HUT75894A - Antibodies to p-selectin and their uses - Google Patents

Abstract

The present invention relates to compositions and methods for treating inflammation and other pathological conditions using novel blocking P-selectin antibodies that inhibit adhesion of leukocytes to activated platelets and/or to activated vascular endothelium in vivo. Both murine and humanized antibodies are provided.

Description

The present invention relates to novel immunoglobulins which react with functional epitopes on the P-selectin cell surface receptor. The invention also relates to the diagnostic and therapeutic use of antibodies.

The ability of cells to adhere to each other plays a critical role in development, normal physiology, and pathological processes such as inflammation. This ability is mediated by adhesion molecules, usually glycoproteins, expressed on cell membranes. Often, an adhesion molecule expressed on one cell type binds to an adhesion molecule expressed on another cell type, forming a receptor-receptor pair. Three important classes of adhesion molecules are members of the integrins, selectins, and immunoglobulin (Ig) superfamily (Springer, Natura 346: 425 (1990); Osborn: Cell 52: 3 (1990); Hynes: Cell 69, 11 (1992)]. The foregoing publications are hereby incorporated by reference in their entirety for all purposes. These molecules are particularly important in the interaction of leukocytes and platelets, in their interaction with the extracellular matrix and in the vascular endothelium.

P-selectin, also known as CD62, is a specialized surface receptor on vascular endothelial cells and platelets, a granule membrane protein-140 (GMP-140) / platelet activation-dependent granule external membrane (PADGEM) / LECCAM-3). , which play a role in recognizing different circulating cells. P-selectin is a surface glycoprotein that has a lectin-like domain, i.e., a region that is homologous to epidermal growth factor and has a region that is homologous to complement regulatory proteins [McEver: Blood Cells 16, 73-83 (1990)]. The structure of P-selectin is similar to that of two other cell surface receptors, the endothelial leukocyte adhesion molecule (ELAM-1) and the lymphocyte homing receptor (LHR). Selectin has been proposed for the general class of these receptors because of the selective nature of their lectin-like domain and their adhesion functions.

P-selectin is present on the surface of platelets and endothelial cells in response to numerous stimuli where it mediates platelet-leukocyte and endothelial-leukocyte interactions. In contrast, ELAM-1 is expressed only by endothelial cells, whereas LHR is expressed by a variety of leukocytes in the endothelial thin veins of the peripheral lymph glands. P-selectin expression is inducible and is not expressed on non-activated endothelial cells or platelets. De novo synthesis is not required for P-selectin expression as it is stored in secretory granules (or Weibel-Palade bodies), both in platelets and endothelial cells. Thus, minutes after activation of any cell type with thrombin, histamine, phorbol esters, or other humoral factors, P-selectin is rapidly distributed on the cell surface, where it is available for attachment to other cells.

Numerous murine antibodies against P-selectin have been described in U.S. Patent No. 4,783,330; WO 91/06632, FR90 / 00565 PCT Application Number; McEver et al., 1984, Journal of Biological Chemistry 259: 97999804; Geng et al., Natur. 343, 757-760; Parmentier et al., Blood 77, 1734-1739 (1991). An additional antibody to P-selectin has been described [presentations at Scripps Institute, La Jolla California; Keystone Colloquium, January 16, 1992]. Some of these antibodies, which block Pselectin-mediated adhesion, are Ca ⁺⁺ -dependent.

In vitro experiments using P-selectin antibodies revealed limited ligand specificity for this receptor. P-selectin has been reported to bind neutrophils, monocytes, and certain carcinoma cells [Bevilacqua et al., Proceedings of the National Academy of Sciences, USA 84,

9238-9242 (1987); Geng et al., 1990, Nat. 343: 757-760; Larsen et al., Cell 63: 467-474 (1990); Polley et al., P-selectin has also been reported to recognize Le ^x as a high affinity ligand (Larsen et al., Cell 63: 467-474 (1990)). Further experiments, however, have demonstrated that oligosaccharides containing the SLe ^{x has} in fact a much more potent inhibitor mediated by P-selectin adhesion than Le ^x (Polley et al .: Proceedings of the National Academy of Sciences, USA 88, 6224-6228 (1991)].

The involvement of P-selectin in in vitro intercellular adhesion suggests that, like other adhesion molecules, P-selectin may participate in cellular interactions that contribute to inflammatory diseases in vivo. However, the specific inflammatory diseases in which P-selectin may play a major role have not yet been identified. Similarly, the epitope specificity of agents that effectively suppress certain P-selectin mediated inflammatory diseases is also unclear. Some of the known antibodies against P-selectin bind to P-selectin but do not block intracellular interactions mediated by P-selectin. Other known antibodies have the capacity to block P-selectin-mediated intercellular binding, but only in the presence of high Ca ⁺⁺ , a condition which is not always in vivo. The absence of high levels of Ca ⁺⁺ under in vivo conditions renders these antibodies inactive as therapeutic agents. However, all anti-P-selectin antibodies produced so far are rodent and therefore induce a human anti-mouse antibody response (HAMA) in clinical use.

From the foregoing, it is clear that new anti-P-selectin agents that have the appropriate epitope specificity are required to stop intercellular interactions leading to in vivo inflammatory diseases. Ideally, such agents do not induce a HAMA response in the treatment of humans. The present invention satisfies this need and other needs.

The present invention relates to blocking antibodies that specifically bind to P-selectin. Such an antibody is labeled mu MAb PB1.3 (produced by cell line HB11041 ATCC). Other antibodies competitively inhibit the binding of mu MAb PB1.3 to Pselectin, as measured in a competitive inhibition assay. Preferred antibodies bind to P-selectin in the absence of Ca ⁺⁺ . Antibodies of the invention also include fragments such as Fab, Fab'F (ab ') 2> Fabc and Fv, and intact antibodies.

The present invention also relates to pharmaceutical compositions. The pharmaceutical compositions contain a P-selectin blocking antibody as described above and a pharmaceutically acceptable carrier.

The invention also relates to medical methods for treating diseases of the immune system. According to the present invention, a therapeutically effective dose of a pharmaceutical composition as described above is administered to a patient suffering from such a disorder. Acute lung injury and ischemic reperfusion injury are good examples of diseases that can be treated according to the present invention.

Some of the antibodies of the present invention are humanized immunoglobulins. Humanized immunoglobulins contain a humanized heavy chain and a humanized light chain. The humanized light chain comprises three complementarity determining regions (CDR1, CDR2, CDR3) having the amino acid sequence derived from the corresponding murine PB1.3 immunoglobulin light chain complementarity determining regions, and a human kappa light chain variable region framework sequence. The humanized heavy chain contains three complementarity determining regions (CDR1, CDR2,

• · ··· «• · ·

CDR3) having the amino acid sequence derived from the heavy chain complementarity determining regions of the corresponding murine PB1.3 immunoglobulin, as well as a human heavy chain variable region framework sequence. Humanized immunoglobulins bind specifically to P-selectin with a lower binding affinity of about 10 ⁷ / mol / l and an upper limit of about five times the binding affinity of mouse PB 1.3 immunoglobulin.

In many humanized immunoglobulins, the framework sequence of the human kappa light chain variable region is substituted at at least one position selected from the first group: L21, L46, L60 and L70, an amino acid equivalent to the mouse PB1.3 immunoglobulin light chain variable region. keretszekvenciájában. Similarly, the human heavy chain variable region framework sequence has at least one position selected from the second group: H1, H2, H71, and H73, an amino acid equivalent to the mouse PB1.3 heavy chain variable region framework sequence. In some humanized immunoglobulins, the humanized heavy chain variable region framework sequence is a 21 / 28'CL heavy chain variable region framework sequence. In some humanized immunoglobulins, the humanized light chain variable region framework sequence is a DEN light chain variable region framework sequence. An example of the mature light chain variable region amino acid sequence is shown in Figures 14-17. Figs. An example of the mature heavy chain variable region amino acid sequence is shown in Figures 11-13. Figs. A preferred humanized immunoglobulin comprises the mature light chain variable region of Figure 17 and the mature heavy chain variable region of Figure 12.

The present invention relates to intact humanized immunoglobulins as well as to humanized immunoglobulin fragments such as Fab, Fab'F (ab ') 2, Fabc and Fv.

• ·

Some humanized immunoglobulins also contain a constant region, with or without effector function.

The present invention also provides nucleic acids encoding the humanized immunoglobulins described above. Some nucleic acids encode heavy chains, some nucleic acids encode light chains.

The present invention also relates to a computer programmed to display a three-dimensional image of the immunoglobulins described above on a monitor or a printer.

The present invention also provides pharmaceutical compositions comprising the above-described humanized immunoglobulins and a pharmaceutically acceptable carrier.

The invention further provides methods for detecting P-selectin using the humanized immunoglobulins described above. A humanized immunoglobulin is administered to a patient or is added to tissue from a patient. Complexes formed by specific binding between the immunoglobulin and the P-selectin present in the sample are detected because they indicate the presence of P-selectin.

The invention also relates to methods of treating patients suffering from immune system disorders using humanized immunoglobulins. According to the present invention, a therapeutically effective dose of a pharmaceutical composition as described above is administered to a patient. Treatable diseases include ischemia-reperfusion injury and acute lung injury. For example, the compositions are useful for treating patients with epidermal, myocardial, renal, cerebral, spleen, liver, spinal, visceral, lung, body or whole body ischemia.

• · · • · · ·

The invention also relates to stable cell lines producing humanized immunoglobulins. Stable cell lines contain two nucleic acid segments. One nucleic acid segment encodes the heavy chain of the humanized immunoglobulin described above, operably linked to a promoter that permits expression of the heavy chain. The other nucleic acid segment encodes the humanized immunoglobulin light chain, the second segment operably linked to a second promoter, which permits expression of the light chain. Preferred cell lines produce about 30 µg of humanized immunoglobulin (/ 10θ cells / day).

The invention further provides fragments of P-selectin. These fragments are up to 100 amino acids in length and contain at least five contiguous amino acids at positions 448-467 of the amino acid sequence shown in Figure 34. Preferred fragments, up to 100 amino acids in size, contain the entire contiguous segment at positions 448-467.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.

Figure 1 shows data demonstrating that the anti-inflammatory immunoglobulins of the invention inhibit the binding of thrombin-activated platelets to neutrophils.

2A. 2B and 2B. Figures 2 to 9 show that the anti-inflammatory immunoglobulins of the invention effectively prevent lung injury caused by infusion of cobram venom factor (CVF), as measured by permeability (Figure 2A) and bleeding (Figure 2B).

3A-3H. Figures 6 to 9 show that P-selectin expression levels in the lungs of animals are increased in response to cobram venom infusion, which can be visualized by horseradish peroxidase staining of mu MAb PB1.3. Panel (a) - (d) shows the expression of Pselectin in the small pulmonary vessels at various time points after the CVF infusion; 0 time (a); Panels 5, 10 and 15, b, c and d (Fig. 3A). 3B. Fig. 2A shows light microscopic and transmission electron microscopy images of rats treated with 200 pg of non-blocking antibody (mu MAb PNB1.6) for CVF-induced acute lung injury (ac frames) or P-selectin blocking antibody with P-selectin (mu MAb PB1.3). treated rats with CVF-induced acute lung injury (bd frames). In PNB1.6-treated animals, vascular injury is indicated by extensive intra-alveolar hemorrhage (? -Frame) associated with neutrophil intravascular aggregates (c-frame, arrows) with close contrast of endothelial cells. In contrast, animals treated with mu MAb PB1.3 show no intra-alveolar hemorrhage (b-frame) and intravascular neutrophil cells show little contact with the endothelium (d-frame, arrows). Frame a and b in plastic-embedded section, painted with toluidine blue, X160; frames c and d painted with uranyl acetate, lead acetate, X2250).

Figure 4 shows the binding of mu MAb PB1.3 monoclonal antibody to Pselectin: effect of chelating divalent cations and EDTA. In the figure, the circles (bottom line) indicate the binding in the presence of Ca ⁺⁺ and Mg ⁺⁺ , while the squares (top line) indicate the binding in the presence of EDTA. The antibody was diluted to 1.6 mg / ml.

Figure 5 shows the effect of CQNRYTDLVAIQNKNE peptide on the binding of mu MAb PB1.3 to P-selectin. The black squares indicate binding in the presence of 0.35 mg / ml peptide and the blank circles indicate binding in the absence of peptide. Labeled dilutions of mu MAb PB1.3 were prepared from a 1.6 mg / ml solution.

6A, 6B. 6C and 6C. Figures 1 to 5 show the ability of monoclonal antibodies to cross-link P-selectin binding. Blocking antibodies are present at a concentration of 50 μg / ml. Dilutions of biotinylated antibodies were 1.6 mg / ml mu MAB PB1.3, 0.87 mg / ml mu MAb PNB1.6 and 0.59 mg / ml mu MAb 84/26.

• · · ·

Figure 7 shows the degree of thrombin-induced platelet aggregation under control conditions in the presence of antibody and in the presence of increasing concentrations (1-100 pg / ml) of purified mu MAb PB1.3 antibody.

Figure 8 shows that the extent of myocardial ischemia, expressed as a percentage of the risk area, is significantly smaller in animals treated with blocking anti-P-selectin antibody (mu MAb PB1.3) than in animals treated with non-blocking anti-P-selectin antibody. In addition, the ability of coronary rings to relax in response to acetylcholine is significantly greater in animals treated with mu MAb PB1.3 than in animals treated with mu MAb PNB1.6. The fact that these events are attributable to a decrease in myocardial leukocyte infiltration is reflected in the reduced number of PMNs in the myocardial tissue of animals treated with mu MAb PB1.3.

Compared to animals treated with PNB1.6.

Figure 9 shows the sequence and translation of the signal peptide and variable region of the PB1.3 heavy chain.

Figure 10 shows the sequence and translation of the signal peptide and variable region of the PB1.3 light chain.

Figure 11 shows the sequence and translation of the CY1748 heavy chain A signal peptide and its variable region.

Figure 12 shows the sequence and translation of the CY1748 heavy chain B signal peptide and variable region.

Figure 13 shows the sequence and translation of the CY1748 heavy chain C signal peptide and variable region.

Figure 14 shows the sequence and translation of the CY1748 light chain A signal peptide and its variable region.

• · · · • ··

Figure 15 shows the sequence and translation of the CY1748 light chain B signal peptide and its variable region.

