PT98935A - Process for preparation of polypeptide analogues from the active site of interleukin-8, antibody for detection of the presence of IL-8, and therapeutic compositions - Google Patents

Abstract

The present invention relates to the process for preparation of polypeptide analogues from the active site of interleukin-8 (IL-8), and of antibodies for the active site, which involves the sequential addition of amino acid residues to the growing peptide chain, the amino terminal or the carboxyl terminal being protected with a suitable selectively removable protecting group until the desired polypeptide analogue has been prepared. The invention also relates to the process for the detection of the presence of IL-8 in a body fluid, which involves: (a) mixing an aliquot of body fluid with an antibody composition which immunoreacts with a segment of the carboxy terminal of IL-8 or with an antibody which immunoreacts with a segment of the amino terminal of IL-8, (b) keeping said immunoreaction mixture under biological assay conditions for a sufficient period of time to form an immunoreaction product; and (c) detecting the presence of said immunoreaction product and thus the presence of IL-8. The invention also relates to the process for preparation of therapeutic compositions from the polypeptide analogues prepared.

Description

73 116 SCRF Case 217.1

DESCRIPTIVE MEMORY

Technical Field The present invention relates to cytokines and their role in inflammatory reactions. More specifically, the present invention relates to the process of preparing an active site IL-8 polypeptide analogue of antibodies to the active site and to methods suitable for inhibiting inflammation by neutralizing the IL-8 receptor site interaction site .

Previous Art

Immuno-competent cells and vascular endothelium interact to produce hemostasis as well as inflammatory and immune reactions. The mediators of these intricate bidirectional interactions between leukocytes and vascular cells are those cytokines that are produced and act on endothelial cells (Immunol, Today 10: 370-375 (1989). with which they release chemo-tracers that induce chemotaxis and extravasation of po-limorphonuclear cells and monocytes [Broudy et al., J. Immunol., 139: 464-468 (1987)], Sieff et al., Blood. 72: 1316-1323 (1988)] This recruitment and activation of leukocytes severely breaks down the architecture of vessel walls and underlying tissues, causing injury.

Belonging to a growing family of lipopolysaccharide (LPS) -induced monomeric peptide cytokines, tumor necrosis factor (TNF), or other cytokines, IL-8 is a putative mediator of tissue injury in septic shock, acute respiratory distress syndrome (ARDS) and, generally, during diseases or inflammatory conditions associated with increased TNF production. IL-8 has been recently detected in bronchoalveolar lavage fluid from patients with premature ARDS. Nomenclature for the new group of cytokines comprising IL-8 has not been established. The intact IL-8 appears to be identical to the monocyte derived neutrophil guanacota factor (MDNCF)

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SCRF Case 217.1P0R

[Matsushima et al., J. Ex. Med., 167: 1883-1893 (1988)], to the neutrophil activating factor (ΝΑΓ) [Lindley et al., Proc. Natl. Acad. Know. USA. 85: 9199-9203 (1988); PCT N, WO 89/04836] and neutrophil chemotactic factor (ncf) (pct Nao 89/10962). Blocking chemotaxis and neutrophil activation during an inflammatory disease is a major therapeutic goal. Therapeutic intervention is necessary at various points in the inflammatory cascade to prevent tissue damage. Induction of IL-1 and TNF production occurs within 30 minutes after exposure, for example, to LPS. Antibodies to the active site of TNF receptor interaction are effective intervenients in various animal models, for example, in a porcine model for septic shock. However, to be effective, the anti-TNF antibodies have to be administered very early in the course of the disease.

Thus, what is needed is a blocking antibody directed against a late acting inflammatory protein. This would allow intervention at the secondary level of the response, subsequent to the production of TNF, resulting in a broad framework for diagnosis and preparation for treatment. In addition, a tandem therapeutic approach using anti-IL-8 antibodies in conjunction with anti-TNF antibodies would increase efficacy in cases where small amounts of TNF, which escape the anti-TNF binding, would be secondarily neutralized.

BRIEF SUMMARY OF THE INVENTION

It has now been found that the intact, native human IL-8 receptor interaction site is located on a segment of 18 amino acid residues at the carboxyl terminus of the 72 amino acid residue mature protein.

One aspect contemplated in the present invention is a polypeptide analogue of the IL-8 receptor interaction site, having an amino acid residue sequence corresponding to the formula: Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val -Glu-Arg-Val-Val-Glu-Lys-Phe-Leu-Lys, designated p50-67.

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The present invention also contemplates a polypeptide having an amino acid residue sequence corresponding to the formula: (3-20), designated p3-20.

Another aspect of the present invention is an antibody which immunoreacts (a) the IL-8 receptor interaction site and (b) with a polypeptide analogue having an amino acid residue sequence corresponding to the formula for 67.

A preferred embodiment of the present invention is a monoclonal antibody that immunoreacts with the IL-8 receptor site of interaction and as the polypeptide analog, described above, designated p50-67.

A related aspect of the present invention is an antibody that immunoreacts (a) with IL-8 and (b) with a polypeptide having an amino acid residue sequence corresponding to the formula: (3-20), designated p3-20 .

A preferred embodiment of this aspect is a monoclonal antibody that immunoreacts with IL-8 and the β 3-20 polypeptide.

One process, for detecting the presence of IL-8 in body fluids, is another aspect contemplated by the present invention. This process comprises mixing an aliquot of body fluid with an antibody composition comprising an antibody molecule that immunoreacts with a carboxyl terminus segment of IL-8 having an amino acid residue sequence corresponding to the formula:

Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-

Val-Glu-Lys-Phe-Leu-Lys. Contact of the body fluid with the antibody composition is maintained for a time sufficient to form an immune reaction product. The formed immunoreaction product, containing IL-8, is then detected.

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SCRF Case 217.1

A related aspect contemplates a method for detecting the presence of IL-8 in body fluids comprising mixing an aliquot of body fluid with an antibody composition comprising antibody molecules that immunoreact with an amino-terminal segment of IL-8 having an amino acid residue sequence corresponding to the formula: (3-20). Contact of the body fluid with the antibody composition is maintained as above and the formed immune reaction product, containing IL-8, is then detected. The present invention further contemplates a diagnostic system in the form of a diagnostic set. The system comprises a package containing, in an amount sufficient to carry out at least one assay, an antibody composition containing a population of antibody molecules that immunoreacts with an antigenic determinant on an IL-8 molecule. This antibody composition may be in a liquid solution or may be attached to a solid phase matrix. The system also contains a marker for indicating the presence of antibody molecules in the formed immuno-reaction product.

In a preferred embodiment, the antibody composition contains a monoclonal antibody.

In the present invention there is also contemplated a therapeutic composition which is suitable for preventing inflammation. Such a composition comprises an antibody that immunoreacts (a) with the IL-8 receptor site of interaction and (b) with a polypeptide analogue having a sequence of amino acid residues corresponding to the formula:

Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-

Val-Glu-Lys-Phe-Leu-Lys, or with their biological equivalents in a pharmaceutically acceptable excipient.

A related therapeutic composition contemplated by the pre-

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The present invention comprises an antibody which immunoreacts with IL-8 and (b) with a polypeptide having an amino acid residue sequence corresponding to the formula: (3-20), or their biological equivalents, in a pharmaceutically acceptable excipient.

Additionally, a method of preventing inflammation in a patient is contemplated. This method comprises administering to the subject a composition containing an antibody which immunoreacts with IL-8 and the above-described polypeptides designated p50-67 or p3-20, or their biological equivalents, in a pharmaceutically acceptable excipient . The present invention has certain advantages, namely, a) intervention at a secondary level in the inflammatory cascade is now possible, allowing additional time for diagnosis and preparation for treatment, and b) more complete and effective intervention is possible by combining the previously known method, using anti-TNF antibodies, and the method of the present invention to secondarily neutralize the TNF molecules that escape the binding of anti-TNF antibodies.

Still further embodiments and advantages of the invention will become apparent to those skilled in the art upon reading all disclosures contained herein.

