WO1995003067A1 - Pharmaceutical with immunomodulating activity - Google Patents

Abstract

Pharmaceutical peptide preparations for inducing a heightened state of anti-microbial cellular or humoral immunity in a subject in need thereof consisting essentially of an L-Lys-L-Glu or L-Glu-L-Trp preparation and a pharmaceutically acceptable carrier.

Description

PHARMACEUTICAL WITH IMMUNOMODULATING ACTIVITY

Field of the Invention The invention relates to peptides useful as pharmaceutical agents having immunomodulatoiy activity and particularly to methods of using therapeutic dipeptides

L-lysyl-L-glutamic acid (L-Lys-L-Glu) and L-glutamyl-L-tryptophane (L-Glu-L-Tip).

Background of the Invention Tissue extracts that affect proliferation and/or differentiation of T-lymphocytes have been reported, including thymus extracts of animal origin, e.g., "thymosine" (1),

"thymaline" (2), "T-activine" (3), "thymosin-α_! (4) and others. Commonly tissue extracts exist as complex mixtures that includes polypeptides. Widespread use of such biological mixtures in medical practice has been hindered by the lack of highly purified and characterized sources, as well as by the low yields obtainable, and, once isolated, the considerable variability of chemical and physical properties potentially effecting potency, stability, toxicity, and safety.

"Thymogen" (5) is reportedly prepared as a synthetic L-Glu-L-Trp product whose design is in accord with a dipeptide in a fraction isolated a thymalin (2) extract of bovine thymus.

T-lymphocytes participate in cellular and humoral immune responses to foreign antigens that are triggered following binding of ligand to cell surface receptors. The CD2 receptor on T-lymphocytes, (previously known as the erythrocyte rosette receptor- abbreviated E-receptor) serves a dual role as both an adhesion molecule and a signal transducing molecule. CD2 binding to LFA-3 may promote adhesive binding of T-lymphocytes to LFA3 (CD58) B-lymphocytes and thymic epithelial cells. CD2 binding to CD59 and CD48 ligands may facilitate binding to other cells. CD2 is a cell surface molecule bound by PHA and involved in PHA-induced lymphocyte blastogenesis. Binding of anti-CD2 antibodies to the CD2 receptor is capable of triggering T-lymphocyte blastogenesis that may be independent of the CD3/T cell receptor complex. CD4 and CD8 define MHC class specificity of T helper and cytotoxic T lymphocytes, respectively. (Paul, W.E. Ed. 1993. "Fundamental Immunology, 3rd. Edition", Raven Press, N.Y. pp.541-5.)

Summary of the Invention Disclosed herein are the results of studies showing that treatments with

L-Lys-L-Glu dipeptide and Thymogen (L-Glu-L-Trp) are capable of increasing expression of CD2 on T-lymphocytes and thymocytes, altering tissue distribution of lymphocyte subpopulations in experimental animal model systems, and stimulating immune cells involved in antimicrobial cellular immunity. In in vitro studies L-Lys-L-Glu and Thymogen also stimulated increased expression of CD4 (but not CD8) on peripheral blood lymphocytes isolated from patients with secondary immunodeficiency syndromes. In experimental animal model systems, treatment with L-Lys-L-Glu and Thymogen altered the tissue distribution of T-lymphocytes and lymphocytes bearing cell surface Fc-receptors, and L-L, and L-Lys-L-Glu increased splenic weight and T-lymphocyte content. When delivered locally (i.e., intraperitoneally), the subject L-Lys-L-Glu and Thymogen treatments increased the activation state of resident macrophages in experimental animal studies (as measured by NBT reduction); and, promoted neutrophil infiltration in response to a sterile inflammatory mediator (proteose peptone). The combined results show that L-Lys-L-Glu and Thymogen treatments stimulated immune cells and reticuloendotheiial tissues participating in antimicrobrial mechanisms of cellular and humoral immunity, and indicate that the subject treatments heighten the state of antimicrobial immunity. For these and other reasons (disclosed herein) L-Lys-L-Glu and Thymogen are termed "immunomodulators" capable of heightening the state anti-microbial cellular or humoral immunity in a treated subject.

Embodiments of the invention provide methods of treatment with L-Lys-L-Glu (KE) and Thymogen to induce a heightened state of anti-microbial cellular or humoral immunity in a subject in need thereof.

Detailed Description of the Preferred Embodiment While conducting comparative in vitro dose-response studies of different synthetic peptides and Thymogen (L-Glu-L-Trp), the L-Lys-L-Glu dipeptide was discovered to upregulate E-rosette formation by T-lymphocytes. Subsequent in vitro and in vivo model studies revealed new therapeutic effects of both dipeptides on the reticuloendotheiial system.

The results of in vitro studies disclosed in EXAMPLES 2-4, below, show that dipeptide L-Lys-L-Glu and Thymogen increased expression of accessory molecules on the surface of thymocytes and mature T-lymphocytes as evidenced by i) increased E-rosette forming cells (E-RFC) in thymocyte cultures after incubation with either dipeptide; ii) increased E-RFC in cultures of thymocytes from aged animals after incubation with either dipeptide; and, iii) increased expression of OKT 4 in cultures of human peripheral blood

T-lymphocytes from patients with secondary immunodeficiency syndromes following incubation with either dipeptide. Increased expression of CD2 and CD4 accessory molecules on T-lymphocytes (EXAMPLES 2-4) is compatible a heighten the state of innate or induced immunity to infection, e.g., by upregulating T-helper and cytotoxic T-lymphocytes to respond to lower levels of antigen. On a weight basis L-Lys-L-Glu appeared to be more than 100-times more effective than Thymogen at increasing cell surface expression of CD2 and CD4 on lymphocytes. Neither dipeptide upregulated CD8 expression on lymphocytes. IΛ vivo studies, disclosed in EXAMPLES 5-10 (below), show immunological effects of both dipeptides in experimental animal models. (In vivo studies are discussed further, below, in regard to the treatment methods using L-Lys-L-Glu and L-Glu-L-Trp.)

As used herein the symbols for amino acids are according to the IUPAC-IUB recommendations published in Arch. Biochem. Biophys. 115: 1-12, 1966 with the following single letter symbols for the amino acids: namely,

L, Leu, Leucine V, Val, Valine Y, Tyr, Tyrosine D, Asp, Aspartic Acid

I, lieu, Isoleucine P, Pro, Praline W, Tip, Tryptophan E, Glu, Glutamic Acid

M, Met, Methionine G, Gly, Glycine N, Asn, Asparagine K, Lys, Lysine

T, Thr, Threonine A, Ala, Alanine 0, Gin, Glutamine R, Arg, Arginine

F, Phe, Phenylalanine S, Ser, Serine C, Cys, Cysteine H, His, Histidine

The symbols for protective groups used in peptide synthesis are described in Schi-udex and Lbbke, "The Peptides", Academic Press, N.Y. 1965, e.g., Boc, t- butyloxycarbonyl and Bzl, benzyl. Other abbreviations used are e.g., HPLC, high pressure liquid chromatography; TFA, trifluoroacetic acid; KD, dissociation constant; Ka, association constant; Keq, equilibrium constant; f.a., fatty acid; E, erythrocyte; E-RFC, E-rosette forming cell; EA, erythrocyte antibody; EAC, erythrocyte antibody complement; APC, antigen presenting cell; PHA, phytohemagglutinin; Con-A, concanavalin A; LPS, lipopolysaccharide; IL, interleukin; CSF, colony stimulating factor; IFN, interferon; CTL, cytotoxic T-lymphocyte; NK-cell, natural killer cell; BM, bone marrow; PBL, peripheral blood leukocyte; LN, lymph node; KLH, keyhole limpet hemocyanin; ELISA, enzyme linked immunosorbent assay; FIA, fluorescence immunoassay; TRF, time resolved florescence assay; and, RIA, radioimmunoassay.

The following terms are intended to have meanings as follows: namely, "CD2" is intended to mean the lymphocyte cell surface accessory molecule that is the homo- and heterotypic receptor mediating binding of heterologous erythrocytes (E; e.g., rabbit or sheep erythrocytes) to lymphocytes to form E-rosettes (as disclosed in Paul, W.E. Ed. "Fundamental Immunology, 3rd. Edition, Raven Press, N.Y. at page 562 ; incorporated herein by reference). Lymphocytes having cell surface CD2, when capable of forming rosettes with rabbit erythrocytes are referred to herein as E-rosette forming cells, abbreviated E-RFC.

"CD4⁺ lymphocyte" is intended to mean a lymphocyte having a plurality of cell surface CD4 molecules as evidenced by binding of an antibody specific for CD4 to the cell surface, e.g., monoclonal antibody OKT4 (Ortho Diagnostics, Piscatawy, N.J.).

"CD8⁺ lymphocyte" is intended to mean a lymphocyte having a plurality of cell surface CD8 molecules as evidenced by binding of an antibody specific for CD8 to the cell surface, e.g., monoclonal antibody OKT8 (Ortho Diagnostics, Piscatawy, NJ).

"Reticuloendotheiial system" is intended to mean immune tissues containing macrophages, lymphocytes, reticular cells, mast cells, basophils, eosinophils, neutrophils, and the like. Representative components of the reticuloendotheiial system include lymph nodes, spleen, thymus, bone marrow, lung, liver, epithelial tissues, lymphatic vessels, blood, and the like.

'Teripheral blood leukocytes", abbreviated PBL, is intended to mean the cellular components of the immune system in blood, e.g., lymphocytes, monocytes, eosinophils, basophils, neutrophils, plasma cells, mast cell precursors, and the like.

"Immune cell" is intended to encompass the cell types of the reticuloendotheiial system, e.g., lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, plasma cells, reticular cells in the spleen, Langerhans cells and γδ-bearing lymphocytes in the epithelia, Kupfer cells in the liver, and the like.

