AU684084B2 - Antibodies to P-selectin and their uses - Google Patents

Description

till D)ATE 21/11/94 AOC0? DATE 05/01/95 APPLN. ID 69063/94 PCT NUMBER PCT/US94/04935 AU9469063 (51) International Patent Classification 5 (11) International Publication Number: WO 94/25067 7/01,5/00(43) International Publication Date: 10 November 1994 (10.11,94) (21) International Application Number: PCr'/S94/04935 Torrey Pines Terrace, Del Mar, CA 92014 JONES, Tarran [GB/GB]; 10 The Close, Radlett, Hertfordshire (22) International Filing Date: 4 May 1994 (04.05.94) WD7 8HA SALDANHA, Jose, W. [GB/GB]; 22A Loncoin Way, Enfield, Middlesex, EN I ITE BENDIG, M~aryj, M. [US/GB]; 64 Solent Road, West Hampstead, Priority Data: London NW6 17X KRIEGLER, Michael [USftUS]; PCTItJS93/04272 4M?&y 1993 (04.05.93) WO P.O. Box 9094, Rancho Santa Fe, CA 92067 PEREZ, (34) Cowatries for which the regional or Carl [US/US]; 7245 Charrnant Drive, #518, San Diego, CA international application was filed: US et al. 92122 BAYER, Robert [US/US]; 6105 Dirac Street 0.3/057,292 5 May 1993 (05.05.93) us San Diego, CA 92122 NUNN, Michael [US/US]; 650 03/202,047 25 February 1994 (25.02.94) us Columbia Street. #308, San Diego, CA 92101 (US).

(74) Agents: LIEBESCH]ET', Joe et al.; Townsend and Townsend Parent Application or Grant Kbourie and Crew, 20th floor, One Market Plaza, Steuart (63) Related by Continuation Stres-t Tower, San Francisco, CA 94105 (US).

us 08/202,047 (CON) Filed on 25 February 1994 (25.02.94) (81) Designated States: AT, AU, BB, BG, BR. BY, CA, CH, CN, CZ, DE, DK, ES, FR GB, HU, JP, KG, KP, KR, KZ, LK, (71) Applicant (for all designated States except US): CYTEL LU, LV, MD, MG, MN, MW, NL, NO, NZ, PL, PT!, RO, CORPORATION [US/US]; 3525 John Hopkins Court. San RU, SD, SE, SL SK, TJ, TiT, UA. US, UZ, VN, European Diego, CA 92121 patent (AT, BE, CH, DE, DK, ES, FR GB, Gil, IE, IT, LU, MC, NL,PT SE), OAPI patent (BU BJ, CF, CG,CL. CMK (72) Inventors; and 1G).

(7S).Inventors/Applicants (for US only): CHESNUT, Robert. W, [US/US]; 1473 Kings Cross Drive, Card if, CA 92007 (US).

POLLEY, Margaret. J. [GB/UJS]; 6621 Avenida Mirola, La Published Jolla, CA 92037 PAULSON, James, C. [US/US]; 209 With internati elr I e,0 (54) Fitliet ANTIBODIES TO P-SELECTIN AND THEIR USES (57) Abstract 'I The-pfesent invention relates to compositions and methods fib'treating inflammation and other pathological conditions using novel blocking P-selcctin antibodies that inhibit adhesion of leukocytes to activated platelets and/or to activated vasrular endothelium In vivo.

Both murine and humtaized antibodies are provided.

WO 94/25067 PCT/US94/04935 1 ANTIBODIES TO P-SELECTIN AND THEIR USES CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of 08/057,292, filed May 5, 1993, which is a continuation-in-part of 07/880,196, filed May 5, 1992, now abandoned. These applications are incorporated by reference in their entirety for all purposes.

TECHNICAL FIELD This application relates generally to novel immunoglobulins reactive with functional epitopes on a cell surface receptor P-selectin. This application also relates generally to diagnostic and therapeutic methods of using the antibodies.

BACKGROUND OF THE INVENTION The ability of cells to adhere to one another plays a critical role in development, normal physiology, and disease processes such as inflammation. This ability is mediated by adhesion molecules, generally glycoproteins, expressed on cell membranes. Often, an adhesion molecule on one cell type will bind to another adhesion molecule expressed on" a different cell type, forming a receptor counter-recepto:c pair. Three important classes of adhesion molecules are the integrins, selectins, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425 (1990); Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992), all of which are incorporated herein by reference in their entirety for all purposes). These molecules are especially vital to the interaction of leukocytes and platelets with themselves and with the extracellular matrix and vascular endothelium.

P-selectin, also known as CD62, granule membrane protein-140 (GMP-140)/platelet activation-dependent granule external membrane (PADGEM)/LECCAM-3 is a specialized cell- SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 2 surface receptor on vascular endothelial cells and platelets that is involved in the recognition of various circulating cells. P-selectin is a surface glycoprotein with a lectinlike domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see McEver, Blood Cells 16:73-83 (1990)). The structure of P-selectin is similar to that of two other vascular cell surface receptors, endothelial leukocyte adhesion molecule (ELAM-1) and lymphocyte homing receptor (LHR). The term "selectin" has been suggested for this general class of receptors, because of their lectin-like domain and the selective nature of their adhesive functions.

P-selectin is present on the surface of platelets and endothelial cells in response to a variety of stimuli, where it mediates platelet-leukocyte and endothelium-leukocyte interactions. By contrast, ELAM-1 is expressed only on endothelial cells and LHR is expressed on a variety of leukocytes in endothelial venules of peripheral lymph nodes.

Expression of P-selectin is inducible and it is not expressed on unactivated endothelial cells or platelets. P-selectin expression does not require de novo synthesis since it is stored in secretory granules (or Weibel-Palade bodies) in both platelets and endothelial cells. Thus, within minutes of activation of either cell type by thrombin, histamine, or phorbol esters or other humoral factors, P-selectin is rapidly redistributed to the surface of the cell where it is accessible for binding to other cells.

A number of mouse antibodies to P-selectin have been reported (see, U.S. Patent No. 4,783,330, WO 91/06632, PCT Application No. FR90/00565, McEver et al., J. Biol. Chem.

259:9799-9804 (1984)); Geng et al., Nature 343:757-760; Parmentier et al., Blood 77:1734-1739 (1991)). An additional antibody to P-selectin has been described in presentations at the Scripps Institute, La Jolla California and at a Keystone Colloquium held January 16, 1992. Certain of these antibodies that block P-selectin mediated adhesion are Ca++ dependent.

In vitro experiments employing antibodies to Pselectin have provided a limited understanding of the ligand SUBSTITUTE.SHEET (RULE 26) WO 94125067 PCT/US94/04935 3 specificity of this receptor. It has been reported that Pselectin binds neutrophils, monocytes and certain carcinoma cells (Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84:9238- 9242 (1987); Geng et al., Nature 343:757-760 (1990); Larsen et al., Cell 63:467-474 (1990); and Polley et al. It has been also been reported that P-selectin recognizes Lex as a highaffinity ligand (Larsen et al., Cell 63:467-474 (1990)).

However, subsequent experiments showed that SLex-containing oligosaccharide is in fact a more potent inhibitor of Pselectin mediated adhesion than Lex (Polley et al., Proc.

Natl. Acad. Sci. USA 88:6224-6228 (1991)).

The participation of P-selectin in intercellular adhesion in vitro suggests that like other adhesion molecules, P-selectin might participate in cellular interactions that contribute to inflammatory diseases in vivo. However, the specific inflammatory diseases in which P-selectin might play a major role have not yet been elucidated. Similarly, the epitope-specificity of agents effective to abort particular Pselectin-mediated inflammatory diseases is also ,nclear. Of the known antibodies to P-selectin, some bind to P-selectin but do not block P-selectin mediated intercellular interactions. Other known antibodies have the capacity to block P-selectin-mediated intercellular binding only in the presence of high concentrations of Ca 2 a condition that may not always exist in vivo. The absence of high concentrations of Ca 2 in vivo would render these antibodies ineffective as therapeutic agents. Moreover, all antibodies to P-selectin produced to-date have been of murine origin and might therefore induce a human-antimouse antibody response (HAMA) in clinical use.

Based on the foregoing it is apparent that a need exists for novel agents targeted against P-selectin that have the appropriate epitope-specificity to abort intercellular interactions leading to inflammatory diseases in vivo.

Ideally such agents would not induce a HAMA response in treatment of humans. The present invention fulfills this and other needs.

SUBSTITUTE SHEET (RULE 26) \a y04/2£vf 4 SUMMARY OF THE INVENTION Blocking antibodies that specifically bind to P-selectin are disclosed herein. One such exemplified antibody is designated mu MAb PB1.3 (produced by a cell line having ATCC accession number HB11041). Other antibodies competitively inhibit the binding of mu MAb PB1.3 to P-i ctin measured by a competitive inhibition assay.

Preferred antibodies bind to P-selectin in the absence of Ca' The antibodies of the invention include fragments such as Fab, Fab' Fabc, and Fv, as well as intact antibodies.

Pharmaceutical compositions are also described. The pharmaceutical compositions comprise a blocking P-selectin antibody as described above and a pharmaceutically acceptable carrier.

The invention also provides therapeutic methods for treating diseases of the immune system. The methods comprise administering to a patient having such a disease a therapeutically effective dose of a pharmaceutical composition 20 as described above. Acute lung injury and ischemic reperfusion injury are examples of diseases treatable by the present invention.

Antibodies of the invention are humanized immunoglobulins. The humanized immunoglobulins comprise a humanized heavy chain and a humanized light chain. The humanized light chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid a 0 sequences from the corresponding complementarity determining regions of a mouse PB1.3 immunoglobulin light chain, and a variable region framework from a human kappa light chain b'e variable region framework sequence. The humanized heavy chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of a mouse PB1.3 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework RAsequence. The humanized immunoglobulins specifically bind to S P-selectin with a binding affinity havine a lower limit of SUBSTTUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 about 107 M 1 and an upper limit of about five-times the binding affinity of the mouse PB1.3 immunoglobulin.

In many humanized immunoglobulins, the human kappa light chain variable region framework sequence is substituted in at least one position selected from a first group consisting of L21, L46, L60 and L70, by an amino acid present in the equivalent position of the mouse PB1.3 immunoglobulin light chain variable region framework sequence. Similarly, the human heavy chain variable region framework sequence is substituted in at least one position selected from a second group consisting of H2, H71 and H73, by an amino acid present in the equivalent position of the mouse PB1.3 heavy chain variable region framework sequence. In some humanized immunoglobulins, the humanized heavy chain variable region framework is a 21/28'CL heavy chain variable region framework sequence. In some humanized immunoglobulins, the humanized light chain variable region framework is a DEN light chain variable rejgon framework sequence. Exemplary mature light chain variable region amino acid sequences are shown in Figures 14-17. Exemplary mature heavy chain variable region amino acid sequences are shown in Figures 11-13. A preferred humanized immunoglobulin comprises the mature light chair variable region shown in Figure 17 and the mature heavy chain variable region shown in Figure 12.

The invention provides intact humanized immunoglobulins and humanized immunoglobulin fragments such as Fab, Fab' F(ab')2 Fabc and Fv. Some humanized immunoglobulins further comprise a constant region, which may or may not have an effector function.

In another aspect of the invention, nucleic acids encoding the humanized immunoglobulins described above are provided. Some nucleic acid encode heavy chains and others encode light chains.

The invention also provides computers programmed to display a three dimensional representation of the immunoglobulins described above on a monitor or printer.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 6 The invention further provides pharmaceutical compositions comprising the humanized immunoglobulins described above and a pharmaceutically acceptable carrier.

In another aspect, the invention provides methods for detecting P-selectin using the humanized immunoglobulins described above. A humanized immunoglobulin is administered to a patient or a tissue sample therefrom. Complexes formed by specific binding between the immunoglobulin and P-selectin present in the target sample are detected to indicate the presence of P-selectin.

The invention also provides methods of treating a patient suffering from a disease of the immune systems using humanized immunoglobulins. The methods comprise administering to the patient a therapeutically effective dose of a pharmaceutical composition as described above. Diseases amenable to treatment include ischemia-reperfusion injury and acute lung injury. For example, the compositions are useful for treating patients suffering from epidermal, myocardial, renal, cerebral, splenic, hepatic, spinal, splanchnic, pulmonary, partial-body, or whole-body ischemia.

In a further aspect of the invention, stable cell lines producing humanized immunoglobulins are provided. The stable cell lines comprise two nucleic acids segments. One nucleic acid segment encodes the heavy chain of a humanized immunoglobulin as described above, the segment operably linked to a promoter to allow expression of the heavy chain. The second nucleic acid segment encodes the light chain of the humanized immunoglobulin, the second segment operably linked to a second promoter to allow expression of the light chain.

Preferred cell lines produce about 30 pg of the humanized 6 cells/day.

In another aspect of the invention, fragments of Pselectin are provided. These fragments have up to 100 amino acids, the up to 100 amino acids comprising at least five contiguous amino acids from between positions 448 and 467 of the amino sequence shown in Fig. 34. In preferred fragments, the up to 100 amino acids comprise the entire contiguous segment of amino acids between positions 448 and 467.

SUBSTITUTE.SHEET (RULE 26) WO 94/25067 PCT/US94/04935 7 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 presents data showing that anti-inflammatory immunoglobulins of the invention inhibit binding of thrombin activated platelets to neutrophils.

Figs. 2A and 2B show that anti-inflammatory immunoglobulins of the invention effectively prevent lung injury induced by infusion of cobra venom factor (CVF) as measured by permeability (Fig. 2A) and hemorrhage (Fig 2B).

Fig. 3A through 3H show that P-selectin expression content in lungs of animals is upregulated in response to cobra venom infusion visualized by horseradish proxidase stain of mu MAb PB1.3 panel show expression of P-selectin in lung venules at various times after infusion of CVF; time 0 5, 10 and 15 min, panels b, c and d, respectively (Fig.

3A). Fig. 3B are light and transmission electron micrographs of CVF-induced acute lung injury in rats treated with 200 Ag non-blocking antibody (mu MAb PNB1.6) (frames a.c) or with blocking P-selectin antibody (mu MAb PB1.3) to P-selectin (frames In animals treated with PNB1.6, vascular injury was indicated by extensive intra-alveolar hemorrhage (frame associated with intravascular aggregates of neutrophils (frame c. arrows) in close contrast with endothelial cells.

In contrast, in animals treated with mu MAb PB1.3 intraalveolar hemorrhage was absent (frame b) and intravascular neutrophils showed little contact with the endothelium (frame d. arrows). (Frames a and b, plastic embedded sections stained with toluidine blue, X160: frames c and d, uranyl acetate, lead citrate stained X2250.) Fig. 4 shows the binding of the monoclonal antibody mu MAb PB1.3 to P-selectin: effect of chelating divalent cations with EDTA. In the plot the open symbols (lower trace) is binding in the presence of Ca'" and Mg* while the filled symbols (upper trace) is binding in the presence of EDTA.

Antibody was diluted shown at 1.6 mg/ml.

Fig. 5 shows the effect of the peptide CQNRYTDLVAIQNKNE on the binding of mu MAb PB1.3 to P-selectin.

Filled symbols are in the presence of 0.35 mg/ml peptide, open s SUBSTImTE SHEET (RULE 26) Vi-^

I

symbols are in the absence of peptides. Indicated dilutions of mu MAb PB1.3 were from a 1.6 mg/ml solution.

Figs. 6A, 6B and 6C show the ability of monoclonal antibodies to cross-block binding to P-selectin. Blocking antibodies were present at 50 pg/ml. Dilutions of biotinylated antibodies shown were from 1.6 mg/ml mu MAb PB1.3, 0.87 mu MAb mg/ml PNB1.6, and 0.59 mg/ml mu MAb 84/26.

Fig. 7 shows that the extent of platelet aggregation induced by thrombin is equivalent under control conditions in the absence of antibody and in the presence of increasing concentrations (1-100 ig/ml) of purified mu MAb PB1.3 antibody.

Fig. 8 shows that the extent of myocardial ischemia, expressed as a percentage of the area at risk, is significantly less in animals treated with the biocking anti- P-selectin'antibody (mu MAb PB1.3) than in animals treated with the non-blocking anti-P-selectin antibody. Furthermore, the ability of coronary artery rings to relax in response to acetylcholine is significantly greater in mu MAb PB1.3 compared with mu MAb PHB1.6 treated animals. That these phenomena are attributable to a reduction in myocardial leukocyte infiltration is suggested by the reduced number of PMNs in myocardial tissue from mu MAb PB1.3 treated animals compared with PNB1.6 treated animals.

25 Pig. 9 shows the sequence and translation of PB1.3 heavy chain signal peptide and variable region (SEQ ID Nos.

2 and 3.

Fig. 10 shows the sequence and translation of PB1.3 light chain signal peptide and variable region (SEQ ID Nos.

30 4 and Fig. 11 shows the sequence and translation of CY1748 heavy chain-A signal peptide and variable region (SEQ ID Nos. 6 and 7).

Fig. 12 shows the sequence and translation of CY1748 heavy chain-B signal peptide and variable region (SEQ ID Nos. 8 and 9).

Fig. 13 shows the sequence and translation of CY1748 heavy chain-C signal peptide and variable region (SEQ ID Nos. 10 and 11).

Fig. 14 shows the sequence and translation of CY1748 light chain-A signal peptide and variable region (SEQ ID Nos. 12 and 13).

Fig. 15 shows the sequence and translation of CY1748 light chain-B signal peptide and variable region (SEQ ID f YL. Nos. 14 and r~p~4 4 C~PP~~ -9 Fig. 16 shows the sequence and translation of CY1748 light chain-C signal peptide and variable region (SEQ ID Nos. 16 and 17).

Fig. 17 shows the sequence and translation of CY1748 light chain-D signal peptide and variable region (SEQ ID Nos. 18 and 19).

Fig. 18 shows physical map of the neo-plasmid expressing the human heavy chain (gamma-i constant region).

Fig. 19 shows the physical map of the neo-plasmid expressing the human light chain (kappa constant region).

Fig. 20 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.

Fig. 21 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.

Fig. 22 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.

Fig. 23 shows the physical map of the dhfr-plasmid expressing the human heavy chain (gamma-i constant region).

Fig. 24 shows the physical map of the dhfr-plasmid expressing the human heavy chain (gamma-4 constant region).

Fig. 25 shows the inhibition of biotinylated-PB1.3 20 binding to rsP-Selectin by PB1.3 and CY1748 RHA/RLA-IgGI.

Fig. 26 shows the inhibition of biotinylated-PBI.3 binding to rsP-Selectin by PB1.3 and CY1748 RHB/RLD-IgG4.

0:6. Fig. 27 shows that antibody PB1.3 reduces swelling associated with ischemia and reperfusion in the rabbit ear.

25 Fig. 28 shows that antibody PB1.3 prevents the development of necrosis following ischemia and reperfusion of the rabbit ear.

Fig. 29 shows that antibody PB1.3 inhibits leukocyte-endothelial cell interactions induced by the mast cell degranulating agent compound 48/80.

Fig. 30 shows the proposed folding pattern of the domains of P-selectin.

Fig. 31 shows a schematic of the deletion mutants of P-selectin used to map the epitope that PB1.3 recognizes.

Fig. 32 shows the binding of PB1.3 and rabbit polyclonal antibodies to P-selectin deletion mutants.

I R Fig. 33 shows the PB1.3 and P6H6 binding to Pselectin anrd rr-selectin AS.CRP which is a deletion mutant of P-selectiyn 4: ts the fifth CRP.

Fig, o'ill the sequences of Eynthetic peptides derived Kamino acid sequence of the human Pselectir SEQ ID No. 20) and the PB1.3 reactivity to these imtmoilized peptides. Amino acids are numbered accordir.qy to the convention of Johnston et al., Cell, 56:1033-1044 (1989) for a full-length P-selectin polypeptide.

DEPINI'IONS

Abbreviations for the twenty naturally occurring amino acids follow conventional usage (XImmunology A Synthesis (2nd ed., E.S. Golub D.R. Gren, eds., sinauer Asae, Sunderland, MA, 1991)). Stereoisomers 0amino acids) of the twenty conventional amino acids, unnatur al amino acids such as a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamateo

,N

trimethyllysine, e-N-acetyllysine, 0-phosphoserinet Nacetylserine, N-f ormylmethionine, 3-methylhistidinet 9 9 hydroxylysine,, w-N-methylarginine, and other similar amino acids and imino acids (eg,4-hydroxyproline). Moreover, V, amino acids may be modified by glycosylation, phosphorylation a 0: Goand the like.

00 to In the polypeptide notation used herein., the lefthand direction is the amino terminal direction and the 30 righthand direction is the carboxy-terminal direction, in accordance with standard usage and conVention, similarly, unless specified otherwise, the lefthand end of singlestranded pc1.ynucleotide sequences is the 5' end; the lefthand direction of double-stranded Oolynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA~ transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 51 WO 94/25067 PCT/US94/04935 11 end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

The phrase "polynucleotide sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes self-replicating plasmids, infectious polymers of DNA or RNA and non-functional DNA or RNA.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of Figs. 9-17, or may comprise a complete DNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each comprise a sequence a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "cpmparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) of percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal SUBSTTUTE. SHEET (RULE 26) WO 94/25067 PCT/US94/04935 12 alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith Waterman, Adv. Appl.

Math. 2:482 (1981), by the homology alignment algorithm of Needleman Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson Lipman, Proc. Natl. Acad.

Sci. (USA) 85:2444 (1988) (each of which is incorporated by reference in its entirety for all purposes), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection, and the best alignment resulting in the highest percentage of sequence similarity over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polynucleotide sequences are identical on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least percent sequence identity, preferably at lsast 90 to percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 13 larger sequence, for example, the sequence shown in Figs. 17.

As applied to polypeptides, the term "sequence identity" means peptides share identical amino acids at corresponding positions. The term "sequence similarity" means peptides have identical or similar amino acids conservative substitutions) at corresponding positions. The term "substantial identity" means that two peptide sequences, when optimal3y aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions. The term "substantial similarity" means that two peptide sequences share corresponding percentages of sequence similarity.

The term "substantially pure" means an object species is the predominant species present on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCTIUS94/04935 14 orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Nonconservative substitutions constitute exchanging a member of one of these classes for another.

Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated Hx and Lxx respectively, where x is a number designating the position of an amino acids according to the scheme of Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991)) (hereinafter collectively referred to as "Kabat et al.," incorporated by reference in their entirety for all purposes).

Kabat et al. list many amino acid sequences for antibodies for each subclass, and list the most commonly occurring amino acid for each residue position in that subclass. Kabat et al. use a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. Kabat et al.'s scheme is extendible to other antibodies not included in the compendium by aligning the antibody in question with one of the consensus sequences in Kabat et al. The use of the Kabat et al. numbering system readily identifies amino acids at equivalent positions in different antibodies. For example, an amino acid at the L50 position of a human antibody occupies the equivalence position to an amino acid position L50 of a mouse antibody.

"Immunoglobulin," "antibody" or "antibody peptide(s)" refers to an intact antibody or a binding fragment thereof that competes with the intact antibody for specific binding to P-selectin.

"Substantial inhibition" means at least about inhibition, preferably about 60% to about 80%, and more usually about greater than 85% or more (as measured in an in vitro competitive binding assay).

Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 different species. For example, the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as 71 and 74. A typical therapeutic chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody, although other mammalian species may be used.

DESCRIPTION OF THE PREFERRED EMBODIMENT This invention provides compositions and methods for inhibiting diseases and conditions of the immune system mediated by P-selectin. Specifically, the invention provides immunoglobulins which have the ability to inhibit P-selectinmediated adhesion of cells in vivo.

I. Characteristics of Immunolobulins A. General The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyterminal portion of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a region of about 10 more amino acids.

(See generally, Fundamental Immunology (Paul, ed., 2nd ed.

Raven Press, 1989), Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibody binding site. The chains all exhibit the SUBSTITUTE SHEET (RULE 26) 16 same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. CDR an FR residues are delineated according to the standard sequence definition of Kabat et al., supra.

