JP2015117214A - Therapeutic target and drug useful in ischemia-reperfusion injury - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent effective in ischemia-reperfusion injury of a heart.SOLUTION: The invention provides a method for treating ischemia-reperfusion injury of a heart using a therapeutic agent selected from a protein, polypeptide, peptide, peptidomimetic, nucleic acid, organic low molecular weight compound, antibody or a fragment or derivative thereof, soluble receptor (soluble CTLA4 molecule, preferably CTLA4Ig fusion protein), and aptamer, which can interact to at least either one of CD80 or CD86 polypeptide, the interaction interfering in the binding of at least either one of CD80 or CD86 polypeptide with CD28 polypeptide.

Description

  The present invention relates generally to the field of medicine, and more specifically to the field of treatment of heart conditions. In particular, the present invention provides therapeutic therapeutics useful in the treatment of ischemia reperfusion injury, more specifically cardiac ischemia reperfusion injury, even more specifically cardiac warm ischemia reperfusion injury. Relates to targets and therapeutic agents.

  Current interventional procedures for acute myocardial infarction (AMI) focusing on restoring blood flow to the myocardium (i.e. cardiac reperfusion) by interventional procedures such as thrombolysis or percutaneous coronary angioplasty are Reducing both size and mortality significantly improves the clinical outcome of AMI. However, cardiac reperfusion paradoxically causes serious adverse effects arising from the cardiac repair pathway that occurs after infarction, which can lead to left ventricular remodeling and heart failure. This injury, often referred to as ischemia-reperfusion injury (IRI), is a major cause of the chronic complications observed in patients who survive from severe AMI.

IRI is also observed in patients undergoing arterial bypass grafting procedures, heart transplantation, and other surgical procedures including temporarily stopping blood flow to the myocardium and then restoring blood flow.
Although the precise mechanism of IRI is not yet fully understood, it appears to involve, among other things, the activation of the systemic inflammatory response, which is already damaged cardiomyocytes and endothelial cells in the ischemic region In particular, leading to cell and tissue damage and ultimately loss of function.

  There is some experimental and clinical evidence suggesting a role for inflammation in cardiovascular disease, and anti-inflammatory agents have been proposed as hypotheses for the treatment of cardiovascular disease. For example, WO2005 / 016266 proposes the treatment of cardiovascular diseases that use soluble CTLA-4 molecules that block endogenous B7 molecules, thereby suppressing T cell responses.

  Proliferation of naive T lymphocytes and their differentiation into effector cells require additional signals provided by antigen recognition and costimulatory molecules expressed on antigen presenting cells (APC). Recognition of the antigen can occur directly when the antigen is associated with the major histocompatibility complex (MHC) on the surface of the foreign cell or tissue, or the antigen is processed and then the antigen-presenting cell (APC). It can occur indirectly when associated with MHC on the surface. Resting T lymphocytes that recognize such antigen-presenting MHC complexes are activated by the association of these complexes with T cell receptors. Such activated T cells require additional signals (ie costimulation) to proliferate and become functional. The best defined costimulatory pathways are the B7: CD28 pathway and the CD40: CD40 ligand (CD40L) pathway. Two known members of the B7 family, B7-1 (CD80) and B7-2 (CD86), are expressed on APC and their expression is enhanced by microbial and innate immune responses. Recognition of B7 by CD28, which is constitutively expressed on the majority of T lymphocytes, is thought to initiate a T cell response. In contrast, binding of CD80 and CD86 to the inhibitory receptor CTLA-4 down regulates the T cell response. CD40 is constitutively expressed on APC and CD40L is induced on T cells upon antigen recognition. The interaction between CD40 and CD40L also enhances the T cell response.

  Total infarct size is considered a critical determinant of the risk of patients developing heart failure, generally considering that cardiovascular disease and especially acute myocardial infarction is the leading cause of death in the Western world, There is an urgent need for additional therapeutic targets useful in the treatment of cardiac ischemia reperfusion injury, more specifically cardiac ischemia reperfusion injury.

WO2005 / 016266

The inventors have realized the unexpected finding that inhibition of the T cell response has a beneficial effect in the treatment of heart warm ischemia reperfusion injury.
In particular, we propose costimulatory pathway B7: CD28 as a useful and promising target for the treatment of cardiac ischemia-reperfusion injury, alone or in combination with the costimulatory pathway CD40: CD40L. . Extending these findings, we have specifically identified the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) as valuable targets for the treatment of cardiac warm ischemia-reperfusion injury. Recognized.

Thus, among others, the following aspects and embodiments are provided according to the present invention.
For use in the treatment of cardiac ischemia-reperfusion injury capable of interacting with at least one of CD80 or CD86 polypeptide, said interaction interfering with binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide Therapeutic agent.

  Administering to a subject in need of treatment a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent can interact with at least one of the CD80 or CD86 polypeptide, said interaction being a CD80 or CD86 polypeptide. A method for treating cardiac warm ischemia reperfusion injury in a subject in need of treatment that interferes with binding of at least one of the peptides to a CD28 polypeptide.

  Treatment of cardiac warm ischemia reperfusion injury wherein the therapeutic agent can interact with at least one of CD80 or CD86 polypeptide, said interaction interfering with binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide The use of therapeutic agents for the manufacture of pharmaceuticals for use.

  In the treatment of cardiac warm ischemia reperfusion injury, which can prevent or inhibit the expression of at least one of CD80 or CD86 polypeptide by a cell, or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the cell surface A therapeutic agent for use.

  Administering a therapeutically effective amount of a therapeutic agent to a subject in need of treatment, said therapeutic agent being capable of preventing or inhibiting expression of at least one of CD80 or CD86 polypeptide by the cell, or cell surface A method for treating cardiac warm ischemia reperfusion injury in a subject in need of treatment capable of preventing or inhibiting presentation of at least one of the above CD80 or CD86 polypeptides.

  A cardiac warm ischemia reperfusion wherein the therapeutic agent can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell or can prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the cell surface Use of a therapeutic agent for the manufacture of a medicament for the treatment of a disorder.

  The above and further aspects and preferred embodiments of the invention are set forth in the following paragraphs and appended claims. The subject matter of the appended claims is hereby specifically incorporated by reference.

An example of infarct size evaluation by creatine kinase (CKMB) measurement in the blood of a subject slightly after the infarction is shown. An example of evaluation of infarct size by measuring cardiac troponin I (cTnI) in the blood of a subject slightly after the infarction is shown. An example of measurement of ejection fraction (EF) over time by magnetic resonance imaging (MRI) is shown. An example of measurement of left ventricular end-diastolic volume (LVEDV) over time by magnetic resonance imaging (MRI) is shown. An example of measurement of left ventricular end systolic volume (LVESV) over time by MRI is shown. An example of scar size measurement by MRI is shown. An example of scar size evaluation at autopsy is shown.

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural references unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of” as used herein include “including”, “includes” or “containing”. ) "," Contains "and is comprehensive or expandable and does not exclude further unmentioned members, elements or method steps. These terms also encompass “consisting of” and “consisting essentially of” which enjoy the established meaning in patent terms.
Reference to numerical ranges by endpoints includes the endpoints mentioned with all numerical values and fractions encompassed within each range.

  The term “about” or “approximate” as used herein when referring to a measurable value, such as a parameter, amount, time period, etc., is +/− 10% of the specific value and from the specific value To include variations of and from a particular value, such as below, preferably +/− 5% or less, more preferably +/− 1% or less and even more preferably +/− 0.1% or less. Meaning (as long as such variations are appropriate for carrying out the disclosed invention). It is understood that values referred to by the modifier “about” or “approximately” are also specifically and preferably disclosed per se.

The term “one or more” or “at least one”, such as at least one member of one or more members or groups of members, is clear per se by further illustration, which term is Includes references to any one of the members, or any two or more of the members, such as any ≧ 3, ≧ 4, ≧ 5, ≧ 6 or ≧ 7 of the member and up to all of the members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
Discussion of the background of the invention herein is included to explain the relationship of the invention. This is not an admission that any of the materials mentioned is a known or partial general consensus published in any country on any priority date of the claims.

  Throughout this disclosure, various publications, patents and published patent specifications are referenced by identifying citations. All documents cited herein are hereby incorporated by reference in their entirety. In particular, the teachings or portions of such documents specifically referred to herein are incorporated by reference.

  Unless defined otherwise, all terms used to disclose the invention, including technical and scientific terms, have the meaning commonly understood by a person skilled in the art to which the invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. When specific terms are defined in connection with specific aspects of the invention or specific embodiments of the invention, unless otherwise defined, That is, it is intended to apply in the context of other aspects or embodiments of the invention.

  In the following text, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment defined in this manner may be combined with any other aspect or embodiment unless explicitly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

  Reference to “one embodiment” or “an embodiment” throughout this specification includes that particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Means that. The appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but all refer to the same embodiment. there's a possibility that. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art from this disclosure. In addition, some embodiments described herein include some features (but not other features) included in other embodiments, but combinations of features from different embodiments will occur to those skilled in the art. As will be appreciated, it is meant to be within the scope of the present invention and forms different embodiments. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

  As stated above, the inventors have described that the costimulatory pathway B7: CD28 alone or in combination with the costimulatory pathway CD40: CD40L is abbreviated as cardiac ischemia-reperfusion injury (abbreviated herein as CWIRI). Recognized as a useful and promising target for the treatment of As used in the art and herein, the term “costimulatory pathway” refers to a T cell costimulatory pathway, which is a non-antigen molecule (ie, a costimulatory molecule) present on the surface of an antigen presenting cell and that on a T cell. Interaction with each binding partner, which is necessary to induce proliferation and differentiation of T cells along with major histocompatibility complex (MHC) -antigen-T cell receptor interactions Say. Thus, the inventors have unexpectedly found that inhibition of these T cell responses has a beneficial effect in the treatment of cardiac warm ischemia-reperfusion injury.

Thus, certain embodiments of the present invention are capable of interacting with at least one of CD80 or CD86 polypeptide, wherein said interaction interferes with binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide. It relates to therapeutic agents for use in the treatment of reperfusion injury.
Another aspect of the invention is capable of preventing or inhibiting the expression of at least one of CD80 or CD86 polypeptide by the cell or preventing or inhibiting the presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell. The present invention relates to a therapeutic agent for use in the treatment of heart temperature ischemia-reperfusion injury.

As used herein, the term `` (agent) '' refers to any chemical substance (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule). ), Combinations or mixtures thereof, samples of undetermined composition, or extracts made from biological material such as bacteria, plant, fungus or animal cells or tissues. Preferred but not limiting `` (drug) agents '' include nucleic acids, oligonucleotides, ribozymes, polypeptides or proteins, peptides, peptidomimetics, antibodies and fragments and derivatives thereof, aptamers, photoaptamers, chemicals, preferably organic molecules, More preferably include small organic molecules, lipids, carbohydrates, polysaccharides, etc., as well as any combination thereof.
As used herein, the term “therapeutic agent” is used interchangeably with “drug” and refers to an agent taught herein for use in the treatment, cure, prevention or diagnosis of a disease.

  As used herein, the terms “treat”, “treating” or “treatment” refer to therapeutic treatment of an already occurring disease or condition, particularly treatment of CWIRI, and prevention or prevention. , Where the aim is to prevent or reduce the opportunity for the appearance of undesired pain, and in particular to prevent CWIRI. Beneficial or desirable clinical results include, but are not limited to, preventing or damaging heart tissue damage or injury, reducing the degree of heart tissue damage or injury, not exacerbating heart tissue damage or injury, Healing received heart tissue, reducing scar size, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

  As used herein, the term “ischemic reperfusion injury” is caused when the blood supply returns to the tissue or organ after a period of ischemia or lack of oxygen in the tissue or organ (ie, reoxygenation). Refers to tissue or organ injury. For illustration, the absence of oxygen and nutrients in the blood during the ischemic period creates a condition in which circulatory recovery results in inflammation and oxidative damage by induction of oxidative stress rather than restoration of normal function. Oxidative stress associated with reperfusion can cause injury to affected tissues or organs. Ischemic reperfusion injury is thus biochemically characterized by oxygen depletion during an ischemic event and subsequent reoxygenation and the generation of reactive oxygen species during the reperfusion.

  Ischemic reperfusion injury is, for example, recovery of blood flow after ischemic infarction, such as cerebral infarction, myocardial or pulmonary infarction, vein or artery occlusion or restenosis; Recovery of blood flow following treatment; recovery of blood flow after cardiovascular arrest; recovery of blood flow after trauma; or one or more to restore blood flow to a tissue or organ that has been depleted of blood supply It can be caused by surgical procedures or the result of other therapeutic interventions. Such surgical procedures include, for example, coronary artery bypass grafting, coronary angioplasty, vascular ligation and reperfusion during organ transplantation.

  The term “warm ischemia reperfusion injury” more specifically refers to ischemia reperfusion injury in a tissue or organ that has been maintained or exposed to a substantially physiological temperature during the period of ischemia. Physiological temperature most typically refers to normal body temperature. For example, in a human subject, normal body temperature may specifically refer to a temperature between about 36.00 ° C. and about 37.50 ° C. Physiological temperature, however, can also include cases where high fever, particularly high fever, is compatible with living organisms. For example, in human subjects, the high fever is between about 37.51 ° C and about 41.00 ° C, preferably between about 37.51 ° C and about 40.00 ° C, more preferably between about 37.51 ° C and about 39.00 ° C, and even more. Preferably, a temperature between about 37.51 ° C and about 38.50 ° C may be specifically mentioned. Physiological temperature may also include hypothermia, particularly where the degree of hypothermia is compatible with living organisms. For example, in human subjects, hypothermia is between about 24.00 ° C. and about 35.99 ° C., preferably between about 28.00 ° C. and about 35.99 ° C., more preferably between about 31.00 ° C. and about 35.99 ° C., More specifically may refer to temperatures of about 32.00 ° C or about 33.00 ° C or about 34.00 ° C and about 35.99 ° C, and even more preferably between about 35.00 ° C and about 35.99 ° C. One skilled in the art can easily ascertain the physiological temperature of warm-blooded animals, particularly mammals, either from the literature or simply by temperature measurement.

  For example, warm ischemia reperfusion injury may occur as a result of reperfusion when tissue or organs are present in the body or not removed from the body during the ischemic period. As another example, warm ischemia reperfusion injury is a reperfusion where the tissue or organ is not refrigerated during the ischemic period, for example when the tissue or organ is temporarily removed from the body during that period. May result. Refrigeration in this context specifically maintains the tissue or organ at a temperature between about 2 ° C. and about 8 ° C., for example between about 4 ° C. and about 6 ° C. (exposing the tissue or organ to said temperature). Can mean.

  The phrase “heart warm ischemia reperfusion injury” thus broadly encompasses warm ischemia reperfusion injury of heart tissue or organs. More specifically, this phrase may refer to warm ischemia-reperfusion injury of the heart muscle tissue (myocardium). The heart warm ischemia reperfusion injury contemplated herein may be the result of, for example, myocardial infarction, which may be chronic myocardial infarction or preferably acute (ie sudden and severe) myocardial infarction (STEMI) (cardiac ischemia). (May have been exacerbated by treatment of myocardial infarction (e.g. anticoagulant therapy) aimed at restoring blood flow to the area), cardiovascular arrest, coronary artery bypass grafting, coronary angioplasty, transient vasospasm, acute Can be coronary syndrome (NSTEMI), cardiac arrest, revascularization during vascular ligation and heart transplantation.

Thus, in certain embodiments, cardiac ischemia reperfusion injury is myocardial infarction, chronic myocardial infarction, acute myocardial infarction (STEMI), myocardial infarction aimed at restoring blood flow to the ischemic region of the heart May be the result of reperfusion during treatment, cardiovascular arrest, coronary artery bypass grafting, coronary angioplasty, transient vasospasm, acute coronary syndrome (NSTEMI), cardiac arrest or vascular ligation and heart transplantation.
Thus, in certain embodiments, heart warm ischemia reperfusion injury can be the result of acute myocardial infarction.

The term “interact”, as used herein, refers to an act or process in which two or more, eg, two, agents affect each other. Interactions between agents such as therapeutic agents described in this application and at least one of CD80 or CD86 polypeptides, or second therapeutic agents described in this application and CD40 polypeptides are covalent (ie, interatomic electrons). Mediated by one or more chemical bonds with pair sharing) or more typically non-covalent (ie non-covalent forces such as hydrogen bridges, bipolar interactions, van der Waals interactions, for example) Mediated by).
Typically, an agent that interacts with another agent (which may be referred to as a “target” for convenience) can thus bind to the other agent (target) and is described, for example, in this application. The therapeutic agent can bind to at least one of CD80 or CD86 polypeptide, or the second therapeutic agent described in the present application can bind to CD40 polypeptide, and more typically, K _A ≧ 1 × 10 ⁵ M ^-1 , preferably K _A ≧ 1 × 10 ⁶ M ⁻¹ , more preferably K _A ≧ 1 × 10 ⁷ M ⁻¹ , even more preferably K _A ≧ 1 × 10 ⁸ M ⁻¹ , even more preferably K _A ≧ 1 × 10 ⁹ M ⁻¹ , and even more preferably K _A ≧ 1 × 10 ¹⁰ M ⁻¹ or K _A ≧ 1 × 10 ¹¹ M ⁻¹ (where K _A = [A_T] / [A] [ T], A represents a drug, and T represents a target), and can bind to the other agent with a binding affinity constant (K _A ). Determination of K _A, for example bio-layer interferometry (BLI), using equilibrium dialysis and Scatchard plot analysis can be carried out by methods known in the art.

  Preferably, the interaction (eg binding) between the agent and the target, eg the interaction between the therapeutic agent described in this application and at least one of the CD80 or CD86 polypeptide, or the second therapeutic agent described in this application Interaction with the CD40 polypeptide is specific or selective, ie, the agent does not substantially interact (eg, bind) to a random or unrelated molecule or substance. In this way, the agent does not substantially alter the activity and level of such random and unrelated molecules or substances. The binding between the target and the agent can be evaluated by, among other methods, conventional methods for examining interactions, such as co-immunoprecipitation, immunoassay, chromatography, gel electrophoresis, yeast two-hybrid method, or combinations thereof.

  Interaction (eg, binding) between an agent and a target, eg, an interaction between a therapeutic agent described in this application and at least one of CD80 or CD86 polypeptide, or a second therapeutic agent described in this application and a CD40 polypeptide Interaction desirably alters the activity and / or level of the target. Reference to “activity” is to be interpreted broadly and any one or more aspects of the biological activity of the target at any level (eg, molecule, cell and / or physiological), eg, without limitation, eg Any one or more aspects of its biochemical activity, enzymatic activity, signal transduction activity, interaction activity, ligand activity, receptor activity or structural activity within a cell, tissue, organ or organism may be encompassed in general. . For example and without limitation, reference to the activity of a CD80 or CD86 polypeptide refers to the activity of the CD80 or CD86 polypeptide as a ligand, ie one or more cognate receptors, in particular a CD28 polypeptide or CTLA-4 polypeptide or CD28. And their ability to bind both CTLA-4 polypeptides and / or the activity of CD80 or CD86 polypeptides as signaling molecules, ie, participate in one or more cell signaling pathways, particularly the B7: CD28 costimulatory pathway These abilities are sometimes referred to specifically. Reference to “level” is broadly interpreted and generally refers to the amount and / or availability of a target (eg, availability to perform its biological activity) within a cell, tissue, organ or organism. Can be included.

  In particular, as contemplated in certain embodiments, the interaction of a therapeutic agent described in this application with at least one of CD80 or CD86 polypeptide may result in binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide. Ie, at least one of the CD80 or CD86 polypeptide interferes with the ability to bind to the CD28 polypeptide. This allows the interaction of the therapeutic agents described in this application with at least one of CD80 or CD86 polypeptide to inhibit or prevent the generation of B7: CD28 costimulatory signals and T cell proliferation and differentiation. The phrase “interfering”, as used herein, is synonymous with terms such as “reduce,” “reduce,” “reduce,” “inhibit,” “disturb,” or “disturb”. Intended to be, indicating a qualitative or quantitative decrease in interfering binding. The term encompasses any degree of such interference. For example, if a decrease in binding affects a determinable or measurable variable, the interference is at least about 10%, such as at least about 20%, at least about 30%, such as at least about a reference situation without said interference. About 40%, at least about 50%, such as at least about 60%, at least about 70%, such as at least about 80%, at least about 90%, such as at least about 95%, such as at least about 96%, 97%, 98%, 99 % Or even 100% (corresponding to a decrease in the binding of at least one of the CD80 or CD86 polypeptide to the CD28 polypeptide) may include a decrease in the value of said variable. Appropriate molecular tests to measure polypeptide binding, such as binding of at least one of CD80 or CD86 polypeptide to a CD28 polypeptide, and to determine the extent to which an agent interferes with such binding are known to those skilled in the art. Is available to In addition, a suitable in vitro cell test for measuring the extent to which an agent interferes with the binding of at least one of CD80 or CD86 polypeptide to a CD28 polypeptide is a one-way allogeneic mixed lymphocyte reaction (some suitable in such tests). Such endpoints may include testing the ability of such agents to interfere with the induction of T cell responses (which are EC50 values).

  Similarly, as contemplated in certain embodiments, the interaction of the second therapeutic agent described in this application with a CD40 polypeptide is the binding of a CD40 polypeptide to a CD40 ligand (CD40L) polypeptide, ie, Interferes with the ability of a CD40 polypeptide to bind to a CD40L polypeptide. Includes any degree of such interaction. For example, if a decrease in binding affects a determinable or measurable variable, the interference is at least about 10%, such as at least about 20%, at least about 30%, such as at least about a reference situation without said interference. About 40%, at least about 50%, such as at least about 60%, at least about 70%, such as at least about 80%, at least about 90%, such as at least about 95%, such as at least about 96%, 97%, 98%, 99 % Or even 100% may include a decrease in the value of said variable (corresponding to a decrease in binding between CD40 polypeptide and CD40L polypeptide). Appropriate molecular tests are available to those skilled in the art to measure polypeptide binding, such as binding of CD40 and CD40L polypeptides, and to determine the extent to which an agent interferes with such binding. . In addition, a suitable in vitro cell test for measuring the extent to which an agent interferes with the binding of CD40 and CD40L polypeptides is a one-way allogeneic mixed lymphocyte reaction (one suitable endpoint in such a test is Testing the ability of such agents to interfere with the induction of T cell responses (which are EC50 values).

