99%.

As used herein, "donor tissue-activated HLA-E⁺T cells "are HLA-E of a recipient activated by donor tissue that has been transplanted into the recipient⁺T cells. Similarly, when the method involves transplantation of cells or organs, "cell-activated HLA-E⁺T cell or organ activated HLA-E⁺T cells "are HLA-E of a recipient activated by a donor tissue or organ, respectively, that has been transplanted into the recipient⁺T cells.

As used herein, an "alloreactive T cell" is a T cell of a transplant recipient subject that reacts with cells of the donor subject when the donor and recipient are of the same species. In one embodiment, the alloreactive T cells react with non-recipient human cells from a human donor, but not with recipient human cells.

In one embodiment, the HLA-E⁺The T cell is CD4⁺/HLA-E⁺T cell, CD8⁺/HLA-E⁺T cells, and the HLA-E binding peptide of type B is an Hsp60sp peptide. In yet another embodiment, the Hsp60sp peptide comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the peptide has the sequence Xaa-Met/Leu-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Leu (SEQ ID NO: 1). In yet another embodiment, the peptide is HLA-E bindingThe monomer (2) of (1). In yet another embodiment, the peptide does not bind to the CD94/NKG2A receptor. In yet another embodiment, the peptide is recognized by regulatory CD8+ T cells upon binding to HLA-E. In yet another embodiment, the peptide for binding to HLA-E is capable of competing with B7 sp.

"agent" shall mean any chemical substance including, but not limited to, glycopolymers (glycomers), proteins, antibodies, hemagglutinin, nucleic acids, small molecules, and any combination thereof, as well as biological entities such as exosomes or liposomes. A "small molecule" is an organic molecule that may be substituted with an inorganic atom or group containing an inorganic atom, the molecule having a molecular weight of less than 1000 Da. Specific non-limiting examples of agents include dendritic cell-derived HLA-E bearing exosomes loaded with Hsp60sp peptide; autologous dendritic T cells loaded with Hsp60sp peptide; a membrane-bound component or liposome loaded with Hsp60sp peptide; HLA-E/IgG fusion proteins; or HLA-E/Hsp60sp tetramer. In an embodiment, the Hsp60sp is a human Hsp60sp peptide.

In embodiments, the agent is a dendritic cell-derived HLA-E-bearing exosome loaded with an HLA-E-binding peptide of type B, or the agent is a dendritic cell-derived HLA-E-bearing exosome loaded with an Hsp60sp peptide, or the agent is an HLA-E/IgG fusion protein, and the agent is an HLA-E tetramer or an HLA-E/Hsp60sp tetramer. Fusion proteins are described in U.S. Pat. nos. 5,116,964 and 5,336,603, both of which are incorporated herein by reference. HLA-E tetramers are described, for example, in Braud et al, Nature.1998Feb 19; 391(6669): 740-1, 743; and garcia et al, eur.j.immunol.2002 Apr; 32(4): 936-44, both of which are incorporated herein by reference. HLA-E protein sequences are described in NCBI accession numbers CAA05527, CAA40172, BAB63328, and BAF31260, which are incorporated herein by reference. In embodiments, the agent is an HLA-E/IgG fusion protein and the agent is an HLA-E tetramer or an HLA-E/Hsp60sp tetramer. Tetramers are described, for example, in saledo et al, eur.j.immunol.2000 Apr; 30(4): 1094-. Reagents suitable for use in the present invention are described in WO/2008/103471 published at 8 months 2008, which is incorporated herein by reference.

Hsp60sp has the sequence QMRPVSRAL (SEQ ID NO: 2) or, in the case of humans, QMRPVSRVL (SEQ ID NO: 3).

"administering" an agent can be accomplished or performed using any of a variety of methods and delivery systems known to those skilled in the art. For example, administration can be by intravenous injection, oral, nasal, via cerebrospinal fluid, via implants, transmucosal, transdermal, intramuscular, and subcutaneous. The following delivery systems employing a variety of conventionally used pharmaceutically acceptable carriers are merely representative of the various embodiments envisioned for administering the compositions according to the methods of the present invention.

Injectable drug delivery systems include solutions, suspensions, gels, microspheres, and polymer injections, and may contain excipients such as agents that alter solubility (e.g., ethanol, propylene glycol, and sucrose) and polymers (e.g., polycaprolactone and PLGA). Implantable systems include strips and sheets, and may contain excipients such as PLGA and polycaprolactone.