"Lymphocytes" is intended to encompass T-lymphocytes, (also referred to as T-cells), B-lymphocytes (also referred to as B-cells), natural killer-lymphocytes (NK-cells), cytotoxic T lymphocytes (CTL), T-helper lymphocytes, as well as precursors of the former and activated derivatives thereof.

"Activated lymphocyte" is intended to mean that subset of lymphocytes which has been i) exposed to a stimulus, and ii) has been triggered to change from a metabolically-quiescent cell into a cell with increased protein synthesis and/or DNA and RNA synthesis and possibly cell division. Illustrative stimuli and triggering pathways leading to activated lymhocytes include interaction of Tcell receptors with antigens, interaction of interleukin receptors with interleukins, interaction of growth factor receptors with growth factors, interaction of lymphocyte cell surface determinants with antibody (e.g., anti-CD2) or mitogens (e.g., PHA), and the like. "Mononuclear phagocyte" is intended to mean cells of the monocyte/macrophage lineage including e.g., monocytes, macrophages, dendritic cells, reticular cells, Langerhans cells, and the like.

"Activated macrophage" is intended to mean a mononuclear phagocyte having an increased capacity for phagocytization and intracellular killing of a microbe. In addition to tests for phagocytic activity, activated macrophages may be recognized for example by biochemical markers (e.g., 5'-nucleotidase), increased oxidative metabolism (e.g., increased ability to reduce nitrotetrazolium blue dye, NBT), and the like.

"Interle kin" is intended to mean an agent released by a first immune cell that affectsa biological activity in a second immune cell. Representative interleukins include cytokines such as tumor necrosis factors (e.g., TNFα and TNFβ), IL-1, IL-2 (also known as T cell growth factor), IL-3, IL-4, IL-5, IL-6, IFN-γ and the like.

"Growth factor" is intended to mean an agent capable of stimulating an increase in cell number in a population of immune cells. Representative growth factors include granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M- CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), and the like. Interleukins that have growth stimulatory activity are included as a subcategory within this usage, (e.g., IL-2, IL-3, IL-6 and the like), as are compounds having a mitogenic effect on immune cells (i.e., PHA, Con-A, LPS, and the like).

"Anti-microbial cellular and humoral immunity" is intended to mean that immunity which ameliorates (makes better) or eliminates one or more clinical or laboratory indicia of disease produced by a microbe.

"Microbe" is intended to mean an agent that when multiplying in a subject causes a disease. Representative microbes include viruses, bacteria, mycoplasma, mycobacteria, parasites, rickettsia, dengue fever agent, prion disease agent, kuru kuru disease agent, and the like. Representative examples of clinical indicia of disease include (but are not limited to) redness and swelling, fever, malaise, septicemia, pyogenic exudates, and the like. Laboratory indicia include (but are not limited to) abnormal values in measurements of: (i) neutrophil or lymphocyte counts in peripheral blood; (ii) hematocrit; (iii) serum alpha globulins or immunoglobulins; (iv) expression of one or more cell surface accessory molecules on an immune cell, for example, molecules indicating a maturation state or activation state of either a T-lymphocyte (e.g., CD2, CD3, CD4, CD28, and the like) or a B-lymphocyte (e.g., B220, surface Ig, expression of rearrangement-recombinase activating genes in Rag 1 and Rag 2, and the like) or a mononuclear phagocyte (e.g., Ia/Mac-1 expression, 5'-nucleotidase activity and the like); (v) synthesis of one or more interleukins (e.g., IL-2, IL-1, TNF, TGF-β, IFN-γ, and the like); (vi) synthesis of antibody specific for the subject microbe (e.g., using a diagnostic immunoassay format); (vii) phagocytic activity in an in vitro assay with mononuclear phagocytes or neutrophils from the subject; and, (viii) splenic mass (e.g., as determined by CAT scan or sonography).

"Cellular immunity" to a subject microbe is intended to mean that immunity which is conferred upon a host by immune cells and which e.g. in an experimental animal model system may be transferred from one animal to another using immune cells that are free of serum, and that is free of B-lymphocytes and/or plasma cells producing an immunoglobulin specifically reactive with the subject microbe.

"Humoral immunity" to a subject microbe is intended to mean that immunity which is conferred upon a host by antibody and which e.g. in an experimental animal model system may be transferred from one animal to another using immune serum that contains antibody that is free of cells, or alternatively, by B-lymphocytes and/or plasma cells that are producing antibody specifically reactive with the subject microbe.

"Innate anti-microbial cellular immunity" is used to mean that immunity which is preexistent in a host before exposure to a microbe such as e.g., that which is conferred by Langerhans cells and T-lymphocytes bearing γδ-T cell receptor molecules in epithelial tissues, or immune cells in biological fluids. In the context of the present disclosure the term is intended to encompass anti-microbial activities of neutrophils, monocytes, platelets, macrophages, dendritic cells, and Langerhans cells, and not the activities of serum proteins such as coagulation factors, complement, lysozyme and the like.

"Subject in need thereof is intended to mean a mammal, e.g., humans, domestic animals and livestock, having one or more clinical or laboratory indicia of infection with a microbe. The subject may exhibit clnical disease activity or may have a subclinical or latent infection.

"Polypeptide" is intended to mean a serial array of amino acids of more than 16 and up to many hundreds of amino acids in length, e.g., a protein.

Embodiments of the invention provide methods for treating a subject to induce a heightened state of anti-microbial cellular or humoral immunity by administering a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp. Preparations containing both L-Lys-L-Glu and L-Glu-L-Trp are also envisaged, as well as preparations containing one or both dipeptides in mixture with one or more antibiotics. The is preferably a patient having a microbial infection. The subject may optionally have an underlying immunodeficiency syndrome that occassions frequent, recurrent, or intermittent microbial infection. The subject underlying syndromes include (but are not limited to) hereditary and acquired immunodeficiency syndromes such as primary immunodeficiency syndromes (e.g., DiGeorge Syndrome, Severe Combined Immunodeficiency Syndrome, Combined Immunodeficiency, X-linked lymphoproliferative disease); syndromes resulting from an underlying autoimmune disease (e.g., diabetes, systemic lupus erythematosus, Sjogrens syndrome); syndromes resulting from an underlying infection (e.g., HIN infection); syndromes resulting from a toxic reaction to a drug or a chemical; or secondary immunodeficiency disorders such as those disclosed in EXAMPLE 4, below). The subject treatment methods of the invention are preferably effective to ameliorate or eliminate one or more clinical or laboratory indicia of disease. For example the subject treatment methods may be useful to decrease redness and swelling, fever, malaise, septicemia, pyogenic exudates, and the like, or to restore one or more measurements of a laboratory indicia of disease to a more normal value or to increase the subject value above the normal range to achieve increased anti-microbial immunity: for example, i) decreasing an elevated neutrophil or lymphocyte count in peripheral blood, ii) increasing an abnormally low hematocrit, iii) decreasing an elevated serum alpha globulin or immunoglobulin level, iv) increasing abnormally low expression of one or more cell surface accessory molecules on an immune cell, such as molecules indicating a maturation state or activation state of either a T-lymphocyte (e.g., CD2, CD3, CD4, CD28, and the like) or a B-lymphocyte (e.g., B220, surface Ig, expression of rearrangement-recombinase activating genes in Rag 1 and Rag 2, and the like) or a mononuclear phagocyte (e.g., Ia/Mac-1 expression, 5'- nucleotidase activity and the like), v) increasing synthesis of one or more interleukins (e.g., EL-2, IL-1, TΝF, TGF-β, IFΝ-γ, and the like), vi) increasing synthesis of antibody specific for the subject microbe (e.g., using a diagnostic immunoassay format), vii) increasing phagocytic activity in an in vitro assay with mononuclear phagocytes or neutrophils from the subject, viii) decreasing an abnormally enlarged spleen or increasing a small spleen to more effectly combat infection (e.g., as determined by CAT scan or sonography), ix) increasing the proportion of Fc-receptor bearing lymphoid cells in spleen, thymus, and bone marrow, x) increasing Fc-receptor bearing lymphoid cells in peripheral blood, xi) increasing T-lymphocytes in spleen, xii) increasing the number of neutrophils in a measured volume of a tissue infiltrate in response to an inflammatory agent, and/or xiii) increasing the percentage of phagocytically active cells in the subject neutrophil infiltrate. In a preferred embodiment a treatment course of about 10 μg kg to about 1 mg kg of a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp is administered to a subject daily over a period of about 3 days to about 10 days and optionally at the discretion of the attending physician the treatment course may be repeated after about 1 to about 6 months intermission. In a preferred treatment course L-Glu-L-Trp is administered by im injection daily to adults at 50-100 μg for 3-10 days and the attending physician prescribes a repeat course of therapy after 1-6 months if additional treatment is required. The subject methods include those in which the L-Lys-L-Glu or L-Glu-L-Trp are administered by injection, e.g., by parenteral, intramuscular, intradermal, subcutaneous, and intraperitoneal injection. In a most preferred embodiment the treatment dose is sufficient to increase a number or percentage of CD2 or Fc-receptor bearing lymphocytes in peripheral blood or in a reticuloendotheiial tissue, or to increase a number of immune cells in an inflammatory infiltrate, or to increase a proportion of phagocytically active cells in an inflammatory infiltrate.