Figure 16 shows the sequence and translation of the CY1748 light chain C signal peptide and its variable region.

Figure 17 shows the sequence and translation of the CY1748 light chain D signal peptide and its variable region.

Figure 18 is a physical map of the neo-plasmid expressing the human heavy chain (gamma-1) constant region.

Figure 19 is a physical map of the neo plasmid expressing the human kappa chain (kappa constant region).

Figure 20 shows the binding curves for chimeric PB1.3 and modified versions of CY1748 to rsP-selectin.

Figure 21 shows the binding curves for chimeric PB 1.3 and the modified versions of CY1748 to rsP-selectin.

Figure 22 shows the binding curves for chimeric PB1.3 and the modified versions of CY1748 to rsP-selectin.

Figure 23 is a physical map of the dhfr plasmid expressing the human heavy chain (gamma-1 constant region).

Figure 24 is a physical map of the dhfr plasmid expressing the human heavy chain (gamma-4 constant region).

Figure 25 shows the inhibition of biotinylated PB1.3 binding to rsP-selectin by PB1.3 and CY1748 RH / \ / RL / \ - IgG1.

Figure 26 shows the inhibition of binding of biotinylated PB1.3 to rsP-selectin by PB1.3 and CY1748 RHB / RI_Q-IgG4.

Figure 27 shows that antibody PB 1.3 reduces ischemia-related swelling and reperfusion in the rabbit ear.

·· ···· • ·. · ···.

Figure 28 shows that PB1.3 prevents the development of necrosis in rabbit ears following ischemia and reperfusion.

Figure 29 shows that PB1.3 inhibits leukocyte-endothelial cell interactions induced by 48/80 other cellular degranulation compounds.

Figure 30 shows the putative three-dimensional structure of the P-selectin domain.

Figure 31 is a schematic of the P-selectin deletion mutants used for mapping epitopes recognized by PB1.3.

Figure 32 shows the binding of PB1.3 and rabbit monoclonal antibodies a

P-selectin for deletion mutants.

Figure 33 shows the binding of PB1.3 and P6H6 to P-selectin and Pselectin A5CRP, a deletion mutant of P-selectin lacking the fifth CRP.

Figure 34 shows the sequences of synthetic peptides derived from the human P-selectin CRP 5 amino acid sequence and the reactivity of PB1.3 with these immobilized peptides. The amino acids are numbered according to the convention established by Johnston et al., 1989 for the full length P-selectin polypeptide (Johnston et al., Cell 56: 1033-1044 (1989)).

Twenty natural amino acids are abbreviated according to standard usage (Immunology - A Synthesis (2nd edition, ES Golub & DR Gren, eds., Sinauer Associates, Sunderland, MA (1991)).) Twenty standard amino acid stereoisomers (e.g. amino acids), non-naturally occurring amino acids such as α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other abnormal amino acids may also be suitable components of the polypeptides of the present invention. ···· may include, for example, 4-hydroxyproline, γ-carboxyglutamate, ε-Ν, Ν, Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, n-N-methylarginine and the like and imino acids (such as 4-hydroxyproline) In addition, the amino acids can be modified by glycosylation, phosphorylation and the like.

In the polypeptide description used below, the N-terminal direction is to the left and the C-terminal direction to the right, according to standard application and convention. Similarly, unless otherwise defined, the left end of single-stranded polynucleotide sequences is the 5 'end; the left side of the double-stranded polynucleotide sequences corresponds to the 5 'direction. The 5 'to 3' insertion direction of the nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand that have the same sequence as the RNA and are located 5 'to the 5' end of the RNA transcript are called upstream sequences; sequence regions on the DNA strand that have the same sequence as the RNA and are located 3 'to the 3' end of the RNA transcript are called downstream sequences.

Polynucleotide sequence refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read in the 5'-3 'direction. These include self-replicating plasmids, infectious polymers of DNA or RNA, and non-functional DNA or RNA.

The following terms are used to describe sequence relationships between two or more polynucleotides: reference sequence, comparator window, sequence identity, percent sequence identity, and substantial identity. The reference sequence is a specific sequence used as a basis for comparing the sequences; a reference sequence may be a subset of a larger sequence, such as a full-length cDNA

or a segment of a gene sequence given in a sequence list, e.g. The polynucleotide sequences shown in FIGS. 1A to 4A may comprise a complete DNA or gene sequence. In general, a reference sequence is comprised of at least 20 nucleotides, generally at least 25 nucleotides, and often at least 50 nucleotides. Because each of the two polynucleotides (1) contains a sequence (i.e., a portion of the entire polynucleotide sequence) that is similar in both polynucleotides, and (2) may also contain a sequence that is different in the two polynucleotides, the two (or more) polynucleotides Comparison is generally performed by comparing the sequences of the two polynucleotides through a comparison window to identify and compare local regions of sequence similarity. A comparison window hereinafter refers to a conceptual sequence comprising at least 20 contiguous amino acid positions where a polynucleotide sequence can be compared to a reference sequence containing at least 20 contiguous nucleotides, and wherein a portion of the polynucleotide sequence in the comparison window contains additions and deletions. i.e., holes), 20% or less compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. For alignment of a comparison window, optimal alignment of sequences can be performed using the Smith and Waterman local homology algorithm [Smith & Waterman, Adv. Appl. Math. 2, 482 (1981)] by Needleman & Wunsch's homology fitting algorithm (Needleman & Wunsch, Journal of Molecular Biology 48, 443 (1970)), Pearson & Lipman Proceedings of the National Academy of Sciences, USA 85, 2444 (1988)] (the references cited hereinbelow are incorporated by reference), using computer-aided application of these algorithms (GAP, BESTFIT, FASTA and TFASTA

Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspecting, and selecting the best fit (i.e., the highest percent sequence similarity in the comparison window) provided by the various methods. "Sequence Identity" means that the two polynucleotide sequences are identical (i.e., based on comparison per nucleotide) in the comparison window. Percentage of sequence identity means that we calculate by comparing two sequences that are optimally matched in the comparison window, determining the number of positions that have the same nucleic acid base (i.e., A, T, C, G, U, or I). in both sequences, to obtain the exact number of matching positions, then dividing the number of matching positions by the total number of positions in the comparator window (i.e., the size of the window) and multiplying the result by 100 to obtain the percent sequence identity. Substantial Identity hereinafter refers to a feature of a polynucleotide sequence wherein the polynucleotide comprises a sequence having at least 85% sequence identity, preferably at least 9095% sequence identity, but generally at least 99% sequence identity. using a comparison window of at least 20 nucleotides in position relative to the reference sequence, and often 25 to 50 nucleotides in size, wherein the percent sequence identity is calculated by comparing the reference sequence to a polynucleotide sequence which may contain deletions or additions that window only 20% or less of the reference sequence. The reference sequence may be part of a larger sequence, e.g.

The sequence shown in FIG.

* «V ·

As used herein, the term "sequence identity" refers to peptides that contain the same amino acids at the corresponding positions. Sequence similarity refers to peptides that contain identical or similar amino acids (i.e., conservative substitutions) at the respective positions. Substantial identity means that two peptide sequences, when optimally aligned, such as GAP or BESTFIT, using default well weights, show at least 80 percent sequence identity, preferably at least 90 percent sequence identity, but more preferably at least 95 percent sequence identity. or greater (e.g., 99%) sequence identity. Positions that are not identical preferably differ in conservative amino acid substitutions. Substantial similarity means that two peptide sequences have a sufficient percentage of sequence similarity.

A substantially pure term means that a single object type is the predominant object type (i.e., more abundant in the composition on a molar basis than any other type), and preferably a substantially purified fraction is a composition in which at least 50 percent of the relevant object is present. on a molar basis) to form macromolecules present. A substantially pure composition contains more than about 80-90 percent of all types of macromolecules present in the composition. Most preferably, the molecule of interest is purified to substantial homogeneity (the impurity species cannot be detected in the composition by conventional detection methods) while the composition comprises essentially a single macromolecule species.

For the purpose of classifying amino acid substitutions into conservative or non-conservative substitutions, amino acids have been divided into the following groups ** «·« «··· Group I (hydrophobic side chains): norleucine, methionine, alanine, valine, leucine, isoleucine; II. group (neutral hydrophilic side chains): cysteine, serine, threonine; III. group (acidic side chains): aspartic acid, glutamic acid; ARC. group (basic side chains): asparagine, glutamine, histidine, lysine, arginine; Group V (groups influencing chain orientation: glycine, proline; and group VI (aromatic side chains): tryptophan, tyrosine, phenylalanine. Conservative substitution means replacing amino acids in the same group. Non-conservative substitution means to substitute an amino acid in one group for an amino acid in another group.

Amino acids derived from the variable regions of the mature heavy chains and light chains of immunoglobulins are denoted by Hx and Lxx, where x is a number representing the position of an amino acid according to the system of Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) (1987 and 1991)], which is hereinafter referred to as Kaba et al., And is hereby incorporated by reference in its entirety. Kaba and coworkers list several amino acid sequences for each subgroup of antibodies, and list the most common amino acids for each amino acid position in that subgroup. Kaba and coworkers use a method of assigning a group sequence number to each amino acid in the listed sequence, and this method has become standard in the art for group sequence assignments. The scheme of Kabat and coworkers can be extended to other antibodies not included in the compendium by inserting the test antibody into one of the consensus sequences developed by Kabat and coworkers. Using the system developed by Kaba and co-workers, it is easy to identify amino acids that are ····, ·. ....

·· »··· ··" ·· • · · · · · _the ' »found in equivalent positions in different antibodies. For example, an amino acid residue at position L50 of a human antibody occupies an equivalent position with an amino acid at position L50 of a mouse antibody.

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

The term substantial inhibition means at least about 50% inhibition, preferably about 60-80% inhibition, and most preferably greater than 85% inhibition (as measured by in vivo competitive binding assay).

Chimeric antibodies are antibodies whose light chain and heavy chain genes are generally generated by genetic engineering from various species of immunoglobulin gene segments. For example, the variable (V) segments of a mouse monoclonal antibody genes can be linked to human constant (C) segments, such as γι or y2. Thus, a typical pharmaceutical chimeric antibody is a hybrid protein comprising the V or antigen-binding domain of a mouse antibody and the C or effector domain of a human antibody, although other mammalian species may be used.

The present invention relates to compositions and methods for inhibiting P-selectin-mediated diseases and conditions of the immune system. More particularly, the present invention relates to immunoglobulins having the ability to inhibit P-selectin-mediated cell adhesion in vivo.

. ·. : ··· .......

· * · * ·. : ·· ··· ....... .. · .. ·

I. Characteristics of immunoglobulins

A. General

It is well known that the basic antibody unit contains a tetramer. Each tetramer consists of two identical polypeptide chain pairs, each pair having a light (about 25 kDa) and a heavy (about 50-70 kDa) chain. The N-terminal portions of each chain contain variable regions of about 100-110 amino acids or more that are primarily responsible for antigen recognition. The C-terminal portion of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. The heavy chains can be classified into gamma, mu, alpha, delta or epsilon groups, and the antibody isotype is IgG, IgM, IgA, IgD, and IgE. Within the light and heavy chains, the variable and constant regions are linked by a J region of about 12 or more amino acids, and the heavy chain also contains a D region of about 10 or more amino acids [Fundamental Immunology, Chapter 7; ed .; Paul, W., 2nd Edition, Raven Press, N.Y. (1989)]. This publication is hereby incorporated by reference in its entirety.

The variable regions of each light / heavy chain pair create the antibody binding site. Each chain has the same general structure, that is, relatively conserved framework regions (FRs) linked to three hypervariable regions, otherwise known as complementarity determining regions or CDRs. The CDRs formed from the two strands of each pair are joined by the framework regions, allowing them to bind to a specific epitope. The CDR and FR groups are assigned to each other based on the standard sequence definition provided by Kaba et al. An alternative structure definition has been suggested by Chothia et al., Journal of Molecular Biology · ♦ ·

196: 901-917; Natur. 342: 878-883 (1989); Molecular Biology 186: 651-663 (1989)]. These publications are hereinafter referred to as Chothia et al., And are hereby incorporated by reference in their entirety.

B, Binding specificity and affinity

The immunoglobulins (or antibodies) of the invention selectively bind to a functional epitope on P-selectin that is associated with a response to tissue injury and inflammation. Binding of antibodies to a functional epitope on Pselectin generally generally effectively inhibits leukocyte attachment to activated platelets and / or activated vascular endothelium, in vivo. jr. antibodies having this property are referred to as blocking antibodies. Preferred blocking antibodies inhibit the adhesion of leukocytes to the activated vascular endothelium, thereby preventing or inhibiting the inflammatory and / or thrombotic state.

Many of the blocking antibodies of the invention compete with mu MAb PB1.3 for exemplary binding to P-selectin. A hybridoma producing the mu MAb PB1.3 antibody was deposited with the American Type Culture Collection (Rockville, Maryland) under the Budapest Convention under accession number HB11041. The production of this antibody is described in Example 1. Other blocking antibodies of the invention compete with another exemplary antibody 84/26.

Competition is determined by an assay in which the immunoglobulin tested inhibits the specific binding of a reference antibody (e.g., mu MAb PB1.3) to an antigenic determinant on the P-selectin molecule. Many different types of 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 (Stahili et al., Methods in Enzymology 9, 242-253 (1983)). )]; solid phase direct biotin-avidin EIA [Kirkland et al., J. Immunol. 137: 3614-3619 (1986)]; solid phase direct labeled sandwich assay (Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using 1-125 [Morei et al., Molec. Immunol. 25 (1): 7-15 (1988)]; solid phase direct biotin-avidin EIA (Cheung et al., 1990, Virology 176, 546-552); and direct labeled RIA [Moldenhauer et al., Scand. J. Immunol. 32: 77-82 (1990)]. Such an assay typically uses purified P-selectin or solid surface-bound P-selectin, a labeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of solid surface-bound label in the presence of the test immunoglobulin. The immunoglobulin tested is generally present in excess. A suitable competitive assay is described in Example 8. Antibodies (competing antibodies) identified by the competition assay include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to a neighboring epitope close enough to the epitope to which the reference antibody binds, in order for steric inhibition to occur. Generally, if a competing antibody is present in excess, it will inhibit the binding of the reference antibody to P-selectin by at least 10, 25, 50, or 75 percent.

