IE83565B1

IE83565B1 - Mixed specificity fusion proteins

Info

Publication number: IE83565B1
Application number: IE1992/0202A
Authority: IE
Inventors: A. Aruffo Alejandro; S. Linsley Peter; A. Ledbetter Jeffrey; K. Damle Nitin; H. Perry Fell, Jr.
Original assignee: Bristol Myers Squibb Company
Filing date: 1992-01-23

Description

PATENTS ACT, 1992 92/0202 MIXED SPECIFICITY FUSION PROTEINS BRISTOL-MYERS SQUIBB COMPANY TECHNICAL FIELD OF INVENTION The present invention is directed to mixed specificity fusion proteins capable of binding to cellular adhesion receptors, as well as their synthesis and use to inhibit inflammatory reactions and to inhibit cellular metastasis in a patient.

BACKGROUND OF INVENTION The ability of circulating leukocytes to migrate across the vascular endothelial lining of the blood vessels (extravasation) is critical for homeostasis and also for effective host responses to infectious organisms and tumors. Lymphocytes continuously recirculate from blood into various lymphoid organs providing immunological surveillance and also serving to disseminate regionally stimulated lymphocytes to distant sites. During diverse inﬂammatory events, other leukocytes such as neutrophils and monocytes also migrate into lymphoid and nonlymphoid tissues. The leukocyte specificity of extravasation during inﬂammation likely assures the accumulation of leukocyte subsets appropriate to the particular stage and nature of the inflammatory response. Leukocyte extravasation is controlled in part by specific interactions with vascular endothelial cells via specific adhesion receptors (Osborn L, 1990, Cell Q23-6).

There are at least three distinct classes of adhesive molecules that leukocytes employ during their adhesive interactions: a) integrins including LEC-CAMS/Selectins (ELAM—1, LAM-1/Ieu8/TQ1, and GMP140/PADGEM); b) those belonging to the immunoglobulin superfamily which include CD2 (LFA-2), CD3/TCR, CD4, CD8, CD28, CD44, CD54 (ICAM-1), ICAM-2, CD58 (LFA-3), VCAM-1, B7; and c) Class I and II MHC (See above cited articles).

There are at least six distinct alpha subunits alpha1 (CD49a), alpha2 (CD49b), alpha3 (CD49e), alpha4 (CD49d), alpha5 (CD49e), and alpha6 (CD49f) capable of associating with beta1 (CD29). nonhematopoietic and leukocyte cell types and are thought to play an active role in The betal integrins are expressed on many tissue organization by binding to extracellular matrix components found in many tissues and in the basement membranes underlying muscles, nervous system, epithelium and endothelium. While the expression of many betal integrins on leukocytes requires consistent activation, their expression on nonhematopoietic cells does not (Hemler, M.E., 1988, Immunol. Today 2:109-113; Patarroyo, M., and Makgoba, M.W., 1989, Scand. J. Immunol. 302129-164). The complexity of the integrin family has been The changes in the expression of various beta2 integrins due to activation appears to be governed also by their preordained genetic programs. On neutrophils and monocytes, stimulation with a number of factors including calcium ionophore, phorbol esters, fMLP, GM-CSF, TNF, C5a, PDGF, LTB4 or even increases in ambient temperature (hyperthemia) rapidly (minutes) results in significantly increased surface expression of both CD11b/CD18 and CD11c/CD18 without appreciable change in the levels of expression of CD11a/CD18 (LFA—1). In contrast, on lymphoid cells (both T and B) which express only CD113/CD18, longer duration (hours) of activation is required to increase expression of CD11a/CD18 required, perhaps due to the fact that there are intracellular storage pools for CD11b/CD18 and CD11c/CD18 in myeloid cells. No such storage pools for CD11a/CD18 have been demonstrated for The CD11b/CD18 molecule also exhibits ability to bind to ICAM—1. In addtion, this molecule is also utilized in binding to Arg—Gly—Asp sequences within iC3b, factor X of the clotting cascade, and fibrinogen, each perhaps contributing to the activation of neutrophils. Although the expression of CDl1c/CD18 is upregulated during activation of neutrophils and monocytes, its interacting ligand still remains elusive (Moller, G.

Editor, 1990, Immunol. Rev. _l1_4:l-217).