Brief Description of Drawings

In the drawings which form part of the present invention: Figure 1 illustrates the effects of IL-8 or synthetic polypeptides, corresponding to the sequence of amino acid residues 50-67 and 57-72 of IL-8, in the activation of neutrophils as they are measured by calcium mobilization. The ratio F, which is equal to the value of intracellular calcium, is plotted on the Y-axis as a function of time, second after treatment with IL-8 or with peptide represented on the X-axis. IL-8 and the synthetic polypeptide 50-67, also referred to as Pep-1, induce calcium mobilization in neutrophils, whereas the polypeptide

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SCRF Case 217.1 POR-7 Synthetic 57-72, also referred to as Pep-2, does not. Calcium mobilization, induced by the treatment of neutrophils with Pep-1, is inhibited by anti-Pep-1 polyclonal antibodies. Figure 2 illustrates the non-saturable binding curve of increasing concentrations of iodinated IL-8 to neutrophils. The specific binding is calculated in the presence of a 100-fold molar excess of unlabeled IL-8 and is subtracted from the total binding to calculate the specific binding represented by filled squares. Each point is the mean ± standard error of the mean (EPM) of three independent experiments. IL-8 binding is inhibited by antibodies directed against synthetic polypeptide 50-67, Pep-1, (shown by solid triangles) related to IL-8. FAB fragments of the anti-Pep-1 antibody are more effective than the intact antibodies in inhibiting the binding of IL-8 to neutrophils (shown by blank squares). Figure 3 is a bar graph illustrating the effects of anti-IL-8, anti-Pep-1 (anti-50-67) and anti-Pep-3 (anti-3-20), immunospecific for the defined polypeptide regions of IL-8, on the binding of iodinated IL-8 (125-I-IL-8) to neutrophils (PMN), as described in Example 5A (2). The anti-11-29 antibody served as a control in the experiment since it can not prevent the inhibition of 125 I-IL-8 binding to PMN as compared to the inhibition seen with the IL-8 specific antibodies. >

Detailed Description A. Definition of Terms

Amino acid: all amino acid residues identified herein are in the natural configuration L. By maintaining the standard nomenclature of the polypeptides, J. Biol. Chem., 243: 3557-59, (1969), abbreviations for amino acid residues are as shown in the following Corresponding Table:

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SCRF Case 217.1ENG -8-

TABLE OF CORRESPONDENCE Symbol Amino acid 1 letter 3 letters Y Tyr L-tyrosine G Gly L-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V Go L-valine P Pro L-proline K Lys L-lysine H His L-histidine Q Gin L-glutamine E GlU L-glutamic acid W Trp L-tryptophan R Arg L-arginine D Asp L- Aspartic acid N Asn L-asparagine C Cys L-cysteine

It will be appreciated that all sequences of amino acid residues are represented herein by formulas whose orientation from left to right is in the conventional sense from the amino terminus to the carboxyl terminus. In addition, it will be appreciated that a trace at the beginning or the end of an amino acid residue sequence indicates a bond to a radical such as H and OH (hydrogen and hydroxyl) at the amino and carboxyl terminus, respectively, or an additional sequence of one or more amino acid residues, up to a total of about fifty residues in the polypeptide chain.

Polynentide and Peptide; Polypeptide and peptide are terms herein which are used interchangeably to a relatively low molecular weight sequence, ie, from about 210 daltons to about 10 kD, and denote a linear series of no more than about 50 amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjuvants residues.

Protein; Protein is a term herein used to denote a linear series of more than 50 amino acid residues linked to each other such as in a polypeptide.

Pharmaceutically acceptable refers to molecular entities and to compositions which do not produce a similar allergic or undesirable reaction, such as gastric disturbances, dizziness and the like, when administered to a human. B. Polypeptide Analog

In the present invention there is contemplated an IL-8-related polypeptide which mimics certain functions of the native IL-8 molecule receptor interacting active site, having an amino acid residue sequence corresponding to the formula:

Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-

Val-Glu-Lys-Phe-Leu-Lys. The polypeptide mimics the functions of IL-8 by promoting calcium mobilization in polymorphonuclear neutrophils, inducing elastase release, and, when injected into rabbits, promoting neutrophil chemotaxis to a concentration of 10 -13 M.M. versus IL -8, the polypeptide does not promote the polymerization of F-actin which results under the occupation of the IL-8 receptor. The polypeptide of the present invention contains about 18 amino acid residues. Additional polypeptides, having a length of less than 18 amino acid residues, are contemplated to more accurately define the precise active site. It is now known, for example, that a polypeptide analog containing amino acid residues 57 to 72 of native IL-8 is inactive. Thus, a portion of the active site must reside or be directed

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Conformationally by the amino acid residues found in the first seven residues of the active site as presently defined.

A polypeptide sequence of the present invention may differ from the native sequence in that the sequence is modified by NH2-terminal acylation, e.g., acetylation or amidation with thioglycolic acid, by carboxyl-terminal amidation, e.g., with ammonia, methylamine and the like. 1. Polypeptide Synthesis

A polypeptide of the present invention may be synthesized by any of the techniques that are known to those skilled in the art of the polypeptides, including recombinant DNA techniques. Synthetic chemistry techniques such as Merrifield type solid phase synthesis are preferred for reasons of purity, antigenic specificity, exemption of unwanted side products, ease of production and the like. An excellent summary of the many techniques available can be found in J.M. Steward and J.D. Young, " Solid Phase Peptide Synthesis ", W.H. Freeman Co., San Francisco, 1969; M. Bodanszky, et al., &Quot; Peptide Synthesis ", John Wiley & Sons, Second Edition, 1976 and J. Meienhofer, " Hormonal Proteins and Peptides ", Vol. 2, p. 46, Academic Press (New York), 1983 for the synthesis of solid phase peptides and Έ. Schroder and K. Kubke, " The Peptides ", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis, each of which is incorporated herein by reference. Suitable protecting groups, usable in these syntheses, are described in the foregoing and J.F.W. McOmie, " Protective Groups in Organic Chemistry ", Plenum Press, New York, 1973, which is hereby incorporated by reference.

In general, these methods comprise the sequential addition of one or more amino acid residues or amino acid residues suitably protected to a growing peptide chain. Typically, either the amino group or the carboxyl group of the first amino acid residue is protected by a suitable selectively removable protecting group. A group

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SCRF Case 217.1 A separate, selectively removable protector is used for amino acids containing a reactive side group such as lysine.

Using solid phase synthesis as an example, the protected amino acid or derivative is attached to an inert solid support through its carboxyl or unprotected amino group. The amino or carboxyl protecting group is then selectively removed and the next amino acid in the sequence having the appropriately protected complementary (amino or carboxyl) group is mixed and reacted under conditions suitable for forming the amide bond with the residue already attached to the support solid. The amino or carboxyl protecting group is then removed from this newly added amino acid residue and the next (suitably protected) amino acid is then added and so on. After all of the desired amino acids have been linked in the appropriate sequence, any remaining terminal protecting groups or side groups (and the solid support) are removed, sequentially or concurrently, to obtain the final polypeptide. 2. Activity Assays

The chemically synthesized polypeptide analogs must have the active character of the IL-8 receptor interaction region. To ensure that this activity is present the following tests are performed. 1) Mobilization of Ca2 + on indo-1-AM-labeled neutrophils, 2) Elastase release on neutrophils using a fluorescence method previously described rj. Biol. Chem. 260: 11461 (1985)], 3) Measurement of neutrophil shape modification by reduction of right angle deviation of light (performed in parallel with Ca2 + determination), 4) Chemotactic capacity measured by injection into the skin of rabbit and light microscopy of a biopsy 4 hours later. 3. Polypeptides

Also contemplated by the present invention is a polypeptide

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SCRF case having an amino acid residue sequence corresponding to the formula: (3-20), designated p3-20. The β 3-20 polypeptide is useful for inducing antibodies of the present invention that immunoreact with IL-8, as described below. C. Antibodies The term " antibody " refers to a receptor molecule, produced by B cells, which immunoreacts with, and binds to, an antigen ligand to form an immunoreactant. It is a member of a family of glycosylated proteins, called immunoglobulins, that can specifically combine with an antigen. The term " antibody " in its various grammatical forms is also used herein to denote immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody or paratopoplast combining site.

Examples of antibody molecules include intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of an immunoglobulin molecule containing the paratopo, including the portions known in the art as Fab, Fab7, F (ab7) 2 and F (v) .

The Fab and F (ab7) 2 portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, with substantially intact antibodies, by methods which are well known. See, for example, U.S. Patent 4,344,256 to Theofilopolous and Dixon. Fab7 antibody moieties are also well known and are produced from the F (ab7) 2 moieties followed by reduction of the disulfide bonds that bind the two heavy chain moieties, such as a mercaptoethanol and followed by alkylation of the mercaptan of the resulting protein with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred and is used as illustrated here. 73 116

SCRF Case 217.1ENG 13-

The phrase " monoclonal antibody " (MoAb) designates an antibody produced by single cell clones, termed "ma hybrid", which secretes only one type of antibody molecule. The hybridoma cell is formed by fusion of an antibody-producing cell and a myeloma cell line or other self-perpetuating cell line. These antibodies were first described by Kohler and Milstein, Nature. 256: 495-497 (1975), the disclosure of which is incorporated by reference.

Polyclonal antibodies " (Pab) are antibodies produced by clones obtained from different cells which secrete different antibodies that bind to a plurality of epitopes of the immunogenic molecule. ) 1. Polypeptide Immunogen The term " immunogen " as used herein describes an entity which induces the production of antibodies in the host animal. In some cases, the antigen and the immunogen are the same entity, while in other cases the two entities are different. The word " inoculum ", in its various grammatical forms, is used herein to describe a composition containing a polypeptide of the present invention, preferably Pep-1 or Pep-3, as shown in Table 1, as the active ingredient used for preparation of antibodies against IL-8. When a polypeptide is used to induce antibodies, it is assumed that the polypeptide may be used alone, attached to a carrier or in the form of a multimer, but for ease of expression, these alternatives will not be expressly referred to herein.