  As used herein, the term “surface antigen classification 80” or “CD80” or B7-1 is predominantly used on antigen presenting cells (APCs) that provide costimulatory signals necessary for T cell activation and survival. Refers to the protein found in The term encompasses CD80 expressed in APC and any other cell type, such as endothelial cells. CD80 is a ligand for two different proteins on the T cell surface, CD28 and CTLA-4.

  The term “antigen-presenting cell” is understood by those skilled in the art and, by further explanation, is a major histocompatibility complex (MHC) molecule (MHC class I or II) or part thereof, or one or more non-classical MHC molecules or their Refers to any cell that presents an antigen (eg, in particular an exogenous (exogenous) antigen) on its surface along with a portion and can present the antigen to T cells in the context of MHC molecules, for example. Antigen presenting cells can therefore also suitably process proteins and polypeptides into peptides that can be presented in the context of MHC molecules. Antigen presenting cells (APCs) can include, but are not limited to, dendritic cells, macrophages and B cells. The term “T cell” is understood by those skilled in the art and, by further explanation, refers to a type of thymus-derived leukocyte that participates in various cell-mediated immune responses. The term includes, but is not limited to, in particular, the ability to lyse target cells or effector or helper functions, such as cytokine secretion, which can result in death of target cells or generation or enhancement of anti-target effector activity. It may refer to CD8 + or CD4 + T cells that can be provided.

  An exemplary but non-limiting CD80 mRNA is from Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_005191.3 (the number after the period indicates the Genbank sequence version). Includes human CD80 mRNA with the nucleic acid sequence annotated below. An exemplary but non-limiting CD80 includes a human CD80 protein (precursor) having an amino acid sequence annotated under Genbank accession number NP_005182.1.

  One skilled in the art can appreciate that in some cases, the sequences referred to herein are precursors (eg, preproteins) and may include portions that are no longer processed from the mature protein or polypeptide. One skilled in the art can also recognize that any and all isoforms of a protein or polypeptide are encompassed herein, even if the sequence referred to exhibits only one such isoform.

  As used herein, the term “surface antigen classification 86” or “CD86” or B7-2 is found primarily on antigen presenting cells that provide costimulatory signals necessary for T cell activation and survival. It means protein. The term includes CD86 expressed in APC and any other cell type, such as endothelial cells. CD86 is a ligand for two different proteins on the T cell surface, CD28 and CTLA-4. Exemplary but non-limiting CD86 genes include the human CD86 gene having a nucleic acid sequence that is annotated under Genbank accession number EF064748.1. Exemplary but non-limiting CD86 mRNA includes human CD86 mRNA having a nucleic acid sequence annotated under Genbank accession number U04343.3. Exemplary but non-limiting CD86 proteins include the human CD86 protein having an amino acid sequence annotated under Genbank accession number ABK41931.1.

  CD80 and CD86 polypeptides are collectively referred to as “costimulatory molecules” and their presence, together with the antigenic peptide (signal 1), is necessary to obtain efficient stimulation of antigen-reactive T cells. (Signal 2). The presence of costimulatory molecules is sensed by receptors on the surface of T cells.

  As used herein, the term “surface antigen classification 28” or “CD28” refers to one of the molecules expressed on T cells that provides the costimulatory signal required for T cell activation. . CD28 is a receptor for CD80 and CD86. An exemplary but non-limiting CD28 gene includes the human CD28 gene having a nucleic acid sequence annotated under Genbank accession number NG — 029618.1. Exemplary but non-limiting CD28 mRNA includes human CD28 mRNA having a nucleic acid sequence annotated under Genbank accession numbers NM_006139.3, NM_001243077.1 or NM_001243078.1. Exemplary but non-limiting CD28 proteins include human CD28 protein (precursor) having an amino acid sequence annotated under Genbank accession number NP_006130.1, NP_001230006.1 or NP_001230007.1.

  As used herein, the terms “cytotoxic T lymphocyte antigen 4”, “CTLA-4”, “CTLA4”, “surface antigen classification 152” or “CD152” are required for T cell activation. Refers to a molecule expressed on the surface of a T cell that provides a costimulatory signal. CTLA4 is a receptor for CD80 and CD86. Exemplary but non-limiting CTLA4 genes include the human CTLA4 gene having a nucleic acid sequence that is annotated under Genbank accession number NG_011502.1. Exemplary but non-limiting CTLA4 mRNA includes human CTLA4 mRNA having a nucleic acid sequence annotated under Genbank accession number NM_001037631.2 or NM_005214.4. Exemplary but non-limiting CTLA4 proteins include human CTLA4 protein (precursor) having an amino acid sequence annotated under Genbank accession number NP_001032720.1 or NP_005205.2.

  As used herein, the term “surface antigen classification 40” or “CD40” refers to a CD40 costimulatory molecule expressed on activated antigen presenting cells. CD40 can bind to CD154 (CD40L) on T cells. Exemplary but non-limiting CD40 genes include the human CD40 gene having a nucleic acid sequence that is annotated under Genbank accession number DQ871604.1. Exemplary but non-limiting CD40 mRNA includes human CD40 mRNA having a nucleic acid sequence annotated under Genbank accession number NM_001250.4 or NM_152854.2. Exemplary but non-limiting CD40 proteins include human CD40 protein (precursor) having an amino acid sequence annotated under Genbank accession number NP_001241.1 or NP_690593.1.

  As used herein, the terms “CD40L”, “CD40 ligand”, “surface antigen classification 154” or “CD154” refer to a protein that is predominantly expressed on T cells that can bind CD40. Exemplary but non-limiting CD40L genes include the human CD40L gene having a nucleic acid sequence that is annotated under Genbank accession number NG — 007280.1. Exemplary but non-limiting CD40L mRNA includes human CD40L mRNA having a nucleic acid sequence that is annotated under Genbank accession number NM — 000074.2. An exemplary but non-limiting CD40L protein includes a human CD40L protein having an amino acid sequence annotated under Genbank accession number NP_000065.1.

  As used herein, a reference to any one nucleic acid or protein corresponds to a nucleic acid, protein, polypeptide or peptide generally known in the art under the respective description. These terms refer to any organisms found, particularly animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals including human and non-human mammals, and even more preferably human nucleic acids, proteins. , Including polypeptides or peptides. These terms include, in particular, nucleic acids, proteins, polypeptides or peptides that have a native sequence, ie, have the same primary sequence as that of a nucleic acid, protein, polypeptide or peptide found in nature or derived from nature. To do. One skilled in the art understands that native sequences can differ between different species due to genetic diversity between species. Furthermore, the native sequence may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Natural sequences may also differ between different individuals of the same species or even within different individuals due to post-transcriptional or post-translational modifications. Any such variant or isoform of a nucleic acid, protein, polypeptide or peptide is contemplated herein. Thus, any sequence of a nucleic acid, protein, polypeptide or peptide found in nature or derived from nature is considered “natural”. These terms are used when forming part of a living organism, organ, tissue or cell, when forming part of a biological sample, and when being at least partially isolated from such sources. Includes nucleic acids, proteins, polypeptides or peptides. These terms also encompass nucleic acids, proteins, polypeptides or peptides when produced by recombinant or synthetic means.

  Unless stated otherwise from the context, references herein to any nucleic acid, protein, polypeptide or peptide include, for example, phosphorylation, glycosylation, lipidation, methylation, cysteineation, sulfonation, glutathioneation Such nucleic acids, proteins, polypeptides or modified forms of the nucleic acid, such as having post-expression or chemical modifications, including acetylation, oxidation of methionine to methionine sulfoxide or methionine sulfone, etc. may also be generally included.

  The nucleic acid, protein, polypeptide or peptide may preferably be human, ie their primary sequence is of a naturally occurring human nucleic acid, protein, polypeptide or peptide or a naturally occurring human nucleic acid, protein , Can be the same as the corresponding primary sequence present in the polypeptide or peptide. Thus, the modifier “human” in this context relates to the primary sequence of the respective nucleic acid, protein, polypeptide or peptide rather than its origin or source. For example, such nucleic acids, proteins, polypeptides or peptides are present in or can be isolated from a sample of a human subject, or other means (e.g., recombinant expression, cell-free translation or non-biological). Peptide synthesis).

  References herein to any nucleic acid, protein, polypeptide or peptide can also encompass fragments thereof. The term “fragment” of a nucleic acid generally refers to a 5′- and / or 3′-terminal deleted or truncated form of said nucleic acid. The term “fragment” of a protein, polypeptide or peptide generally refers to an N-terminal and / or C-terminal deleted or truncated form of said protein, polypeptide or peptide. Without limitation, a nucleic acid, protein, polypeptide or peptide fragment may comprise at least about 5% or at least about 10% of the nucleotide sequence of the nucleic acid or the amino acid sequence of the protein, polypeptide or peptide, such as ≧ 20%, ≧ It may represent 30% or ≧ 40%, such as preferably ≧ 50%, such as ≧ 60%, ≧ 70% or ≧ 80% or more preferably ≧ 90% or ≧ 95%.

  References herein to any nucleic acid, protein, polypeptide or peptide can also encompass their variants. The term `` variant '' of a nucleic acid, protein, polypeptide or peptide means that the sequence (i.e. the nucleotide sequence or amino acid sequence, respectively) is substantially identical (i.e. not completely identical, but not completely identical) to the nucleic acid, protein or polypeptide sequence. Identical), e.g. at least about 80% identical or at least about 85% identical, e.g. preferably at least about 90% identical, e.g. at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g. at least 94% Nucleic acids, proteins, polypeptides or the same, even more preferably at least about 95% identical, such as at least 96% identical, even more preferably at least about 97% identical, such as at least 98% identical, most preferably at least 99% identical It refers to peptides. Preferably, the variant is this against the nucleic acid, protein, polypeptide or peptide described when the entire sequence of the described nucleic acid, protein, polypeptide or peptide is queried for sequence alignment (i.e., overall sequence identity). This degree of identity can be shown. Also included in nucleic acid, protein, polypeptide or peptide fragments and variants are fusion products of nucleic acids, proteins, polypeptides or peptides with other normally unrelated nucleic acids, proteins, polypeptides or peptides.

  Sequence identity can be determined using a suitable algorithm known per se to perform sequence alignment and determine sequence identity. An exemplary but non-limiting algorithm is based on the Basic Local Alignment Search Tool (BLAST) first described by Altschul et al., 1990 (J Mol Biol 215: 403-10), e.g., Tatusova and Madden, 1999. The “Blast 2 sequence” algorithm described by (FEMS Microbiol Lett 174: 247-250) (eg using the published default settings or other suitable settings (eg for the BLASTN algorithm: gap open cost = 5, gap extension cost = 2, mismatch penalty = -2, match reward = 1, gap x_drop-off = 50, expected value = 10.0, word size = 28, or BLASTP algorithm: matrix = Blosum62, gap open cost = 11, gap extension cost = 1, Expected value = 10.0, Word size = 3).

  A variant of a nucleic acid, protein, polypeptide or peptide may be a homologue (eg ortholog or paralog) of said nucleic acid, protein, polypeptide or peptide. As used herein, the term “homology” is a structural similarity between two macromolecules from the same or different taxa, in particular between two nucleic acids, proteins or polypeptides, due to a common ancestor. Generally speaking the similarity.

  Where this specification refers to or encompasses a nucleic acid, protein, polypeptide or peptide fragment and / or variant, this is preferably “functional”, ie the respective nucleic acid, protein, poly Refers to variants and / or fragments that at least partially retain a peptide or biological activity or intended functionality of the peptide. For example and without limitation, a functional fragment and / or variant of an antisense agent or RNAi agent at least partially retains its functionality, i.e. its ability to reduce or eliminate expression of the target molecule (gene). To do. As another example and without limitation, a functional fragment and / or variant of a CD80 or CD86 polypeptide may be a biological activity of CD80 or CD86, respectively, such as one or more cognate receptors, more specifically CD28. And / or retain at least in part the ability to bind CTLA-4, or one or more cellular pathways, more specifically the ability to participate in the generation of costimulatory signals necessary for T cell activation. . Preferably, a functional fragment and / or variant is where a biological activity can be expressed as a determinable or measurable variable, for example by using an appropriate quantitative test to determine or measure the biological activity. At least about 20%, such as at least about 30%, or at least about 40%, or at least about 50%, such as at least, of the intended biological activity or functionality compared to the corresponding nucleic acid, protein, polypeptide or peptide Retain at least 60%, more preferably at least about 70%, such as at least 80%, even more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95% or even about 100% or more obtain.

  The term “expression” of a polypeptide by a cell refers to the production of said polypeptide by the cell. As is generally known, the expression of a polypeptide by a cell can involve several sequential molecular mechanisms, including, but not limited to, transcription of the gene encoding the polypeptide into RNA, polyadenylation And, where applicable, splicing of RNA to mRNA and / or other post-transcriptional modifications, localization of mRNA to the cytoplasm, other post-transcriptional modifications of mRNA, if applicable, translation of mRNA into a polypeptide chain, If applicable, involves post-translational modification of the polypeptide as well as folding of the polypeptide chain into the mature conformation of the polypeptide. For compartmentalized polypeptides, such as secreted and transmembrane polypeptides, the production process involves the passage of the polypeptide, i.e. compartments or organelles where the polypeptide is below the appropriate cellular level, membranes such as cell membranes or extracellularly. It is further accompanied by a cellular mechanism to be transported.

  The term “preventing or inhibiting the expression of a polypeptide” thus refers to achieving a qualitative or quantitative reduction in the production of the polypeptide by the cell. Such prevention or inhibition can affect any one or more stages of the expression process as long as the amount of mature biologically active polypeptide produced by the cell is reduced. The term encompasses any degree of such prevention or inhibition. For example, the term can be used to describe a reduction in the amount of polypeptide expressed by a cell as a determinable or measurable variable, for example by using an appropriate quantitative test to determine or measure the amount of polypeptide. At least about 10%, such as at least about 20%, at least about 30%, such as at least about 40%, at least about 50%, such as at least about 60%, at least about 70%, compared to a reference situation without said prevention or inhibition Including reduction of the amount of polypeptide expressed by, for example, at least about 80%, at least about 90%, such as at least about 95%, such as at least about 96%, 97%, 98%, 99% or even 100% Can do. Many suitable tests for measuring the amount of polypeptide expressed by cells are available, such as those based on quantitative biochemical assays, immunoassays, mass spectrometry or chromatographic methods, or combinations thereof It is.

  Preferably, the prevention or inhibition of the expression of the polypeptide, for example the prevention or inhibition of the expression of at least one of the CD80 or CD86 polypeptide, or the prevention or inhibition of the expression of the CD40 polypeptide is specific or selective, i.e. An agent that prevents or inhibits does not substantially prevent or inhibit the expression of a random, unrelated polypeptide.

  Among them, polypeptides such as receptors or ligands including CD80, CD86, CD28, CD40 and CD40L are typically presented on the surface of cells expressing them (i.e. on the cell membrane) and their biological Performs a function (binding to a cognate receptor or ligand and performing a signaling activity). Thus, the term “preventing or inhibiting the presentation of a polypeptide on the surface of a cell” achieves a qualitative or quantitative reduction in the presentation of a mature biologically active polypeptide on the surface of the cell. It shows that. Such prevention or inhibition can affect any one or more stages of the passage or migration process as long as the amount of mature biologically active polypeptide presented on the surface of the cell is reduced.

  For example, determine prevention or inhibition of the amount of polypeptide presented on the surface of a cell as a measurable variable, for example by using an appropriate quantitative test to determine or measure the amount of polypeptide on the cell surface Where possible or expressable, the term is at least about 10%, such as at least about 20%, at least about 30%, such as at least about 40%, at least about 50%, for example, compared to a reference situation without prevention or inhibition. On the surface of at least about 60%, at least about 70%, such as at least about 80%, at least about 90%, such as at least about 95%, such as at least about 96%, 97%, 98%, 99% or even 100% A reduction in the amount of polypeptide presented in Many suitable tests for measuring the amount of polypeptide expressed on the surface of cells, such as those based on quantitative biochemical assays, immunoassays, quantitative fluorescence activated cell sorting methods or combinations thereof Is available.

  Preferably, prevention or inhibition of presentation of the polypeptide on the surface of the cell, such as prevention or inhibition of presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell, or presentation of CD40 polypeptide on the surface of the cell. Prevention or inhibition is specific or selective, i.e., the agent that prevents or inhibits does not substantially prevent or inhibit the presentation of random, unrelated polypeptides on the surface of the cell.

In certain embodiments, a therapeutic agent capable of interacting with at least one of CD80 or CD86 polypeptide may be a therapeutic agent capable of interacting with CD80 polypeptide, wherein said interaction is with CD80 polypeptide. Interferes with binding to CD28 polypeptide.
In certain embodiments, the therapeutic agent that can interact with at least one of CD80 or CD86 polypeptide may be a therapeutic agent that can interact with CD80 polypeptide but not with CD86 polypeptide, wherein The interaction interferes with the binding of CD80 and CD28 polypeptides.

In certain embodiments, the therapeutic agent capable of interacting with at least one of CD80 or CD86 polypeptide may be a therapeutic agent capable of interacting with CD86 polypeptide, wherein said interaction is with CD86 polypeptide. Interferes with binding to CD28 polypeptide.
In certain embodiments, the therapeutic agent that can interact with at least one of CD80 or CD86 polypeptide may be a therapeutic agent that can interact with CD86 polypeptide but not with CD80 polypeptide, wherein The interaction interferes with the binding of CD86 polypeptide and CD28 polypeptide.

  In certain embodiments, the therapeutic agent capable of interacting with at least one of CD80 or CD86 polypeptide may be a therapeutic agent capable of interacting with CD80 polypeptide and CD86 polypeptide, wherein said interaction is Interfers with the binding of CD80 and CD86 polypeptides with CD80 and CD86 polypeptides.

In certain embodiments, the therapeutic agent can interact with at least one of CD80 or CD86 polypeptide, wherein said interaction is with at least one of CD80 or CD86 polypeptide, CD28 polypeptide and CTLA-4. Interferes with binding to the polypeptide.
In certain embodiments, the therapeutic agent can interact with at least one of the CD80 or CD86 polypeptide, wherein the interaction involves binding of at least one of the CD80 or CD86 polypeptide to the CD28 polypeptide. Interfers but does not interfere with binding to CTLA-4 polypeptide.

  In certain embodiments, a therapeutic agent that can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell is May be a therapeutic agent that can prevent or inhibit the expression of CD80 polypeptide by the cell or prevent or inhibit the presentation of CD80 polypeptide on the surface of the cell.

  In certain embodiments, a therapeutic agent that can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell is Can prevent or inhibit the expression of CD80 polypeptide by cells but cannot prevent or inhibit the expression of CD86 polypeptide or can prevent or inhibit the presentation of CD80 polypeptide on the surface of cells It may be a therapeutic agent that cannot be prevented or inhibited.

  In certain embodiments, a therapeutic agent that can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell is May be a therapeutic agent that can prevent or inhibit the expression of CD86 polypeptide by the cell or prevent or inhibit the presentation of CD86 polypeptide on the surface of the cell.

  In certain embodiments, a therapeutic agent that can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell is Can prevent or inhibit the expression of CD86 polypeptide by cells but cannot prevent or inhibit the expression of CD80 polypeptide, or can prevent or inhibit the presentation of CD86 polypeptide on the surface of cells, It may be a therapeutic agent that cannot be prevented or inhibited.

  In certain embodiments, a therapeutic agent that can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell is A therapeutic agent that can prevent or inhibit the expression of CD80 and CD86 polypeptides by the cell or that can prevent or inhibit the presentation of CD80 and CD86 polypeptides on the surface of the cell.

In a further embodiment, at least one of the CD80 or CD86 polypeptide may be contained in a cell and the CD28 polypeptide may be contained in another cell.
In particular, at least one of the CD80 or CD86 polypeptide may be contained in an antigen presenting cell (APC) and the CD28 polypeptide may be contained in a T cell, thereby interacting with at least one of the CD80 or CD86 polypeptide. The effects of the therapeutic agents disclosed herein that can interfere with the costimulatory pathway B7: CD28 and result in inhibition of the T cell response. Accordingly, such embodiments provide a therapeutic agent for use in the treatment of heart warm ischemia-reperfusion injury, wherein the therapeutic agent comprises at least one of CD80 or CD86 polypeptide contained in antigen presenting cells. The interaction can interfere with the binding of at least one of the CD80 or CD86 polypeptide contained in the antigen-presenting cell and the CD28 polypeptide contained in the T cell.

  In embodiments, at least one of the CD80 or CD86 polypeptide may be contained in an endothelial cell and the CD28 polypeptide may be contained in a T cell, thereby allowing the book to interact with at least one of the CD80 or CD86 polypeptide. The action of the therapeutic agents disclosed herein interferes with T cell adhesion to the endothelium. Accordingly, such embodiments provide a therapeutic agent for use in the treatment of cardiac warm ischemia-reperfusion injury, wherein the therapeutic agent interacts with at least one of CD80 or CD86 polypeptide contained in the endothelial cells. The interaction can interfere with the binding of CD28 polypeptide contained in T cells and at least one of CD80 or CD86 polypeptide contained in endothelial cells.

  In a further embodiment, at least one of CD80 or CD86 polypeptide may be included in an antigen presenting cell (APC), thereby preventing or inhibiting expression of at least one of CD80 or CD86 polypeptide by the cell, Alternatively, the action of a therapeutic agent disclosed herein that can prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of a cell interferes with costimulatory pathway B7: CD28 and inhibits T cell response. Bring. Accordingly, such an embodiment provides a therapeutic agent for use in the treatment of heart warm ischemia-reperfusion injury, wherein the therapeutic agent comprises expression of at least one of CD80 or CD86 polypeptide by antigen presenting cells. It can be prevented or inhibited, or it can prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of antigen presenting cells.

  In embodiments, at least one of CD80 or CD86 polypeptide may be contained in endothelial cells, thereby preventing or inhibiting expression of at least one of CD80 or CD86 polypeptide by the cell, or on the surface of the cell. The action of the therapeutic agents disclosed herein that can prevent or inhibit the presentation of at least one of the CD80 or CD86 polypeptides interferes with T cell adhesion to the endothelium. Accordingly, such embodiments provide a therapeutic agent for use in the treatment of heart warm ischemia reperfusion injury, wherein the therapeutic agent prevents expression of at least one of CD80 or CD86 polypeptide by endothelial cells. Alternatively, it can be inhibited, or the presentation of at least one of CD80 or CD86 polypeptide on the surface of endothelial cells can be prevented or inhibited.

  In certain embodiments, a therapeutic agent contemplated herein comprises a protein, polypeptide, peptide, peptidomimetic, nucleic acid, small organic molecule and compound or any combination of two or more thereof, or Or can be selected from the group consisting of.