The beta2 integrins are also intricately involved in the functions of neutrophils and also other granulocytes such as eosinophils, basophils and mast cells. Predominant function of polymorphonuclear leukocytes is to sense the existence of inﬂammatory foci and in response to the inflammatory stimuli emigrate across the endothelial barrier to the inflammed sites to carry out the scavenger role. As a result, interaction of neutrophils with vascular endothelial cells are considered crucial in host defense against infections and also the subsequent repair process. Neutrophils are the predominant leukocytes at the inflammed site with the peak of emigration occurring within the first Within 12-24 hours, however, mononuclear cells including lymphocytes and monocyte / macrophages become the most several hours after the onset of inflammation. threatening bacterial infections.

Although a vast majority of reports dealt with the inhibition of various adhesion- dependent functions of leukocytes in vitro by mAb directed at beta2 integrins, a few The monoclonal antibody MAb 60.3 directed at beta2 integrin (CD18) (Beatty, P. G., Ledbetter, J. A., Martin, P. J., Price, T. H., and Hansen, J. A., 1983, J. Immunol. @2913-2918) was shown to reduce organ injury and improve survival from hemorrhagic shock and resuscitation in rabbits by attenuating both the liver and gut injuries Caused by generalized ischemia and reperfusion. The above tissue injury is considered to be the consequence of damage caused by activated neutrophils to the endothelium and the surrounding tissue (Vedder, N. B., Winn, R. K., Rice, C. L., Chi, E. Y., Arfors, K.-E., and Harlan, J. M., 1990, Proc. Natl. Acad. Sci. USA, 812643-2646).

In another model, myocardial injury (myocardial infarction) caused by activated neutrophils in ischemic and reperfused dogs was significantly reduced by the anti-CD18 mAb 60.3 (Patarroyo, M., and Makgoba, M.W., 1989, Scand. J. Immunol. ;(_):129-164; Moller, G. Editor, 1990, Immunol. Rev. _1_ﬂ:1—217; Beatty, P. G., Ledbetter, J. A., Martin, P. J., Price, T. H., and Hansen, J. A., 1983, J. Immunol. _1_3_1_:2913-2918). In humans, mAb to the CD11a/CD18 (LFA—1) was reported to prevent allogeneic graft- failure in HLA-mismatched bone marrow transplantation.

Mab 60.3 recognizes an epitope on the CD18 (beta2 integrin) molecule (Beatty, P. G., Ledbetter, J. A., Martin, P. J., Price, T. H., and Hansen, J. A., 1983, J. Immunol. 13_1:2913—2918) which is a constituent of all the three beta2 integrins (CD11a, CD11b, CD11c) critically involved in all functions mediated via beta2 integrins (Springer T.A., 1990, Nature _3g1_6:425-434; Patarroyo, M., and Makgoba, M.W., 1989, Scand. J.

SUMMARY OF THE INVENTION The present invention is directed to substantially pure chimeric molecules and their use to inhibit inflammatory and metastatic processes. These chimeric molecules are fusion proteins that contain at least two separate binding regions. Each of these regions has binding specificity for a cellular adhesion molecule. Each of these binding regions has the specificity of a different cell surface receptor extracellular domain or represents the variable region of an antibody directed to an adhesion molecule. The binding regions are operatively attached to a polypeptide to produce the chimeric molecule of the present invention.

One class of molecules of the present invention are immunog1obulin—like fusion The immunoglobulin constant region of these fusion proteins can substantially correspond proteins having a mixed specificity containing such binding regions. to a constant region of IgG. Binding regions of the fusion proteins can comprise binding portions of the extracellular domains of cell surface receptors, such as ELAM- 1, GMP140, and ICAM-1. invention include a fusion protein of a human IgG constant region attached to regions of the extracellular domains of ELAM-] and GMPl40, a fusion protein of an IgG constant region attached to regions of the extracellular domains of ICAM-1 and Specific fusion proteins contemplated by the present ELAM-1, a fusion protein of an IgG constant region attached to regions of the extracellular domains of ELAM—1 and VCAM~1, and a fusion protein of an IgG constant region attached to regions of the extracellular domains of ICAM-1 and GMP140.

Compositions of the fusion proteins of the present invention are further contemplated, together with methods of inhibiting inﬂammation and metastasis in a patient by administering a therapeutically effective amount of the fusion protein of the present invention to the patient.