"L-Lys-L-Glu treatment" or "L-Glu-L-Trp treatment" is intended to mean a method of delivering to a subject in need thereof a pharmaceutical preparation of L-Lys-L-Glu or L-Glu-L-Trp with the aim of treating or preventing one or more clinical or laboratory indicia of disease in the subject. The subject methods include delivering the preparation to a patient i) before the disease has been diagnosed, e.g., prophylactic protocols delivered with the aim of preventing development of the disease, as well as, ii) after the disease has been diagnosed, e.g., therapeutic protocols. That the subject treatments have fulfilled the intended aim of treating or preventing the microbial infection in the subject will be evident by a change (increase or decrease) or complete elimination of one or more clinical or laboratory indicia of disease as set forth above. Representative illness, diseases, and conditions have been classified and codified ("International Classification of Diseases; ICD-9-CM, Washington DC, 1989. The methods of the invention find use in treatment of a variety of disease conditions (above) where it is advantageous to stimulate an immune response. For example, embodiments of the invention find us in treatment of infections, (e.g., for stimulating immunity to the microbe. Infections treatable by the methods of the invention include those caused by the following microbes: e.g. namely, Mycobacteria gn., gram negative bacteria (e.g., E. coli), gram positive bacteria (i.e., staphlococci), Pseudomonas gn., Hemophilus gn., Mycoplasma gn., Pneumocystis gn., influenza, rhinovirus, and the like. The subject methods of the invention find a variety of prophylactic and therapeutic uses in treatment of immune pathophysiologic conditions in man and domestic animals. In certain embodiments the methods of the invention find use during in vitro maintenance and expansion of bone marrow, peripheral blood leukocytes, CD34⁺ lymphocytes, and other immune cells such as may occur prior to autologous or allogenic transplantation. In other illustrative embodiments the KE- and EW-dipeptides of the invention may be administered in an intravenous bolus injection (or by perfusion) during (or after) surgery as a prophylactic treatment to prevent infection and the like

The dipeptides disclosed herein have the advantage of providing the desired effects at very low dosage levels and without toxicity. Thus, a purpose of therapy in an acute setting may be to rapidly increase the concentration of an KE or EW in a tissue, e.g., by bolus intravenous injection or infusion. Alternatively, in other cases it may desirable to deliver the KE or EW over a longer period of time. Although the compounds themselves are water-soluble at the low concentrations at which they are usually employed, they are preferably used in the form of their acid salts with pharmaceutically acceptable salts, e.g., acetic, citric, maleic, or succinic acid (as disclosed in greater detail below). Freely-soluble salts of the KE or EW of this invention may also be converted to salts of low solubility in body fluids by modification with a slightly water-soluble pharmaceutically acceptable salt, e.g., tannic or palmoic acid, or by inclusion in a time-release KE or EW formulation such as covalently coupled to a larger carrier protein or peptide, or in timed-release capsules and the like. In general, the acid addition salts of the subject KE and EW dipeptides with pharmaceutically acceptable acids will be biologically equivalent to the subject dipeptides themselves. Multimers of KE and EW, (e.g., di- or tri-dipeptides),are also envisaged, such as easily hydrolyzable polypeptides (e.g., anhydrous chlorides or fluorides and the like) which when introduced into aqueous solution are hydrolized to monomeric KE. "Multimer" as used herein is intended to mean a molecule containing two or more KE or EW. Representative multimers include molecules in which two or more KE or EW covalently bonded to a common linker, or in which the two or more KE or EW dipeptides are non-covalently linked together through the linker (e.g., through ion or hydrophobic interactions).

The route of delivery of the KE or EW dipeptides and the like is determined by the disease and the site where treatment is required. For topical application it may be desirable to apply the KE or EW, analogs, agonists, and antagonists at the local site (e.g., by placing a needle into the tissue at that site) or by placing an impregnated bandage during surgery); while for more advanced diseases it may be desirable to administer the compositions systemically. For other indications the KE or EW dipeptides, analogs, agonists, antagonists, derivatives and the like may be delivered by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal injection, as well as, by intrabronchial instillation (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier in a skin patch), or gastrointestinal delivery (e.g., with a capsule or tablet).

The preferred therapeutic compositions for inocula and dosage will vary with the clinical indication. The inocula is typically prepared from a dried peptide (or peptide conjugate) by suspending the peptide in a physiologically acceptable diluent such as water, saline, or phosphate-buffered saline. Some variation in dosage will necessarily occur depending upon the condition of the patient being treated, and the physician will, in any event, determine the appropriate dose for the individual patient. The effective amount of peptide per unit dose depends, among other things, on the body weight, physiology, and chosen inoculation regimen. A unit does of peptide refers to the weight of peptide without the weight of carrier (when carrier is used). An effective treatment will be achieved when the concentration of KE or EW dipeptide at a tissue site in the microenvironment of the cells approaches a concentration of 10^"5 M to 10^"9 M. Skilled practitioners can make use of clinical and laboratory indicia (above) to monitor patient response to the subject therapy and adjust the dosage accordingly. Since the pharmacokinetics and pharmacodynamics of KE and EW dipeptides, agonists, antagonists, and the like will vary in different patients, a most preferred method for achieving a therapeutic concentration in a tissue is to gradually escalate the dosage and monitor the clinical and laboratory indicia (above). The initial dose, for such an escalating dosage regimen of therapy, will depend upon the route of administration. For intravenous administration, of a KE or EW dipeptide with an approximate molecular weight of 200 to 400 daltons, an initial dosage of approximately 0.1 mg/kg body weight is administered and the dosage is escalated at 10-fold increases in concentration for each interval of the escalating dosage regimen.

L-Lys-L-Glu is commercially available (6), and is conveniently synthesize in solution reactions using classical methods of peptide synthesis such as the illustrative stepwise synthesis set forth in EXAMPLE 1, below. Conveniently, the subject KE- and EW-dipeptides are synthesized by any of a number of automated techniques that are now commonly available. Generally speaking, these techniques involve stepwise synthesis by successive additions of amino acids to produce progressively larger molecules. The amino acids are linked together by condensation between the carboxyl group of one amino acid and the amino group of another amino acid to form a peptide bond. To control these reactions, it is necessary to block the amino group of one amino acid and the carboxyl group of the other. The blocking groups should be selected for easy removal without adversely affecting the peptides, i.e., by racemization or by hydrolysis of the formed peptide bonds. Amino acids with carboxyl- groups (e.g., Asp, Glu) or hydroxyl- groups (e.g., Ser, homoserine, and tyrosine) also require blocking prior to condensation. A wide variety pf procedures exist for synthesis of peptides, solid-phase synthesis usually being preferred. In this procedure an amino acid is bound to a resin particle, and the peptide generated in a stepwise manner by successive additions of protected amino acids to the growing chain. Modifications of the technique described by Merrifield are commonly used (Merrifield, R.B., J. Am. Chem. Soc, 96: 2989-2993, 1964.) In an exemplary automated solid-phase method, peptides are synthesized by loading the carboxy- terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethyl phenylacetamidomethyl) covalently attached to an insoluble polystyrene resin that is cross-linked with divinyl benzene. Blocking with t-Boc is used to protect the terminal amine, and hydroxyl- and carboxyl- groups are commonly blocked with O-benzyl groups. Synthesis is accomplished in an automated peptide synthesizer (Applied Biosystems, Foster City, CA, e.g., Model 430-A). Following synthesis the product may be removed from the resin and blocking groups removed using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods (Bergot, B.J. and S.N. McCurdy, Applied Biosystems Bulletin, 1987). A routine synthesis can produce 0.5 mmole of peptide-resin. Yield following cleavage and purification is approximately 60 to 70%. Purification of the product peptides is accomplished for example by crystallizing the peptide from an organic solvent such as methyl-butyl ether, followed by dissolving in distilled water, and dialysis (if greater than about 500 molecular weight) or reverse HPLC (e.g., using a C18 column with 0.1% trifluoroacetic acid and acetonitrile as solvents) if less than 500 molecular weight. Purified peptide is lyophilized and was stored in a dry state until use.

Pharmaceutically acceptable salts may be conveniently prepared from KE or EW dipeptides or analogs by conventional methods. Thus, such salts may be, for example, prepared by treating a KE or EW dipeptide with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or other metallic base and evaporating the resulting solution to dryness, preferably under reduced pressure in a nitrogen atmosphere. Alternatively, a solution of a KE or EW dipeptide may be mixed with an alkoxide to the desired metal, and the solution subsequently evaporated to dryness. The pharmaceutically acceptable hydroxides, bases, and alkoxides include those with cations for this purpose, including (but not limited to), potassium, sodium, ammonium, calcium, and magnesium. Other representative pharmaceutically acceptable salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valarate, oleate, laurate, borate, benzoate, lactate, phosphate, tosulate, citrate, maleate, furmarate, succinate, tartrate, and the like.

The compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents. The pharmaceutical compositions formed by combining KE- or EW-dipeptide with a pharmaceutically acceptable carrier and an optional antibiotic. The subject combination therapeutic agents are then readily administered in a variety of dosage forms such as tablets, lozenges, syrups, injectable solutions, and the like. Combination therapeutic agents may also include both KE- and EW-dipeptide in the same unit dosage form. Pharmaceutical carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be employed along with various disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates, together with binding agents such as polyvinylpyrolidone, sucrose, gelatin, and acacia. Additionally, lubricating agents, such as magnesium sterarate, sodium lauryl sulfate, and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed as fillers in salt and hard-filled gelatin capsules. Preferred materials for this purpose include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions of elixirs are desired for oral administration, the essential active KE- or EW-dipeptide ingredients therein may be combined with various sweetening or flavoring agents, colored matter or dyes, and if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof. For parenteral administration, solutions of the KE or EW, analog, or receptor fragment in sesame or peanut oil or in aqueous polypropylene glycol may be employed, as well as sterile aqueous saline solutions of the corresponding water soluble pharmaceutically acceptable metal salts previously described. Such an aqueous solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection. The sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art. Additionally, it is possible to administer the aforesaid compounds topically (e.g., through a placed catheter) using an appropriate solution suitable for the purpose at hand.