For a polypeptide containing less than about 35 amino acid residues, it is preferable to use the peptide attached to a carrier for the purpose of inducing the production of antibodies.

When coupled to a carrier to form what is known in the art as a carrier-hapten conjugate, a polypeptide of the present invention is capable of inducing antibodies that

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Immunoreactivity with and neutralize IL-8. Useful carriers are well known in the art and are generally the proteins themselves. Examples of such carriers include keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA), red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyamino acids, such as poly (D-lysine: D-glutamic acid), and the like.

As is also well known in the art it is often beneficial to attach a synthetic polypeptide to its carrier using a linker intermediate group. As was previously mentioned, glutaraldehyde is one of these linking groups. However, when cysteine is used, the intermediate linking group is preferably m-maleimidobenzoyl-N-hydroxysuccinimide (MBS).

In addition, MBS may first be added to a carrier by an ester-amide exchange reaction, as described by Liu et al., Supra. The addition can then be followed by the addition of a blocked mercapto group, such as thiolacetic acid (CH3COSH), to the maleimido double bond. After cleavage of the blocking acyl group, a disulfide bond is formed between the unblocked linking mercaptan group and the mercaptan of the added cysteine residue of the synthetic polypeptide.

Other means of immuno-potentiation include the use of liposomes and immunostimulating complex particles (ISCOM). The unique versatility of the liposomes lies in the fact that their size can be adjusted, in their surface characteristics, in the lipid composition and in the various ways in which they can accommodate anions. In ISCOM particles the cage-like matrix is composed of Quil A, extracted from the bark of a South American tree. A strong immune response is evoked by antigenic proteins or peptides bound by a hydrophobic interaction to the surface of the matrix .

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The choice of carrier is more dependent on the end use of the immunogen than on the immunogen's determining portion, and is based on criteria not particularly involved in the present invention. For example, if an inoculum is to be used in animals, a carrier which does not generate an adverse reaction in the animal should be selected.

In a preferred embodiment of the present invention, the inoculum comprises the above-described polypeptide coupled to KLH via MBS. 2. Polyclonal Antibodies

To induce IL-8 neutralizing antibodies, an immunogenic amount of an inoculum of the present invention, preferably containing Pep-1 or Pep-3 peptide, is administered as the active immunogen, typically by subcutaneous or intramuscular injection, to an mammal such as a mouse, rabbit, goat, horse, human and others. In a preferred embodiment of the present invention, administration was performed by subcutaneous injections, at multiple sites, into rabbits on days 1, 14 and 21. The administered (inoculated) mammal is then maintained for a period of time sufficient for the polypeptide, present as the active ingredient in the inoculum, induces the formation of neutralizing antibodies, anti-IL-8. If desired, antibodies formed may then be harvested using well-known techniques and used in preparations for passive immunization (therapeutic administration of neutralizing antibodies) against the active site of IL-8, or in assays or in diagnostic systems to detect IL-8 in body samples. The anti-peptide antibody thus produced is oligoclonal with respect to 11-8 and thus has a restricted epimer-specificity over anti-IL-8 polyclonal antisera.

Preferably, a polyclonal antibody of the present invention is in an immuno-purified form. Immunofluorescent polyclonal antibody compositions are produced by immunoreactivity

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(Adsorption) of a polyclonal antiserum on a solid phase containing the polypeptide immunogen used to induce the polyclonal antisera, removing by washing the solid phase the non-immunoreactive or weakly immunoreactive antibodies, and subsequently eluting and collecting the immunoreacted antibodies specifically to form the immunopurified antibody. Immuno-purification is a process generally well known in the art and may be performed under various conditions designed to produce a composition of polyclonal antibodies having a higher net affinity for the immuno-purifying solid phase immunogen, than affinity of the starting polyclonal antisera. Example 3 describes an example of immuno-purification. 3. Monoclonal Antibodies

Suitable antibodies in the monoclonal form typically whole antibodies may be prepared using the hybridoma technology described by Nimam et al., Proc. Natl. Acad. Know. USA. 80: 4949-4953 (1983), the disclosure of which is hereby incorporated by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma cell line or other self-pertaining cell line is fused to lymphocytes obtained from the spleen of a hyper- immunized with a polypeptide of the present invention.

It is preferred that the myeloma cell line be of the same species as the lymphocytes. Typically, a 129 G1X + strain is the preferred mammal. Mouse myelomas suitable for use in the present invention include the hypoxanthine-aminopterin-thymidine (HAT), P3X63-Ag8.653 and Sp2 / 0-Agl4-sensitive cell lines which are available from the American Type Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively.

Typically, splenocytes are fused to myeloma cells using polyethylene glycol (PEG) 1500. The fused hybrids are selected for their sensitivity to HAT. The hybridomas secreting the antibody molecules of the present invention are identified

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(RIA) assay described in Example 4.

A monoclonal antibody composition of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules with the appropriate polypeptide specificity. The culture is maintained under conditions and for a period of time sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be isolated by well-known techniques.

Useful media for the preparation of such compositions are both well known in the art and are commercially available, and include synthetic culture media, inbred mice and the like. An example of a synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol 8: 396 (1959)) supplemented with 4.5 g / l glucose, 20 mg / l glutamine and 20% of fetal calf serum. An example of an inbred mouse strain is Balb / c.

The monoclonal antibody compositions produced by the foregoing process may be used, for example, in diagnostic and therapeutic modalities in which the formation of an IL-8 containing immune reaction product is desired. D. Diagnostic Processes The present invention contemplates any method that results in the detection, in a body fluid such as blood plasma or serum, of an IL-8 receptor and thus IL-8 receptor interaction site, or of IL-8, using an antibody composition of the present invention. The method for detecting IL-8 by these means comprises forming an immunoreactant product between a carboxyl-terminal segment of IL-8 in the harvested vascular fluid and an active anti-IL-8 antibody site molecule, such as

of the product of 73 116

Described herein, and the immuno-reaction detection thus formed.

Alternatively, the method for detecting IL-8 comprises forming an immunoreactant product between an amino-terminal segment of IL-8, namely, the region defined p 3-20 in the harvested vascular fluid and a molecule of anti-IL-8 antibody as described herein, and subsequent detection of the thus formed immuno-reaction product.

Those of skill in the art will appreciate that there are numerous well known clinical diagnostic chemistry procedures which can be used to form detectable immuno-complexes. Thus, although examples of test procedures are described herein, the present invention is not limited thereto.

A method contemplated for detecting the presence of IL-8 in a body fluid comprises the steps of: a) mixing an aliquot of body fluid with an antibody composition that immunoreacts with a carboxy terminus segment of IL-8 having a amino acid residue sequence corresponding to the formula:

Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-

Gln-Arg-Val-Val-Glu-Lys-Phe-Leu-Lys. b) maintaining the immune reaction mixture under biological test conditions for a period of time sufficient to form an immunoreactant; and c) detecting the presence of said immuno-reaction product and, thus, the presence of IL-8.

In a related IL-8 detection process, the foregoing steps are performed except that the antibody composition, admixed in step (a), contains antibody molecules that immunoreact with an amino-terminal segment of IL-8 having an amino acid residue sequence corresponding to the formula: (3-20).

To carry out the diagnostic tests as previously

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As described, diagnostic systems are contemplated in the form of a diagnostic set. E. Diagnostic Systems A diagnostic system of the present invention is contemplated for the in vitro detection of the presence of IL-8 in vivo, the system comprises a composition containing antibody molecules that immunoreact with an active site of receptor interaction IL-8. The antibody molecules are linked to in vitro indicator media. such as enzyme indicator means. The diagnostic system comprises a package containing antibody molecules that immunoreact with an epitope present on a segment of the carboxyl terminus of IL-8. the most preferred antibodies are antibodies capable of immunoreacting with an epitope present in a sequence of not more than 18 amino acids in length and including the amino acid residue sequence of the native IL-8 molecule, from residue 50 to residue 67.

A diagnostic system of the present invention, in the form of an assembly, includes, in an amount sufficient to carry out at least one assay, a composition containing active, anti-IL-8, polyclonal or monoclonal receptor interaction site molecules , or fragments thereof, in the form of a separately packaged reagent, together with a label indicating the presence of an immunoreactant. Typically, instructions for using the packaged reagent are also included.

A related diagnostic system useful for in vitro detection of the presence of IL-8 comprises a composition containing antibody molecules that immunoreact with an epitope present on the amino-terminal segment of IL-8 defined by the β- 20.