An alternative structural definition has been proposed by Chothia et al., J. Mol. Biol.196:901-917 (1987); Nature 342:878-883 (1989); and J. Mol. Biol. 186:651-663 (1989) (hereinafter collectively referred to as "Chothia et al." and incorporated by reference in their entirety for all purposes).

B. Binding Specificity and Affinity The immunoglobulins (or antibodies) of the invention selectively bind a functional epitope on P-selectin associated with a response to tissue injury and inflammation. Usually, binding of the antibodies to a functional epitope on P-selectin effectively inhibits adhesion of leukocytes to activated platelets and/or to the activated vascular endothelium in vivo. Antibodies showing this property are referred to as "blocking" antibodies. Preferred blocking antibodies impair the adhesion of leukocytes to the activated vascular endothelium to prevent or inhibit an inflammatory and/or thrombotic condition.

Many of the blocking antibodies of the invention compete with an exemplary antibody designated mu MAb PB1.3 for specific binding to P-selectin. Hybridoma CY 1747.1 producing the mu MAb PB1.3 antibody has been deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A. under the Budapest Treaty on 16 May 1992 and given the Accession No.

HB11041. The production of this monoclonal antibody is described in Example 1. Cell line CHO-48B4 which expresses a modified form of MAb PB1.3 was also deposited with the American Type Culture Collection (ATCC), S Rockville, Maryland, U.S.A. under the provisions of the Budapest Treaty on 30 March 1994 and given Accession No.

S:24100A 16A CRL 11596. Other blocking antibodies of the invention compete with a second exemplified antibody designated 84/26.

Competition is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody mu MAb PB1.3) to an antigenic determinant on a P-selectin molecule.

Numerous types of S:24100A WO 94/25067 PCT/US94/04935 17 competitive binding assays are known for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Molec. Immunol. 25(1):7- (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990)).

Typically, such an assay involves the use of purified Pselectin or cells bearing P-selectin bound to a solid surface, an unlabelled test immunoglobulin and a labelled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. An example of a suitable competitive binding assay is presented in Example 8.

Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance tu occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to P-selectin by at least 10, 25, 50 or The epitope bound by mu MAb PB1.3 ha% been mapped to an epitope between amino acids 448 and 467 of '-,uman Pselectin. See Figure 34 and Johnson et al., Cell 56:1033-1044 (1989) (hereby incorporated by reference in its entirety for all purposes). The epitope occurs withis the fifth CRP domain of P-selectin. See Figure 30. Antibodies that compete with mu MAb PB1.3 usually bind to a segmet, of amino acids between 448 and 467 or to another segment within the fifth CRP domain.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 18 The binding specificity of many blocking antibodies of the invention is further defined by their capacity to bind P-selectin in the complete or substantial absence of Ca++ in the presence of 25 mM EDTA (a calcium chelator) and the absence of Ca in an in vitro assay). Antibodies rdquiring a Ca cofactor for blocking activity may not be effective in in vivo conditions where levels of Ca++ are expected to fluctuate.

Antibodies that compete with the mu MAb PB1.3 antibody are further characterized by the observation that their capacity to specifically bind to P-selectin is not inhibited by a fragment of P-selectin having the amino acid sequence CQNRYTDLVAIQNKNE. A molar excess of this fragment usually inhibits specific binding of such antibodies by less than Mu MAb PB1.3 and competing antibodies are thereby distinguished from reported mouse antibodies designated GI, G2 and G3 (Geng et al., The Journal of Biologicl31 Chemistry 266:22313-22318 (1991)) whose binding to P-selectin is partially or completely blocked by the above peptide.

Some antibodies of the invention are further characterized by their capacity to selectively inhibit some Pselectin mediated responses while leaving others unimpaired.

For example, some antibodies are capable of blocking specific binding of neutrophils to activated endothelial cells in vivo, without inhibiting platelet aggregation. Such antibodies are particularly useful for treating inflammatory disorders without impairing a patient's capacity to initiate a response to conditions wounding) in which platelet aggregation is desirable.

The antibodies of the invention usually exhibit a specific binding affinity for P-selectin of at least 107, 108, 109, or 1010 M" 1 Usually the upper limit of binding affinity of the antibodies for P-selectin is within a factor of about three, five or ten of that of the mu MAb PB 1.3. Often the lower limit of binding affinity is also within a factor of about three, five or ten of that of mu MAb PB1.3. In this context, the term "about" encompasses the small degree of SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 19 experimental error that may typically occur in the measurement of binding affinities.

II. Production of Imnunoglobulins A. Nonhuman Antibodies The production of non-human monoclonal antibodies, murine, lagomorpha, equine is well known and can be accomplished by, for example, immunizing an animal with a preparation containing cells bearing P-selectin thrombin-activated platelets), isolated P-selectin molecules or fragments thereof, such as extracellular domains.

Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to P-selectin, and then immortalized. See Harlow and Lane, supra. Alternatively, substantially monospecific antibody populations can be produced by chromatographic purification of polyclonal sera.

B. Human Antibodies Aaainst P-Selectin In another aspect of the invention, human antibodies against P-selectin are provided. Some human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as mu MAb PB1.3. Such antibodies are particularly likely to share the useful therapeutic properties demonstrated for mu MAb PB1.3. Human antibodies to P-selectin can be produced by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). Antibodies binding to Pselectin or a frag.nt thereof are selected. Sequences encoding such antibodies (or a binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047 (each of which is incorporated by reference in its entirety for all purposes). In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces.

Antibodies are usually displayed as Fv or Fab fragments.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 Phage displaying antibodies with a desired specificity are selected by affinity enrichment to an P-selectin polypeptide or fragment thereof.

In a variation of the phage-display method, human antibodies having the binding specificity of a selected murine antibody can be produced. See Winter, WO 92/20791. In this method, either the heavy or light chain variable region of the selected murine antibody mu MAh PB1.3) is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members displays the same light chain variable region the murine starting material) and a different heavy chain variable region. The heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding for P-selectin at least 108 and preferably at least 109 M 1 is selected. The human heavy chain variable region from this phage then serves as a starting material for constructing a further phage library.

In this library, each phage displays the same heavy chain variable region the region identified from the first display library) and a different light chain variable region.

The light chain variable regions are obtained from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding for L-selectin are selected. These phage display the variable regions of completely human anti-P-selectin antibodies. These antibodies usually have the same or similar epitope specificity as the murine starting material mu MAb PB1.3).

SC. Humanized Antibodies The invention provides humanized immunoglobulins having variable framework regions substantially from a human immunoglobulin (termed an acceptor immunoglobulin) and complementarity determining regions substantially from a mouse immunoglobulin termed mu MAb PB1.3 (referred to as the donor immunoglobulin). The constant region(s), if present, are also substantially from a human immunoglobulin. The humanized SUBSTITUTE SHEET (RULE 26) 21 antibodies of the present invention offer several advantages over the mouse donor antibody, which has already shown to be effective in animals models: 1) The human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against'a totally foreign mouse antibody or a partially foreign chimeric antibody.

LO 2) Because the effector portion of the humanized antibody is human, it may interact better with other parts of the human immune system.

3) Injected mouse antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of normal human antibodies (Shaw at al., J.

IImunl. 138:4534-4538 (1987)). Injected humanized antibodies have a half-life essentially equivalent to naturally occurring human antibodies, allowing smaller and less frequent doses.

i. Cloning and Sequencing Variable Domains of mu Mab 20 PB1.3 The cloning and sequencing of cDNA encoding the mu Mab PBI.3 antibody heavy and light chain variable regions is described in Example 10, and the nucleotide and predicted amino acids sequences are shown in Figs. 11 and 12. Tables 1 (mouse IIA=SEQ ID No. 21, human I=SEQ ID No. 22, human 21/28'CL SEQ ID No. 23) and 2 (mouse k-V SEQ ID No. 24, human k-I SEQ ID No. 25, human DEN SEQ ID No. 26) illustrate the subdivision of the amino acid coding 4 sequence into framework and complementarity determining domains. From N-terminal to C-terminal, both light and heavy chains comprise the domains FRI, CDR, FR2, CDR2, 4.e.

FR3, CDR3 and FR4. The assignment of amino acids to each 4".4 domain is in accordance with the numbering convention of Kabat et al., supra.

Selection of Human Antibodies to Supply Framework Residues The substitution of mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable \domain WO 94/25067 PCT/US94/04935 22 framework adopts the same or similar conformation to the mouse variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

Suitable human antibody sequences are identified by computer comparisons of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies.

The comparison is performed separately for heavy and light chains but the principles are nimilar for each. This comparison reveals that the mu MAb PB1.3 light chain shows greatest sequence identity to human light chains of subtype kappa-1 and that the mu PB1.3 heavy chain shows greatest sequence identity to human heavy chains of subtype 1, as defined by Kabat et al., supra. Thus, light and heavy human framework regions are usually derived from human antibodies of these subtypes, or from consensus sequences of such subtypes.

The preferred heavy and light chain human variable regions showing greatest sequence identity to the corresponding regions from mu MAb PB1.3 are from antibodies 21/28'CL and DEN respectively.

Computer Modelling The unnatural juxtaposition of murine CDR regions with human variable framework region can result in unnatural conformational restraints, which, unless corrected by substitution of certain amino acid residues, lead to loss of binding affinity. The selection of amino acid residues for substitution is determined, in part, by computer modelling.

Computer hardware and software for producina three-dimensional SUBSTITUTE SHEET (RULE 26) WO 94125067 PCT/US94/04935 23 images of immunoglobulin molecules are widely available. In general, molecular models are produced starting from solved structures for immunoglobulin chains or domains thereof. The chains to be modelled are compared for amino acid sequence similarity with chains or domains of solved three dimensional structures, and the chains or domains showing the greatest sequence similarity is/are selected as starting points for construction of the molecular model. For example, for the light chain of mu MAb PB1.3, the starting point for modelling was the human light chain DEN. The solved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modelled, and those in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from one another and that bond lengths and angles are within chemically acceptable limits. Such a model can in turn serve as a starting point for predicting the three-dimensional structure ol an antibody containing the mu MAb PB1.3 complementarity determining regions substituted in human framework structures.

Additional models can be constructed representing the structure when further amino acid substitutions to be discussed infra, are introduced.

Substitution of Amino Acid Residues As noted supra, the humanized antibodies of the invention comprise variable framework regions substantially from a human immunoglobulin and complementarity determining regions substantially from a mouse immunoglobulin termed mu MAb PB1.3. Having identified the complementarity determining regions of mu MAb PB1.3 and appropriate human acceptor immunoglobulins, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody.

In general, substitution of human amino acid residues with murine should be minimized, because introduction of murine residues increases the risk of the antibody elicitina a HAMA SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/IJS94/04935 24 response in humans. Amino acids are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modelling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.

When an amino acid differs between a mu MAb PB1.3 variable framework region and an equivalent human variable framework region, the human framework amino acid should usually be substituted by the equivalent mouse amino acid if it is reasonably expected that the amino acid: noncovalently binds antigen directly amino acids at positions H1, H2, and L60 of mu MAb PB1.3) is adjacent to a CDR region, is part of a CDR region under the alternative definition proposed by Chothia et al., supra, or otherwise interacts with a CDR region is within about 3 A of a CDR region) H73 of mu MAb PB1.3, Tables 1 and or participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of more typical human immunoglobulins. Alternatively, amino acids from equivalent positions in mu MAb PB1.3 can be introduced into the human framework regions when such amino acids are typical of human immunoglobulin at the equivalent positions.

In general, substitution of all or most of the amino acids fulfilling the above criteria is desirable.

Occasionally, however, there is some ambiguity about whether a particular amino acid meets the above criteria, and alternative variant immunoglobulins are produced, one of which has that particular substitution, the other of which does not.

Surprisingly, an exemplified humanized antibody designated reshaped MAb PB1.3 (HgLD) contains only one substitution of the human heavy-chain variable region framework residues and SUBSTITUTE SHEET (RULE 26) WO 94125067 PCT/US94/04935 only two substitutions of the human light-chain variable region framework residues and yet exhibits substantially similar (within a factor of two) binding affinity to a murine or chimeric antibody having intact murine variable regions fused to a human constant region (designated chi MAb PB1.3).

Some humanized antibodies of the invention contain a substitution of a human light chain framework residue with a corresponding mu MAb PB1.3 residue in at least 1, 2 or 3, and sometimes 4 of the following positions: L21, L46, L60 and (See Tables 2 and Some humanized antibodies usually contain a substitution of a human heavy chain framework residue in at least 1, 2 or 3 and sometimes 4 of the following positions HI, H2, and H71 and H73. (See Tables 1 and 3) Many humanized antibodies contain substitutions of both light and heavy chain variable framework regions. Preferred antibodies contain a substitution of a human heavy chain framework residue at least at position H71, and substitutions of human light chain framework residues at least at positions 21 and 46.

Usually the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the mu MAb PB1.3 antibody.

Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Occasionally, substitution of CDR regions can result in enhanced binding affinity.

Other than for the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 26 immunoglobulin. However, in general, such substitutions are undesirable.

Production of Variable Regions Having conceptually selected the CDR and framework components of humanized immunoglobulins, a variety of methods are available for producing such immunoglobulins. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each immunoglobulin amino acid sequence.

The desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide.

Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983).

Briefly, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a singlestranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer, and encodes the selected alteration in the target polypeptide DNA.

Selection of Constant Region The variable segments of humanized antibodies produced as described supra are typically linked to at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B-cells (see Kabat et al., supra, and W087/02671) (each of which is incorporated by reference in its entirety for all purposes). Ordinarily, the antibody will contain both light chain and heavy chain constant regions. The heavy chain constant region usually includes CH1, hinge, CH2, CH3, and CH4 regions.

The humanized antibodies include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4.

SUBSTITUTE SHEET (RULE 26) SUB~fh1J~SHEE (RUE 26 WO 94/25067 PCT/US94/04935 27 When it is desired that the humanized antibody exhibit cytotoxic activity, the constant domain is usually a complement-fixing constant domain and the class is typically IgG 1 When such cytotoxic activity is not desirable, the constant domain may be of the IgG 4 class. The humanized antibody may comprise sequences from more than one class or isotype.

Expression Systems Nucleic acids encoding humanized light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, U.S.

Patent 4,704,362).

E. coli is one prokaryotic host useful particularly for cloning the polynucleotides of the present invention.

Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell an origin of replication). In addition, any number of a variety of wellknown promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with SUBSTITUTE SHEET (RULE 26) 1111 I I WO 94-/25067 PCT/US94/04935 28 an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

Other microbes, such as yeast, may also be used for expression. Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see Winnacker, From Genes to Clones (VCH Publishers, 1987).

Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed. Preferred suitable host cells for expressin't nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol.

36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); african green monkey kidney cells' (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-46 (1982)); baculovirus cells.

The vectors containing the polynucleotide sequences of interest the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for Drokarvotic cells.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 29 whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated by reference in its entirety for all purposes). When heavy and light chains are clqned on separate expression vectors, the vectors are cotransfected to obtain expression and assembly of intact immunoglobulins. After introduction of recombinant DNA, cell lines expressing imrnunoglobulin products are cell selected.

Cell lines capable of stable expression are preferred undiminished levels of expression after fifty passages of the cell line).

Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, 1982). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.

The recombinant techniques described above can also be used for expression of native sequences encoding human or murine antibodies. This approach is particularly advantageous for expression of human antibodies that are isolated as unstable cell lines.

III. Antibodies Fragments In another embodiment of the invention, fragments of the intact antibodies described above are provided.

Typically, these fragments compete with the intact antibody from which they were derived for specific binding to Pselectin, and bind with an affinity of at least 107, 108, 109

M

1 or 10 10

M

1 Antibody fragments include separate heavy chains, light chains Fab, Fab' F(ab') 2 Fabc, and Fv.

Fragments can be produced by enzymic or chemical separation of intact immunoglobalins. For example, a fragment can SUBSTITUTE SHEET (RULE 26)

-E

WO 94/25067 PCT/US94/04935 be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988). Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. (See id.) Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids encoding selected fragments are produced by digestion of fulllength coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. This manner of expression is advantageous for affinity-sharpening of antibodies as discussed in Section IV.

IV. Affinity-Sharpened Antibodies Many of the immunoglobulins described above can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions, or intolerable reduction of binding affinity below about 107 M" 1 Usually, immunoglobulins incorporating such alterations exhibit substantial sequence identity to a reference immunoglobulin from which they were derived.

occasionally, a mutated immunoglobulin can be selected having the same specificity and increased affinity compared with a Sterence immunoglobulin from which it was derived. Phage- U, ay technology offers powerful techniques for selecting such immunoglobulins. See, Dower et al., WO 91/17271 McCafferty et al., WO 92/01047; and Huse, WO 92/06204.

V. Hybrid Antibodies The invention also provides hybrid antibodies that share the specificity of blocking antibodies against Pselectin but are also capable of specific binding to a second moiety. In hybrid antibodies, one heavy and light chain pair is usually from an anti-P-selectin antibody and the other pair from an antibody raised against another epitope. This results UBSTITUTE. SHEET (RULE 26) WO 94/25067 PCT/US94/04935 31 in the property of multi-functional valency, ability to bind at least two different epitopes simultaneously, where at least one epitope is the epitope to which the blocking Pselectin antibody binds. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques.

Immunoglobulins can also be fused to functional regions from other genes enzymes) to produce fusion proteins immunotoxins) having novel properties.

VI. Nucleic Acids The intact antibodies and antibody fragments described above are often produced by expression of nucleic acids. All nucleic acids encoding any antibody or antibody described in this application are expressly included in the invention. Modifications of nucleic acids are readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see Gillman and Smith, Gene, 8:81- 97 (1979) and Roberts, et al., Nature, 328:731-734 (1987)).

Many of the nucleic acids of the invention show substantial sequence identity to nucleic acids encoding the heavy and light chains of mu MAb PB1.3 or the exemplified humanized derivatives thereof.

VII. Computers In another aspect of the invention, computers programmed to display three dimensional images of antibodies on a monitor or printer are provided. For example, a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the molecular modelling package QUANTA (Polygen Corp. USA) is suitable. Computers are useful for visualizing models of variants of humanized antibodies. In general, the antibodies of the invention already provide satisfactory binding affinity. However, it is likely that antibodies with even stronger binding affinity could be identified by further variation of certain amino acid residues. The three dimensional image will also identify many noncritical amino acids, which could be the subject of SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 32 conservative substitutions without appreciable affecting the binding affinity of the antibody.

VIII. Large-Scale Production of Antibodies For large-scale production of antibodies, stable cells are produced.containing multiple copies of the genes encoding heavy and light chain immunoglobulins in their chromosomes. Multiple copies are obtained by amplification of isolated region(s) of a cell's chromosomal DNA. Amplification is achieved using a selection agent, methotrexate (MTX), that inactivates a cellular enzyme DHFR) that is essential under certain growth conditions. Amplification, the accumulation of multiple copies of the DHFR gene, results in greater amounts of DHFR being produced in response to greater amounts of MTX. Amplification pressure is applied notwithstanding the presence of endogenous DHFR, by adding ever greater amounts of MTX to the media. Amplification of desired genes (here genes encoding immunoglobulin heavy and light chains) is achieved by linking each gene to a copy of a DHFR gene on the same or separate plasmid(s), and cotransfecting the plasmid(s). Amplification of the DHFR genes in response to MTX results in a concomitant increase in copy number of the desired immunoglobulin genes. Increased copy number of the desired genes results in greater expression of the desired hetevologous protein. Stable cell lines expressing about 10-100, 20-50 or about 30 pg humanized immunoglobulin per 106 cells per day are preferred.

IX. P-Selectin Fragments In another aspect of the invention, fragments of Pselectin are provided. The fragments comprise amino acids from the fifth C3b-C4b regulatory domain of P-selectin (fifth CRP). The fragments contain the epitope bound by mu MAb PB 1.3 and/or proximal epitopes bound by competing antibodies.

The fragments usually contain up to 5, 10, 20, 25, 50, 75, 100 or 200 amino acids in total. Most fragments contain some or all of the amino acids between positions 448 and 467 of the human P-selectin sequence shown in Fig. 34. These amino acids SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 33 define the epitope specifically bound by mu MAb PB1.3. In many fragments, at leist five contiguous amino are from between positions 448 and 467 of the amino acid sequence shown in Fig. 34. Other fragments contain the entire contiguous segment of amino acids between positions 448 and 467. Some fragments consist essentially of this contiguous segment of amino acids. In these fragments, no other amino acids are present that contribute to the binding specificity or affinity of the fragment. Often, no other amino acids of any description are present.

The polypeptide fragments of the invention have a variety of uses. The fragments are useful as immunogens for generating antibodies that compete with mu MAb PB 1.3 for specific binding to P-selectin. The use of defined fragments Is immunogens reduces the extent of screening required to isolate an antibody of desired specificity. The polypeptide fragments are also useful in the diagnostic and therapeutic methods described infra. Some fragments compete with fulllength P-selectin molecules for binding to activated neutrophils. Thus, these fragments are useful for aborting diseases and conditions of the immune system mediate by interactions of P-selectin with its ligand(s) on activated neutrophils. Some fragments are also useful for monitoring the presence of activated neutrophils by binding to ligands to P-selectin present on the surface of these cells. The small size of the fragments renders them particularly suitable for effective delivery following in vivo administration. The exclusively human origin of the fragments reduces the risk of side-effects that might occur with some other agents.

Therapeutic and Diagnostic Methods The therapeutic methods employ the antibodies (whole and binding fragments), and fragments of P-selectin discussed above as therapeutic agents for treatment of various diseases.

The therapeutic agents are useful for prophylactic and therapeutic treatment of a wide variety of diseases and disorders of the immune system. Such diseases and disorders include transplant rejection, graft versus host disease, autoimmune diseases such as insulin-dependent diabetes SUBSTITUTE SHEET (RULE 26)"' WO 94/25067 PCT/US94/04935 34 mellitus, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis and lupus erythematosus, and inflammatory disorders. The agents are also useful for preventing tumor metastasis inhibiting the adhesion of circulating cancer cells, such as carcinomas of the colon and melanoma. Some therapeutic agents function by blocking or otherwise antagonizing the action of a P-selectin molecule with its ligand. Other therapeutic agents function by killing cells bearing a polypeptide against which the agent is targeted.

The therapeutic agents are particularly suitable for treatment of inflammatory and thrombotic conditions including post-ischemic leukocyte-mediated tissue damage (reperfusion injury) arising from traumatic shock, stroke, myocardial infarction, acute transplantation rejection, frost-bite injury, compartment syndrome, and pathophysiologic conditions associated with cardio-pulmonary bypass, acute leukocytemediated lung injury adult respiratory distress syndrome), septic shock, wound associated sepsis secondary to viral infection by herpes simplex virus, IgE-mediated allergic reactions such as acute phase asthmatic disease, and chronic inflammatory conditions, including rheumatoid arthritis, atopic dermatitis and psoriasis.

Ischemia/reperfusion injury is an inflammatory condition that occurs on restoring blood flow to organs suffering from an obstructed supply causing ischemia (oxygen deprivation). Unless rapidly relieved by reperfusion, ischemia causes death of surrounding cells, and eventually, death of a whole organ or patient. However, accumulating evidence suggests that reperfusion may itself exert deleterious effects on surrounding tissue. The deleterious effects of reperfusion are believed to result at least in part from.an inflammatory response mediated by activated neutrophils in the restored blood flow. Some patients have whole-body ischemia, whereas in other patients ischemia is confined to particular parts or organs of the body. For example, a patient may suffer from epidermal, myocardial, renal, cerebral, splenic, hepatic, spinal, splanchnic, SUBSTITUTE.SHEET (RULE 26) WO 94/25067 PCT/US94/04935 pulmonary, partial-body, or whole-body ischemia. The therapeutic agents of the invention function by antagonizing the interaction of such lymphocytes with P-selectin.