The epitope bound by mu MAb PB1.3 antibody has been mapped between amino acid 448 and 467 of human P-selectin (Johnson et al., Cell, 56, 1033-1044 (1989)), which is hereby incorporated by reference in its entirety. for purposes of reference. The epitope occurs in the fifth CRP domain of P-selectin (see Figure 30). Antibodies competing with mu MAb PB1.3 are generally a

448-467, or to another segment in CRP 5 domain.

The binding specificity of many of the blocking antibodies of the invention is further defined by their capacity to bind P-selectin in the complete or partial absence of Ca ⁺⁺ ions (i.e., 25 mmol / L EDTA in the presence of a calcium chelator), and Ca ⁺⁺ ions. absent in an in vitro study. Antibodies requiring Ca ⁺⁺ cofactor for blocking activity may not be effective under in vivo conditions where Ca ⁺⁺ levels may fluctuate.

Antibodies competing with mu MAb PB1.3 are further characterized by the observation that their specific binding capacity to P-selectin is not inhibited by a P-selectin fragment having the amino acid sequence CQNRYTDLVAIQNKNE. The molar excess of this fragment generally inhibits the specific binding of these antibodies by less than 5 percent. Mu MAb PB1.3 and competing antibodies are thus distinguished from the described murine antibodies designated G1, G2 and G3 (Geng et al., 1991, Journal of Biological Chemistry 266, 22313-22318), which bind to Pselectin. it is partially or completely inhibited by said peptide.

Some of the antibodies of the invention are characterized by their capacity to selectively inhibit some P-selectin-mediated responses while not inhibiting others. For example, some antibodies are capable of inhibiting in vivo the specific binding of neutrophils to activated endothelial cells without inhibiting platelet aggregation. These antibodies are particularly useful in the treatment of inflammatory diseases without interfering with the patient's ability to induce a response to conditions (e.g., injury) in which platelet aggregation is desired.

- 23 - ............

The antibodies of the invention generally have specific binding affinities for P-selectin at a concentration of at least 10 µm, 10 µm, 10θ → 10 µmol / L. The upper limit of binding affinity for anti-P-selectin antibodies is within about three, five, or ten times the affinity of mu MAb PB1.3. The lower limit of binding affinity is often also within the triple, five or ten fold affinity of mu MAb PB1.3. In this context, about refers to the small number of experimental errors that often occur when measuring binding affinities.

II. Preparation of immunoglobulin

A. Non-human antibodies

Non-human, such as murine, lagomorpha, equine antibodies are well known and can be prepared, for example, by immunizing an animal with a composition comprising either P-selectin-bearing cells (e.g., thrombin-activated platelets) or isolated P -selectin molecules or fragments thereof, such as extracellular domains. Antibody-producing cells from immunized animals are immortalized and screened, or conversely, first screened for the production of anti-P-selectin antibody and subsequently immortalized (Harlow and Lane, Antibodies, Laboratory Manual, Cold Spring Harbor Press, 1988). ]. Alternatively, a substantially monospecific antibody population can be generated by purifying the polyclonal serum by chromatography.

B. Human antibodies to P-selectin

The invention also relates to the production of human antibodies against P-selectin.

Some human antibodies are selected by competitive binding assay or with the same epitope specificity as a specific murine antibody, such as mu MAb PB1.3. These antibodies are particularly well thought to have the same useful therapeutic properties as those exhibited for mu MAb PB1.3. Antibodies against P-selectin can be made by screening a DNA library derived from human B cells according to the general description of Huse et al., Science 246: 1275-1281 (1989). Antibodies that bind to P-selectin or fragments thereof are selected. The sequences encoding such antibodies (or a binding fragment thereof) are then cloned and amplified. The protocol described by Huse et al. Can be made more effective by phage display technology [Dove et al., WO 91/17271; McAfferty et al., WO 92/01047]. These publications are hereby incorporated by reference in their entirety. In these methods, phage libraries are prepared in which members display various antibodies on their surface. Antibodies are generally presented as Fv or Fab fragments. Phages displaying antibodies with the desired specificity can be selected by affinity enrichment of a P-selectin polypeptide or fragment thereof.

In a variation of the phage display method, an antibody having a binding affinity for a selected murine antibody can be prepared (Winter, WO 92/20791). In this method, the heavy or light chain variable region of the murine antibody of choice is used as starting material. For example, if a light chain variable region is selected as the starting material, a phage library is created in which the members show the same light chain variable regions (i.e., rodent starting materials) and show different heavy chain variable regions. A phage exhibiting a strong specific binding to P-selectin (e.g., at least 10θ and preferably at least 10θ mol / l) is selected. The human heavy chain variable region derived from this phage is then used as a starting material for the construction of an additional phage library. In this directory, each phage represents the same heavy chain variable region (i.e., 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, phages showing strong specific binding to P-selectin are selected. These phages show variable regions of fully human anti-P-selectin antibodies. These antibodies generally have the same or similar epitope specificity as the murine starting material (e.g., mu MAb PB1.3).

C. Humanized antibodies

The present invention relates to humanized immunoglobulins having variable framework regions essentially derived from a human immunoglobulin (referred to as acceptor immunoglobulin) and essentially a complementarity determining region derived from a murine immunoglobulin PB1.3 (called donor immunoglobulin). The constant region (s), if present, are also derived from a human immunoglobulin. The humanized antibodies of the present invention have several advantages over the mouse donor antibody which have been shown to be effective in animal models:

1) The human immune system should not recognize a framework or constant region of a humanized antibody, and therefore the response to such an injected antibody should be less than that of a fully foreign mouse antibody or a partially foreign chimeric antibody.

2) Because the effector part of the humanized antibody is human, it may interact more closely with other parts of the human immune system.

• *

3) Injected mouse antibodies have been reported to have a much shorter half-life in human circulation than normal human antibodies [Shaw et al., J. Immunol. 138: 4534-4538 (1987)]. The half-life of injected humanized antibodies is substantially equivalent to that of naturally occurring antibodies, which allows for much lower doses.

1, Cloning and sequencing of mu MAb PB1.3 variable domains

Cloning and sequencing of the cDNA encoding the heavy and light chain variable regions of the mu MAb PB1.3 antibody is described in Example 10 and the nucleotide and calculated amino acid sequences are shown in Figures 11 and 12. 1 and 2.

Tables 1 to 4 show the subdivision of the amino acid coding sequence and domains that determine complementarity. From the N-terminus to the C-terminus, both light and heavy chains contain the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The assignment of amino acids to each domain is carried out according to the numbering convention developed by Kaba et al. (1987 and 1991, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD).

(2) Selection of human antibodies for the provision of framework groups

Replacement of mouse CDRs with a human variable frame most likely results in maintaining their correct spatial orientation when the human variable domain frame adapts to the same or a similar conformation as that of the mouse variable frame region from which the CDRs come. This is accomplished by taking the human variable domains from human antibodies having framework sequences that are highly sequenced.

- 27 - · ^: · ...... '' ·· * 'show identity to the rodent variable framework domains from which the CDRs are derived. The light and heavy chain variable framework regions may be derived from the same or another human antibody sequence. Human antibody sequences may be sequences of naturally occurring human antibodies or may be consensus sequences derived from a number of human antibodies (Kettleborough et al., 1991, Protein Engineering 4, 773; Kolbinger et al., 1993, Protein Engineering 6: 971-7.

Suitable human antibody sequences are identified by computerized comparison of the amino acid sequence of mouse variable regions with known human antibodies. The comparison is made separately for the heavy and light chains, but the principles are the same in both cases. This comparison reveals that the light chain of the mu MAb PB1.3 shows greater sequence identity to the human heavy chains of the kappa-1 subtype, and the mu MAb PB1.3 heavy chain shows the highest sequence identity in the human heavy chain of subtype 1. chains according to the system of Kábát et al. (1987 and 1991, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD). Thus, light and heavy human framework regions are generally derived from human antibodies of this subtype, or from consensus sequences corresponding to such subtype. Preferred heavyweight and light chain human variable regions with greater sequence identity to the corresponding regions from mu MAb PB1.3 are derived from 21/28'CL and DEN antibodies.

(3) Computer Modeling

The unnatural juxtaposition of murine CDR regions and the human variable framework region results in unnatural conformational constraints, which, unless corrected for certain amino acids instead, · · ··

- 28 - .............

This results in a loss of binding affinity. The amino acids to be replaced are partially determined by computer modeling. The computer hardware and software needed to make a three-dimensional image of immunoglobulin molecules are widely available. Molecular models are generally determined from the solution structure of immunoglobulin chains or their domains. The chains to be modeled are compared based on the similarity of their amino acid sequences to the chains or domains of the three-dimensional structures in solution, and the chain (s) or domain (s) with the highest sequence similarity are selected as the starting point for the molecular model. See e.g.! the starting point for modeling for the mu MAb PB1.3 light chain is the human DEN light chain. The initial structure generated in solution is modified to allow differences between the actual amino acid sequence in the immunoglobulin chains or domains to be modeled and the amino acid sequence in the parent structure. The modified structures are then brought together in a composite structure. Finally, the model is refined by minimizing energy and verifying that each atom is at a sufficient distance from the other atoms and that bond lengths and angles are within chemically acceptable limits. Such a model may also serve as a starting point for predicting the three-dimensional structure of an antibody containing a mutation determining region of mu MAb PB1.3 substituted in human framework constructs. Further models can be constructed that characterize the structure in which additional amino acid substitutions, discussed below, are made.

(4) Substitution of amino acid residues

As noted above, the humanized antibodies of the invention essentially contain framework regions derived from human immunoglobulin, as well as complementarity determining regions in a human immunoglobulin.

MAb PB1.3 mouse from immunoglobulin. After identifying the complementarity determining regions of mu MAb PB1.3 and the corresponding human acceptor immunoglobulins, the next step is to determine whether these components should be substituted and, if so, which ones, to optimize the properties of the resulting humanized antibody. Generally, the replacement of human amino acids with murine residues should be minimized, since the introduction of murine residues increases the risk that the antibody will elicit a HAMA response in humans. The amino acids to be replaced are selected based on their putative influence on CDR conformation and / or antigen binding. Such putative influences are examined by modeling, by examining the characteristics of the amino acids at particular points, or by observing empirically the substitution or mutagenesis of a given amino acid.

If an amino acid differs between a mu MAb PB1.3 variable framework region and an appropriate human variable framework region, the amino acid residue in the human framework region will generally be replaced by the equivalent mouse amino acid if it is realistically expected that the amino acid:

(1) binds non-covalently directly to the antigen (i.e., the amino acids at positions H1, H2 and L60 of mu MAb PB1.3), (2) adjacent to a CDR region, is part of a CDR region according to an alternative definition by Chothia et al. Chothia et al., 1987, Journal of Molecular Biology 196: 901-917; Natur. 342: 878-883 (1989); Journal of Molecular Biology 186, 651-663 (1989)] (i.e., less than 3 Å from a CDR region) (i.e., amino acids H1 to H73 of Table 1 and Table 3 in mu MAb), or (3) in the V | _-V {- (interface.

Other candidates for substitution are the acceptor human framework amino acids which are unusual in a human immunoglobulin at that position. These amino acids can be replaced by amino acids at the appropriate positions of the more typical human immunoglobulins. Alternatively, amino acids at the PB1.3 equivalent positions of the mu MAb may be introduced into the human framework regions if these amino acids are specific for the human immunoglobulin at the equivalent position.

In general, replacement of all or most of the amino acids that meet the above conditions is desirable. However, it is sometimes unclear whether a given amino acid meets the above requirement and alternative immunoglobulins need to be produced, one of which contains the substitution in question and the other does not. Surprisingly, a transformed human antibody PB1.3 (HgL0) contains only one substitution in the framework of the human heavy chain variable region, has only two substitutions in the framework of the human light chain variable region, and yet exhibits substantially similar (within two fold) binding affinities. a murine or chimeric antibody having intact murine variable regions fused to the human constant region (referred to as chi MAb PB1.3).

Some of the humanized antibodies of the invention contain a human light chain framework group substitution with the corresponding mu MAb PB1.3 group at least 1, 2 or 3, and sometimes 4 at positions L21, L46, L60 and L70 (see Figures 2 and Table 4). Some humanized antibodies generally contain a human heavy chain framework group substitution with the corresponding mu MAb PB1.3 group, at least 1, 2 or 3, and sometimes 4, at positions H1, H2, H71 and H73 (see Tables 1 and 3). ). Many humanized antibodies contain substitutions in both light and heavy chain variable framework regions. Preferred antibodies are one

human heavy chain framework substitution at least at position H71; and a human light chain framework substitution at least at positions 21 and 46.

In humanized antibodies, the CDR regions are generally substantially the same and, more often, the corresponding CDR regions in the mu MAb PB1.3 antibody. Although generally undesirable, it is sometimes possible to perform one or more conservative amino acid substitutions in the CDR groups without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Occasionally, substitution of CDR regions results in increased binding affinity.

Except for the specific amino acid substitutions discussed above, the framework regions of the humanized antibodies are generally substantially the same, and more often, the framework regions of the human antibody from which they are derived. Of course, many of the amino acids in the framework region do not contribute directly, or only marginally, to the specificity or affinity of an antibody. Thus, many conservative substitutions of the framework groups are tolerated without any appreciable change in the specificity or affinity of the resulting humanized immunoglobulin. However, these substitutions are generally undesirable.

(5) Production of variable regions

After selecting the CDR and framework components of humanized immunoglobulins on a conceptual basis, there are several different methods for preparing such immunoglobulins. Due to the degeneracy of the code, each immunoglobulin amino acid sequence is encoded by a number of different nucleic acid sequences. The desired nucleic acid sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of a previously produced variant of a desired polynucleotide.

Oligonucleotide-mediated mutagenesis is a preferred method for the preparation of substitution, deletion and insertion variants of the target polypeptide DNA (Adelman et al., 1983, DNA 2: 183). Briefly, the DNA of the target polypeptide is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize a complete second complementary strand of the template containing the oligonucleotide primer and encoding the desired change in the DNA of the target polypeptide.

(6) Select a constant region

The variable segments of the humanized antibodies prepared as described above are generally linked to at least a portion (Fc) of an immunoglobulin constant region, usually a portion of a human immunoglobulin. DNA sequences of the human constant region can be isolated from a variety of human cells using well-known techniques, but preferably from immortalized B cells (Kábát et al., 1987 and 1991, Sequences of Proteins of Immunological Interest, Bethesda, MD). , which are hereby incorporated by reference in their entirety for all purposes. Under normal conditions, the antibody contains both light chain and heavy chain constant regions. The heavy chain constant region generally includes the CH1, wrist, CH2, CH3 and CH4 regions.