This related diagnostic system in the form of an assembly, includes, in an amount sufficient to carry out at least one assay, a composition containing polyclonal or monoclonal, anti-IL-8 antibody molecules, or fragments thereof, 73 116

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immuno-specific for the β 3-20 polypeptide, together with a marker indicating the presence of an immunoreactant. Typically, instructions for use of the packaged reagent of this embodiment are also included. " Instructions for use " typically include a tangible expression describing the reagent concentration or at least one parameter of the assay process, such as the relative amounts of reagent and sample to be mixed, the maintenance time periods for reagent / sample mixtures, temperature, buffer conditions and the like. Also included in one form or another are charts, graphs and the like of predetermined concentration levels correlating specific physiological conditions with IL-8 levels

A diagnostic system of the present invention also includes a label or indicator means capable of signaling the formation of a specifically bound complex containing an antibody molecule of the present invention.

In addition, those in which the antibody molecules are attached to enzyme indicator media, such as horseradish peroxidase (HRPO), are preferred.

As used herein, the terms " marker " and " indicator means " in their various grammatical forms refer to simple atoms and molecules which are involved either directly or indirectly in the production of a detectable signal to indicate the presence of a complex. Any marker or reporter means may be attached or incorporated into an antibody molecule that is part of an antibody or a monoclonal antibody composition of the present invention, or may be used separately, and such atoms or molecules may be used alone or together with additional reagents. These labels are themselves well known in clinical diagnostic chemistry and are part of the present invention only in that they are used with novel processes and / or systems.

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The binding of labels, i.e. labeling of polypeptides and proteins is well known in the art. For example, antibody molecules produced by a hybridoma can be labeled by the metabolic incorporation of radioisotope-containing amino acids provided as a component of the culture medium. See, for example, Galfre et al., Meth. Enzol., 73: 3-46 (1981). Protein conjugation or coupling techniques by activated functional groups are particularly applicable. See, for example, Avrameas, et al., Scand. J. Immunol. Vol. 8, Suppl. 7: 7-23 (1978), Rodwell et al., Biotech., 3: 889-894 (1984), and U.S. Patent No. 4493795.

Diagnostic systems may also preferably include, in the form of a separate package, a specific binding agent. A " specific binding agent " is a molecular entity capable of selectively binding to a reactant species of the present invention, but which is not itself an antibody molecule of the present invention. Examples of specific binding agents include antibody molecules, complement proteins or fragments thereof, protein A and the like. Preferably, the specific binding agent may bind to the antibody molecule of the present invention when it is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled. However, where the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as the medium or amplification reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding to the amplification medium when the amplification medium is attached to a complex containing the reactant species.

The diagnostic kits of the present invention may be used in an " ELISA " for detecting, for example, the presence or amount of IL-8, by determining the immunoreactive molecules in a sample of body fluid, such as serum or

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SCRF Case 217.1ENG -22- plasma. " ELISA " refers to a bound enzyme immunosorbent assay using an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantitate the amount of an antigen or an antibody present in a sample. A description of the ELISA technique can be found in Chapter 22 of the 4th edition of Basic and Clinical Immunolocrv of DP Sites et al., Published by Lange Medical Publications of Los Altos, CA, USA, 1982, and U.S. Patents 3654090 ; No. 3850752; and 4016043, which are incorporated herein by reference.

Thus, in preferred embodiments, the antibody or antigen reagent component may be affixed to a matrix | solid to form a solid support which is packaged separately in said diagnostic systems. Typically, the reagent is affixed to the solid matrix by adsorption from an aqueous medium, although other display modes, well known to those skilled in the art, may be used. Useful solid matrices are well known in the art. These materials include crosslinked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NY, U.S.A.); agarose; polystyrene beads having a diameter of about 1 micron to about 5 millimeters, available from Abbott Laboratories of North Chicago, IL, U.S.A.; polyvinyl chloride based screens, polystyrene, crosslinked polyacrylamide, nitrocellulose or "nylon", such as sheets, ribbons or cushions; or tubes, plates or the wells of a microtiter plate such as those made of polystyrene or polyvinyl chloride. The reagent species, the labeled specific binding agent, or the amplification reagent of any diagnostic system described herein may be provided in solution as a liquid dispersion or in the form of a substantially dry powder, eg, in the form lyophilized. When the reporter medium is an enzyme, the enzyme substrate may also be provided in a separate package of a system. A solid support such as the

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The microtitration described above and one or more buffers may also be included as separately packaged elements in this diagnostic assay system.

The packages described herein for diagnostic systems are those commonly used in diagnostic systems. Such packages include glass and plastic bottles (e.g., polyethylene, polypropylene and polycarbonate), vials, plastic and sheet plastic sheeting, and the like. F. Therapeutic Compositions Also contemplated in the present invention is a therapeutic composition suitable for inhibiting IL-8 binding and thereby preventing inflammation, comprising an antibody that immunoreacts with (a) the IL receptor interaction site -8 and with (b) a polypeptide analogue having an amino acid residue sequence corresponding to the formula:

Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-

Val-Glu-Lys-Phe-Leu-Lys, or their biological equivalents, in a pharmaceutically acceptable excipient. This composition achieves its therapeutic effect by combining and neutralizing the active site of IL-8. A therapeutically related composition is contemplated which is suitable for inhibiting IL-8 binding and thus for the prevention of IL-8 mediated events such as inflammation, comprising an antibody that immunoreacts ) with IL-8 and with (b) a polypeptide having an amino acid residue sequence corresponding to the formula: (3-20), or their biological equivalents, in a pharmaceutically acceptable excipient. This composition achieves its therapeutic effect by immunoreacting with IL-8 and thereby interfering with (neutralizing) the active site of IL-8. Since op 3-20 is located at the amino terminus of IL-8, compared to the p-50 polypeptide analogue 50-67 and which was not effective as an IL-8 active site analog, it is believed that 3- 20 does not represent an analog of the active site. Thus, anti-peptide immunospecific antibodies

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SCRF Case 217.1ENG -24- p3-20 do not immuno-react with the active site, per se. but bind (immuno-react) with IL-8 in an interfering manner and thereby neutralize IL-8 binding activity.

Thus, in another embodiment, a therapeutic composition is contemplated which is suitable as an IL-8 analogue, having the ability to function as defined herein for a polypeptide analogue, comprising a polypeptide analogue having a corresponding amino acid residue sequence to the formula: (50-67), or their biological equivalents in a pharmaceutically acceptable excipient. This composition achieves its therapeutic effect by meticulating the active site of IL-8 and thereby exhibiting the biological activity of IL-8 as defined herein for a polypeptide analogue. The preparation of therapeutic compositions containing antibody molecules or polypeptides, as active ingredients, is well known in the art. Typically, these compositions are prepared as injectables either as liquid solutions or as suspensions. However, solid forms suitable for dissolution or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. Further, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, which enhance the efficacy of the active ingredient.

An antibody or polypeptide may be formulated into a therapeutic composition as a form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or

73 116 SCRF Case 217.1ENG 25-

organic acids such as acetic, oxalic, tartaric, mandelic and the like. The salts formed with the free carboxyl groups may also be obtained from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine , procaine and others. G. Processes for Inhibiting the Inflammatory Response Also contemplated by the present invention is a method for inhibiting the activity of IL-8 and thus preventing inflammation in a patient comprising administering to a patient a composition containing an antibody which immuno- reacts with the IL-8 receptor site of interaction and (b) with a polypeptide analogue having an amino acid residue sequence corresponding to the formula:

Cy -Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Glu-Arg-Val-Val-Glu-Lys-Phe-Leu-Lys, or their biological equivalents, in a pharmaceutically acceptable excipient.

In a related embodiment, the invention contemplates a method for inhibiting IL-8 activity and thus preventing inflammation in a patient comprising administering to the patient a composition containing an antibody that immunoreacts with IL- 8 and with (b) a polypeptide having an amino acid residue sequence corresponding to the formula: (3-20) or its biological equivalents, in a pharmaceutically acceptable excipient.

Therapeutic compositions containing antibody molecules are conventionally administered intravenously, for example, by injection of a unit dose. The term " unit dose ", when used in connection with a therapeutic composition of the present invention, relates to physically discrete units suitable as unitary dosages each containing a predetermined amount of active material, calculated to produce the desired therapeutic effect, in combination with the diluent

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SCRF Case 217.1ENG -26- required; i. carrier or vehicle.

The compositions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the subject being treated, the ability of the subject's system to utilize the active ingredient without adverse side effects, and the degree of inhibition of the desired interaction between IL-8 and the appropriate cell type. The precise amounts of active antibody required for administration depend on the judgment of the physician and are particular to each individual. However, suitable dosage ranges are in the range of 0.1 to 40 milligrams, preferably 10 to 20 milligrams, and most preferably, about 15 milligrams, of antibody per kilogram of body weight of the subject, and depend on the route of administration. administration.

A single dose of about 15 mg / kg body weight of an individual will be expected to be effective. Suitable regimens for booster injections, if required, are also variable but typically include initial administration followed by repeated doses at predetermined intervals, by sub-hot injection or by other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of 15 milligrams per kilogram of body weight in the blood is contemplated.