One major component of acute "llergic conditions, including asthma, is the degranulation of mast cells following challenge of subjects with antigens to which they are specifically sensitized. The consequences of mast cell degranulation include a bronchoconstrictor response and also an inflammatory response characterized in part by leukocyte accumulation. Histamine, which is contained together with other inflammatory mediators in mast cells, can induce the expression of P-selectin on vascular endothelial cells, indicating that degranulation of mast cells may also result in P-selectin expression and subsequent leukocyte accumulation.

Because mast cell degranulation is a key element in the pathogenesis of allergic conditions, administration of antibodies to P-selectin will be useful for the treatment of human allergic conditions.

The methods are particularly useful for humans, but may also be practiced on veterinary subjects. The therapeutic methods are usually applied to organs present in living subjects. However, some methods, such as ischemia-reperfusion therapy, are equally applicable to dissected organs, particularly when such organs are awaiting transplant to a recipient patient. Therapeutic methods can also be performed ex vivo.

Therapeutic agents and compositions targeted against P-selectin can also be used in combination with agents targeted against other molecules, particularly humanized or human antibodies reactive with different adhesion molecules.

Suitable immunoglobulins include those specific for CDlla, CD1b, CD18, E-selectin, L-selectin and ICAM-1. The immunoglobulins should bind to epitopes of these adhesion molecules so as to inhibit binding of leukocytes, particularly neutrophils, to endothelial cells. Other suitable antibodies for use in combination therapies are those specific for lymphokines, such as IL-1, IL-2 and IFN-y, and their receptors. Such antibodies serve to block activation of SUBSTT SHEET (RULE 26) WO 94/25067 PCTIUS94/04935 36 endothelial cells, and thereby prevent their interaction with neutrophils in an inflammatory response.

The blocking P-selectin antibodies and pharmaceutical compositions of this invention are particularly useful for parenteral administration, subcutareously, intramuscularly or intravenously. A number of new drug delivery approaches are being developed, the pharmaceutical compositions 'f the present invention are suitable for administration using these new methods, as well. See Langer, Science, 249:1527-1533 (1990).

The blocking P-selectin antibodies can be directly or indirectly coupled to the chemotherapeutic agent. The coupling, which may be performed by means, generally known in the art, should not substantially inhibit the ability of the immunoglobulin to bind the receptor nor should it substantially reduce the activity of the chemotherapeutic agent. A variety of chemotherapeutics can be coupled for targeting. For example, anti-inflammatory agents which may be coupled include immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenise inhibitors, lipoxygenase inhibitors, and leukotriene antagonists. Some preferred moieties include cyclosporin A, indomethacin, naproxen, FK- 506, mycophenolic acid, and the like. Similarly, antioxidants, superoxide dismutase, are useful in treating reperfusion injury. Likewise, anticancer agents, such as daunomycin, doxorubicin, vinblastine, bleomycin, and the like can be targeted.

The P-selectin targeting may also be accomplished via amphipaths, or dual character molecules (polar:nonpolar) which exist as aggregates in aqueous solution. Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are generically referred to herein as liposomes. In these preparations, the drug to be delivered is incorporated as part of a liposome in which an anti-P-selectin immunoglobulin is embedded. In this SUBSTITUTE SHEET (RULE 261 WO 94/25067 PCT/US94/04935 37 embodiment, the immunoglobulin need not bind a functional epitope on the P-selectin molecule, so long as the immunoglobulin effectively targets the liposome to P-selectin molecules. When the liposomes are brought into proximity of the affected cells, they deliver the selected therapeutic compositions.

A variety of methods are available for preparing liposomes, as described in, Szoka et al., Ann. Rev.

Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, incorporated herein by reference.

Targeting of liposomes using a variety of targeting agents ligands, receptors and monoclonal antibodies) is well known in the art. (see, U.S. Patent Nos. 4,957,773 and 4,603,044, both of which are incorporated herein by reference). Standard methods for coupling targeting agents to liposomes can be used. Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen, et al., J. Blol. Chem., 265:16337- 16342 (1990) and Leonetti et Proc. Natl. Acad. Sci. (USA) 87:2448-2451 (1990).

Pharmaceutical compositions for parenteral administration usually comprise a solution of a therapeutic agent an antibody against P-selectin (intact or binding fragment or a P-selectin fragment) or a cocktail of several such agents dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, from less than about usually at or at least about 0.1% to as much as 1.5% or SUBSTITUTE SHEET (RULE 26) WO 94/25067 IPCT/US94/04935 38 by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and ate described in more detail in, for example, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pennsylvania (1985), which is incorporated herein by reference.

The antibodies of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed. Lyophilization and reconstitution can lead to varying degrees of antibody activity loss with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.

The compositions containing the present antibodies or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatments. In therapeutic application, compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about .05 mg/kg body weight to about 5 mg/kg body weight, preferably between about .2 mg/kg body weight to about 1.5 mg/kg body weight.

The materials of this invention may generally be employed in serious disease states, that is, life-threatening or potentially life-threatening situations. In such cases, in view of the minimization of extraneous substances and the lower probability of "foreign substance" rejections HAMA) which are achieved by human or humanized antibody forms of this invention, it is possible and may be felt desirable by SUBSTITE SHEET ULE 26) WO 94/25067 PCT/US94/04935 39 the treating physician to administer substantial excesses of these antibodies.

In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but is generally in the ranges described above.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the immunoglobulins of this invention sufficient to treat the patient effectively.

The antibodies of invention (whole and binding fragments) and fragments o' P-selectin can also be used for diagnostic purposes. An amount sufficient for these purposes is defined to be a "diagnostically effective dose." In diagnostic uses, the precise amounts will depend upon the patient's state of health, mode of administration, and the like. The antibodies may either be labeled or unlabeled.

Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as antibodies specific for the particular immunoglobulin constant region. Alternatively, the antibodies can be directly labeled. A wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens).

The antibodies (whole and binding fragments) are useful for detecting the presence of cells bearing the Pselec~in receptor. The presence of such cells is diagnostic of an inflammatory condition or disease and may signal the need for commencement of a therapeutic method discussed supra.

Diagnosis can be accomplished by removing a cellular sample from a patient. The amount of expressed P-selectin receptor SUBSTITUTE SHEET (RULE 26) WO 941/25067 PCTIUS94/04935 in individual cells of the sample is then determined, by immunohistochemical staining of fixed cells or by Western blotting of a cell extract with an antibody of the invention.

Fragments of P-selectin are useful for monitoring the presence of activated neutrophils, a presence that can be indicate of an undesirable immune response.

Diagnosis can also be achieved by in vivo administration of a labelled antibody (preferably a humanized or human antibody) and detection by in vivo imaging. The concentration of MAb administered should be sufficient that the binding to cells having the target antigen is detectable compared to the background signal. The diagnostic reagent can be labelled with a radioisotope for camera imaging, or a paramagnetic isotope for magnetic resonance or electron spin resonance imaging.

A change (typically an increase) in the level of Pselectin protein in a cellular sample or imaged from an individual, which is outside the range of clinically established normal levels, may indicate the presence of an undeoirable inflammatory response reaction in the individual from whom the sample was obtained, and/or indicate a predisposition of the individual for developing (or progressing through) such a reaction. P-selectin can also be employed as a differentiation marker to identify and type cells of certain lineages and developmental origins. Such cell-type specific detection can be used for histopathological diagnosis of undesired immune responses.

Kits can also be supplied for use with the subject antibodies. Thus, the subject antibody composition of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type. The antibodies, which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, serum albumin, or the like, and a set of instructions for use. Generally, these materials will be present in less than about 5% wt. based on the amount of SUBSTITUTE SHEET (RULE 26) WO 94125067 PCT/US94/04935 41 active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt.

of the total composition. Where a second antibody capable of binding to an anti-P-selectin antibody is employed in an assay, the second antibody will usually be pr.esent in a separate vial. The second antibody is typically conjugated to a label and formulated as described above.

The following examples are offered by way of illustration, not by limitation.

Example 1 Preparation of Reagents This example describes preparation or isolation of reagents used for the immunizations in Examples 2 and 3 and the ELISAs in Example 4.

A. "OUTDATED PLATELETS" A preparation of platelets which was purified from "outdated platelets" obtained from the San Diego Blood Bank.

The method used was a modification of the method described in Moore et al., J. Cell. Biol. 112:491-499 (1991), which is incorporated herein by reference. Briefly, 25 units of outdated platelet-rich plasma were centrifuged twice for min at 1200rpm (300xg) to remove contaminating non-platelet blood cells. The purified platelet preparation was then washed three times with a buffer containing 0.1M NaCl, Tris and 5mM Benzamidine. It also contained 3.8% sodium citrate. The pH was adjusted to 7.5 with lN HC1. The purified platelets were stored at 70°C and were washed once in PBS before injection.

B. PURIFIED P-SELECTIN Fractionation of Platelets: Washed outdated platelets were fractionated essentially according to Moore et al., 1991, supra, 25 units washed platelets were made 100 uM SUBSTITUTE SHEET (RULE 26) WO 94/25067 P'CT/US94/04935 42 in leupeptin and 1 mM in 4-(2aminoethyl)-benzenesulfonylfluoride (AEBSF), freeze-thawed three times in dry ice/methanol, and homogenized ten strokes with a Dounce homogenizer. The suspension was then centrifuged in a Beckman Ti 70 rotor at 4 0 C at 35,000 rpm for 60 min. The supernatant from this spin was the starting material for the purification of "soluble" P-selectin. The pellet was resuspended in 30 ml 1 mM MnCl 2 1 mM CaCl 2 5 mM benzamidine-HCl, 0,1M NaCl, 20 mM Tris, 2% Triton X-100, pH 7.5, homogenized 10 strokes with a Dounce homogenizer, and incubated for 1 h at 4 C. The suspension was then centrifuged for 1 h in a Beckman Ti rotor at 4 0 C at 35,000 rpm. The supernatant from this spin was the starting material for the isolation of "membrane-bound" P-selectin.

Isolation of P-Selectin from Fractionated Platelets: "Soluble" and "'membrane-bound" P-selectin were then further purified separately by some or all of the following procedures. The non-ionic detergent Rennex 30 (Accurate Chemical and Scientific Corp.) was included at a concentration of 0.1% in all buffers for isolation of the membrane-bound form of P-selectin. All buffers also included 1 mM CaCl 2 and 1 mM MnCl 2 Fractions containing P-selectin were detected by Western blotting with the monoclonal antibody PNB1.6.

Lentil.Lectin Chromatoqraphv: "Soluble P-selectin" supernatant or "membrane-bound P-selectin" detergent extract from 25 units of platelets ware passed over a column (1.5 x cm) of lectin from Lens culinaris (lentil lectin) coupled to Sepharose 4B (Sigma Chemical Co., L-0511). The column was then washed with 150 ml 20 mM Tris, 0.1M NaCl, pH 7.5. Bound glycoproteins were then eluted from the column with 10 x 3 ml aliquots of 20 mM Tris, 0.5M NaCl, 0.5M alpha-methyl Dmannopyranoside, pH 7.5. Fractions containing P-selectin were concentrated to 1.5-2 ml in an Amicon Centriprep Gel Filtration Chromatography: Concentrated fractions from lentil lectin chromatography were injected onto a Toso Haas G3000 SW HPLC column (21.5 mm x 30 cm) equilibrated with 20 mM Tris, 0.1M NaCl, pH 7.5. The column was eluted with the same buffer at a flow rate of 0.75 ml/min.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 43 fractions (1.5 ml) were collected, and analyzed by Western blotting. Fractions containing P-selectin were pooled and concentrated to 1.5-2 ml in an Amicon Centriprep 30, followed by several buffer exchanges with 20 mM Tris, pH Heparin-Aqarose Chromatoqraphv: The sample, in mM Tris, pH 7.5, was applied to a 1 ml column of Heparin- Agarose, the column was washed with the same buffer, and eluted with 20 mM Tris, 1.5M NaCl, pH Anion Exchange Chromatoaraphy: Fractions were dialyzed into 20 mM Tris, pH 7.5, and injected onto a Tcso Hans DEAE-5PW column (7.5 mm x 7.5 cm) that had been equilibrated with 20 mM Tris, pH 7.5, and eluted with a salt gradient to 1M NaCl. Fractions containing P-selectin were pooled, and concentrated in an Amicon Centriprep-30, and stored at -80 0

C.

Cation Exchange Chromatoraphv: Samples were dialyzed into 10 mM phosphate, pH 7, and injected onto a Poros CM/P (4.6/100) column that had been equilibrated in the same buffer, and eluted with a salt gradient to IM NaCl.

C. ISOLATION AND ACTIVATION OF FRESH HUMAN PLATELETS All chemicals used throughout the platelet isolation procedure were obtained from Sigma Chemical Company, St.

Louis, MO.

1. 42 ml of blood was drawn from a human volunteer blood donor into a syringe containing 7 ml Acid Citrate Dextrose anticoagulant (ACD).

Preparation of ACD anticoaqulant Dextrose 2.Og Sodium Citrate 2.49g Citric Acid 1.25g Bring to 100 ml with distilled water 2. The needle was removed and the blood was transferred to two sterile 50 ml tubes.

3. The tubes were centrifuged in an IECC-6000 centrifuge equipped with a 921 head (radius=17.2 ca.) at 800 rpm (approx. 90xg) for 15 min at room temperature with the brake off.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 4. The supernatant was removed using a plastic pipette The supernatant was removed as close as possible to the buffy layer.

The supernatant was centrifuged at 1200 rpm (approx 300xg) for 6 min.

6. The supernatant was removed.

7. The supernatant was centrifuged at 2000 rpm (approx 1200xg) for 10 min. The platelets appeared as a cell button at the bottom of the tube. The supernatant was discarded.

8. The platelets were washed as follows: 2 ml Tyrode- Hepes Buffer pH 6.5, containing Prostaglandin,

E

1

(PGE

1 at a final concentration of 100nM was added and the platelets were gently resuspended. A further 10 ml of the same buffer was added and the sample was centrifuged at 3000 rpm for 10 min.

Preparation of Tyrode-Hepes Buffer NaCl KCl 0.2g NaH 2

PO

4 H20 0.057g MgCl 2 6H 2 0 0.184g NaHC03 0.1g Dextrose HEPES 2.383g Bring to 1 liter with distilled water. Adjust pH tc with IN NaOH.

9. Step #8 was repeated once more and then the platelets were washed once in the same buffer without PGE 1 The platelets were counted in a Coulter Counter and diluted to 2x10 8 /ml.

11. The pH of the platelet suspension was adjusted to 7.2, and the amount of thrombin (Human thrombin- Sigma) required for maximal activation was determined. There is a certain amount of donor variation, but optimal activation is normally obtained in the range of 0.25 0.5 Units/ml.

Maximal activation maximal thrombin-induced SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 aggregation appears to correspond to maximal expression of P-selectin.

12. The required amount of thrombin was added to the platelet preparation and the mixture was allowed to stand at room temperature for 20 min without stirring to prevent aggregation.

Example 2 Preparation of Blocking P-selectin Antibody This example describes preparation of mu mAB PB1.3, a monoclonal antibody to P-selectin that inhibits binding of thrombin-activated platelets to neutrophils.

One RBF/DnJ male mouse, received from Jackson Laboratories, was used as a source of antibody producing cells and was immunized according to the following schedule (all injections were performed intraperitoneally): 1. Month 1 200 Ml of packed "outdated platelets" containing approximately 6x10 9 platelets 2. Month 2 250 Al of packed "outdated platelets" (8x10 9 platelets) 3. Month 4 250 Al of packed "outdated platelets" (8x10 9 platelets) 4. Month 5 "Soluble" fraction of P-selectin, isolated on lentil lectin.

5. Month 6 "Soluble" P-selectin, purified by l'ntil lectin, gel filtration, heparin-agarose, and ion-exchange chromatography.

Details of the preparation of all agents used for immunization are presented in Example 1.

A. MYELOMA FUSION CELL LINE FOX-NY, a mouse myeloma cell line deficient for adenosine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) was obtained from the American Type Culture Collection (CRL 1732) and maintained in RPMI 1640 containing 10% fetal bovine serum (Hyclone) and 1% L-glutamine.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PC'r/US94/04935 46 B. CELL FUSION PROCEDURE Four days after the final boost the spleen was removed and 1.2x10 8 splenocytes were recovered. These were fused with FOX-NY myeloma cells using PEG 1500(BMB) using the following protocol: The isolated splenocytes were washed twice in serum-free cell culture medium. Splenocytes and myeloma cells were combined at a ratio of 1:4.8 (myelomas to spleen cells). The combined cell pellet was washed twice in serum free medium, then aspirated to dryness. The cell pellet was resuspended by gentle tapping and heated in a waterbath at 37°C for 1 min. The pellet was distributed around the sides and bottom of a 50 ml conical centrifuge tube. One ml of PEG 1500 (50% w/v in 75mM HEPES, BMB Lot #14702800) previously warmed to 37 0 C was added over 60 seconds while rotating the tube to maintain a thin layer of cells. One ml of serum free medium was added slowly over 60 seconds. An additional 1ml of medium was added slightly faster. A further 8 ml of medium was added and the tube was allowed to stand undisturbed for 8 min and was then centrifuged for 5 min at 300xg. The final pellet was resuspended in RPMI 1640 containing 10% fatal bovine serum Hyclone), 1% L-glutamine, 1% Sodium Pyruvats, and IxAAT (Sigma). The cells were plated in 10 flat bottom 96 well microtiter plates (COSTAR). No feeder cells were used.

Examnle 3 Preparation of Non-blocking Anti-P-selectin antibody This example describes preparation of mAB PNB1.6 a monoclonal antibody to P-selectin that does not inhibit binding of thrombin-activated platelets to neutrophils.

One RBF/DnJ male mouse, received from Jackson Laboratories, was used as a source of antibody producing cells and was immunized according to the following schedule (all injections were performed intraperitoneally): 1. Month 1 100 of thrombin-activated freshly isolated platelets (approximately 3x10 9 platelets) 2. Month 2 20 0 pl of thrombin-activated fresh platelets (6x10 9 platelets) SUBSTITUTE SHEET (RULE 26) WO 94/25067 VC'rTUS94/04935 47 3. Month 4 100l of thrombin-activated fresh platelets (3x10 9 platelets) Four days post final boost, the spleen was removed and the splenocytes were recovered. They were fused with FOX- NY myeloma cells using PEG 1500(Sigma) generally as described in Oi et al., "Immunoglobulin-Producing Hybrid Cell Lines" in Selected Methods in Cellular Immunology, eds. Mishell and Shiigi, pp 351-372, 1980, which is incorporated herein by reference.

Exampl 4 Screening for Antibody This example describes screening of supernatant media from fused cells produced in Examples 2 and 3.

Supernatants from Example 2 were tested by ELISA assay against thrombin-activated platelets, purified P-selectin and recombinant P-selectin. Supernatants from Example 2 were tested by ELISA assay against thrombin-activated platelets.

Each of these assays is described below. Preparation of the reagents used in each of the assays is described in Example 1.

A. THROMBIN-ACTIVATED PLATELETS 1. A 96 well flat bottom COSTAR plate was coated with 0.1% gelatin gelatin-Sigma) by adding 100pl/well and incubating at 37°C for 15 min.

2. The gelatin was removed and 100 Al of thrombin activated platelets (10 8 /ml) were added to each well.

3. The plate was incubated at 37 0 C for 15 min.

4. The plate was centrifuged at 800 rpm (90xg) for 2 min.

The unbound platelets were removed and the plate was washed x2 with PBS.

6. l00l of PBS containing 1% BSA were added to each well and the plate was allowed to stand at room temperature for 60 min.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PICT/US94/04935 48 7. 100l of supernatant were added to each well. The plate was placed on a shaker at room temperature for min.

8. The plate was washed x4 with PBS.

9. 1004l of peroxidase-conjugated goat anti-mouse IgG diluted 1/1000 in PBS containing 1% BSA was added to each well. The plate was allowed to stand at room temperature for 30 min.

The plate was washed x7 with PBS.

11. 100l of ABTS substrate system (Kirkegaard and Perry, Laboratories, Inc.) was added to each well.

12. The plate was allowed to develop at room temperature for up to 30 min.

13. The OD was measured at 414nm in a Titertek Multiskan MCC/34.0.

B. PURIFIED P-selectin 1. "Soluble" or "membrane-bound" P-selectin, isolated by chromatography on lentil lectin, gel filtration, and DEAE columns, was diluted (1:50 to 1:1000) in DPBS (Dulbecco's Phosphate Buffered Saline is Phosphate-buffered saline (PBS) containing CaCl 2 (1mM) and MgSO 4 (0.5mM)) and coated overnight at 4 0 C onto Falcon 96 well microtiter plates.

2. The plates were then blocked for 1 h or longer with DPBS+1%BSA.

3. Supernatants to be assayed were then added to the wells, and incubated for 30-60 min at room temp.

4. The plates were washed with DPBS, and then sheep anti-mouse IgG horseradish peroxidase conjugate (Sigma A-6782, 1:1000 in DPBS+1%BSA) was added to the wells, and the plates were incubated at room temp. for 1 hr.

The plates were then washed with DPBS, and developed with the TMB Microwell Substrate System (Kirkegaard and Perry Laboratories, Inc.) SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 49 C. RECOMBINANT P-selectin 1. Ninety six well flat bottom plates were coated overnight at 4 0 C with 1001 of P-selectin which had been shed into the serum free medium of a pool of 293 call clones transfected with the recombinant Pselectin gene. This supernatant contained at least 8ng/ml of P-selectin and was coated onto COSTAR plates either undiluted or diluted 1:8 with media.

2. The plate was washed once with Dulbecco's PBS (DPBS) and blocked with 200pl DPBS containing 1% BSA for min at room temperature.

3. The plate was then processed for the ELISA Assay according to steps 7-13 in Section A, Page 9.

D. ISOTYPING ANALYSIS OF mABs to P-Selectin Isotype analysis of mu MAbs PB1.3 and PNB1.6 indicated that both monoclonal antibodies were of the mouse immunoglobulin subclass, IgG1. Isotyping was performed using the capture method of Boehringer Mannheim Mouse-Hybridoma-Subtyping Kit.

Detection of P-selectin This example describes the detection of P-selectin by western blotting. Samples containing P-selectin were mixed with an equal volume of SDS-PAGE sample buffer (non-reducing), heated at 100 0 C, run on a Novex 8-16% gradient gel, and transferred electrophoretically to nitrocellulose. The nitrocellulose membrane was then blocked for at least 1 h in phosphate-buffered saline (PBS) bovine serum albumin (BSA). The membrane was then incubated at room temperature for 1 h or longer with either tissue culture supernatant from hybridomas expressing the appropriate monoclonal antibody or purified antibody (5 gg/ml) in PBS+1%BSA. The membrane was then washed several times in PBS+1%BSA, and incubated for 1 h with a 1 to 1000 dilution of sheep anti-mouse IgG horseradish peroxidase conjugate (Sigma A-6782) in PBS+1%BSA. The membrane was then washed, and bands visualized with SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/049: tetramethylbenzidine/membrane enhancer (Kirkegaard and Perry Laboratories, Inc.) For tissue culture supernatants from -th mu MAbs PB1.3 and PNB1.6 a protein was detected having a ^lecular weight of 140Kd. This was shown in preparations of solubilized platelet membranes as well as in purified Pselectin.

Example 6 In Vivo Inhibition of Acute Inflammatory Injurv by Blocking P-selectin Antibody This example provides data demonstrating the ability of blocking P-selectin antibody of the invention to treat acute lung injury. In this example, systemic complement activation was produced by vascular infusion of the cobra venom factor (CVF) into rats. Injury in this model develops rapidly and is known to be dependent on toxic oxygen products generated from neutrophils.

For those studies, the two monoclonal antibodies described in Examples 2 and 3 were used. Of particular importance tfo this study, the blocking P-selectin antibody mu MAb PB1.3 ;iso inhibited adhesion of thrombin-activated rat platelets to human neutrophils (Fig. These data suggest that mu MAb PB1.3 recognizes a conserved functional P-selectin epitope and that a blocking P-selectin antibody might be useful in the treatment of other inflammatory injuries.