It may be desirable to stabilize the KE or EW dipeptides, analogs, receptor fragments, and the like to increase their shelf life and pharmacokinetic half-life. Shelf life stability is improved by adding excipients such as: a) hydrophobic agents (e.g., glycerol); b) sugars (e.g., sucrose, mannose, sorbitol, rhamnose, xylose); c) complex carbohydrates (e.g., lactose); and/or d) bacteriostatic agents. Pharmacokinetic half-life of peptides is modified by coupling to carrier peptdes, polypeptides, and carbohydrates by chemical derivatization (e.g., by coupling side chain or N- or C-terminal residues), or chemically altering the amino acid to another amino acid (as above).

Pharmacokinetic half-life and pharmacodynamics may also be modified by: a) encapsulation (e.g., in liposomes); b) controlling the degree of hydration (e.g.,. by controlling the extent and type of glycosylation of the peptide); and, c) controlling the electrostatic charge and hydrophobicity of the peptide.

Experimental animal trials (disclosed herein in EXAMPLES 5-10, below) determined the effects of Thymogen and L-Lys-L-Glu at dosages of about 10 μg kg to about 1 mg/kg on different lymphocyte subpopulations and lymphoid organs in vivo. The results of the studies disclosed in EXAMPLES 5-10 are summarized in TABLES A-E, below. The results indicate that:

In an experimental animal model treatments with L-Lys-L-Glu were effective to i) increase CD2 lymphocytes in peripheral blood by about 3-fold (TABLE A); ii) increase Fc receptor bearing lymphoid cells in spleen, thymus, and bone marrow (BM) by about 1.2-fold to 1.4-fold, and in peripheral blood by about 3-fold (TABLE B); iii) increase splenic mass by about 1.2-fold (TABLE C); iv) increase activity of macrophages by about 2-fold (TABLE D); and, increase neutrophil infiltration into tissues in response to an inflammatory agent by about 2-fold with about a 1.5-fold increase in phagocytically active cells in the infiltrate.

Treatment with L-Glu-L-Trp at a dosage of about 0.1 mg kg to about 1 mg/kg was effective to i) increase CD2 lymphocytes in peripheral blood by about 3-fold (TABLE A); ii) increase Fc receptor bearing lymphoid cells in spleen and bone marrow (BM) by about 1.3-fold and about 1.7-fold, respectively (TABLE B); and, increase neutrophil infiltration into tissues in response to an inflammatory agent by about 2-fold with about a 1.7-fold increase in phagocytically active cells in the infiltrate.

TABLE A CD2⁺ Lymphocytes in Treated Guinea Pigs

TABLE B

Immune Cells Bearing Fc receptors

Splenic and Thymic Mass in Treated Mice*

Lymphoid L-Lys*L-GIu L-Ghi-L-Trp Organ Treatment Treatment

Thymus ns 44% I (pO.Ol)

Spleen 1.2 X t p<0.05) 1 15% I (p<0.05)

*ns, no statistically significant change; results presented in EXAMPLE 6 (TABLE 5), below.

TABLE D

State of Activation of Resident Tissue

Macrophages in Treated Mice* -Lysi-L-Gl-u *Gl«-L»Trp

Activated Treatment Treatment

Non-activated 2.2 X t (p<0.05) ns

Activated 2 X t (p<0.05) ns

*ns, no statistically significant change; results presented in EXAMPLE 6 (TABLE 7 ), below. TABLE E

Neutrophils Induced by a

Sterile Inflammatory Agent in Treated Mice*

Neutrophil L*Lys«L«Gltι L-Glu-L-Trp Index Treatment Treatment

Total cells X10° 2 X t (p<0.05) 2 X t (p<0.05)

Phagocytic cells (%) 1.5 X t (p<0.05) 1.7 X t p<0.05)

Phagocytic Index ns ns

*ns, no statistically significant change; results presented in EXAMPLE 6 (TABLES 9-10 ), below. The disclosed effects of L-Lys-L-Glu and Thymogen on macrophages and neutrophils (EXAMPLES 2-6) are compatible with the interpretation that the subject treatments of the invention heighten the state of innate immunity to infection by i) activating anti-microbial activity of resident macrophages; and, ii) promoting neutrophil infiltration into tissues in response to inflammatory agents.

In certain therapeutic uses it may prove useful to monitor dipeptide levels in bodily fluids (supra) while escalating the dose delivered to the patient, as described below. The representative KE and EW dipeptides are non-toxic to human and guinea pig lymphocytes in vitro, and non-toxic when in injected intraperitoneally into guinea pigs and mice as disclosed in the EXAMPLES, below.

Knowledge of the amino acid sequence of the KE and EW dipeptides also permits the preparation of appropriate nucleotide sequences (e.g., by standard techniques, and incorporation of these sequences into bacterial, yeast, and insect plasmid DNA's, as well as into mammalian cell viral vectors (e.g., retroviral vectors.) Expression systems that may be used to produce the peptides of the invention include prokaryotic, eukaryotic, yeast, and insect cells. It is presently believed highly likely that KE and EW are cytomedines released from hydrolysis of tissue polypeptides, at a rate homeostatically determined (at least) by tissue pH and the enzyme activity in the tissues. The present disclosure serves as a useful basis for constructing derivatized and covalently modified KE or EW analogs, antagonists, and the like. For example, the KE or EW may be used for preparation of an analog that is e.g. a) covalently modified by adenylation, methylation, acylatioή, phosphorylation, uridylation, fatty-acylation, glycosylation, and the like; b) a sterioisomer of an L-Lys-L-Glu or L-Glu-L-Trp, e.g., replacing a D- for an L- sterioisomer, and the like; c) a derivative of KE or EW, wherein one amino acid is substituted for another of like properties, i.e., a neutral nonpolar amino acid for another neutral nonpolar (e.g., W replaced by S, T, Y, N, Q, or C), an acidic amino acid for another acid (e.g., E replaced by D), or a basic amino acid for another basic (e.g., K replaced R or H); d) a chemically modified form of KE or EW, e.g., a C-terminal (or gamma-carboxyl) group modified to a carbonyl, or an N-terminal group modified to an amide, or N- or C-terminal extension with Sar or gamma-amino butyric acid ( GABA); e) a chemically derivatized form of KE or EW, e.g., covalent coupling of the IM peptide to a larger peptide (or polypeptide) carrier, or an N- or C-terminal extended peptide; or, f) replacing one amino acid with another of slightly different properties, e.g., changing the hydrophobicity of the dipeptide.

Bifunctional KE or EW dipeptides may also be synthesized to combine advantageous properties of both KE and EW into a single molecule (e.g., a molecule according to Formula π, below), or as a mixture of KE and EW.

The KE and EW dipeptides disclosed herein also provide basis for organic synthesis of molecules that include derivatives, analogues, agonists and the like, that have immunomodulatory activity. The subject organic molecules conform to the following general Formula I: namely,

O H

H II I H

i i i

¹ 2 I R I

1 I I ■

R3 I R₅ I

(aai)

Formula I wherein, Rι is a neutral polar, neutral nonpolar, or basic residue selected from a functional group containing a halogen atom such as an amine, amide, amido- and the like, or alternatively, R} is selected from a functional group containing a straight or branched peptide chain, or straight or branched chain alkyl-, alkoxy-, or cyclic hydrocarbon, such as methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, cyclohexyl-, phenoxy-, glycol- and the like, or alternatively, R₅ is selected from a hydrophobic or amphipathic residue such as a glycerol-phosphatide or fatty-acyl chain (e.g., phosphatidyl-ehtanolamine, phosphatidyl-choline, phosphatidyl-inositol, butyryl-, lauryl-, myristyl-, undecylyl-, and the like) or a glycolipid (e.g., cerebroside, ceramide dihexoside and the like), or one or more hexosyl- residues, (e.g., sucrosyl-, glucosyl-, glucosaminyl-, galactosyl-, galactosaminyl-, lactosyl-, mannosyl-, sorbitolyl-, glycerolyl-, amylosyl- and the like);

R₂ and R₄ are straight or branched alkyl chains selected from methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-; isopropyl-, isobutyl-, isopentyl-, isohexyl-, and the like; R₃ is a positively charged residue such as an amine, amide, amido, residue and the like, or alternatively, R₃ is a negatively charged residue such as a modified carbonyl- residues such as carboxylic acids, carboxylic acid amides, acyl-modified carboxylic acids, carbamates, di-alcohols, aldehydes, and the like;

R₅ and R_$ are electron donor residues such as acetyl-, alkyl-, or modified carbonyl- residues such as carboxylic acids, carboxylic acid amides, acyl-modified carboxylic acids, carbamates, di-alcohols, aldehydes, or alternatively, one or more cyclic or heterocyclic hydrocarbon rings such as phenyl-, phenoxy-, and the like;

Derivatives may also be constructed as lipopeptides constructed with multiple KE or EW residues (e.g., di-, tri-, and tetra-lipopeptides and the like). One illustrative example of a glycerol-phosphatide multimer is provided in Formula II, below, as a phosphatidyl fatty acid (abbreviated fatty acyl in Formula II) of glycerol having one "aaιaa₂" dipeptide (e.g., KE) and a second "aa₃aa₄" dipeptide (e.g., EW). R_x and R₂ are "non-interfering residues", as set forth below, that may used to stabilize or enhance the biological activity of compound of Formula II.

"Analog" is intended to mean a chemical compound according to Formula I, above, that mimics or improves on the electronic, steric, hydrophobic, and 3-dimensional space filling requirements of the constituent residues in KE or EW that are involved in binding to the ligand-receptor (e.g., a mimetic chemical KE or EW compositions). Representative analogs include chemical mimetic compounds that are capable of antagonizing binding of KE or EW to a ligand-receptor, i.e., antagonists (as defined below),and other ligands that are capable of binding to a ligand receptor and exerting effects similar to KE or EW, i.e., agonists (as defined below).