The following examples are intended to be illustrative and not limiting of the present invention.

EXAMPLES

The following examples are intended to be illustrative but not limiting of the present invention. 1. Synthesis of polypeptides

The polypeptides corresponding to the sequence of amino acid residues 3-20, 50-67 and 57-72, hereinafter referred to respectively as Pep-3, Pep-1 and Pep-2 of IL-8, were synthesized by

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(1969) adapted for use with a Model 430A automatic peptide synthesizer (Applied Biosystems, Foster City, CA, USA) USA). The polypeptide resins were cleaved using hydrogen fluoride, extracted and analyzed for purity by high performance liquid chromatography on a reverse phase C18 column manufactured by Water Associates, Milford, MA, U.S.A.

The amino acid residue sequences of the above-mentioned polypeptides are shown in Table 1. TABLE 1

Name of Formula1 2

Sequence Sequence of origin of IL-8 amino acid

Pep-1 Pep-2 Pep-3 50-67 57-72 3-20

The Pep-1, Pep-2 and Pep-3 polypeptides are also referred to herein as p50-67, p57-72 and p3-20, respectively. 2. Activation of Neutrophils by a Synthetic Polyoletide Related to IL-8 A. In Vitro Assays 2

Preparation of cells

Blood was collected, after informed consent, from normal volunteers free from medication and anti-coagulated with a mixture of 0.14 M citric acid, 0.2 M tri-sodium citrate and 0.22 de dextrose. The anti-coagulated blood was centrifuged at 800 x g for 15 minutes at room temperature and the platelet-rich plasma supernatant was discarded. The sedimented erythrocytes and mononuclear and polynuclear cells were resuspended, and diluted with a volume equal to the volume of

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SCRF Case 217.1P0R-28-blood with 0.14 M cold PBS, pH 7.4. Peripheral blood mononuclear cells (PBMC) were then removed from the cell suspension diluted by centrifugation in low endotoxin Ficoll-Hypaque (Sigma Chem. Corp., St. Louis, MO, USA) at 400 xg for 10 minutes to 18 degrees C (18Â ° C).

Polynuclear cells (neutrophils) in the cell suspension devoid of PBMC were then recovered by sedimentation in dextran. The resulting cell pellet containing neutrophils at a concentration of 1.5 x 107 cells / milliliter (ml) was resuspended in Hanks Balanced Salt Solution (HBSS) (Sigma Chem. Corp.) calcium free. The resuspended cell suspension was maintained on ice and used within two hours following isolation. (2) Calcium Mobilization The isolated cell suspension containing neutrophils expressing the IL-8 receptor, isolated, was fluorescently labeled with Indo-1-AM (Molecular Probes, Eugene, OR, U.S.A.). A five micromolar (Mm) solution of Indo-1-AM dissolved in a final concentration of 0.1% dimethylsulfoxide (DMSO) with 1x10 7 neutrophils / ml in calcium-free HBSS prepared in the above step (1) , and the mixture was maintained for 30 minutes at 37 ° C to form fluorescently labeled neutrophils. After this period, the Indo-1-AM labeled cell suspension was washed twice with calcium-free HBSS. The fluorescently labeled, resultant neutrophils were resuspended in HBSS containing 10 mM Hepes, 1.5 mM calcium and 1 gram of glucose, to a final concentration of 2 × 10 6 cells / ml. The cell suspension was then preheated by maintenance at 37 ° C for 2 minutes.

Aliquots of the preheated fluorescence-labeled neutrophil cell suspension aliquots were mixed with 1 x 10 qu of the synthetic polypeptide, Pep-1, produced in Example 1 (TABLE 1), IL- 8 obtained either from endothelial cells (Biosource, Westlake Village, CA,

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Or from 1 x 10-8 M of the synthetic polypeptide, Pep-2 produced in Example 1. Each mixture was subjected to cell fluorometry , by which the effects of synthetic peptides or IL-8 on calcium mobilization in neutrophils were evaluated. Calcium mobilization was measured in each mixture by detecting fluorescence variations over time on a SIM 8000 fluorometer. The fluorometer was equipped with an excitation filter having a wavelength of 340 nanometers (nm), with a emission filter with a wavelength of 400 nm on channel A, representing calcium-bound Indo-1-AM, and with an emission filter having a wavelength of 485 nm on channel B representing the free Indo-1-AM . The ratio between the detected fluorescence in channel A and that detected in channel B (represented by the ratio F) is equal to the value of the amount of intracellular calcium.

The results of this assay, shown in Figure 1, indicate that calcium mobilization on Indo-1-AM-labeled neutrophils increased ten seconds after treatment with the synthetic polypeptide, Pep-1, as measured by fluorometry and calculating the F ratios as previously described. The results are compared to the F ratios calculated from neutrophils treated with either recombinant human IL-8 or the synthetic peptide Pep-2. Both Pep-1 and recombinant human IL-8 induce calcium mobilization that saturates 20 seconds after treatment. In contrast, the synthetic peptide, Pep-2, does not promote calcium mobilization. The effect of Pep-1 on calcium mobilization in neutrophils mimics human recombinant IL-8 Intacta, whereas Pep-2 does not promote the same response.

The results of this study indicate that the first seven amino acid residues of the amino terminus of Pep-1 are responsible for Pep-1's ability to bind to the IL-8 receptor. Thus, additional polypeptides of the present invention have an amino acid residue sequence length in the range of 7 to 30, and preferably no more than 20 to 25 residues, and contain the sequence CLDPKEN. Preferably, these peptides have an overall sequence corresponding to, and which is,

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SCRF Case 217.1AG-30- is preferentially identical to that of IL-8. It is also preferred that these peptides bind to the IL-8 receptor, as evidenced by their ability to competitively inhibit the binding of IL-8 to the receptor, but do not induce neutrophil activation, as described in section B2 previous, Activity Assays. Examples of peptides are shown in Table 2 below. TABLE 2

CLDPKEN

CLDPKENW

CLDPKENWV

CLDPKENWVQ

CLDPKENWVQR

CLDPKENWVQRV

CLDPKENWVQRW

CLDPKENWVQRWE

CLDPKENWVQRWEK

CLDPKENWVQRWEKF

CLDPKENWVQRWEKFL

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(4) Elastase Release Elastase release was determined with the fluorescence substrate oMe-succinimide-Ala-Ala-Pro-Val-methyl-coumarilamide (Peninsula Laboratories). Aliquots of isolated 2 x 106 neutrophils / ml prepared as in step (1) above were maintained at 37Â ° C for 2 minutes in the presence of 2 x 10-5 M of the fluorescent substrate dissolved in 0.1% and 1 microgram (æg / ml) cytochalasin B. The substrate treated neutrophil aliquots were then mixed separately with the synthetic polypeptides prepared in Example 1. The resulting mixture was then subjected to analysis fluorometer, performed as in step (2) above on a SLM 8000 fluorometer equipped with an excitation filter having a wavelength of 380 nm and a 4900 angstrom emission filter. The elastase released from the peptide-treated neutrophils was measured kinetically over a period of 2 minutes in a " cuvette " stirred at 37 ° C. The total neutrophil elastase content was determined by lysing the cells with 1% Triton X-100. Spontaneous release of elastase was subtracted from the total to obtain elastase release corrected.

Elastase release assay results indicate that Pep-1, at a concentration of 1 x 10-9 M, promoted elastase release in neutrophils comparable to that seen with intact human recombinant IL-8. These results further support the fact that Pep-1 contains the active site of IL-8, thereby mimicking the activation of neutrophils by IL-8. B. In Vivo Assays The Pep-1, prepared in Example 1, was dissolved in lipopolysaccharide-free phosphate buffered saline (PBS) and the solutions in concentrations ranging from 1 x 10 9 Mel x 10 11 11 M were injected into the skin of a rabbit. The similarly prepared human recombinant IL-8 was injected into a skin area separated from the same rabbit. Injections of PBS alone served as a negative control. A second rabbit received similar injections of the solutions.

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Skin corresponding to the areas of the injections with Pep-1, IL-8 and PBS was excised from the rabbits at 4 hours post-injection. Excised skin samples were analyzed by light microscopy to evaluate chemotaxis of neutrophils at the injection site. Pep-1 injections resulted in dose-dependent neutrophil chemotaxis, which was indistinguishable from the influx induced by the same concentrations of IL-8. Neutrophils did not prevail in areas that received PBS injections. 3. Preparation of Polyclonal Antisera Against an IL-8 Related Synthetic Polypeptide A. Preparation of Immunogen The synthetic polypeptide, Pep-1, prepared in Example 1, was coupled to keyhole limpet hemocyanin (KLH) by the residue Pep-1 N-terminal cysteine to form Pep-1-KLH conjugates. The KLH was dialyzed against a 10 mM PBS solution and the resulting dialyzed KLH solution was adjusted to a concentration of 20 milligram (mg) / ml. 3 mg of m-maleimino-dobenzoyl-N-hydroxysuccinimide ester (MBS) (Pierce Biochemicals, Rockford, IL, U.S.A.) were dissolved in 5 ml of dimethylformamide (Pierce Biochemicals). 85 microliter (μ) of the resulting MBS solution was mixed with 200 μl KLH solution to form a mixture of KLH-MBS which was maintained at 25 ° C for 30 minutes.