For the in vivo experiments 20 units CVF/kg body weight were infused intravenously into 300 gm adult male Long- Evans rats. This results in rapid intrapulmonary, intravascular sequestration of blood neutrophils, with resulting damage of interstitial capillary endothelial cells at points of physical contact of endothelial cells and neutrophils. When employed, mu MAbs PB1.3 and PNB1.6.were infused intravenously in the amounts indicated together with CVF, in total volume of 0.5 ml. Negative control animals received 0.5 ml intravenous infusion of phosphate buffered saline (PBS). In all cases the infusion material also contained trace amounts of 12 s)-bovine serum albumin and SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 51 homologous 51 Cr)-red blood cells (RBC). The parameters of lung injury (leakage of albumin and extravasation of RBC) were determined at 30 min. according to established techniques Mulligan, et al., J. Clin Invest. 88:1396 (1991).

The results of Laese experiments are shown in Figs.

2A and 2B. Coinfusion of 200 Ag PNB1.6 (the non-blocking anti-P-selectin antibody) together with CVF failed to cause any reduction in lung injury when compared to the values of untreated CVF positive controls. Thus animals receiving CVF PNB1.6 in PBS served as the reference positive control values.

When 100 Ag mu MAb PB1.3 antibody was infused with the CVF permeability was reduced by 19.2% (p=.002) and hemorrhage was reduced by 37.5% (p=.001) (data now shown). When 200 gg mu MAb PB1.3 was added, permeability was reduced by 50.8% (p<.001) (Fig. 2A) and hemorrhage by 70.0% (p<.001) and 70.1% respectively (Fig. 2B).

Lungs from companion sets of animals were homogenized sonicated and the myeloperoxidase activity (MPO) measured in order to obtain an estimate of the neutrophil content in lung. MPO was determined by the decomposition of H202 in the presence of o-dianisidine according to standard procedures, Warren et al. J. Clin. Invest. 84:1873 (1989). As shown in Fig. 3A, treatment with 100, 200 or 400 Ag mu MAb PB1.3 reduced MPO content below the values in the re'ererue (untreated) positive control group by 26% 41% (p<.001) and 50% respectively. In animals injected with 200 Ag of PNB1.6 there was no reduction in MPO content (Fig. 3A), consistent with the inability of this antibody to protect against CVF-induced lung injury. Thus, the protective effects of the blocking antibody mu MAb PB1.3, correlato with its ability to interfere with neutrophil accumulation in lung tissue. Transmission electron microscopic examination of lung sections confirmed that treatment with mu MAb PB1.3 resulted in reduced accumulation of neutrophils within the pulmonary interstitial capillaries, diminished adherence of neutrophils to endothelial cells, and reduced evidence of damaged endothelial cells (Fig. 3B).

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 52 In order to investigate lung expression of Pselectin following intravenous injection of CVF, an additional group of rats was infused with CVF and animals sacrificed at times 0, 5, 10, 15, 20 and 60 min later. Lungs were inflated with snap frozen, and sections obtained and examined for presence of P-selectin by immunohistochemical techniques, using mu MAb PB1.3. Little detectable reactivity in the pulmonary vasculature was found at time 0, whereas staining was clearly evident at 5 min and was increased at 15 and min after infusion of CVF. The pattern of staining involved pulmonary venules and septal areas in a pattern consistent with staining of interstitial capillaries. At 60 min.

staining had largely disappeared (data not shown). Although P-selectin is presumably present in intracellular storage granules prior to CVF infusion, it is apparent that mobilization of P-selectin to the surface of the endothelium dramatically enhanced its reactivity with mu MAb PB1.3 antibody.

Exaple 7 Competitive Binding Assay Methods of isolation of platelets from human blood, activation of isolated platelets with thrombin and isolation of PMNs (neutrophils) are descri ed in Example 1 above.

INCUBATION OF THROMBIN ACTIVATED PLATELETS WITH MONOCLONAL ANTIBODIES TO P-SELECTIN.

a. Place 20 Al thrombin-activated platelets (2xl0 8 /ml) into each of 24 Eppendorf tubes (1.5 ml) in duplicate.

b. To 8 of these tubes, add 20 Al of the IgG fraction purified from PNB1.6 at a concentration of 10 ug/ml in Tyrode-Hepes Buffer, pH 7.2 containing 1 mM CaCl 2 To a second row of 8 tubes, add 20 pl of the same IgG preparation at a concentration of 1 Ag/ml.

To the third row of 8 tubes add 20 Al of the buffer alone.

c. Mix and stand at room temperature for 20 min.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 53 d. To each row of 8 tubes add 20 Al of the IgG fraction purified from mu MAb PB1.3 at concentrations ranging from 10 Ag/ml to 0.03 Ag/ml.

e. Mix and stand at room temperature for 20 min.

f. Add 20 Al neutrophils at 3x10 6 /ml.

g. Mix and stand at room temperature for 20 min.

h. Evaluate adhesion microscopically as follows: Count 100 neutrophils in each sample. Score a cell as positive if it has bound 2 or more platelets, and as negative if it has bound less than 2. Calculate the percentage of positive cells.

As shown.in Fig. 1, pretreatment of thrombin activated platelets with mu MAb PNB1.6 did not affect the ability of mu MAb PB1.3 to block their P-Selectin mediated adhesion to neutrophils.

Example g Characterization ofBdin of Blocking P-selectin Antibodies I(a) Purification of Moclonal Antibodie: The monoclonal antibodies PNB1-6 nd 84/2were isolated from tissue culture supernatants by passage over a column of protein G Sepharose 4 Fast Flow (Pharmacia). The column was washed extensively with phosphate-buffered saline (PBS), and bound antibody was eluted with 0.1M glycine-HCl pH 2.7 into tubes containing 0.2 to 0.5 volume 1M Tris, pH 8.8. The procedure used for isolating mu MAb PB1.3 was identical except that the antibody bound to protein G was eluted with 0.1M acetate-HCl pH 2.5 into tubes containing 0.3 volumes 2M Tris, pH 10. Antibody containing fractions were pooled, and dialyzed against several changes of PBS at 49C.

Preparation of mu MAb PB1.3 Affinity Column: For use in the purification of P-selectin, mu MAb PB1.3 was coupled to tresyl activated agarose according to the manufacturer's affinity instructions (Schleicher and Schuell).

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 54 Purification of P-Selectin: to washed, outdated human platelets, prepared as described above, was added 1/100th volume 5 mg/ml leupeptin, and 1/100th volume 35 mg/ml AEBSF (Calbiochem). After mixing, the platelets were freeze-thawed three times in either a dry-ice/methanol bath or liquid nitrogen, homogenized ten strokes with a Dounce homogenizer, and centrifuged for 1 h at 35,000 rpm at 4°C in a Beckman 70TI rotor. The pellet was resuspended in Dulbecco's phosphate-buffered saline (DPBS, Whittaker Bioproducts, which contains approximately 0.9 uM CA and 0.5 mM and made 5 mM in benzamidine-HCl, 100 uM in leupeptin, and 2% in Triton X-100. The suspension was resuspended, and homogenized strokes with a Dounce homogenizer. After a 1 h incubation at 4 0 C, the solution was again centrifuged in a Beckman 70TI rotor for 1 h at 40C at 35,000 rpm. The supernatant was passed over a column of mu MAb PB1.3agarose that had been equilibrated with DPBS+0.05% Rennex (Accurate Chemical and Scientific Corp.). The column was washed extensively with DPBS+0.05% Rennex 30, and bound P-selectin was eluted with 0.1M triethylamine-HCL, 0.05% Rennex 30, pH 11.5, into tubes containing 0.1 volume 1 M phosphate, pH 6.8. Fractions containing Pselectin were pooled, dialyzed against DPBS+0.05% Rennex 30, concentrated using either a Centricon 30 or Centriprep 30 spin concentrator (Amicon), aliquoted, and stored at Preparation of the Peptide CONRYTDLVAIONKNE (Cvs-Gln-Asn- Arq-Tvr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lvs-Asn-Glu-NH 2 The peptide was synthesized on an Applied Biosystems (Foster City, CA) 430A peptide synthesizer using Fmoc protected amino acids and 2-(1H-Benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBATU) esters for amino acid activation. Each amino acid was routinely double coupled. Fmoc protected amino acids and Hydorxybenzotriazole were purchased from Applied Biosystems. All solvents were purchased from Burdick and Jackson. HBTU was purchased from Richelieu SUBSTITUTE SHEET (RULE 26) WO 94/25067 IPCT/US94/04935 Biotechnologies (St. Hyacinthe, Canada). Piperidine and trifluoroacetic acid, acetic anhydride, thioanisole, phenol and ethanedithiol were purchased from Sigma Chemical Corporation.

Fmoc-Amide resin (Bachem Biosciences) was loaded into the peptide synthesis reaction vessel and washed one time with N-methylpyrolidone (NMP). The following operations were then sequentially performed: 1. The Fmoc protecting group was removed by treatment of the resin bound amino acid with piperidine in NMP.

2. The resin was washed 5 times with NMP.

3. A mixture containing N-a-FPoc-L-Glutamic acid 7-t-butylester, diisopropylethylamine, HBTU and NMP was added to the reaction vessel and allowed to react for 30 min, under vortex agitation.

4. The solvent was drained, and the resin was washed three times with NMP.

5. Steps and were repeated two more times.

6. The resin was washed four more times with NMP.

Steps 1-6 were repeated for each amino acid of the peptide.

Following the final coupling cycle, the resin-bound peptide was deprotected by reaction with 25% piperidine in NMP, washed 7 times with NMP, and washed 2 times with dichloromethane.

The resin was dried in vacuo for 24 h. The peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% ethanedithiol, 5% thioanisole, 7.5% phenol, and 5% water. The polystyrene resin was separated from the trifluoroacetic acid solution by filtration. Trifluoroacetic acid was removed by evaporation in vacuo. The crude peptide was triturated with diethylether and dissolved in water. The water was removed by lyophilization. The peptide was then purified by reverse phase HPLC on a C 8 column (VYDAC) using a gradient of acetonitrile, water, each containing 0.1% TFA as modifier.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCTIUS94/04935 56 Biotinvlation of Monoclonal Antibodies: Antibodies were dialyzed against 0.1M sodium bicarbonate, pH 8.5. NHS-LC Biotin (Pierce) was added to 0.3 mg/ml. After 2 hrs at room temperature, antibodies were dialyzed against several changes of PBS.

Coating of Microtiter Plates for ELISA: 96 well microtiter plates (Falcon Microtest III) were coated overnight at 4 C with 50 il/well of 2 pg/ml affinity purified P-selectin in DPBS.

Western Blotting: mu MAbs PB1.3, PNB1.6, and 84-26 all bind to a single band of 140 KD when platelets are dissolved in SDS-PAGE running buffer (unreduced), subjected to .SDS-PAGE, and transferred to nitrocellulose (data not shown). We have also found that mu MAbs PB1.3 and PNB1.6 no longer recognize P-selectin on western blots when the saaples have been reduced with 3mercaptoethanol.

II. Effect of Chelatine Divalent Cations on Binding of Monoclonal Antibodies to P-Selectin: Two microtiter plates were coated with affinity purified P-selectin as above.

Plate 1 was blocked with 200 Ml/well PBS+1% BSA, while plate 2 was blocked with 200 Mi/well DPBS+1% BSA (DPBS contains Ca and After 1 hr, the plates were washed (all washes for plate 1 were PBS, for plate 2, with DPBS. 25 Al of 25 mM EDTA in PBS+1% BSA was added to the wells of plate 1, and 25 Al of DPBS+1% BSA was added to plate 2. After 1 hr, 25 Al of dilutions of the appropriate antibody were added to the wells of either plate. Dilutions for plate 1 were made in PBS+1% BSA, for plate 2 in DPBS+1% BSA. After 1 hr, the plates were washed, and 50 Al of a 1 to 1000 dilution of sheep anti-mouse IgG horseradish peroxidase conjugate (dilutions for plate 1 were made in PBS+1% BSA, for plate 2 in DPBS+1% BSA) was added. After 1 hr, the plates were washed, and 50 pl of tho peroxidase substrate TMB (3,3',5,5'-tetramethylbenzidine, Kirkegaard and Perry Laboratories, Inc.) was added. After the color had developed to the appropriate level, the substrate SUBSTITUTE SHEET (RULE 26) WO 94/2507 PCT/US94/04935 57 reaction was quenched by the addition of 25 Al of 1M phosphoric acid and the absorbance at 450 nm was read.

Fig. 4 shows the binding of the anti-P-selectin monoclonal antibodies to P-selectin in the buffer DPBS, which contains CA and Mg and in PBS 25 mM EDTA, a chelator of divalent metal cations. The concentration of divalent cations present in DPBS is more than sufficient to support neutrophil adhesion to P-selectin (Geng et al., 1991), while the chelator_ _EDTA at 25 mM is more than sufficient to block neutrophil adhesion to P-selectin. The binding of all of the monoclonal antibodies to P-selectin is little affected by the presence of EDTA, demonstrating the Ca is not reeired to be foese r binding to occur. Mu MAb PB1.3 is a blocking antibody, that it is able to block the binding of neutrophils P-selectin, for example on activated platelets. The PNB1.6 is a nonblocking antibody. The ability of 84/26 to block the binding of neutrophils to P-s~e-eI-n-.~.i..e~eiflly -character i ed. In"t"thenti-P- eEThtin monoclonal antibodies described by Geng et al., only the non-blocking antibody S12 is able to bind in the absence of Ca* III. Effect of the Peptide CONRYTDLVAIONKNE on the bindin of mu MAb PB1.3 to P-Selectin: A microtiter plate was coated with affinity purified P-selectin as above, blocked with 200 il/well DPBS+1% BSA for 1 hr, and washed. Dilutions of mu MAb PB1.3 in DPBS+1% BSA were mixed with either 0.35 mg/ml CQNRYTDLVAIQNKNE in PBS, or as a control, PBS alone. After 1 hr, 50 1 of each antibody dilution was added to the microtiter plate, and incubated for 1 hr. The plate was washed with DPBS, and a 1 to 1000 dilution of sheep anti-mouse IgG horseradish peroxidase conjugate in DPBS+1% BSA was added.

After 1 hr, the plate was washed, the substrate TMB was added, and the color development was stopped and the plates read as above.

Fig. 5 shows that the peptide CQNRYTDLVAIQNKNE, homologous to residues 19-34 of the lectin domain of Pselectin, has no effect on the binding of mu MAb PB1.3 to Pselectin when the peptide is present at a concentration of SUBSTITUTE SHEET (RULE 26) WO 94/25067 ICT/US94104935 58 0.35 mg/ml. This distinguishes this particular monoclonal antibody from the monoclonal antibodies G1, G2, and G3, whose binding to P-selectin is partially or completely blocked by this peptide.

IV. Ability of Cvtel Mu MAbs PB1.3. PNB1.6. and 84/26 to Block Binding of Each Other to P-Selectin: Microtiter plates coated with affinity purified P-selectin as above were blocked for 1 h with DPBS+1% BSA, and washed. 25 Al of a 50 ug/ml solution of mu MAbs PB1.3, PNB1.6, or 84/26 in DPBS+1% BSA were added to appropriate wells, and incubated for 1 hr.

Then, 25 Al of a dilution (in DPBS+1% BSA) of biotinylated antibody was added. The plate was then incubated on a platform shaker for 1 hr. After washing, 50 Al of a 1 to 1000 dilution of a streptavidin horseradish peroxidase conjugate was added, and incubated for 1 hr. After washing with DPBS, the plate was developed with the substrate TMB as above.

Fig. 6 shows the ability of the mu MAbs PB1.3, PNB1.6, and 84/26 to interfere with the binding of each other to P-selectin. Fig. 6A shows that only mu MAb PNB1.6 is able to block the binding of biotinylated mu MAb PNB1.6 to Pselectin, demonstrating that mu MAbs PB1.3 and 84/26 must recognize different epitopes from mu MAbs PNB1.6. Likewise, In Fig. 6B, mu MA) PB1.3, but not mu MABs PNB1.6 or 84/26, is able to block the binding ,f biotinylated PB1.3 to selectin, demonstrating that the other two monoclonals must recognize S" different epitopes. Fig. 6C, bothnu, MAbs 84/26 and PB1.3, but not mu MAb PNB1.6, are able to block the binding of J biotinylated mu MAb 84/26 to P-selectin.

V. Blocking P-selectin antibodies do not inhibit thrombininduced aagrecation of human platelets: This example demonstrates that the P-selectin antibodies, mu MAbs PB1.3 and PNB1.6 do not inhibit thrombininduced aggregation of platelets. Fresh human platelets were isolated as previously described and suspended in Tyrode-Hepes Buffer, ph 7.2 at a concentration of 2xl0 8 /ml.

hJMJ SUBSTITUTE SHEET (RULE 26) I 1. -n WO 94/25067 PCT/US94/04935 59 Platelet aggregometry was performed in a Lumi aggregometer (Chrono-Log Corp.). For the assay, 0.45 ml of the platelet suspension was placed in a standard siliconized cuvette. To this was added 10 Al of the antibody or vehicle control. After 5 min. at 379C, 0.1 unit of thrombin (Human, Sigma) was added and aggregation was recorded.

When platelet aggregation was measured under control conditions, in the absence of antibody, an aggregation response of 71 units was obtained. An equivalent extent of aggregation was obtained in the presence of 10 Al of undiluted culture supernatants from cells producing mu MAb PB1.3 antibody (Fig. Different concentrations of mu MAb PB1.3 (1-100 pg/ml) were added to the platelet suspension. At no concentration of mu MAb PB1.3 was platelet aggregation different from the reponse determined in the absence of antibody (Fig. 7).

Example 9 In Vivo Protective Effects of Blockin P-Selectin Antibody.

mu MAb PB1.3 in Feline Mvocardial Ischemia and ReDerfusion A model of myocardial ischemia and reperfusion injury in cats was performed according to the methods of Tsao et al. (Circulation 82:1402-1412 (1990)). Briefly, male cats (2.5-3.5 Kg) were anesthetized with sodium pentobarbital mg/Kg, An intratracheal tube was inserted through a mid-line incision and all cats were given intermittent positive pressure ventilation by a Harvard small animal respirator. A polyethylene catheter was inserted into the external jugular vein and the right femoral vein was cannulated and connected to a Statham P23Ac pressure transducer for the measurement of arterial blood pressure. A midline thoracotomy was performed, the pericardium was opened and the heart was exposed. A 2-0 silk suture was carefully placed around the left anterior descending artery (LAD) 10-12 mm from its origin. After a 30 min period of stabilization myocardial ischemia was initiated by complete ligation of the LAD for 1.5 h of ischeia followed by 4.5 h of reperfusion.

Blocking (mu MAb PB1.3) and non-blockina (mu MAb PNB1.6) anti- SUBSTITUTE SHEET (RULE 26) WO 94/25067 PlCT/US94/04935 P-selectin antibodies were administered intravenously at a dose of 1 mg/kg 10 min prior to the initiation of reperfusion.

The ischemic myocardium was determined as that portion of tissue which did not stain with nitroblue tetrazolium and was expressed as a percentage of the area at risk. Area at risk was determined by reocclusion of the LAD at the end of the reperfusion period followed by the injection of Evans blue dye into the left atrium. The area of risk was, therefore, determined by negative staining.

Endothelial dependent relaxation of coronary artery rings was determined by measuring the acetylcholine-induced relaxation of rings previously contracted with the thromboxane A2 mimetic U46619. Data are expressed as percentage relaxation.

Neutrophil accumulation in the myocardium was measured as the myeloperoxidase activity of polytron homogenized myocardial tissue.

The results of these experiments are shown in Fig. 8. In cats treated with the non-blocking anti-P-selectin antibody mu MAb PNB1.6 the extent of myocardial ischemia was 33 5% of the area at risk. In contrast, the extent of ischemia in PB1.3 treated animals was significantly (P<0.01) less at 15 3% of the area at risk. Endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries taken from cats treated with mu MAb PB1.3 compared to mu MAb PNB1.6 (67 6 vs 11 P<0.01). That these phenomena are attributable to a reduction in myocardial leukocyte infiltration is suggested by a significant reduction in the number of PMNs in myocardial tissue from mu MAb PB1.3-treated animals (64 7 PMN/mm 2 compared with mu MAb PNB1.6 treated animals (136 12 PMN/mm 2 SUBSTTUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 61 Example Production of Humanized Antibodies to P-Seecti A. Cloning and Sequeicing of Mu MAb PB1.3 Variable Regions The isolation of mu MAb PB1.3 is described in Example 2. Total RNA was isolated from hybridoma cells producing mu MAb PB1.3. The cDNls encoding the variable regions of the heavy and light chains of mu MAb PB1.3 were amplified by polymerase chain reaction (PCR). The entire mouse variable region was amplified using a mixture of degenerate primers that hybridized within the leader sequence and a single primer that hybridized to the mouse constant region near the V-C junction. See Jones Bendig, Bio/Technology 9:88-89 (1991) and Bio/Technology 9:579 (1991).

The PCR fragments were cloned and sequenced. The nucleotide sequences and the corresponding amino acid sequences for the variable regions of the heavy and light chains of mu MAb PB1.3 are given in Figs. 9 and 10, respectively.

B. Modelling the Structure of the Mu MAb PB1.3 Variable Regions A molecular model of the VL and Vy regions of mu MAb PB1.3 was built. The model was built on a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the molecular modelling package QUANTA (Polygen Corp., USA). The amino acid sequence of the mu MAb PB1.3 variable regions were compared with the sequences of structurally-solved immunoglobulin variable regions. See Tables 1 and 2.

C. Design of Reshaped MAb PB1.3 Variable Recions Human variable regions were selected that have framework regions most similar to those found in the mu MAb PB1.3 variable regions. The human mAb DEN light chain variable region and the human mAb 21/28'CL heoavy chain variable region were selected as frameworks to join the corresponding complementarity determining regions (CDRs) of mu MAb PB1.3. The human framework regions were modified using the methodology described in Monoclonal Antibodies, 2: SUBSTITUTE SHEET (RULE 26) AppL:. cations in Clinical Onco.logy (Editor A. Epenetos: Chapman Hall, 1992, which is incorporated herein by reference; see especially, Bendig et al.t The Humanization Of Mouse Monoc.lonal. Antibodies by CDR-Grafting: Exam ples with Anti-Viral and Anti-Tumor Cell Antibodies contained therein, wh~ich is incorporated herein by reference). The alignment of amino acids~ of the variable regions leading to the design of the reshaped humanized PBI. 3 variable regions are given in Tables I and 2.

Three versions of the heavy chain variable region were modelled and were designated CY1748lHA, CY1748lHB, and CY31748RH%. The nucleotide. sequences and corresponding amino acid sequences of these variable regions and signal peptides are given in Figs. lit. 1.2, and 13, respectively. in the case of CY1748ERH 5 the first two mouse residues conserved in CY1 74 BIU{A (glutaxicit id and alanine) were changed back to the original human amino acids (glutamine and valine) found in the donor human FRI.. In the case of CY1748Rflc, computer modelling 0:00 suggested that the muxine lysine at position 73 might form a 20 salt bridge with residue 55 (aspartic acid) in CDR2. If this were the case, then the lysine would be important for the stabilization of the H12 loop. The human threonine residue at 9. this position would be unable to form such a salt bridge.

Therefore, version CY1748P'J1C substitutes the murine lysine at *o 25 position 73 of CY1.740RHX. Version CY1748RHU contains the highest number of human amino acid residues. A summary of the amino acid differences among these three versions of the heavy chain is given in Table 3.