BRIEF DESCRIPTION OF THE FIGURES In the drawings: FIGURE 1 illustrates the SDS-PAGE banding patterns for supernatants from transfected cells under reducing and nonreducing conditions (lane 1 and 2); supernatants from cells transfected with a plasmid encoding the GMP140-IgG fusion protein under reducing and nonreducing conditions (lane 3 and 4); supernatants from cells transfected with a plasmid encoding the ELAM—1-IgG fusion protein under reducing and ,nonreducing conditions (lane 5 and 6); supernatants from cells cotransfected with a mixture of plasmids encoding the GMP140-IgG and ELAM—1-IgG fusion proteins under reducing and nonreducing conditions (lane 7 and 8); and molecular weight markers (lane 9).

FIGURE 2 illustrates the purification of the ELAM-1/GMP14O IgG fusion protein. Panel A shows an elution profile for the separation of COS cell supernatant proteins upon a hydroxyapatite column. Elution is with a KHZPO4/KZHPO4 (pH6.8) gradient from 10mM to 350mM, at a flow rate of 1 ml/min., 1 ml fractions were collected.

Panel B illustrates a SDS-PAGE analysis of the fractions obtained from the hydroxyapatite separation of Panel A.

Five-fraction groups, starting at fraction 1 and extending to fraction 50, were pooled and concentrated on a sephacryl protein A matrix. The concentrated fractions were applied to SDS-PAGE and the protein bands resolved.

DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is directed to mixed specificity fusion proteins that are capable of binding to cellular adhesion proteins. Particular fusion proteins of the present invention contain a polypeptide or an immunoglobulin-like protein region, such as an IgG constant region, operatively linked, or attached, to at least two specific binding regions. Each binding region preferably corresponds to either a variable region of an antibody directed to an adhesion molecule or a region of the extracellular domain of a cell surface receptor, such as ELAM-1, VCAM—1, GMP140 and ICAM-1. A particularly preferred antibody variable region is the variable region corresponding to the specificity of mAb 60.3 which is directed against beta 2 integrin (CD18).

As used herein the term "extracellular domain" refers to a region of the extracellular portion of a cell surface receptor that retains binding specificity for a cellular adhesion molecule. Such an extracellular domain is capable of inhibiting binding between target cells such as neutrophils and vascular endothelium.

As used herein, the term "cellular adhesion molecule" refers to specific inflammatory cell surface molecules that are recognized and bind to vascular endothelium and / or granulocytes.

As used herein, the term "operatively attached" refers to the linkage of groups in a manner such that the binding affinity of the group is not inhibited by the attachment.

As used herein, the term "IgG" constant region" refers to domains of the gamma chain of the IgG molecule that are adjacent to the variable region that corresponds to the first 107 amino acids of the gamma chain or fragments thereof. The four domains within the gamma chain constant region are designated CH1, H, CH2, and CH3. CH1 is adjacent to the variable region and encompasses amino acid residues 114 through 223. H (hinge; residues 224-245) is adjacent to CH1 and contains the cysteine residues that form the disulfide bonds which covalently link the two immunoglobulin heavy chains. CH2 is adjacent to the hinge and encompasses amino acid residues 246 through 361, followed by CH3 which contains amino acid residues 362 through 496.

The extracellular domains of at least two different cell surface receptors are thus fused in the present invention to give hybrid fusion proteins having multiple specificities and functional properties. The fusion proteins are capable of binding to natural ligands on target cells, such as endothelial cells and neutrophils, and blocking adhesion and /or cellular activation. The proteins of the present invention are thus contemplated to be effective in blocking neutrophil-mediated endothelial cell injury, such as in ischemia— reperfusion, by blocking CD18 mediated neutrophil aggregation and adherence to endothelium.

The mixed specificity receptor fusion proteins of the present invention are preferably directed against the neutrophil cell surface proteins responsible for neutrophil-endothelial binding, and thus they can block the binding of neutrophils to endothelium.

In a preferred embodiment, the fusion proteins of the present invention are produced by fusing the cDNA fragments encoding the extracellular domains of the endothelial and granulocyte surface receptors responsible for neutrophil-endothelium binding, such as ICAM—1/ICAM—2, VCAM—1, ELAM—1 and GMP140, to a genomic fragment encoding the human IgG constant region. Combinations of these constructs are then transfected into mammalian cells. The mixed specificity receptor- immunoglobulin fusion proteins are thereby assembled in these cells and secreted side by side with the single specificity immunoglobulin fusion proteins. In the present invention mixed specificity fusion proteins, such as ICAM-1/ELAM-1, ICAM-1/GMP- 140, VCAM-1/GMP-140 etc., have been produced and can be tested alone and in combination for their ability to bind neutrophils and alleviate reperfusion injury.