Oral delivery systems include tablets and capsules. These delivery systems comprise excipients such as binders (e.g. hydroxypropylmethylcellulose, polyvinylpyrrolidone, other cellulosic materials and starch), diluents (e.g. lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrants (e.g. starch polymers and cellulosic materials) and lubricants (e.g. stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels, and creams, and may contain excipients such as solubilizing agents and enhancers (e.g., propylene glycol, bile salts, and amino acids) and other carriers (e.g., propylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropyl methylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, hydrogels and non-hydrogels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon matrices, and powders, and may contain excipients such as solubilizers, permeation enhancers (such as fatty acids, fatty acid esters, fatty alcohols, and amino acids), and hydrophilic polymers (such as polycarbophil and polyvinylpyrrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a dermal penetration enhancer.

Solutions, suspensions, and powders of the reconstitutable delivery system contain carriers such as suspending agents (e.g., gums, xanthan gum, cellulosic materials, and sucrose), humectants (e.g., sorbitol), solubilizing agents (e.g., ethanol, water, PEG, and propylene glycol), surfactants (e.g., sodium lauryl sulfate, spans (Span), tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering a compound of the invention to an animal or human. The carrier may be a liquid, aerosol, gel or solid and is selected according to the intended mode of administration.

An "antibody" shall include, but is not limited to, an immunoglobulin molecule comprising two heavy chains and two light chains and recognizing an antigen. The immunoglobulin molecules may be derived from any type known in the art, including, but not limited to, IgA, secretory IgA, IgG and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG 4. "antibody" includes, e.g., naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; a human or non-human antibody; fully artificially synthesized antibodies; and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Methods for humanizing antibodies are well known to those skilled in the art. "antibody" also includes, but is not limited to, fragments or portions of any of the immunoglobulin molecules described above and includes monovalent and divalent fragments or portions. Antibody fragments include, for example, Fc fragments and antigen binding fragments (Fab).

A "monoclonal antibody" (also designated mAb) is an antibody molecule whose primary sequences are substantially identical and which exhibits the same antigen specificity. Monoclonal antibodies can be produced by hybridomas, recombinant, transgenic, or other techniques well known to those skilled in the art.

"humanized" antibodies refer to antibodies in which some, most, or all of the amino acids outside of the CDR regions are substituted with corresponding amino acids from human immunoglobulin molecules. In one embodiment of a humanized form of the antibody, some, most, or all of the amino acids outside of the CDR regions have been substituted with amino acids from a human immunoglobulin molecule, while some, most, or all of the amino acids within one or more of the CDR regions have not been altered. Minor additions, deletions, insertions, substitutions or modifications of amino acids are also permissible as long as they do not abrogate the binding capacity of the antibody to a given antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA, IgE and IgM molecules. "humanized" antibodies retain antigen specificity similar to the original antibody.

In one embodiment, the anti-CD 40 ligand is the 5c8 monoclonal antibody produced by the 5c8 hybridoma, ATCC accession No. HB 10916, described in U.S. patent No. 5,474,771 (incorporated herein by reference in its entirety) issued 12.12.1995.

The skilled person knows how to prepare the humanized antibodies for use in the present invention. Various publications, some of which are incorporated herein by reference, also describe how to make humanized antibodies. For example, the method described in U.S. Pat. No. 4,816,567 involves the preparation of chimeric antibodies having the variable region of one antibody and the constant region of another antibody.

U.S. Pat. No. 5,225,539 describes another method for producing humanized antibodies. This patent describes the use of recombinant DNA technology to produce humanized antibodies in which the CDRs of the variable region of one immunoglobulin are replaced by CDRs of an immunoglobulin of different specificity, such that the humanized antibody recognizes the desired target but is not recognized in an obvious manner by the human subject's immune system. Specifically, the CDRs are grafted onto the framework using site-directed mutagenesis.

Other methods for humanizing antibodies are described in U.S. Pat. nos. 5,585,089(73) and 5,693,761(74), as well as WO 90/07861, which both describe methods for producing humanized immunoglobulins. These humanized antibodies have one or more CDRs and possibly additional amino acids from a donor immunoglobulin and framework regions from an accepted human immunoglobulin. These patents describe methods for increasing the affinity of an antibody to a desired antigen. Some amino acids within the framework are selected to be identical to those at those positions in the donor, but not the recipient. Specifically, these patents describe the preparation of humanized antibodies that bind to the receptor by combining the CDRs of a mouse monoclonal antibody with human immunoglobulin framework and constant regions. The human framework regions can be selected to maximize homology to the mouse sequence. Computer models can be used to identify amino acids within the framework regions that are likely to interact with the CDRs or specific antigen, and mouse amino acids can then be used at these positions to generate humanized antibodies.