Four versions of the light chain variable region were modelled and 'Were designated CY1.740RL&t CY1.740RLB# CY1.740RL., and CY2.748M.L. The nucleoiide sequences and corresponding amino acid sequences of these variable regions and signal peptides are given in Figs. 1.4, 2.5, 16 and 2.7, respectively. Computer modelling suggested that the murine sidue glutamic acid at position G0 might be important in P- $FAZ selectin binding. This residue was incorporated in version 1' t CY1748RLA but replaced with the original human framework residue serine in version CYl74hRLa. The murine aspartic 63 residue might have a charge interaction with residue 24 in loop 1 (Li) therefore CY1748RLc is identical to CY1748RLA except for the replacement of the mouse residue aspartic acid at position 70 for the human framework amino acid glutamic acid. CY1748RLD incorporates both these amino acid replacements and of the four versions contains the greatest number of human amino acid residues. A summary of theJ amino acid differences among these four versions of the light chain is given in Table 4.

D. Subclonjir3r 2f -OA Qjr-Encoding 1jeav- -andLiahtChain VarirAble _Reions Into EIwressipn Vectr II, Constructio 0of Exoress~ion3 Vectorp nUIM-1748RLT -neg is The expression vector contains the human cytomegalovirus (HCHV) enhancer and promoter to drive transcription of the recombinant iminunoglobulin light chain, the coding sequences of the bacterial neo gene to act as a dominant selectable markcet during stable transf ormation, and 9:06 20 the SV40 origin of replication to give high levels of transient expression in cos cells. To construct the vactor, a HindX-SacXi fragment from a pUCS vector containing the PstIm fragment of HcHV (Boshart et al., Call 41:521-530 (1985)) was converted to an EcoRi:-Hindlll fragment using appropriate adaptor oligonucleitides. in a three-way ligation, the 1.2 kd' EcOltt-Hindfll fragment containing the HCKJ enhancer-promoter 0 was ligated to a 5.05 kb Bam}1-EcoRI fragment from pSV2neo (Southern and Berg, J. Mol. App. Genet. 1:327-341 (1982) and a 0.5 kb HindIII-BamHI fragment containing the V~lys 30 kappa light variable region (Foote and Winter, J. Mo..

Biol. 224:487-499 (1992)). The HindIII zite in pSV2neo had ~.ipreviously been removed by filling-in with Klenow polymerase producing a vector designated pFICMV-VLlys-neO.

A 2.6 kb EcoRI fragment of the genomic human kappa region was cloned (Rabbitts et al., Curr. Top. Microbiol.

.rznzunol. 113:166-171 (1984)) and subcloned into a pUC vector, pUCKR17 (Mengle-Gaw and Rabbitts, E'MBO J, 6:1959- 1965 (1987)). BamHI sites were added to both ends of the fragment using EcoRI-BamBI adapters. This fragment was then inserted into the BamHI site of pHCMV-VLlys-neo, in the appropriate orientation, to create pHCIV-VLIys-KR-neo. To facilitate insertion of reshaped human variable regions, the BamHI site on the 3'-side of the human kappa constant region was removed by filling-in with Klenow polymerase. In the resulting vector, designated pHCMV-1748RL- KR-neo, the HindIII-BamHI fragment containing the VLlys is easily replaced with the VL of a reshaped antibody.

DHCMV-1748-1C-neo The construction of this veetor began with the same three way ligation as described above for generation of pHCHV- VLlys-neo above save for the insertion of a 0.7 kb HindIII- BamHI fragment containing the VuIs heavy variable region (Verhoeyen et al., Science 239:1534-1536 (1988)); Foote and Winter, supra) in place the 0.5 Kb HindIlI-BamKI fragment containing the VLlys kappa lignt chain. The cDNA coding for human 71 constant region was inserted into the BamHI site to produce a vector designated pHCMV-Vhlys-ylC-neo. cDNA coding for human yl constant region was cloned from a human cell line secreting a human yl antibody by PCR amplification. BamHI sites were created at each end of the cDNA, and a splice acceptor site and a 65bp intron sequence were introduced at the 5'-end of the cDNA sequence. The BamHI fragment (1176bp) containing the human yl cDNA plus the splice acceptor site and intron sequence was then cloned into the expression vector. As with the light chain 30 expression vector, the BamHI site at the 3'-side of the human yl constant region was removed by filling-in with Klenow polymerase. With this vector, designated pHCMV- 1748-ylC-neo, the HindIII-BamHI fragment containing the unwanted Vhlys is easily replaced with the VH of a reshaped 35 antibody.

Insertion of Immunoglobulin Coding Sequences The cDNAs encoding the heavy chain variable region of mu Mab PB1.3 and the three humanized versions were inserted ?Ai into the expression vector HCMV-ylC-neo by standard S techniques WO 94/25067 PCT/V"94/04935 generating expression plasmids HCMV-1747CH-7lC-neo, HCMV- 1748RHA-l7C-neo, HCMV-1748RHB-l1C-neo, and HCMV-1748RHc-y7Cneo. The nucleotide sequence of the human IgGi constant region cDNA and corresponding protein sequence is given in Ellison et al., Nuc. Acids Res., 10:4071-4079 (1987). The cDNAs encoding the light chain variable regions of CY1747(PB1.3) and the four humanized versions were inserted into the expression vector HCMV-KR-neo generating plasmids HCMV-1747CL-KR-neo, HCMV-1748RLA-KR-neo, etc. The nucleotide sequence of the human kappa constant region gene and corresponding protein sequence is shown in Hieter et al., Cell 22:197-207 (1980) (incorporated by reference in its entirety for all purposes) Schematic maps of the heavy chain and light chain expression vectors are given in Figs. 18 and 19.

By coexpressing appropriate combinations of heavy and light chains, several antibodies can be expressed. For example, coexpression of HCMV-1748RHA-7lC-neo and HCMV-1748RLA-KR-neo gives rise to the reshaped MAb PB1.3 (HA/LA). Coerpression of HCMV-1747CH-7lC-neo and HCMV-1747CL-KR-neo gives rise to the chi MAb PB1.3 chimeric antibody comprising murine variable domains and human constant domains).

E. Electroporation of COS Cells.

The DNA constructions were tested in COS cells for the transient expression of chi MAb PB131.3 and various reshaped versions of MAb PB1.3 antibodies. DNA was introduced into the COS cells by electroporation using the Gene Pulser apparatus (Biorad). COS cells were trypsinized and washed once in phosphate buffered saline (PBS). DNA (10 pg of each heavy chain plasmid and appropriate light chain plasmid) and a 0.8 ml aliquot of 1 x 107 cells/ml in PBS were placed in a sterile Gene Pulser Cuvette (Biorad, 0.4 cm gap). A pulse was delivered at 1900 volts, 25 microfarads capacitance. After a minute recovery period at ambient temperature, the electroporated cells were added to 20 ml of DMEM media (GIBCO/BRL) containing 10% fetal bovine serum. After a 48 h incubation, the medium was collected, centrifuged to remove SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 66 cellular debris, and stored under sterile conditions at 4°C for short periods of time.

Example 1: Analysis of Humanized Antibodies Media from the transfected COS cells were assayed by ELISA to quantify levels of human antibody produced and analyze capacity to bind to recombinant soluble P-selectin (rsP-selectin). Immulon microtiter plates (Dynatech, #011- 010-3355) were coated with 100 pl/well of a solution of goat anti-human IgG (whole molecule, Sigma #1-1886) to a final concentration of 12.5 pg/ml or a solution of rsP-selectin (1.2 mg/ml) diluted in Dulbecco's phosphate buffered saline (DPBS).

Plates were coated overnight at 4 0 C or for 2 h at 37°C. The plates were then washed three times with DPBS. The plates were then blocked with 400 ~l/well of DPBS 1% bovine serum albumin (BSA, Sigma, #A-7888) for one h or longer at room temperature. The plates were then washed three times with

DPBS.

Serial dilutions of the monoclonal antibodies in DPBS 1% BSA were added to the plates (100 pl/well), and the plates were incubated for 1 h at room temperature. The plates were then washed three times with DPBS. 100 pl/well of a second antibody (goat anti-human IgG peroxidase conjugate, Sigma #A--8867) diluted 1 to 1000 in DPBS 1% BSA was added to the plates, and the plates were incubated for one h at room temperature. The plates were then washed three times with DPBS. 100 pl/well of tetramethylbenzidine peroxidase substrate/H 2 0 2 (TMB, Kirkegaard and Perry Laboratories #50-76- 00) was added to the plates, and the color was allowed to develop for an appropriate period of time (usually 3 to min), and the reaction was quenched by the addition of 100 Cl/well of 1-M phosphoric acid. The plates were read at 450 nm in a Titertek Multiskan MCC/340.

The concentration of the antibodies produced in all COS cell supernatants was determined by comparing the IgG ELISA results to a human IgG1 antibody (kappa light chain, Sigma #1-3889) of known concentration.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 67 P-selectin binding was plotted as optical density against the concentration of IG. Binding curves of chi PB1.3 and various reshaped versions of reshaped MAb PB1.3 are given in Figs. 20, 21, and 22. The objective was to select a humanized antibody showing substantially indistinguishable binding characteristics from chi MAb PB1.3. If more than one humanized antibodies showed such characteristics, the one containing the most human amino acid residues (or the fewest murine amino acids) would be preferred, because this version would be least likely to induce a HAMA response in clinical use. All four of the reshaped light chains coexpressed with heavy chain CY1748RHA generated antibodies that bound to rsPselectin as well as the chi MAb PB1.3. Of these, light chain variable region CY1748RL was selected for further experiments, because it contains the most human amino acid residues. The heavy chain variable regions CY1748RHA and CY1748RHB when coexpressed with light chain variable regions CY1748RLA and CY1748RLP also generated antibodies that bound to rsP-selectin to the same extent as chi MAb PB1.3 (Fig. 22).

Of these heavy chains, the CY1748RHB heavy chain is preferable because it contains the most human amino acid residues. Thus, a preferred humanized antibody is formed from light chain D and heavy chain B and is designated reshaped MAb PB1.3

(HB/LD).

Example 12 Stable Expression of Reshaped MAb PB1.3 (Hg/LD) in CHO Cells A. Introduction of Humanized Immunoglobulin Chains in CHO cells Construction of Vectors pHCMV-1748RH-y1C-dhfr This vector is identical to pHCMV-1748RH-ylC-neo described in Example 11 except that the neo gene is replaced by the dihydrofolate reductase (dhrf) gene linked to a defective SV40 promoter-enhancer sequence. In order to remove the enhancer sequence from the SV40 early promoter, the A7 1 plasmid DNA, pSV2-dhfr (Subramani et al., Mol. Cell. Biol.

1:854-864 (1981)) was digested with SphI and PvuII, filled-in SSUSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 68 with Klenow polymerase, and self-ligated to yield pSV2-dhfr- AE. A -3.7 kb EcoRI fragment containing the HCMV promoter, the VHlys heavy chain variable region, and a genomic DNA clone of the human 71 constant region was excised from the plasmid pHCMV-VHlys-71-neo with EcoRI. This fragment was ligated to EcoRI-digested pSV2-dhfr-AE to create pHCMV-VHlys-71-dhfr.

The genomic clone of the 71 constant region was replaced with a BamHI fragment containing the cDNA clone of the human 71 constant region-described in Example 11. The BamHI site on the 3'-side of the human 71 constant region was then removed by filling in with Klenow polymerase as in Example 11. The resulting vector was designated pHCV-174RH-7C-dhfr. The HindIII-BamHI fragment of this vector containing the unwanted VHlys is easily replaced with the VH of a reshaped antibody.

PHCMV-1748RH-74-dhfr This vector is idential to pHCMV-1748RH-ylC-dhfr described above, but for the replacement of the cDNA clone of the human 71 constant region with a genomic clone of the human 74 constant region. To construct the vector, a 7.0 HindIII fragment of DNA containing the genomic clone of the human 74 constant region (BrUggemann et al., J. Exp. Med. 166:1351-1361 (1987)) was subcloned into the HindIII site of pUC19 to create a plasmid called 428D. Because the human 74 fragment already had a BamHI site near its end, the subclone was inserted into pUC19 in the orientation that placed this site distal to the BamHI site in the polylinker of pUC19. The 7.0 kb human 74 fragment was excised from 428D using BamHI and ligated into pHCMV-Vylys-neo to create the plasmid pHCMV-VHlys-74-neo.

The final plasmid was constructed by a three-way ligation of a 5.4 kb BamHI-HindIII fragment containing the HCMV enhancer-promoter and the pSV2-dhfr-AE plasmid sequence, a 0.5 kb HindIII-BamHI fragment containing the VH of reshaped human 1748 antibody and the 7.0 kb BamHI-BamHI fragment containing the genomic clone of the human 74 constant region.

SUBSTITUTE SHEET (RULE 26) 69 Insertion of Irniunogl~obuli.flCoding Seqouences The cDNA encoding l7438RH, heavy chain variable region was cloned into expression vector H-CMvV-ylC-dhfr generating HCMVl748RIA-YlC-dhfr. The cDNA encoding 1748RHB1 was cloned into expression vectors HCMV-ylC-dhfr and HCI4V-y4C-dhfr generating the plasmids HCMV-1748RHB-ylC-dhfr and HCVM- 1748RHB-~y4C-dhfr, respectively. The nucleotide sequence of the human IgG4 constant region gene and corresponding amino acid sequence is given by Ellison et al., DNA, 1:11-18 (1981) .The vector HCMV-ylC-dhfr expresses an IgGl isotype antibody and vector HCMV-y4C-dhfr expresses an IgG4 isotype antibody when they are each coexpressed with the kappa light chain vector.

The schematic maps Of the dhfr-expression vectors are given in Figs. 23 and 24, respectively.

CIo dhfr-deficient cells were grown in cxafl nucleosides, GIBCO/BRL) and 10t fetal -bovine serum. Plasmid DNA was. introduced into the Cos cells by electroporation using the Gene Pulser apparatus. COclswr rpiie n wased ncein hoshat bffered saline (PBS). DNA (10 j~ of 20 each heavy chain plas-mid and appropriate light chain plasmid) and a 0.8 ml aliquot of 1 X 107 cells/mi in PBS were placed in a sterile Gene PUlser Cuvette (0.4 CM gap). A pulse was delivered at 1900 volts, 25 microfarads; capacitance. After a 10 minute recovery Period at room temperature, the 25 eleCtroporated cells were added to 20 ml of alHEM nucleosides),'lo% FES. After a 24-48 h incubation cells were trypsinized and plated into 100 Zm dishes in (XHE nuclcosides)/10* dialyzed FES (to select for the expression of the dhfr-containing Plasmid) supplemented with 500 Msg/ml G418 (GXBCO/BRL? to select for the expression of the neo-containing plasmid). Hedia was changed everY 3-4 days until colonies emerged. Single clones were isolated via cloning cylinders, expanded and analyzed for tgG production via JELISA.

Three sets Of DRAs were introduced into the CHO dhfr-deficient cells: 1) HcMV1-748RHA-lC-dhfr and HCI(V- 1740R~LA-M-neo expressing reshaped HAb FBI. 3 (HA/LA),I IgG1 isotype; Z)HCMV'-1748a~a-1c..dhfr and HCHV-1748RLb-KR-neo expressing reshaped KAB PBI.3 1gGI isotype; and HCHV-

S.

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i-~ \4JNTO WO 94/25067 PCTIUS94/04935 1748RHB-74C-dhfr and HCMV-1748RLD-KR-neo expressing a reshaped MAb PB1.3 IgG4 isotype.

CHO cells were expanded and seeded into (6000 cm 2 total surface area) Nunc cell factories. The medium was harvested at 72 h and passed over a protein-A Sepharose Fast Flow (Pharmacia) column. The column was washed and bovine IgG was eluted at pH 4.5. The humanized antibodies were eluted at pH 3.5, dialyzed in PBS or 20 mM acetate, 0.15 M NaCl, pH 5.5. Antibody concentration was determined spectrophotometrically and confirmed by an ELISA for human IgG. (A purified human IgG4 antibody (kappa, Sigma #1-4639) was used as the control for mu MAb PB1.3 IgG4 isotype.) Purity was confirmed by SDS-PAGE.

B. Competitive Binding Assay The relative binding affinities of the various versions of reshaped MAb PB1.3 to P-selectin were determined by competitive ELISA assay. The assay measures the concentration of unlabelled antibody that inhibits by 50% the binding of labelled mu MAb PB1.3 to P-selectin.

Two ml of purified MAb PB1.3 (1 mg/ml) was mixed with 100.8 Al of NHS-LC-biotin (1 mg/ml, Pierce) and incubated overnight at 4 0 C. The 2.1 ml sample was loaded on a NAP column (size exclusion column, Pharmacia). The column was eluted with 0.3 ml fractions with PBS. Each fraction was read at 280 nm. The highest O.D.280 nm fractions were pooled. The biotinylated-MAb PB1.3 was titered against rsP-selectin to select an appropriate concentration to use for the competitive binding assay.

A 96-well plate (Microtest III, Falcon #3912) was coated with 50 il/well of rsP-selectin (diluted to 2 Ag/ml in DPBS) overnight at 4 0 C or 90 min at 370C. The plate was inverted and residual fluid was tapped away.

The coated plate and an additional blank plate were blocked with 200 gl/well of DPBS 1% bovine serum albumin (BSA, Sigma #A-7888), for 1 h at room temperature. The blank plate was inverted and the residual blocking fluid was tapped away. The rsP-selectin coated plate was allow to continue SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 71 blocking during the antibody dilution period. Serially diluted sample antibodies (in DPBS 1% BSA) were added to the blank plate at 50 ~l/well. The initial concentrations of the antibodies were 100 pg/ml. Biotinylated-PB1.3 (diluted to 0.3 pg/ml in DPBS 1% BSA) was added to the sample antibodies in the blank plate, 50 pl/well, and mixed by pipetting up and down. The rsP-selectin coated plate was washed three times with DPBS. The sample antibody/biotinylated -PB1.3 mixture was pipetted into the plate coated with rsPselectin, 50 Cl/well, and allowed to incubate for one h at room temperature. The plate was then washed three times with

DPBS.

Streptavidin horse radish peroxidase conjugate (Pierce #21124) diluted 1:2000 in DPBS BSA was added to the plate, 50 pl/well and allowed to incubate for 1 h at room temperature. The plate was then washed three times with DPBS, pl/well of tetramethylbenzidine peroxidase substrate/H 2 0 2 (TMB, Kirkegaard and Perry Laboratories #50-76- 00) was added to the plates, and the color was allowed to develop for an appropriate period of time (usually 3 to min), and the reaction was quenched by the addition of pl/well 1 M phosphoric acid. The plates were read at 450 nm in a Titertek Multiskan MCC/340.

Apparent binding affinity is expressed as the concentration of antibody that binds rsP-selectin at one-half maximum optical density at 450 nm. The ratio of concentrations at one-half maximum binding of the sample antibody to that of the control (unlabelled PB1.3) antibody provides an estimate of the relative binding affinity of a test antibody to rP-selectin.

Results from two typical competitive binding experiments are given in Figs. 25 and 26. In the first experiment, twice as much reshaped MAb PB1.3 (HALA), IgGi isotype (2.0 pg/ml) as PB1.3 (1.0 pg/ml) is needed to inhibit the binding of labelled PB1.3 to 50% of maximum. Therefore this humanized antibody binds rsP-selectin 50% as well as PB1.3. Similarly, in the second experiment PB1.3/(HBLD) (IgG4 isotype) binds to rsP-selectin 50% as well as PB1.3.

SUBSTITUTE SHEET (RULE 26) WO 94125067 I'CT/US94/04935 72 Example 13 Increasing Antibody Production in CHO Cells Via DHFR Amplification CHO cells were plated into flasks and allowed to reach confluence before changing media (8 ml for a T-25 cm 2 flask or 20 ml for a T-75cm 2 flask). After incubating the cells for 48-72 h the media was harvested and assayed for IgG production by ELISA. The cells were trypsinized and counted.

Antibody production rate was expressed as the amount of antibody in micrograms secreted by one million cells in a 24 h period.

The three to six highest-producing clones were amplified separately, and the remaining seven best clones were pooled and amplified. Cells were plated at a density of 1 x 5 cells/100 mm dish in selection media. For the first round of amplification cells were plated in three sets of dishes containing aMEM(- nucleosides)/10% dialyzed FBS supplemented with 500 pg/ml G418 and either 10, 20 or 50 nM methotrexate (Sigma #A-6770). Cultures were fed every four days. At 10-14 days single colonies were isolated by cloning cylinders, expanded, and assayed for IgG production by ELISA. Additional rounds of amplification were performed on single clones or pools of clones at methotrexate concentrations 5-10 times the initial methotrexate concentration.

Table 5 provides a summary of the antibody production for the CHO cells expressing three humanized versions of PB1.3. One amplified clone expressing reshaped MAb PB1.3 (HBLD) (IgG4 isotype) arose from a pool of clones that expressed less than 0.2 pg/10 6 cells/day, amplified to pg/10 6 cells/day in the first round, and reached 33.0 6 cells/day after the third round. This clone was selected to be grown for large scale production of reshaped MAb PB1.3.

SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT/US94/04935 73 Bioreactor Run 160-g collagen-coated microcarrJer heads (150-200 microns, JRH Biosciences #60142-100) were hydrated in 800-ml of sterile water for 30 min and the water decanted. The beads were resuspended in 800-ml water and autoclaved for 30 min.

After cooling to room temperature the beads were washed twice with sterile serum free AMEM(-nucleosides) and resuspended in 1-1 complete media aMEM(-nucleosides)/5% dialyzed FBS.

The cell line CHO-48B4, which expresses reshaped MAb PB1.3 (HBLD) (IgG4 isotype) at 33.0 Ag/10 6 cells/day, was expanded into 36 T-225 cm 2 in aMEM(-nucleosides)/10% dialyzed FBS/500 nm methotrexate. When the cells reached confluence they were trypsinized and resuspended in 2-1 complete media (no methotrexate) at a final concentration of 5.0 8.0 x 10 4 cells/ml.

1-1 beads, 2-1 cell suspension, and 2-1 fresh complete media were aseptically added to the 10-1 autoclaved vessel of a Proteus 2000 Bioreactor (Wheaton). The headspace was continuously perfused with air and 5% CO 2 Cells were allowad to attach to the microcarriers by programming the Proteus to stir the cell/microcarrier suspension at 70 rpm for 1 min and 0 rpm for 59 min. After a total of 48 cycles, 5-1 media was added to the bioreactor, which was programmed to maintain the suspension at 370C, pH at 7.0, dissolved oxygen at 30% that of air, and stirred continuously at 50 rpm.

After 3 days the bioreactor was programmed to replace complete media at 5-1/day utilizing a gravity filter attached to the harvest port (to leave microcarriers in the bioreactor). The harvested media was stored at 4 0 C until processing. The bioreactor was run for 5-7 days until accumulated cell debris clouded the media harvest (due to aeration damage).

The media harvest was clarified by passing though a Millipak-60 0.45 micron filter (Millipore). 20-1 filtered supernatant was passed over a column (2.5 x 12 cm) of protein- A Sepharose Fast Flow (Pharmacia) at a flow rate of 0.55 1/hr.

After all of the supernatant had passed through the column, SUBSTITUTE SHEET (RULE 26) 1 WO 94/25067 PCT/US94/04935 74 the column was washed with 1.5-1 PBS (BioWhittaker, #17-516Y).

The column was pre-eluted with 8 x 40-ml 0.2-M sodium acetate, pH 4.5, and eluted with 8 x 40-ml 0.2-M glycine, 0.5-M NaCL, pH 3.5, both into tubes containing 10 ml 2-M Tris-HCl, pH The single fraction with the highest absorbance at 280 nm was dialyzed into 20-mM acetate, 0.15-M NaC1, pH 5.5, and stored at 4 0

C.

200-mg antibody was harvested per day for 5-7 days.

The purification yield was approximately 50%. Therefore, one bioreactor run yields 500-700 mg purified antibody.

