Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Antibody synthesis is a defense response of higher vertebrates.
• the molecular entities which stimulate antibody synthesis e.g., a virus particle
• antigens e.g., a virus particle
• the introduction of an antigen into the body of a higher vertebrate stimulates specific white blood cells, B lymphocytes, to produce antibodies that combine specifically with the antigen to prevent its further multiplication., or to otherwise inactivate it.
• B lymphocytes specific white blood cells
• the study of antibodies and their action with antigens is a branch of immunology.
• Antibodies which circulate in blood or other body fluids are termed humoral antibodies, as distinguished from “membrane antibodies" which remainbound to their parent lymphocytes.
• immunoglobulin is used to generically refer Co all antibodies. In humans, all immunoglobulins are divided into five classes termed IgG, IgA, IgM, IgD and IgE. Each immunoglobulin molecule consists of two pairs of identical polypeptide chains. The larger pair termed “heavy chains” and designated gamma ( ⁇ ), alpha ( ⁇ ), mu ( ⁇ ) , delta ( ⁇ ) and epsilon ( ⁇ ), respectively, are unique for each immunoglobulin class and are linked together by disulfide (s-s) bonds between each chain.
• s-s disulfide
• Each heavy chain consists of about 400 to 500 amino acid residues linked together by polypeptide bonds.
• Each light chain by contrast, consists of about 200 amino acids and are usually linked to a heavy chain by a single disulfide bond.
• Gerald Edelman first determined the amino acid sequence of an entire human IgG molecule.
• V domains are also organized into structurally similar domains havin different amino acid sequences.
• the first or aminoterminal domain of both light and heavy chains have discrete regions within which considerable variation in amino acids occur. These domains are therefore termed variable (V) domains, and are designated V H in heavy chains and V L in light chains.
• V L and V H domains The molecular association of a V L and V H domain within an intact immunoglobulin forms an antigencombining site which may bind to a specific antigen with high affinity.
• the domain s gagtur e of all light chains is identical regardless of the associated heavy chain class. Each light chain has two domains, one V L domain and one domain with a relatively invariant amino acid sequence termed constant, light or C L .
• Heavy chains by contrast may have either three (IgG, IgA, IgD) or four (IgM, IgE) constant or C domains termed C H 1, C H 2, C H 3, and C H 4 and one variable domain, termed V H .
• C domains may be designated according to their heavy chain class; thus C ⁇ 4 indicates the C H 4 domain of the IgE (epsilon) heavy chain.
• C ⁇ 4 indicates the C H 4 domain of the IgE (epsilon) heavy chain.
• Visualization of antibodies by electron microscopy or by x-ray diffraction reveals that they have a "Y" shape.
• IgA and IgM antibodies in addition, combine in groups of two and five, respectively, to form dimers and pentamers of the basic Y shaped antibody monomer.
• fragments When antibodies are exposed to proteolytic enzymes such as papain or pepsin, several major fragments are produced.
• the fragments which retain antigen-binding ability consist of the two "arms” of the antibody's Y configuration and are termed Fab (fragment-antigen binding) or Fab '2 which represent two Fab arms linked by disulfide bonds.
• the other major fragment produced constitutes the single "tail” or central axis of the Y and is termed Fc (fragmentcrystalline) for its propensity to crystallize from solution.
• the Fc fragment of IgG, A, M, and D consists of dimers of the two carboxy-terminal domains of each antibody (i.e., C H 2 and C H 3 in IgG, IgA and IgD, and C H 3 and C H 4 in IgM.)
• the IgE Fc fragment by contrast, consists of a dimer of its three-carboxyterminal heavy chain domains (C ⁇ 2, C ⁇ 3 and C ⁇ 4).
• the Fc fragment contains the antibody's biologically "active sites” which enable the antibody to "communicate” with other immune system molecules or cells and thereby activate and regulate immune system defensive functions. Such communication occurs when active sites within antibody regions bind to molecules termed Fc receptors.
• Fc receptors are molecules which bind with high affinity and specificity to molecular active sites within immunoglobulin Fc regions. Fc receptors may exist as integral membrane proteins within a cell's outer plasma membrane or may exist as free, "soluble” molecules which freely circulate in blood plasma or other body fluids.
• IgE Fc receptors bind with high affinity to only IgE Fc regions or to isolated IgE Fc fragments. It is known that different types of classspecific Fc receptors exist which recognize and bind to different locations within the Fc region. For example, certain IgG Fc receptors bind exclusively to the second constant domain of IgG (C H 2), while Fc receptors mediating other immune functions bind exclusively to IgG's third constant domain (C H 3). Other IgG Fc receptors bind to active sites located in both C H 2 and C H 3 domains and are unable to bind to a single, isolated domain.
• Fc receptors mediate a variety of importantimmune killing and regulatory functions.
• Certain IgG Fc receptors mediate direct killing of cells to which antibody has bound via its Fab arms (antibody-dependent cell mediated cytotoxicity(ADCC)).
• Other IgG Fc receptors when occupied by IgG, stimulate certain white blood cells to engulf and destroy bacteria, viruses, cancer cells or other entities by a process known as phagocytosis.
• Fc receptors on certain types of white blood cells known as B lymphocytes regulate their growth and development into antibody-secreting plasma cells.
• Fc receptor for IgE located on certain white cells known as basophils and mast cells, when occupied by antigenbridged IgE, trigger allergic reactions characteristic of hayfever and asthma.
• Certain soluble Fc receptors which are part of the blood complement system trigger inflammatory responses able to kill bacter ia , viruses and cancer cells .
• Other Fc receptors stimulate certain white blood cells to secrete powerful regulatory or cytotoxic molecules known generically as lymphokines which aid in immune defense. These are only a few representative examples of the immune activities mediated by antibody Fc receptors.
• Fc receptors are, therefore, the critical link between antibodies and the remainder of the immune system. Fc receptor binding to antibody Fc region active sites may thus be characterized as the "final common pathway" by which antibody functions are mediated. If an antigen-bound antibody does not bind to an Fc receptor, the antibody is unable to activate the other portions of the immune system and is therefore rendered functionally inactive. Any peptide with the ability to bind to immunoglobulin Fc receptors has therapeutic usefulness as an immunoregulator by virtue of the peptide's ability to regulate binding to the receptor. Such an Fc receptor "blocker” occupies the immunoglobulinbinding site of the Fc receptor and thus "short circuits" the immunoglobulin's activating ability.