The KLH-MBS mixture was then run through a Sephadex G-25 column (Pharmacia, Piscataway, NJ, U.S.A.) and 1 ml fractions of MBS treated KLH were collected. 1 ml of the chromatographed KLH was then mixed with 1 ml of a 5 mg / ml solution of Pep-1, previously dissolved in PBS, to form a Pep-1-KLH conjugate immunoassay. The pH of the Pep-1-KLH mixture was then adjusted to pH 7.0, and then the mixture was maintained at 25 ° C for three hours.

A Pep-3-KLH conjugate immunogen was prepared as above, except that the synthetic Pep-3 polypeptide was used in place of Pep-1. B. Immunization and Harvest of Polyclonal Antisera Pep-1-KLH immunogen, prepared in Example 3A, was 73 116

SCRF Case 217.1ENG 33-

emulsified in complete Freund's adjuvant (CFA) and Pep-1-KLH antigens were incorporated into the emulsion at a concentration of 200æg / 1.5 ml. Two rabbits were each injected with 1.5 ml of the emulsion prepared after serum samples were collected prior to immunization. The total volume of the emulsion dose was divided equally into subcutaneous administration on the back, shoulder and hip of a rabbit.

At day 14, the rabbits received a booster injection of Pep-1-KLH immunogen emulsified in incomplete Freund's adjuvant (IFA). On day 21, each rabbit received an intraperitoneal injection of 1 ml of a 10 mg / ml solution of free Pep-1 dissolved in 1.2 ml of PBS containing 0.8 ml of aluminum hydroxide. Serum samples were collected from each immunized rabbit 7 and 14 days after the third injection and analyzed by Western blotting as described below in C for the detection of Pep-1 specific antibodies.

Similarly, serum samples were prepared using rabbits immunized with the Pep-3-KLH immunogen from Example 3A to form Pep-3 specific antibodies which were characterized as described below in CC Analysis of Polyclonal Antisera by Analysis of

Western transfer

Human recombinant IL-8, Pep-1 and Pep-3 samples were collected at a concentration of 100 ng / ml by gel electrophoresis, and transferred to nitrocellulose for subsequent immuno-transfer analysis. The nitrocellulose filter was mixed with antibody blocking buffer (20 mM sodium phosphate, pH 7.5, 0.5 M sodium chloride, 1% bovine serum albumin and 0.05% Tween 40) for 3 to 12 hours at room temperature. The sera collected from rabbits, prepared in Example 3B, which are believed to contain Pep-1 or Pep-3, 1: 500 immunoreactive antibodies in the antibody blocking buffer were diluted and mixed with nitrocellulose . The resulting mixture was kept for 12 hours at ambient temperature to allow the formation of a product of

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Immuno-reaction on the solid phase.

The preimmune sera from the same rabbits were tested simultaneously on separate samples to serve as a negative control. The nitrocellulose was then washed three times with excess volumes of antibody blotting buffer. The washes were followed by mixing the nitrocellulose with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chem Corp.) in a 1: 500 dilution in antibody blocking buffer for one hour at room temperature to allowing the HRP-labeled IgG to bind to any immuno-reaction product present in the solid phase on the nitrocellulose. The nitrocellulose was then washed as described herein and developed by treatment with 4-chloro-1-naphthol as a substrate in order to visualize the label and thus any immunoreactive product on the nitrocellulose.

Polyclonal antisera containing antibodies immunoreactive with IL-8, Pep-1 and Pep-3 were then evaluated for their ability to inhibit IL-8 function as described in Example 5. D. Immunoaffinity Purification of Polyclonal Antibodies

Anti-Pep-1 and anti-Pep-3 antibodies, prepared with polyclonal antisera as described in Example 3C, were immunoaffinity purified using solid phase immobilized polypeptide as follows.

Pep-1 and Pep-3 polypeptides were coupled separately to cyanogen bromide (CNBr) -sepharose 4B activated (Pharmacia), at a concentration of 3 mg of protein to 1 ml of gel, in coupling buffer ( 0.5 M NaCl and 0.05 M borate, pH 8.5) overnight at 4 ° C to form a polypeptide-Sepharose suspension. The suspension was packed onto a column and washed with coupling buffer to remove unbound polypeptide according to the manufacturer's instructions. Polyclonal antisera were mixed with a buffer solution containing NaCl

73 116 SCRF Case 217.1ENG-35-

0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM benzamidine, 0.02% NaN 3, 0.02% Tween-20 and Tris-HCl at pH 7.4. The buffered sera were then passed over the prepared polypeptide-Sepharose column to immobilize the anti-polypeptide antibodies on the antibody column and separate the polypeptide-specific antibodies from the non-specific or weakly specific antibodies and other serum components . The polypeptide column containing immobilized antibody was washed, subsequently to remove further unbound antibodies and other proteins. The immobilized antibody was then eluted from the column with 3M sodium thiocyanate (NaSCN) in 1.0 M NaCl, 4 mM benzamidine, 2 mM EDTA, 0.02% NaN 3 and 0.05 M Tris-HCl at pH 7.0 . The eluted and purified antibody was found to have a purity greater than 95% when analyzed using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 4. Preparation of Monoclonal Antibodies Against a Synthetic Polypeptide Related to IL-8 A. Generation of Anti-Pep-1 Hybridomas The synthetic polypeptide, Pep-1, is prepared as an immunogen according to Example 3. Immunize (ip), Balb / c ByJ mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, CA, USA) were mixed with 50 μg prepared Pep-1-KLH immunogen in CFA followed by a second and a third immunization, using the same Pep-1-KLH immunogen, each separated from about three weeks, in IFA. Mice are given a 50 μg boost of Pep-1-KLH immunogen prepared intravenously (i.v.) in normal saline 4 days before fusion and a second booster infusion similar a day later.

The animals thus treated are sacrificed and the spleen of each mouse is harvested. A suspension of spleen cells is then prepared. The spleen cells are then removed from the spleen cell suspension by centrifugation for about 10 minutes at 1000 rpm at 23øC. After removal of the supernatant, the cell pellet is resuspended in 5 ml of NH 4 Cl lysis buffer and incubated for about 10 minutes.

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To the suspension of lysed cells is added 10 ml of Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO) and HEPES [4- (2-hydroxyethyl) -1-piperidineethanesulfonic acid] buffer and this mixture is centrifuged for about 10 minutes at 1000 rpm at 23øC.

The supernatant is decanted, the pellet resuspended in 15 ml of DMEM and HEPES, and centrifuged for about 10 minutes at 1000 rpm at 23 ° C. The above procedure is repeated.

The pellet is then resuspended in 5 ml of DMEM and HEPES. An aliquot of the spleen cell suspension is then removed for counting. The fusions are performed as follows, using the non-segregating mouse myeloma cell line P3X63Ag8.653.1, a subclone of the cell line P3X63Ag8.653 (ATCC 1580). Using a ratio of myeloma cells to spleen cells of about 1 to 10 or about 1 to 5, a sufficient amount of myeloma cells are centrifuged to form a pellet, washed twice in 15 ml of DMEM and HEPES, and centrifuged for 10 minutes at 1000 rpm at 23 ° C.

Spleen cells and myeloma cells are combined in 15 ml round bottom tubes. The cell mixture is centrifuged for 10 minutes at 1000 rpm at 23øC, and the supernatant is removed by aspiration. 200 μΐ of a 50 percent (w / v) aqueous solution of 4000 molecular weight polyethylene glycol (PEG; ATCC Baltimore, MD, USA) is then blended at about 37øC using a 1 ml pipette, with vigorous stirring, for the breakdown of the sediment, and the cells are gently mixed for a period of between 15 and 30 seconds. The cell mixture is centrifuged for 4 minutes at 700 rpm.

About 8 minutes before PEG is added, 5 ml of DMEM plus HEPES buffer are slowly mixed with the pellet without disturbing the cells. After 1 minute, the resulting mixture is cleaved with a 1 ml pipette and incubated for an additional 4 minutes. This mixture is centrifuged for 7 minutes at 1000 rpm. The supernatant is decanted, 5 ml of HT medium (hypoxanthine / thymidine) is slowly mixed with the pellet, and the mixture is maintained

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SCRF Case 217.1ENG -37- undisturbed for 5 minutes. The pellet is then broken into large pieces and the final suspension of cells is placed in T75 flasks (2.5 ml per flask) in which 7.5 ml of HT medium have been previously placed. The resulting cell suspension is incubated at 37 ° C to develop the molten cells. After 245 hours, 10 ml of HT medium is added to the flasks, followed by 0.3 ml of 0.04 mH aminopterin, 6 hours later. Forty-eight hours post-fusion, 10 ml HAT medium (hypoxanthine / aminopterin / thymidine) is added to the flasks.