Fusion proteins of the present invention are preferably produced by the fusion of human proteins and, as such, would be less immunogenic than non—human monoclonal antibodies that may have related specificity to one or more adhesion molecules. The multiple specificity of these fusion proteins enables the simultaneous binding of several of the neutrophil proteins responsible for neutrophil—endothelial binding, and thus will be potent blockers of the neutrophil—endothelial adhesion that is associated with reperfusion injury and inflammation.

Preferred embodiments of the present invention are the mixed specificity fusion proteins described herein, the pharmaceutically acceptable salts thereof and related variants thereof. The phrase "pharmaceutically acceptable salts", as used herein, refers to non-toxic alkali metal, alkaline earth metal and ammonium salts used in the pharmaceutical industry, including the sodium, potassium, lithium, calcium, magnesium and ammonium salts and the like that are prepared by methods well-known in the art.

The phrase also includes non-toxic acid addition salts that are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.

Representative salts include the hydrochloride, hyrdrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, vorate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate and the like.

Compositions of the present invention contain mixed specificity fusion proteins, as described hereinabove, together with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a physiologically tolerable, non-toxic material in which the fusion proteins of the present invention can be dissolved or dispersed. Illustrative pharmaceutically acceptable carriers can be solid or liquid materials and can include water, saline, phosphate—buffered saline, Ringer's solution, dextrose, cornstarch, lipid emulsions and the like.

The fusion proteins and compositions of the present invention can be effectively utilized in a method for inhibiting inflammation in a patient. A therapeutically effective amount of a mixed specificity fusion protein, as described herein, is adminstered to a patient for a time period sufficient to a ameliorate or inhibit inﬂammatory processes and/or reactions in the patient by inhibiting the attachment of inflammatory cells, such as neutrophils, to vascular endothelium.

The fusion proteins and compositions of the present invention can also be effectively utilized for the inhibition of metastasis in a patient. Colon carcinoma cells are known to have glycosylated surface proteins which are recognized by cellular receptors such as VCAM-1 and ICAM-1. In a method of the present invention, a therapeutically effective amount of a mixed specificity fusion protein, as described herein, is administered to a patient for a time period sufficient to inhibit the metastasis of responsive tumor cells.

The present invention is further described by the following Examples which are intended to be illustrative and not limiting.

EXAMPLE 1: Preparation of ELAM-1/GMP140 Fusion Proteins Expression plasmids containing CDNA fragments encoding the complete extracellular domain of ELAM—1 and the four amino terminal domains of the GMP140 protein fused to a genomic fragment encoding the human IgG constant region were mixed in equal amounts and cotransfected into COS monkey cells by the DEAE- dextran method of Seed, B. and Aruffo, A., 1987, Proc. Natl. Acad. Sci. USA, $3365- 3369.

Twenty four hours after transfection the cells were washed with phosphate- buffered saline (PBS, 5 milliliters(ml)/ 100 mm dish), and the serum—containing medium (Dulbeccos's Modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS)) was replaced with serum—free medium (DMEM, 10 ml/100 mm dish). Four days following transfection additional serum-free DMEM was added to the transfected cells (10 ml/dish) and six days later the COS cell supernatant was harvested and cellular debris were removed by low speed centrifugation.

The recombinant proteins obtained were analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS—PAGE). illustrate the banding for proteins obtained from mock-transfected COS cells (control), COS cells transfected with DNA encoding the ELAM-1 immunoglobulin fusion protein (ELAMIgG) or COS cells transfected with DNA encoding the GMP140 The results shown in Figure 1 immunoglobulin fusion protein (GMP140-IgG). It can be seen that the supernatant obtained from the COS cells transfected with the mixture of DNAs contain three proteins corresponding to the ELAM—l-IgG and the GMP140—IgG hornodimeric fusion proteins and the ELAM-1/GMP140 mixed specificity receptor fusion protein.