• the antibody's active sites when isolated and synthesized as peptides, can perform the immunoregulatory functions of the entire antibody molecule.
• the peptide may either stimulate or inhibit immune functions. Stimulation may occur if the Fc receptor is of the type that becomes activated by the act of binding to an Fc region or, alternatively, if an Fc active site peptide stimulates the receptor.
• the type of stimulation produced may include, but is not limited to, functions directly or indirectly mediated by antibody Fc region-Fc receptor binding.
• Examples of such functions include, but are not limited to, stimulation of phagocytosis by certain classes of white blood cells (polymorphonuclear neutrophils, monocytes and macrophages; macrophage activation, antibody-dependent cell mediated cytotoxicity (ADCC); natural killer (NK) cell activity; growth and development of B and T lymphocytes and secretion by lymphocytes of lymphokines (molecules with killing or immunoregulatory activities).
• white blood cells polymorphonuclear neutrophils, monocytes and macrophages
• ADCC antibody-dependent cell mediated cytotoxicity
• NK natural killer
• B and T lymphocytes and secretion by lymphocytes of lymphokines molecules with killing or immunoregulatory activities.
• the ability to stimulate immune system functions including those listed above, is known to be therapeutically useful in treating diseases such as infectious diseases caused by bacteria, viruses or fungi, conditions in which the immune system is deficient due either to congenital or acquired conditions, cancer and many other afflictions of human beings or animals
• Such immunostimulation is also useful to boost the body's protective cellular and antibody response to certain injected or orally administered substances administered as vaccines.
• This list is not intended to be all inclusive and merely provides representative examples of diseases or conditions in which immune stimulation has established therapeutic usefulness. Inhibition of immune system functions may occur if an active site peptide binds to a particular Fc receptor which is not activated by the mere act of binding to an Fc region.
• Fc receptors normally become “activated” only when several Fc regions withinan antigen-antibody aggregate or immune complex simultaneously bind to several Fc receptors, causing them to become “crosslinked”.
• Such Fc receptor crosslinking by several Fc regions appears to be the critical signal required to activate certain types of Fc receptors.
• an active site peptide By binding to and blocking such an Fc receptor, an active site peptide will prevent Fc regions within immune complexes or antigen-antibody aggregates from binding to the receptor, thus blocking Fc receptor activation.
• the ability to inhibit immune system functions is known to be therapeutically useful in treating diseases such as allergies, autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus, certain types of kidney diseases, inflammatory bowel diseases such as ulcerative colitis and regional enteritis (Crohn's disease), certain types of inflammatory lung diseases such as idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, certain types of demyelinating neurologic diseases such as multiple sclerosis, autoimmune hemolytic anemias, idiopathic (autoimmune) thrombocytopenic purpura, certain types of endocrinological diseases such as Grave's disease or Hashimoto's thyroiditis and certain types of cardiac disease such as rheumatic fever.
• diseases such as allergies
• Immunosuppression is also therapeutically useful in preventing the harmful immune "rejection" response which occurs with organ transplantation or in transplantation of bone marrow cells used to treat certain leukemias or aplastic anemias. This list is not intended to be all inclusive but merely provides representative examples of diseases or conditions in which immunosuppression is known to be therapeutically useful.
• amino acid components of the peptides are identified as abbreviations for convenience. These abbreviations are intended to include both the D- and L-forms although the L-form is preferred:
• the classical pathway for complement activation consists of a group of blood plasma proteins which are activated to form a biochemical cascade beginning with the first complement component, C1 and ending with a cellular "attack complex" consisting of C5, C6, C7, C8 and C9.
• the pattern of activation is highly specific and begins when C1 binds to Fc region active sites within antibody-antigen complexes of either IgG or IgM.
• a molecular subunit of C1, termed Clq contains six identical Fc receptors for IgG or IgM.
• Clq is an example of a "soluble" Fc receptor that exerts its bioactivity (complement activation resulting in cell lysis and inflammation) without being anchored to a cell surface membrane.
• one peptide with the aa was about as effective in activating Clq-mediated cell lysis as immune complexes formed by heat aggregated IgG.
• the aforementioned researchers attributed this activity to the peptide ' s ability to act as an active binding site for the Clq Fc receptor.
• Other synthetic peptides with sequences derived from th is region of IgG or from the aa 487-491 region of C H 4 of IgM (Glu-Trp-Met-Gln-Arg), e.g Lys -Phe-Asp-Trp-Ala-Val-Asp-Gly
• Trp-Leu and Tyr-Glu-Ala-Gly were inactive.
• peptides identical to IgG C H 2 residues 281-290 (Gly-Val-Gln-Val-His-Asn-Ala-Lys-Thr-Lys) and aa282292 (Val-Gln-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-OH) were approximately as active as inhibitors as intact monomeric IgG.
• Other peptides viz.
• aa272-290 Phe-Asn-Trp-Tyr-Val-Asp-Gly-Val-Gln-Val-His-Asn-AlaLys-Thr-Lys
• aa275-279 Ac-Phe-Asn-Trp-Tyr-Val
• aa289-292 Thr-Lys-Pro-Arg
• Tuftsin is a tetrapeptide, with sequence Thr-Lys-Pro-Arg, and is present in the second constant domain of all human IgG subclasses and in guinea pig IgG at aa 289-292. It was originally isolated from proteolytic digests of IgG by Najjar who found it to stimulate phagocytosis by granulocytes, monocytes and macrophages in vitro and is described in U.S. Patent No. 3,778,426. Subsequent studies have shown Tuftsin to be active at nanomolar concentrations in many species including humans , cows , dogs , rabbits , guinea pigs and mice.
• Tuftsin In addition to its phagocytosis stimulating ability, Tuftsin has been shown to stimulate ADCC, Natural Killer (NK) cell activity, macrophage-dependent-T-cell education and antibody synthesis to T-cell-dependent and independent antigens in vitro and in vivo. Tuftsin is believed to act by binding to stereospecific receptors on granulocytes, macrophages and lymphocytes. Analysis of these receptors indicate that they resemble IgG Fc receptors in both number and ligand dissociation constants. Recent studies by Ratclfffe and Stanworth (Immunol.