Humanized antibodies include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype including IgG1, IgG2 , IgG-3 and IgG-4. If the humanized antibody is required to exhibit cytotoxic activity, the constant domain is usually a complement-fixing domain, and the class is typically IgG1. If such cytotoxic activity is not required,

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The humanized antibody may comprise sequences of one or more isotypes or classes.

(7) Expression system

Nucleic acids encoding humanized light and heavy chain variable regions, optionally linked to constant regions, are incorporated into expression vectors. The light and heavy chains may be cloned into the same or different expression vectors. DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector (s) to ensure expression of the immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer sequence, and a transcription termination sequence. Expression vectors generally replicate either as an episome or as an integral part of the host chromosomal DNA. Expression vectors generally also contain selectable markers, such as tetracycline or neomycin resistance, to detect cells transformed with the desired DNA sequences (U.S. Patent No. 4,704,362).

Escherichia coli is a prokaryotic host that is particularly suitable for cloning the polynucleotides of the present invention. Other suitable microbial hosts include bacillus such as Bacillus subtilis and other enterobacteria such as Salmonella, Serratia and various species of Pseudomonas. In these prokaryotic hosts, expression vectors can be prepared which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, a variety of well known promoters may be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the lambda promoter system. Promoters generally ··· ··· ·· * regulate expression, optionally with an operator sequence, have a ribosome binding site and the like, to initiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. The preferred host is Saccharomyces with suitable vectors containing control sequences such as promoters, including 3 phosphoglycerate kinase or other glycolytic enzymes, as well as an origin of replication, termination sequences and the like.

In addition to microorganisms, cell cultures of mammalian tissues can also be used to express and produce the polypeptides of the present invention. (1987)]. At present, eukaryotic cells are preferred because they have developed a number of appropriate cell lines capable of expressing complete immunoglobulins. Preferred host cells expressing the nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293) [Graham et al., J. Gene. Virol. 36, 59 (1977)]; baby hamster kidney cells (BHK, ATCC CCL 10); hamster ovary cell DHFR [CHO; Urlaub and Chasin, 1980, Proceedings of the National Academy of Sciences, USA 77, 4216; mouse sertoli cells [TM4; Mather: Bioi. Repr. 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 CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (# 138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast tumor (MMT 060562, ATCC CCL51); and TR1 cells [Mather et al., Annals N.Y. Acad. Sci. 383: 44-46 (1982)]; baculovirus cells.

Vectors containing polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) of interest may be transferred to a host cell by known methods, which will depend on the type of host. For example, calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment or electroporation can be used in other cellular hosts (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, NY, USA (1989). This publication is hereby incorporated by reference in its entirety. When the heavy and light chains are cloned in another expression vector, the vectors are co-transfected to achieve expression and formation of complete intact immunoglobulins. Following the introduction of recombinant DNA, cell lines expressing immunoglobulin products are selected. Preference is given to cells in which expression is stable (i.e., expression does not disappear even after fifty passages of the cell line).

Once expressed, whole antibodies, dimers, individual light and heavy chains, and other forms of immunoglobulin of the present invention may be purified by standard techniques known in the art, including ammonium sulfate precipitation, affinity chromatography, general gel chromatography and gel electrophoresis. : Scopes: Protein Purification, Springer-Verlag, NY (1982)]. Homogeneous pure immunoglobulins of at least 90-95% homogeneity are preferred, with a homogeneity of 98-99% or higher for pharmaceutical applications being most preferred.

The recombinant techniques described above can be used to express native sequences encoding human or murine antibodies. This approach is particularly advantageous for the expression of human antibodies isolated from unstable cell lines.

III, Antibody fragments

In another embodiment, fragments of the intact antibodies described above are produced. These fragments typically compete for specific binding to P-selectin with intact antibodies from which they are derived, at a concentration of at least 1θΛ 10θ, 10θ, ΙΟ ^ θ mol / L. Antibody fragments include the separate heavy chain, light chain Fab, Fab'F (ab ') 2, Fabc and Fv fragments. The fragments may be prepared by enzymatic or chemical treatment of intact immunoglobulin. For example, the F (ab ') 2 fragment can be prepared from the IgG molecule by proteolytic digestion with pepsin at pH 3.0 to 3.5 using standard methods [Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Pubs. N.Y. (1988)]. Fab fragments may be produced by limited reduction from F (ab ') 2 fragments or by papain digestion of whole antibody in the presence of reducing agents [Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Pubs. N.Y. (1988)]. The fragments can also be produced by recombinant DNA technology. Nucleic acid segments encoding the selected fragments can be prepared by restriction enzyme digestion of the full length coding sequences or by de novo synthesis. Fragments are often expressed as phage coat fusion proteins. Such expression is beneficial for the affinity sharpening of antibodies (see section IV).

ARC. Affinity-sharpened antibodies

Many of the immunoglobulins described above may undergo non-critical amino acid substitution, addition, or deletion, both in the variable and constant regions, without losing binding specificity or effector function or unacceptably decreasing binding affinity (i.e., 10 µM). kon37 • «

centered). Immunoglobulins containing such changes generally have substantial sequence identity with the reference immunoglobulin from which they originate. It is sometimes possible to select a mutated immunoglobulin that has the same specificity and enhanced affinity as the reference immunoglobulin from which it is derived. Phage display technology offers very suitable techniques for the selection of such immunoglobulins [Dower et al., WO 91/17271; McAfferty et al., WO 92/01047; Huse: WO 92/06204],

V. Hybrid Antibodies

The invention further relates to hybrid antibodies having the same specificity as blocking antibodies against P-selectin, but also capable of specific binding to a second moiety. In hybrid antibodies, a pair of heavy and light chains is generally derived from an anti-P-selectin antibody, and the other pair is derived from an antibody directed against another epitope. This results in polyvalence, i.e. the ability to bind to at least two different epitopes simultaneously, wherein at least one epitope is the epitope to which the blocking P-selectin antibody binds. Such hybrids may be prepared by fusion of hybridomas producing the appropriate component antibodies or by recombinant techniques.

Immunoglobulins may be fused to functional regions (e.g., enzymes) from other genes to produce fusion proteins (e.g., immunotoxins) that have novel properties.

VI. Nucleic acids

The intact antibodies and antibody fragments described above are often prepared by expression of nucleic acids. Any ellena38 · * · *

The substance or any of the antibodies disclosed in the present invention are expressly included within the scope of the invention. Modification of nucleic acids is readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (Gillma and Smith, Gene 8: 81-97 (1979); Roberts et al., Nature 328: 731-734 (1987)]. Many of the nucleic acids of the invention have substantial sequence identity to nucleic acids encoding the heavy and light chains of the mu MAb PB1.3, or nucleic acids encoding exemplified humanized derivatives derived therefrom.

VII. computers

The invention also relates to computers that are programmed to display a three-dimensional image of antibodies on a monitor or printer. An example is a Silicon Graphics IRIS 4D workstation running on a UNIX operating system using the QUANTA (Polygen Corp. USA) molecular modeling package. Computers can be well used to display models of variants of humanized antibodies. The antibodies of the present invention generally have sufficient binding affinity. However, it may be possible to identify antibodies with even greater binding affinity by further varying certain amino acid residues. Many non-critical amino acids can also be identified in the three-dimensional image, which can then be subjected to conservative substitution without substantially affecting the binding capacity of the antibody.

Vili. Production of large quantities of antibodies

To produce large quantities of antibodies, stable cells are produced that contain multiple copies of the genes encoding the heavy and light chain immunoglobulins in their chromosomes. Multiple copy numbers are obtained by amplifying the isolated region (s) of the chromosomal DNA of a cell. Amplification is achieved using a selection agent, such as methotrexate (MTX), which inactivates a cellular enzyme (e.g., DHFR) that is vital under certain growth conditions. Amplification, i.e., the accumulation of multiple copies of the DHFR gene, results in the production of a greater amount of DHFR in response to a greater amount of MTX. Amplification pressure was applied irrespective of the presence of endogenous DHFR, adding an increasing amount of MTX to the medium. Amplification of selected genes (i.e., genes encoding the heavy and light chains of the immunoglobulin) is achieved by linking each gene to a DHFR gene copy on the same or different plasmid (s) and co-transfecting the plasmid (s). Amplification of DHFR genes in response to MTX results in a concomitant increase in copy number of selected immunoglobulin genes. The increased copy number of the selected genes results in a higher level of expression of the heterologous protein of interest to us. Preferred stable cell lines, from which approximately 10 ⁶ cells ΙΟΙ 00, 20-50 or about 30 pg humanized immunoglobulin is expressed in a day.

IX. P-selectin fragments

The invention also relates to the production of P-selectin fragments. The fragments contain amino acids derived from the fifth C3b-C4b regulatory domain of P-selectin (fifth CRP). The fragments contain the epitope bound by mu MAb PB1.3 and / or adjacent epitopes bound by competing antibodies. The fragments generally contain a total of 5, 10, 20, 25, 50, 75, 100 or 200 amino acids. Most fragments contain all the amino acids between positions 448-467 of the Pselectin sequence shown in Figure 34.

These amino acids define the epitope specifically bound by mu MAb PB1.3. Many fragments contain at least five contiguous amino acids from positions 448 to 467 of the P-selectin sequence shown in Figure 34. Other fragments contain the entire contiguous amino acid segment between positions 448-467. Some fragments consist essentially of this contiguous amino acid segment. Only fragments that contribute to the binding specificity or affinity of the fragment are present in these fragments. Often no other amino acid is present.

The polypeptide fragments of the present invention have many different applications. The fragments are useful immunogens for the production of antibodies that compete with mu MAb PB1.3 for specific binding of P-selectin. The use of defined fragments as immunogens reduces the screening required to isolate an antibody with the required specificity. In addition, polypeptide fragments are useful in the diagnostic and therapeutic methods described below. Some fragments compete with full-length P-selectin molecules for binding to activated neutrophils. Thus, these fragments are useful in disrupting immune system diseases and conditions mediated by the interaction of P-selectin with its ligands on activated neutrophils. In addition, some fragments are useful for monitoring the presence of activated neutrophils by binding to Pselectin ligands on the surface of these cells. The small size of the fragments makes them particularly suitable for effective delivery after in vivo administration. The exclusive human origin of the fragments reduces the risk of side effects that may occur with other agents.

• ί. * ·. : ···. ·· • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • were were were were were were were were were founding on by Eden.

X. Therapeutic and diagnostic methods

Therapeutic methods employ antibodies (whole or binding fragments) and the P-selectin fragments discussed above as agents for treating various diseases. Therapeutic agents are useful for the prophylactic and therapeutic treatment of many different diseases and disorders of the immune system. Examples of such disease or disorder are graft rejection, graft versus cold disease, autoimmune diseases such as insulin dependent diabetes mellitus, multiple sclerosis, stiff manus syndrome, rheumatoid arthritis, muscle wasting and lupus erythematosus, and inflammatory disorders. The agents may also be used to prevent cancer metastases by preventing the adherence of circulating cancer cells, i.e., colon and melanoma carcinomas. Some therapeutic agents work by blocking or otherwise antagonizing the reaction of the P-selectin molecule with its ligand. Other therapeutic agents work by killing cells that contain the polypeptide that the agent targets.

Therapeutic agents are particularly useful for the treatment of inflammatory and thrombotic conditions, including post-ischemic leukocyte-mediated tissue injury (reperfusion injury), stroke, myocardial infarction, acute transplant rejection, heart failure, frostbite injury, pathophysiological conditions, acute leukocyte-mediated lung injury (i.e. adult respiratory distress syndrome), septic shock such as herpes simplex virus infection, wound-related sepsis, IgE-mediated allergic reactions such as acute phase asthmatic diseases and chronic inflammatory conditions including rheumatoid arthritis, atopic dermatitis and psoriasis.

.:: ··· ·· «. ϊ. · ···. ··. : »· ··· ....... ·„ ·., ·

Ischemic / reperfusion injury is an inflammatory condition that occurs when the blood flow in the interrupted organs causing ischemia (lack of oxygen) is restored. Unless quickly improved by reperfusion, ischemia causes the destruction of surrounding cells and ultimately the death of the entire organ or patient. However, there is increasing evidence that reperfusion itself may have deleterious effects on surrounding tissue. The deleterious effects of reperfusion are thought to result, at least in part, from an inflammatory response mediated by activated neutrophils in restored blood flow. Some patients have full-body ischemia, while in other patients, ischemia is limited to a limited part of the organs or body. For example, a patient may have epidermal, myocardial, renal, cerebral, spleen, liver, spinal cord, visceral, lung, body part, or whole body ischemia. The therapeutic agents of the invention work by antagonizing the interaction of such lymphocytes with P-selectin.

A major component of acute allergic conditions, including asthma, is the degranulation of breast cells following stimulation with antigens to which they are specifically sensitized. The consequences of mammary cell degranulation include a bronchoconstrictor response and an inflammatory response, which is partially characterized by leukocyte accumulation. Histamine, which is found in breast cells along with other inflammatory mediators, can induce P-selectin expression on vascular endothelial cells, indicating that breast cell degranulation can result in P-selectin expression and subsequent leukocyte accumulation. Since breast cell degranulation is a key element in the pathogenesis of allergic conditions, administration of antibodies against P-selectin may be useful in the treatment of human allergic conditions.

The methods are particularly useful in humans, but can also be practiced in veterinary subjects. Therapeutic methods are usually applied to the living subject · **. ; ···. ·· «...:. · ... ...: .. • ·· .... ... ·,. ·.,» Is applied to existing organs. However, some methods, such as ischemia reperfusion therapy, are equally applicable to excised organs, especially when these organs are awaiting transplantation into a recipient patient. Therapeutic methods may also be performed ex vivio.

Therapeutic agents and compositions against P-selectin can be used in combination with agents against other molecules, in particular humanized or human antibodies capable of reacting with various adhesion molecules. Suitable immunoglobulins include immunoglobulin specific for CD11a, CD11b, CD18, E-selectin, L-selectin and ICAM-1. Immunoglobulins must bind to the epitopes of these adhesion molecules in such a way as to prevent leukocytes, especially neutrophils, from binding to endothelial cells. Other suitable antibodies useful in the combination include, for example, antibodies specific to lymphokines, i.e., IL-1, IL-2 and IFN-γ, and their receptors. Such antibodies block the activation of endothelial cells, thereby preventing their interaction with neutrophils in an inflammatory response.