ExamPle The Effect of Mu MAb PB1.3 on Tissue Iniurv Produced by Ischemia Reperfusion Following Replantation of the Rabbit Ear New Zealand White rabbits were anesthetized with a combination of ketamine and xylazine followed by infiltration of lidocaine into the base of the ear. The left ear was partially amputated leaving the central artery, vein and a bridge of cartilage intact. All nerves were divided to render the ear totally anesthetic. The ear was then reattached and a microvascular clip was placed across the artery to produce total ischemia. Rabbits were kept in a room maintained at 23.5 0 C for 6 h then the microvascular clamp was removed and the ear allowed to reperfuse. Treatment was administered immediately prior to reperfusion with either mu MAb PB1.3(2 mg/kg) or with either saline or an isotype (IgGI) matched murine monoclonal antibody designated PNB1.6 (2 mg/kg). Ear volume was measured daily for 7 days by water displacement to quantify tissue edema. On day 7 necrosis was estimated as a percentage of total surface area.

A significant increase in ear volume was determined in control animals administered either saline or PNB1.6 following reperfusion of the ears (Fig. 27). Because no differences between the two control groups were noted, their data have been combined. An increase in edema was also seen in animals treated with MAb PB1.3, the magnitude of the increase was significantly less than that observed in the control group at all time points measured. The extent of SUBSTITUTE SHEET (RULE 26) WO 94/25067 PICT/US94/04935 necrosis (Fig. 28), measured on day 7, was also significantly reduced in mu MAb PB1.3-treated animals compared with control animals.

Thus, ischemia followed by reperfusion of the replanted rabbit ear produces an edematous response initially followed by extensive tissue necrosis. These two manifestations of tissue injury are substantially and significantly reduced in animals pre-treated with the anti-Pselectin antibody mu MAb PB1.3.

Example 16 The Effect of Mu MAb PB1.3 on Vascular Patencv Followina Ischemia and ReDerfusion of the Canine Skeletal Muscle This experiment utilizes a model of ischemia and reperfusion of the isolated canine gracilis muscle. One aspect of the reperfusion injury is a phenomenon referred to as the "no-reflow" phenomenon in which blood flow through the microcirculation of a previously ischemic tissue is absent or severely reduced following reperfusion.

An isolated canine gracilis muscle preparation was utilized as described previously (Carden et al. circ. Res.

66:1436-1444 (1990)). Briefly, adult mongrel dogs were anesthetized with pentobarbital sodium. The skin overlying the gracilis muscle was divided and the muscle was freed from connective tissue by blunt dissection. The obturator nerve was sectioned. The proximal caudal artery and vein were cannulated and all other collateral vessels ligated. The femoral artery cannula was connected to a constant flow perfusion pump and the outflow from the femoral vein drained in to a reperfusion reservoir. Arterial and venous pressures were monitored via side branches and blood flow was adjusted to maintain perfusion pressure at 100 mm Hg. Vascular isolation was assumed complete if arterial perfusion pressure decreased to less than 20 mm Hg when the perfusion pump was switched off.

Microvascular patency was assessed by perfusion of the isolated gracilis muscle with contrast media (india ink) at the end of the experimental protocol. Computerized video SUBSmUTE SHEET (RULE 26) WO 94/25067 PCTIUS94/04935 76 imaging was used to guantitate the number of ink-containing microvessels 10 pm diameter) per muscle fiber in histologic sections obtained from isolated canine gracilis muscles subjected to 4.5 h of continuous perfusion, 4 h of ischemia followed by 0.5 h of reperfusion or ischemia reperfusion in the presence of the anti-P-selectin antibody mu MAb PB1.3 at a concentration of 40 pg/ml in the perfusate.

The ischemia and reperfusion protocol reduced the number of patent microvessels to 36 3 of the value determined in control animals. When mu MAb PB1.3 was included in the perfusate the number of patent microvessels following ischemia and reperfusion was 119 18 of control.

Thus, the anti-P-selectin antibody, mu MAb PB1.3, completely prevented the development of the "no-reflow" phenomehon, determined by the number of patent microvessels, in the ischemic and reperfused canine gracilis muscle.

Exfmlge 17 The Effect of Mu MAb PB1.3 on Leukocvte Endothelial Cell Interactions Induced by the Dearanulationof Tissue Mast Cells I. Mthos a) Intravital microasov Male Wistar rats (200-250g) were obtained from Harlan Sprague Dawley, Indianapolis, and were fasted for 12-24 hrs before the surgical procedure. Surgical anaesthesia was induced by an intramuscular injection of ketamine/rompun/ acepromazine. A catheter was placed in the left jugular vein, and a tracheostomy was performed to facilitate spontaneous breathing. The abdomen was shaved and washed, and a 3/4 inch long incision was made through the midline into the peritoneal cavity, taking care to avoid any bleeding. The rat was placed on a microscope stage specifically designed for the intravital microscopy procedure. A wall-vascularized posterior loop of the ileum was exteriorized, and the mesentery draped over a viewing pedestal heated to 376C. Throughout the experiment, the mesentery was superfused at 2 ml/min with a heated bicarbonate-buffered saline solution (370C, pH 7.4).

Additionally, small pieces of Saran-Wrap (previously soaked in SUBTTUTE SHEET (RULE 26) 'r- WO 94/25067 PCTIUS94/04935 77 alcohol and rinsed in saline) were placed over the ileum and mesenteric areas peripheral to the viewed area, in order to keep these areas moist. The microcirculatory beds were observed through an intravital microscope (Mikron Instruments, San Diego) equipped with 10x oculars and a 20x objective (water immersion, numerical aperture A high-resolution video system, consisting of a video camera (Hitachi color CCD) clamped onto the microscope, a video timer (American Video Equipment), a VCR (Sony SVO-9500MD) and a monitor (Sony Trinitron), permitted video recordings of the microscope image. In each mesentery preparation, 3 postcapillary venular segments of approximately 180 Am length were selected for repeated recordings according to the following criteria: A true postcapillary venule receiving blood from confluent capillaries, diameter 15-30 Am; Brisk blood flow, so that individual red blood cells could not be distinguished; Some baseline rolling of leukocytes (less than 8 leukocytes present at any given time in the vascular segment), but no firmly adhering leukocytes. A leukocyte was defined as being firmly adhering if it stayed immobile at the vessel wall for more than 30 seconds.

b) Experimental protocol One-minute video recordings of the baseline leukocyte interaction in the selected venules were made at and 25 min after completion of the surgical procedure. All treatments were administered intravenously before the second baseline recording. At 30 min after surgery, infusion of compound 48/80 (Sigma, catalog #C4257) into the superfusion buffer was begun, and continued throughout the rest of the experiment. This yielded a final concentration of 10 jg/ml at the mesentery, and was done in order to induce mast cell degranulation. Venules were recorded again at 20 and 30 min after the beginning of 48/80 application. The extent of leukocyte-endothelial cell interactions was determined at the end of the experiment by analyzing the recorded video tapes.

The number of clearly visible leukocytes cells interacting with the vessel wall) was determined from frames SUBSTITUTE SHEET (RULE 26) I WO 94/25067 PCT/US94/04935 78 frozen at exactly 0, 15, 30, 45, and 60 seconds of the recorded sequence, and the mean from these five determinations was calculated. Hence, counted leukocytes included rolling cells as well as those firmly adherent.

c) Statistics Each experimental group contained 5-6 animals with 2-3 venules from each animal. The mean and its standard error is given. Significant differences among group means were ±0 tested through analysis of variance followed by the Tukey- Kramer's HSD test. Differences before and after treatments in individual venules were studied by paired t-tests. All tests were run using JMP software running on a Macintosh IIsi, and significance was accepted at p<0.05.

II. Results Topical application of compound 48/80 to the rat mesentery resulted in visible degranulation of more than of the mast cells, which took place within 3 min after application. Subsequently, an increase in leukocyte rolling and adhesion was seen. No significant difference in leukocyte accumulation was detected between the 20 and 30 minute timepoints post 48/80 application, the mean of these two timepoints was therefore used in the statistical analysis.

The venule-to venule variability in the model was quite high, with baseline interaction ranging from 0 to 4.8 leukocytes present in individual venules, and mast cell-induced interaction ranging from 2.8 to 25.6 leukocytes present in the PBS-treated group (coefficient of variability, CV, 37%).

Animal means showed much lower variability (CV, hence, each individual venule was treated as a separate experimental entity for statistical purposes.

As seen in Fig. 29, intravenous treatment with mu MAb PB1.3 inhibited the mast cell-induced intravascular leukocyte accumulation by 90% when compared with the formulation buffer control group. The non-reactive antibody P6H6 did not have any effect in this model.

SUBSTITUTE SHEET (RULE 26) t 79 Thus, administration of the anti-P-selectin antibody, mu MAb Pbl.3, to rats completely prevents the interaction of leukocytes with the vascular endothelium and their subsequent migration into the tissues induced by application of the mast cell degranulating agent 48/80.

Mapping the Epitope of Mu MAb PB1.3 P-selectin is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, P-selectin is rapidly' redistributed to the plasma membrane. The mature molecule is a protein with multiple domains, including a "lectin" region, "EGF" region, nine tandem consensus "C3b-C4b Regulatory Protein" (CRP)repeats, a transmembrane region, and a cytoplasmic domain. See Johnston et al., Cell, 56:1033-1044 (1989) (herein incorporated by reference in its entirety for all purposes) for the cloning and description of P-selectin.

Fig. 30 shows a proposed folding pattern of the domains of P- 20 selectin.

P-Selectin Deletion Analyses In order to map the epitope specifically bound by mu H MAb PB1.3, mutants of recombinant P-selectin were constructed 25 by deleting consecutive CRP domains (Fig. 31). This sitespecific mutagenesis was accomplished by PCR amplification using primers specific to each CRP domain (Table 6 SEQ ID Nos. 27-45). The template used for PCR-mutagenesis was a pUC DNA clone of P-selectin which was truncated and mutated at DNA sequence position 2354 (sequence positions are in accordance with the published sequence of P-selectin of Johnston et al., supra. This template plasmid pGAl, T which was derived initially by in vitro mutagenesis, lacks the trans-membrane domain and the cytoplasmic carboxyterminus of P-selectin, and encodes instead the carboxy- Sterminal 11 amino acid residues of SV40 Large T antigen.

RA This SV40-derived C-terminus is recognisea by the S W monoclonal antibody KT-3. (See MacArthur et al., Journal Sof Virology 52:483-491 (1984)). After PCR amplification, the WO 94/25067 PCTUS94/04935 deleted DNA molecules were purified, ligated, transformed into bacteria, cloned and sequenced as described below. DNAs with the appropriate linkage of CRP to the T-antigen tag sequence were subcloned into the pcDNA1 expression vector (Invitrogen) and transfected into COS cells.

PCR mutagenesis was performed on plasmid pGMPAl,T.

After phosphorylation at the 5'-end, the primers in Table 6 were added to individual PCR reactions at 1 MM, together with the T-antigen sequence primer lys-l. The PCR was performed in a Perkin Elmer-Cetus machine for 30 cycles at 95°C for 50 sec, 0 C for 1 min. and 73°C for 4 min. using Pfu polymerase (Stratagene) according to the manufacturer's recommended conditions. After purification by agarose gel electrophoresis and elution by Qiaex (Qiagen Corp.), the mutated pUC DNAs were ligated and transformed into E. coli InvF'a cells (Invitrogen). Cloned DNAs carrying the desired mutation were identified by DNA sequence analysis and the SalI-Hpal fragments containing the mutated P-selectin gene cloned into the Xhol and Xbal sites of the expression vector, pcDNAI (Invitrogen). The pcDNA clones were transformed into E. coli strain MC1061 (Invitrogen) and purified plasmid DNAs used for transfection into COS.

DEAE-Dextran Transfection Method This method is described in Kriegler, Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman and Company (1990). COS cells were seeded at 1 x 106 cells/100 mm dish in DMEM (Biowhittaker), 10% fetal bovine serum (FBS). On day two, plasmid DNA was ethanol precipitated, and resuspended at a concentration of 20 pg/mL in sterile TE (10 mM Tris, pH 8.0, 1 mM EDTA). 150 AL of DNA was mixed with 300 Al of sterile TBS (Tris Buffered Saline, 140 mM NaCl, 5 mM KCl, 1.4 mM Na 2

HPO

4 25 mM Tris-base, pH 7.5, 1.0 mM CaC1 2 and 0.5 mM MgC1 2 and with 300 Al of sterile DEAE dextran (Sigma, #D- 9885, 1 mg/ml in TBS). The growth media was aspirated, and the cell monolayers were washed once with PBS, and once with TBS. 750 Al of the DNA/DEAE dextran/TBS mixture was added to the monolayer. The dish was incubated at ambient temperature inside a laminar flood hood rocking the dish every 5 min for SUBSTITUTE SHEET (RULE 26) r IIIL r I WO 94/25067 PCT/US94/04935 81 1 h. After the 1 h incubation, the DNA solution was aspirated and the cells were washed once with TBS and then once with PBS. The cells were incubated in a complete medium supplemented with 100 pM chloroquine (Sigma, #C-6628), 37 0

C,

5% CO 2 After 4 h, the medium was replaced with complete media, and the cells were incubated at 37 0 C and 5% CO 2 After 48 h post-transfection, the cells were fed with DMEM growth medium lacking serum. 24 h later the medium was harvested, the cell debris removed by centrifugation at 1500 rpm for 5 min in a tabletop clinical centrifuge.

Capture ELISA to Analyze Secreted P-Selectin Mutant Proteins A 96-well Costar plate was coated with KT-3 antibody (produced in ascites and purified by Protein G Sepharose, Pharmacia) 100 Ai/well at 40 Ag/ml at 4 0 C overnight. The plate was washed 3x with DPBS, blocked for 1 h at ambient temperature with 250 pl/well of DPBS 1% BSA. The plate was washed 3x with DPBS, and incubated with COS cell supernatants 100 Il/well for 2 h at ambient temperature. After washing the plate 3x with DPBS the bound P-selectin was detected by adding either 100 il/well of 1 ng/ml rabbit anti-P-selectin polyclonal Ig, or biotinylated mu MAb PB1.3, or (c) biotinylated P6H6 diluted in DPBS/1% BSA for 1 h at ambient temperature. After washing the plate 3x with DPBS the rabbit antibody was detected using 100 Al/well of a 1:1000 dilution of HRP-conjugated goat anti-rabbit antibody (Biorad), blocked with tissue culture media from the KT-3 cell line in DPBS/1% BSA for 30 min at ambient temperature. The biotinylated antibodies were detected using 100 pl/well of a 1:1000 dilution of HRP-streptavidin (Pierce) for 30 min at ambient temperature. The bound HRP was incubated with TMB and quenched with 1 M phosphoric acid. Figure 32 shows the signal less the background from supernatants of mock transfected COS cells. The "Mock" column is the supernatant from cells transfected with a full length form of P-selectin, which lacks the SV40 T-antigen tag, and therefore is not detected in this KT-3 capture ELISA. The X-axis specifies the number of CRPs expressed by the various mutant P-selectin cDNAs, from all 9 SUBSTITUTE SHEET (RULE 26) ii WO 94/25067 PCT/US94/04935 82 CRPs in column 2 to one CRP in column 10. The results show that, although P-selectin mutants lacking the carboxy-terminal CRPs can be detected by the polyclonal rabbit Ig, these molecules do not react with PB1.3. This suggests that the binding site for PB1.3 lies within the fifth CRP repeat domain.

In order to confirm the results from epitope mapping, the deletion analysis was extended to delete only the fifth CRP domain, leaving the carboxy-terminal sequences of Pselectin intact (see Fig. 30). PCR amplimers were designed to remove the region of P-selectin encoding amino acids 407 to 468 (Table 6) using the same protocols described above. Cell supernatants were analyzed directly by ELISA with antibodies Mu MAb PB1.3 and P6H6 (Fig. 33). Although mu MAb PB1.3 binds the intact P-selectin, it fails to bind the deleted molecule.

P6H6 iinds both molecules equivalently.

Synthetic Peptide Analyses The mu MAb PB1.3 epitope was further characterized by synthesizing peptides spanning the 62 amino acid region of the fifth CRP of P-selectin (Fig. 34) and screening for mu MAb PB1.3 binding by ELISA. The fifth CRP extends from amino acid sequence 407 to 468 according to the nomenclature of Johnston, et al. The peptide sequences are 968.01, a.a. 408-426; 968.02, a.a. #418-436; 968.04, a.a. #408-433; 968.05, an oxidized version of 968.04; 968.06, a.a. #428-447; and 968.08, a.a. #448-467. Each peptide was dissolved to 2mg/ml in dimethyl sulfoxide (DMSO) and coated onto a 96-well microtiter plated by dilution of 1 microgram into 100 Al PBS and dried at 37 0 C overnight. Coated wells were washed with PBS and blocked with 250 Al/well of a solution of PBS/1% BSA for 30 min at ambient temperature. The wells were washed with PBS and incubated with 100pl/well of PB1.3 diluted to 1ig/ml in PBS/1%BSA for 1 h at room temperature. Bound mu MAb PB1.3 was detected with HRP-conjugated goat anti-mouse antibody reagent and TMB substrate as described before. Fig. 34 shows a comparison PB1.3 reactivity with the peptides. The carboxyterminal peptide, number 968.08 reacted strongly with PB1.3.

SUBSTITUTE SHEET (RULE 26) I I- WO 94125067 PCT/US94/04935 83 Therefore the epitope recognized by PB1.3 maps to amino acids 448 to 467 in the fifth CRP of human P-selectin.

For the purposes of clarity and understanding, the invention has been described in these examples and the above disclosure in some detail. It will be apparent, however, that certain changes and modifications may be practiced within the scope of the appended claims. All publications and patent applications cited above are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.

SUBSTITUTE. SHEET (RULE 26) PCT(US94/04935 WO 94/25067 84 TABLE 1(1 of 4) 1MJZIENT OF L.24:T1O ACD SEUENCES LEADING 7o :HE DESCN OF RESHAPED LUMA ?B131 HEAVY CHAIN'.ARIABLE REGION i FR cir -Du humuse bhrn muis RH Va Comm CDR 1 47 II 1 17 47 II FM E E PCA cbo m HI.

pombla law ta angta A IV V v ujasiIAA.

omo wHl.

pomzblr M binding L. L L L L Qc I I Q iv v v__ 16 0 Q Q Q S S S S a I a Ia ja a a c 19 p p AJ A Po I I E E E 1I L L v v V I IK :3 K K IK I.

I p p Ip p p I!I I' 'a ac a Ia a I 116 1 1 1A IA A A I s s IIs Is I v v v v v K19 1 K K K K :o V t v v IsI S S I.S C i c c C i K K K K K :r:4 A 1 'A A a a a' V S.

F

suBsTIT SSHEET4RULE 2) L- PCT/IUS94/04935 WO 94125067 TABLE 1 (2 of 4) 30_ FRI T T IT T 31 31 ('DRI D s ss 32 32 YIY Y Y Y 33 331 v y A A v 34M I M Nil 3Sfl H N T H 351R) CDR1 36 36 F_ w w w 37 37 I v Iv v v 38 38 K K R rR 39 39 Q Q Q IQ Q K js A A A _1 4 _p p 42 42 0 C; a 0 G 13 4 3 Q Q K Q Q Q a a t A14 IL L L L L 46 6 E E Ea A7 47 w w w W W 14 1 1 M "A m 19 IFR. IMa Ic c I so 50 Js CDo F D w 51 I s I I I jI 52 1 IN I 52A 53 I p P A I- 53 I s 0 B a a a a is 56 D a a G 54 5 D Is fs !8 p 51 5 K K I_ 59 w y y I Ty Ii

I

F )A II

KR

SUBSTITUTE SHEET (RULE 26) WO 94/25067 WO 9425067PCT(US94/04935 66 TABLE 1 (3 of 4) 67K KRR

JR

71- T T s3J A H2 A,, iso~~~m bey J J vI 32 *~IIs I s s s 329 Js Is Is Ts AsI 36 _j 7s s Is Is 77 119 "2 T21 21 -9 IA A IA A IA .9 30I y ly A2 A4 s ss 19 E E E WOb 94/25067 PCTIUS94/04935 87 TABLE 1 (4 of 4) 94 :00 It x P R- 97 101 p Y r p 99 102 a_ y a I m F S a F too ICA I s D 1005 y I0 I I I K 1000 cI I I IOOE Y I F Ix Y 1000 Ix a v 10014 I I. Iv In Ia 1001 has y Y s w lowK l II Y A I o 101 D 0 I I 102 Ilog IcUi Iv v Iv v 103 :10 FR4 w w 1w 1 w 104 111 Ia a a a I lMs 112 I I o I 106 11 Ia Ia h a Ia 107 1 I IT I r__r lot II I IT IL L L 109 1116 Iv Iv Iv v I110 I Ir I Ito 117 T s I 2 119 m '.13 :0 1 4 s SUBSTITUTE SHEET (RULE 26) WO 94/25067 PCT1US9404935 TABLE 2 (1 of 4) ALIGNMENT OF AMINO ACID SEQUENCES LEADING TO 7HE DESIGN OF RESHAPED PB1.3 LIGHT CHAIN VARIABLE REGION F& or 184" h.bu H VL Cowmax ICDR I1747 1 1 v 1.1 IDEN 1747 FRI D D D D_ 2 2 I I I I 3 3 V Q Q Q Q 4 4 M M M M M ST T T T T 6 Q Q Q Q S 7 s s Is Js 8 Is I. I p 'p p Ip -t SS S 5 9 I' S I9lsss 0 loI I F s s TT Il I I M L L L L I: 12 Ii I I s 13 13 T AAA A I- A 14Is s s Is_ is isI Iv L. Iv v 16 16 0 Ic 1 I 17 17 D D_ II Is I IR IR IR R 19 9i v v v v Iv -0 Is IT IT II IT I lupponing L. I my IT IT T T 23 FRI c Ic c .4 24 I K R R K J I A A A A I 26 126 s I Is IS 1 17 I IQ IQ 0K :7A D-s Iis IwE Is I I.78 II. L I SUBSTITUTE SHEET (RULE 26)

I

WO 94/25067 PCTIUS94/04935 89 TABLE 2 (2 of 4) 21 :9 v D Is .9 A s A'S 31 T s- 31 32 N N NIS W N 32 33 V E yI L I V 34 I~ iV L L A V 34 CDORI N A

I

35 IFRQ w 1w w w Iw J6 16 Iv Iv I Iv I IQ r IQ IQQ is is1 o 7 Q QI 39 139 I: f K YK K IK Ip p P Ip 1 41 I:a aI A2 42 Q J Q K E 43 J AS S i 4 45 K 1K TK K K K6 f6A L jL L ryb L 2 L (ma y 11tar the 47 47 :IL I IL IL I $0 iso ICDPL Tr Iv A IG T- i I- 1A IA- IA IA A- 2L Is Is I s I s Is S 53 s3 y f s .I 1 4 4 IR L IL IL f IF H I. CO-, s Is Is

S

p SUBSTITUTE SHEET (RULE 23) WO 94/25067 PCT[UJS9404935 TABLE 2 (3 of 4) 0 0 5s 5 Clow ka I rwat.4 !a U.on LM may bind Ar 61 61 I R R t R 62 62 I F F F F F 63 63 Is s 64 64 I a o 165 s 66 66 a a_ 67 167 I Is s [s s I s 68 5 I a Ia Ia,; 69 69L I7 IT T T Dl 0 D E 2ay have charge inwatimac wuh rendu 24 in Ll.

11 71 F Y F F FO I Iv IFJFr 13 73 IL IL L T 7__14_ T IT T IT T_ 176 I6 T S s Is I77 I I Is Is s 78 -8 V L L L L _9 1 I7 I9 Q I 1s0 I I s 1 92 I I I a I a D- DD 183 L I F F F 4 I: IA IA A A A I T Ir T S6 S6 Y y y y 87 17 F F Y Iv '88 I Iu Ic c c I 49 9 IcRJ 'p '0 N 'I SSTT y (RULE 26) ,11 *3 V T suBSTITME. SHEET (RULE 126) WO 94/25067 WO 9425067PCT[US9410493$ TABLE 2 (4 of 4) 1'IY L L F Y 9S9s p p p p P, 96 96 y R WyY 97 97 ICDR3 T IT Tr TT 98 98 I Fv Fv F

F

99 0 a a a 100 100 G a Q 101 101 I:I o aaJ 100h2 T Ti Tr Tr Tr (73 im0 I K K 'K 1K K 104 10 IL I L IL j106 106 I I 1 1i IiI 107 J107 1 FR4 JQ K IK 'K SUBSTITUTE SHEET (RULE 26) 92 TALBE 3 'l-NCES IN AMINO ACID SEQUENCES FOR THE "11VY CHAIN VARIABLE DOMAINS FOR THE THREE RESHAPED VERSIONS OF PB1.3 CONSTRUCT AMINO ACID 2 Note: In addition, all versions contained an Arg7lSer modification to the frameworks of the human donor 2l/283'CL variable region.