• Tuftsin does bind to IgG Fc receptors since it competitively inhibits human IgG binding to human monocyte IgG Fc receptors. Tuftsin's immunostimulatory abilities are thus attributed to its ability to bind to immunoglobulin Fc receptors.
• Rigin is a tetrapeptide analogue of Tuftsin located in the peptide region of human IgG which spans the C H 2 and C H 3 domains at aa 341-345 having sequence Gly-Gln-Pro-Arg. (Veretennikova, et al., Int. J. Peptide Protein Res., 17, 430 (1981)). It has phagocytosis-stimulating abilities similar to those of Tuftsin and is described in U.S. Patent No. 4,353,823.
• This peptide (Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-ArgGlu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Ser-Arg-Glu-Glu-Met) and the 23 residue peptide lacking the carboxy-terminal methio ⁇ ine probably acts by binding to lymphocyte Fc receptors for IgG.
• This invention describes the sequences of new and useful peptides that can block the binding of human IgG immune complexes to IgG Fc receptors on human polymorphonuclear neutrophils (PMNs), of IgG and IgE immune complexes to IgG and IgE Fc receptors on monocytes and macrophages (MMs) and other white blood cells.
• PMNs human polymorphonuclear neutrophils
• MMs macrophages
• a further object is to describe how such new and useful peptides and their analogs and derivatives may be used to treat certain human diseases in which immune complexes contribute to disease pathogenesis.
• diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, serum sickness, polyarthritis nodosa and other forms of vasculitis, and other diseases described under the generic category of "autoimmune diseases", idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, asthma and other diseases and conditions.
• a still further object is to describe how such peptides can prevent destruction of erythrocytes (red blood cells) and platelets (blood clotting cells) as occurs in certain autoimmune diseases such as autoimmune hemolytic anemias and idiopathic thrombocytopenic purpura, respectively.
• a still further object is to describe how certain of these peptides bind to Fc receptors on other classes of white blood cells (lymphocytes and basophils) which enable such peptides to perform additional therapeutically useful functions.
• PMNs and MMs monocytes are immature macrophages
• PMNs contain cellular organelles known as lysosomes which contain proteolytic enzymes and inflammatory mediators sequestered in a latent, inactive form.
• lysosomes cellular organelles known as lysosomes which contain proteolytic enzymes and inflammatory mediators sequestered in a latent, inactive form.
• PMNs and MMs are stimulated to release the contents of their lysosomes to the external cellular millieu, resulting in considerable tissue inflammation and cell death.
• Such inflammation and cell death are mediated by a variety of biologically active substances.
• the lysosomal granules of PMNs and MMs contain many different proteolytic and glycolytic enzymes (viz. collagenase which degrades collagen, elastase which degrades elastin, lysozyme which degrades certain types of carbohydrate polymers, etc.) which cause direct destruction of cartilage, connective tissue and cells. Certain of these enzymes may also activate the blood clotting system or inflammatory peptide mediators such as bradykinin or complement. Other important inflammatory mediators released by PMNs and MMs include prostaglandins (PGs) and leukotrienes (LTs) which have a variety of potent inflammatory and immunomodulatory effects. Some of these effects, together with the elicting substance are enumerated below.
• proteolytic and glycolytic enzymes viz. collagenase which degrades collagen, elastase which degrades elastin, lysozyme which degrades certain types of carbohydrate
• Prostaglandin E2 (PGE2)- PGE2 produces vasodialation, erythema, increased vascular permeability, edema (swelling), potentiates the inflammatory and pain producing actions of histamine and bradykinin and stimulates bacterial endotoxin-induced collagenase production by leukocytes. (Kuehl, et al., Science, 210, 978 (1980)).
• PGE2 also has potent immunosuppressive effects and can suppress NK and ADCC-mediated killing of cancer cells, T cell colony growth, clonal proliferation of the committed granulocyte-macrophage stem cell and antigen and mitogen-induced B-lymphocyte maturation into antibody-secreting plasma cells. PGE2 also directly activates short-lived T suppressor lymphocytes which can, in turn, suppress other protective immune functions. Such PGE2-mediated suppression of the immune system is thought to be important in producing the immunosuppression that frequently accompanies various forms of cancer. (Goodwin, Clin. Immunol. Immunopath., 15, 106 (1980); Stenson, et al., Immunol.
• Leukotrienes C4 (LTC4), D4 (LTD4) and E4 (LTE4) -
• SRS-A anaphylaxis
• LTB4 Leukotriene B4
• LTB4 is one of the most potent chemotatic substances known for certain human leukocytes. It is produced by preparations of human peripheral leukocytes, neutrophils, lung tissue and other cells. LTB4 attracts neutrophils, and eosinophils, both of which are present in high numbers at sites of inflammation. LTB4 also stimulates the release of lysosomal enzymes, including lysozyme, from neutrophils which directly mediates tissue destruction. LTB4 is probably involved in the pathogenesis of many inflammatory conditions, including rheumatoid arthritis in which LTB4 is found in elevated concentrations in affected joints. (Samuelsson, Science, 220, 568 (1983); Science, 215, 1382 (1982)).
• Immune complexes also cause PMNs and MMs to release highly reactive free radicals such as the superoxide anion (O 2 -) which directly damages tissue which it contacts.
• O 2 - superoxide anion
• IgG. and IgE containing immune complexes, aggregates or objects to which IgG or IgE is attached via Fab-mediated binding or by passive surface adsorption are potent stimulators of phagocytosis (engulfment and digestion of complexes, particulate matter and cells) by PMNs and MMs and subsequent enzyme and inflammatory mediator release.
• Such stimulation is known to depend on the binding of IgG or IgE Fc regions within immune complexes to Fc receptors located on the PMN or MM cell surface.