"Agonist" as used herein means a chemically modified KE, EW, or organic chemical molecule according to Formula I or Formula II, above, that is capable of spacially conforming to the molecular space filled by a KE or EW ligand and that is further capable of combining with the subject ligand receptors to initiate an action that is initiated by KE or EW following binding to their specific ligand receptor(s) on cells in vitro or in vivo. Representative examples of actions initiated by KE and EW may be: i) upregulation of lymphocyte cell surface determinants such as CD2, CD4, CD28, CD45, CD58, CD59,- LFA 1, ICAM 1, ICAM 2, ICAM 3, and the like; ii) macrophage activation; iii) stimulating (or inhibiting) lymphocyte blastogenesis in response to antigen (or mitogen), iv) stimulating release of interleukins from lymphocytes (e.g., IL2, IL4, IL5, IL6, IL10, IFNγ, TGFβ and the like); v) mobilization of intracellular calcium (e.g., through membrane calcium channels) and cyclic AMP; vi) stimulating cell interactions between T-helper lymphocytes, B-lymphocytes and antigen presenting cells in initiation of primary and secondary humoral (antibody-mediated) immune responses; vii) stimulation of macrophages to increase their antibacterial and cytotoxic activities to microbial and mammalian target cells, and the like. Agonists possess binding affinity for ligand- receptor(s) and intrinsic activity for inducing the immune effects that are induced when KE or EW bind to their ligand receptor.

"Antagonist" as used herein means a chemical molecule according to Formula I or Formula π, above, that spacially conforms to the molecular space filled by KE or EW and is further capable of combining with the subject ligand receρtor(s), as set forth above, to inhibit, neutralize, impede or reverse, at least in part, an action initiated following binding of EW or KE to the subject ligand receptor on a lymphocyte or a macrophage.

"Non-interfering residue" as used herein means any chemical residue that when present in position R_! or 1^ of Formula I, or position Rj or R₂ of Formula II does not interfere with binding of the subject ligand of the respective formula to a ligand receptor. For example, non-interfering amino acid residues or glycosyl residues may be useful in positions Rj and Rg of Formula I (or positions R_j or R₂ of Formula II) to stabilize or enhance the biological activity of the subject compound.

The biological activities of chemical analogs according to Formula I or Formula II may be compared with those of KE or EW in vivo in experimental animal model systems, or alternatively, it may prove convenient to use in vitro assays to monitor intracellular second messenger pathways triggered in the test cells by interaction of a ligand with a ligand receptor. Biological activity of a compound according to Formula I or Formula II may be determined by testing for second messenger pathways triggered by receptor binding the subject ligand. Second messenger pathways may be monitored e.g. by testing intracellular levels of Ca^**, cAMP, cGMP, adenyl cyclase, tyrosine kinase, guanylate cyclase, Protein kinase C; or, cellular release of interleukins, or mediators such as arachidonic acid metabolites, prostaglandins, and the like. The present disclosure of significant biological effects of KE and EW on immune cells also serves as basis for isolating cell surface receptors binding KE and EW, and for identifying other ligands in biological fluids which may bind to the subject receptors.

"Ligand" as used herein refers to a compound that is capable of filling the three- dimensional space in a receptor binding site so that electrostatic repulsive forces are minimized, electrostatic attractive forces are maximized, and hydrophobic and hydrogen bonding forces are maximized. Representative ligands include EW and KE. Ligands bind to their specific receptors in a specific and saturable manner, e.g., specificity may be determined by the ability of the subject ligand to bind to the ligand receptor in a manner that is not competitively inhibited in the presence of an excess (e.g., 1000-fold molar excess) of KE or EW.

"Ligand receptor" is used herein to refer to a receptor capable of binding a ligand, according to the definition above, in a specific and saturable manner. In an illustrative assay for identifying a ligand receptor in a sample of cells, a labeled ligand (e.g., radiolabeled or biotin-labeled KE or EW at a concentration selected from within the range of O.lnM to lOmM) is incubated at room temperature, 37°C, and 4°C with an aliquot of about 10^-10' cells, the cells are washed (e.g., by centrifugation through an isobutylpthalate or sucrose cushion) and the amount of labeled ligand associated with the cell pellet is determined (e.g., by quantifying radioactivity or reacting the sample with enzymatically-labeled avidin, washing to remove unbound avidin, and then adding substrate to visualize the enzyme-bound-avidin-biotin receptor complex). The data obtained in this manner may be used to conduct a Scatchard binding analysis of the data from which association constant and the relative binding affinity of the ligand-receptor for the ligand may be determined. The association constant for binding of a ligand (e.g., KE or EW) is about O.OlnM to about ImM, and most preferably about InM to about O.lmM. Methods for purifying ligand receptors by affinity chromatography e.g. on solid phase resins containing immobilized KE or EW ligands may be useful for preparing substantially purified preparations of the subject receptors and their fragments. Ligand receptors may also be conveniently prepared and may be used as pharmacological inhibitors, antagonists, and agonists of KE or EW binding to lymphocyte receptors. For example, receptor (or its binding domains) may conveniently be isolated by affinity chromatography of receptor preparations (and fragments) in tissue and cell extracts on chromatographic resins containing bound KE or EW, or alternatively, by binding the receptor to such a resin and then treating with one or more selected proteases to create receptor fragments that remain bound to the resin. The receptor (or fragments) may be eluted from the resin either at high salt and/or low pH, or by competing the binding of the receptor (or fragments) to the resin with excess soluble KE or EW ligand. The amino acid sequence of the receptor is conveniently determined by automated amino acid sequencing, and the sequence may be used to construct synthetic peptides, and nucleotide probes (e.g., degenerate probes) for cloning the subject receptor.

"Ligand-receptor fragments" is a term used to refer to portions of the subject ligand receptor that are smaller in size than a ligand receptor isolated from a natural source, e.g., tissue, biological fluids and the like. Fragments may be prepared from a ligand receptor isolated from a tissue and then subjected to proteolytic degradation or treatment with a chemical such as cyanogen bromide. In the latter case, the subject fragments of the receptor are conveniently purified before use, e.g., by reverse-phase HPLC or immoaffinity chromatography. Alternatively, fragments of the ligand receptor may be prepared by expressing a portion of a nucleotide sequence of a genomic or cDNA clone capable of expressing the subject ligand receptor, e.g., a portion of ligand receptor nucleotide sequence in an expression plasmid or vector introduced into a cell, wherein the cell manufactures the subject receptor fragment and the fragment can be purified (as above). Fragments of the subject ligand receptor may be soluble in biological fluids and aqueous solutions and may bind KE or EW with a greater or lesser KD than a complete (non-degraded) ligand receptor.

"Substantially purified" as used herein refers to a preparation from a natural source that contains a "peptide" (i.e., intended to mean about 2 to about 9 amino acids), a ligand according to criteria set forth above, or a ligand receptor or receptor fragment that is i) enriched greater than about 100-fold from a natural source material, e.g., a membrane preparation of a cell, and that ii) contains less than 5% peptide or polypeptide impurities detectable by reverse-phase HPLC.

The disclosure herein of substantial biological effects for KE and EW also serves as basis for isolating proteins that proteolytically inactivate KE and EW, or influence binding of these dipeptides to their respective receptors. "KE-peptidase" and "EW-peptidase" are used herein to refer to dipeptidylpeptidases capable of catalyzing hydrolysis of a Lysyl-Glutamic acid peptide bond in KE or a Glutamyl-Tryptophane peptide bond in EW. The subject enzymes inactivate KE and EW peptides and render them biologically inactive. KE- and EW-peptidases may conveniently be purified from tissues and cells using conventional purification methods and colorimetric or fluorescent peptide substrates in protease test assays. In turn, inhibitors of KE- and EW-peptidases may be identified in protease test assays. The subject inhibitors of KE- and EW-peptidases may have biological effects similar to KE or EW, since they may decrease proteolytic degradation and increase the biological half life of an endogenous (i.e., natural) KE or EW in a tissue.

The present disclosure further provides basis for diagnostic immunoassays, antibody reagents and the like for measuring levels of KE and EW in biological fluids. While KE and EW are relatively nonimmunogenic, (falling into the class of low molecular weight haptens), when conjugated to a carrier (e.g., KLH) antibodies may be induced in experimental animals. Polyclonal and monoclonal antibodies to KE and EW find uses in a variety of immunoassay formats for diagnostic monitoring the levels of the subject dipeptides in bodily fluids in health and disease. Representative immunoassay formats include: enzyme linked immunoabsorbent assays (ELISA), radioimmunoassays (RIA), fluorescence immunoassay (FIA), time-resolved fluorescence assays (TRF), and cascade assay formats that are routinely used in the art for increasing low-end sensitivity of assays. Individuals with high levels of the KE or EW dipeptide may be at decreased relative risk of infectious disease, while those with decreased levels may be at an increased relative risk. The KE and EW dipeptides find uses as positive and negative controls in the subject diagnostic assays. The subject assays may be assembled for use in a reagent immunoassay test kit. The antibodies disclosed herein specific for the KE or EW may be useful in a variety of competitive and non-competitive direct and indirect immunoassay formats as will be apparent to skilled artisans. The level of KE or EW in a biological sample is commonly determined by comparison with positive and negative controls and assay calibrators. The term "biological fluids" is used herein to mean tissue fluids (such as joint fluid, cerebrospinal fluid and the like), plasma and serum, fluids in body cavities (i.e., peritoneal fluid, lung lavage fluid, urogenital mucus secretions, and the like), urine, feces, sputum, sweat, and the like. _s

While not wishing to be tied to any particular molecular mechanisms of action it is presently believe highly likely that Thymogen (L-Glu-L-Trp) and L-Lys-L-Glu may exert their effects in stimulating expression of CD2 on thymocytes by direct ligand-receptor interaction of cell surface CD2 receptors.