*6a 4* 400 *6# (4 S:.24100A 93 TABLE 4 DIFFERENCES IN AMINO ACID SEQUENCES FOR THE LIGHT CHAIN VARIABLE DOMNINS FOR THE FOUR RESHAPED VERSIONS OF PB1.3

CONSTRUCT

AMINO ACID 1748RLA GLU ASP 1748RL 8 3 SER ASP 1748RLc GLU GLU 1748RLD SER GLU Note: In additional, all versions contained an Ile2lVa.

and a Leu46Ala modification to the frameworks of the donor DEN variable region.

0 0e*~ 6 6066 60 V* 6 66 9 0066 6 0

SO

4S 0* 0e 64 6 6 6699 66 6 960 4 9 SS 9, 66 60 06 I 66 0 St *566

I.

60 69 0I 6 *0 06 6 S:241 OOA WO 94/25067 WO 9425067PCT(US94/04935 TABLE ANTIBODY PRODUCTION RATES FOR AMPLIFIED CHO CELL LINES PRODUCING THREE DIFFERENT VERSIONS OF HUMANIZED P81.3 (CYl 748) (micrograms/I 106 cell Is/day) Anti body- Unapl~iflPJLJ.d 1it Lr.und. 2nd rounfld--.r ~tro~nd 1748A-IgGl 0.36 2.1 8.2 29.6 0.36 2.1 13.7 21.8 0.36 0.90 9.9 19.7 1748 B-IgGl 0.10 0.24 0.2 0.10 1.6 1.5 7.3 1.6 1 7488-IgG4 0.3 0.3 2.0 0.75 2.6 0.3 3.0 0.80 6.5 2.0 0.78 2.5 0.78 8.8 SUBSTITUTE SHEET (RULE 26) 23.9 21.1 18.6 17.8 26.9 23.2 20.8 20.8 19.4 18.6 17.8 WO 94/25067 PTU9/43 PCT/US94/04935 TABLE S DNA SEQUENCE OF THE PCR AMPLIMERS USED TO GENERATE HUMAN P-SELECTIN CRP DELETION MUTANTS POR 3'-amplimer to remove QRP Number: 9 8-9 7-9 6-9 5-9 4-9 3-9 2-9 5'-TTrCACAGCTCTGCATG C-3' 5'-TGCTATGCCTTTGCAGGT-3' 5'-CTTGATGGCTTCACACAT-3' 5'-GGGAATGGCTTGGCATTC-3' 5'-CTGCAAAGCTTGACAGAC-3' 5'-CGAAATAGCCTCACAGGT-3' 5'-CTG CA CAG CTTTA CAC AC-3' 5'-CTGG GCAG CTAAACACTG-3' Predicted aminoacid seguence-of mmutn CRP TAg CRAVK TlQEA LK CKGIA TIQEA LK CEAlK TIQEA LK CQAiP TIQEA LK CQALQ TlQEA LK CEAIS TlOEA LK CKAVQ TIOEA LK CLAAQ TIQEA lys-1 CCTCC CAC ACC CC CT-3' To Delete only CRP#5 these two amplimers were used: 5'-TGCACACCTTTGCTAAGC-3' 3'-amplimer 5'-CTGCAAAGCTTGACAGAC-3' SUBSTITUTE SHEEI (RULE 26) SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: CHESNUT, Robert W.

POLLEY, Margaret J.

PAULSON, James C., JONES, S. Tarran SALDANHA, Jose W.

BENDIG, Mary M.

(ii) TITLE OF INVENTION: Antibodies (iii) NUMBER OF SEQUENCES: to P-Selectin and Their Uses

OO

*0 0 S0* 0 o• 3* 0 *s 9000 0* C.

OB

o 0 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gin Asa Lys Asn Glu 1 5 10 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs ATTACHMENT B TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..417 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..417 OTHER INFORMATION: /note- "Sequence encoding CY1747(PB1.3) Heavy Chain Signal Peptide and Variable Regionn (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 000.

0 0..

000 0 0 6*0* 0000 00 00 G O *c *9*0 0 0 9* 9.

0 0 0 0 0 0000 0 0

ATG

Met 1

GTC

Val

CCT

Pro

ACC

Thr

GAG

Glu

GAG

Glu

ACA

Thr

TAT

Tyr

TGG

Trp GGA TGG AGC Gly Trp Ser CAC TCT GAG His Ser Glu GGG GCT TCA Gly Ala Ser AAC TAT GTT Asn Tyr Val TGG ATT GGA Trp Ile Gly AGG TC AAA Arg Phe Lys GCC TAC ATG Ala Tyr Met 100 TTC TGT GCA Phe Cys Ala 115 GGC GCA GGG Gly Ala Gly 130 GGG GTC TTT CTC TTT Gly Val Phe Leu Phe GCC CAG CTG CAG CAG Ala Gin Leu Gin Gin GTG AAG GTG TCC TGC Val Lys Val Ser Cys 40 ATG CAC TGG GTG AAG Met His Trp Val Lys 55 rrT ATT AAC CCA TCC Phe Ile Asn Pro Ser 70 AAC AAG GCC ACA CTG Asn Lye Ala Thr Leu GAG CTC AGC AGC CTG Glu Leu Ser Ser Leu 105 AGA GCC CGC CCG GGG Arg Ala Arg Pro Gly 120 ACC ACG GTC ACC GTC Thr Thr Val Thr Val 135 CTC CTG TCA GGA ACT Leu Leu Ser Gly Thr

GGA

Gly

GCT

Ala

AAG

Lys

CAT

Asp

TCA

Ser

TCT

Ser

GAC

Asp

TCA

Ser

OCT

Pro

TCT

Ser

CCT

Pro

GGT

Gly

GAC

Asp

GAG

Glu

TGG

Trp

GAG

Glu

GGA

Gly

GGG

Gly

OCT

Pro

AAA

Lys

GTC

Val

TAO

125 96 144 192 240 GCA GGT Ala Gly INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid TOPOLOGY: iinr2r (ii) MOLECULE TYPE: protein 98 DESCRIPTION: SEQ ID NO:3: (Xii SEQUENCE Met Gly Trp Ser 1.

Val Pro Thr Glu Giu Thr Tyr Trp His Ser Giu Gly Ala Ser Asn Tyr Val Trp Ile Gly Arg Phe Lys Ala Tyr Met 100 Phe Cys Ala 115 Gly Ala Gly 130 Giy 5 Ala Val Met Phe Asn Giu Arg Thr Val Phe Leu Phe Leu Leu Ser Gly Thr Leu Val Trp 55 Asn Ala Ser Arg Val 135 1.0 Ser Gly Lys Ala Gin Lys Asn Asp Thr Ser 90 Thr Ser Phe Asp Ser Ser Pro Ser Pro Gly Asp Glu Trp Giu Gly Gly Pro Lys Val Tyr 125 Leu Tyr Gin Lys Ser Ser 110 Phe Aia is Val Thr Gly Tyr Ser Ala

ASP

V.

*0 of a. o 0 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 393 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: ODS LOCATION: 1...393 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1...393 OTHER INFORMATION: /note. "Sequence encoding CY1747(PBl.3) Light Chain Signal Peptide and Variable Region"~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATG GGC ATC AAG ATG GAG TCA CAG ATT CAG GTC 'iTT GTA TAC ATO TTG Met Gly Ile Lys Met Glu Ser Gin Ile Gin Val Phe Val Tyr Met Leu 1. 5 10 CTO TGG TTG TOT GGT OTT OAT GGA GAC ATT GTG ATG ACC CAG TOT CIA Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gin Ser Gin 2S AAA TTC ATO TCC ACA TCA GTA GGA GAO AGG GTC AGC GTC ACC TOO AAO Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val 5cr Val Thr Cys Lys 40 GCC AGT CAG AAT GTG GCT ACT AAT GTA GTC TGG TAT CAA CAG AGA CCA 192 Ala Ser Gin Asn Val Ala Thr Aen Val Val Trp Tyr Gin Gin Arg Pro 55 GGA CAA TCT CCT AAA GCG CTT ATI TAT ACG GCA TCC TAC CGG TIC AGT 240 Gly Gin Ser Pro Lys Ala Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser 70 75 GGA GTC CCT GaA CGC TTC TCA GGC AGT GGA TCT GGG ACA GAT T'TC ACT 288 Gly Val Pro Glu Arg Phe Ser Giy Ser Gly Ser Gly Thr Asp Phe Thr 90 CTC ACC ATC ACC AAT GTG CAG TCT GAA GAC TIG GCA GAO TAT TTC TOT 336 Leu Thr Ile Thr Asn Val Gin Ser Giu Asp Leu Ala Asp Tyr Phe Cys 100 105 lin CAA CAA TAT AAC AAC TAT CCC TAC ACG TTC GGA GGG GGG ACC AAG GTG 384 ,Gin Gin Tyr Asn Asn Tyr Pro Tyr Thr Phe Giy Giy Gly Thr Lye Val 115 120 125 GAA ATT CAA 393 Glu Ile Gin 130 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 131 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Ile Lys Met Glu Ser Gin Ile Gin Val Phe Val Tyr Met Leu 1 5 10 Leu Trp Leu Ser Gly Val Asp Giy Asp Ile Val Met Thr Gin Ser Gin 25 Phe Met Ser Thr Ser Val Gly Asp Arg Val 5cr VaL Thr Cys Lys 40 Ala ser Gin Asn Val Ala Thr Asn Val Val Trp Tyr Gin Gin Arg Pro 55 Gly Gin Ser Pro Lys Ala Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser 65 70 75 Gly Val Pro Glu Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 90 Leu Thr Ile Thr Asn Val Gin Ser Giu Asp Leu Ala Asp Tyr Phe Cys 100 105 110 Gin Gin Tyr Aen Asn Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val 115 120 12S Glu Ile Gin 130 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .417 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..417 OTHER INFO MTION: /note. "Sequence encoding CY1748 (PBl.3-Humanized) Heavy Chain-A. Signal Peptide and Variable RegionO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATO GAG T GGG CTG AGC TGG CTT TITr CTT GTG GCT ATT ?1rA AAA Met Giu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu cc,.

e.

C.

C C CC A cc..

C C

C

cc..

C

C S

S.

A

C C 9*A#

CC

C

o so,

C

cci.

C.

C c

C.

ec

C

C. A

S.

CCC.

*0 C CC .9 CC C A 0 *9 9 9 1

GTC

Val

CCT

Pro

ACC

Thr

GAG

Giu 65

GAG

Glu

ACC

Thr

TAT

Tyr

TGG

Trp,

CAG

Gin

GG

Gly

AAC

Asn 50

TGG

Trp

AGG

Arg

GCC

Ala

TAC

Tyr

OGA

Gly 130

TOT

Cys

OCT

Ala 35

TAT

Tyr

ATG

Met

TTC

Phe

TAC

Tyr

TOT

Cys 115

CAG

Gin GGA, OCT Oly Ala OCT TCT Ala Ser GCT CCC Ala Pro GAT GOT Asp Gly 75 TCA GAC Ser Asp TCC GAG Ser Glu GAC TG Asp Trp

TCA

GAG

Glu

GGA

Gly

GG

Gly

CT

Pro

ACA

Thr

GAO

Asp

TAO

Tyr 125 Lys is

AAA

Lys

ACA

Thr

AGG

Arg

TAC

Tyr

GCC

Ala

GCA

Ala

GAT

Asp

GGT

Gly

AAG

Lys

TTC

Phe Leu

AAT

Asn

AGC

GTC

Val

GTC

Val 96 144 192 240 288 336 384 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid TOPOLOGY: 'linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Glu Phe Gly Leu Gin Cys Glu Ala Gly Ala Ser Val Asn Tyr Val Met Trp Met Gly Phe Arg Phe Lys Asn Ala Tyr Met Glu 100 Tyr Cys Ala Arg 115 Gly Gln Gly Thr 130 101 DESCRIPTION: SEQ ID Ser Trp Leu Phe Leu Gin Leu Val Gin Ser Lys Val Ser Cys Lys 40 His Trp Val Arg Gin 55 Ile Asn Pro Ser Asn Arg Val Thr Ile Thr Leu Ser Ser Leu Arg 105 Ala Arg Pro Gly Phe 120 Leu Val Thr Val Ser 135 NO:7: Val Ala Gly Ala Ala Ser Ala Pro Asp Gly Ser Asp Ser Glu Asp Trp Ser Ile Glu Gly Gly Pro Thr Asp Tyr 125 669A ede a 4 ee 0 0

A.

e A S r a INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATORE: NAME/KEY: CDS LOCATION: 1..417 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..417 OTHER INFORMATION: /note- "Sequence encoding CY1748 (PB1.3-Humanized) Heavy Chain-B Signal Peptide and Variable Region"

ATG

Met 1

GTC

Val

CCT

Pro (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAG TTT GGG CTG AGC TGG CTT TTT CTT GTG GCT ATT TTA AAA GGT Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 5 10 CAG TGT CAG GTC CAG CTG GTG CAG TCT GGA GCT GAG GTG AAA AAG Gln Cys Gin Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 25 GGG G CT TCA GTG AAG GTG TCC TGC AAG GCT TCT GGA TAC ACA TTC Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 40

ACC

Thr

GAG

Glu

GAG

Giu

ACC

Thr

TAT

Tyr

TGG

Trp CAC TGG His Trp ATi' AAC Ile Aen 70 AGG GTC Arg Val CTG TCC Leu Ser GCA CC Ala Arg Crr GTC Leu Val 135

GTG

Val

CCA

Pro

ACA

Thr

AGC

5cr

CCG

Pro 120

ACC

Thr 102 CGC CAA GCT Arg Gin Ala TCC AAT GAT Ser Asn Asp 75 ATC ACT TCA le Thr Ser CTG CGC TCC Leu Arg Ser 105 GGG rrC GAC Giy Phe As;.

GTC AGT TCA Val Ser Ser

CCC

Pro

GGT

Gly

GAC

Asp

GAG

Glu

TGG

Trp CAG AGG CTT Gin Arg Leu AAG TAC AAT Lys Tyr Asn so TCC GCC AGC Ser Ala Ser ACT GCA GTC Th2r Ala Vai 110 TTC GAT GTC Phe Asp Val INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARCTERISTICS: LWNGTH: 139 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein *999 .9 9 9 9, 9

S

S S .9

S.

9.

9 5 9. 9 .9 9 099

S

9.

59

S.

9, *9 5 99 9 *9 .9.9 *9 99 .9 59 9 9.

9* 9 met Val Pro Thr Giu 65 Giu Thr Tyr Trp (xi) SEQUENCE Giu Phe Gly Leu 5 Gin Cys Gin Val 20 Gly Ala Ser Val.

35 Aen Tyr Val Met so Trp Met Giy Phe Arg Phe Lys Asn 85 Ala Tyr Met Giu 100 Tyr Cys Ala Arg 115 Gly Gin Gly Thr 130 DESCRIPTION: SEQ ID Ser Trp Leu Phe Leu Gin Leu Vai Gi1n Ser Lys Val 5cr Cys Lys 40 His Trp Val Arg Gin 55 Ile Asn Pro Ser Asn 70 Arg Vai Thr :le Thr Leu Ser Ser Leu Arg 105 Ala Arg Pro Gly Phe 120 Leu Val Thr Val 5cr 13S NO.-9: Val Ala Gly Ala Ala 5cr Ala Pro Asp Gly Ser Asp Ser Glu Asp Trp Ser Ile Glu Gly Gly pro Thr

ASP

Tyr 125 INFORMATION FOR SEQ ID NOtl0: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs %l91u TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (Ui) MOLECULE TYPE: cONA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .417 (iX) FEATURE: NAME/KEY: misc feature LOCATION: 1. .417 O7rIER INFORMATION: /note= "Sequence encoding CY1748 (PB1.3-Humanized) Heavy Chain-C Signal Peptide and Variable Region* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0: ATG GAG 'rrr GGG CTG Met Clu Phe Gly ft. ft.

ft ft..

ft.

ft ft ft ft. ft ft. ft.

ft ft ft ft ft...

ft...

ft ft.

ft ft ft ft.

ft.

ft ft ft ft ft ft Oftftft ft.

ft.

ft oft.

ft ft...

ft ft.

ft ft ft ft.

ft ft.

ft. ft ft. ft ft ft.

ft...

ft ft.

ft ft ft.

I

GTC

Val

CCT

Pro

ACC

Thr

GAG

Giu 65

GAG

Glu

ACC

Thr

TAT

Tyr

TG

Trp

CAG

Gin

CG

Gly

AAC

Asn 50

TG

Trp

AGG

Arg

CC

Ala

TAC

CGA

Gly 130

GAG

Glu 20

TCA

Ser

GTT

Val

GGA

Gly

AAA

Lys

ATC

met 100

CC

Ala

GT

Gly Leu 5

CC

Ala

GTG

Val

ATC

Met Phe

AAC

Asn 85

GAA

Glu

AGA

Arg

ACC

Thr

CRC

Gln

AAG

Lys

CAC

His

AT

Ile 70

AGO

Arg

CTC

Leu

CCA

Ala CTr Lou

CTC

Lou

GTG

Val

TG

Trp 55

AAC

Aen

GTC

Val

TCC

Ser

CC

Arg

GTC

Val.

13S GTC CRC Val Gin 25 TCC TOC Ser Cys 40 GTG CC Val Arg CCA TCC Pro Ser ACA ATC Thr Ile AGC CTG Ser Leu 105 CCC COO Pro Cly 120 ACC GTC Thr Val 10

TCT

Ser

AAG

Lys

CAA

Gin

AAT

Asn

ACT

Thr 90

CC

Arg

TTC

Phe

AGT

Ser GGA GCT Cly Ala GCT TCT Ala Ser GCT CCC Ala Pro GAT GOT Asp Cly TCA GAC Ser Asp TCC GAG Ser Glu GAC TGO Asp Trp

TCA

Ser

GAG

Clu

GGA

Gly

CG

Gly

CCT

Pro

A

Lye

GCC

Asp

TAC

12S AUC TCG CT TID CTT GMG GCT Arr Ser Trp Lou Phe Lou Val. Ala Ile TTA AMA Lou Lys CTC AMA Val Lys TAC ACA Tyr Thr CRC AGO Gin Arg AAC TAC Lys Tyr TCC CC Ser Ala ACT GCA Thr Ala 110 TTC CAT Phe Asp INFORMATION FOR SEQ ID NO:il: SEQUENCE CHtARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein

RAI

met Val Pro Thr Glu Giu Thr Tyr TrP (xi) SEQUENCE Glu Phe Gly Leu Gin Cys Giu Ala Gly Ala Ser Val Asn Tyr Val Met Trp Meat Gly Phe Arg Phe Lys Asn Ala Tyr Met Glu 100 Tyr Cys Ala Arg 115 Gly Gin Gly Thr 130 104 DESCRIPTION: SEQ ID Ser Trp Leu Phe Leu Gin Leu Val Gin Ser 25 Lye Val Ser Cys Lys 40 His Trp Val Arg Gin 55 Ile Aan Pro Ser Asn 70 Arg Val. Thr Ile Thr Leu Ser 5cr Leu Arg 105 Ala Arg Pro Gly Phe 120 Leu Val. Thr Val Ser 135 NO: 11: Val Ala Gly Ala Ala Ser Ala Pro ASP Gly 75 Ser Asp Ser Glu Asp Trp Ser Ile Leu Glu Val Gly Tyr Gly Gin Pro Lye Lye Ser Asp Thr 110 Tyr Phe 125 Gly Lys Phe Leu Asn Ser Val Val a ha..

a.

a a *a a *a.a a a a.

a.

S.

a.

a. a a. a a.

a.

a..

a a aa a a a Oa a a.

a a. a a a.

a a.

a Ia a. a a ae a a INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 378 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAM/KEY: CDS LOCATION: 1. .378 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .3"78 OTHER INFORMATION: /note- "Sequence encoding CY1748 (P11.3-Humanized) Light Chain-A Signal Peptide and Variable Region"

ATG

met

I

GTC

Val

AGC

Sker (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 5 10 CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA TCC ACC CTG TCT GCA His Ser Asp Ile Gin Met Thr Gin Ser Pro 5cr Thr Leu 5cr Ala 25 GTA GGA GAC AGA GTC ACC GTC ACC TOC AAG GCC AGT CAG AAT GTG Val Gly ASP Arg Val Thr Val Thr Cys Lye Ala Ser Gin Asn Val 40 C -1 I 105 GCT ACT AAT GTA GTC TGG TAT CAA CAG AAA Ala Thr Asn Val Val Trp Tyr Gin Gin Lye I 55 GCG CTr ATT TAT ACG GCA TCC TAC CGG TTC J Ala Leu Ile Tyr Thr Ala Ser Tyr Arg Phe 70 TTC TCA GGC AGT GGA TCT GGG ACh GAT TTC I Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 90 CTG CAG TCT GAT GAC TT GCA ACT TAT TAC Leu Gin Ser Asp Asp Phe Ala Thr Tyr Ty'r 100 105 TAC CCA TAC ACG TrC GGC CAA GG ACC AAG I Tyr Pro Tyr Thr Phe Giy Gin Gly Thr Lye i 115 120 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear

GGA

Gly

GGA

Gly

CTC

Leu

CA

Gin

GAA

Glu CCT AAA Pro Lye GAA CGC Glu Arg AGC AGT Ser Ser AAC AAT Asn Ann (ii) MOLECULE TYPE: protein n e

C

0*0.

0 0 0* 00 *0a 0e

C

*00 0a 00 0*a 00

S.

*0a

C

0 0 0 0000 00 0* *0a 0 (xi) SEQUENCE Met Gly Trp Ser Cys 1 5 Val His Ser Asp Ile Ser Val MUy Asp Arg Ala Thr Asn Val Val 50 Ala Leu lie Tyr Thr Phe Ser Gly Ser Gly 85 Leu Gin Ser Asp ",sp 100 Tyr Pro Tyr Thr Phe 115 DESCRIPTION: SEQ ID Ile Ile Leu Phe Leu 10 Gin Met Thr Gin Ser 25 Val Thr Val Thr Cys 40 Trp Tyr Gin Gin Lys 55 Ala Ser Tyr Arg Phe 70 Ser Gly Thr Asp Phe 90 Phe Ala Thr Tyr Tyr 105 Gly Gin Gly Thr Lys 120 NO:13: Val Ala Pro Ser Lye Ala Pro Gly Ser Gly 75 Thr Leu Cys Gin Leu Glu Thr Ala Thr Leu Ser Gln Giu Ala Val Pro Thr Ile Gin Tyr 110 Ie Lys 125 Thr Ser Asn Pro Glu Ser Asn Gly Ala Val Lys A~g Ser Asn INFORMATION FORSEQ ID NO:14: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 378 base pairs TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

-I

(ix) FEATURE: NAME/KEY: CDS LOCATION: 1..378 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..378 OTHER INFORMATION: /note- "Sequence encoding CY1748 (PBl.3-Humanized) Light Chain-B Signal Peptide and Variable Region" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATG GGA TGG AGC TGT ATC ATC CTC TrC ITG GTA GCA ACA Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr 1

GTC

Val

AGC

Ser

GCT

Ala

GCG

Ala

TTC

Phe

CTG

Leu

TAC

Tyr

TCC

Ser

GGA

Gly

AAT

Aen ATr Ile

GGC

Gly

TCT

Ser

TAC

Tyr 115 10 AGC CCA Ser Pro TGC AAG Cys Lys AAA CCA Lys Pro TrC AGT Phe Ser 75 TTC ACT Phe Thr 90 TAC TGT Tyr Cys AAG CTC Lys Leu

ACC

Thr

AGT

Ser

GAG

Glu

GTC

Val

ACC

Thr

CAG

Gin

ATC

lie 125 GCT ACA GGT Ala Thr Gly CTG '1C4T oCA Leu Ser Ala CAG AAT GTG Gin An Val GCT CCT AAA Ala Pro Lys CT TCA CGC Pro Ser Arg ATC AGC AGT Ile Ser Ser TAT AAC AAT Tyr Asn Asn 110

AAA

Lys 4 44.