• IgG or IgE antigen complexes heat aggregated IgG or IgE, IgG or IgE passively adsorbed to cell or particulate surfaces
• IgG or IgE antigen complexes heat aggregated IgG or IgE, IgG or IgE passively adsorbed to cell or particulate surfaces
• the critical factor in common to these stimulatory forms of IgG or IgE is that multiple Fc regions in a fixed, relatively immobilized form be present to simultaneously bind to multiple PMN or MM Fc receptors Such multiply engaged Fc receptors then trigger lysosomal enzyme or inflammatory mediator release.
• RA rheumatoid factors
• RFs rheumatoid factors
• Idiopathic Pulmonary Fibrosis is a condition in which the normally thin, gas permeable wall of the lung's respiratory unit, the alveolus, is greatly thickened and replaced with large amounts of relatively gas-impermeable, fibrous connective tissue. This greatly reduces the lung's ability to respire and may lead to chronic pulmonary incapacitation and death. IPF frequently accompanies idiopathic interstitial pneumonias and the interstitial pneumonias of rheumatoid arthritis, systemic lupus erythematosus, scleroderma and polymyositis-dermatomyositis. (Dreisin, et al., N. Engl. J. Med.
• IPF tissue destruction in IPF
• the final common pathway leading to the tissue destruction in IPF is believed to involve IgG-immune complexes produced either systemically or locally within the lung parenchyma.
• Localization of immune complexes within the lung leads to an influx of PMNs and monocytes from blood which accumulate in large numbers within the interstitium and within alveolar structures. Monocytes then develop into mature macrophages and join the normally present pulmonary macrophages.
• IgG Fc regions within the immune complexes then combine with PMN and MM Fc receptors causing lysosomal enzyme and inflammatory mediator release, inflammation and alveolar destruction.
• Some of the peptides detailed in the present invention can block IgG-immune comp-lex binding to IgG Fc receptors on PMNs, monocytes and macrophages and can thereby reduce or prevent inflammation and tissue destruction of idiopathic pulmonary fibrosis and other immune complex-mediated diseases.
• the glomeruli of kidneys are the filtration devices which separate from blood the plasma ultrafiltrate that ultimately becomes urine. Glomeruli are easily damaged by the inflammatory processes initiated by immune complexes which accumulate as a result of the blood filtration process.
• monocytes and macrophages accumulate in the glomerular mesangium where they encounter immune complexes.
• IgG Fc regions within these complexes bind to IgG Fc receptors of the monocytes and macrophages and are thereby stimulated to release lysosomal enzymes and the inflammatory mediators previously discussed. These substances produce glomerular inflammation, (glomerulonephritis) which may lead to kidney failure and subsequent death.
• Immune complex-mediated glomerulonephritis and resultant kidney failure is the single leading cause of death in patients with systemic lupus erythematosis.
• Many other conditions such as rheumatoid arthritis, other autoimmune diseases, infectious diseases such as streptococoal or hepatitis virus infection and others are accompanied by glomerulonephritis caused by immune complexes.
• Some of the peptides detailed in the present invention can block IgG immune complex binding to monocyte and macrophage IgG Fc receptors and can thereby reduce or prevent inflammation and tissue destruction of immune complex-mediated glomerulonephritis .
• Hypersensitivity Pneumonitis includes a spectrum of conditions characterized by granulomatous interstitial and alveolar-filling lung diseases associated with exposure to a wide range of inhaled organic dusts and particles. Affected individuals synthesize relatively large amounts of IgG directed against the offending inhaled dust and produce IgG immune complexes within the lung parenchyma. These complexes bind to IgG Fc receptors of PMNs, monocytes and pulmonary macrophages which, in a manner similar to that previously discussed, are stimulated to release lysosomal enzymes and inflammatory mediators which produce an accute pneumonia.
• the lung damage may become permanent in the form of chronic granulomatous interstitial pneumonitis.
• Some of the peptides detailed in the present invention can block IgG-immune complex binding to IgG Fc receptors of PMNs, monocytes and macrophages and can thereby reduce or prevent inflammation and tissue destruction of hypersensitivity pneumonitis and other immune-complex mediated diseases.
• Atopic (IgE-mediated) asthma is an inflammatory lung disease in which IgE bound to pulmonary mast cells and circulating basophil Fc receptors causes them to release inflammatory mediators upon exposure to the sensitizing allergan (antigen). It is also known that IgE not already bound to cellular Fc receptors may also bind to the sensitizing allergan to form IgE-allergan immune complexes. These circulating IgE immune complexes may then bind to monocyte or macrophage IgE Fc receptors causing them to release the various inflammatory mediators previously discussed. Additionally, IgG directed against the sensitizing allergan may be present and may also produce IgG-allergan immune complexes.
• IgG Fc receptors on PMNs and monocytes and macrophages in the lungs and thereby contribute to lysosomal enzyme and inflammatory mediator release.
• Some of the peptides detailed in the present invention can block IgE-immune complex binding to IgE Fc receptors on monocytes and macrophages and can thereby reduce or prevent inflammation characteristic of asthma and other disease pathogenesis.
• This autoimmune disease arises when certain immune system cells recognize antigens on erythrocytes (red blood cells) as foreign and cause the synthesis of antibodies (usually IgG) directed toward the erythrocytes.
• This "anti-erythrocyte Ig” then binds to the erythrocyte via its Fab antigen-binding arms, leaving the Fc region exposed to the erythrocyte exterior.
• Macrophages, and to a lesser extent monocytes then bind to such IgG-sensitized erythrocytes via cell surface IgG Fc receptors. Most such binding occurs in the spleen and results in phagocytosis and destruction of erythrocytes and subsequent anemia.
• Some of the peptides detailed in the present invention can block IgG receptors on monocytes and macrophages and can therefore reduce or prevent IgG Fc-mediated destruction of erythrocytes and subsequent anemia.
• ITP Idiopathic (Autoimmune) Thrombocytopenic Purpura
• ITP is a syndrome characterized by chronic thrombocytopenia (low platelet count) caused by a circulating antiplatelet antibody that results in platelet destruction by phagocytic leukocytes.
• the antibody is of the IgG class and is directed toward a normally present platelet-associated antigen.
• IgG-coated platelets encounter macrophages and monocytes, especially in the spleen, the exposed IgG Fc regions on the platelets bind to IgG Fc receptors oh macrophages and monocytes and stimulate platelet phagocytosis and destruction.