  The term “protein” as used herein generally encompasses a macromolecule comprising one or more polypeptide chains, ie a polymer chain of amino acid residues linked by peptide bonds. The term can encompass naturally occurring, recombinantly, semi-synthetic or synthetically produced proteins. The term includes modifications that occur simultaneously with or after expression of one or more of the polypeptide chains, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N Also included are proteins having terminal Met removal, conversion of proenzymes or prehormones to active forms. The term further includes protein variants or variants that have amino acid sequence variations, such as amino acid deletions, additions and / or substitutions, relative to the corresponding native protein. The term contemplates both full-length proteins and protein portions or fragments, eg, naturally occurring protein portions that result from processing of such full-length proteins.

  The term “polypeptide” as used herein generally encompasses a polymer chain of amino acid residues linked by peptide bonds. Thus, as long as the protein is composed only of a single polypeptide chain, the terms “protein” and “polypeptide” are used interchangeably herein to refer to such a protein. The term is not limited to any minimum length of the polypeptide chain. The term can encompass naturally occurring, recombinantly, semi-synthetic or synthetically produced polypeptides. The term includes modifications that occur simultaneously with or after expression of one or more of the polypeptide chains, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N Polypeptides having terminal Met removal, conversion of proenzymes or prehormones to active forms, etc. are also included. The term further includes polypeptide variants or variants that have amino acid sequence variations, such as amino acid deletions, additions and / or substitutions, relative to the corresponding native polypeptide. The term contemplates both full-length polypeptides and portions or fragments of polypeptides, eg, naturally occurring polypeptide portions that result from the processing of such full-length polypeptides.

  The term “peptide” as used herein preferably has 50 amino acids or less, such as 45 amino acids or less, preferably 40 amino acids or less, such as 35 amino acids or less, more preferably 30 amino acids or less, such as 25 Hereinafter, it means a polypeptide used herein consisting essentially of 20 or less, 15 or less, 10 or less, or 5 or less amino acids.

  The term “peptidomimetic” refers to a non-peptide agent that is a topological analog of the corresponding peptide. Methods for rationally designing peptidomimetics of peptides are known in the art. For example, as a guide, the rational design of three peptidomimetics based on the sulfated 8-mer peptide CCK26-33 and two peptidomimetics based on the 11-mer peptide substance P and the related peptidomimetic design principles are described in Horwell 1995 (Trends Biotechnol 13: 132-134).

  The term “nucleic acid” as used herein typically refers to a polymer of any length (preferably a linear polymer) consisting essentially of nucleoside units. The nucleoside unit contains a heterocyclic base and a sugar group in common. Heterocyclic bases can include, among others, purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are naturally occurring. Widely used in nucleic acids, naturally occurring bases (e.g. xanthine, inosine, hypoxanthine) and chemically or biochemically modified (e.g. methylated), non-natural or derivatized bases Yes. Exemplary modified nucleobases include, without limitation, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (2-aminopropyladenine, 5-propynyluracil and 5-propynyl). Including cytosine). In particular, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability, for example in antisense agents, even more specifically when combined with 2'-O-methoxyethyl sugar modifications. It may be a preferred base substitution. Sugar groups are among others pentose (pentofuranose) groups, such as ribose and / or 2-deoxyribose or arabinose, preferably common to naturally occurring nucleic acids, 2-deoxyarabinose, threose, or hexose sugar groups, and modified or Substituted sugar groups (eg, without limitation, 2′-O-alkylated, such as 2′-O-methylated or 2′-O-ethylated sugar, such as ribose; 2′-O-alkyloxyalkylated, For example 2'-O-methoxyethylated sugars such as ribose; or 2'-O, 4'-C-alkylene linkages such as 2'-O, 4'-C-methylene linkages or 2'-O, 4'- C-ethylene linked sugars such as ribose; 2′-fluoro-arabinose and the like). Nucleoside units include, among other things, phosphodiester linkages common in naturally occurring nucleic acids, and linkages based on further modified phosphates or phosphonates, such as phosphorothioates, alkyl phosphorothioates, such as methyl phosphorothioates, phosphorodithioates, alkylphosphonates, For example, methyl phosphonate, alkyl phosphonothioate, phosphotriester, such as alkyl phosphotriester, phosphoramidate, phosphoropiperadidate, phosphoromorpholide, bridged phosphoramidate, bridged methylene phosphonate, bridge Phosphorothioates; and also siloxanes, carbonates, sulfamates, carboalkoxys, acetamidates, carbamates such as 3'-N-carbamates, It may be linked to each other by any one of a number of known internucleoside linkages, including ruphorino, borano, thioether, 3'-thioacetal and sulfone internucleoside linkages. Preferably, the internucleoside linkage may be a phosphate-based linkage including a modified phosphate-based linkage, such as more preferably a phosphodiester, phosphorothioate or phosphorodithioate linkage or combinations thereof. The term `` nucleic acid '' refers to any other nucleobase containing polymer, such as, but not limited to, peptide nucleic acid (PNA), peptide nucleic acid having a phosphate group (PHONA), locked nucleic acid (LNA), morpholino phosphorodiamine. See dating backbone nucleic acids (PMO), cyclohexane nucleic acids (CeNA), tricyclo-DNA (tcDNA) and nucleic acids with backbone compartments with alkyl or amino linkers (e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644) Nucleic acid mimetics including “Alkyl” as used herein refers to lower hydrocarbon moieties such as C1-C4 linear or branched, saturated or unsaturated hydrocarbons such as methyl, ethyl, ethenyl, propyl, 1-propenyl, Specifically included are 2-propenyl and isopropyl. The nucleic acids contemplated herein can include naturally occurring nucleosides, modified nucleosides, or mixtures thereof. Modified nucleosides can include modified heterocyclic bases, modified sugar moieties, modified internucleoside linkages, or combinations thereof. The term `` nucleic acid '' specifically includes hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides and synthetic (e.g. chemically synthesized) DNA, RNA and DNA / RNA hybrids, including DNA / RNA and RNA / DNA / Further preferred are RNA hybrid molecules. Nucleic acids are naturally occurring, e.g., naturally occurring or can be isolated from nature, are recombinantly produced, i.e., by recombinant DNA technology, and / or partially or completely chemically or biochemically. Can be synthesized. A “nucleic acid” can be double-stranded, partially double-stranded or single-stranded. If single stranded, the nucleic acid can be the sense strand or the antisense strand. Furthermore, the nucleic acid can be circular or linear. RNAi agents comprising nucleic acids and in particular antisense oligonucleotides or small interfering RNAs may be referred to herein as containing uracil (U) bases. It will be appreciated that U may be optionally substituted with thymine (T) in such (at least some) nucleic acids and agents. For example, 2′-O-methyl phosphorothioate antisense oligonucleotides are more “RNA-like” and may be used and indicated with U in such molecules. Using other antisense chemistries, such as peptide nucleic acids or morpholino backbones, T bases are preferably shown and used.

  The term “small organic molecule” encompasses organic compounds having a size comparable to that of organic molecules commonly used in pharmaceuticals. This term excludes biological macromolecules (eg, proteins, nucleic acids, etc.). Preferred small organic molecules are up to about 5000 Da, such as up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, such as about 900, 800, 700, 600 or about 500. The size range up to Da.

  In certain embodiments, a small organic molecule suitable as a therapeutic agent herein is a heterocyclic compound described in WO2005116033, WO2005046679, WO2003004495, WO2004048378 and WO 2004281011 that binds CD80, such as RhuDex® (Medigene ).

In embodiments, the small molecule may be a compound of formula (I) or (II), or any pharmaceutically acceptable salt, hydrate or solvate thereof:
(Where
X is a bond or the formula-(Z) _n- (Alk)-or-(Alk)-(Q) _n- ; -NH-C (O) -Alk-, -NH-C (O) -Alk- A divalent group of O—Alk— or —C (O) —NH, wherein Q is —O—, —S— or —NH—, n is 0 or 1, and Alk is An optionally substituted divalent linear or branched C ₁ -C ₁₂ alkylene or C ₂ -C ₁₂ alkenylene or C ₂ -C ₁₂ alkynylene group, or a divalent C ₃ -C ₁₂ carbon A cyclic group, preferably Alk is C ₁ -C ₆ alkylene, any of these groups being one or more —O—, —S— or —N (R ₈ ) — , R ₈ may represent H or C ₁ -C ₄ alkyl, C ₃ -C ₄ alkenyl, C ₃ -C ₄ alkynyl or C ₃ -C ₆ cycloalkyl))
Y represents —O—, —S— or —N (R ₅ ) — in formula (I), and Y represents —O—, —S—, N-oxide or —N (R) in formula (II). ₅ )-(wherein R ₅ represents H or C ₁ -C ₆ alkyl);
R ₁ is a C ₁ -C ₆ alkyl optionally substituted with H; F; Cl; Br; -NO ₂ ; -CN; F or Cl in formulas (I) and (II); or If C ₁ substituted by -C ₆ alkoxy;
R ₂ represents H or optionally substituted C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl or optionally substituted phenyl in formulas (I) and (II) ;
R ₃ is C ₁ -C ₆ alkyl optionally substituted with H; F; Cl; Br; -NO ₂ ; -CN; F or Cl in formulas (I) and (II); or If C ₁ substituted by -C ₆ alkoxy;
R ₄ in formula (II) is -C (= O) NR ₆ R ₇ , -NR ₇ C (= O) R ₆ , -NR ₇ C (= O) OR ₆ , -NHC (= O) NHR ₆ Or -NHC (= S) NHR ₆ (wherein
R ₆ represents H or a group of the formula — (Alk) _b —Q, wherein
b is 0 or 1,
Alk, when divalent may be substituted by linear or branched C ₁ -C ₁₂ alkylene, is C ₂ -C ₁₂ alkenylene or C ₂ -C ₁₂ alkynylene group (these, one or more non Adjacent —O—, —S— or —N (R ₈ ) — group wherein R ₈ is H or C ₁ -C ₄ alkyl, C ₃ -C ₄ alkenyl, C ₃ -C ₄ alkynyl or C ₃ ~C represents a ₆ cycloalkyl) and may be interrupted),
Q is H; —CF ₃ ; —OH; —SH; —NR ₈ R ₈ , wherein each R ₈ may be the same or different and is an ester group; or optionally substituted phenyl, represents a C ₃ -C ₇ cycloalkyl, a heterocyclic ring having a C ₅ -C ₇ cycloalkenyl or 5-8 ring atoms)),
R ₇ represents H or C ₁ -C ₆ alkyl, or when taken together with one or more atoms to which they are attached, R ₆ and R ₇ are optionally substituted, Forming a heterocyclic ring having ˜8 ring atoms)).

In embodiments, the small molecule may be a compound of formula (III), or a pharmaceutically acceptable salt, hydrate or solvate thereof:
(Wherein R is OH, W is F, Z is H and X is CH).

Further examples of small molecules suitable for use in the present invention include:
[[3- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenylcarbamoyl] -methoxy] -acetic acid,
N- [4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenyl] -succinamic acid,
4- [4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenylcarbamoyl] -butyric acid,
[[4-(-2-Oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenylcarbamoyl] -methoxy] -acetic acid,
4- [4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenylcarbamoyl] -2-phenyl-butyric acid,
N- [3- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenyl] -succinamic acid,
2-[[4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenylcarbamoyl] -methyl] -benzoic acid,
2-chloro-4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (6,8-dimethyl-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (8-methoxy-6-methyl-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (6,8-dimethoxy-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (7,9-dimethoxy-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (6-methyl-3-oxo-8-trifluoromethyl-3,5-dihydropyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
4- (7,9-dichloro-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
[4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenyl] -propionic acid,
[4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -phenyl] -acetic acid,
4- (4-Methyl-3-oxo-3H-chromeno [4,3-c] pyrazol-2-yl) -benzoic acid,
4- (3-oxo-3H-thiochromeno [4,3-c] pyrazol-2-yl) benzoic acid,
4- (5-methyl-3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) -benzoic acid,
2- [4- (3-oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) benzoylamino] -3-phenyl-propionic acid, and 2- [4- (3- Oxo-3,5-dihydro-pyrazolo [4,3-c] quinolin-2-yl) benzoylamino] -2-acetic acid.

In embodiments, the small molecule is a compound of formula (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt, hydrate or solvate of any of them. You can:

Wherein R ₁ is H in formula (IV); F; Cl; Br; —NO ₂ ; CN; C ₁ -C ₆ alkyl optionally substituted with F or Cl; or Represents C ₁ -C ₆ alkoxy optionally substituted by: R ₁ represents H; F in formula (V); F represents methyl, trifluoromethyl, methoxy or trifluoromethoxy; R ₁ represents formula (VI ) And (VII) represent H or C ₁ -C ₆ alkyl;
R ₂ represents H or C ₁ -C ₆ alkyl in formula (VII);
R ₃ is C ₁ -C optionally substituted with H; F; Cl; Br; —NO ₂ ; CN; F or Cl in formulas (IV), (V), (VI) and (VII). C ₆ alkyl; or C ₁ -C ₆ alkoxy optionally substituted with F;
R ₄ represents a carboxylic acid group (—COOH) or an ester thereof, or —C (═O) NR ₆ R ₇ , —NR ₇ C (═ in formulas (IV), (V), (VI) and (VII). O) R ₆ , -NR ₇ C (= O) OR ₆ , -NHC (= O) NR ₇ R ₆ or -NHC (= S) NR ₇ R ₆ (wherein
R ₆ represents H or a group of formula-(Alk) _m -Q, wherein
m is 0 or 1,
Alk is an optionally substituted divalent linear or branched C ₁ -C ₁₂ alkylene or C ₂ -C ₁₂ alkenylene or C ₂ -C ₁₂ alkynylene group, or a divalent C _3- A C ₁₂ carbocyclic group (any of these groups may be one or more —O—, —S— or —N (R ₈ ) — linkages, wherein R ₈ is H or C ₁ -C ₄ alkyl; C ₃ -C a ₄ alkenyl may contain C ₃ -C represents a ₄ alkynyl or C ₃ -C ₆ cycloalkyl)),
Q is H; —NR ₉ R ₁₀ , wherein R ₉ and R ₁₀ are independently H; C ₁ -C ₄ alkyl; C ₃ -C ₄ alkenyl; C ₃ -C ₄ alkynyl; C _3- C ₆ cycloalkyl; an ester group; represents an optionally substituted carbocyclic or heterocyclic group, or R ₉ and R ₁₀ form a ring when combined with the nitrogen to which they are attached. And the ring is optionally substituted)),
R ₇ represents H or C ₁ -C ₆ alkyl, or when taken together with one or more atoms to which they are attached, R ₆ and R ₇ are optionally substituted, Forming a monocyclic heterocyclic ring having 6 or 7 ring atoms),
X is a bond in formulas (IV), (V), (VI) and (VII) or a formula-(Z) _n- (Alk)-or-(Alk)-(Q) _n- (wherein Q Represents —O—, —S— or —NH—, Alk is as defined for R ₆ and n is 0 or 1),
Y is —CH ₂ —, —CH ₂ CH ₂ — or —CH ₂ CH ₂ CH ₂ — in formula (V),
Z is N or CR _z in formulas (IV) and (V), wherein R _z is H or optionally substituted C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl or Is phenyl optionally substituted by
Ar represents a monocyclic or bicyclic aromatic or heteroaromatic group having 5 to 10 ring atoms which may be optionally substituted in formulas (VI) and (VII).

  Small molecules can also be in any tautomeric form of any one of structures (I), (II), (III), (IV), (V), (VI) and (VII).

Unless otherwise specified in the context in which it occurs, the term “substituted” as used in any part of formulas (I) and (II) means substituted with one or more of the following substituents, ie , (C ₁ -C ₆ ) alkyl, trifluoromethyl, (C ₁ -C ₆ ) alkoxy (including the special case where the ring is substituted on the adjacent ring C atom by methylenedioxy or ethylenedioxy), tri trifluoromethoxy, (C ₁ ~C ₆₎ alkylthio, phenyl, benzyl, phenoxy, (C ₃ ~C _{8) cycloalkyl, -OH, -SH, -NH 2,} -F, -Cl, -Br, -CN, -NO _2, oxo (= O), - COOH, -SO 2 OH, -CONH 2, -SO 2 NH 2, -COR A, -COOR A, -SO 2 OR A, -NHCOR A, -NHSO 2 R ^A , -CONHR ^A , -SO ₂ NHR ^A , -NHR ^A , -NR ^A R ^B , -CONR ^A R ^B or -SO ₂ NR ^A R ^B (wherein R ^A and R ^B are independently (C ₁ -C ₆₎ alkyl group).

Unless otherwise specified in the context in which it occurs, the term “substituted” as used in any part of formulas (IV) and (V) is for example (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkenyl, (C ₂ ~C ₆₎ alkynyl, fluoro-substituted (C ₁ ~C ₆₎ alkyl, fluorine-substituted (C ₁ ~C ₆₎ alkenyl, fluoro-substituted (C ₂ ~C ₆₎ alkynyl, (C ₁ -C ₆ ) Alkoxy and fluorine substituted (C ₁ -C ₆ ) alkoxy (including the special case where the ring is substituted on the adjacent ring C atom by alkylenedioxy, such as methylenedioxy or ethylenedioxy), (C ₁ -C ₆₎ alkylthio, phenyl, benzyl, phenoxy, _{benzyloxy, -OH, -SH, -NH 2,} -F, -Cl, -Br, -CN, -NO 2, oxo (= O), - COOH, -SO ₂ OH, -CONH ₂ , -SO ₂ NH ₂ , -COR ^A , -COOR ^A , -SO ₂ OR ^A , -NHCOR ^A , -NHSO ₂ R ^A , -CONHR ^A , -SO ₂ NHR ^A , -NHR ^{A , -NR A R B, -CONR A} R B During -SO ₂ NR ^A R ^B (wherein another, R ^A and R ^B are independently, (C ₁ ~C ₆₎ alkyl or (C ₂ ~C ₆₎ alkoxy or 5- to 7-membered ring, Monocyclic carbocyclic or heterocyclic groups, or R ^A and R ^B form a ring when combined with the nitrogen to which they are attached) substituted with at least one substituent selected from Means that

Unless otherwise specified in the context in which it occurs, the term “substituted” as used in any part of structures (VI) and (VII) is for example (C ₁ -C ₆ ) alkyl, trifluoromethyl, (C ₁ ˜C ₆ ) alkoxy (including the special case where the ring is substituted on the adjacent ring C atom by alkylenedioxy, such as methylenedioxy or ethylenedioxy), trifluoromethoxy, (C ₁ -C ₆ ) alkylthio, phenyl, benzyl, phenoxy, _{benzyloxy, -OH, -SH, -NH 2,} -F, -Cl, -Br, -CN, -NO 2, oxo (= O), - COOH, -SO 2 OH, - CONH ₂ , -SO ₂ NH ₂ , -COR ^A , -COOR ^A , -SO ₂ OR ^A , -NHCOR ^A , -NHSO ₂ R ^A , -CONHR ^A , -SO ₂ NHR ^A , -NHR ^A , -NR ^A R ^B , —CONR ^A R ^B or —SO ₂ NR ^A R ^B (wherein R ^A and R ^B are independently (C ₁ -C ₆ ) alkyl or C ₂ -C ₆ alkoxy groups, or 5 ~ 7-membered monocyclic carbocyclic ring Or or a heterocyclic group, or R ^A and R ^B, which means that it is substituted with at least one substituent which is selected from a ring) when taken together with the nitrogen to bind .

When “substituted” means substituted by (C ₃ -C ₈ ) cycloalkyl, phenyl, benzyl, phenoxy or benzyloxy, the phenyl ring is itself (C ₃ -C ₈ ) cycloalkyl, It may be substituted with any of the above except phenyl, benzyl, phenoxy or benzyloxy.

As used herein, the term “alkylene” refers to a linear or branched alkyl chain having two unsatisfied valences, such as —CH ₂ —, —CH ₂ CH ₂ —, —CH _2. It refers to CH ₂ CH ₂ —, —CH (CH ₃ ) CH ₂ —, —CH (CH ₂ CH ₃ ) CH ₂ CH ₂ CH ₃ and —C (CH ₃ ) ₃ .
As used herein, the term “alkenylene” refers to a linear or branched alkenyl chain having two unsatisfied valences, such as —CH═CH—, —CH ₂ CH═CH—, — C (CH ₃ ) ═CH— and —CH (CH ₂ CH ₃ ) CH═CHCH ₂ —.
As used herein, the term “alkynylene” refers to a linear or branched alkynyl chain having two unsatisfied valences, such as —C≡C—, —CH ₂ C≡C— and — It means CH (CH ₂ CH ₃ ) C≡CCH _2- .

As used herein, the unrestricted term “carbocyclyl” or “carbocyclic” includes aryls, cycloalkyls, and cycloalkenyls, wherein the ring atoms are all carbon ring systems (monocyclic, bicyclic, Tricyclic or bridged).
As used herein, the non-limiting term “cycloalkyl” refers to a carbocyclic ring system containing only a single bond between ring carbons.

As used herein, the unrestricted term “heterocyclyl” or “heterocyclic” includes “heteroaryl”, in particular, for example, pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl , Including pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzofuranyl, pyranyl, quinuclidinyl, aza-bicyclo [3.2.1] octanyl, benzimidazolyl, methylenedioxyphenyl, maleimide and succinimide groups, and N Means a 5- to 8-membered aromatic or non-aromatic heterocyclic ring containing one or more heteroatoms selected from O.
As used herein, the term “heteroaryl” refers to a 5- or 6-membered aromatic ring containing one or more heteroatoms. Examples of such groups are thienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.

In certain embodiments, a therapeutic agent disclosed herein, particularly a therapeutic agent capable of interacting with at least one of CD80 or CD86 polypeptide, comprises an antibody or fragment or derivative thereof, a soluble receptor and an aptamer, They can consist essentially of or be selected from the group consisting of them.
As used herein, the term “antibody” is used in its broadest sense and includes, without limitation, chimeric, humanized, human, recombinant, transgenic, grafted and single chain antibodies, etc., or of interest Generally refers to any immunological binding agent, such as a whole antibody, including any fusion antibody, conjugate, fragment or derivative thereof that contains one or more domains that selectively bind to an antigen. The term “antibody” thus includes whole immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, or immunologically effective fragments of any of these. The term thus includes intact monoclonal antibodies, polyclonal antibodies, multivalent (eg, valences of 2, 3 or more) and / or multispecific antibodies (eg, bi- or antibody) formed from at least two intact antibodies. Antibodies of greater specificity), and antibody fragments as long as they exhibit the desired biological activity (especially the ability to specifically bind the antigen of interest), and multivalent and / or multiplex of such fragments Specific inclusions are specifically included. The term “antibody” is intended to encompass at least one complementarity-determining region (CDR) that can specifically bind to an epitope on an antigen of interest, rather than only antibodies produced by methods involving immunization. Also included are any polypeptides produced, such as recombinantly expressed polypeptides. Thus, the term is used for such molecules regardless of whether they were generated in vitro, cell culture or in vivo.