Three days after fusion, the viable cells are plated on 96-well tissue culture plates with about 2 x 10 5 viable cells per well (a total of 768 wells) in HAT buffer medium as described in Kennett et al., Curr Top. Microbiol. Immunol., 81: 77 (1978). Cells are fed seven days after fusion with HAT medium and then at approximately 4-5 day intervals as required with HT medium. The growth is followed microscopically, and the culture supernatants are harvested about two weeks later and analyzed for Pep-1 specific antibody by solid phase radioimmunoassay (RIA).

Briefly, 50 μl of PBS, containing 5 μg / ml prepared Pep-1-KLH immunogen, is added to the wells of microtiter plates. The plates are maintained overnight (about 16 hours) at 4Â ° C, to allow Pep-1-KLH immunogen to adhere to the walls of the wells. After washing the wells, four times with SPRIA buffer (2.68 mM KCl, 1.47 mM KH2 PO4, 137 mM NaCl, 8.03 mM Na2HPO4, 0.05% Tween-20, 0.1 ΚΙϋ / ml Traysol , 0.1% BSA and 0.015% NaN 3), 200 μl of SPRIA buffer containing 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA) were added to each well, to block the excess protein binding locias. The plates are maintained for 30 minutes at 20Â ° C, the wells are leached by shaking and dried by absorption to form a solid support, i.e., a solid matrix to which Pep-1-KLH immunogen is operatively attached.

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In each well 50 μΐ of hybridoma tissue culture supernatant is then added to form a solid-liquid-phase immuno-reaction mixture. The mixture is maintained for 2 hours at 37 ° C to allow the formation of solid phase immuno-reaction products. After washing the wells as described hereinbefore, 50 μl of labeled goat anthra-mouse IgG (Cappel Laboratories, Downington, PA, USA), with 0.25 μg protein per ml, to each well, to form a labeling reaction mixture. This mixture is maintained for 1 hour at 37 ° C to allow the formation of solid phase immunoreaction products labeled with After washing the wells as described above, the amount of 125 I-labeled product, bound to each well, is determined by gamma detection.

Hybridomas are selected from hybridoma cultures that secrete anti-Pep-1 antibodies to their culture media, and further characterized as described herein.

Hybridomas are produced and selected from hybridoma cultures which secrete anti-Pep-3 antibodies, using the procedures described above except that Pep-3 is used in place of Pep-1, as the immunizing polypeptide and of research. B. Production and Purification of Pep-1 Monoclonal Antibody Pep-1 hybridoma is grown in a humidified atmosphere with 5% Co 2 at 37øC in DMEM containing 2 mM L-glutamine, 50 μg gentamycin per ml, 10% fetal bovine serum and 10% horse serum, all from Grand Island Biological Co., Lawrence, MA, USA, 10% NCTC medium from Microbiological Associates, Ro-kville, MD, USA, hypoxanthine, 1mM and 0.3 mM thymidine, both from Sigma Chemical Corp. The concentration of cells is maintained in the range of about 1-2 x 105 cells per ml of medium to about 1-2x106 cells per ml of medium, for growth and division of cells and production of antibody.

To produce ascites tumor fluid containing

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Anti-Pep-1 antibody, 10-week-old Balb / c mice were immunized immunologically by intraperitoneal injection with 0.3 ml of mineral oil and subsequently injected intraperitoneally with 3-5 x 105 anti-Pep-1 hybridoma cells. The inoculated mice are then maintained for a period of time sufficient to accumulate tumor-containing ascites fluids, e.g., antibodies for about 10 to about 21 days. The ascites fluid is collected and clarified by centrifugation at 15,000 x g for 1 hour at 4 ° C and stored frozen at -20 ° C.

The anti-Pep-1 antibody molecules are isolated from the ascites fluid, by subjecting the fluid to Fast Protein Liquid Chromatography (FPLC) on a Pharmacia Mono QHR 5/5 anion exchange column in a Pharmacia FPLC system (both from Pharmacia, Inc.) using a 0-0.5 M NaCl gradient in 10 mM Tris, pH 8.0, and following the instructions provided with the column. The isolated anti-Pep-1 antibody molecules can then be transferred to any physiologically tolerable diluent by dialysis.

Altematively, the anti-Pep-1 antibody molecules can be isolated from the tumor ascites fluid by precipitation with ammonium sulfate according to the procedure described by Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, p 100-101 (1983). Briefly, this process comprises slowly mixing saturated ammonium sulfate solution with the ascites fluid until an ammonium sulfate concentration of about 45% to about 50% is reached. The precipitated immunoglobulins are then collected by centrifugation at 2000 x g, preferably at 10,000 x g. the precipitate is washed 2 or 3 times in 40% saturated ammonium sulfate solution. the precipitated anti-Pep-1 antibody molecules are then dialysed against 500-1000 volumes of phosphate buffered saline (PBS) or any other physiologically tolerable diluent desired to remove the ammonium sulfate. The dialysis fluid is changed several times at intervals of a few hours. The protein concentration of the recovered dia-lysed anti-Pep-1 antibody solution is determined by the method of Lowry [Lowry et al.

Î ± 1 '

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SCRF Case 217.1 PG -40 al., J. Biol. Chem., 193, 265-275 (1951)] using a bovine serum albumin standard. Confirmation of the functional specificity of the an-ti-Pep-1 antibodies, either in a serum sample obtained in Example 3, or in the hybridoma culture supernatant, is determined, in neutrophil binding assays expressing IL-8 receptors, in competition assays described below in Example 5. The anti-Pep-3 hybridoma, prepared as described in Example 4A, is cultured as described above for the anti-Pep-1 hybridoma. Anti-Pep-3 antibody molecules are purified from the anti-Pep-3 hybridoma as described above for the anti-Pep-1 antibody molecules. Confirmation of the functional specificity of the anti-Pep-3 antibodies, either in a serum sample obtained in Example 3 or in the hybridoma culture supernatant, is determined in the Western blot analysis of Example 3C and in the neutrophil binding assays expressing the IL-8 receptor, as described in Example 5. 73 116

SCRF Case 217.1

neutrophil bound IL-8. The total radioactivity associated with the cells was determined by gamma counter detection. Specific binding of the cell associated IL-8 was calculated by subtracting the cell-associated radioactivity which resulted in mixtures maintained in the presence of a 100-fold molar excess of unlabeled IL-8, at the total radioactivity. The net or specific neutrophil iodinated IL-8 binding reaction results are shown in Figure 2, each dot representing the mean ± SEM of three independent experiments. The results show that neutrophil suspensions, containing IL-8 receptors, bind to iodinated IL-8 in a specific and unsaturated reaction. B. Inhibition Assays (1) Anti-Pep-1 Polyclonal Antibodies The binding of iodinated IL-8 to the IL-8 receptor interaction site on neutrophils was confirmed by performing inhibition assays with anti-Pep-1 polyclonal antibodies prepared in Example 3. IL-8 was iodinated as described in A as above, and increasing concentrations of the labeled IL-8, ranging from 3 x 10- 10 M to 1 x 10- 9, were mixed separately. M, with 2 mg / ml of anti-Pep-1 polyclonal antibody to form an immunoreaction mixture which was maintained at 37 ° C for 30 minutes. FAB fragments of anti-Pep-1 antibodies were prepared and also evaluated in the inhibition assays following the same procedure. Fab fragments were prepared from sera using the manufacturer's protocols and the preparation kit for the preparation of Fab, Immunopure (Pierce Chemical Co., Rockford, IL, U.S.A.).

After the maintenance period, the immuno-reaction mixtures were separately mixed with aliquots of 2 x 106 neutrophils / ml prepared in Example 1 to form a mixture of cellular immuno-reaction. The resulting mixture was maintained as in the IL-8 binding assays. The ligation reaction was terminated as previously described. The specific binding of IL-8 to the re-

73 116 SCKF Case 217.1GB -42-

anti-Pep-1 polyclonal antibodies were calculated as described in A. The binding of IL-8 to the IL-8 receptor interaction site on neutrophils was inhibited by treatment with either intact anti-Pep-1 antibodies either with anti-Pep-1 Fab fragments, for all concentrations of iodinated IL-8 evaluated in the assay, as shown in Figure 2. The results indicate that polyclonal antibodies directed against the amino acid residue sequence 50-67 of IL -8, Pep-1, are effective at binding to the active site on IL-8, thereby preventing the binding of IL-8 to the IL-8 (receptor) ligand interaction site on neutrophils. (2) Immuno-Affinity Purified Anti-Peo-1 and Anti-Peo-3 Polyclonal Antibodies The ability of anti-IL-8 and immuno-affinity purified antibody compositions, prepared in Example 3D, to inhibit the binding of IL-8 to neutrophils containing IL-8 receptors. For the inhibition assays, neutrophils were isolated as described in Example 1. Inhibition assays were performed by first mixing separate aliquots of neutrophils at a concentration of 1 x 10 4 cells / ml with 50æg / ml of Fab fragments of anti-Pep-1 and anti-Pep-3 antibodies, purified by immunoaffinity, to form antibody-cell blends. The Fab fragments were prepared as described in step (1) above. In this assay anti-IL-8 and a control antibody, anti-IL-29, were also tested. The resulting mixtures were maintained for 5 minutes at 37 ° C prior to the addition of approximately 10 μl of IL-8 at 40,000 cpm, prepared as described in Example 5A. The IL-8-containing mixtures were then maintained for 30 minutes at 37 ° C to allow IL-8 to bind to IL-8 receptors on neutrophils if the IL-8 receptor binding site was not blocked by the IL-8 receptors. antibodies tested.