• Some of the peptides detailed in the present invention can block macrophage and monocyte IgG Fc receptors and thus represent a significant improvement over intervenous IgG since peptides are relatively inexpensive to produce and do not carry with them the risk of infectious disease transmission (e.g., hepatitis) which accompanies human blood products.
• the peptides disclosed in this invention may have other important effects on the immune system which may prove to be therapeutically efficacious in treating human diseases.
• monocytes when IgG complexes bind to monocyte IgG Fc receptors, monocytes secrete prostaglandin E2 (PGE2) which inhibits many important immune defense functions. These inhibited functions include Natural Killer (NK) cell and ADCC killing of cancer cells, growth and development of T lymphocytes, and granulocyte-macrophage stem cells and antigen and mitogen-induced B-lymphocyte maturation into protective antibody-secreting plasma cells. Additionally, PGE 2 also directly activates short-lived suppressor lymphocytes which may in turn, suppress other protective immune functions.
• the peptides described in this invention which block IgG immune complexes to monocyte IgG Fc receptors may, therefore be expected to act as immunostimulants by virtue of their ability to inhibit immune complexmediated PGE 2 secretion.
• Some of the peptides described in the present invention also demonstrate the ability to block IgG and/or IgE immune complex binding to lymphocyte Fc receptors. Such abilities may be expected to be therapeutically useful in several ways:
• Immunostimulation All antibodies are synthesized by plasma cells which develop from B lymphocytes. B lymphocyte-toplasma cell development is, therefore, critical for the production of protective antibody synthesis. Substances which inhibit this development may result in an inadequate concentration of antibodies and may render a person susceptible to infection. Of particular importance to the present invention, is the fact that complexes or aggregates of antigen and antibody, termed immune complexes, are potent inhibitors of B lymphocyte development. Such inhibition is thought to occur by several mechanisms, all of which require that Fc regions of antibodies within the immune complex bind to lymphocyte Fc receptors.
• IgG or IgE Fc receptorblocking peptides may stimulate antibody synthesis involves immunoglobulin-binding factors (IBFs).
• IBFs are soluble Fc receptors released from activated T lymphocytes, PMNs and possibly monocytes and macrophages. IgG IBF is a potent suppressor of B lymphocyte development into antibody-secreting plasma cells.
• IgG IBF In order to produce such suppression, it is believed that IgG IBF must first bind to Fc regions of IgG molecules and to molecular structures on the surface of B lymphocytes. It is known that IgG IBFmediated suppression is abrogated if IBF is prevented from binding to IgG Fc regions. (Fridman, et al., Immunol. Rev., 56, 51 (1981); Bich-Thuy, J. Immunol., 129, 150 (1982)). It is therefore expected that peptides which bind to and thus block IBF Fc receptors should also block IBF-mediated B lymphocyte suppression These peptides should cause stimulation of protective antibody synthesis.
• TRF T cell Replacing Factor
• TRF directly stimulates B lymphocyte development and prevents immune complexes from inhibiting development.
• Some of the peptides described in the present disclosure can, like TRF, bind to B lymphocyte Fc receptors and are therefore expected to share some of TRFs immunostimulatory activities.
• T lymphocytes proliferate and differentiate into functionally distinct cells with helper, suppressor, inducer or killer activities. These cells may then, in turn, directly participate in immune defense or may activate the defensive functions of other leukocytes such as B lymphocytes or monocytes/macrophages.
• AMLR autologous mixed lymphocyte reaction
• IgG immune complexes are potent inhibitors of the AMLR. Immune complexes are thought to directly exert their inhibitory . effects on T lymphocytes by binding to IgG Fc receptors of the T lymphocytes which normally participate in the AMLR reaction.
• the AMLR is greatly suppressed in patients with a variety of autoimmune or neoplastic diseases including systemic lupus erythematosus (Sakane, et al., Proc. Natl. Acad. Sci. U.S.A., 75, 3464 (1978)), Sjögren's syndrome (Miyasaka, et al., J. Clin. Invest., 66, 928 (1980)), primary biliary cirrhosis (James, et al., J. Clin. Invest., 66 , 1305 (1980)), Hodgkin's lymphoma (Engleman, et al., J. Clin.
• Fc receptors may also be expected to stimulate cellular or delayed type hypersensitivity (DTH) which is known to be important in defense against cancer and certain infectious diseases such as tuberculosis.
• DTH delayed type hypersensitivity
• Immune complexes can significantly inhibit DTH in experiments using mice (Efouvas, Ann. Immunol. Inst. Pasteur, 132C, 307 (1981)). Such inhibition is known to be dependent on the presence of Fc regions within the immune complex which bind to cellular Fc receptors.
• Antibody-dependent cell-mediated cytotoxity is a process by which T lymphocytes, monocytes/ macrophages and polymorphonuclear neutrophils destroy foreign or infectious cells.
• IgG antibodies must first bind to antigens on the target cell which sensitizes the cell for recognition by ADCC cells.
• IgG Fc receptors on the ADCC cell bind to exposed Fc regions on the surface of the target cell. Such Fc receptor binding activates the ADCC cell to directly lyse the target cell, causing its death.
• ADCC Inappropriate cell killing via. ADCC is thought to mediate some of the inflammation and organ destruction which occurs in chronic active hepatitis (Cochrane, et al., Lancet, 1, 441 (1976)), Ulcerative colitis (Hibi, et al., Clin. Exp. Immunol., 49, 75 (1982)). Hashimoto's thyroiditis (Calder, et al., Clin. Exp. Immunol., 14, 153 (1973)), and other conditions. Some of the peptides disclosed in the present invention can block lymphocyte IgG Fc receptors and are therefore useful to block ADCC-mediated killing. Such inhibition is expected to prevent much of the inflammation and tissue destruction characteristic of the above diseases. Peptides Which Exert A Direct Anti-Allergic Effect
• IgE-mediated allergies viz. allergic rhinitis (hayfever), types of asthma, allergic reactions to insect stings
• IgE Fc receptors located on mast cells and basophils.
• mast cell and basophil IgE Fc receptors may be blocked by administering either chemically isolated IgE Fc fragments or certain peptides that are described in the United States and foreign patents discussed above. Such compounds have affinity for the IgE Fc receptor and thus bind to it and prevent IgE from binding.