The blocking β-selectin antibodies and pharmaceutical compositions of the present invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular or intravenous administration. Many new drug delivery methods have been developed, and the pharmaceutical compositions of the present invention are also suitable for delivery by these new methods (Langer, Science 249: 1527-1533 (1990)).

The blocking P-selectin antibodies can be directly or indirectly coupled to the chemotherapeutic agent. The coupling, which can be accomplished by methods known in the art, should not substantially interfere with the ability of the immunoglobulin to bind to the receptor and to bind to the receptor.

- 44 - ^·: · no activity ..... 'chemotherapeutic agent inhibiting significantly. Many different chemotherapeutic agents can be combined to achieve the targeted effect. For example, switchable anti-inflammatory agents include immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors and leukotriene antagonists. Some preferred substances include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid and the like. Similarly, antioxidants such as superoxide dismutase are useful in the treatment of reperfusion injury. Similarly, anticancer agents such as daunomycin, doxorubicin, vinblastine, bleomycin and the like can be used for targeted treatment.

Targeting P-selectin can be accomplished with amphipathic or double-character (polar: apolar) molecules that exist as aggregates in aqueous solution. The amphipathic molecules include apolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules exist in the form of emulsions, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are commonly referred to as liposomes. In these formulations, the drug to be delivered is delivered as part of this liposome to which an anti-β-selectin immunoglobulin is added. In this insertion, the immunoglobulin does not need to bind to a functional epitope of P-selectin as long as the immunoglobulin effectively targets the liposome to the P-selectin molecule. When introduced into the vicinity of the affected cells, the liposomes release the selected pharmaceutical composition.

There are many different methods for preparing liposomes known in the art. Port. Biophys. Bioengineering. 9, 467 (1980); U.S. Patent Nos. 4,235,871; 4,501,728; 5,837,028, which are incorporated herein by reference. Targeting liposomes with a variety of targeting agents (i.e., ligands, receptors, and monoclonal antibodies) is well known in the art (see U.S. Patent Nos. 4,957,773 and 4,603,044, which are incorporated herein by reference). Standard methods for blindly coupling targeting agents to liposomes are used. For example, antibody-directed liposomes can be prepared using, for example, liposomes incorporating protein A (Rennisen et al., 1990, Journal of Biological Chemistry 265: 16337-16342; Leonetti et al., 1990, Proceedings of the National Academy of Sciences, USA 87: 2448-2451.

Pharmaceutical compositions for parenteral administration generally comprise a solution of a therapeutic agent (e.g., an antibody to P-selectin (intact or binding fragment, or a P-selectin fragment)) or a solution of a mixture of several such agents in a suitable carrier, preferably an aqueous carrier. . A variety of aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally do not contain particulate matter. These formulations may be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable excipients as necessary to maintain appropriate physiological conditions, such as pH adjusting and buffering agents, osmotic pressure adjusting agents and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, lactate and the like. The concentration of antibody in these formulations may vary widely, e.g. from less than 0.5% in general or at least about 0.1%, up to 1.5 or 2.0% by weight, and in particular fluid volume, viscosity, etc. selected according to the route of administration chosen. Current methods of preparing parenteral formulations are known to those skilled in the art and are described in detail in several publications (e.g., Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pennsylvania (1985), which is incorporated herein by reference).

The antibodies of the present invention may be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective for conventional immunoglobulins and to employ lyophilization and dissolution techniques known to those skilled in the art. Lyophilization and dissolution may result in the loss of some of the activity of the antibody (e.g., conventional immunoglobulins, IgM antibodies can lose more of their activity than IgG antibodies) and the level of use should be adjusted accordingly.

Formulations containing the present antibodies, or mixtures thereof, may be administered to humans for prophylactic and / or therapeutic purposes. In medical applications, the compositions are administered to a patient in an amount sufficient to cure or at least partially stop the infection and complications. An amount sufficient to accomplish this is referred to as a therapeutically effective dose. Suitable amounts for this purpose will vary depending on the severity of the disease and the general condition of the patient's own immune system, but will generally range from 0.05 mg / kg to about 5 mg / kg, preferably 0.2-1.5 mg / kg. changes.

The substances of the present invention are generally used in the treatment of serious illnesses, that is, in life-threatening or potentially life-threatening situations. In such cases, given the minimal amount of foreign matter and the lower likelihood of foreign matter rejections (such as HAMA) that can be achieved with the human or humanized antibody forms of the present invention, it is possible and considered necessary by the attending physician to: these substances are administered in significant excess.

In prophylactic applications, compositions comprising the antibodies or mixtures thereof of the present invention are administered to a patient who is no longer in a condition such that the patient's resistance can be enhanced. This amount is defined as the prophylactically effective dose. In this application, the exact amount will depend on the patient's state of health and general immunity, but will generally fall within the aforementioned range.

Single or multiple administrations of the formulations may be accomplished at dosage levels and frequency selected by the attending physician. In all cases, the pharmaceutical compositions should provide an amount of the immunoglobulin of the present invention sufficient to effectively treat the patient.

Antibodies (whole or binding fragments) of the present invention as well as fragments of P-selectin can also be used for diagnostic purposes. An amount sufficient for these purposes is defined as a diagnostically effective dose. In diagnostic applications, the exact amount will depend upon the patient's health, the mode of administration, and the like. The antibodies can be labeled and unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibody) that are capable of reacting with the antibody, that is, specific to the constant region of a given immunoglobulin. Alternatively, the antibodies may be directly labeled. A variety of labels can be used, such as radioactive atoms, fluorescence markers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens).

Antibodies (whole or binding fragments) are useful in detecting the presence of cells bearing the P-selectin receptor. The presence of such cells is useful in diagnosing an inflammatory condition or disease and may indicate the need to initiate one of the therapeutic treatments described above. Diagnosis can be made using a cell sample removed from the patient. The amount of P-selectin receptor expressed by individual cells of the sample is determined, for example, by immunohistochemical staining of fixed cells or by Western blotting of the cell extract with the antibody of the present invention. Pselectin fragments are useful in detecting the presence of activated neutrophils, suggesting an undesired immune response.

The diagnosis can be made by in vivo administration of a labeled antibody (preferably humanized or human antibody) and imaging in vivo. The concentration of MAb administered should be sufficient to detect binding to cells containing the target antigen relative to the background noise. The diagnostic reagent may be labeled with a radioactive isotope for camera imaging or may be labeled with a paramagnetic isotope for nuclear magnetic resonance or electrospun resonance imaging.

In a cellular sample or imaged from an individual, a change (usually an increase) in P-selectin protein levels outside the clinically accepted range may indicate the presence of an adverse inflammatory response in the sampled patient and / or indicate to the patient the tendency to develop (or undergo) such a reaction. P-selectin can also be used as a differentiating marker to identify and typify cells belonging to a particular cell line and origin. Such cell-specific detection can be used to histopathologically diagnose unwanted immune responses.

Kits can also be prepared using the antibodies of the present invention so that the antibody composition of the present invention can be prepared, usually in lyophilized form, in a container, either alone or with additional antibodies specific for the selected cell type. Antibodies, which may be conjugated to a label or a toxin, or may not be conjugated, may be included in the kit with buffers such as TRIS, phosphate, carbonate, etc. buffers, stabilizers, biocides, inert proteins such as serum albumin and the like, and may include instructions for use. These substances are generally present in an amount of less than 5% by weight, based on the active antibody, and generally present at least 0.001% by weight, again based on the concentration of the antibody. It is often desirable to include an inert filler for diluting the active ingredient, wherein the filler is present in an amount of from 1 to 99% by weight based on the total composition. Where an antibody that is capable of binding to an anti-P-selectin antibody is used as the second antibody in the assay, the second antibody will generally be in a separate container. The second antibody is generally conjugated to a label and formulated as above.

The following examples are provided by way of illustration and are not intended to limit the scope of the invention.

Example 1

Preparation of reagents

This example describes the preparation of reagents used in the ELISAs in Examples 2 and 3 and in Example 4.

A. Expired platelets

Platelet preparation purified from San Diego Blood Bank expired platelets. The method used is a modification of the method described by Moore et al., J. Cell Biol. 112, 491-499 (1991)], which is incorporated herein by reference. Briefly, 25 units of expired, platelet-rich plasma is centrifuged twice at 1200 rpm (300 x g) for 10 min to remove contaminating non-platelet blood cells. The purified platelet preparation is then washed three times with a buffer containing 0.1 M NaCl, 20 mM TRIS and 5 mM benzamide. The solution also contains 3.8% sodium citrate. The pH is adjusted to 7.5 with 1 M hydrochloric acid. Purified platelets were stored at -70 ° C and washed once with PBS before injection.

B. Purified P-selectin

Fractionation of platelets

The washed expired platelets are essentially fractionated by the method described by Moore et al., J. Cell Biol. 112, 491-499 (1991)], then 25 µl of washed platelet suspension is adjusted to 100 µL of leupeptin and 1 mM of 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF), then frozen three times and thawed in dry ice. / methanol, then homogenize with a Dounce homogenizer in ten turns. The slurry was then centrifuged on a Beckman Ti 70 rotor at 4,000 C at 35,000 rpm for 60 minutes. The supernatant from this centrifugation is the starting material for purification of soluble Pselectin. The pellet was dissolved in 30 mM MnCl2, 1 mM CaCl2, 5 mM benzamidine-HCl, 0.1 M NaCl, 20 mM TRIS, 2% Triton X-100, pH 7.5 slurry, homogenize by 10 pulls with a Dounce homogenizer, and incubate for 1 hour at 4 ° C. The suspension is centrifuged for 1 hour on a Beckman Ti 70 rotor at 4,000 rpm. The centrifuge from this! supernatant from the starting material for the isolation of membrane-bound P-selectin.

Isolation of P-selectin from fractionated platelets

Soluble and membrane-bound P-selectin is further purified separately, by the following procedures, or some of them. Non-ionic Rennex 30 detergent (Accurate Chemical and Scientific Corp.) is also added to all buffers used to isolate membrane-bound P-selectin. Each buffer also contains 1 mM CaCl2 and 1 mM MnCl2. Fractions containing P-selectin were detected by Western blotting using PNB 1.6 monoclonal antibody.

Lens lectin chromatography

Plasma soluble P-selectin supernatant or membrane-bound P-selectin detergent extract from 25 units is passed through a column (1.5 x 5.5 cm) containing lensin from Lens culinaris (lentil lectin) bound to Sepharose 4B ( Sigma Chemical Co., L-0511). The column was then washed three times with 150 mL of 20 mM TRIS, 0.1 M NaCl, pH 7.5. The bound glycoproteins are then eluted with 10 x 3 ml aliquots of 20 mM Tris, 0.5 M NaCl, 0.5 M alpha-methyl-D-mannopyranoside, pH 7.5. The fractions containing P-selectin were concentrated to 1.5-2 ml using an Amicon Centriprep 30 apparatus.

Gel filtration chromatography

Concentrated fractions from lens lectin chromatography were injected onto a Toso Haas G3000 SW HPLC column (21.5mm x 30cm) equilibrated with 20mM TRIS, 0.1M NaCl, pH 7.5. The column is equilibrated with the same buffer at a flow rate of 0.75 ml / min.

1.5 ml fractions were collected and analyzed by Western blot. The fractions containing P-selectin were pooled and concentrated to 1.5-2 ml with an Amicon Centriprep 30 centrifuge and several buffer exchanges (buffer composition 20 mM TRIS pH 7.5).

Heparin-Agarose chromatography

This sample was applied to a 1 ml Heparin-Agarose column in 20 mM TRIS, pH 7.5, and the column was washed with the same buffer and 20 mM TRIS, 1.5 M NaCl , pH 7.5.

Anion-exchange chromatography

The fractions were dialyzed into 20 mM TRIS pH 7.5 buffer and applied to a Toso Haas DEAE-5PW column (7.5 mm x 7.5 cm) loaded with 20 mM TRIS pH 7.5 and equilibrated with a salt gradient up to 1 M NaCl. The fractions containing P-selectin were combined and concentrated on an Amicon Centriprep-30 and stored at -80 ° C.

Cation exchange chromatography

Samples were dialyzed into 10 mM phosphate pH 7 buffer and loaded on a Poros CM / P (4.6 / 100) column equilibrated with the same buffer and eluted with a gradient of up to 1 M NaCl.

C. Isolation and activation of fresh human platelets

All chemicals used in platelet isolation were purchased from Sigma Chemical Company (St. Louis, MO).

1. Take 42 ml of blood from a voluntary blood donor into a syringe containing 7 ml of Acid Citrate Dextrose Anticoagulant (ACD).

Preparation of ACD anticoagulant

Dextrose 2.0 g

Sodium citrate 2.49 g

Citric acid 1.25 g

Make up to 100 ml with distilled water.

2. Remove the needle and transfer the blood to two sterile 50 ml tubes.

3. Centrifuge the tubes with an IECC-6000 centrifuge used with a 921 head (radius = 17.2 cm) at 800 rpm (approximately 90 x g) for 15 minutes at room temperature with the brake off.

4. Remove the supernatant with a plastic pipette. The supernatant is removed so as to be as close as possible to the transition layer.

5. Centrifuge the supernatant at 1200 rpm (about 300 x g) for 6 minutes.

6. Remove the supernatant.

7. Centrifuge the supernatant at 2000 rpm (approximately 1200 x g) for 10 minutes. Platelets appear as cellular cells at the bottom of the tube. The supernatant was discarded.

8. Wash the platelets as follows: 2 ml Tyrode-Hepes buffer, pH 6.5, containing Prostaglandin Eq (PGE-) at a final concentration of 100 nmol / L, and carefully resuspend the platelets. An additional 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 8.0 g KCI 0.2 g NaH2PO4 χ H2O 0.057 g MgCl 2 x 6 H2O 0.184 g NaHCO> 3 0.1 g saccharose 1.0 g HEPES 2.383 g

Make up to 1 liter with distilled water and adjust the pH to 6.5 with 1 M NaOH.