  The term “immunoglobulin sequence”, whether used herein to refer to a heavy chain antibody or a conventional four chain antibody, refers to a full size antibody, its individual chains and all parts, domains or fragments thereof. It is used as a general term that includes (but is not limited to) an antigen binding domain or fragment, eg, a VHH domain or a VH / VL domain, respectively. Furthermore, the term “sequence” as used herein (eg in terms such as “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”) Unless the context requires a limited interpretation, it is generally understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence that encodes it.

  The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitope determinants may include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryls, sulfonyls, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that binds to an antibody. An antibody is said to specifically bind an antigen when it preferentially recognizes the target antigen in a complex mixture of proteins and / or macromolecules.

  The term “binding region”, “binding site” or “interaction site” as used herein has the meaning of the site, part, domain or chain of specific amino acid residues responsible for binding to the antigen of interest. . Such binding regions consist essentially of the specific amino acid residues of the antibodies described herein, and these residues make contact with the target molecule.

The term “specificity” refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein (eg, antibody) molecule can bind. The specificity of the antigen binding protein can be determined based on affinity and / or binding activity. The affinity, expressed as the equilibrium constant (KD) for the dissociation of the antigen with the antigen binding protein, is a measure for the strength of the binding between the antigenic determinant and the antigen binding site on the antigen binding protein, KD The smaller the value of, the stronger the binding between the antigenic determinant and the antigen-binding molecule (alternatively, affinity can be expressed as an affinity constant (KA) that is 1 / KD). As will be apparent to those skilled in the art, affinity can be determined in a manner known per se, depending on the specific antigen of interest. Binding activity is a measure of the strength of binding between an antigen binding molecule (eg, an antibody) and a related antigen. Binding activity is related to both the affinity between the antigenic determinant and its antigen binding site on the antigen binding molecule and the number of related binding sites present on the antigen binding molecule. Typically, the antigen binding protein (eg, antibody) is 10 ⁻⁵ to 10 ⁻¹² mol / liter (M) or less, preferably 10 ⁻⁷ to 10 ⁻¹² mol / liter (M), more preferably 10 ^−. A dissociation constant (KD) of ⁸ to 10 ⁻¹² mol / liter and / or at least 10 ⁷ M ⁻¹ , preferably at least 10 ⁸ M ⁻¹ , more preferably at least 10 ⁹ M ⁻¹ , eg at least 10 ¹² M It binds with an association constant (KA) of ^-1 . Any KD value greater than 10 ⁻⁴ M is generally considered to indicate non-specific binding. Preferably, the antibody binds the desired antigen with a KD of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant is, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays and as such in the art It can be determined in any suitable manner known per se, including their various variants known.

A naturally occurring full length antibody is an immunoglobulin molecule comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The amino terminal portion of each chain contains a variable region of about 100-110 amino acids that is primarily responsible for antigen recognition by the complementarity determining region (CDR) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions.
CDRs are interspersed with more conserved regions called framework regions (FR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and 4 arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. Consists of two FRs. The three CDRs of the light chain are referred to as “LCDR1, LCDR2 and LCDR3”, and the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2 and HCDR3”. CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions follows the well-known Kabat numbering convention, which is more variable than other amino acid residues in the heavy and light chain regions of antibodies. Refers to a numbering system for certain (ie hypervariable) amino acid residues (Kabat et al., Ann. NYAcad. Sci. 190: 382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication 91-3242 (1991)). CDR positioning in the variable region of the antibody follows Kabat numbering or simply “Kabat”.

Light chains are classified as kappa or lambda and are characterized by specific constant regions known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses such as IgG1, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region having a sequence known in the art.
In some embodiments, the antibody may be any of the IgA, IgD, IgE, IgG and IgM classes, preferably an IgG class antibody.

In some embodiments, the antibody may be a polyclonal antibody, such as an antiserum or an immunoglobulin purified therefrom (eg, affinity purified).
In other embodiments, the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target specific antigens or specific epitopes within antigens with greater selectivity and reproducibility.

  As used herein, the term “monoclonal antibody” refers to an antibody derived from a single copy or clone, including for example any eukaryotic, prokaryotic or phage clone, and not the method by which it is generated. . The monoclonal antibody is preferably present in a homogeneous or substantially homogeneous population. The monoclonal antibodies and antigen-binding fragments thereof of the present invention can be generated, for example, by recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting or combinations of such techniques, or other techniques known in the art.

For example and without limitation, monoclonal antibodies can be produced by the hybridoma method first described by Kohler et al., 1975 (Nature 256: 495) or by recombinant DNA methods (such as in US 4,816,567). it can. Monoclonal antibodies should be generated using phage antibody libraries using techniques such as those described by Clackson et al., 1991 (Nature 352: 624-628) and Marks et al., 1991 (J Mol Biol 222: 581-597). You can also.
These latter technologies are based on the phage display technology described in US5837500, US5571698, US5223409, US7118879, US7208293 and US7413537, among others. Briefly, therapeutic candidate molecules, such as human antibody fragments (eg, Fabs), peptides and small proteins are displayed on the surface of small bacterial viruses called bacteriophages (or phages). A series of presented molecules is known as a library. Phage display can search these libraries to identify molecules that bind to a target of interest, eg, a therapeutic target, preferably with high specificity and / or affinity. Non-limiting examples of phage antibody libraries include HuCAL® (human combinatorial antibody library, Morphosys), Ylanthia® (Morphosys), the human Fab fragment library described in WO200070023 and WO 1996040878. Contains the described macaque antibody library. The human combinatorial antibody library (Morphosys) has been prepared using synthetic consensus sequences covering the structural repertoire of antibodies encoded in the human genome as described in WO 199708320.

Non-limiting examples of monoclonal antibodies suitable for use as therapeutic agents in the present invention include monkey monoclonal antibodies 7B6, 16C10, 7C10 and 20C9 (these are CD80 with CD28 polypeptide as described in WO 1996040878. And / or inhibits the interaction of CD86 polypeptides); anti-human CD80 monoclonal antibody 5B5D1 deposited under EC950 collection under 9502611, anti-human CD86 deposited under EC950 collection under 9506210 Monoclonal antibody 1G10H6D10, monoclonal antibody B7-24 described in WO199401547 (which specifically binds to the B7-1 molecule but does not bind to B7-2).
Further examples of anti-CD80 antibodies include IDEC114 (Biogen Idec) and galiximab (Cancer and Leukemia Group B).

In certain embodiments, the therapeutic agent may be an antibody fragment.
The term “antibody fragment” or “antigen-binding portion” includes a portion or region of a full-length antibody, generally the antigen-binding or variable domain thereof. Examples of antibody fragments are Fab, Fab ′, F (ab) 2, Fv, sFv fragment, single domain (sd) Fv, such as V _H domain, _VL domain and V _HH domain, diabody, linear antibody, Single chain antibody molecules, particularly heavy chain antibodies; and multivalent and / or multispecific antibodies formed from antibody fragments, such as dibodies, tribodies and multibodies. The above indications Fab, Fab ′, F (ab ′) 2, Fv, scFv and the like are intended to have the meaning established in the art.

  The term “antigen-binding portion” or “antigen-binding region” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. These may be bispecific, dual specific or multispecific formats that specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody are: (i) Fab fragments, ie monovalent fragments consisting of VL, VH, CL and CHI domains; (ii) F (ab ′) 2 fragments A bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CHI domains; (iv) consisting of the VL and VH domains of a single arm of the antibody Fv fragment, (v) dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCT brochure WO 90/05144) (which contains a single variable domain); and (vi) isolated complementarity Includes a decision region (CDR). In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, which can be combined into a single protein chain in which the VL and VH regions form a monovalent molecule. Can be joined using recombinant methods (known as single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. , 85: 5879-5883 (1988)) such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody, including other forms of single chain antibodies, such as diabodies. Include.

  Diabodies are bivalent, bispecific antibodies, where the VH and VL domains are expressed on a single polypeptide chain, but allow pairing between two domains on the same chain. It uses linkers that are too short, so these domains are forced to pair with the complementary domains of another chain, creating two antigen binding sites (Holliger et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994)). Such antibody binding moieties are known in the art (Edited by Kontermann and Dubel, Antibody Engineering (2001) Springer-Verlag. New York. 790 (ISBN 3-540-41354-5)).

  Further, the antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by a covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to generate tetrameric scFv molecules (Kipriyanov, SM et al., Human Antibodies and Hybridomas, 6: 93-101 (1995)), and bivalent Use of cysteine residues, marker peptides and a C-terminal polyhistidine tag (Kipriyanov et al., Mol. Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab and F (ab ′) 2 fragments, can be prepared from whole antibodies using conventional techniques, eg, papain or pepsin digestion, respectively, of whole antibodies. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.

In certain embodiments, the therapeutic agent may be Nanobody®. The terms “Nanobody®” and “Nanobodies®” are trade names of Ablynx NV (Belgium). The term “nanobody” is known in the art and, as used herein, in its broadest sense, (1) a naturally occurring heavy chain antibody, preferably a heavy chain antibody derived from a camelid. Isolating a V _HH domain, (2) expression of a nucleotide sequence encoding a naturally occurring V _HH domain, (3) “ _humanizing ” a naturally occurring V _HH domain or such a humanized V _HH Expression of nucleic acids encoding the domain, (4) "camelization" of naturally occurring V _H domains from any animal species, particularly mammalian species, eg humans, or encoding such camelid V _H domains Expression of nucleic acids, (5) "camelization" of "domain antibodies" or "dAbs" described in the art, or expression of nucleic acids encoding such camelid dAbs, (6) proteins known per se , Polypeptide Or using synthetic or semi-synthetic techniques to prepare other amino acid sequences, (7) preparing nucleic acids encoding Nanobodies using techniques known per se for nucleic acid synthesis, and then doing so And / or (8) an immunological binding agent obtained by any combination of one or more of the above. `` Camelidae '' as used herein refers to Old World Camelidae (Camelus bactrianus and Camelus dromaderius) and New World Camelidae (e.g. Lama paccos), Including Lama glama and Lama vicugna). Exemplary Nanobodies suitable for use in the present invention are the B7-1 and / or B7-2 described in Table B-1 on pages 371-372 and Table B-2 on pages 372-374 of the WO2008071447 pamphlet. In contrast, it is a nanobody produced.

  The amino acid sequence and structure of Nanobodies can be considered to be composed of, but not limited to, four framework regions or “FR”, which are referred to in the art and herein as “Framework Region 1”, respectively. "Or" FR1 "," framework area 2 "or" FR2 "," framework area 3 "or" FR3 "and" framework area 4 "or" FR4 ". These framework regions are interrupted by three complementarity determining regions or “CDRs”, which in the art are referred to as “complementarity determining region 1” or “CDR1” and “complementarity determining region 2”, respectively. Alternatively, they are referred to as “CDR2” and “complementarity determining region 3” or “CDR3”. The total number of amino acid residues in the Nanobody may range from 110 to 120, preferably 112 to 115. However, Nanobody parts, fragments, analogs or derivatives satisfy the further requirements outlined by such parts, fragments, analogs or derivatives, and are preferably suitable for the purposes described herein. Note that the length and / or size are not particularly limited.

  Preferred CDR and framework sequences for Nanobodies for B7-1 and / or B7-2 are shown in Table A-1a on pages 148 and 149 of WO2008071447. Nanobodies suitable for use in the present invention are a group consisting of the CDR1, CDR2 and CDR3 sequences listed in Table A-1a, respectively, or at least one of the CDR1, CDR2 and CDR3 sequences listed in Table A-1a, respectively. A group consisting of CDR1, CDR2 and CDR3 sequences each having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with one and / or Table A- Selected from the group consisting of CDR1, CDR2 and CDR3 sequences each having at least one, two, or only one amino acid difference from at least one of the CDR1, CDR2 and CDR3 sequences listed in 1a respectively, preferably from Table A- At least one, preferably at least two of the CDR1, CDR2 and CDR3 sequences selected from the group consisting of the CDR1, CDR2 and CDR3 sequences respectively listed in 1a; Preferably it may have all three also. Preferably, a Nanobody suitable for use in the present invention may have at least a CDR3 sequence selected from the group consisting of the CDR3 sequences listed in Table A-1a above. Preferred combinations of CDR sequences for Nanobodies to B7-1 and / or B7-2, preferred combinations of framework sequences, and preferred combinations of framework and CDR sequences are listed in Table A-1a on pages 148 and 149 of WO2008071447. Is shown in

  Nanobody amino acid residues are numbered according to the general numbering for VH domains set forth by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). The According to this numbering, Nanobody FR1 contains amino acid residues 1-30, Nanobody CDR1 contains amino acids 31-35, and Nanobody FR2 amino acids 36-49. Nanobody CDR2 contains amino acid residues 50-65, Nanobody FR3 contains amino acids 66-94, and Nanobody CDR3 contains amino acids 95-102 , Nanobody FR4 contains amino acid residues 103-113. [In this connection, as is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and corresponds to the total number of amino acid residues indicated by Kabat numbering. Note that one or more positions by Kabat numbering may not be occupied in the actual sequence, or the actual sequence may be more than the number allowed by Kabat numbering. May contain many amino acid residues). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. However, in general, according to Kabat numbering and regardless of the number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the start of FR1 and vice versa, and vice versa. The 36th position according to the number corresponds to the start of FR2, and vice versa, and the 66th position according to Kabat numbering corresponds to the start of FR3, and vice versa, the 103rd position according to Kabat numbering corresponds to the FR4 position. Corresponds to the beginning and vice versa. ]. An alternative method for numbering amino acid residues in VH domains that can be applied in a similar manner to VHH domains and Nanobodies from camelids is described by Chothia et al. (Nature 342, 877-883 (1989)). The so-called “AbM definition” and the so-called “contact definition”. However, in the description, claims and drawings, unless otherwise stated, the numbering according to Kabat applied by Riechmann and Muyldermans to the VHH domain is followed.

  Nanobodies may be “humanized”. For example, humanized Nanobodies suitable for use in the present invention can bind to B7-1 and / or B7-2, and i) of the amino acid sequences listed in Table B-1 on pages 371-372 of WO2008071447 One humanized variant, and / or ii) at least 80% amino acid identity with at least one of said amino acid sequences (amino acids forming a CDR sequence for the purpose of determining the degree of amino acid identity) Ignore residues), iii) preferably amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering One or more are Nanobodies selected from the Hallmark residues mentioned in Table A-3 on page 259 of WO2008071447. In addition or alternatively, other possibly useful humanized substitutions may compare the framework region sequence of a naturally occurring VHH sequence to the corresponding framework sequence of one or more closely related human VH sequences. After which one or more possible useful humanized substitutions (or combinations thereof) thus determined are introduced into the VHH sequence (in any manner known per se). ), The resulting humanized VHH sequences can be tested for target affinity, stability, ease and level of expression, and / or other desired properties. In this manner, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art with a limited degree of trial and error. Also, based on the above, the nanobody (the framework region thereof) can be partially or fully humanized.

  For a general description of heavy chain antibodies and their variable domains, reference is made, inter alia, to the following references (these are referred to as general background art): WO 94/04678, Vrije Universiteit Brussel, WO 95/04079 and WO 96/34103; Unilever's WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 by Algonomics NV and the applicant; National WO 01/90190 by Research Council of Canada; WO 03/025020 (= EP 1 433 793) by Institute of Antibodies; and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863 by applicant, WO 04/062551 and further published patent applications by the applicant; Hamers-Casterman et al., Nature, June 3, 1993; 363 (6428): 446-8; Davies and Riechmann, FEBS Lett. 19 February 21, 1994; 339 (3): 285-90; Muyldermans et al., Protein Eng. September 1994; 7 (9): 1129-3; Davies and Riechmann, Biotechnology (NY) May 1995; 13 (5): 475-9; Gharoudi et al., 9th Forum of Applied Biotechnology, Med. Fac. Landbouw Univ. Gent. 1995; 60 / 4a Part I: 2097-2100; Davies and Riechmann, Protein Eng. June 1996 9 (6): 531-7; Desmyter et al., Nat Struct Biol. September 1996; 3 (9): 803-11; Sheriff et al., Nat Struct Biol. September 1996; 3 (9): 733- 6; Spinelli et al., Nat Struct Biol. September 1996; 3 (9): 752-7; Arbabi Ghahroudi et al., FEBS Lett. September 15, 1997; 414 (3): 521-6; Vu et al., Mol. Immunol. 1997-November 1997; 34 (16-17): 1121-31; Atarhouch et al., Journal of Camel Practice and Research 1997; 4: 177-182; Nguyen et al., J. Mol. Biol. 23; 275 (3): 413-8; Lauwereys et al., EMBO J. July 1, 1998; 17 (13): 3512-20; Frenken et al., Res Immunol. July-August 1998; 149 (6 ): 589-99;

Transue et al., Proteins, Sep. 1, 1998; 32 (4): 515-22; Muyldermans and Lauwereys, J. Mol. Recognit. March-April 1999; 12 (2): 131-40; van der Linden et al. Biochim. Biophys. Acta April 12, 1999; 1431 (1): 37-46 .; Decanniere et al., Structure Fold. Des. April 15, 1999; 7 (4): 361-70; Ngyuen et al., Mol. Immunol. June 1999; 36 (8): 515-24; Woolven et al., Immunogenetics October 1999; 50 (1-2): 98-101; Riechmann and Muyldermans, J. Immunol. Methods 1999 12 May 10; 231 (1-2): 25-38; Spinelli et al., Biochemistry February 15, 2000; 39 (6): 1217-22; Frenken et al., J. Biotechnol. February 28, 2000; 78 (1): 11-21; Nguyen et al., EMBO J. March 1, 2000; 19 (5): 921-30; van der Linden et al., J. Immunol. Methods June 23, 2000; 240 (1 ~ 2): 185-95; Decanniere et al., J. Mol. Biol. 30 June 2000; 300 (1): 83-91; van der Linden et al., J. Biotechnol. 14 July 2000; 80 (3): 261-70; Harmsen et al., Mol. Immunol. August 2000; 37 (10): 579-90; Perez et al., B iochemistry January 9, 2001; 40 (1): 74-83; Conrath et al., J. Biol. Chem. March 9, 2001; 276 (10): 7346-50; Muyldermans et al., Trends Biochem Sci. 2001 April; 26 (4): 230-5; Muyldermans S., J. Biotechnol. June 2001; 74 (4): 277-302; Desmyter et al., J. Biol. Chem. July 13, 2001 ; 276 (28): 26285-90; Spinelli et al., J. Mol. Biol. 3 Aug 2001; 311 (1): 123-9;

Conrath et al., Antimicrob Agents Chemother. October 2001; 45 (10): 2807-12; Decanniere et al., J. Mol. Biol. October 26, 2001; 313 (3): 473-8; Nguyen et al., Adv Immunol. 2001; 79: 261-96; Muruganandam et al., FASEB J. February 2002; 16 (2): 240-2; Ewert et al., Biochemistry March 19, 2002; 41 (11): 3628-36; Dumoulin et al., Protein Sci. March 2002; 11 (3): 500-15; Cortez-Retamozo et al., Int. J. Cancer. March 20, 2002; 98 (3): 456-62; Su et al., Mol. Biol. Evol. March 2002; 19 (3): 205-15; van der Vaart JM., Methods Mol Biol. 2002; 178: 359-66; Vranken et al., Biochemistry July 9, 2002; 41 (27): 8570-9; Nguyen et al., Immunogenetics April 2002; 54 (1): 39-47; Renisio et al., Proteins June 1, 2002; 47 (4): 546-55; Desmyter et al., J Biol. Chem. June 28, 2002; 277 (26): 23645-50; Ledeboer et al., J. Dairy Sci. June 2002; 85 (6): 1376-82; De Genst et al., J. Biol. Chem. August 16, 2002; 277 (33): 29897-907; Ferrat et al., Biochem. J. September 1, 2002; 36 6 (Pt 2): 415-22; Thomassen et al., Enzyme and Microbial Technol. 2002; 30: 273-8; Harmsen et al., Appl. Microbiol. Biotechnol. December 2002; 60 (4): 449-54; Jobling Nat Biotechnol. January 2003; 21 (1): 77-80; Conrath et al., Dev. Comp. Immunol. February 2003; 27 (2): 87-103; Pleschberger et al., Bioconjug. Chem. 2003. 3-4 April; 14 (2): 440-8; Lah et al., J. Biol. Chem. 18 April 2003; 278 (16): 14101-11; Nguyen et al., Immunology. May 2003; 109 (1): 93-101; Joosten et al., Microb. Cell Fact. January 30, 2003; 2 (1): 1; Li et al., Proteins July 1, 2003; 52 (1): 47-50 Loris et al., Biol Chem. 25 July 2003; 278 (30): 28252-7;

Van Koningsbruggen et al., J. Immunol. Methods. August 2003; 279 (1-2): 149-61; Dumoulin et al. Nature. August 14, 2003; 424 (6950): 783-8; Bond et al., J. Mol. Biol. 19 September 2003; 332 (3): 643-55; Yau et al., J. Immunol. Methods. 1 October 2003; 281 (1-2): 161-75; Dekker J. Virol. November 2003; 77 (22): 12132-9; Meddeb-Mouelhi et al. Toxicon. December 2003; 42 (7): 785-91; Verheesen et al., Biochim. Biophys. Acta 2003 December 5; 1624 (1-3): 21-8; Zhang et al., J Mol Biol. January 2, 2004; 335 (1): 49-56; Stijlemans et al., J Biol Chem. 2004 1 May 9; 279 (2): 1256-61; Cortez-Retamozo et al., Cancer Res. August 15, 2004; 64 (8): 2853-7; Spinelli et al., FEBS Lett. April 23, 2004; 564 (1-2): 35-40; Pleschberger et al., Bioconjug. Chem. 2004-June; 15 (3): 664-71; Nicaise et al., Protein Sci. July 2004; 13 (7): 1882-91; Omidfar et al., Tumor Biol. July-August 2004; 25 (4): 179-87; Omidfar et al., Tumor Biol. September-December 2004; 25 (5 ~ 6): 296-305; Szynol et al., Antimicrob Agents Chemother. 2004 September; 48 (9): 3390-5; Saerens et al., J. Biol. Chem. 10 December 2004; 279 (50): 51965-72; De Genst et al., J. Biol. Chem. December 17, 2004; 279 (51): 53593-601; DoIk et al., Appl. Environ. Microbiol. January 2005; 71 (1): 442 -50; Joosten et al., Appl Microbiol Biotechnol. January 2005; 66 (4): 384-92; Dumoulin et al., J. Mol. Biol. February 25, 2005; J Immunol Methods. February 2005; 297 (1-2): 213-24; De Genst et al., J. Biol. Chem. April 8, 2005; 280 (14): 14114-21; Huang et al. Eur. J. Hum. Genet. April 13, 2005; DoIk et al., Proteins. May 15, 2005; 59 (3): 555-64; Bond et al., J. Mol. Biol. May 2005. 6; 348 (3): 699-709; Zarebski et al., J. Mol. Biol. April 21, 2005.