• the allergic response can be abrogated independent of the par ticular ant igen wh ich el ic i ted IgE synthes is .
• Certain of the peptides described in the present invention can bind to IgE Fc receptors on human basophils and can block subsequent IgE binding.
• Thes e peptides therefore have antiallergic properties which may be useful in the treatment of allergy in animals and in humans.
• this invention is concerned with a method of blocking immune-complex-mediated inflammation, new peptides having therapeutic value in various areas, therapeutic compositions containing these peptides, and methods for use thereof.
• the present invention provides active site peptides having the following sequences:
• these functional groups include such normal substitution as acylation on the free amino group, particularly by formyl or acetyl groups, and amidation on the free carboxylic acid group, as well as the substitution of amino acids such as the, D-isomers of the naturally occuring amino acids.
• the peptides of this invention are highly unusual since they are able to bind to and block cellular Fc receptors in the same manner as the much larger parent IgG or IgE molecules, a portion of which they resemble. It is especially unusual and surprising that some of these peptides can bind to and block (i) IgG receptors of PMNs; ( ⁇ ) IgG and IgE receptors of lymphocytes; and
• peptides to retain their biological activity and natural folding is illustrated by the fact that they have Fc receptor binding activity similar to the parent antibody molecules. While the peptides of the present invention are believed to act by "blocking" Fc receptor activity as.described herein, it is not intended that the present invention be limited to any particular mechanism of action.
• the Fc receptor blocking activity of the subject peptides was assessed using well established techniques and procedures.
• a rosette assay was employed which uses either monomeric, chemically or heat aggregated human IgG or IgE obtained from human myeloma sera.
• the IgG or IgE is then adsorbed to ox-erythrocytes which has been previously treated with trypsin and pyruvic aldehyde.
• Fresh human cell PMNs, monocytes and basophils from a patient with basophilic chronic myelogenous leukemia or human cell lines were then incubated in the presence or absence of inhibitors (monomeric IgG or IgE or peptides) at equal concentrations for fifteen minutes before addition of the indicator oxerythrocytes.
• inhibitors monomeric IgG or IgE or peptides
• the cells expressing surface Fc receptors formed rosettes, clusters of three or more indicator erythrocytes that bound to the cells by virtue of exposed Fc regions on the erythrocyte surface.
• inhibitor myeloma proteins or inhibitor peptides produced a reproducible reduction of rosette formation, calculated by dividing the percentage of rosettes formed when the diluent, phosphate buffered isotonic saline, was used alone as control and then multiplying the calculated quotient by 100.
• Table 1 presents the inhibition observed by representative peptide inhibitors calculated as a percentage of the inhibition observed with intact, monomeric myeloma proteins. Thus, 50 percent inhibition implies that the peptide was 50% as inhibitory as the intact myeloma protein to which it was compared.
• Table 2 demonstrates representative immunostimulatory and immunomodulatory activity of one of the subject peptides, viz. Thr-Ile-Ser-Lys-Ala-LysGly-Gln-Pro-Arg when incubated with normal human mononuclear cells (lymphocytes and monocytes) separated by ficoll-metronidizole gradient.
• Mitogenic lectins are thought to mimic some of the cellular events that accompany antigenic stimulation of leukocytes and are useful indicators of cellular immune reactivity, in vitro. Following lectin addition, the cells were cultured for several days and, at specified intervals, "pulsed" with radioactive Iododeoxyuridine which becomes incorporated into newly synthesized DNA of dividing cells.
• the amount remaining cell-associated radioactivity may be used to estimate the degree to which the cells were stimulated to proliferate.
• Table 2 shows that the peptide strongly inhibited the mitogenic effects of the six mitogens indicating that the peptide can substantially modulate the proliferative ability of immunologically important mononuclear cells and thus has use as an immunomodulator.
• the peptide alone also stimulated mitogenesis (proliferation) of mononuclear cells by 50% on day 3, 331% on day 5 and 283% on day 7. This indicates that the peptide can act as an immunostimulant by enhancing proliferation of mononuclear cells.
• Other tests which can be used to check the activities of the peptides are described by
• an effective amount of a polypeptide or derivative thereof, or a pharmaceutical composition containing same, as defined above is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents such as antihistamines, corticosteroids, and the like.
• These compounds or compositions can thus be administered orally, sublingually, topically (e.g. on the skin or in the eyes), parenterally (e.g., intramuscularly, intravenously, subcutaneously or intradermally), or by inhalation, and in the form of either solid, liquid or gaseous dosage including tablets, suspensions, and aerosols, as discussed in more detail hereinafter.
• the administration can be conducted in single unit dosage form with continuous therapy or in single dose therapy ad libitum.
• the method of the present invention is practiced when the relief of symptoms is specifically required or perhaps imminent; in another preferred embodiment, the method hereof is effectively practiced as continuous or prophylactic treatment.
• the effective dosage in .accordance herewith can vary over a wide range.
• an effective systemic dosage in accordance herewith can best be described as between 2 x 10 3 and 2 x 10 6 times the Fc receptor content, on a molar scale. For an average subject this would be between about 0.5 and 500 mg/kg/day, depending upon the potency of the compound. Of course, for localized treatment, e.g., of the respiratory system, proportionately less material will be required.
• compositions hereof can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, powders, enterically coated or other protected formulations (such as by binding on ion exchange resins or other carriers, or packaging in lipid-protein vesicles or adding additional terminal amino acids or replacing a terminal amino acid in the L-form with one in the D-form), sustained release formulations, solutions (e.g., opthalmic drops), suspensions, elixirs, aerosols, and the like.
• the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like.
• Suitable liquid carriers include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
• compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like.
• conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like.
• Suitable pharmaceutical carriers and their formulation are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
• Such compositions will, in any event, contain an effective amount of the active compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the host.
• the therapeutic agents be relatively non-toxic, nonantigenic and non-irritating at the levels in actual use.