EXAMPLE 1 Synthesis of L-Lys-L-Glu L-Lys-L-Glu was synthesized according to the following solution-phase stepwise method: namely,

1. Preparation of N^N^-dibenzyloxicarbonyllysyl-γ-benzylglutamic acid (1): 0.154 g (0.65mmol) of γ-dibenzylglutamic acid was suspended in 3 ml of dimethylformamide. While mixing, 0.091 ml (0.65 mol.) of triethylamine was added, followed by 0.300 g (0.59 mmol.) of N-oxisuccinimide ether N°, IsT-dibenzyloxicarbonyllysine. The reaction was allowed to proceed for 12 hours at room temperature, with mixing., after which solvent was evaporated under vacuum at 40°C. To the residue was added 10 ml of IN H₂SO and the product was extracted with ethylacetate twice (30 ml; two times). The organic layer was washed with IN H₂SO₄ in distilled water until neutrality was obtained, and then dried over Na₂SO₄. Using redistilled ethylacetate (distilled under vacuum at 40°C), the residue was dissolved in l-2ml and the product then precipitated with hexane. Recrystalization was achieved in the same ethylacetate/hexane system. The product of the recrystalization was filtered and dried in vacuum over P₂O₅. Yield from this synthesis was 0.330 gm (88%). R_g=0.81 (benzene:acetone 1:1, siluphol).

2. Preparation of L-Lysyl-L-Glutamic Acid:

0.33 grams of protected dipeptide from step 1, above, was dissolved in 10 mg of methanol, water (3 mg) was added, and the mixture hydrated over palladium with heat. Thin layer chromatography was used to monitor the time needed for the hydration reaction. After hydration was completed the palladium catalyst was removed by filtration, the residue dissolved in a minimal volume of distilled water, and the product precipitated in methanol. The precipitated product was collected by filtration, washed with ethanol, and dried in vacuum over P₂O₅. Yield was 0.110 g (85%). The melting temperature of the product was 194-196°C, [α]_γ ²⁰= +20.0° (c=3.0; H₂O) R_g = 0.54 (acetonitrile: water 1:3, Merck). Electrophoresis: E_GIy = 1.96; E_his = 0.98 (1400N, 45 min., 2% acetic acid, Wattman 3MM paper).

EXAMPLE 2

Effects of Dipeptide L-Lys-L-Glu on

Expression of E-receptors on Thymocytes Thymus cells isolated from male guinea pigs (180-200 g) under standard conditions of tissue mincing and differential centrifugation in medium 199. Isolated thymocytes were treated with trypsin to remove cell surface E-receptors capable of binding rabbit erythrocytes (E). The percentage of cells capable forming E-rosettes (E-RFC) was decreased by about two-fold and under these conditions. Thymogen has been reported to restore T-cell receptors and E-RFC following incubation at 37°C. Studies were conducted in which the activity of L-Lys-L-Glu dipeptide and Thymogen were compared in the latter assay.

Thymocytes were prepared from 48 guinea pigs, treated with trypsin, and tested for

E-RFC according to Nekam et al. (7), incorporated herein by reference. Dipeptide

L-Lys-L-Glu and Thymogen were dissolved in 0.9% (w/v) NaCl and added to cultures to at test concentrations of 1 mg/ml, 0.01 mg/ml, and 0.0001 mg/ml. The results of these experiments are summarized in TABLE 1, below.

TABLE 1 Effects of L-Lys-L-Glu and Thymogen on

Expression of 5-receptors on Thymocytes (X±S.D.)

The results presented in TABLE 1 show that addition of dipeptide L-Lys-L-Glu at concentrations of 0.0001-1 mg/ ml and Thymogen at 0.01-1 mg/ ml increased the percentage of E-RFC in trypsin-treated thymocyte cultures. The results suggest that L-Lys-L-Glu stimulates expression of E-receptors on thymocytes over a wide range of concentrations, perhaps by direct ligand-receptor interaction of L-Lys-L-Glu with the E-rosette receptor. On a weight basis L-Lys-L-Glu was more about 100-times more effective than Thymogen (L-Glu-L-Trp).

EXAMPLE 3 Effects on Thymocytes from Aged Animals

Effects of L-Lys-L-Glu on expression of E-receptors on "aged" thymocytes was investigated in a manner similar to that in EXAMPLE 2, above, but using thymocytes obtained from old male guinea pigs (700-800 g). In aged animals it is recognized that the percentage of thymocytes forming rosettes with rabbit erythrocytes is reduced, and cell surface E-receptors are likewise reduced. For these studies aged thymocytes were isolated in medium 199 from 42 guinea pigs, and E-RFC determined as described by Stadecker et al. (8), incorporated herein by reference. Thymogen and L- Lys-L-Glu dipeptide were dissolved in 0.9% (w/v) NaCl and added to cultures of thymocytes at concentrations of 1,

0.01, and 0.0001 mg/ml. The results of these experiments are summarized in TABLE 2, below.

TABLE 2

Effects of L-Lys-L-Glu and Thymogen on

The

the percentage of E-RFC in populations of aged thymocytes, i.e., from 66 ± 5% (TABLE 1) to 45.3 ±

3.7% (TABLE 2). Thymogen fostered a statistically significant increase in the percentage of E-RFC at 1 mg/ml, and L-Lys-L-Glu induced a statistically significant increase at all three test dosages. The results in TABLE 2 suggest that on a weight basis L-Lys-L-Glu exerts statistically significant effects at 100-10,000 times lower concentrations than

Thymogen.

EXAMPLE 4 Effects of Dipeptide L-Lys-L-Glu on

T-Lymphocyte Subsets in Human Peripheral Blood The effects of L-Lys-L-Glu on human peripheral blood T-lymphocytes was investigated using indirect immunofluorescence microscopy and OKT4 and OKT8 monoclonal antibodies (Ortho, USA) directed to lymphocyte cell surface differentiation antigens. For these studies lymphocytes were isolated from heparinized peripheral blood

(25 units heparin/ml) isolated from 18 different donors with inflammatory lung diseases and chronic purulent bronchitis accompanied by a secondary immunodeficiency state.

Lymphocytes were prepared by centrifugation on Ficoll-Hypaque, washed, and then incubated in vitro for 45 minutes at 37°C in the presence (or absence) of L-Lys-L-Glu or

Thymogen at concentrations of 1 mg/ml, 0.01 mg/ml, 0.0001 mg/ml. Following incubation the cells were washed with medium 199 and the percentage of OKT4

(T-helper) and OKT8⁺ (T-suppressor) lymphocytes determined by indirect immunofluorescent microscopy. The results presented in TABLE 3, below, show the mean percentages of OKT4 and OKT8 cells in this group of patients after incubation with medium 199 (Control), or with Thymogen or L-Lys-L-Glu. The OKT4⁺/OKT8⁺ ratios for this group of patients is also shown in TABLE 3.

TABLE 3

Effects of L-Lys-L-Glu and Thymogen on T-helper (OKT4⁺) and T-suppressor (OKT8*) Lymphocytes in Patients with S econdary mmunodeficiency (X+S.D.)

The results

increased the percentage of detectable OKT4 T-helper lymphocytes after only 45 minutes at 37°C at all concentrations tested. Thymogen increased the percentage of detectable T-helpers at concentrations of 1 and 0.01 mg/ml. Neither Thymogen nor L-Lys-L-Glu altered the percentage of detectable OKT8 T-suppressor cells. On a weight basis, L-Lys-L-Glu was about 100-times more effective than Thymogen in inducing the observed in vitro increase in detectable T-helper lymphocytes.

EXAMPLE 5 Effects of Dipeptide L-Lys-L-Glu on Immunity in Experimental Animals Immune parameters were investigated in healthy male guinea pigs (250-300 g) following administration of test or control agents. Each test and control group consisted of

10 animals. Test agents were administered to animals once daily by the intramuscular (im) route over a period of 5 days and at the following doses: namely, dipeptide L-Lys-L-Glu-

0.1 mg/kg; and, Thymogen - lmg/kg. Physiological saline was administered im to the animals of the control group. The effects of these treatments was determined on day 10 (i.e., 5 days after the last injection) by preparing cells from peripheral blood (PBL), thymus (TYM), lymph nodes (LN), spleen (SPL), and red pulp of bone marrow (BM). Cells were tested for antibody Fc receptors (EA-RFC), complement receptors (EAC-RFC), T-lymphocytes, "active" T-lymphocytes (E-RFC), B-lymphocytes (EA-RFC and EAC-RFC; according to the method of citation #10). The results of these analyses are shown in TABLES 4A-4C, below, where data are expressed either as the number of RFC xlO⁹ per liter of blood, or RFC x 10³ per milligram (mg) of tissue.

TABLE 4A

Effect of L-Lys-L-Glu and Thymogen on

Immune Indices: E-RFC (X±S.D.)

Cell Index per Control Lys-Glu Thymogen Population 10* cells

PBL 107L 0.53 ± 0.10 1.64 ± 0.49* 1.59 ± 0.78

TYM TOTrng 440.4 ± 82.3 493.0 ±50.7 448.1 ±51.4

LN 107mg 119.1 ± 19.3 37.8 ± 4.1* 81.4 ± 7.6*

SPL 107mg 69.5 ± 6.6 81.2 ± 12.3 75.9 ± 6.8

BM 107mg 22.5 ± 3.9 18.6 ±2.1 11.2 ±2.3*

* statistically significant at the p<0.05 level m comparison with the indices in the control.