  In accordance with the terms used in the above references, the variable domain present in naturally occurring heavy chain antibodies is the heavy chain variable domain present in conventional four chain antibodies (this is referred to herein as the “VH domain”). And also referred to as “VHH domains” to distinguish them from light chain variable domains present in conventional four chain antibodies (referred to herein as “VL domains”). As mentioned in the prior art above, VHH domains have several unique structural and functional properties that allow them to be isolated VHH domains (and naturally occurring VHH domains and their structures). Nanobodies based on it that share common functional and functional properties) and proteins containing them would be very advantageous for use as functional antigen binding domains or proteins. In particular, and without limitation, VHH domains (originally designed to functionally bind antigens in the absence of light chain variable domains and without any interaction with light chain variable domains) And Nanobodies can function as a single relatively small functional antigen-binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies (which are themselves generally suitable for practical applications as single antigen binding proteins or domains). (But not in the form of a functional antigen-binding unit (e.g., a conventional antibody fragment, e.g., a Fab fragment; such as in a ScFv fragment consisting of a VH domain covalently linked to a VL domain) in some form or in combination with another) .

  In certain embodiments, the therapeutic agent may be a domain antibody (dAb). For the term “dAb”, see, for example, Ward et al. (Nature Oct. 12, 1989; 341 (6242): 544-6), Holt et al. Trends Biotechnol., 2003, 21 (11): 484-490; and Domantis Ltd. Reference is made to WO 06/030220, WO 06/003388 and other published patent applications. Single domain antibodies or single variable domains can be derived from certain sharks (e.g. the so-called "", although not necessarily in the context of the present invention since they are not of mammalian origin). It should also be noted that “IgNAR domains”, see eg WO 05/18629). Exemplary dAbs suitable for use in the present invention are dAbs described in US 13 / 021,127, more particularly domain antibodies that bind to the same epitope as domain antibody 1 h-239-891 (D70C), including D70C Specifically, a dAb comprising a first single variable domain having binding specificity for the first antigen and a second single variable domain having binding activity for the second antigen (where the first antigen is CD28 And the second antigen is an antigen other than CD28, and dAb is described in claim 7 of US 13 / 021,127).

  In further embodiments, the antibody or antibody fragment comprises at least 2 (eg, 2, 3, etc.) binding sites (each directed to a different antigen or antigenic determinant) (eg, bispecific, Antibodies such as trispecificity). Exemplary multispecific antibody fragments suitable for use as therapeutic agents in the present invention are trispecific diabodies capable of binding to both CD40 and CD80 and CD86 as described in WO 1998003670, binding to human CD40 and human CD86 Bispecific diabodies that can be made, and trispecific triabodies that can bind CD40, CD80 and CD86.

  In some embodiments, the therapeutic agent may be a dual variable domain immunoglobulin (DVD-Ig ™).

  The term antibody includes antibodies comprising one or more moieties originating from or derived from any animal species, preferably vertebrate species including, for example, birds and mammals. Without limitation, the antibody may be a chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Again, without limitation, antibodies may include humans, mice (e.g. mice, rats, etc.), pigs, donkeys, rabbits, goats, sheep, guinea pigs, monkeys (e.g. cynomolgus monkeys), camels (e.g. Bactrianus and Cameras Doromadelius), llamas that also contain llama heavy chain antibodies (eg llama pacos, llama gramma or llama vicuna), or horses.

  The term antibody as used herein also encompasses “chimeric antibodies” originating from at least two animal species. More specifically, the term “chimeric antibody” or “chimeric antibody” includes heavy and light chain variable region sequences from one species and constant region sequences from another species. Antibody, eg, an antibody having murine heavy and light chain variable regions linked to human, non-human primate, dog, horse or cat constant regions. A chimeric antibody comprises a portion of a heavy and / or light chain that is derived from a particular species or is identical or homologous to the corresponding sequence from an antibody belonging to a particular antibody class or subclass, with the remainder of the chain Identical or homologous to corresponding sequences in antibodies from other species or belonging to another antibody class or subclass and fragments of such antibodies exhibiting the desired biological activity (e.g., U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies are made by combining a portion of a monoclonal antibody from one species, such as a mouse or monkey, such as DNA encoding an Fv region, with antibody-producing DNA from another species, such as a human.

  Non-limiting examples of chimeric antibodies suitable for use as therapeutic agents in the present invention include monkey monoclonal antibodies specific for human CD80 and / or CD86, particularly 16C10, 7C10, 20C9 and 7B6, described in WO 1996040878. Primatized antibodies derived from, which are primatized antibodies derived from the 7C10 monkey monoclonal antibody (this primatized antibody has the amino acid sequence shown in FIGS. 8a and 8b of WO 1996040878); 7B6 monkey monoclonal A primatized antibody derived from an antibody (this primatized antibody has the amino acid sequence shown in FIGS. 9a and 9b of WO 1996040878); and a primatized antibody derived from the 16C10 monkey monoclonal antibody (this primatized antibody is , Having the amino acid sequence shown in FIGS. 4a and 4b of WO 1996040878). These primatized antibodies comprise the variable heavy and light chain domains of monkey antibodies and human constant domains.

  In certain embodiments, the therapeutic agent may be a “fully human antibody”. As used herein, the term “fully human antibody” refers to an antibody whose encoded genetic information is of human origin. Thus, the term “fully human antibody” refers to an antibody having variable and constant regions derived solely from human germline immunoglobulin sequences. The term “fully human antibody” thus does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, are grafted onto human framework sequences. Fully human antibodies are derived from phage human antibody libraries as described above or they are as described in Lonberg and Husznar, 1995 (Int. Rev. Immunol. 13 (1): 65-93). Can be obtained by immunization of transgenic mice engineered to replace the mouse immunoglobulin coding region. A fully human antibody produced using phage display is preferably generated by recombinant expression in a human cell line that yields an antibody having a human sugar chain pattern. A non-limiting example of a fully human antibody is the HuCAL® antibody (Morphosys). Gene information for constructing HuCAL® antibodies is extracted from the HuCAL® antibody library (Morphosys) and introduced into human PER.C6® cells in the form of vectors (i.e., transfection). ). Transfected cells translate genetic information into proteins. The protein is further modified by glycosylation and the resulting antibody molecules are finally secreted into the culture medium by the cells.

  The term antibody, as used herein, is an antibody that is derived from a non-human species and whose protein sequence has been modified to increase similarity to naturally occurring antibodies in humans. Antibody ". More specifically, the term “humanized antibody” includes heavy and light chain variable region sequences from non-human species (eg, mice), but at least a portion of the VH and / or VL sequences is more “human-like”. ", Ie, antibodies that have been altered to be more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.

  A humanized antibody immunospecifically binds to an antigen of interest, a framework (FR) region having substantially the amino acid sequence of a human antibody, and a complementarity determining region having substantially the amino acid sequence of a non-human antibody. (CDR) or a variant, derivative, analog or fragment thereof. A humanized antibody has all or substantially all CDR regions equivalent to those of a non-human immunoglobulin (i.e., a donor antibody), and all or substantially all framework regions of a human immunoglobulin consensus sequence. Substantially all or at least one and typically two variable domains (Fab, Fab ′, F (ab ′) 2, FabC, Fv). Humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanized antibody may contain both the light chain and at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. Alternatively, a humanized antibody may contain only a humanized light chain or only a humanized heavy chain. Exemplary humanized antibodies contain a light chain humanized variable domain and a heavy chain humanized variable domain.

Also, for example, humanized antibodies can be conventional antibodies from camelids, particularly llamas (eg, Lama Paccos, Lama Grama or Lama Vicuna) (ie, two heavy chains (H) interconnected by disulfide bonds) and An immunoglobulin molecule comprising two light chains (L), the variable domains of which exhibit a high degree of amino acid sequence identity with the variable domains of human antibodies. A suitable platform for the production of such humanized antibodies is the SIMPLE Antibody ™ platform (ArGEN-X) described in WO 2011080350. Thus, a humanized antibody may comprise a camelid VH domain and a camelid VL domain of either a lambda light chain class (Vλ) or a kappa light chain class (Vκ), wherein at least one amino acid substitution is defined as described above. Characterized in that it is present in the VH domain and / or the VL domain, the amino acid substitution is selected from:
(a) Amino acid substitution in the camelid VH domain, according to Kabat, H1, H5, H7, H11, H12, H13, H14, H29, H30, H37, H40, H48, H67, H68 of the camelid VH domain , H69, H71, H74, H77, H78, H80, H81, H82b, H83, H84, H85, H86, H89, H93, H94, and an amino acid at one or more positions selected from the group consisting of H108 and H108 is a human VH domain Replaced with an amino acid found at the corresponding position in the sequence, and
(b) Amino acid substitutions in the camelid VL domain, for the camelid VL domain of lambda light chain class (Vλ), according to Kabat, L1, L2, L3, L7, L8, L9, L11 of the camelid Vλ domain , L14, L15, L17, L18, L19, L20, L38, L39, L40, L42, L44, L46, L47, L49, L58, L59, L60, L66, L67, L69, L70, L71, L72, L74, L76 , L78, L80, L84 and L103, wherein one or more amino acids selected from the group consisting of the amino acids found at the corresponding positions in the human Vλ domain sequence are replaced, and the camelaceae of the kappa light chain class (Vκ) For VL domains, according to Kabat, C1, K3, K4, K7, K9, K11, K12, K13, K14, K15, K18, K19, K36, K42, K43, K45, K46, K58, K63, An amino acid at one or more positions selected from the group consisting of K70, K77, K78, K79, K80, K83, K100, K103, K104 and K106 is in the human Vκ domain sequence. It is replaced with an amino acid that is found in the response to the position. In particular, for humanization towards human VH3, VH1 or VH4, one or more of the respective amino acid substitutions described in Table 4 on page 35 of WO 2011080350 can be performed, and human Vκ1, Vκ2, Vκ4, Vλ1, For humanization towards Vλ2, Vλ3, Vλ5 or Vλ6, the respective amino acids described in Table 5 on pages 38-41 of the WO 2011080350 pamphlet can be performed. One or more or any combination of CH1 domain, hinge region, CH2 domain, CH3 domain, optionally CH4 domain and CL domain in such an antibody may be fully or substantially human with respect to its amino acid sequence.

  In certain embodiments, the therapeutic agent may be an intrabody. The term “intrabody” generally refers to an intracellular antibody or antibody fragment. Antibodies, particularly single chain variable antibody fragments (scFv), can be modified for subcellular localization. Such modifications, for example, necessarily involve the fusion with a stable intracellular protein, such as a maltose binding protein, or the addition of an intracellular traffic / localized peptide sequence, such as the endoplasmic reticulum retention or recovery signal sequence KDEL. There is.

  One skilled in the art understands that an antibody can include one or more amino acid deletions, additions and / or substitutions (eg, conservative substitutions) (as long as such changes preserve binding of the respective antigen). . For example, as described in US Pat. No. 8,323,962, mutations can be introduced into antibodies, particularly in the Fc region, to increase in vivo half-life without compromising immunogenicity.

An antibody may comprise one or more natural or artificial modifications (eg, glycosylation, etc.) of constituent amino acid residues.
Methods for producing polyclonal and monoclonal antibodies and fragments thereof are known in the art, as are methods for producing recombinant antibodies or fragments thereof (see, eg, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring. Harbor Laboratory, New York, 1988; Harlow and Lane, `` Using Antibodies: A Laboratory Manual '', Cold Spring Harbor Laboratory, New York, 1999, ISBN 0879695447; `` Monoclonal Antibodies: A Manual of Techniques '', edited by Zola, CRC Press 1987 ISBN 0849364760; “Monoclonal Antibodies: A Practical Approach”, edited by Dean and Shepherd, Oxford University Press 2000, ISBN 0199637229; Methods in Molecular Biology, Volume 248: “Antibody Engineering: Methods and Protocols”, edited by Lo, Humana Press 2004 , ISBN 1588290921).

  An immunizing antigen (e.g., a CD80 or CD86 polypeptide disclosed herein) optionally fused or covalently or non-covalently linked, adsorbed or adsorbed to a presentation carrier is used to produce an animal, e.g., a non-human animal, e.g., an experiment. Methods for immunizing animals or livestock, as well as the preparation of antibodies or cellular reagents from immune sera are known per se and are described in the literature referred to elsewhere herein. The animal to be immunized may include any animal species, preferably warm-blooded species, more preferably vertebrate species including, for example, birds and mammals. Without limitation, the antibody may be a chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Again, without limitation, the antibody may be human, murine (eg, mouse, rat, etc.), pig, donkey, rabbit, goat, sheep, guinea pig, camel, llama or horse. The term “presentation carrier” or “carrier” generally refers to an immunogenic molecule that, when bound to a second molecule, enhances the immune response to the second molecule, usually by providing additional T cell epitopes. To say. The presentation carrier may be a (poly) peptide structure or a non-peptide structure, in particular glycans, polyethylene glycols, peptidomimetics, synthetic polymers and the like. Exemplary non-limiting carriers include human hepatitis B virus core protein, multiple C3d domains, tetanus toxin fragment C or yeast Ty particles. Following immunization, antibody-producing cells from the animal may be isolated and used to generate monoclonal antibody-producing hybridoma cells using techniques known in the art.

  Methods for producing recombinant antibodies or fragments thereof require a host organism or cell. The terms “host cell” and “host organism” may suitably refer to cells or organisms that include both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoa, plants and animals. As host organisms or cells for the production of antibodies, inter alia unicellular organisms such as bacteria (eg E. coli) and (cultured) animal cells (eg mammalian or human cells) are contemplated. The advantages of generating antibodies in bacteria are, among other things, that the handling of bacterial cells is relatively safe and simple, and the microbial replication cycle is rapid. Bacteria are particularly suitable for the production of antibody fragments with a simple structure. For the production of full-length immunoglobulins or more complex antibody fragments in prokaryotic cells, bacterial cells are treated with at least two nucleic acids (each of which is an antibody fragment or a different part of an immunoglobulin as described in WO 2009021548 for full-length immunoglobulins). For example, encoding a heavy or light chain). In bacterial cells, the genetic information encoding the antibody is read and translated into protein. The resulting antibody accumulates in the periplasmic space and can be collected by lysis of bacterial cells. An additional separation step may be performed to purify the antibody. WO 2009021548 describes an E. coli-based secretion system in which bacteria release antibodies to the surrounding culture medium by introducing a signal sequence into the antibody-encoding construct. This allows the antibody to be purified easily and conveniently from the cell culture medium. An exemplary mammalian cell line that can be used for the production of antibodies is the Chinese hamster ovary (CHO) cell line. Exemplary human cell lines suitable for the production of antibodies include the PER.C6® cell line or derivatives thereof deposited with ECAC under 96022940 and described in WO 2000063403. Human cell lines are particularly suitable for the production of fully human antibodies because they produce antibodies with human glycosylation patterns.

  Unless otherwise stated, all methods, steps, techniques and operations not specifically described in detail can be performed and performed in a manner known per se, as will be apparent to those skilled in the art. . For example, here again, reference is made to standard references as well as the general background art referred to herein and the further references cited therein.

  In certain embodiments, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a primatized antibody, a human antibody, a Nanobody, or a mixture thereof.

The term “soluble receptor” generally refers to a cell surface molecule, eg, a soluble form of a cell surface receptor (ie, circulating, not bound to a cell), or a fragment or derivative thereof. For example, a cell surface molecule can be solubilized by binding a soluble fusion partner, such as an immunoglobulin (Ig) moiety or portion thereof, to the extracellular domain or by removing its transmembrane domain.
Of particular interest in the present invention are “soluble CTLA-4 molecules”, which are cell surface non-binding (ie circulating) CTLA-4 molecules or CTLA-4 binding to CD80 and / or CD86. Any functional fragment of a molecule, referred to as a CTLA4Ig fusion protein (e.g., encoded by DNA deposited with ATCC accession number 68629) (where the extracellular domain of CTLA-4 is the immunoglobulin (Ig ) Moieties, e.g. IgC gamma 1, IgC gamma 2, IgC gamma 3, IgC gamma 4, IgC mu, IgC alpha 1, IgC alpha 2, IgC delta or IgC epsilon or fragments and derivatives thereof); organisms A portion of a chemically active or chemically active protein such as papillomavirus E7 gene product (CTLA4-E7), melanoma-associated antigen p97 (CTLA4-p97) or HIV env protein (CTLA4-env gpl20) (US Pat. Recorded in No. 5,844,095 Proteins having the extracellular domain of CTLA-4 fused or linked to them, or fragments and derivatives thereof; hybrid (chimeric) fusion proteins such as CD28 / CTLA4Ig (as described in US Pat. Or) fragments and derivatives thereof; including but not limited to CTLA-4 molecules from which the transmembrane domain has been removed (Oaks, MK et al., 2000. Cellular Immunology 201: 144-153) or fragments and derivatives thereof. Examples of CTLA4Ig molecules are described in WO2005 / 016266, among others. “Soluble CTLA-4 molecule” also includes fragments, portions or derivatives thereof and soluble CTLA-4 mutant molecules having CTLA-4 binding activity (eg, L104EA29YIg). `` L104EA29YIg '' is a wild-type with amino acid changes of A29Y (a tyrosine amino acid residue replaces alanine at position 29) and L104E (a glutamic acid amino acid residue replaces leucine at position +104) linked to an Ig tail. A soluble CTLA4 mutant molecule containing the extracellular domain of type CTLA4 or a fusion protein that is part of it that binds to the B7 molecule (DNA encoding L104EA29YIg was deposited on June 20, 2000 under ATCC number PTA-2104 ).

Specific examples of soluble CTLA-4 molecules include molecules composed of the Fc region of immunoglobulin IgG1 fused to the extracellular domain of CTLA-4, such as Abacept (sold as Orencia®, Bristol Myers Squibb), sold as Belatacept (Nulojix®, Bristol Myers Squibb) and XPro9523 (Xencor).
For example, the CTLA4-KDEL fusion protein described in Tan et al. (2005. Blood 106: 2936-2943) contains the extracellular domain of CTLA-4 fused to the endoplasmic reticulum retention or recovery signal sequence KDEL. CTLA4-KDEL fusion proteins bind to newly synthesized CD80 / 86 molecules in the ER and prevent them from reaching the cell surface.

  In certain embodiments, the soluble receptor may be a soluble CTLA4 molecule, preferably a CTLA4Ig fusion protein.

The term “aptamer” refers to single- or double-stranded oligo DNA, oligo RNA or oligo DNA / RNA, or any analog thereof that can specifically bind to a target molecule. Advantageously, aptamers can exhibit fairly high specificity and affinity (eg, a K _{A on the} order of 1 × 10 ⁹ M ⁻¹ ) for their target. Aptamer generation is among others US 5,270,163; Ellington and Szostak 1990 (Nature 346: 818-822); Tuerk and Gold 1990 (Science 249: 505-510); or `` The Aptamer Handbook: Functional Oligonucleotides and Their Applications '', Klussmann. Ed., Wiley-VCH 2006, ISBN 3527310592 (incorporated herein by reference). The term also includes photoaptamers, ie, aptamers that contain one or more photoreactive functional groups that can be covalently linked or crosslinked with a target molecule.

In certain embodiments, the present specification can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by a cell or prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of a cell. Can be selected from the group comprising, consisting essentially of, or consisting of intrabodies or fragments or derivatives thereof, antisense agents, and RNA interference (RNAi) agents. Antisense agents capable of binding (annealing) with a sequence region in the CD80 or CD86 pre-mRNA or mRNA sequence are particularly contemplated. Such RNAi agents configured to target CD80 or CD86 mRNA can be specifically contemplated.
In some embodiments, the therapeutic agent is an intrabody directed to a CD80 and / or CD86 polypeptide that binds these molecules intracellularly and prevents these molecules from reaching the cell surface. It can be an intrabody.

  The term `` antisense '' generally refers to an agent (e.g., an oligonucleotide) that is configured to specifically anneal (hybridize) with a predetermined sequence in a target nucleic acid, e.g., target DNA, hnRNA, pre-mRNA or mRNA. In particular, it typically comprises, consists essentially of, or consists of a nucleic acid sequence that is complementary or substantially complementary to the target nucleic acid sequence. Antisense agents suitable for use in the present invention can anneal (hybridize) with the respective target nucleic acid sequences under high stringency conditions and can specifically hybridize with the target under physiological conditions.

  The terms “complementary” or “complementarity” as used herein refer to a single strand by base pairing, preferably Watson-Crick base pairing, under permissive salt (ionic strength) and temperature conditions. This refers to normal binding between nucleic acids. For example, complementary Watson-Crick base pairing occurs between A and T, A and U, or G and C. For example, the sequence 5′-AGU-3 ′ is complementary to the sequence 5′-ACUU-3 ′.

  The terms “bind” or “binding” as used herein preferably refers to specific binding, ie, one agent is one or more targets of interest, eg one or more pre-mRNAs. When binding (annealing) a molecule or fragment or variant thereof substantially with other random or unrelated molecules and optionally with other structurally related molecules. Say. The binding of an agent to a target can be confirmed, for example, using conventional methods for investigating interactions, such as in silico sequence analysis or nucleic acid hybridization experiments, for example, specific hybridization, for example, under high stringency conditions. And can be evaluated.

  Specific binding does not necessarily require that an agent bind exclusively to its intended target. For example, an agent has an affinity under binding conditions for an intended target that is at least about 2 times greater, preferably at least about 5 times greater, and more preferably at least about 10 times greater than an affinity for a non-target molecule. Even more preferably at least about 25-fold greater, even more preferably at least about 50-fold greater, even more preferably at least about 100-fold or at least about 1000-fold greater than a given pre-mRNA of interest or fragment thereof or It can be said that it specifically binds to a variant.