Patents 5,585,089 and 5,693,761, and WO 90/07861(75) also disclose four criteria that can be used to design humanized antibodies. The first proposal is to use, for the recipient, a framework from a particular human immunoglobulin that is usually homologous to the donor immunoglobulin to be humanized, or a consensus framework from multiple human antibodies. The second proposal is that if the amino acids in the human immunoglobulin framework are not conventional and the donor amino acid at that position is typical for human sequences, the donor amino acid can be selected instead of the acceptor amino acid. A third proposal is that donor amino acids can be selected within the humanized immunoglobulin chain at positions immediately adjacent to the 3 CDRs rather than acceptor amino acids. A fourth proposal is to use donor amino acid residues at framework positions that predict that amino acids have side chain atoms within 3A of the CDR in a three-dimensional model of the antibody and that are presumed to be able to interact with the CDR. The above methods are merely illustrative of some of the methods that can be employed by those skilled in the art to make humanized antibodies. Wu et al (1999) j.mol.biol.284: 151 and U.S. Pat. nos. 6,165,793, 6,365,408, and 6,413,774 can improve the binding affinity and/or specificity of the humanized antibody.

Hsp60sp has the sequence QMRPVSRAL (SEQ ID NO: 2) or, in the case of humans, QMRPVSRVL (SEQ ID NO: 3).

All combinations of the various elements of the methods, compositions, and processes described herein are also within the scope of the invention.

The present invention may be better understood by reference to the following experimental details, but those skilled in the art will readily appreciate that the specific experimental details are merely illustrative of the invention, which is more fully described in the claims.

Details of the experiment

The affinity model emphasizes that in the context of a reduced T cell pool lacking high affinity T cells, selective down-regulation of specific intermediate affinity T cells of any antigen is the biological basis for the immune system to achieve peripheral self-tolerance. Based on this property of the regulatory system, acute transplant rejection is usually mediated by high-affinity alloreactive T cells that are tolerant to downregulation, while chronic transplant rejection is mainly mediated by medium-affinity alloreactive T cells that are downregulated. The present invention discloses the deletion of high affinity alloreactive T cells or the conversion of said alloreactive T cells from high affinity to intermediate affinity by employing agents known to reduce the affinity of T cells responding to alloantigens. The resulting pool of alloreactive T cells will lack high affinity alloreactive T cells, but is enriched for intermediate affinity alloreactive T cells that induce regulatory suppressor cells and are also susceptible to such suppression.

Certain non-specific immunosuppressive agents currently used to prevent acute transplant rejection will engineer T cell banks against transplants by preferentially irradiating the majority of activated clones, which may have high affinity and thus be enriched in intermediate affinity T cells. For the composition of allo-responding T cell banks, this will place the transplanted graft in a unique position between the foreign and self-antigens. This creates a situation that enables the immune system to treat the graft as if it were a self-antigen (treat) according to the affinity model. For example, grafts may be made to survive acute rejection by using an immunomodulator such as "anti-CD 40L mAb" which can specifically modify the alloreactive T cell bank by eliciting a moderate affinity response from alloreactive T cells lacking high affinity. Residual alloreactive T cells may primarily mediate the subsequent moderate-affinity chronic rejection of continued activation of the transplant received in vivo. An effective way to down-regulate these cells is to reactivate the HLA-E restricted CD8+ T cell mediated regulatory pathway that may be disrupted by prior anti-rejection therapies during the acute rejection phase.