9. Step 8 is repeated once more and the platelets are washed once with the same buffer without PGE.

10. Platelets are counted with a Coulter Counter and diluted to 2x108 / ml.

11. Adjust the pH of the platelet suspension to 7.2 and determine the amount of thrombin required for maximum activity (Human Thrombin, Sigma). There is some degree of donor variation, but optimum activation is obtained in the range of 0.25-0.5 units / ml. Maximum activation, i.e. maximum thrombin-induced aggregation, appears to correspond to maximal Pselectin expression.

12. The desired amount of thrombin is added to the platelet preparation, and the mixture is allowed to stand at room temperature for 20 minutes without agitation to prevent aggregation.

• «· ···

Example 2

Preparation of blocking P-selectin antibody

This example describes the preparation of mu MAb PB1.3, a monoclonal antibody to Pselectin that inhibits the binding of thrombin-activated platelets to neutrophils.

An RBF / DnJ male mouse (from Jackson Laboratories) was used as a source of antibody-producing cells and immunized (all injections intraperitoneally) as follows:

1. Month 1 - 200 μΙ packed expired platelet containing approximately 6x10 ^ platelets,

2. Month 2 - 250 μΙ expired platelet (8x10θ platelet),

3. Month 4 - 250 μΙ expired platelets (8x10 ^ platelets),

4. Month 5 - Soluble P-selectin fraction isolated on lens lectin,

5. Month 6 - Soluble P-selectin fraction, lectin lens, purified by gel filtration, heparin agarose, and ion exchange chromatography.

Preparation of all materials used for immunization is described in Example 1.

A. Myeloma fusion cell line

FOX-NY is a murine myeloma cell line deficient in adenosine phosphoribosyl transferase (APRT) and hypoxanthine phosphoribosyl transferase (HPRT) from the American Type Culture Collection (CRL 1732) and maintained in RPMI 1640, It contains 10% calf serum (Hyclone) and 1% L-glutamine.

• · · · · ·

B. Cell fusion procedure

Four days after the last excitation, the spleen was removed and 1.2 x 10 3 splenocytes were recovered. They were fused with FOX-NY myeloma cells using PEG 1500 (BMB) as follows: The isolated splenocytes were washed twice in serum-free cell culture medium. Splenocytes and myeloma cells in a ratio of 1: 4.8 (myeloma spleen cell). The pooled cell pellet was washed twice in serum-free medium and aspirated to dryness. The cell pellet was resuspended by gentle tapping and the temperature was raised to 37 ° C for 1 minute in a water bath. The pellet was distributed along the sides and bottom of a 50 ml conical centrifuge tube. One ml of PEG 1500 (50% w / v in 75 mM HEPES, BMB Batch No. 14702800) previously heated to 37 ° C was added over 60 seconds while rotating the tube to maintain the thin layer of cells. Serum-free medium is added slowly over 60 seconds. An additional 1 ml of medium was added, a little faster. Another 8 mL of medium was added, and the tube was allowed to stand undisturbed for 8 minutes and centrifuged at 300xg for 5 minutes. The final pellet was resuspended in RPMI 1640 containing 10% fetal calf serum (Hyclone), 1% L-glutamine, 1% sodium pyruvate and 1xAAT (SIGMA). Cells were plated in 10 flat bottom 96-well microtiter plates (COSTAR). We do not use nourishing cells.

Example 3

Preparation of non-blocking ant-P-selectin antibody

This example describes the preparation of a monoclonal antibody to mAB PNB1.6 against P-selectin, which does not inhibit the binding of thrombin-activated platelets to neutrophils.

• · ·· ί —— τ _ Σ _ »·» ·

- 57 _ · * · ♦ ** · ·· «·· ··.

An RBF / DnJ male mouse (from Jackson Laboratories) was used as a source of antibody producing cells and immunized as follows (all injections intraperitoneally):

1. Month 1 - 100 μΙ thrombin-activated, freshly isolated platelet containing approximately 3x10 ⁹ platelets,

2. Month 2 - 200 μΙ thrombin-activated fresh platelets (6x10 ⁹ platelets),

3. Month 4 -100 μΙ thrombin-activated fresh platelets (3x10 ⁹ platelets).

Four days after the last excitation, the spleen is removed and the splenocytes are harvested. FOX-NY is fused with myeloma cells using PEG 1500 (SIGMA), generally as described by Oi et al., (1980) ImmunoglobulinProducing Hybrid Cell Lines, Selected Methods in Cellular Immunology, 351-372. , the publication of which is hereby incorporated by reference.

Example 4

Screening for antibodies

In this example, a screening assay of the supernatant from the fused cells prepared in Examples 2 and 3 is described. The supernatants from Example 2 were assayed by ELISA for (a) thrombin-activated platelets, (b) purified P-selectin, and (c) recombinant P-selectin. Supernatants from Example 2 were assayed for thrombin-activated platelets by ELISA. Each such assay is described below. The preparation of reagents used in each assay is described in Example 1.

· «·

A. Thrombin-activated platelets

1. Cover a 96-well flat bottom COSTAR plate with 0.1% gelatin (2% gelatin Sigma), add 100 μΙ per well, and incubate for 15 minutes at 37 ° C.

2. Gelatin is removed and 100 µl of thrombin-activated platelet (ΙΟθ / ml) is added to each well.

3. Incubate the plate for 15 minutes at 37 ° C.

4. Incubate the plate at 800 rpm (90 x g) for 2 minutes.

5. Unbound platelets are removed and the plate is washed twice

PBS.

6. Add 100 µl PBS containing 1% BSA to each well and allow the plate to stand at room temperature for 60 minutes.

7. Add 100 μΙ of supernatant to each well. Place the plate on a shaker for 60 minutes at room temperature.

8. Wash plate four times with PBS.

9. Add 100 μΙ peroxidase-conjugated goat anti-mouse IgG diluted 1/1000 in PBS to each cell. Allow the plate to stand for 30 minutes at room temperature.

10. Wash plate seven times with PBS.

11. Add 100 μΙ ABTS substrate system (Kirkegaard and Perry, Laboratories, Inc.) to each well.

12. Allow the plate to develop at room temperature for 30 minutes.

13. OD was measured at 414 nm with a Titertek Multiskan MCC / 340.

B. Purified P-selectin

1. Soluble or membrane-bound P-selectin in DPBS (Dulbecco's phosphate-buffered saline is a solution of phosphate-buffered saline (PBS) containing 1 mmol / l CaCl2 / 0.5 mmol / l) isolated by chromatography on lens lectin, gel filtration and DEAE columns. 1M MgCl2) was diluted 1:50 to 1: 1000 and covered with Falcon wells overnight at 4 ° C.

2. The plates are then blocked with DBPS + 1% BSA for 1 hour or more.

3. The supernatants to be assayed are then added to the wells and incubated for 30-60 minutes at room temperature.

4. The plates are incubated with DBPS, then sheep anti-mouse IgG horseradish peroxidase conjugate (Sigma A-6782, 1: 1000 in DBPS + 1% BSA) is added to the wells and the plates are incubated for 1 hour at room temperature.

5. Plates are then washed with DBPS and TMB Microwell Substrate

System (Kirkegaard and Perry Laboratories, Inc.).

C, Recombinant P-selectin

1. A ninety-six-well flat bottom plate was coated overnight at 4 ° C with 100 μΙ P-selectin obtained by combining 239 cell clones previously transfected with the recombinant P-selectin gene and introducing it into serum-free medium. This supernatant contained at least 8 ng / ml P-selectin and was coated with COSTAR plates either undiluted or diluted in medium at T.8.

2. Wash plate once with Dulbecco's PBS (DPBS) and block at room temperature for 30 minutes with 200 μΙ DPBS containing 1% BSA.

3. The disk is then set out in section A, pages 7-13. Prepare for the ELISA according to step.

D. Isotype analysis of MABs against P-selectin

Analysis of mu MAb PB1.3 and PNB1.6 shows that both monoclonal antibodies belong to the murine IgG1 immunoglobulin subclass. Isotype analysis was performed using the Boehringer Mannheim Mouse-Hybridoma-Subtyping Kit.

Example 5

Detection of P-selectin

In this example, the detection of P-selectin by Western blot is described. Samples containing Pselectin were mixed with an equal volume of SDS-PAGE sample loading buffer (non-reducing), heated to 100 ° C, run on an Npvex 8-16% gradient gel and electrophoretically transferred to nitrocellulose. The nitrocellulose membrane is then blocked with phosphate buffered saline + 1% bovine serum albumin (BSA) for at least 1 hour. The membrane is then incubated at room temperature for 1 hour or more, either with tissue culture supernatants of hybridomas expressing the appropriate monoclonal antibody or with purified antibody (5 pg / ml) in PBS + 1% BSA. The membrane was then washed several times with PBS + 1% BSA and incubated for 1 hour in PBS + 1% BSA diluted 1: 1000 in sheep anti-mouse IgG horseradish peroxidase conjugate (Sigma A-6782). The membrane is then washed and the bands are visualized with tetramethylbenzidine / membrane enhancer (Kirkegaard and Perry Laboratories, Inc.).

A protein of 140 kDa was detected in both mu MAb PB1.3 and mu MAb PB1.6 tissue culture supernatants. This could be detected in both solubilized platelet membrane formulations and purified P-selectin.

Example 6

In vivo inhibition of acute inflammatory injury with blocking P-selectin antibody

In this example, it is reported that the blocking antibody P-selectin of the invention can be used to treat acute lung injury. In this example, systemic complement activation is obtained by infusion of cobram venom factor (CVF) into rats. In this model, the lesion develops rapidly, and is known to be dependent on the toxic oxygen products produced by neutrophils.

Two monoclonal antibodies described in Examples 2 and 3 were used for these assays. Of particular importance for this assay is that the mu MAb PB1.3 blocking P-selectin antibody also inhibits the binding of thrombin-activated rat platelets to human neutrophils (Figure 1). These data suggest that mu MAb PB1.3 recognizes a conserved Pselectin epitope, and that a blocking P-selectin antibody may be useful in the treatment of other inflammatory diseases.

For in vivo experiments, 20 units of CVF / kg body weight were infused intravenously into 300 g adult male Long-Evans rats. This results in rapid intrapulmonary, intravascular seizure of blood neutrophils, resulting in damage to the intercellular endothelial cells at the physical contact points of ·· ··· endothelial cells and neutrophils. When used, the mu MAb PB1.3 and PNB1.6 monoclonal antibody was infused intravenously in the indicated amount, together with CVF, in a total volume of 0.5 ml. Negative control animals received 0.5 ml intravenous infusion of phosphate buffered saline (BPS). In each case, the infused material contained trace amounts (125). h _{bovine serum} albumin and homologous (51Cr) -red blood cells (RBC). Lung injury parameters (albumin leakage and RBC extravasation) were determined after 30 minutes using the technique of Mulligan et al., J. Clin. Invest. 88, 1396 (1991)].

The results of these experiments are shown in Figure 2A. 2B and 2B. Figures 3 to 5 are shown. If 200 pg PNB1.6 (the non-blocking P-selectin antibody) is co-infused with CVF, it cannot reduce the rate of lung injury compared to untreated CVF positive controls. Thus, animals receiving CVF PNB1.6 in PBS give reference positive control values. Co-infusion of 100 pg mu MAb PB 1.3 with CVF resulted in a 19.2 percent (p = 0.002) reduction in permeability and a 37.5 percent (p = 0.001) reduction in bleeding (unpublished data). When 200 pg mu MAb PB1.3 was added, permeability was reduced by 50.8 percent (p <0.001) (Figure 2A), bleeding by 70.0 percent (p <0.001) and 70.1 percent (p < 0.001) (Figure 2B).

Lungs from animals in this group are homogenized and myeloperoxidase activity (MPO) is measured to provide information on the lung neutrophil cell content. MPO is determined by the decomposition of H2O2 in the presence of o-dianizidine using standard procedures [Warren et al., J. Clin. Invest. 84, 1873 (1989)]. As shown in FIG. As shown in Figures 1 to 4, treatment with 100, 200 or 400 pg mu MAb PB1.3 was 26 percent (p = 0.024), 41 percent (p <0.001), and 50 percent (p <0.001) compared to the reference (untreated) positive control group ) reduces MPO content. MPO content was not reduced in 200 pg PNB1.6 treated animals (Fig. 3A), consistent with the fact that this antibody is unable to protect against CVF-induced lung injury. Thus, the protective effects of mu MAb PB1.3 blocking antibody are consistent with its ability to interfere with the accumulation of neutrophils in lung tissue. Transmission electron microscopic examination of lung sections confirms that treatment with mu MAb PB1.3 results in decreased accumulation of neutrophil cells in the interstitial capillaries of the lungs, fails to adhere neutrophils to endothelial cells, and reduces evidence of damaged endothelial cells. ).

To test for P-selectin expression in the lungs after intravenous injection of CVF, an additional group of rats was infused with CVF and animals were sacrificed at 0, 5, 10, 15, 20 and 60 minutes later. The lungs were inflated with O.T.C, frozen immediately, sections were harvested, and immunohistochemistry assayed for the presence of P-selectin using mu MAb PB1.3. At time 0, very little detectable reactivity was found in the pulmonary vessels, while staining was clearly evident after 5 minutes and increased 15 and 20 minutes after infusion of CVF. The staining pattern includes the small lungs and the septal areas, with a pattern that is similar to that of the interstitial capillaries. After 60 minutes, the staining largely disappeared (data not shown). Although P-selectin is probably present in intracellular storage granules prior to CVF infusion, it is clear that mobilization of P-selectin to the surface of the endothelium drastically enhances the reactivity of mu MAb with PB1.3 antibody.

··· * · «• · · · · · · · · · · · · · · · ·

Example 7

Comparative binding test

Isolation of platelets from human blood, activation of isolated platelets with thrombin, and isolation of PMNs (neutrophils) are described in Example 1 above.

INCUBATION OF THROMBIN ACTIVATED BLOOD PLATES WITH MONOCLONAL ANTI-P-SELECTIN

the. 20-20 µL of thrombin-activated platelet (2x10 µl / ml) were added to 24 Eppendorf tubes (1.5 ml) in duplicate.

b. To eight of these tubes, 20 µl of the IgG fraction isolated from PNB1.6 was added at a concentration of 10 µg / ml in Tyrode-HEPES buffer, pH 7.2, containing 1 mM CaCl 2. To the second tube is added 20 µl of the same IgG preparation at a concentration of 1 pg / ml. To the third eight tubes, only 20 µl of buffer is added.

c. Mix and allow to stand at room temperature for 20 minutes.

d. To each of the eight tube groups was added 20 µl of the IgG fraction purified from mu MAb PB1.3 in a concentration range of 10 pg / ml to 0.03 pg / ml.

e. Mix and allow to stand at room temperature for 20 minutes.

f. 20 µl of neutrophil cells are added at 3 x 10 6 / ml.

g. Mix and allow to stand at room temperature for 20 minutes.

h. The microscope is evaluated as follows; 100 neutrophils were counted in each sample. Record a cell as positive if two or more platelets are attached, and negative if less than two platelets are attached. Calculate the percentage of positive cells.