The cells were then centrifuged through a mixture of silicon oils for 15 minutes as described in Example 5A. The radioactivity in the resulting supernatant and the

73 116

SCRF Case 217.1P0R -43- was measured by gamma detection. The cell-specific binding of IL-8 to neutrophils was calculated as described in Example 5A, except that a 200-fold molar excess of unlabeled IL-8 was used to calculate non-specific binding. The percent inhibition was then determined by calculating the difference between the specific binding associated with the cells in the presence and absence of added antibodies.

The results of the inhibition assays in which both the anti-IL-8 and anti-Pep-1 antibodies completely inhibited the binding of iodinated IL-8 to IL-8 receptors on neutrophils are shown in Figure 3. the anti-Pep-3 antibody was approximately 100% effective. No inhibition was observed with the control antibody, 11--29. The results indicated that immunoaffinity purified polyclonal antibodies, Pep-1 and Pep-3, directed against the amino acid residue sequence 50-67 and against the amino acid residue sequence 3-20, respectively, of IL-8, were both effective at binding to the active site of IL-8, thereby avoiding the binding of IL-8 to IL-8 receptors on neutrophils. (3) Anti-Pep-1 Monoclonal Antibodies

The purified anti-Pep-1 monoclonal antibodies prepared in Example 4 were also evaluated as competitive inhibitors of 125β-IL-8 binding at the site of IL-8 receptor interaction in neutrophils in assays performed as described for the polyclonal antibodies. 6. Inhibition of Neutrophil Activation with IL-8 Related Synthetic Polynucleotide Antibodies A. Anti-Pep-1 Polyclonal Antibodies

Anti-Pep-1 polyclonal antibodies prepared in Example 3 were evaluated for their ability to inhibit neutrophil activation as measured by the calcium mobilization performed as in Example 2.A. (2). Pep-1, prepared in Example 1, was mixed with anti-Pep-1 polyclonal antibodies to form a peptide immunoreaction admixture as described in Example 5A. Mistu- 73 116

P

An isolated allus of neutrophils was expressed by expressing the IL-8 receptor prepared in Example 1 with Pep-1 / anti-Pep-1 mixture, and maintained as described in Example 2.A. (2). The mixture was subjected to fluorometry for measurement of calcium mobilization, performed as described in Example 2.A. (2).

Anti-Pep-1 polyclonal antibodies inhibited the mobilization of Pep-1-induced calcium, as shown in Figure 1. The results indicate that polyclonal antibodies directed against the amino acid residues 50-67 of IL-8, Pep-1, are effective at binding to the active site of IL-8, thereby preventing the binding of IL-8 to site of interaction of IL-8 receptor on neutrophils and avoiding the consequent activation of neutrophils measured by calcium mobilization. B. Anti-Pep-1 Monoclonal Antibodies

The purified anti-Pep-1 monoclonal antibodies prepared in Example 4 were also evaluated for their ability to inhibit neutrophil activation in calcium mobilization assays. Assays are performed as described for the polyclonal antibodies at point A above.

Although the present invention has now been described in terms of certain preferred embodiments, and exemplified with respect to these embodiments, it will be understood by one skilled in the art that various modifications, variations, omissions and substitutions may be made without departing from the spirit of the present invention. Accordingly, it is intended that the present invention be limited only by the scope of the following claims.

Claims (11)

A method of preparing a polypeptide analog of the receptor interacting site of interleukin-S (IL-8), which interacts with the receptor, having an amino acid residue sequence corresponding to The present invention provides a process for the preparation of a compound of the formula: ## STR1 ## in which the amino acid residues are sequentially added to the the amino terminus or carboxyl terminus being protected with a suitable selectively removable protecting group until the desired polypeptide analogue has been prepared. A method of preparing a monoclonal antibody which immunoreacts with the IL-8 receptor site of interaction and with a polypeptide analogue having an amino acid residue sequence corresponding to the formula: Cys-Leu-Asp-Pro-Lys-Glu -Asn-Trp-Val-Gln-Arg-Val-Val-Glu-Lys-Fe-Leu-Lys, characterized by culturing a hybridoma obtained by fusing a myeloma cell, or other auto- and maintained with an antibody producing cell in a suitable nutrient medium under conditions and for a period of time suitable for the hybridoma to secrete the antibody molecules into the medium, to collect the medium containing the antibody and to isolate the antibody. A method for detecting the presence of IL-8 in a body fluid comprising the steps of: (a) mixing an aliquot of body fluid with an antibody composition that immunoreacts with a carboxy-terminal segment of IL-8 having a sequence of amino acid residues corresponding to the formula: Cis-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-Val-Glu-Lys-Fe-Leu-Lys, maintaining said immunoreaction admixture under biological assay conditions for a sufficient period of time To form an immunoreaction product; and (c) detecting the presence of said immunoreaction product and thus the presence of IL-8. A method of producing a diagnostic system in the form of a diagnostic set for determining the presence of a site of interaction with the IL-8 receptor, characterized in that it is packaged in an amount sufficient to achieve at least one determination: ( a) an antibody comprising an antibody molecule which immunoreacts with a carboxy terminal segment of IL-8 having an amino acid residue sequence corresponding to the formula: Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val -Gln-Arg-Val-Val-Glu-Lys-Fe-Leu-Lys, (b) a label for indicating the presence of antibody molecules in the formed immunoreaction product. A method of preparing a therapeutic composition suitable for the prevention of inflammation characterized in that an antibody is immunoreacted with (a) the site of IL-8 interacting with the receptor and (b) a polypeptide analog having a sequence of amino acid residues corresponding to the formula: Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-Val-Glu-Lys-Fe-Leu-Lys, or their biological equivalents, with a pharmaceutically acceptable excipient. A process for the preparation of a polypeptide having an amino acid residue sequence corresponding to the formula: LysGluLeuArgCisGlnCisIleLisThrTirSerLisProFeHisProHis characterized in that the amino acid residues are sequentially added to the growing peptide chain, the amino terminus or the carboxyl terminus being protected with a selectively removable protective group , until the desired polypeptide analogue has been prepared. SCRF Case 217.1P0R -47- R * v s * P A method of preparing a monoclonal antibody which immunoreacts with IL-8 and polyimidazole having a sequence of amino acid residues corresponding to the formula: LysGluLeuArgCisGlnCisIleLisThrTirSerLisProFeHisProHis characterized by culturing a hybridoma obtained by fusing a myeloma cell or another self-perpetuating cell line with an antibody producing cell in a suitable nutrient medium under conditions and for a period of time suitable for the hybridoma to secrete the antibody molecules into the medium, collection of the medium containing the antibody, and isolation of the antibody. antibody. A method for detecting the presence of IL-8 in a body fluid sample comprising the steps of: (a) mixing an aliquot of body fluid with an antibody that immunoreacts with an amino-terminal segment of IL-8 having a sequence of amino acid residues corresponding to the formula: LisGluLeuArgCisGlnCisIleLisThrTirSerLisProFeHisProHis to form an immunoreaction mixture? (b) maintaining said immunoreaction admixture under biological assay conditions for a period of time sufficient to form an immunoreaction product; and (c) detecting the presence of said immunoreaction product and thus the presence of IL-8. A diagnostic system for determining the presence of IL-8 in a sample, characterized in that it is packaged in an amount sufficient to effect at least one determination: (a) an antibody comprising an antibody molecule which immunoreacts with an amino-terminal segment of IL-8 having an amino acid residue sequence corresponding to the formula: LysGluLeuArgCisGlnCisIleLisThrTirSerLisProFeHisProHis, and (b) a marker for the presence of molecules -48- 73 116 SCRF Case 217.1 antibody to the immunoreaction product formed. A method of preparing a therapeutic composition suitable for the prevention of inflammation, characterized in that an antibody which immunoreacts with (a) IL-8 and (b) a polypeptide having an amino acid residue sequence corresponding to the formula: or their biological equivalents, with a pharmaceutically acceptable excipient. Lisbon '11. SET 1991 By THE SCRIPPS RESEARCH INSTITUTE = 0 0FICIAL AGENT =