• the peptides of this invention are well tolerated. They are distinguished by low toxicity, particularly by the absence of cytotoxic effects, and may, f.e., be used for the treatment of patients with depressed neutrophil functions, f.e., neonatal, aged, or immunosuppressed patients; for suppressing the immunologic rejection of transplanted organs; for treating allergies, autoimmune diseases like rheumatoid or psoriatric arthritis , lupus erythematosus , ulcerative colitis, regional enteritis (Crohn's disease), sero-negative spondyloarthropathies, spondylitis ankylosa, Reiter's syndrome, hypersensitivity angiitis, hemolytic anemia, Grave's disease, demyelinating neurological diseases such as multiple sclerosis, postviral encephalomyelitis, contact dermatitis, psoriasis, diabetes mellitus (type I), rheumatic fever, certain types of leuk
• B is Val
• C is Leu
• D is His
• E is Gin
• F is Asn
• G Trp
• H Leu
• I Asp
• J Gly
• K Lys
• L is Glu
• M is Tyr and N is Val.
• Z is Asn, A is Asn, B is Gly, C is Gin, D is Pro,
• E is Glu
• F is Asn
• G is Asn
• H is Tyr
• I is Lys
• 3 is Thr
• K is Thr.
• Z is Asn, A is Glu, B is Val, C is Gin, D is Leu,
• E Pro
• F Asp
• G Ala
• H Arg
• I His
• J is Ser
• K is Thr
• L is Thr
• M is Gin
• N is Pro
• O is Arg
• P is Lys
• Q is Thr
• R is Lys.
• B is Val
• C is Gly
• D is Thr
• E is Arg
• F is Asp
• G is Ala
• H is He
• I is Glu
• J is Gly
• K is Glu
• L is Thr.
• X is Ser
• Y is Lys
• Z is Lys
• A is Thr
• B is lie
• C is Ser, D is Lys, E is Ala, F is Lys, G is Gly,
• H is Gin
• I Pro
• J is Arg
• K i s Gl u L is P ro ,
• M is Gin , N is Val and 0 is Tyr.
• B is Ser, C is Thr, D is Thr, E is Lys, F is Thr,
• G is Ser
• H is Gly
• I is P ro
• J i s Ar g is Al a
• L is Ala , M is Pro, N is Glu, 0 is Val and P is Tyr SYNTHESIS OP PEPTIDES
• Peptides of this invention may be synthesized by any techniques that are known to those in the peptide art, f.e. those described in Houben-Weyl, Methoden der Organischen Chemie, Vol. 15/11, pages
• Peptides of this invention are preferably synthesized by the solid phase peptide synthesis (or Merrifield) method. This established and widely used method is described, including the experimental procedures, in the following references:
• the C-terminal end of the growing peptide chain is covalently bound to a resin particle and amino acids having protected amino groups are added in the stepwise manner indicated above.
• a preferred amino protecting group is the t-BOC group, which is stable to the condensation conditions and yet is readily removable without destruction of the peptide bonds or racemization of chiral centers in the peptide chain. At the end.
• the final peptide is cleaved from the resin, and any remaining protecting groups are removed, by treatment under acidic conditions such as, for example, with a mixture of hydrobromic acid and trifluoroacetic acid or with hydrofluoric acid, or the cleavage from the resin may be effected under basic conditions, for example, with triethylamine, the protecting groups then being removed under acid conditions.
• acidic conditions such as, for example, with a mixture of hydrobromic acid and trifluoroacetic acid or with hydrofluoric acid
• the cleavage from the resin may be effected under basic conditions, for example, with triethylamine, the protecting groups then being removed under acid conditions.
• the cleaved peptides are isolated and purified by means well known in the art such as, for example, lyophilization followed by either exclusion or partition chromatography on polysaccharide gel media such as Sephadex G-25, or countercurrent distribution.
• the composition of the final peptide may be confirmed by amino acid analysis after degradation of the peptide by standard means.
• Salts of carboxyl groups of the peptide may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as for example sodium carbonate or sodium bicarbonate; or an amine base such as for example triethylamine, triethanolamine, and the like.
• a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
• a metal carbonate or bicarbonate base such as for example sodium carbonate or sodium bicarbonate
• an amine base such as for example triethylamine, triethanolamine, and the like.
• Acid addition salts of the polypeptides may be prepared by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid.
• Esters of carboxyl groups of the polypeptides may be prepared by any of the usual means known in the art for converting a carboxylic acid or precursor to an ester.
• One preferred method for preparing esters of the present polypeptides when using the Merrifield synthesis technique described above, is to cleave the completed polypeptide from the resin in the presence of the desired alcohol either under basic or acidic conditions, depending upon the resin.
• the C-terminal end of the peptide when freed from the resin is directly esterified without isolation of the free acid.
• Amides of the polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide.
• a preferred method for amide formation at the C-terminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
• N-acyl derivatives of an amino group of the present polypeptides may be prepared by utilizing an N-acyl protected amino acid for the final condensation or by acylating a protected or unprotected peptide.
• O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and Oacylation may be carried out together, if desired.
• the coupling, deprotection/cleavage reactions and preparation of derivatives of the subject polypeptides are suitably carried out at temperatures between about -10 and +50°C, most preferably about 20-25°C.
• the exact temperature for any particular reaction will of course be dependent upon the substrates, reagents, solvents and so forth, all being well within the skill of the practitioner. Illustrative reaction conditions for these processes may be gleaned from the examples.
• the first step of purification was by CCD (counter current distribution) using the solvent system nrbutanol: acetic acid: water: ethanol in 4:1:5:0.02 ratio. After 200 transfers tubes 39-59 were pooled to yield 1.2 g material. It showed some tailing by TLC (thin layer chromatography) in the solvent system acetic acid : ethyl acetate : water : n:butanol
• Each of the peptides of the present invention may be prepared by an analogous procedure by the stepwise addition of the desired amino acid to the growing peptide chain which is bound by a covalent bond to the solid resin.