*r 4 44 44 .4, .4 44 4 4. 4 4..

4.

4.

44 4r .4.

4 .4r 4 44 4.~ 4 44r 44 4 .4 4 *o 4 44 4 .4 .4 4 4 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 5 10 His Ser Asp Ile Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala 25 Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gin Asn Val 40 Ala Ala Phe Leu Tyr (2) 1 107 Thr Asn Val Val Trp Tyr Gin Gin LYS Pro Gly Glu Ala Pro Lys 55 Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg 70 75 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 90 Gin Ser Asp Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Asn 100 1.05 110 Pro Tyr Thr Phe 'Mly Gin Gly Thr Lys Leu Giu Ile Lys 115 120 125 INFORMATION FOR SEQ ID NO.:i6: SEQUENCE CHARACTERISTICS: LENGTH: 378 base pairs TYPE: nucleic acid STRAUDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .478 (ix) FEATURE: NAMEKEY: misc feature LOCATION: 1. .378 OTHER INFORMATION: /notem "Sequence encoding CY1748(PBi.3-Hwnanized) Light Chain-C Signal Peptide and Variable Region" a *4 a a S* a a a 9 a a a a.

a.

a.

a. a a. a a.

9a a a..

0**a a.

a.

a.

a.

a. a a a.

aa a a.

a. a a.

SO

a

ATG

Met 1

GTC

Val

AGC

Ser

OCT

Ala

C

Ala 65

TTC

Phe (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iE: OGA TGO AGC TOT ATC ATC CTC TrC TTG GTA Gly Trp Ser Cys Ile Ile Leu Phe Leu Val 5 10 CAC TCC GAC ATC ChO ATG ACC CAG AGC CCA His Ser Asp Ile Gin Met Thr Gin Ser Pro 25 GTA OGA GAC AGA GTC ACC GTC ACC TOC AAG Val Gly Asp Arg Val Thr Val Thr Cys Lys 35 40 ACT AAT GTA GTC TGG TAT CAA CAG AAA CCA Thr Asn Val Val Trp Tyr Gin Gin Lys Pro CTI' AT' TAT ACO GCA TCC TAC COG T1'C AGT Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser 70 TCA GGC AOT GGA TCT COG ACA GAA TI'C ACT Ser Gly Ser Cly Ser Gly Thr Glu Phe Thr 90 ACh OCT ACA Thr Ala Thr ACC CTG TCT Thr Leu Ser AGT CAG AAT Ser Gin Asn GAG OCT CCT Giu Ala Pro GTC CCT OAA Val Pro Glu ACC ATC AC Thr Ile Ser L 108 CTG CAG TCT GAT GAC TTT GCA ACT TAT TAC TGT CAA CAG TAT AAC AAT 336 Leu Gln Ser Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gin Tyr Asn Asn 100 105 110 TAC CCA TAC ACG TTC GGC CAA GGG ACC AAG CTC GAA ATC AAA 378 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala 25 Ser Val Gly Asp Arg Val Thr Val Thr Cys Lys Ala Ser Gln Asn Val 40 Ala Thr Asn Val Val Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys 55 Ala Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser Gly Val Pro Glu Arg 70 75 Phe Ser Gly Ser G y Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser 85 90 4 Leu Gln Ser Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn 4* 4100 105 110 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 378 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..378 (ix) FEATURE: NAME/KEY: misc feature LOCATION 1..378 OTHER INFORMATION: /note- "CY1748 (PB1.3-Humanized) Light Chain-D Signal Peptide and Variable Region" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATO GGA Met Gly TGG AGC TGT ATC ATC Trp Ser Cys Ile Ile 109 CTC TTC TTG Leu Phe Leu 10 GTA GCA ACA GCT Val Ala Thr Ala 1 5

GTC

Val

AGC

Ser

GCT

Ala

GCG

Ala

TTC

Phe

CTG

Leu

TAC

Tyr

CAC

His

GTA

Val

ACT

Thr

CTT

Leu

TCA

Ser

CAG

Gin

CCA

Pro TCC GAC Ser Asp GGA GAC Giy Asp AAT GTA Aen Val ATT TAT Ile Tyr GGC AGT Gly Ser TCT GAT Ser Asp 100 TAC ACG Tyr Thr 115

ATC

Ile

AGA

Arg

GTC

Val

ACG

Thr

GGA

Gly

GAC

Asp TrC Phe ATG ACC CAG Met Thr Gin 25 ACC GTC ACC Thr Val Thr 40 TAT CAA CAG Tyr Gin Gin 55 TCC TAC CGG Ser Tyr Arg GGG ACA GMA Gly Thr Glu GCA ACT TAT Ala Thr Tyr 105 CAA GGG ACC Gin Gly Thr 120

CCA

Pro

AAG

Lys

CCA

Pro

AGT

Ser 75

ACT

Thr

TGT

Cys

CTC

Leu

TCC

Ser

GCC

Ala

GGA

Gly

GGA

Gly crC Leu

CAA

Gin

GAP.

Glu

ACC

Thr

AGT

Ser

GAG

Glu

GTC

Val

ACC

Thr

CAG

Gin

ATC

Ile 125

CTG

Leu

CAG

Gin

GCT

Ala

CCT

Pro

ATC

Ile

TAT

Tyr 110

AAA

Lye ACA GGT Thr Gly TCT GCA Ser Ala T GTG Asn Val CCT AAA Pro Lye TCA CGC Ser Arg AGC AGT Ser Ser AAC AAT Asn Asn INFORMATION FOR SEQ ID NO:19: too* a. a a a *0 0 0 SS 0 *000 S ol *r S *5S* a.

400 S a a. a 6000

S

o a s a.r a a Met Val Ser Ala Ala 65 Phe Leu Tyr SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: proteia (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i9: Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala 10 His Ser Asp Ile Gin Met Thr Gin Ser Pro Ser 20 25 Val Gly Asp Arg Vai Thr Val Thr Cys Lye Ala 35 40 Thr Aen Val Val Trp Tyr Gin Gin Lye Pro Gly 55 Leu Ile Tyr Thr Ala Ser Tyr Arg Phe Ser Gly 70 75 Ser Gly Ser Gly Ser Gly Thr Giu Phe Thr Leu 90 Gin Ser Asp Asp Phe Ala Thr Tyr Tyr Cys Gin 100 105 Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu 115 120 Thr Thr Ser Glu Val Thr Gin Ile 125 Ala Thr Gly is Leu Ser Ala Gin Aen Val Ala Pro Lys Pro Ser Arg Ile Ser Ser Tyr Asn Asn 110 Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 62 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Gin Asp Leu Pro Val Pro Aen Giu Ala Arg Val Aeui Cys Ser His to1 5 10 Pro Phe Giy Ala Phe Arg Tyr Gin Ser Val Cys Ser Phe Thr Cys Asn 25 Giu Gly Leu Leu Leu Val Gly Ala Ser Val Leu Gin Cys Leu Ala Thr 40 Gly Aen Trp Asn Ser Val Pro Pro Glu Cys Gin Ala Ile Pro s 55 INFORMATION~ FOR SEQ ID NO:2i: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid STR~ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 0:60 (ix) FEAT1URE: too: NAME/KBY: Protein LOCATION: 1.-128 OTHER INFORMATION: /label. MOUSE h1A (xi SEQUENCE DESCRIPTION: SEQ ID NO:21: Giu Val Gin Leu Gin Gin Ser Gly Pro Giu Leu Val Lys Pro Gly Ala 1 510 1 Ser Val Lye Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 25 Tyr Met Asn Trp Val Lys Gin Ser Pro Gly Lye Ser Leu Giu Trp Ile 40 Gly Asp Ile Asn Pro Gly Asn Gly Gly Thr Ser Tyr Asn Gin Lye Phe s0 55 to Lys Gly Lys Ala Thr Leu Thr Val Asp Lye Ser Ser Ser Thr Ala Tyr 70 75 Met Gin Leu Ser Ser Leu Thr Ser Giu Asp Ser Ala Val Tyr Tyr CYs 90 Ala Arg Gly Xaa Tyr Tyr Ser Ser Ser Tyr Met Xaa Ala Xaa Xaa Tyr 100 105 110 Tyr Ala Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:22: Wi SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 128 OTHER INFORMATION: /label- HUMAN. I (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Gin Val Gin Leu Val Gin Ser Gly Ala Giu Val Lys Lys Pro Gly Ala 1. 5 10 is Ser Val Lys Val Ser Cys Lys Ala Ser Giy Tyr Thr Phe Thr Ser Tyr 25 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 40 Gly Trp Ile Asn Pro Tyr Gly Asn Gly Asp Thr Ann Tyr Ala Gin Lys 55 Phe Gin Gly Arg Val 'rhr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala 70 75 Tyr Met Giu Leu Ser Ser Leu Arg Ser Giu Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Ala Pro Gly Tyr Gly Ser Gly Gly Gly Cys Tyr Arg Giy 100 105 110 Asp Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: TYPE: amino acid STRANDEDNESS:- single 6. TOPOLOGY: linear 4 S (ii) MOLECULE TYPE: protein (ix) FEATURE NAME/KEY: Protein LOCATION: 1..12i OTHER INFORMATION: /label. HUMAN2i/28'CL (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 112 Pro Cys Ala Val Gin Leu Val Gin Ser Gly Ala Giu Val Lys Lys Pro 1 5 10 Gly Ala Ser Val Lye Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 25 Ser Tyr Ala Met His Trp Val Arg Gin Ala Pro Gly Gin Arg Leu Giu 40 TrP Met Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Thr Ser Gin so 55 Lys Phe Gin Gly Arg Val Thr Ile Thr Axg Asp Thr Ser Ala Ser Thr 70 75 Ala Tyr Met Giu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 90 Tyr Cys Ala Arg Gly Gly Tyr Tyr Gly Ser (fly Ser Asn Tyr Trp Gly 100 105 110 Gin Gly Thrc Leu Val Thr Val Ser Ser 115 120 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1. .108 OTHER INFORMATION: /iabeiw mouse kappa-V (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Asp Ile Gin met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 555 1 5 10 is Asp Arg Val Thr Ile Thr Cys Arg Ala Ser GIn Asp Asp Ile Ser Aen 25 Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu 40*4 I le Tyr Tyr Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser so 55 Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu 70 75 Gin Giu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro 85 90 o of Arg Thr Phe Gly Gly Gly Thr Lye Leu Giu Ile Lys 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 114 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1.-114 OTHER INFORMATION: /labelv, human-kappa_1 (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Len Ser Ala Ser Val Gly 1 5 10 Asp Arg Val Thr Ilie Thr Cys Arg Ala Ser Gin Ser Leu Val Xaa Xaa 25 Ser Ile Ser Aen Tyr Len Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala 40 Pro Lys Len Len Ile Tyr Ala Ala Ser 5cr Len Glu 5cr Gly Val Pro so 55 Arg Phe 5cr Giy 5cr Gly 5cr Gly Thr Asp Phe Thr Leu Thr Ile 70 75 Ser Ser Leu Gin Pro Giu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr 90 Asn 5cr Leu Pro Gin Ie Trp Thr Phe Gly Gin Gly Thr Lye Val Gin 100 105 110 SIle Lye see: INFORMATION FOR SEQ ID NO:26: 0 *go (i SEQUENCE CHARACTERISTICS: 000:. LENGTH: 106 amino acids 04.06 0(B) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein fix) FEATURE: NAME/KEY: Protein LOCATION: 1..106 OTHER INFORMATION: /label. human-DEN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: eggs Asp Ile Gin Met Thr Gin Ser Pro 5cr Thr Leu Ser Ala 5cr Val Gly 1 S 10 Aip Arg Val Thr Ile Thr Cys Arg Thr Ser Gin cr Ile Ser Arg Trp, 25 Len Ala Trp Tyr Gin J~ln Lys Pro Gly Glu Ala Pro Lye Leu Le Ile 40 114 Tyr Gly Ala Ser Asn Leu Giu Ser Gly Val Pro Arg Phe Ser Gly Ser 55 Gly Ser Gly Thr Giu Phe Thr Leu Thr Ile Ser Ser Leu Gin Ser Asp 70 75 Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Asp Ser Phe Pro Tyr Thr 90 Phe Gly Gin Gly Thr Lye LeU Gu Ile Lys 100 105 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH,- 18 base pairs TYPE: nucleic acid STRANDEDNESS:- single TOPOLOGY: linear 'i)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TI'TCACAGTC CTGCATGC 1 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRAMDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:, DNA (genomic) *000 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: :TGCTATGCCT T1'GCAGGT 18 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs O TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear to (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CCTGATGGCT TCACACAT 1 INFORMATION FOR SEQ ID 0: SEQUENCE CHA.RACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAATGGCT TGGCATTC 18 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid (C)-STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CTGCAAAGCT TGACAGAC 18 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STR.ANDEDNESS:- single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CGAAATAGCC TCACAGGT 18 INFORMATION FOR SEQ ID 211O033: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid 00:0 STRANDEDNESS: single 0 TOPOLOGY: linear (ii) MOLECULE TYPE, DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:.33: :::CTGCACAGCT TTACACAC 1 0(2) INFORMATION FOR SEQ ID NO:34:# i)SEQUENCE~ CHARACTERISTICS: 7: LENGTH: 18 base pairs TYPE: nucleic acid 04 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:- DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CTGGGCAGCT AAACACTG 18 INFORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENGTH: 18 baqe pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE. DNA (genomic) (Xci) SEQUENCE DESCRIPTION: SEQ ID AAACCTCCCA CACCCCCT is INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: IS base pairs TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TGCACACCTT TGCTAAGC 18 INFORMATION FOR SEQ ID NO:37: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 18 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37t CTGCAAAGCT TGACAGAC 1 INFORMATION FOR SEQ ID NO:08: Ci) SEQUENCE CHARACTERISTICS, LENGTH: 12 amino acids TYPE: amino acid to CC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Cys Arg Ala Val Lys Thr Ile Gin Giu Ala Leu Lys 1 5 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Cys Lys Gly Ile Ala Thr Ile Gin Giu Ala Leu Lys 1 510 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Cya Giu Ala Ile Lys Thr Ile Gin Glu Ala Leu Lys 1 5 INFORMATION FOR SEQ ID NO:41: ()SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS:- single TOPOLOGY: linear *set0 (ii) MOLECULE TYPE: protein (x)SQENEDSRPTO:SQ DN:1 *y Gl l0l*roTrIeGnGl l e y (xi) SEQUENCEION DESCRIPI O: SQ D O INFORM TOOY SEQinar (i TYPCLETE: amiotci (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

I

Cys Gin Ala Leu Gin Thr Ile Gin Glu Ala Leu Lys 1 5 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Cys Glu Ala Ile Ser Thr Ile Gin Giu Ala Leu Lys 1 5 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acidr, TYPE: amino acid STRANDEDNESS: singlft TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION- SEQ ID NO:44: Cys Lye Ala Val Gin Thr Ile Gin Giu Ala Leu Lys 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGT: 11 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID V. Cys Leu Ala Ala Gin Thr 1ie Gin Glu Ala Len *1 S.

Claims (3)

1. 2. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 2 3 1 2 3 1 2 2 1 1. A humanized immunoglobuliri comprising a 2 1iumanit'ei heavy chain and a humanized light chain* 3 (1 the humanized light chain comprising three 4 complementarity determining regions (CDRI, CDR2 and COR3) having amino acid sequences from the corresponding 6 complementarity determining regions of a mouse PBI.3 S7 immunoglobulin light chain, and a variable region framework 8 from a human kappa light chain variable region framework 9 sequence; and the humanized heavy chain cow-rising three
2. 12. complementarity determining regions (CDRl,0 CDR2 and CDR3) 12 having amino acid sequences from the corresponding 13 complementarity determining regions of t. mouse FBI. 3 *sea 14 immunoglobulin heavy chain,, and a variable region framework to:. 15 from a human heavy chain variable region framework sequence; 16 wherein the humanized immunoglobulin specifically 17 binds to P-selectin with a binding affinity having a lower 18 limit of about 107 W1 and an upper limit of about five-times 19 the binding atfinity of the mouse PBI. 3 immunglobulin. 1 2. The hummnize~t immunoglobulin of claim 1, 2 wherein: 3 the human kappa light ahain variable region 4 (AQ4 s framework sequence is substituted in at least one position I F- 120 selected from a r1rst group consisting of L21, L46, L60 and by an amino acid present in the equivalent position of the mouse PB1.3 immunoglobulin light chain variable region framework sequence; and the human heavy chain variable region framework sequence is substituted in at least one position selected from a second group consisting of HI, H2, H71 and H73, by an amino acid present in the equivalent position of the mouse PB1.3 heavy chain variable region framework sequence: The humanized immunoglobulin of claim 2, wherein- the humanized heavy chain variable region framework is a 21/28'CL heavy chain variable recrion framework sequence. 0* 4 S S 4. S. 4 *4*S S. S 4 I 4*S* 4 40 *0 *i 0 S S 4 4. 4 4. wherein the a DEN light wherein 'the substituted 6. whereih -the substituted The humanized immunoglobulin of claim 3, humanized light chain variable region framework is chain variable region framework sequence. The humanized immunoglobulin of claim 4, humanized light chain variable region framework is in at least two positions from the first group. The humanized immunoglobulin of claim humanized heavy chain variable region framework is in at least two positions fro the second group. 7. The humanized immunoglobulin of claim 1, whereia the humanized light chain comprises the 1748RLA mature light chain variable reion amino acid sequence shown in Figure 14 (SEQ. ID. No. 13). 8. The humanized immunoglobulin of claim 1 wherein-the humanized light chain comprises the 1748RLB mature light chain variable region anino acid sequence shown in Figure 15 (SEQ. ID. No. 121 1 9-The humanized immunoglobulin of claim 1, 2 where~i the- numanized light chain comprises the 1748RLC mature 3 light chain variable region amino acid sequence shown in 4 Figure 16 (SEQ. ID. NO. IL7),* 1 1.The humanized izmunoglobulin Of plaim 1, 2 wherein ttm numanized light chain comprises the 1748RLD mature 3 light chain variable region amino acid sequence shown in 4 Figure 17 (SEQ. ID. No. 19). 1. 11 The humanized immunoglobulin Of claim 1, 2 wherein. tb~a humanized heavy chain comprises the 1748RH{A mature 3 heavy chain variable region amino acid sequence shown in 4 Figure 13. (SEQ. ID. No. 1 12. The humanized immunoglobulin of 'claim 1, 2 whereift the numanized heavy chain comprises the 1748RH{B heavy 3 chain variable region aznino acid sequence shown in Figure 12 4 (SEQ. ID. No. 9) boo13. The humanized immunoglobulin Of claim 1, 2 whereiI3 the humanized heavy chain comprises the 174S1Ut mature 60004 3 heavy chain variable region amino acid sequence shown in 4 Figure 13 (SEQ. ID. No. 11). 1 hri 14. The humanized immunoglobulin of claim 2 hre* th~e numanized heavy chain comprises the 174ftIIf mature 3 heavy chain variable region amino acid sequence shown in 4 Figure 12 (SEQ. ID. No.9). b3.o 159. The humanized immunoglobu3.in Of claim 1, boo, 2 is a PaU, tab', F(ab') 2 rahc or Fv fragment. 3. 16. The humanized iimmnoglobulin Of claim 14, 2 furthe~r comprising a constant region domain.
3. 17. The humanized immunoglobulin of claim 16, frP I' whereih~ tivhe constant region domain has .an effector function. 122 1 1. The humanized immunoglobulin of claim 14 'ii) 2 wherein the constant region domain lacks an effector function. 1 19. The humanized immunoglobulin of claim 17, 2 whereiin the effector function is capable of complement 3 fixation or antibody dependent cellular toxicity. 1 20. A nucleic acid encoding the heavy chain of the' 2 humanized immulnoglobulin of claim 1. 1 21. A nucleic acid entcoding the light chain of the 2 humakvl!d immunoglobulin of claim 1. 1 22. A computer programmed to display a three- 2 dimenitional, xapresentation of the humanized immunoglobulin of 3 claim 1 on a monitor or a prinj-r 1 23. A pharmaceuti.dal comjosition comprising the 2 hunianieea antibody of claim 1.4 and a pharmaceutically 3 acceptable carrier theref or. 1 2. A rlt4.rmaceutical comnosition comprising the S24 2 humaniz-Oad immunoglobulin of claim 1 and a pharmaceutically 3 acceptable carrier therefor. 1 2. The pharmaceutical composition of claim 24, 2 wherein the humanized immunoglobulin is a Fab fragment. 1 26. A method for detecting P-selectin, the method S. 2 comprisiaqn: 3 Administering the humanized immunoglobulin of 4 claim 14 to a patient or a tissue sample therefrom; and detecting complexes formed by specific binding S. 6 between the iinmunoglobulin and P-selectin present in the 7 target sample. 123 1 27. A method of inhibiting adhesion of a leukocyte 2 to an endothelial cell or platelet, the method comprising 3 administering a therapeutically effazet4vp amount of the 4 pharmaceutical composition of claim 24. 1 28. A method of treating a patient suffering from a 2 disease of the immune system, the method comprising 3 administering to the patient having the disease a 4 therapeutically effective amount of the pharmaceutical composition of claim 24. 1 29. The method of claim 28, pherein the disease is 2 a rea.Lt of acute lung injury. 1 30. The method of claim 28, Wherein the disease is 2 ischemia-reperfusion injury. 1 31. The method of claim 30, y h erein the patient is 2 s£fering from epidermal, myocardial, renal, cerebral, 3 splenic, hepatic, spinal, splanchnic, pulmonary, partial-body, 4 or whole-body ischemia. e 1 32. A method of treating a patient suffering from a 2 condition of the immune system, the method comprising 3 administering to the patient having the conaition a 4 therapeutically effective amount of the pharmaceutical 0 5 composition of claim 24. 1 33. A stable cell line comprising: 2 a nucleic acid segment encoding the hoavy chain of 3 the humanized immunoglobulin of claim 14, the segment operably 4 linked to a promoter to allow expression of the heavy chain? a second nucleic acid segment encodi!g t4e light 6 chain of the humanized immunoglobulin of claim 14, tht second 7 segment operably linked to a second promoter tc allow 8 expression of the light chain; .9 wherein the stable cell linp i- capable of producing the humanized immunoglobulin of claim 14. SUBSTITUTE SHEET (RULE 26) 124 1 3. The cell line of claim 33 tthat is capable of 2 producing about 30o pg of the humanize' immunoglobulin/ 106 3 cells/day. The cell line of claim 34 d~signated CHO-48B4 deposited with the ATCC under Accession No. CRL 11596. 1 36. A purified P-selectin polyyeptide having up to 2 100 amino acids, the up to 100 amino acids comprising at least 3 five contiguous amino acids from between position's 448 and 467 4 of the amino sequence shown in Fig. 34 (SEQ. ID. NO- 2 0 1 3. The pu.rified polypeptide of claim 36, wherein 2 the up tEo 100 amino acids comprise the contiguo'ms segment of 3 amino acids between positions 448 and 467 of the amino acid 4 sequence shown in Fig. 34 (SEQ. ID- NO. 1 38. The purified polypeptide of claim 37 consisting 2 essentially of the contiguous segment of amino acids between 3 positions 448 and 467 of the amino acid sequence shown in 4 Fig. 34 (SEQ. ID. No. 0 0 '1 ByterPtetAtre oil* S 0#140