.:. · *. : · ". ··: ··· • · *** · * · ··· •« «« · ··> * * ·· * ·· *

As shown in Figure 1, pre-treatment of thrombin-activated platelets with mu MAb PB1.3 does not affect the ability of mu MAb PB1.3 to block platelet binding to P-selectin.

Example 8

Characterization of the binding of blocking P-selectin antibodies (a) Purification of monoclonal antibodies: PNB1.6 and 84/26 monoclonal antibodies were isolated from tissue culture supernatants by passage through a protein G Sepharose 4 Fás Flow (Pharmacia) column. The column was washed extensively with phosphate buffered saline (PBS) and the bound antibody was eluted with 0.1 M glycine-HCl, pH 2.7, 0.2-0.5 volumes of 1 M TRIS. (pH 8.8). The procedure used for the isolation of mu MAb PB1.3 is the same except that the protein G-bound antibody is eluted with 0.1 mol / L acetate-HCl, pH 2.5, at 0.3 volumes of 2 mol / L Tubes containing TRIS (pH = 10). The antibody-containing fractions were pooled and dialyzed against PBS at 4 ° C with multiple buffer exchanges.

(b) Preparation of mu MAb PB 1.3 affinity column: For use in purification of P-selectin, mu MAb PB1.3 was coupled to tresyl-activated agarose according to the manufacturer's affinity description (Schleicher and Schuell).

(c) Purification of P-selectin: 1/100 volumes of 5 mg / ml leupeptin and 1/100 volumes of 35 mg / ml AEBSF (Calbiochem) for the washed, expired human platelets prepared above. After mixing, the platelets are freeze-thawed three times in succession, or homogenized in a dry ice / methanol bath or liquid nitrogen in ten turns in a Dounce homogenizer, and centrifuged at 35,000 rpm for 1 hour at 4 ° C in a Beckman 70 TI rotor. The pellet was in Dulbecco's phosphate buffered saline (DPBS, Whittaker Bioproducts containing approximately 0.9 pmol / l Ca ⁺⁺ and 0.5 mmol / l Mg ⁺⁺ ) followed by 5 mM benzamidine-HCl, 100 pmol / l leupeptin and 2% Triton X-100 are adjusted. The suspension is resuspended and homogenized by 10 pulls in a Dounce homogenizer. After incubation for 1 hour at 4 ° C, the solution is centrifuged again in a Beckman 79 TI rotor (1 hour, 4 ° C, 35,000 rpm). The supernatant was passed through a mu MAb PB1.3 agarose column previously equilibrated with DPBS + 0.05% Rennex 30 (Accurate Chemical and Scientific Corp.). The column is washed extensively with DPBS + 0.05% Rennex 30, and the bound P-selectin is eluted with 0.1 mol / L triethylamine-HCl, 0.05% Rennex 30 pH 11.5. Pipes containing 1 M phosphate buffer (pH 8.6). The fractions containing P-selectin were pooled, dialyzed against DPBS + 0.05% Rennex solution, concentrated in a Centricon 30 or Centriprep 30 centrifugal concentrator (Amicon), aliquoted and stored at -80 ° C.

(d) Preparation of CQNRYTDLVAIQNKNE Peptide (Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-lle-Gln-Asn-Lys-Asn-Glu-NH?)

The peptide was synthesized using Applied Biosystems (Foster City, CA) using a peptide synthesizer 430A using Fmoc protected amino acids and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBATU) esters. to activate the amino acids. Each amino acid is routinely coupled twice. Fmoc-protected amino acids and hydroxybenzotriazole are purchased from Applied Biosystems.

All solvents are purchased from Burdick and Jackson. HBTU is purchased from Richelieu Biotechnologies (St. Hyacinthe, Canada). Piperidine and trifluoroacetic acid, acetic anhydride, thioanisole, phenol and ethanedithiol are purchased from Sigma Chemical Corporation.

• · · · · · ·

• · · · ·

Fmoc-Amide resin (Bachem Biosciences) was charged into the peptide synthesis reaction vessel and washed once with N-methylpyrrolidone (NMP). Then do the following:

1. Removal of the Fmoc protecting group by treatment with a resin-bound amino acid solution in 25% piperidine in NMP.

2. Wash the resin five times with NMP.

3. Add a mixture of N-α-Fmoc-L-glutamic acid, γ-t-butyl ester, diisopropylamine, HBTU and NMP, and allow to react for 30 minutes with vortexing.

4. The solution is aspirated and the resin is washed three times with NMP.

5. Steps (3) and (4) are repeated two more times.

6. Wash the resin four times with NMP.

Steps 1-6 are repeated for each amino acid of the paptide. Following the final coupling cycle, the resin-bound peptide is deprotected with 25% piperidine in NMP, washed 7 times with NMP, and washed twice with dichloromethane. The resin was dried under reduced pressure for 24 hours. The peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% ethanediol, 5% thioanisole, 7.5% phenol and 5% water. The polystyrene resin is separated from the trifluoroacetic acid solution by filtration. The trifluoroacetic acid was removed by evaporation under reduced pressure. The crude peptide was triturated with diethyl ether and dissolved in water. The water is removed by lyophilization. The peptide was then purified by reverse phase HPLC on a Cq column (VYDAC) using a gradient of acetonitrile in water, each containing 0.1% TFA as modifier.

(e) Biotinylation of the monoclonal antibody: Antibodies are dialyzed against 0.1 M sodium bicarbonate, pH 8.5. 0.3 mg / ml Concentration 68 -.: ..... · · .. · ....

NHS-LC Biotin (Pierce) was added. After 2 hours at room temperature, the antibodies were dialyzed against several times exchanged PBS.

(f) Coating microtiter plates for ELISA: 96-well microtiter plates (Falcon Microtest III) were coated overnight at 4 ° C with 50 μΙ / well in 2 μρ / ηηΙ affinity-purified P-selectin (DPBS solution).

(g) Western blotting: mu MAb PB1.3, PNB1.6, and monoclonal antibodies 84-86 bind to a single 140 kDa band, when platelets are dissolved in SDSPAGE run buffer (non-reducing) and plated on SDS-PAGE. and transferred to a nitrocellulose membrane (unpublished data). It was also found that mu MAb PB1.3 and PNB1.6 no longer recognize P-selectin on Western biota when samples are reduced with β-mercaptoethanol.

II. Effect of chelating divalent cations on the binding of monoclonal antibodies to P-selectin: Two microtiter plates are covered with affinity purified P-selectin. Plate 1 is blocked with 200 μΙ / well PBS + 1% BSA and second plate is blocked with 200 μΙ / well DPBS + 1% BSA (DPBS also contains Ca ⁺⁺ and Mg ⁺⁺ ). After 1 hour the plates are washed (plate 1 always washed with PBS, plate 2 always washed with DPBS). 25 μΙ 25 mM EDTA (PBS + 1% BSA) was added to wells of plate 1 and 25 μΙ 25 mM EDTA (DPBS + 1% BSA) was added to wells 2. plate for holes. After one hour, dilute 25 μΙ of the appropriate antibody to the wells of each plate. Plate 1 dilutions were performed in PBS + 1% BSA, and Plate 2 was diluted in DPBS + 1% BSA. After one hour, the plates are washed and 50 μΙ of a 1: 1000 dilution of sheep anti-mouse IgG horseradish peroxidase conjugate is added (dilution in PBS + 1% BSA, dilution in DPBS + 1% for Plate 2). BSA). After 1 hour, the plates were washed and 50 μΙ peroxidase substrate (TMB, 3,3 ', 5,5'-tetramethylbenzidine, Kirkegaard and Perry Laboratories, Inc.) was added. After sufficient color development, the substrate reaction is stopped by the addition of 25 μΙ 1 M phosphoric acid and the absorbance at 450 nm is read.

Figure 4 shows the binding of anti-P-selectin monoclonal antibodies to P-selectin in DPBS buffer containing Ca ⁺⁺ and Mg ⁺⁺ ions and PBS + 15 mM EDTA, which forms chelates with divalent metal ions . Concentrations of divalent cations in DPBS are more than sufficient to confirm binding of neutrophils to P-selectin [Geng et al. (1991)], while chelator EDTA at 25 mM is more than sufficient to prevent neutrophil cells from binding to P-selectin. The binding of all monoclonal antibodies to P-selectin is slightly affected by the presence of EDTA, indicating that the presence of Ca ⁺⁺ is not required for binding to occur. Mu MAb PB1.3 is a blocking antibody that is capable of blocking the binding of neutrophils to Pselectin, such as activated platelets. PNB1.6 is a non-blocking antibody. The ability of 84/26 to block neutrophil cell binding to P-selectin has not yet been fully characterized. In contrast, of the anti-P-selectin monoclonal antibodies disclosed by Geng et al., Only the non-blocking S12 antibody is capable of binding in the absence of Ca ⁺⁺ .

III. Effect of CQNRYTDLVAIQNKNE peptide on binding of mu MAb to PB1.3 P-selectin: A microtiter plate was coated with affinity-purified P-selectin, blocked with 200 μ 200 / well DPBS + 1% BSA for 1 hour and washed. The mu MAb PB1.3 in DPBS + 1% BSA was mixed with either 0.35 mg / ml CQNRYTDLVAIQNKNE peptide in PBS or control with PBS alone. After one hour, 50 μΙ of each antibody dilution was added to the microtiter plate and incubated for one hour. The plate is washed with DPBS and then diluted 1: 1000 in sheep anti-mouse IgG horseradish peroxidase conjugate in DPBS + 1% BSA. After 1 hour, the plate is washed, TMB substrate is added, color development is stopped, and plates are read as above.

Figure 5 shows that the CQNRYTDLVAIQNKNE peptide homologous to groups 19-34 of the lectin domain of P-selectin has no effect on binding of mu MAb PB1.3 to Pselectin when present at a concentration of 0.35 mg / ml. This distinguishes this monoclonal antibody from the G1, G2 and G3 monoclonal antibodies, the binding of which to P-selectin is partially or completely inhibited by this peptide.

ARC. Ability of Cytel PB1.3, PNB1.6 and 84/26 Mu Monoclonal Antibodies to Block P-Selectin Binding: As described above, affinity-purified P-selectin-coated microtiter plates were blocked for 1 hour with DPBS + 1% BSA and then wash. 25 µl of 50 µg / ml PB1.3, PNB1.6 or 84/26 mu MAb solutions were added to the appropriate wells and incubated for 1 hour. Subsequently, 25 µl of biotinylated antibody was diluted in DPBS + 1% BSA and incubated for 1 hour. After washing with DBPS, the plate is developed with TMB as above.

Figure 6 shows the ability of PB1.3, PNB1.6, and 84/26 mu MAbs to interfere with each other's binding to P-selectin. 6A. Figure 1A shows that only mu MAb PNB1.6 can inhibit the binding of biotinylated mu MAb PNB1.6 to P-selectin, indicating that PB1.3 and 84/26 mu MAbs recognize other epitopes than PNB1.6 mu MAb. Similarly, FIG. In Figure 1B, PB1.3 mu MAb is able to inhibit the binding of biotinylated PB1.3 to Pselectin, whereas PNB1.6 or 84/26 does not,

- ^: ..... '··' that the other two monoclonal antibodies must recognize different epitopes. 6C. Figure 8A shows that both 84/26 and PB1.3 mu MAb are capable of inhibiting the binding of biotinylated 84/26 mu MAb to P-selectin, whereas PNB1.6 mu MAb is unable.

V. Blocking P-selectin antibodies do not inhibit thrombin-induced aggregation of human platelets:

In this example, it is demonstrated that mu MAb PB1.3 and PNB1.6 P-selectin antibodies do not inhibit thrombin-induced platelet aggregation. Fresh human platelets were isolated as above and resuspended in Tyrode-HEPES buffer (pH 7.2) at 2x1 Οθ / ml.

Platelet aggregometry was performed with a Lumi aggregometer (ChronoLog Corp.). For the assay, 0.45 mL of platelet suspension is placed in a standardized siliconized cuvette. To this is added 10 μΙ of antibody or vehicle as a control. After 5 minutes, 0.1 unit of thrombin (Human, Sigma) was added at 37 ° C and the aggregation was recorded.

When platelet aggregation is measured under control conditions in the absence of antibody, 71 units of aggregation response are obtained. An equivalent degree of aggregation was obtained with 10 μΙ of undiluted supernatant of mu MAb PB 1.3 producing cells (Fig. 7). Different concentrations of mu MAb PB1.3 (1-100 pg / ml) are added to the platelet suspension. There is no concentration of mu MAb PB1.3 at which platelet aggregation other than that observed in the absence of antibody is observed (Figure 7).

• · · · · · · · · ·

9, Example

In vivo protective effect of mu MAb PB1.3 blocking antibody in feline myocardial infarction and reperfusion

The cat model of myocardial infarction and reperfusion injury was set up according to the methods of Tsao et al., Tsao et al., Circulation 82: 1402-1412 (1990). Briefly, 2.5-3.5 kg male cats (30 mg / kg, i.v.) were anesthetized with sodium pentobarbital. An intratracheal tube is introduced through a midline incision and each cat is given intermittent positive pressure ventilation with a Harvard pet ventilator. A polyethylene catheter was inserted into the external cervical vein and cannulated into the right femoral vein and then connected to a Statham P23Ac pressure sensor to measure arterial blood pressure. A midline chest incision is made, the pericardium is opened, and the heart is exposed. A 2-0 silk suture is carefully placed around the left first descending artery (LAD), 10-12 mm from the origin. After a 30-minute stabilization period, myocardial ischemia is initiated, with complete closure of the LAD, 1.5 hours ischemia followed by 4.5 hours of reperfusion. Blocking (mu MAb PB1.3) and non-blocking (mu MAb PNB1.6) anti-P-selectin antibodies were administered intravenously at a dose of 1 mg / kg 10 minutes prior to initiation of reperfusion.