Thus, it has been demonstrated that anti-CD 40L mAb 5C8 induces long-term transplant tolerance in non-human primates after a relatively short-term allograft rejection therapy (20-24). Indeed, a single course of treatment over a period of one month results in years of allograft tolerance. The induction of long-term tolerance without drug use is due in part to the modification of the alloreactive T cell repertoire by initially receiving anti-CD 40L therapy, which allows selective down-regulation of intermediate affinity alloreactive T cells by HLA-E-restricted regulatory CD8+ T cells. However, the use of anti-CD 40L mAb to treat immune-related clinical problems has been in a state of suspension because the drug is known to have side effects that induce thromboembolic phenomena in a small number of patients but over time in a significant proportion of patients. Thus, the affinity model provides the theoretical basis for using once anti-CD 40L to modify the alloreactive T cell repertoire and subsequently reactivate the HLA-E restricted CD8+ T cell regulatory pathway to establish long-term transplant tolerance. The use of a single anti-CD 40L mAb can significantly reduce the chance of thromboembolic events and also allow for close monitoring of patients during hospital administration of anti-CD 40L. In addition, short-term treatment regimens allow the use of antithrombotic treatments including aspirin and the antiplatelet drug bolivit (Plavix) commonly used to prevent thromboembolism. For example, anti-platelet drugs used during insertion of cardiac stents for myocardial insufficiency may be similarly employed to prevent thromboembolic complications in anti-CD 40L mAb therapy.

The method of inducing transplantation tolerance is as follows. The treatment consists of two phases: anti-CD 40L mAb and immunosuppressive drugs were used once (weeks) during donor-specific infusion (DST) to modify the anti-allogeneic T cell bank, followed by reactivation of HLA-E restricted CD8+ T cells.

Phase 1 modification of the same reaction pool to remove high affinity donor-resistant specific T cells

DST is designed to elicit a primary anti-allogenic immune response to provide a time window for modification of the alloreactive T cell pool to remove high affinity alloreactive T cells prior to graft transplantation. By intravenous injection of 10X 10 at day 1 and at day 7 (as boosters) into recipients⁶Individual irradiated donor cells (PBMC from human or monkey) achieve DST. anti-CD 40L mAb will be injected at 20mg/kg to the receptor on days-1, 0, 3,7 and weekly (for 8 weeks) (25). Immunosuppressive drugs conventionally used in transplant patients (including cyclosporine A, FK506 or rapamycin) are administered within 8 weeks. Graft transplantation (secondary anti-allogenic response) will be performed 2-3 weeks after the start of treatment.

During the course of administration of anti-CD 40L, the recipient will be given the antiplatelet drugs borrelidin and aspirin, and will be required to be hospitalized and closely monitored for any signs of thromboembolic complications.

Induction of HLA-E restricted CD8+ T cell pathway to prevent chronic rejection in stage 2

Stage 2 should be performed after stage 1 is completed. The following agents may be administered to the patient:

an HLA-E tetramer: HLA-E-Hsp60sp tetramers can be used as specific antigens to induce HLA-E-restricted CD8+ T cells in vivo.

2. Exosomes: HLA-E bearing exosomes loaded with selected HLA-E binding peptides can be used to activate this pathway in vivo (26). It is known that dendritic cell-derived exosomes carrying functional MHC class I and class II molecules capable of carrying a selected synthetic peptide can be used as peptide-based vaccines (26).

3. Molecularly engineered complexes consisting of HLA-E/Hsp60sp can also be used as antigens for specifically activating HLA-E restricted CD8+ T cells. Thus, as part of artificial antigen-presenting cells, MHC multimers are known to be potentially useful tools for stimulating and analyzing antigen-specific T cells in immune responses (27-32).

4. The recipient was vaccinated with autologous dendritic T cells loaded with Hsp60 sp.

To load cells with Hsp60sp, for example, recipient dendritic cells, cells from, for example, PBMCs, were cultured in vitro for 6 days, then loaded with 50uM Hsp60sp at 37 ℃ for 2 hours, and then administered intravenously to the recipient.

This technique reduces or eliminates the need for continuous immunosuppressive therapy by inducing tolerance to the donor tissue.

Examples

Prior to transplanting an organ into a recipient subject, administering to the subject: (i) a plurality of cells from a donor subject, wherein the donor cells have been irradiated; and (ii) a monoclonal antibody that specifically binds to a CD40 ligand to inhibit activation of alloreactive T cells in a recipient subject. Performing a transplant surgery on the subject. After transplantation, the recipient subject is administered with HLA-E restricted CD8⁺HLA-E with T cells enhancing donor tissue activation⁺Down-regulation of T cellsAgents whereby the activated HLA-E of the donor tissue is enhanced⁺Down-regulation of T cells, thereby preventing rejection of the recipient subject's immune system against transplanted tissue in the recipient subject. In yet another embodiment, the recipient subject is further administered an immunosuppressive drug.