  The sequence of the antisense agent need not be 100% complementary to the target sequence in order to specifically bind or hybridize with the target sequence. Binding of the antisense agent to the target nucleic acid molecule interferes with the normal function of the target nucleic acid sequence to achieve the intended result (e.g. loss of availability) and under conditions where specific binding is desired, i.e. in vivo A degree sufficient to avoid non-specific binding of the antisense agent to the non-target sequence under physiological conditions in the case of an assay or therapeutic treatment, and in the case of in vitro assays under the conditions under which the assay is performed. When there is complementarity, it can be said that an antisense agent can specifically hybridize. Thus, “specifically hybridizable” and “complementary” indicate a sufficient degree of complementarity or precise pairing such that stable specific binding occurs between the antisense agent and the nucleic acid target. Sometimes.

Preferably, the antisense agent sequence is at least about 80% complementary to the respective target sequence, preferably at least about It may be 90% complementary, more preferably at least about 95% complementary, for example up to about 96%, about 97%, about 98%, about 99% and 100% complementary.
Antisense agents contemplated herein preferably comprise or represent an antisense molecule, such as more preferably an antisense nucleic acid molecule or an antisense nucleic acid analog molecule. Preferably, an antisense agent may refer to an antisense oligonucleotide or an antisense oligonucleotide analog.

  The term “oligonucleotide” as used herein refers to a nucleic acid (including nucleic acid analogs and mimetics) oligomer or polymer as defined herein. Preferably, the oligonucleotide, eg specifically the antisense oligonucleotide, is (substantially) single stranded. Oligonucleotides contemplated herein preferably have a length of between about 10 and about 100 nucleoside units (i.e., nucleotides or nucleotide analogs), preferably between about 15 and about 50, more preferably about It may be between 20 and about 40, also preferably between about 20 and about 30. Preferably, the oligonucleotides contemplated herein are one or more or all non-naturally occurring heterocyclic bases and / or one or more or all non-naturally occurring sugar groups and / or one or more or all naturally occurring. May include unlinked internucleoside linkages, which may improve properties such as enhanced cellular uptake, increased stability in the presence of nucleases and increased hybridization affinity, increased tolerance to mismatches, etc. Can do. Furthermore, the oligonucleotides contemplated herein can be configured to not activate RNase H according to known techniques (see, eg, US Pat. No. 5,149,797).

  Antisense agents, such as the oligonucleotides taught herein, can be further conjugated with one or more moieties or conjugates that enhance oligonucleotide activity, cell distribution or cellular uptake (eg, covalent or non-covalent, Directly or via a suitable linker). Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids, For example di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, or adamantaneacetic acid, palmityl moiety, or octadecylamine or hexylamino -Contains a carbonyl-oxycholesterol moiety.

  It is not necessary for all positions of an agent to be uniformly modified, and in fact more than one of the above modifications can be incorporated into even a single nucleoside within a single agent or oligonucleotide. Further included are antisense compounds that are chimeric compounds. A “chimeric” antisense compound or “chimera” is an antisense molecule that contains two or more chemically distinct regions, each made of at least one monomer unit, ie, a nucleotide in the case of an oligonucleotide compound, in particular It is an oligonucleotide. These oligonucleotides typically contain at least one region, where the oligonucleotides are used to increase resistance to nuclease degradation, increase cellular uptake, and increase binding affinity for the target nucleic acid. Modified to grant additional territory.

RNAi (RNA interference) agents typically comprise or essentially consist of a double-stranded portion or region of the annealed complementary strand (despite the presence of any and potentially preferred single-stranded overhangs). One of the annealed complementary strands (sense strand) consists of or consists of a sequence corresponding to the target nucleotide sequence of the target gene that is down-regulated (ie, at least a portion of the mRNA). The other strand (antisense strand) of the RNAi agent is complementary to the target nucleotide sequence.
The number of mismatches between the target sequence and the nucleotide sequence of the RNAi agent, although the sequence of the RNAi agent, such as more specifically the sequence of its sense strand, need not be completely identical to the down-regulated target sequence Is preferably 1 or less per 5 bases, or 1 or less per 10 bases, or 1 or less per 20 bases, or 1 or less per 50 bases.

  Preferably, in order to ensure the specificity of the RNAi agent for the desired target over unrelated molecules, the sequence of said RNAi agent, such as more specifically the sequence of its sense strand, is at least It may be about 80% identical, preferably at least about 90% identical, more preferably at least about 95% identical, eg up to about 96%, about 97%, about 98%, about 99% and 100% identical.

  The RNAi agent may be formed with separate sense and antisense strands, or alternatively a common strand that results in a folded stem-loop or hairpin design in which the two annealed strands of the RNAi agent are covalently linked May be formed.

siRNA molecules may typically be generated, eg, synthesized, as separate, substantially complementary strand double-stranded molecules, wherein each strand is about 18 to about 35 bases in length, preferably about 19 to about 30 bases, more preferably about 20 to about 25 bases, even more preferably about 21 to about 23 bases.
shRNA is in the form of a hairpin structure. shRNA can be synthesized exogenously or can be formed by in vivo transcription from an RNA polymerase III promoter. Preferably, the shRNA can be engineered in the host cell or organism to ensure continuous and stable repression of the desired gene. It is known that siRNA can be generated by processing hairpin RNA in cells.
The RNAi agents contemplated herein can include any of the modifications described herein for nucleic acids and oligonucleotides to improve their therapeutic properties.

  In embodiments, at least one strand of the RNAi molecule may have a 3 ′ overhang of about 1 to about 6 bases in length, such as 2-4 bases, more preferably 1-3 bases. For example, one strand may have a 3 ′ overhang and the other strand may be blunt ended or may have a 3 ′ overhang. The length of the overhang may be the same or different for each chain. The 3 'overhang can be stabilized against degradation. For example, RNA can be stabilized by including purine nucleotides, such as A or G nucleotides. Instead, substitution of pyrimidine nucleotides by modified analogs, eg, substitution of the 3 ′ overhang of U with 2′-deoxythymidine is tolerated and does not affect the efficiency of RNAi.

Exemplary but non-limiting siRNA molecules can be characterized by any one or more of the following criteria, preferably all:
-At least about 80% sequence identity, such as more specifically sequence identity to the target mRNA of the sense strand of the siRNA molecule, more preferably at least about 90% or at least about 95% or at least about 97 to the target mRNA. % Sequence identity;
-Having a sequence that targets a region of the target gene present in the mature mRNA (e.g. exons or alternatively spliced introns);
-Shows preference to target the 3 'end of the target gene.

Exemplary siRNAs can be further characterized by one or more of the following criteria:
-Has a length of double-stranded nucleic acid of 16-30 bases, preferably 18-23 bases, preferably 19 nucleotides;
-Having a GC content between about 30 and about 50%;
-Has a TT (T) sequence at the 3 'end;
-Show no secondary structure when taking the form of double strands;
-Has a Tm (melting temperature) of less than 20 ° C;
The nucleotide sequence has the nucleotide shown below (where “h” is A, C, T / U but not G, “d” is A, G, T / U but C But “w” is A or T / U but not G or C):

  RNAi agents contemplated herein include RNAi nucleic acid molecules or RNAi nucleic acid analog molecules, such as preferably small interfering nucleic acids and small interfering nucleic acid analogs (siNA), such as small interfering RNA and small interfering RNA analogs (siRNA). May specifically contain or indicate (ie can be selected from the group comprising or consisting of), among others, double-stranded RNA and double-stranded RNA analogs (dsRNA), microRNA and microRNA analogs ( miRNA), as well as small hairpin RNA and small hairpin RNA analogs (shRNA).

  Generation of antisense and RNAi agents can be accomplished by any process known in the art, for example, among others, by partial or complete chemical synthesis (e.g., conventionally known solid phase synthesis, on a modified solid support). An exemplary, non-limiting method for synthesizing oligonucleotides is described in US 4,458,066; in another example, Beaucage et al., 1981 (Tetrahedron Letters 22: 1859) using diethyl phosphoramidite as the starting material. Nucleic acid constructs (templates) using appropriate polymerases such as T7 or SP6 RNA polymerase, for example, by partial or fully biochemical (enzymatic) synthesis. ) Or by recombinant nucleic acid techniques, such as expression from a vector in a host cell or host organism. Nucleotide analogs can be introduced by in vitro chemical or biochemical synthesis. In certain embodiments, antisense agents of the invention are synthesized in vitro and do not include biologically derived antisense compositions or gene vector constructs designed to drive in vitro synthesis of antisense molecules.

In certain embodiments, can interact with at least one of CD80 or CD86 polypeptide, or can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell, or CD80 or CD86 polypeptide on the surface of the cell. A therapeutic agent contemplated herein that can prevent or inhibit presentation of at least one of the peptides can interact with at least the CD80 polypeptide or can prevent or inhibit at least expression of the CD80 polypeptide by the cell or on the surface of the cell. A therapeutic agent capable of preventing or inhibiting the presentation of at least a CD80 polypeptide, and at least a CD86 polypeptide capable of interacting with at least the CD86 polypeptide or preventing or inhibiting the expression of at least the CD86 polypeptide by the cell Prevent the presentation of Or it may be a composition comprising a therapeutic agent capable of inhibiting.
Preferably, the composition comprises a therapeutic agent capable of interacting with at least a CD80 polypeptide, a therapeutic agent capable of interacting with at least a CD86 polypeptide, or a therapeutic agent capable of preventing or inhibiting expression of at least a CD80 polypeptide by a cell, A therapeutic agent that can prevent or inhibit the expression of at least a CD86 polypeptide by or a therapeutic agent that can prevent or inhibit the presentation of at least a CD80 polypeptide on the surface of a cell and prevent the presentation of at least a CD86 polypeptide on the surface of a cell Or a therapeutic agent that can be inhibited.

  As used herein, at least CD80 or CD86 for use in the treatment of cardiac ischemia-reperfusion injury comprising a therapeutic agent capable of interacting with at least a CD80 polypeptide and a therapeutic agent capable of interacting with at least a CD86 polypeptide. Also disclosed are compositions capable of interacting with the polypeptide. As used herein, a therapeutic agent capable of preventing or inhibiting at least expression of a CD80 polypeptide by a cell and a therapeutic agent capable of preventing or inhibiting expression of at least a CD86 polypeptide by the cell. Also disclosed are compositions that can prevent or inhibit expression of at least a CD80 or CD86 polypeptide by a cell for use in treatment. As used herein, a cardiac agent comprising a therapeutic agent capable of preventing or inhibiting the presentation of at least CD80 polypeptide on the surface of a cell and a therapeutic agent capable of preventing or inhibiting the presentation of at least a CD86 polypeptide on the surface of the cell. Also disclosed are compositions that can prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the surface of a cell for use in the treatment of ischemia-reperfusion injury.

  In certain embodiments, can interact with at least one of CD80 or CD86 polypeptide, or can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell, or CD80 or CD86 polypeptide on the surface of the cell. A therapeutic agent contemplated herein that can prevent or inhibit presentation of at least one of the peptides can be co-administered with a second therapeutic agent, wherein the second therapeutic agent can interact with the CD40 polypeptide and the interaction Interferes with the binding of the CD40 polypeptide to the CD40 ligand (CD40L) polypeptide.

  Preferably, a therapeutic agent contemplated herein capable of interacting with at least one of CD80 or CD86 polypeptide can be co-administered with a second therapeutic agent, wherein the second therapeutic agent is capable of interacting with a CD40 polypeptide. The interaction interferes with the binding of the CD40 polypeptide to the CD40 ligand (CD40L) polypeptide.

  The therapeutic agent and second therapeutic agent may be provided as a combined preparation for simultaneous, separate or sequential (in any order) administration. Preferably, the therapeutic agent and the second therapeutic agent may be administered simultaneously. For example, a therapeutic agent and a second therapeutic agent may be included in a composition, preferably a pharmaceutical composition, and the composition may be administered to a subject. Accordingly, a pharmaceutical combination or composition for use simultaneously, separately or sequentially (in any order) in the treatment of cardiac warm ischemia-reperfusion injury comprising the therapeutic agent taught herein and a second therapeutic agent Are also disclosed herein.

  Thus, a subject in need of treatment for cardiac warm ischemia-reperfusion injury can be treated with a therapeutically effective amount of a therapeutic agent (which can interact with at least one of CD80 or CD86 polypeptide or Can prevent or inhibit the expression of at least one of the CD86 polypeptides or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the surface of a cell) and a therapeutically effective amount of a second therapeutic agent (the second therapeutic agent). Two therapeutic agents are capable of interacting with the CD40 polypeptide, wherein the interaction interferes with the binding of the CD40 polypeptide to the CD40L polypeptide). A method for treating a disorder is also provided.

  Can interact with at least one of CD80 or CD86 polypeptide or can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell or prevent presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell Or a second therapeutic agent capable of interacting with a CD40 polypeptide, wherein the interaction is a second therapeutic agent that interferes with the binding of the CD40 polypeptide to the CD40L polypeptide. Also provided is the use for the manufacture of a combination preparation for simultaneous, separate or sequential use (in any order) in the treatment of blood reperfusion injury.

In certain embodiments, can interact with at least one of CD80 or CD86 polypeptide, or can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell, or CD80 or CD86 polypeptide on the surface of the cell. A therapeutic agent contemplated herein that can prevent or inhibit presentation of at least one of the peptides can prevent or inhibit expression of the CD40 polypeptide by the cell or can prevent or inhibit presentation of the CD40 polypeptide on the surface of the cell. Can be administered in combination with a second therapeutic agent.
Preferably, the therapeutic agent contemplated herein capable of preventing or inhibiting the expression of at least one of CD80 or CD86 polypeptide by the cell or preventing or inhibiting the presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell Can be administered in combination with a second therapeutic agent that can prevent or inhibit the expression of the CD40 polypeptide by the cell or can prevent or inhibit the presentation of the CD40 polypeptide on the surface of the cell.

  The therapeutic agent and second therapeutic agent may be provided as a combined preparation for simultaneous, separate or sequential (in any order) administration. Preferably, the therapeutic agent and the second therapeutic agent may be administered simultaneously. For example, a therapeutic agent and a second therapeutic agent may be included in a composition, preferably a pharmaceutical composition, and the composition may be administered to a subject. Accordingly, also disclosed herein are pharmaceutical combinations or compositions for use in the treatment of cardiac warm ischemia reperfusion injury comprising a therapeutic agent and a second therapeutic agent as taught herein.

  Thus, a subject in need of treatment for cardiac warm ischemia-reperfusion injury can be treated with a therapeutically effective amount of a therapeutic agent (which can interact with at least one of CD80 or CD86 polypeptide or Can prevent or inhibit the expression of at least one of the CD86 polypeptides or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the surface of a cell) and a therapeutically effective amount of a second therapeutic agent (the second therapeutic agent). 2 therapeutic agent can prevent or inhibit the expression of CD40 polypeptide by cells or prevent or inhibit the presentation of CD40 polypeptide on the surface of cells). A method for treating blood reperfusion injury is also provided.

  Can interact with at least one of CD80 or CD86 polypeptide or can prevent or inhibit expression of at least one of CD80 or CD86 polypeptide by the cell or prevent presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell Or a therapeutic agent capable of inhibiting and a second therapeutic agent capable of preventing or inhibiting the expression of CD40 polypeptide by the cell or preventing or inhibiting the presentation of CD40 polypeptide on the surface of the cell. Also provided is the use for the manufacture of combined preparations for simultaneous, separate or sequential use (in any order) in the treatment of.

In further embodiments, the CD40 polypeptide may be contained in one cell and the CD40L polypeptide may be contained in another cell.
In particular, the CD40 polypeptide may be contained in an antigen presenting cell (APC) and the CD40L polypeptide may be contained in a T cell, thereby enabling a second therapeutic agent disclosed herein that can interact with the CD40 polypeptide. The action of interferes with the costimulatory pathway CD40: CD40L and contributes in an additive or synergistic manner to the inhibition of T cell responses.

  In further embodiments, the CD40 polypeptide is contained in an antigen presenting cell (APC), which can prevent or inhibit the expression of the CD40 polypeptide by the cell or prevent the presentation of the CD40 polypeptide on the surface of the cell or The action of the second therapeutic agent disclosed herein that can be inhibited interferes with the costimulatory pathway CD40: CD40L and contributes in an additive or synergistic manner to the inhibition of T cell responses.

  In certain embodiments, a second therapeutic agent contemplated herein is a protein, polypeptide, peptide, peptidomimetic, nucleic acid, small organic molecule, and as defined elsewhere herein. It can be selected from the group comprising, consisting essentially of or consisting of compounds or any combination of two or more thereof.

In certain embodiments, a second therapeutic agent disclosed herein, particularly a second therapeutic agent capable of interacting with a CD40 polypeptide, is an antibody or fragment thereof, as defined elsewhere herein. Alternatively, it can be selected from the group comprising, consisting essentially of or consisting of derivatives, soluble receptors and aptamers.
In certain embodiments, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a primatized antibody, a human antibody, a Nanobody, or a mixture thereof.

  Suitable anti-CD40 antibodies include, for example, XmAb® 5485 (Xencor), PG102 (PanGenetics UK Limited, WO2007 / 129895), dacetuzumab (SGN-40, Seattle Genetics, Inc./ Genentec), HCD122 (Novartis), ASKP1240 (Astellas Pharma), 5D12 or functional equivalents thereof (DeBoer et al., 1992, J. Immunol. Methods 152: 15-23) and FFP106, FFP104 and FFP102 (FFPharma).

In a particular embodiment, the anti-CD40 antibody may be an antibody as defined in any one of claims 17 to 22 of WO2007 / 129895. More specifically, the anti-CD40 antibody has an amino acid sequence:
FSX ₁ SRYSVYWX ₂ RQPPGKGX ₃ EWX ₄ GMMWGGGSTDYSTSLKSRLTISKDTSKSQVX ₅ LKMNSLRTDDTAMYYCVRTDGDY (SEQ ID NO: 1), preferably
QVKLQESGPGLVKPSETLSITCTVSGFSX ₁ SRYSVYWX ₂ RQPPGKGX ₃ EWX ₄ GMMWGGGSTDYSTSLKSRLTISKDTSKSQVX ₅ LKMNSLRTDDTAMYYCVRTDGDYWGQGTTVTVSS (sequence number 2)
(Where:
X ₁ is G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H, preferably G, A, V, L, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;
X ₂ is G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;
X ₃ is G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;
X ₄ is G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;
X ₅ is G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R, or H)
Including
Amino acid sequence:
X ₆ LGX ₇ X ₈ ASISCRSSQSLX ₉ NSNGNTYLHWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDX ₁₀ GVYX ₁₁ CSQSTHVPWT (SEQ ID NO: 3), preferably
ELQLTQSPLSLPVX ₆ LGX ₇ X ₈ ASISCRSSQSLX ₉ NSNGNTYLHWYLQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDX ₁₀ GVYX ₁₁ CSQSTHVPWTFGGGTKLEIKR (SEQ ID NO: 4)
(Where:
X ₆ is N, Q, S, T, Y, W or C;
X ₇ is D, E, N, Q, S, T, Y, W or C;
X ₈ is N, Q, S, T, Y, G, A, V, L, I, P, F, M, W or C;
X ₉ is G, A, V, L, I, P, F, M;
X ₁₀ is G, A, V, L, I, P, F, M;
X ₁₁ is N, Q, S, T, Y, G, A, V, L, I, P, F, M, W or C)
A polypeptide may further be included.
For example, X1 may be L, X2 may be V, X3 may be L, X4 may be L, and X5 may be F. In a further example, X6 may be T or S, X7 may be D or Q, X8 may be Q or P, X9 may be V or A, and X10 is V or L. X11 can be F or Y, preferably X6 can be T, X7 can be Q, X8 can be P, X9 can be A, and X10 can be V and X11 may be Y.

  In certain embodiments, a second therapeutic agent disclosed herein that can prevent or inhibit expression of a CD40 polypeptide by a cell or prevent or inhibit presentation of a CD40 polypeptide on the surface of a cell comprises As defined elsewhere in the specification, an intrabody or fragment or derivative thereof, an antisense agent and an RNA interference (RNAi) agent can be included, consist essentially of, or can be selected from the group consisting of them. Antisense agents that can bind (anneal) with a sequence region in the CD40 pre-mRNA or mRNA sequence are specifically contemplated. Such RNAi agents configured to target CD40 mRNA can be specifically contemplated.

If the therapeutic agent or second therapeutic agent is an expressible molecule, such as an antibody or fragment or derivative thereof, protein or polypeptide, peptide, nucleic acid, antisense agent or RNAi agent, can the agent itself be introduced into the subject? Or by a recombinant nucleic acid comprising a sequence encoding an agonist operably linked to one or more regulatory sequences that allow expression of the sequence encoding the agent (eg, gene therapy or cell therapy).
Thus, an agent allows the expression of one or more sequences encoding a protein, polypeptide, peptide, antisense agent or RNAi agent, for example in vitro, host cells, host organs and / or host organisms (expression constructs) 1 A recombinant nucleic acid comprising a sequence encoding one or more desired proteins, polypeptides, peptides, antisense agents or RNAi agents operably linked to the above regulatory sequences may be included. Such recombinant nucleic acid may be included in a suitable vector.

By “encoding”, a nucleic acid sequence or portion thereof is made specific to the genetic code of the organism in question, such as the amino acid sequence of one or more desired proteins or polypeptides, or a template-transcription product (eg, RNA or RNA It means to correspond to another nucleic acid sequence in the relationship of analog).
Preferably, the nucleic acid encoding one or more proteins, polypeptides or peptides may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides. An “open reading frame” or “ORF” begins with a translation initiation codon known per se, ends with a translation termination codon, does not include any internal in-frame translation termination codon, and encodes a protein, polypeptide or peptide This refers to a continuous coding nucleotide triplet (codon). Thus, the term can be synonymous with “coding sequence” as used in the art.

  An “operable linkage” is a linkage in which a regulatory sequence and a sequence to be expressed are connected in such a manner as to allow said expression. For example, sequences such as promoters and ORFs do not interfere with the ability of the promoter to drive transcription of the ORF, (1) the nature of the linkage between the sequences (1) does not result in the introduction of frameshift mutations, (2) If it does not interfere with the ability of the ORF to be transcribed from the promoter sequence, it can be said to be operably linked.

The exact nature of the regulatory sequences or elements required for expression may vary depending on the expression environment, but typically includes promoters and transcription terminators and optionally enhancers.
Reference to “promoter” or “enhancer” is understood to be its broadest relationship, for precise transcription initiation and, where applicable, for precise spatial and / or temporal control of gene expression or its response. Include required transcriptional regulatory sequences such as internal or external (eg exogenous) stimulators. More specifically, a “promoter” may refer to a region on a nucleic acid molecule, preferably a DNA molecule, to which RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, located upstream of the sequence that it controls transcription, ie, 5 '. Typically, in prokaryotes, the promoter region may contain both the promoter itself and a sequence that signals the initiation of protein synthesis when transcribed into RNA (eg, a Shine-Dalgarno sequence).
In embodiments, a promoter contemplated herein may be constitutive or inducible.