Analysis of the proteins under reducing conditions showed two bands corresponding to the ELAM-l—IgG and the GMP140-IgG monomeric fusion proteins, This result indicates that the mixed specificity ELAM-1/GMP140 receptor fusion protein is assembled by the transfected cell and held together by disulfide bonds located within the hinge region of the IgG Fc region. respectively.

EXAMPLE 2 Purification of ELAM—l/GMP140 Fusion Protein The ELAM—1/GMPl40 mixed specificity receptor proteins in the crude COS cell supernatant described in EXAMPLE 1 were purified by passage over a hydroxyapatite column. Material bound to the column was then eluted using a KHZPO4/KZHPO4 (pH 6.8) gradient starting at 10 mM and ending at 350 mM with a flow rate of 1 ml/min. The elution profile is shown in FIGURE 2A. Groups of five one—milliliter fractions (5 ml) at a time were removed during the elution (from Fraction 1 to Fraction 50), pooled, concentrated on a sephacryl protein A matrix and analyzed by SDS—PAGE (FIGURE 2B). The results show that the mixed specificity ELAM- 1/GMP140 IgG heterodimer can be separated from the ELAM—1-IgG and the GMPl40-IgG homodimers.

Claims

Claims:

l. A substantially pure chimeric molecule comprising a fusion protein having a mixed specificity for cellular adhesion molecules and containing at least two separate binding regions operatively attached to a polypeptide.
2. The chimeric molecule according to Claim 1 wherein each binding region has the specificity of a different cell surface receptor extracellular domain.
3. The chimeric molecule according to Claim 1 wherein at least one binding region has the specificity of a variable region of an antibody directed against a cell adhesion molecule.
4. The chimeric molecule according to Claim 3 wherein the binding region corresponds to the specificity of the variable region of a monoclonal antibody specific for CD18.
5. The chimeric molecule according to Claim 1 wherein said fusion protein comprises an immunoglobulin constant region operatively attached to at least two binding regions, each binding region comprising an extracellular domain for a different cell surface receptor for cell adhesion molecules.
6. The chimeric molecule according to Claim 5 wherein said immunoglobulin constant region is a constant region of human IgG.
7. The chimeric molecule according to Claim 5 wherein one binding region comprises an extracellular domain of ELAM-l, GMPl40, ICAM-l, or VCAM—l.
8. The chimeric molecule according to Claim 5 comprising a fusion protein of an lgG constant region attached to regions of the extracellular domains of ELAM-l and GM}:-’l40, ICAM-l and ELAM-l, ICAM-l and GMPl40, or VCAM—l and GMP140.
9. A pharmacological composition comprising a fusion protein having a mixed specificity for cellular adhesion molecules, and containing at least two separate binding regions operatively attached to a polypeptide.
10. The composition according to Claim 9 wherein at least one binding region comprises a variable region of an antibody directed against a cell adhesion molecule.
11. The composition according to Claim 9 wherein said fusion protein comprises an immunoglobulin constant region operatively attached to at least two binding regions, each binding region comprising an extracellular domain of a different cell surface receptor for cell adhesion molecules.
12. The composition according to Claim ll wherein said immunoglobulin constant region is a constant region of human IgG.
13. The composition according to Claim 11 wherein one binding region comprises an extracellular domain of ELAM—1, GMPl40, ICAM—l, or VCAM—l.
14. A fusion protein having a mixed specificity for cellular adhesion molecules and comprising a polypeptide region operatively attached to at least two binding regions having specificity for cell adhesion molecules for use in inhibiting inflammation or inhibiting cellular metastasis.
l5. The protein according to Claim 14 wherein each binding region corresponds to a region of the 15 extracellular domain of a different cell surface receptor for a cell adhesion molecule.
16. The protein of Claim 14 wherein at least one binding region comprises a variable region of an antibody directed to a cell adhesion molecule.
17. The protein according to Claim 14 wherein said fusion protein comprises an lgG constant region operatively attached to regions of the extracellular domains of ELAM—1 and GMP140, ICAM-1 and ELAM-1, ICAM-1 and VCAM—1, VCAM—l and GMP140, or lCAM—1 and GMP140.
18. A pharmacological composition comprising a therapeutically effective amount of a fusion protein according to any one of Claims 1 to 8 and a pharmaceutically acceptable carrier.
19. Use of the fusion protein according to any one of Claims 1 to 8 in the manufacture of a medicament for the treatment of inflammation or cellular metastasis. E. R. KELLY & CO., AGENTS FOR THE APPLICANTS