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Claims (21)

1. A method of preventing rejection of a transplanted tissue from a donor subject into a recipient subject, the method comprising:

a) administering to the recipient subject prior to the tissue transplant the following agents so as to inhibit activation of alloreactive T cells in the recipient subject: (i) a plurality of Peripheral Blood Mononuclear Cells (PBMCs) from the donor subject, wherein the PBMCs have been irradiated such that they cannot proliferate in vivo, and (ii) a monoclonal antibody that specifically binds to a CD40 ligand, thereby inhibiting activation of alloreactive T cells in the recipient subject; and

b) administering to the recipient subject after the tissue transplantation a CD8 restricted by HLA-E⁺HLA-E with T cells enhancing donor tissue activation⁺An agent for down-regulation of T cells, thereby enhancing activated HLA-E of said donor tissue⁺The down-regulation of T-cells is achieved,

thereby preventing rejection of the tissue transplanted into the recipient subject.

2. The method of claim 1, further comprising administering an immunosuppressive drug to the recipient subject during step a), during step b), or during both step a) and step b).

3. The method of claim 1 or 2, wherein the agent is an HLA-E/IgG fusion protein or an HLA-E/Hsp60sp tetramer.

4. The method of claim 1, 2 or 3, wherein the HLA-E⁺The T cell is CD4⁺/HLA-E⁺T cells or CD8⁺/HLA-E⁺T cells.

5. The method of any one of claims 1-4, wherein the agent is an HLA-E bearing exosome loaded with a type B self peptide derived from dendritic cells.

6. The method of claim 5, wherein the type B self peptide has the amino acid sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1-6, wherein the agent is an HLA-E bearing exosome loaded with Hsp60sp peptide derived from dendritic cells.

8. The method of any one of claims 1-7, wherein the agent is an autologous dendritic T cell loaded with Hsp60sp peptide.

9. The method of claim 3,7, or 8, wherein the Hsp60sp peptide comprises a peptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a sequence of contiguous amino acids.

10. The method of claim 8, wherein, prior to administering the autologous dendritic T cells to the recipient subject as the agent, the autologous dendritic T cells have been removed from the recipient subject, cultured ex vivo, and contacted with an Hsp60sp peptide to load the cells with an Hsp60sp peptide.

11. The method of any one of claims 1-10, wherein the agent is administered intravenously, intramuscularly, or orally.

12. The method of any one of claims 1-11, wherein in step a) the monoclonal antibody directed against CD40 ligand is administered to the recipient subject for a period of up to 10 weeks.

13. The method of any one of claims 1-12, wherein in step a), the plurality of cells from the donor subject is administered to the recipient subject in two separate fractions, first a first fraction and then a second fraction, wherein the second fraction is administered to the recipient subject about 7 days after administration of the first fraction.

14. The method of claim 13, wherein in step a) the monoclonal antibody directed against CD40 ligand is administered to the subject (1) on the first 1 day of the first part of the day of administration of the plurality of cells, and (2) on the day of administration of the first part of the day of administration of the plurality of cells, and (3) on the first 1 day of administration of the first part of the day of administration of the plurality of cells, and (4) on the second 3 days of administration of the first part of the day of administration of the plurality of cells, and (5) on the first 7 days of administration of the first part of the day of administration of the plurality of cells, and (6) on the first 14, 21, 28, 35, 42, 49, and 56 days of administration of the first part of the day of administration of the plurality of cells.

15. The method of claim 2, 13, or 14, wherein the immunosuppressive drug is administered daily for up to 10 weeks after administering the plurality of cells or after administering the first portion of the plurality of cells.

16. The method of claim 15, wherein the immunosuppressive drug is administered daily for 56 days.

17. The method of any one of claims 1-16, wherein said tissue is transplanted into said recipient subject 2 to 3 weeks after administering said plurality of cells or said first portion of said plurality of cells.

18. The method of any one of claims 1-17, further comprising administering an anti-thromboembolic agent to the recipient subject during administration of the monoclonal antibody directed to CD40 ligand.

19. The method of any one of claims 1-18, wherein the transplanted tissue is lung tissue, heart tissue, kidney tissue, liver tissue, retinal tissue, corneal tissue, skin tissue, pancreatic tissue, intestinal tissue, genital tissue, ovarian tissue, bone tissue, tendon tissue, or vascular tissue.

20. The method of any one of claims 1-19, wherein the transplanted tissue is transplanted as a whole organ.

21. The method of any one of claims 1-20, wherein the PBMCs have been irradiated with 2000-4000 rads for a sufficient time such that they cannot proliferate in vivo.