• the dry peptide resin (6.8 g) was placed into the reaction vessel of the HF cleavage apparatus and was treated with 60 ml of liquid HF and 6 ml of an-isole at 0°C for 60 minutes. After extraction and lyophilization 1.5 g crude peptide was isolated. The entire batch was applied to purification by CCD (counter current distribution) using the solvent system n-butanol : acetic acid : water in 4:1:5 ratio. After 200 transfers 1.35 g material was collected from tubes 6-24. Rf 0.27. EXAMPLE 3
• HF cleavage was effected by addition of 5.0 ml anis-qle, a few drops of dimethylsulfide and 40 ml of liquid HF and stirring the mixture at 0°C for 60 minutes. The HF was removed and the residual material was washed with diethyl ether and extracted with 60 ml of 50% aqueous acetic acid followed by 240 ml water. The solution was lyophilized to give 1.8 g crude material, which was submitted to purification by counter current distribution using the solvent system n-butanol : acetic acid : water in ratio 4:1:5 in 270 transfers.
• the main fraction (tubes 26-37) were further purified by carboxymethyl cellulose ion exchange chromatography with a linear gradient (0.01-0.3 M) of ammonium acetate buffer of pH 4.5.
• the material corresponding to the main peak was purified by partition chromatography on a Sephadex G25 gel column. Rf 0.55.
• Thr-Arg-Ala-Glu 300 mg was purified by a G-25 partition column- using n-BuOH: HOAc:H 2 O 4:1:5 upper layer as the eluent to afford partially purified Thr-Arg-Ala-Glu (163 mg).
• This peptide was further purified by reversed phase liquid chromatography (C-18, 40 micron) using 0.1% HOAc as the eluting solvent to afford pure Thr-Arg-Ala-Glu (54 mg). Rf 0.31.
• the dry peptide resin (4.1 g) was placed into the reaction vessel of the HF cleavage apparatus and was treated with 40 ml of liquid HF and 5.0 ml of anisole at 0oC for 60 minutes. After extraction and lyophilization 750 mg crude peptide was isolated.
• the dry peptide resin (7.3 g) was placed into the reaction vessel of the HF cleavage apparatus and was treated with 60 ml of liquid HF and 8 ml anisole at 0°C for 60 minutes. After extraction and lyophilization approximately 1 g crude peptide was isolated.
• N-acyl derivatives being prepared by replacing the terminal t-BOC-amino acid with the appropriate N-acylamino acid, e.g. N-acetyl-glutamine (Ac-Gin):
• this peptide may be converted to other metallic salts, e.g., K, Li, Ba, Mg, ammonium, Fe(II), Zn, Mn(II) and Al salts by substitution of the appropriate base.
• metallic salts e.g., K, Li, Ba, Mg, ammonium, Fe(II), Zn, Mn(II) and Al salts by substitution of the appropriate base.
• this peptide may be converted to other acid addition salts, e.g., the hydrobromide, sulfate, phosphate, nitrate, acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, malate ascorbate, and benzoate salts, by substituting the appropriate acid for hydrogen chloride.
• acid addition salts e.g., the hydrobromide, sulfate, phosphate, nitrate, acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, malate ascorbate, and benzoate salts, by substituting the appropriate acid for hydrogen chloride.
• esters of the polypeptides of Examples 1 to 9 may be prepared.
• Asp-Lys-Ser-Arg-OMe is treated with a saturated solution of ammonia in methanol at room temperature for 2 days. The solvent is removed in vacuo to afford Asp-Lys-Ser-Arg-NH 2 .
• Asp-Lys-Ser-Arg yields an N,N,0-triacetyl derivative.
• the corresponding acyl derivatives, particularly N-terminal acetyl derivatives, of the peptides of Examples 1 to 9 may be prepared.
• an active site peptide is a peptide with amino acid sequence derived from an immunoglobulin active site.
• An immunoglobulin active site is a portion of an immunoglobulin molecule which physically binds to an immunoglobulin Fc receptor, thereby triggering Fc receptor functions.
• An active site peptide by virtue of its resemblance to a portion of an intact immunoglobulin molecule, is able to bind to an immunoglobulin Fc receptor and thereby mimic the intact immunoglobulin. Such binding may either stimulate or inhibit the specific immune function normally mediatedby the receptor.
• Lymphocytes are a class of leukocytes which constitute a portion of the "cellular arm" of the immune system. They are found circulating in peripheral blood, lymph fluid and in lymphoid tissues throughout the body including the spleen, thymus, lymph nodes, bone marrow and in other organs.
• Lymphocytes have a central role in immune system development and defense. They function as regulators of the growth and development of leukocytes, as "killer” cells which directly attack infectious or foreign cells and as producers of immunoglobulins. Lymphocytes may be divided into three broad classes; thymus-derived T lymphocytes (T cells), B lymphocytes (B cells) and lymphocytes lacking the characteristics of T and B cells, "null cells.” B cells are the developmental precursors of immunogiobulinproducing plasma cells which synthesize all of the body's antibodies. T ceils have two broadly defined functions which involve immune system regulation and direct killing of infectious or foreign cells. Regulatory T cells (T helper, T suppressor,
• T amplifier/inducer regulate the growth and development of T and B lymphocytes and possibly other leukocytes.
• T lymphocyte secretion of lymphokines potent activating and growth regulatory molecules.
• Certain classes of T cells also have direct cell killing abilities. Such killing may be aided by IgG bound antigens on the offending cell (antibodydependent cell-mediated cytotoxicity (ADCC)) or may occur in an antibody-independent manner as in delayedtype hypersensitivity.
• ADCC antibody dependent cell-mediated cytotoxicity
• Null cells include those lymphocytes which can recognize and kill many cancerous cells without prior exposure to the cells.
• the null cells which mediate such spontaneous killing are termed natural killer cells (NK cells) and are thought to be important in protection against cancer.
• Lymphocytes are also important activators and enhancers of the defensive function of leukocytes. Certain substances secreted by antigen-stimulated lymphocytes, for example, greatly enhance the bacteriocidal and tumoricidal activities of macrophages other lymphocyte-derived substances attract various classes of leukocytes to the site of an immune response thus enhancing the local defensive response.
• Lymphocyte growth, development and activity is also regulated by regulatory molecules secreted by various lymphoid and non-lymphoid cells. These molecules include thymic hormones, interleukins, immunoglobulins and other molecules. Of central importance to the present invention is the ability of the various immunoglobulins to regulate the growth, development and activity of lymphocytes by inter.action with lymphocyte Fc receptors.

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