  The term “terminator” or “transcription terminator” generally refers to a sequence element at the end of a transcription unit that signals the termination of transcription. For example, the terminator is usually located downstream of the ORF encoding the polypeptide of interest, ie, 3 ′. For example, if the recombinant nucleic acid contains two or more ORFs that are, for example, arranged side by side and together form a multicistronic transcription unit, the transcription terminator may be located 3 'to the most downstream ORF. May be advantageous.

  The term “vector” generally refers to a nucleic acid molecule, typically DNA, into which a nucleic acid segment can be inserted and cloned. Thus, a vector typically contains one or more unique restriction sites and can self-replicate in a defined host or vehicle organism so that it can replicate the cloned sequence. Vectors can include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PACs, BACs, linear nucleic acids such as linear DNA, viral vectors and the like as appropriate. Expression vectors are generally configured to allow and / or perform expression of a nucleic acid or ORF introduced therein in a desired expression system, such as in vitro, in a host cell, host organ and / or host organism. The For example, the expression vector may advantageously include appropriate regulatory sequences.

  As stated elsewhere, the agent may comprise a protein, polypeptide or peptide. This includes expression in a host cell or host organism transformed with an expression construct that encodes and expresses said protein, polypeptide or peptide in a host cell or host organism, followed by subsequent protein, polypeptide Or it can obtain suitably by refine | purifying a peptide.

  The term “host cell” or “host organism” suitably refers to cells or organisms including prokaryotes such as bacteria and eukaryotes such as yeast, fungi, protists, plants and animals. There is. Contemplated as host cells include, among others, unicellular organisms such as bacteria (e.g., E. coli, Salmonella tymphimurium, Serratia marcescens or Bacillus subtilis), yeast (e.g., Saccharomyces Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g. from Arabidopsis thaliana) or tobacco (Nicotiana tobaccum) and (cultured) animal cells (e.g. vertebrate cells, mammalian cells) Primate cells, human cells or insect cells). Contemplated as host organisms are, among others, multicellular organisms such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrates, even more preferably mammals, even more preferably primates Of particular interest are animals and classes of animals that are non-human.

  Such proteins, polypeptides or peptides may be suitably isolated. The term “isolated” with respect to a particular component (eg, nucleic acid, protein, polypeptide or peptide) exists such that such component is separated from one or more other components of its natural environment (eg, It is generally indicated that it has been separated from the other components or prepared and / or maintained separately from the other components). For example, an isolated human or animal protein or complex may exist separated from the naturally occurring human or animal body. The term “isolated” as used herein preferably also includes the modifier “purified”. For example, the term “purification” with respect to nucleic acids, proteins, polypeptides or peptides does not require absolute purification. Instead, this means that such nucleic acids, proteins, polypeptides or peptides have their abundance (expressed simply in terms of mass or weight or concentration) relative to other nucleic acids, proteins, polypeptides or peptides. Indicates that it is in a separate environment that is larger than in a biological sample. A separate environment represents a single medium, such as a single solution, gel, precipitate, lyophilizate and the like. Purified nucleic acids, proteins, polypeptides or peptides can be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, and the like.

  The various therapeutic agents of the present disclosure, or pharmaceutically acceptable derivatives thereof, such as pharmaceutically acceptable salts thereof, which can be collectively referred to as the active substance for convenience, include one or more pharmaceutically acceptable salts. May be formulated and administered as a pharmaceutical composition or formulation with a carrier / excipient. Such a pharmaceutical composition or formulation may be included in the kit.

The term “pharmaceutically acceptable” as used herein means consistent with that in the art, compatible with other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.
As used herein, “carrier” or “excipient” refers to any and all solvents, diluents, buffers (eg, neutral buffered saline, phosphate buffered saline or, optionally, Tris HCl, Acetic acid or phosphate buffer), solubilizer (e.g. Tween 80, polysorbate 80), colloid, dispersion medium, excipient, filler, chelating agent (e.g. EDTA or glutathione), amino acid (e.g. glycine), protein, Disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, fragrances, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives (e.g. Thimerosal (trademark), benzyl alcohol), antioxidants (eg, ascorbic acid, sodium metabisulfite), isotonic agents, absorption delaying agents, adjuvants, bulking agents (eg, lactose, mannitol) and the like. The use of such media and agents (excipients) for pharmaceutically active substances is known in the art. Except where any conventional medium or agent is incompatible with the active agent, its use in a therapeutic composition is contemplated. Suitable pharmaceutical carriers are described, inter alia, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990).

Illustrative, non-limiting carriers for use in the formulation of pharmaceutical compositions are aqueous, with or without, for example, an oil-in-water or water-in-oil emulsion, an organic cosolvent suitable for intravenous (IV) use. Compositions, liposomes or surfactant-containing vesicles, especially particulate preparations containing polymer compounds such as polylactic acid or polyglycolic acid, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories , Aqueous suspensions, aerosols and other carriers apparent to those skilled in the art.
Pharmaceutical carriers may include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

  A pharmaceutical composition may be essentially any route of administration, including, but not limited to, buccal administration (e.g., buccal ingestion or inhalation), nasal administration (e.g., nasal inhalation or intranasal mucosal administration), lung (e.g., By aerosol inhalation or insufflation), parenteral administration (e.g. subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, or intrasternal injection or infusion, or intracranial, e.g. intrathecal or intraventricular), intraosseous Also prescribed for intralesional, epidermal and transdermal or transmucosal (e.g. oral, sublingual, intranasal), topical (including ophthalmic), rectal, vaginal or intratracheal instillation it can. In this way, the therapeutic effects that can be achieved by the methods and active substances, compositions or formulations described herein are dependent on the specific needs of a given application, e.g. systemic, local, tissue specific, etc. It can be.

  For example, for buccal administration, the pharmaceutical composition may comprise pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, It can be formulated in the form of a syrup, emulsion or suspension. For example, without limitation, oral dosage form preparations blend an appropriate amount of the active compound in powder form, uniformly and thoroughly, optionally with one or more finely divided solid carriers. Appropriately achieved by formulating into pills, tablets or capsules. Exemplary but non-limiting solid carriers are calcium phosphate, magnesium stearate, talc, sugars (e.g. glucose, mannose, lactose or sucrose), sugar alcohols (e.g. mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone. , Low melting wax and ion exchange resin. A compressed tablet containing the pharmaceutical composition is obtained by uniformly and thoroughly mixing the active ingredient with a solid carrier as described above to obtain a mixture having the required compression characteristics, and then mixing the mixture in the desired shape in a suitable device. And can be prepared by compression to size. Molded tablets can be made by molding in a suitable apparatus a mixture of the powdered compound moistened with an inert liquid diluent. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils and the like.

  For example, for oral or nasal aerosol or inhalation administration, the pharmaceutical composition can be combined with exemplary carriers, for example, saline, polyethylene glycol or glycol, DPPC, solutions containing methylcellulose, or benzyl alcohol or other suitable storage. In addition, agents, absorption enhancers to enhance bioavailability, fluorocarbons and / or other solubilizers or dispersants known in the art can be used to formulate in a mixture containing a powder dispersant. Pharmaceutical formulations suitable for administration in the form of an aerosol or spray are, for example, solutions, suspensions or emulsions of the active substance in a pharmaceutically acceptable solvent, such as ethanol or water or a mixture of such solvents. It is. If desired, the formulation can further contain other pharmaceutical adjuvants such as surfactants, emulsifiers and stabilizers and propellants. For illustration purposes, it can be delivered by using a disposable delivery device, a nebulizer, a breath-actuated powder inhaler, an aerosol metered dose inhaler (MDI), or any other numerous nebulizer delivery devices available in the art. In addition, direct administration by mist tent or endotracheal tube can also be used.

  Examples of carriers for administration through mucosal surfaces depend on the specific route, such as the oral cavity, sublingual, intranasal and the like. For buccal administration, illustrative examples include pharmaceutical grade mannitol, starch, lactose, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc., with mannitol being preferred. For intranasal administration, illustrative examples include or include polyethylene glycol, phospholipids, glycols and glycolipids, sucrose and / or methylcellulose, bulking agents such as lactose and preservatives such as benzalkonium chloride and EDTA Contains no powder suspension. For example, phospholipid 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) may be used as an isotonic aqueous carrier.

  For example, for parenteral administration, the pharmaceutical composition may be advantageously formulated as a solution, suspension or emulsion containing suitable solvents, diluents, solubilizers or emulsifiers. Suitable solvents include, without limitation, water, saline or alcohols such as ethanol, propanol, glycerol, and sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned above. It is. Injectable solutions or suspensions are suitable non-toxic parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersion Excipients or wetting agents and suspending agents such as sterile, non-irritating fixed oils containing synthetic mono- or diglycerides and fatty acids including oleic acid can be used as formulated in the art. The active substance can also be lyophilized and the resulting lyophilizate is used, for example, for the production of injectable or injectable preparations. For example, one illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and balanced USP water for injection (WFI). Other illustrative carriers for intravenous use are 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1 Contains an oil-in-water emulsion of ~ 10% squalene or parenteral vegetable oil. Water or saline solutions and aqueous dextrose and glycerol solutions can preferably be used as carriers, preferably carriers for injectable solutions. Illustrative examples of subcutaneous or intramuscular carriers are phosphate buffered saline (PBS), 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% chloride in USP WFI. Acceptable isotonic solutions such as sodium or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and balanced 5% dextrose or 0.9% sodium chloride; or 0.01-0.2 in USP WFI Contains an oil-in-water emulsion of 1% dipalmitoyl diphosphatidylcholine and 1-10% squalene or parenteral vegetable oil.

  Where aqueous preparations are preferred, such aqueous preparations may contain one or more surfactants. For example, the composition can be in the form of a micelle dispersion comprising at least one suitable surfactant, such as a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerol, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG) and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines such as dimyristoyl phosphatidyl choline (DPMC), Palmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids such as dimyristoylphosphatidic acid (DPMA), dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as diacyl Myristoylphosphatidylethanolamine (DPME), dipalmitoylphosphatidylethanolamine (DPPE) and Distearoylphosphatidylethanolamine including (DSPE). Typically, the molar ratio of surfactant to active substance in the aqueous formulation is from about 10: 1 to about 1:10, more typically from 5: 1 to about 1: 5, Any effective amount of surfactant most suitable for specific purposes may be used in the aqueous formulation.

  In certain embodiments, the pharmaceutical composition or formulation may be configured for bolus intravenous infusion and delivered as a bolus intravenous infusion.

  When administered rectally in the form of suppositories, these formulations contain the active substance in a suitable nonirritating excipient, such as cocoa butter, a synthetic glyceride ester or polyethylene glycol, which are solid at ordinary temperatures, Liquefaction and / or dissolution in the rectal cavity to release the drug) and mixing.

  Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.

  Those skilled in the art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulation techniques and pharmaceutically acceptable excipients and carrier solutions are known in the art and are suitable for use with the specific compositions described herein in various treatment regimes. The same is true for the development of various dosing and treatment regimes.

  In addition, there are several known methods for introducing nucleic acids (eg, antisense and RNAi agents) into animal cells, any of which may be used in the present invention. Most simply, the nucleic acid can be injected directly into the target cell / target tissue. Other methods include fusion of recipient cells with bacterial protoplasts containing nucleic acids, use of compositions such as calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes, or receptor-mediated Includes endocytosis, particle injection with a particle gun (“gene gun” method), viral vector infection, electroporation and the like. Other techniques or methods suitable for delivering nucleic acid molecules to target cells include continuous delivery of NA molecules from poly (lactic acid-Co-glycolic acid) polymer microspheres, or to micropumps that deliver products. Protection (stabilization) Includes direct injection of NA molecules. Another possibility is the use of implantable drug release biodegradable microspheres. Various types of liposomes (immunoliposomes, PEGylated (immunoliposomes)), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic acid-Co-glycolic acid) polymer microspheres, implantable drug release biodegradable microspheres Encapsulation of NA into the sphere and concomitant injection of NA with a protective agent, such as nuclease inhibitor aurintricarboxylic acid, are also envisioned. It is also clear that combinations of the different delivery forms or methods described above can also be used.

Additional methods for delivery of nucleic acids, such as antisense agents and RNAi agents, may use previously published methods. For example, intracellular delivery of nucleic acids may be by a composition comprising a mixture of nucleic acid molecules and an effective amount of a block copolymer. An example of this method is described in US 2004/0248833.
Other methods for delivery of nucleic acids to the nucleus are described in Mann et al., 2001 (Proc Natl Acad Science 98 (1): 42-47) and Gebski et al., 2003 (Human Molecular Genetics 12 (15): 1801-1181). Have been described.
Methods for introducing nucleic acid molecules into cells by expression vectors, either naked DNA or complex with lipid carriers, are described in US 6,806,084.

It may be desirable to deliver nucleic acid molecules in a colloidal dispersion system. Colloidal dispersions include lipid-based systems including macromolecular complexes, nanocapsules, microspheres, beads and oil-in-water emulsions, micelles, mixed micelles and liposomes or liposome formulations. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. These formulations may have net cation, anion or neutral charge characteristics and are useful characteristics of in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles (LUVs) in the range of 0.2-4.0 PHI.m can encapsulate a substantial percentage of aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated in an aqueous interior and delivered to cells in a biologically active form (Fraley et al., 1981 (Trends Biochem ScL 6: 77).
In order for liposomes to be effective gene transfer vehicles, the following characteristics should be present: (1) Encapsulating nucleic acid molecules of interest with high efficiency without compromising biological activity; (2) preferentially and substantially bind to target cells compared to non-target cells; (3) deliver aqueous content of vesicles to the cytoplasm of target cells with high efficiency; and (4) genetic information. Is accurately and efficiently expressed (Mannino et al., 1988 (Biotechniques 6: 682).

  The composition of the liposome is usually a combination of phospholipids, particularly phospholipids with a high phase transition temperature, and usually steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

  Alternatively, the nucleic acid molecule can be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, buccal or transdermal administration.

  The administration route described is intended to be guidance only. This is because one of ordinary skill in the art can readily determine the optimal route of administration and any dosage for any particular animal and condition. Several approaches have been attempted to introduce new genetic material that is functional to cells both in vitro and in vivo (Friedmann 1989 (Science 244: 1275-1280)). These approaches include integration of genes expressed in modified retroviruses (Friedmann 1989, supra; Rosenberg. Cancer Res. 1991, 51 (18), 5074S-79S); integration into nonretroviral vectors (Rosenfeld et al. Cell, 1992, 68, 143-55; Rosenfeld et al., Science, 1991, 252, 431-4); or delivery of a transgene linked to a heterologous promoter-enhancer element by liposomes (Friedmann 1989, supra) Brigham et al., Am. J. Med. Sci., 1989, 298, 278-81; Nabel et al., Science, 1990, 249, 1285-8; Hazinski et al., Am. J. Resp. Cell. Molec Biol., 1991, 4, 206-9; Wang and Huang. Proc. Natl. Acad. Sci. USA., 1987, 84, 7851-5); and a ligand-specific cation-based transport system. Conjugation (Wu and Wu. J. Biol. Chem., 1988, 263, 14621-4)) or use of naked DNA, expression vectors (Nabel et al., 1990, supra; Wolff et al. Science, 1990, 247 pp., Including the 1465-8). Direct injection of the transgene into the tissue results only in local expression (Rosenfeld. 1992, supra; Rosenfeld et al., 1991, supra; Brigham et al., 1989, supra; Nabel 1990, supra; Hazinski et al., 1991, supra). The group of Brigham et al. (Am. J. Med. Sci., 1989, 298, 278-81; Clin. Res., 1991, 39, summary) is a DNA liposome complex intravenous or intratracheal Reported in vivo transfection of mouse lungs only after administration. An example of a review document of human gene therapy procedures is Anderson. Science, 1992, 256, 808-13.

  The pharmaceutical formulations disclosed herein that can be conveniently presented in unit dosage form can be prepared according to conventional techniques known in the pharmaceutical industry. Such techniques typically can include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

  The dosage or amount of active agent used, optionally in combination with one or more other active compounds administered, will depend on the individual case and will achieve the optimum effect, as is customary In order to be adapted to individual circumstances. Thus, the nature and severity of the disorder being treated, and the sex, age, weight, general health, diet, form and time of administration and individual responsiveness of the treated human or animal, route of administration of the compound used , Efficacy, metabolic stability and duration of action, depending on whether the treatment is acute or chronic or prophylactic, or other active compounds are administered in addition to the agents of the present invention.

  Without limitation, depending on the type and severity of the disease, typical daily dosages can range from about 1 ng / kg to 100 mg / kg body weight or more, depending on the above factors. For repeated administrations over several days depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. Preferred dosages for the active agents described herein can range from about 0.05 mg / kg to about 10 mg / kg body weight. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, eg every week or every 2 or 3 weeks.

  In certain embodiments, the pharmaceutical composition is between about 10 nM and about 1 μM, preferably between about 20 nM and about 600 nM, such as about 100 nM or about 200 nM or about 300 nM or about 400 nM or about 500. nM antisense agents or RNAi agents as taught herein may be included.

  In certain embodiments, the active agents described herein may be administered daily during treatment. In certain embodiments, an active agent described herein may be administered at least once daily during treatment, eg, an active agent described herein is administered at least 1 day during treatment. For example, the active agent described herein may be administered at least three times daily during treatment. In certain embodiments, the active agents described herein may be administered continuously during treatment, for example, in an aqueous drinking fluid.

  For the treatment of cardiac ischemia-reperfusion injury caused by acute myocardial infarction, treatment with the therapeutic agents disclosed herein is as early as possible after acute myocardial infarction, for example, about 0.5 hours after acute myocardial infarction, Any treatment of myocardial infarction aimed at restoring blood flow to the ischemic region of the heart as early as 1 hour, about 2 hours, about 6 hours, about 12 hours or about 24 hours later ( For example, at least about 0.5 hours, about 1 hour or about 2 hours before treatment aimed at restoring blood flow to the ischemic region of the heart, as soon as possible, for example, before or simultaneously with (eg anticoagulation) Or, for example, about 0.5 hours, about 1 hour, about 2 hours, about 6 hours, about 12 hours or about 24 hours after a treatment aimed at restoring blood flow to the ischemic region of the heart. It may be desirable to do so. Suitable anticoagulant therapeutic agents include, without limitation, antithrombotic agents such as heparin and coumarin, thrombolytic agents such as streptokinase, urokinase and tissue plasminogen activator, and antithrombocytics.

Accordingly, a pharmaceutical combination or composition for use simultaneously, separately or sequentially (in any order) in the treatment of cardiac ischemia-reperfusion injury comprising the therapeutic agent described herein and an anticoagulant Are also disclosed herein.
Thus, in certain embodiments, is it capable of interacting with at least one of CD80 or CD86 polypeptides that are co-administered with an anticoagulant, e.g., co-administered simultaneously, separately or sequentially (in any order)? Also disclosed are therapeutic agents contemplated herein that can prevent or inhibit the expression of at least one of CD80 or CD86 polypeptide by the cell or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell. Is done. In certain embodiments, a second therapeutic agent combination that can interact with CD40 or that can prevent or inhibit the expression of CD40 polypeptide by the cell or that can prevent or inhibit the presentation of CD40 polypeptide on the surface of the cell. Administration, eg, simultaneous, separate or sequential (in any order) combined administration is also contemplated.

  For the treatment of cardiac warm ischemia reperfusion injury as a result of therapeutic interventions such as surgical procedures such as coronary artery bypass grafting, coronary angioplasty, vascular ligation and reperfusion during heart transplantation It may be preferable to administer the therapeutic agent described in to a subject undergoing treatment prior to therapeutic intervention. For example, the therapeutic agents described herein can be used to treat a subject undergoing treatment, for example, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 5 hours, It can be administered 12 hours, about 24 hours or about 48 hours. The therapeutic agents described herein may be administered to a subject to be treated about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes or about 45 minutes before therapeutic intervention. Can also be administered. The therapeutic agents described herein can also be administered to a subject undergoing treatment during a therapeutic intervention. The therapeutic agents described herein may be used to treat a subject after treatment intervention (ie, after intervention), for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes after therapeutic intervention. Minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours It can also be administered after hours or after about 48 hours.

The therapeutic agents described herein may be used alone or in combination with any one or more active agents useful in the treatment of heart warm ischemia reperfusion injury known in the art, such as one or more immunosuppressive agents. (Combination therapy) may be used.
Accordingly, a pharmaceutical combination or composition for simultaneous, separate or sequential use (in any order) in the treatment of cardiac ischemia-reperfusion injury comprising a therapeutic agent as described herein and an immunosuppressive agent Are also disclosed herein.

  Thus, in certain embodiments, is it capable of interacting with at least one of CD80 or CD86 polypeptides that are co-administered with an immunosuppressive agent, e.g., co-administered simultaneously, separately or sequentially (in any order)? Also disclosed are therapeutic agents contemplated herein that can prevent or inhibit the expression of at least one of CD80 or CD86 polypeptide by the cell or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the surface of the cell. Is done. In certain embodiments, a second therapeutic agent combination that can interact with CD40 or that can prevent or inhibit the expression of CD40 polypeptide by the cell or that can prevent or inhibit the presentation of CD40 polypeptide on the surface of the cell. Administration, eg, simultaneous, separate or sequential (in any order) combined administration is also contemplated.

  The term “immunosuppressive agent” as used herein is meant to include compounds or compositions that suppress the immune response. Exemplary immunosuppressive agents include azaliopurines, macrolides and cyclosporine, in particular macrolides such as pimecrolimus, tacrolimus and sirolimus and cyclosporine such as cyclosporin A. Alternative immunosuppressive agents include muromonab CD3, daclizumab, basiliximab, alemtuzumab and other biological immunosuppressive agents (eg, targeting CD3, CD4 or CD8).

  Except as otherwise noted, “subject” or “patient” is used interchangeably and refers to an animal, preferably a warm-blooded animal, more preferably a vertebrate, even more preferably a mammal, and even more preferably a primate. Specifically, including human patients and non-human mammals and primates. Preferred patients are human subjects. Thus, in certain embodiments, the subject is a human.