TABLE 4B

Effect of L-Lys-L-Glu and Thymogen on

Immune Indices: EA-RFC (X±S.D.)

Cell Index per Control Lys-Glu Thymogen Population 10^* cells

PBL 107L 0.50 ± 0.09 1.60 ± 0.54* 0.82 ± 0.09

TYM 107mg 345.6 ± 63.2 431.7 ± 542.6 ± 42.3 36.2*

LN 107mg 78.7 ± 8.3 52.9 ± 7.4 61.7 ± 8.2

SPL 107mg 48.5 ± 6.4 69.3 ± 6.1* 81.2 ± 7.4*

BM 107mg 19.1 ± 2.2 26.5 ± 2.1* 25.1 ± 1.9*

* statistically significant at the p<0.05 level in comparison with the indices in the control.

TABLE 4C

Effect of L-Lys-L-Glu and Thymogen on

Immune Indices: EAC-RFC (X+S.D.)

Cell Index per Control Lys-Glu Thymogen Population 10^* cells

PBL 107L 0.30 ± 0.09 0.35 ± 0.07 0.79 ± 0.08*

TYM 107mg 3.4 ± 0.7 3.4 ± 1.2 0

LN 107mg 146.5 ± 12.4 151.7 ± 12.3 173.3 ± 12.7

SPL 107mg 140.2 ± 15.6 189.7 ± 141.5 ± 22.6 21.4*

BM 107mg 19.7 ±3.2 13.5 ±2.4 13.9 ±2.1

* statistically significant at the p<0.05 level in comparison with the indices in the control. The results presented in TABLES 4A-4C show that animals treated for 5 days with dipeptide L-Lys-L-Glu or Thymogen had 3-fold more T-lymphocytes in peripheral blood (i.e., PBL; E-RFC) than control animals treated with saline. T-lymphocytes in LN and BM decreased (i.e., 17% for Lys-Glu and 50% for Thymogen) while splenic and thymus T-cells increased by about 11-16% for Lys-Glu and 2-9% for Thymogen. At 10 days, animals who received L-Glu-L-Trp showed an increased number of T-lymphocytes in peripheral blood, spleen and marrow.

L-Lys-L-Glu increased in a statistically significant manner the number of Fc-receptor bearing cells isolated from blood, thymus, spleen, and bone marrow (TABLE 4B). Numbers of complement receptor bearing cells (EAC-RFC) were increased 2-fold in blood by L-Glu-L-Trp treatment, but not by L-Lys-L-Glu treatment. However, L-Lys-L-Glu stimulated a statistically significant increase in splenic complement receptor bearing cells (TABLE 4C).

The results indicate that L-Glu-L-Trp and L-Lys-L-Glu stimulated proliferation, differentiation, and/or migration of T- and B-lymphocytes in vivo.

EXAMPLE 6 Effects of L-Lys-L-Glu Dipeptide on Indices of Innate Immunity The effects of L-Lys-L-Glu on innate mechanisms of immunity was investigated in male CBA mice (18-20 g). Eighty mice were divided into 4 groups of 20 mice each. The animals in each of the groups were injected intraperitoneally (ip) once daily over a period of 6 days with either: i) lmg/kg Thymogen, ii) 0.01 mg/kg Thymogen, iii) 0.01 mg/kg

L-Lys-L-Glu, or iv) non-pyrogenic physiological saline. Next (on day 6), each group of animals was sub-divided into two subgroups of 10 animals each: i.e., one subgroup of animals was injected ip with 10% sterile proteose peptone to induce neutrophils (induced), while the other subgroup were non-induced (non-induced). The animals of the four induced-subgroups were sacrificed 2.5 hours after the ip injection. Thymus and spleen were weights were determined in the non-induced animals; cell suspensions were prepared from each thymus and spleen; and, resident peritoneal exudate cells (PEC) were harvested by lavage with medium 199 from each animal. The percentage content of T- and

B-lymphocytes in the splenic cell populations from the non-induced animals was determined by phase-contrast and indirect immunofluorescence (11) using rabbit antisera specific for murine Thy-1 and immunoglobulin (Ig). (Rabbit anti-Thy-1 was raised by immunization with a murine brain homogenate (12) and anti-Ig by immunization with an ammonium sulfate precipitate of murine serum. The percentage macrophages in the resident PEC populations were determined in Romanowsky stained smears of cells, the cells then collected by centrifugation at 150 x g/10 minutes, and macrophage activity measured by reduction of nitroblue tetrazolium (NBT; 12) before and after induction with complement-opsonized zymosan (guinea pig complement activated by zymosan; 14). NBT reduction was quantified spetrophotometrically at 540 nm. Pinocytic activity of the resident peritoneal macrophages was assessed by measuring uptake of neutral red dye

(measured spectrophotometrically). The percentage of neutrophils in the PEC population was determined using phase contrast microscopy, and in response to induction with complement-opsonized zymosan. Phagocytic activity of neutrophils was determined by incubating the cells with 2.5 x 10 Staphylococcus aureus per milliliter (S. aureus from a

24 hour culture). The data are expressed as both the percentage of phagocytic cells (% of the total cell population phagocytizing 1 or more staphylococci and the phagocytic index

(expressed as the mean number of intracellular bacteria inside each of the phagocytically active neutrophils), both measurements being made by microscopic examination following

Romanowsky-Gϊemsa staining of cell smears (16).

The results of these respective experimental measurements on cells contributing to innate mechanisms of immunity are summarized in TABLES 5-10, below.

TABLE 5 Effect of L-Lys-L-Glu and Thymogen on

Thymic anc Spleen Mass (X±S.D. )

No. Dose Thymus Thymus Spleen Splenic

Group Animals (mg/kg) (mg) Change (%)t (mg) Change (%)

Saline Control 10 0 25.4±1.6 0 81.7±3.4 0

Lys-Glu 10 0.01 23.4±1.2 4- 8 96.5±5.7** T 18

Thymogen 10 1.0 14.1±1.3* 4- 44 88.4±4.4 t 8 10 0.01 14.8±1.6* 4- 42 69.8±2.6** 4- 15

*, statistically significant at the p<0.01 level when compared with saline control; **significant at the p<0.05 level (compared with saline controls); "f, percentage change of the mean value (%) relative to the mean value of the saline control. The results presented in TABLE 5 show that L-Lys-L-Glu did not significantly alter thymic weight, but did increase splenic weight (i.e., by about 18%). In contrast,

Thymogen induced a statistically significant decrease in thymic weight at both dosages, and had opposing effects on splenic weight at different dosages. Interestingly, the 1 mg/kg dosage of Thymogen administered in these studies approximates a clinically effective dosage in humans for immune stimulation.

The results presented in TABLES 6-10, show the effects of these agents on individual cell populations and their activities.

In TABLE 6 are shown the effects of L-Lys-L-Glu and Thymogen on the mean percentage of T- and B-lymphocytes isolated in the cell populations from the spleens of treated animals.

TABLE 6

Effects of L-Lys-L-Glu and Thymogen on the

Percentage of Splenic T- and B-lymphocytes (X±S.D.)

Dose ip Dose Lymphocytes (%)

Group (μg/ml) (mg/kg) Tcells (%) Changef Bcells (%) Changef

Saline Control 0.1 0 37.0±1.28 0.0 50.0±1.71 0.0

Lys-Glu 0.1 0.01 44.0±1.71* t l.2 50.6±2.57 1.0

Thymogen 10 1.0 34.8*2.5 4- 0.9 52.4±1.71 1.0 0.1 0.01 41.2±2.1* t l.l 50.8±3.0 1.0

*, statistically significant at the p<0.01 evel in comparison with the saline control group results; f , Change= % peptide treated/ % control. The results presented in TABLE 6 show that L-Lys-L-Glu and Thymogen slightly altered the percentage of splenic T-lymphocytes, but only the L-Lys-L-Glu effect was statistically significant. In these studies the B-lymphocyte content of the spleen did not seem to be affected by either agent.

In TABLE 7, below, are shown the results of studies with peritoneal macrophages and their state of activation as measured by the ability to reduce NBT.

TABLE 7

Effects of L-Lys-L-Glu and Thymogen on the

Innate State of Activation of Resident Peritoneal Macrophages as

Determined by NBT Reduction

Dose NBT Reduction (OD*,₀)*

Group (mg/kg) Spontaneous Inducedf

Saline Control 0 0.055±0.002 0.105±0.005

Lys-Glu 0.01 0.119±0.003** 0.208±0.017**

Thymogen 1 0.059±0.003 0.116±0.008 0.01 0.101±0.002** 0.205±0.012**

*, mean ± S.D. of four determinations for each group; **, statistically significant at the p<0.05 level from the values recorded in the control group; , macrophages induced with complement opsonized zymosan. The results presented in TABLE 7 show, i) as expected, complement-opsonized zymosan activated resident peritoneal macrophages (saline controls) by about 1.9-fold; ii) Treatment of animals with a six day ip treatment course of L-Lys-L-Glu (0.01 mg/kg) or Thymogen (0.01 mg/kg) resulted in a spontaneous level of macrophage activity that approximated that observed with the induced resident peritoneal macrophages (i.e., in the saline treated controls); iii) When macrophages from the L-Lys-L-Glu (0.01 mg/kg) or

Thymogen (0.01 mg/kg) treated animals were induced with complement-opsonized zymosan, 1.75-fold and 2-fold increases, respectively, in NBT reducing activity were observed. Interestingly, peritoneal macrophages from L-Lys-L-Glu treated animals appeared to be about 1.3-times more pinocytically active than those from

Thymogen-treated animals in reduction of neutral red dye (TABLE 8, below).

In TABLE 8, below, are shown the results of experiments designed to examine the pinocytic activity of resident peritoneal macrophages from the respective treatment groups.