  As used herein, phrases such as “subject in need of treatment” include subjects who may benefit from treatment of a condition, particularly treatment of cardiac ischemia-reperfusion injury. Such subjects include, but are not limited to, subjects diagnosed with the condition, subjects who are likely to be affected by the condition or are likely to develop the condition, and / or subjects who are trying to interfere with the condition. obtain. As used herein, a subject suffering from or susceptible to cardiac ischemia, or subject to cardiac ischemia, such as a subject suffering from myocardial infarction, or a temporary cessation of blood flow to the myocardium Specifically intended for subjects undergoing cardiac surgery with

The term “therapeutically effective amount” as used herein includes, among other things, alleviation of symptoms of a disease or condition being treated in a subject being examined by a researcher, veterinarian, physician or other clinician. The amount of active compound or drug that elicits a biological or medical response to obtain. Methods for determining a therapeutically effective dose for the agent are known in the art.
The term “prophylactically effective amount” refers to the amount of active compound or agent in a subject that inhibits or delays the onset of a disorder being investigated by a researcher, veterinarian, physician or other clinician. The term “therapeutically effective amount” as used herein may include alleviation of symptoms of the disease or condition being treated, among others, in a subject being examined by a researcher, veterinarian, physician or other clinician. The amount of active compound or drug that elicits a biological or medical response. Methods for determining a prophylactically effective dose for the agent are known in the art.

The following items provide further explanation of certain aspects and embodiments disclosed in the present invention.
(i) in the treatment of cardiac warm ischemia reperfusion injury, capable of interacting with at least one of CD80 or CD86 polypeptide, wherein said interaction interferes with the binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide. A therapeutic agent for use.
(ii) The treatment of cardiac warm ischemia reperfusion injury according to item (i), wherein at least one of CD80 or CD86 polypeptide is contained in an antigen-presenting cell (APC) and CD28 polypeptide is contained in a T cell. Therapeutic agent for use in.
(iii) Heart temperature ischemia reperfusion injury that can prevent or inhibit the expression of at least one of CD80 or CD86 polypeptide by the cell, or can prevent or inhibit presentation of at least one of CD80 or CD86 polypeptide on the cell surface Therapeutic agents for use in the treatment of
(iv) The therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to item (iii), wherein the cell is an antigen-presenting cell (APC).
(v) any one of items (i) to (iv) selected from the group consisting of proteins, polypeptides, peptides, peptidomimetics, nucleic acids, organic small molecules and compounds or any combination of two or more thereof A therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to Item.
(vi) Use in the treatment of cardiac warm ischemia reperfusion injury according to any one of items (i) to (v) selected from the group consisting of an antibody or a fragment or derivative thereof, a soluble receptor and an aptamer For the treatment.
(vii) The heart temperature ischemia-reperfusion injury according to item (vi), wherein the antibody is a polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, primatized antibody, human antibody, Nanobody or a mixture thereof A therapeutic agent for use in treatment.
(viii) The therapeutic agent for use in the treatment of cardiac ischemia reperfusion injury according to item (vi), wherein the soluble receptor is a soluble CTLA4 molecule, preferably a CTLA4Ig fusion protein.
(ix) Treatment of cardiac warm ischemia reperfusion injury according to any one of items (iii) to (v) selected from the group consisting of intrabodies or fragments or derivatives thereof, antisense agents and RNAi agents Therapeutic agent for use in.
(x) a therapeutic agent is administered in combination with a second therapeutic agent, the second therapeutic agent can interact with the CD40 polypeptide, and the interaction results in binding of the CD40 polypeptide to the CD40 ligand (CD40L) polypeptide. The therapeutic agent for using in the treatment of the heart warm ischemia reperfusion injury of any one of item (i)-(ix) which interferes.
(xi) The therapy for use in the treatment of cardiac ischemia-reperfusion injury according to item (x), wherein the CD40 polypeptide is contained in an antigen-presenting cell (APC) and the CD40L polypeptide is contained in a T cell. Agent.
(xii) Items (i) to (ix) administered in combination with a second therapeutic agent capable of preventing or inhibiting the expression of CD40 polypeptide by the cell or preventing or inhibiting the presentation of CD40 polypeptide on the surface of the cell A therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to any one of the above.
(xiii) The therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to item (xii), wherein the cell is an antigen-presenting cell (APC).
(xiv) Items (x) to (x) wherein the second therapeutic agent is selected from the group consisting of proteins, polypeptides, peptides, peptidomimetics, nucleic acids, small organic molecules and compounds or any combination of two or more thereof. A therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to any one of xiii).
(xv) The heart temperature ischemia reactivation according to any one of items (x) to (xiv), wherein the second therapeutic agent is selected from the group consisting of an antibody or a fragment or derivative thereof, a soluble receptor and an aptamer. A therapeutic agent for use in the treatment of perfusion disorders.
(xvi) The heart temperature collapse according to any one of items (xii) to (xiv), wherein the second therapeutic agent is selected from the group consisting of an intrabody or a fragment or derivative thereof, an antisense agent, and an RNAi agent. A therapeutic agent for use in the treatment of blood reperfusion injury.
(xvii) The therapeutic agent for use in the treatment of cardiac ischemia reperfusion injury according to any one of items (i) to (xvi), which is administered in combination with an anticoagulant.
(xviii) The therapeutic agent for use in the treatment of cardiac ischemia / reperfusion injury according to any one of items (i) to (xvi), which is administered in combination with an immunosuppressive agent.
(xix) Cardiac warm ischemia reperfusion injury is myocardial infarction, chronic myocardial infarction, acute myocardial infarction (STEMI), treatment of myocardial infarction aimed at restoring blood flow to the ischemic region of the heart, cardiovascular arrest, Items (i) to (xviii) of coronary artery bypass grafting, coronary angioplasty, transient vasospasm, acute coronary syndrome (NSTEMI), myocardial protection or vascular ligation and reperfusion during heart transplantation A therapeutic agent for use in the treatment of cardiac warm ischemia / reperfusion injury according to any one of claims
(xx) The therapy for use in the treatment of cardiac ischemia reperfusion injury according to any one of items (i) to (xix), wherein the cardiac warm ischemia reperfusion injury is a result of acute myocardial infarction Agent.

It is clear that products, methods and uses are provided in accordance with the present invention that provide the above substantial advantages. Although the invention has been described with specific embodiments, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in view of the above description. Accordingly, it is intended to embrace all such alternatives, modifications and variations as follows in the spirit and broad scope of the appended claims.
The above aspects and embodiments are further supported by the following non-limiting examples.

Example 1: Screening for CD80, CD86 and / or CD40 blockers Binding of test blockers (eg peptides, antibodies, antibody fragments, soluble receptors, small organic molecules, etc.) to human CD80, human CD86 and human CD40 molecules Are screened by biolayer interferometry (BLI) to identify blocking agents that may be useful as therapeutic agents in various embodiments of the invention. Briefly, the fusion proteins, CD86-Fc, CD80-Fc and CD40-Fc (R & D systems, purchased from Minnesota) are immobilized on an anti-human IgG Fc capture biosensor (Fortebio, California). The test blocker is then added to the well and binding to the target molecule is recorded in real time using the Octet® RED96 system (Fortebio, Calif.). Real-time kinetic measurements also make it possible to accurately determine in vitro binding properties such as k _a (association rate constant), k _d (dissociation rate constant), K _D (dissociation constant). Test blockers that are considered suitable candidates for further development is to bind the target by K _D (dissociation _{constant) = k d / k a ≦} 10 -7 M.

Example 2: Inhibition of binding between CD80 / CD86 and CD28 To assess whether test blockers can interfere with binding between CD80 and CD86 and CD28, Raji cells (10 ⁵ cells / well) were administered at increasing concentrations. Incubate with CD80 and CD86 test blockers (eg, peptides, antibodies, antibody fragments, soluble receptors, small organic molecules, etc.) for 1 hour. Next, a fusion protein consisting of the extracellular domain of recombinant human CD28 fused with mouse IgG2 Fc (purchased from Ancell, Minnesota, USA) is added to the wells at a saturating concentration (cells are not washed in between). After overnight incubation, cells are washed and stained with PE-conjugated anti-mouse IgG2a antibody (purchased from BD Biosciences, Belgium). The cells are then washed, fixed in paraformaldehyde and measured by flow cytometry using a BD FACSCanto ™ II. The percentage of binding inhibition is calculated based on the mean fluorescence intensity values of the positive control obtained without addition of blocking agent and the negative control obtained without addition of CD28 fusion protein. The EC50 for each test blocker is calculated from the binding curve. Test blockers that are considered suitable candidates for further development are preferably those with an EC50 of ≦ 1 nM.

Example 3: Inhibition of the interaction between CD40 and CD40L To assess whether test blockers can interfere with the binding of CD40 and CD40 ligand, CD40-expressing CHO cells (10 ⁵ cells / well) were added at increasing concentrations. Incubate with CD40 test blocker (eg, peptides, antibodies, antibody fragments, soluble receptors, small organic molecules, etc.) for 1 hour. Next, the extracellular domain of recombinant human CD40L fused to the His tag (Acris Antibodies, purchased from California, USA) is added to the wells at a saturating concentration (cells are not washed in between). After overnight incubation, cells are washed and stained with PE-conjugated anti-his tag antibody (purchased from Miltenyi Biotec, The Netherlands). The cells are then washed, fixed in paraformaldehyde and measured by flow cytometry using a BD FACSCanto ™ II. The percentage of inhibition of binding is calculated based on the mean fluorescence intensity values of the positive control obtained without addition of blocking agent and the negative control obtained without addition of CD40 ligand protein. The EC50 for each test blocker is calculated from the binding curve. Test blockers that are considered suitable candidates for further development are preferably those with an EC50 of ≦ 1 nM.

Example 4: Cross-reactivity of human targeted blockers Non-human primate models are suitable to validate the functionality of blockers targeting human costimulatory molecules (CD80, CD86, CD40) in the context of myocardial infarction To prove this, the binding of candidate blockers to primate antigen presenting cells (APC) is assessed by flow cytometry.

Dendritic cells are isolated from rhesus monkeys and human blood by magnetically activated cell sorting using a commercially available kit (purchased from Miltenyi Biotec, The Netherlands). The isolated dendritic cell fraction is incubated in the presence of the blocking agent selected in the preceding example, such as Example 1. Human APC is used as a positive control. The binding between the blocking agent and dendritic cells is revealed by the use of an appropriate detection system. When the blocking agent is an antibody, an appropriate secondary antibody conjugated with a fluorescent dye-like phycoerythrin is used. When the blocking agent is Nanobody (registered trademark) having a c-myc tag, an anti-c-myc tag monoclonal antibody is used, and then a fluorescent dye-labeled antibody against the c-myc tag antibody is used. When using a soluble receptor-derived blocking agent such as CTLA4-Ig, a monoclonal mouse anti-human IgG1 antibody (Sigma, # I2513) is added as a secondary antibody followed by an anti-mouse fluorescent dye-labeled antibody. Blocker binding is analyzed by flow cytometry using BD FACSCanto ™ II. Fluorescence intensity and percentage of labeled cells are assessed using BD FACSDiva ™ software.
These experiments indicate that many blocking agents that target human costimulatory molecules (CD80, CD86, CD40) that may be useful as therapeutic agents in various embodiments of the present invention have been shown to be non-human primate antigen presenting cells (APCs). ). Thus, the rhesus monkey model is suitable for confirming the functionality of such blocking agents.

Example 5: One-way allogeneic mixed leukocyte reaction (MLR)
In vitro functionality of blocking agents that target human costimulatory molecules (CD80, CD86, CD40) that may be useful as therapeutic agents in various embodiments of the invention, ie, the ability of blocking agents to interfere with the induction of T cell responses. Are assessed by a one-way allogeneic mixed lymphocyte reaction. Both human and rhesus monkey models are tested.

Peripheral blood mononuclear cells (PBMC) from two different donors, termed stimulator and responder population, are each co-cultured in 200 μL RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) for at least 3 days To do. After the stimulation population is inactivated by treatment with mitomycin, it is added to the wells to simply provide foreign alloantigens to the response population. The blocking agents selected in Examples 1 and 2 are added to the wells either alone or in combination (ie targeting different costimulatory molecules). Each condition is repeated in triplicate. Proliferation of CD4 + and CD8 + T cells from the response population is measured by [ ³ H] -thymidine incorporation added to the culture medium over the period of co-culture. IL-2 secretion is measured in parallel by performing ELISA on the culture supernatant. For this purpose, commercially available kits from R & D systems (Minneapolis, Minnesota) are used according to the manufacturer's recommendations. Cyclosporine is used as a positive control in the assay. Increasing doses of each blocking agent are added to the co-culture medium to determine the EC50.

  Blockers with the lowest EC50 are expected to be the most potent costimulatory pathway inhibitors in vivo. Blocking agents that have an EC50 in the nanomolar or preferably subnanomolar range and show comparable results in non-human primates and human models are selected for performing in vivo experiments.

Example 6: In vivo treatment of cardiac ischemia-reperfusion injury in non-human primate models of myocardial infarction Materials and methods Animal models After approval of the Institutional Animal Care and Use Committee, 21 Macaca Mulatta rhesus monkeys are included in the study. The animals are fully grown at the time of infarction and have an average body weight of 8-10 kg.

Myocardial infarction and test substance delivery Following induction of sensory loss, myocardial infarction is induced by 90-minute balloon inflation in the left coronary rotator. Blood flow obstruction is confirmed by fluoroscopy. Animals are defibrillated as necessary. At the end of the ischemic period, the balloon is contracted and reperfused into the ischemic area. Animals are then randomly divided into 4 treatment groups: negative control (n = 6), cyclosporine (n = 5), CTLA4-Ig (purchased from R & D systems) (n = 5) and monoclonal anti-CD40 antibody ( CTLA4-Ig (n = 5) combined with R & D systems). Shortly before reperfusion, the animals were given a bolus intravenous injection of saline or water for injection (n = 3 per vehicle), 2.5 mg cyclosporine / kg body weight dissolved in saline at a concentration of 25 mg / mL (cyclosporine group) (Piot et al., 2012) or 10 mg CTLA4-Ig per kg body weight (CTLA4-Ig group) diluted with sterile water for injection to a final concentration of 25 mg / ml or 10 mg per kg body weight diluted as above A combination of CTLA4-Ig and 10 mg of anti-CD40 antibody (CTLA4-Ig + CD40 Ab group) is given.

Measurement of myocardial enzymes Blood samples are collected repeatedly from the time of reperfusion to 72 hours after reperfusion. Blood levels of cardiac biomarkers, particularly creatine kinase MB (CKMB) and cardiac troponin I (cTnI) are measured using the Meso Scale Discovery assay kit according to the manufacturer's recommendations. These measurements are used to assess infarct size after myocardial infarction.

Magnetic resonance imaging (MRI)
Study design includes four MRI analyses. A control MRI is performed before the induction of myocardial infarction, a baseline MRI is then performed 3 ± 1 days after the infarction, a follow-up MRI is performed 14 ± 1 days after the infarction, and a final MRI is performed 42 ± 2 after the infarct. Sacrifice in 2 days. A Siemens Magnetom Sonata 1.5 Tesla MRI machine is used. Attach MRI active electrode, SpO2 and pulse sensor. Attach the CP Body Array Flex Coil to the animal's chest. Capture images using syngo MR A30 software. Cine short-axis images are captured for functional analysis of the heart using retrogated arrays. This is repeated with about 8 mm slices from the base to the top. A 0.2 mmol / kg dose of gadoteridol is injected intravenously for image capture at a concentration of 0.05 mM / ml at a rate of 2 ml / s. Blood oxygen saturation, mucosal color and pulse rate are regularly monitored. The resulting images are exported to the Medis QMass MR Research Edition software and analyzed for left ventricular (LV) volume, left ventricular ejection fraction (LVEF), wall motion, wall thickening and infarct size.

Necropsy and tissue collection At the end of the final MRI (42 days after infarction), the animals were given propofol intravenously (IV) until the level of surgical anesthesia was reached, followed by a lethal injection of saturated potassium chloride as a rapid bolus. Euthanize by administering IV. Death is confirmed by observation of cardiac arrest or ventricular fibrillation on an electrocardiogram (ECG).

Scar weight estimation After sacrifice, the heart is removed. The atrium and aorta are separated from the ventricle at the atrioventricular groove. The ventricles are then rinsed and cut into slices approximately 1 cm thick starting from the top. The heart slice is then sandwiched between glass panels to obtain an image of the slice. The slice containing the infarct area is analyzed by manually delineating the area and total cross-sectional area of the infarct area using image analysis software. For each image, the infarct area and total ventricular cross section are measured and the ratio between these surfaces is calculated. Infarct weight and volume estimates are derived by combining this ratio with flake weight and thickness.

result
The in vivo efficacy of CTLA4-Ig is evaluated in a non-human primate model of acute myocardial infarction. From Examples 4 and 5, it is expected that CTLA4-Ig targeting human CD80 and CD86 molecules and anti-CD40 antibodies will successfully target primate molecules. Cyclosporine is used as a positive control because it has already been shown to induce a reduction in scar size in patients suffering from acute myocardial infarction.

  The result of the Example about the combination of CTLA4-Ig or CTLA4-Ig and an anti-CD40 antibody which can be expected in some experiments regarding the evaluation items to be evaluated is shown in FIGS. Primary endpoints were blood levels of myocardial enzymes cardiac troponin I (Figure 2) and creatine kinase (Figure 1) or longer time intervals (up to 2 months) shortly after reperfusion (up to 72 hours) (Figure 6). ) Reduction of infarct size as measured by magnetic resonance imaging (MRI). This is also done at the expense of histological evaluation (Figure 7). Secondary endpoints focus on improving cardiac function as assessed by MRI by measuring left ventricular end-diastolic volume (FIG. 4), left ventricular end-systolic volume (FIG. 5) and ejection fraction (FIG. 3).

  Thus, according to some embodiments of the invention, in an illustrative background of acute myocardial infarction, CTLA4-Ig administered in combination with CTLA4-Ig (by blockade of costimulatory pathway B7: CD28) and anti-CD40 antibody Ig can treat heart warm ischemia reperfusion injury, thus inducing improved cardiac function, reducing cardiac remodeling and reducing scar size.

Claims (20)

  For use in the treatment of cardiac ischemia-reperfusion injury capable of interacting with at least one of CD80 or CD86 polypeptide, said interaction interfering with binding of at least one of CD80 or CD86 polypeptide and CD28 polypeptide Therapeutic agent.   The at least one of CD80 or CD86 polypeptide is contained in an antigen-presenting cell (APC), and the CD28 polypeptide is contained in a T cell. Therapeutic agent.   In the treatment of cardiac warm ischemia reperfusion injury, which can prevent or inhibit the expression of at least one of CD80 or CD86 polypeptide by a cell, or can prevent or inhibit the presentation of at least one of CD80 or CD86 polypeptide on the cell surface A therapeutic agent for use.   The therapeutic agent for using in treatment of the heart warm ischemia reperfusion injury of Claim 3 whose cell is an antigen presentation cell (APC).   Heart temperature according to any one of claims 1 to 4, selected from the group consisting of proteins, polypeptides, peptides, peptidomimetics, nucleic acids, small organic molecules and compounds or any combination of two or more thereof. A therapeutic agent for use in the treatment of ischemia-reperfusion injury.   The therapeutic agent for use in the treatment of heart warm ischemia reperfusion injury according to any one of claims 1 to 5, selected from the group consisting of an antibody or a fragment or derivative thereof, a soluble receptor and an aptamer.   The antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a primatized antibody, a human antibody, a nanobody or a mixture thereof, for use in the treatment of heart warm ischemia reperfusion injury according to claim 6. Therapeutic agent.   The therapeutic agent for use in the treatment of heart warm ischemia reperfusion injury according to claim 6, wherein the soluble receptor is a soluble CTLA4 molecule, preferably a CTLA4Ig fusion protein.   The therapeutic agent for use in the treatment of heart warm ischemia reperfusion injury according to any one of claims 3 to 5, selected from the group consisting of intrabodies or fragments or derivatives thereof, antisense agents and RNAi agents. .   A therapeutic agent is administered in combination with a second therapeutic agent, the second therapeutic agent can interact with the CD40 polypeptide, said interaction interfering with the binding of the CD40 polypeptide and the CD40 ligand (CD40L) polypeptide; The therapeutic agent for using in treatment of the heart warm ischemia reperfusion injury of any one of Claims 1-9.   The therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to claim 10, wherein the CD40 polypeptide is contained in antigen-presenting cells (APC) and the CD40L polypeptide is contained in T cells.   10. The administration according to any one of claims 1 to 9, wherein the administration is in combination with a second therapeutic agent capable of preventing or inhibiting the expression of CD40 polypeptide by the cell or preventing or inhibiting the presentation of CD40 polypeptide on the surface of the cell. A therapeutic agent for use in the treatment of the described heart temperature ischemia reperfusion injury.   13. The therapeutic agent for use in the treatment of cardiac ischemia reperfusion injury according to claim 12, wherein the cell is an antigen-presenting cell (APC).   14. The any one of claims 10-13, wherein the second therapeutic agent is selected from the group consisting of a protein, polypeptide, peptide, peptidomimetic, nucleic acid, organic small molecule and compound or any combination of two or more thereof. A therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to Item.   The second therapeutic agent is used in the treatment of cardiac ischemia reperfusion injury according to any one of claims 10 to 14, wherein the second therapeutic agent is selected from the group consisting of an antibody or a fragment or derivative thereof, a soluble receptor and an aptamer. For the treatment.   The treatment of cardiac warm ischemia reperfusion injury according to any one of claims 12 to 14, wherein the second therapeutic agent is selected from the group consisting of intrabodies or fragments or derivatives thereof, antisense agents and RNAi agents. Therapeutic agent for use in.   The therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to any one of claims 1 to 16, which is administered in combination with an anticoagulant.   The therapeutic agent for use in the treatment of cardiac ischemia-reperfusion injury according to any one of claims 1 to 16, which is administered in combination with an immunosuppressive agent.   Heart warm ischemia reperfusion injury is myocardial infarction, chronic myocardial infarction, acute myocardial infarction (STEMI), treatment of myocardial infarction aimed at restoring blood flow to the ischemic region of the heart, cardiovascular arrest, coronary artery bypass grafting 19. The result of any one of claims 1-18, which is the result of reperfusion during surgery, coronary angioplasty, transient vasospasm, acute coronary syndrome (NSTEMI), myocardial protection or vascular ligation and heart transplantation A therapeutic agent for use in the treatment of heart temperature ischemia-reperfusion injury.   The therapeutic agent for use in the treatment of heart warm ischemia reperfusion injury according to any one of claims 1 to 19, wherein the heart warm ischemia reperfusion injury is a result of acute myocardial infarction.