TABLE 8

Effect of L-Lys-L-Glu and Thymogen on Uptake of Neutral Red by Resident Peritoneal Macrophages (X±S.D.)'

Dose Neutral Red Uptake

Group (mg/kg) No. of Assays Absorbance

Saline Control 0 5 0.488±0.026

Lys-Glu 0.01 5 0.802±0.006*

Thymogen 1 5 0.469±0.016 0.01 4 0.604±0.031** , mean absorbance values ± standard deviation of the mean; *, statistically significant at the p<0.01 level in comparison with the control values; **, p<0.05 The results presented in TABLE 8 show a significant 1.6-fold elevation of neutral red uptake by resident peritoneal macrophages from animals treated with ip injections of

L-Lys-L-Glu. Although a 75% increase in neutral red uptake was also observed with macrophages from Thymogen treated animals (0.01 mg/kg), this difference was not statistically different from the control values.

In TABLE 9, below, show the results of experiments designed to investigate the tissue response of neutrophils to an inflammatory agent, i.e., induced in the peritoneum by injection of sterile proteose peptone.

TABLE 9

Effects of L-Lys-L-Glu and Thymogen Intraperitoneal Pretreatment on Neutrophil Response to Proteose Peptone (X±S.D.)

Thymogen (0.1 mg/kg) doubled the number of neutrophils that could be induced following injection of proteose peptone.

It is interesting that the total volume of exudate obtained from Thymogen treated animals (0.01 mg/kg) was increased relative to the volume obtained from controls and the cell concentration was correspondingly decreased, i.e., 0.9 x 10⁶ for Thymogen versus

1.9 x 10⁶ for control. Exudate induced in L-Lys-L-Glu treated animals did not exhibit this property, nor did Thymogen at the test dose of 1 mg/kg, (i.e., L-Lys-L-Glu= 1.9 x 10⁶; and, Thymogen at 1 mg/kg= 1.8 x 10⁶). In TABLE 10, below, are shown the results of experiments designed to test the phagocytic activity of the neutrophil induced by the proteose peptone injection.

TABLE 10

Effect of L-Lys-L-Glu Pretreatment on

Phagocytic Activity of Neutrophils Responding to Induction

Phagocytic Activity

Dose ip Dose Phagocytes Phagocytic Index

Group (μg/ml) (mg/kg) (%) (No. bacteria/phagocyte)

Saline Control 0 0 18.8±0.29 2.05±0.10

Lys-Glu 0.1 0.1 28.7±0.51* 2.01±0.05

Thymogen 10 1 31.5±0.4* 1.89±0.03 0.1 0.1 17.8±1.13 1.90±0.05

*, statistically significant at the p<0.01 level in comparison with the values recorded in controls. The results presented in TABLE 10 show that the six day ip pretreatment course of

L-Lys-L-Glu and Thymogen (1 mg/kg) induced statistically significant 1.5-fold and

1.7-fold increases, respectively, in the percentage of phagocytic cells induced into the peritoneal cavity following injection of a sterile inflammatory challenge with proteose peptone. The phagocytic capacity of the neutrophils induced in this manner was finite, i.e., about two staphylococci per neutrophil in saline control or peptide treated animals.

Thymogen pretreatment at a dose of 0.01 mg/kg did not appear to stimulate neutrophil emigration in response to the sterile proteose peptone inflammatory stimulus (TABLE 9), nor the phagocytic activity of the cells so induced (TABLE 10).

The results presented in EXAMPLES 2-6, above, indicate the following: namely that: i) L-Lys-L-Glu and Thymogen treatment can cause redistribution of lymphoid cell populations in animals; ii) L-Lys-L-Glu treatment stimulated activity of resident peritoneal macrophages (while Thymogen did not); iii) L-Lys-L-Glu and Thymogen treatment stimulated both the number of neutrophils entering the tissue in response to a sterile inflammatory challenge and the percentage of phagocytically active cells in the cell exudate population; iv) Thymogen appeared to exhibit dosage-dependent differential effects, i.e., at the 1 mg/kg dose neutrophils were stimulated, but at the lower 0.01 mg/kg dose macrophages, and not neutrophils, were stimulated; vi) On a weight basis,

L-Lys-L-Glu appears to have a 100-1000 fold greater effect on lymphocyte tissue distribution than Thymogen, with a possible mobilization of thymic and bone marrow lymphocytes and increased T-lymphocytes in the spleen at the conclusion of the 6 day ip treatment regimen. The combined results indicate that treatment with L-Lys-L-Glu and

Thymogen can induce changes in distribution of lymphocyte cell populations, and changes in distribution and activities of macrophages and neutrophils, both of which changes favor a heightened state of innate immunity in the treated animals. For these and other reasons L-Lys-L-Glu and Thymogen are termed "immunomodulators".

Citations

1. Goldstein, A.L., Guha, A., Zatz, M.M. et al. 1972. Purification and biological activity of thymosin, a hormone of the thymus gland. Proc. Natl. Acad. Sci. USA 69 (7): 1800-1803.

2. Mashkovskii, M.D. 1988. "Medicinal Remedies, 2nd. Ed.", Chapter 9, Russia, pp. 168-175.

3. Arion, V.Y., Khavinson, V. Kh., Morozov, V.G. 1988. Comparative investigation of biological activity of Thymalin and synthetic peptide. Scientific conference "Biochemistry-Medicine", Russia, pp . 217-218.

4. Goldstein, A.I., Guha, A., Zatz, M.M., Hardy, M.K. and White. 1972. Proc. Natl.

Acad. Sci. USA. 69: 1800.

5. Khavinson, N.K., Morozov, N.G., Sery, S.N. and Yakolev, G.M. Treatment of immune-deficiency, immunodepressed or hyperactive immune states using the peptide L-Glu-L-Trp (Thymogen) for allergies, inflammatory diseases, etc. WO 92/217,191 (921015).

6. SERNA, Catalog 1987/88, Heidelberg, Germany, PE1-PE40.

7. Νekam, K., H.H. Fudenberg, AJ. Strelkanskas. 1982. Identification of "active"

T-lymphocytes among effector cells in guinea pigs. Immunopharmacol. 5 (1): 85-94.

8. Stadecker, M.J., Bishop, G., Wortis, H.H. 1973. Rosette formation by guinea pig thymocytes and thymus-derived lymphocytes with rabbit red blood cells. J. Immunol. 111(8): 4061-4065.

9. Reinherz, E ., Kung, P.C., Goldstein, G., Schlossman, S.F. 1979. Separation of functional subsets of human T cells by a monoclonal antibody. Proc. Νatl. Acad. Sci. USA 76 (8): 4061-4065.

10. Bianco, C, Patrick, R., Nussenzwieg, V. 1970. A population of lymphocytes bearing a membrane receptor for antigen-antibody complexes. I. Separation and characterization. J. Exp. Med. 132 (4): 702-720.

11. Shtorkh, V., Emmrikh, I.M. 1987. Determination of cell markers by the method of membrane immunofluorescence. Immunological methods, Medicine, Russia, pp. 254-268.

12. Jolyb, E.S. 1971. Brain-associated antigen: reactivity rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2: 353-361.

13. Rook, J.A.W. et al. 1985. J. Immunol. Methods 82: 161-167. 14. Methodological recommendations for evaluation of pharmaceutical preparations immunological properties. M., Russian Federation, 1992.

15. Methods of research with phagocytic cells during the evaluation of human Immune status. L., Russian Federation, 1986.

16. Laboratory methods of research in the clinic. M., Russian Federation, 1987, p. 310.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A pharmaceutical peptide preparation for inducing a heightened state of anti-microbial cellular or humoral immunity in a subject in need thereof consisting essentially of an L-Lys-L-Glu or L-Glu-L-Trp preparation and a pharmaceutically acceptable carrier.

2. The use of a peptide preparation for making a pharmaceutical preparation for inducing a heightened state of anti-microbial cellular or humoral immunity in a subject in need thereof, wherein the peptide preparation comprising L-Lys-L-Glu or L-Glu-L-Trp.

3. The pharmaceutical preparation of claim 2, wherein the immunity so induced is effective to eliminate a microbial infection selected from among a bacterial infection, a viral infection, or a parasitic infection.

4. The pharmaceutical preparation of claim 2, wherein the immunity so induced is manifest as a change in one or more laboratory indicia of disease activity selected from among a neutrophil or a lymphocyte cell count in peripheral blood, a hematocrit value, an elevated serum alpha globulin or immunoglobulin level, a value for a maturation and activation state marker of an immune cell, a ratio of percentages of immune cells having expression of two of said markers, a value for a synthetic activity of the immune cell, a value for an immunoglobulin specifically reactive with the microbe, a value for a phagocyte cell number in an immune cell exudate, a phagocytic index of the immune cell, a percentage of a lymphocyte subpopulation in a reticuloendotheiial tissue.

5. The method of claim 4, wherein the value for the cell surface molecule on the immune cell is selected from the group of maturation and activation state markers consisting essentially of CD2, CD3, CD4, CD28, B220, surface Ig, Rag 1, Rag 2, Ia Mac- 1, and 5'-nucleotidase, or the ratio of percentages of immune cells expressing the two marker is a CD4⁺/CD8⁺ ratio, or the synthetic activity of the immune cell is selected from the group consisting essentially of an immunoglobulin, an interleukin, an enzyme, an integrin, and an adhesin.

6. The method of claim 2, further the use of L-Lys-L-Glu to increase CD2⁺ lymphocytes in peripheral blood, or increase Fc receptor bearing lymphoid cells in spleen or in bone marrow, or increase a number of neutrophils in an infiltrate in a tissue in response to an inflammatory, or increase a percentage of phagocytically active cells in a neutrophil infiltrate.