Ischemic heart muscle is defined as the tissue portion that is not stained with nitro blue tetrazolium and is expressed as a percentage of the area at risk. The at-risk area was determined by re-occlusion of LAD after the reperfusion period by injecting Evans blue dye into the left ventricle. The area at risk is thus determined by negative staining.

Endothelium-dependent relaxation of coronary artery rings was determined by measuring acetylcholine-induced relaxation of rings previously contracted with U46619 that mimic thromboxane A2. Data are expressed as percent relaxation.

Neutrophil cell accumulation in the myocardium is determined by measuring myeloperoxidase activity in polytrone-homogenized myocardial tissue.

The results of these experiments are shown in Figure 8. In cats treated with mu MAb PNB1.6 non-blocking anti-P-selectin antibody, the extent of myocardial ischemia is 33 ± 3% of the risk area. In contrast, in PB1.3 treated animals, the extent of ischemia was significantly (P <0.01) smaller, 15 ± 3% of the risk area. Endothelium-dependent relaxation on acetylcholine was also substantially maintained in the ischemic-reperfusion coronary arteries of cats treated with mu MAb PB1.3 compared to cats treated with mu MAb PNB1.6 (67 ± 6 versus 11 ± 3%, P <0, 01). The fact that these phenomena are attributable to a decrease in myocardial leukocyte infiltration suggests that there is a significant (P <0.1) decrease in the number of PMNs in myocardial tissue from animals treated with mu MAb PB1.3 (67 ± 7 PMN / mm ^) as compared to animals treated with mu MAb PNB1.6 (136 ± 12 PMN / mm 2).

Example 10

Production of humanized antibodies against P-selectin

A. Cloning and sequencing of mu MAb PB1.3 variable regions

Isolation of mu MAb PB1.3 is described in Example 2. Total RNA was isolated mu

MAb from PB1.3-producing hybridoma cells. The cDNAs encoding the heavy and light chain variable regions of the mu MAb PB1.3 were amplified by polymerase chain reaction (PCR). The entire mouse variable region is amplified using a mixture of degenerate primers that hybridize within the leader sequence and a single primer that hybridizes to the mouse constant region close to V-C binding (Jones and Bendig, Bio / Technology 9: 88-89 (1991)); Bio / Technology 9, 579 (1991)]. The PCR fragments are cloned and sequenced. The nucleic acid sequences and the corresponding amino acid sequences of the heavy and light chains of the mu MAb PB1.3 heavy and light chains are shown in Figures 9 and 10.

B. Modeling the structure of the mu MAb PB1.3 variable region

A molecular model of the PB1.3 V | _ and Χ / μ regions of the mu MAb was prepared. The model is made on a Silicon Graphics IRIS 4D workstation under UNIX operating system using the QUANTA (polygen Corp. USA) molecular modeling software package. The amino acid sequences of the mu MAb PB1.3 variable regions are compared with the structurally resolved immunoglobulin variable regions (Tables 1 and 2).

C. Design of the transformed mu MAb PB1.3 variable regions

We select human variable regions whose framework region is most similar to that found in mu MAb PB1.3 variable region. The light chain variable regions of human DEN mAb and the heavy chain variable region of human 21/28 'CL mAb were selected as frames to bind the corresponding complementarity determining regions (CDRs) of mu MAb PB1.3. Human framework regions were modified by the method described in Monoclonal Antibodies 2, Applications in Clinical Oncology (ed. A. Epenetos), Chapman and Hall (1992); In: Bendig et al., The Humanization of Mouse Monoclonal Antibodies by CDR-Grafting: Examples with Anti-Viral and Anti-Tumor Cell Antibodies]. The foregoing publication is hereby incorporated by reference. The amino acid alignment of the variable regions leading to the design of the transformed humanized PB1.3 variable regions is shown in Tables 1 and 2.

• · ·

Three versions of the heavy chain variable region are modeled and designated CY1748RH / \, CY1748RHg and CY1748RHq. The nucleic acid sequence and the corresponding amino acid sequence of these variable regions and signal peptides are shown in Figures 11, 12 and 13, respectively. In the case of CY1748RHb, the first two groups of mice (glutamic acid and alanine) retained in CY1748RH / 1 were changed back to the original human residues (glutamine and valine), which are found in the donor human FR1. For CY1748RHq, computer modeling suggests that the rodent lysine at position 73 may form a salt bridge with group 55 (aspartic acid) in CDR2. If this were the case, lysine would be important for stabilizing the H2 loop. Human threonine in this position would not be able to form a salt bridge. Therefore, the rodent lysine at position CY1748RH / \ in CY1748RHQ is replaced. CY1748RHg contains the largest number of human amino acids. The amino acid differences in these three versions of the heavy chain are summarized in Table 3.

Four versions of the light chain variable region are modeled, designated CY1748RL / \, CY1748RI_b, CY1748RL_c, and CY1748RI_o. The nucleic acid sequence of these variable regions and the corresponding amino acid sequence are shown in Figures 14, 15, 16 and 17. Computer modeling suggested that murine glutamic acid at position 60 may be important for binding to P-selectin. This group is contained in C1748LR / \, but in the CY1748LRg version it is replaced by serine in the original human framework region. Rodent aspartic acid charge may interact with group 24 in loop 1 (L1), resulting in CY1748LRq being identical to CY1748LR / γ, except that mouse aspartic acid at position 70 is replaced by human framework glutamic acid . CY1748LRd contains both of these amino acid substitutions and contains the largest number of human amino acid residues in the four versions. The amino acid differences between the four versions are summarized in Table 4.

D. Subcloning of Heavy and Light Chain Variable Regions into Expression Vectors (1) Preparation of Expression Vectors (a) pHCMV-1748RL-KR-neo

The expression vector contains the human cytomegalovirus (HCMV) enhancer sequence and promoter to drive transcription of the recombinant immunoglobulin light chain, the coding sequence of the bacterial neo gene acts as the dominant selection marker for stable transformation, and the SV40 origin of replication provides a high level of transient expression. cos cells. To construct the vector, the HindIII-Sac11 pUC8 fragment containing the Pstl-m fragment of HCMV (Boshart et al., Cell 41: 521-530 (1985)) was converted to an EcoRI-Hind111 fragment using the appropriate adapter oligonucleotide. By three-way ligation, the 1.2 kb EcoRI-HindIII fragment containing the HCMV enhancer promoter structure was ligated to a 5.05 kb BamHI-EcoRI fragment derived from plasmid pSV2neo (Southern and Berg, J. Mol. App. Gener. 1, 327-341 (1982)] and ligated a 0.5 kb HindIII-BamHI fragment containing the Vllys kappa light chain variable region (Foote and Winter, 1992, Molecular Biology 224: 487-499). )]. The Hindlll cleavage site in pSV2neo was previously removed by Klenow polymerase loading to give a plasmid pHCMV-V1 / neo.

A 2.6 kb Eco RI fragment of the human genomic kappa region was cloned [Rabbits et al., Curr. Top. Microbiol. Immunol. 113, 166-171 (1984)] and then subcloned into a pUC vector, pUCKR17 [Mengle-Gaw: EMBO J. 6,

1959-1965 (1987)]. BamHI cleavage sites were added to both ends of the fragment using EcoRI-BamHI adapters. This fragment is then inserted into the BamHI site of plasmid pHCMVV-llys-KR in the appropriate orientation to give plasmid pHCMV-Vlys-KR-neo. To facilitate the insertion of the engineered human variable regions, the BamHI site on the 3 'side of the human kappa constant region was removed using Klenow polymerase loading. In the resulting vector (designated pHCMV-1748RL-KR-neo), the HindIII-BamHI fragment containing Vlllys can be easily replaced by the VL of the transformed antibody.

(b) pHCMV-1748-y1C-neo

The construction of this vector begins with the same triple ligation as described above for the preparation of the aforementioned pHCMVVJys-neo, to incorporate a 0.7 kb HindIII-BamHI fragment containing the V? -Jlys heavy chain variable region [Verhoeyen et al., Science 239: 1534-1536 (1988); Foote and Winter, Journal of Molecular Biology 224: 487-499 (1992)] instead of the 0.5 kb HindIII-BamHI fragment containing the Vllys kappa light chain. The cDNA encoding the human γ1 constant region was inserted into the BamHI site to obtain the vector pHCMV-Vj-jlysine. The cDNA encoding the human γ1 constant region was cloned by PCR amplification from a human cell line that secretes a human γ1 antibody. At both ends of the cDNA, BamHI cleavage sites were created, and then a splice acceptor site and a 65 bp intron sequence were inserted at the 5 'end of the cDNA sequence. The BamHI fragment (1176 bp) containing the human γ1 cDNA, the splice acceptor site and the intron sequence was then cloned into the expression vector. As for the light chain expression vector, the BamHI cleavage site on the human γ1 constant region3] is removed using Klenow polymerase loading. With this *··· ·? ···· • · · · · 9 V • · <·· <·· * ··

vector, designated pHCMV-1748-y1 C-neo, the HindIII-BamHI fragment containing the unwanted Vp) lys region can easily be replaced by the Ia of the engineered antibody.

(2) Insertion of the Immunoglobulin Coding Sequence The cDNAs encoding the mu MAb PB1.3 heavy chain variable region and the three humanized versions were inserted into the HCMV-y1 C-neo expression vector using standard techniques to produce the HCMV-1747CH- y1 C. neo-y1 HCMV1748RHA the C-neo, HCMV-y1 1748RHB C-neo and HCMV-1748RHc ^Y-1 C-neo plasmids. The cDNA of the human IgG1 constant region and the corresponding protein sequence are described in Edison et al. (Ellison et al., 1982, Nucleic Acids Research 10: 4071-4079). The cDNAs encoding the light chain variable regions of CY1747 (PB1.3) and the four humanized versions were incorporated into the HCMV-KRneo expression vector to obtain the HCMV-1747CL-KR-neo, HCMV1748RLA-KR-neo, and so forth. Plasmids. The nucleotide sequence of the human kappa constant region gene and the corresponding protein sequence are described by Hieter et al., Cell 22: 197-207 (1980). This publication is hereby incorporated by reference in its entirety and for all purposes. A schematic map of the heavy chain and light chain expression vectors is shown in Figures 18 and 19. By co-expressing an appropriate combination of heavy and light chains, many different antibodies can be expressed. For example, co-expression of the HCMV-1748RHA-γ1 C-neo and HCMV-1748RRL-KR-neo plasmids yields the transformed monoclonal antibody PB1.3 (Ha / La). By co-expressing HCMV-1747CH-γ1 C-neo and HCMV1747CL-KR-neo, chi MAb PB1.3 (i. E., A chimeric antibody comprising murine variable domains and human constant domains).

··· · ·· «···« · * * · * 9 • ··· · ·····

E. Electroporation of COS cells

DNA constructs were examined in COS cells for transient expression of achi MAb PB1.3 and various transformed versions of MAb PB 1.3. DNA is introduced into COS cells by electroporation using a Gene Pulser (BioRad). COS cells were treated with trypsin and washed once with phosphate buffered saline (PBS). An aliquot of 10 to 10 µg of each heavy chain plasmid and corresponding light chain plasmid and 1x10 5 cells / ml suspension (in PBS) was placed in a sterile Gene Pulser cuvette (BioRad, 0.4 cm slot). We pass a 1900 volt, 25 microfaradic pulse through the cuvette. After a 10-minute regeneration period at room temperature, the electroporated cells were added to 20 ml of DMEM medium (GIBCO / BRL) containing 10% fetal calf serum. After incubation for 48 hours, the medium is harvested, centrifuged to remove cell debris, and stored under sterile conditions at 4 ° C for a short time.

Example 11

Analysis of humanized antibodies

Culture media from transfected COS cells were assayed by ELISA to determine the level of human antibody produced and the ability of recombinant soluble P-selectin (rsP-selectin) to bind. Immulon microtiter plates (Dynatech, # 011-010-3355) were coated with 100 µl / well of goat anti-human IgG (whole molecule, Sigma # 1-1886) at a final concentration of 12.5 pg / ml or in Dulbecco's phosphate buffered saline (DPBS). ) With rsPselectin diluted to 1.2 mg / ml. The plates were covered overnight at 4 ° C and 2 hours at 37 ° C. The plates are then washed three times with DPBS. The plates were then blocked with 400 μΙ / well DPBS + 1% bovine serum albumin (BSA, Sigma # A7888) for one hour or more at room temperature. The plates are then washed three times with DPBS.

Serial dilutions of monoclonal antibodies in DPBS + 1% BSA were added to the plates (100 μΙ / well) and incubated for 1 hour at room temperature. The plates are then washed three times with DPBS. A second DPBS + 1% BSA diluted 1: 1000 antibody (goat anti-human IgG peroxidase conjugate, Sigma # A-8867) was added to each well at 100 μΙ / well and incubated for 1 hour at room temperature. The plates are then washed three times with DPBS. 100 μΙ / well of tetramethylbenzidine peroxidase substrate / H 2 O 2 (TMB, Kirkegaard and Perry Laboratories # 50-76-00) was added to the plates and allowed to develop for a sufficient time (usually 3 to 15 minutes), followed by 100 μl / well. μΙ is stopped by the addition of 1 M phosphoric acid. Read the plates at 450 nm in a Titertek Multiskan

MCC / 340.

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

P-selectin binding is plotted as an optical density versus IgG concentration. The binding curves for the various transformed versions of chi PB1.3 and the transformed MAb PB1.3 are shown in Figures 20, 21 and 22. The aim is to select a humanized antibody whose binding characteristics are essentially indistinguishable from those of chi MAb PB1.3. If more than one humanized antibody shows such characteristics, we prefer the one that contains most of the human amino acid residues, as this version * · · «>« «

· · · · ················································································································································································································································································ careing evasive to the HAMA in clinical use. All four transformed light chains, co-expressed with the CY1748RH / 1 heavy chain, produced antibodies that bound to rsP-selectin as well as chi MAb PB1.3. From these, the CY1748LR0 light chain variable region is selected for further experiments as it contains most of the human amino acid residues. The heavy chain variable regions of CY1748RH / \ and CY1748RHb, when co-expressed with the light chain variable regions of CY1748RL / \ and CY1748RL0, also produce antibodies that bind to the same extent as rsP-selectin (chi MAb PB1.3. figure). Of these heavy chains, the CY1748RHg heavy chain is preferred as it contains most of the human amino acid residues. Thus, a preferred humanized antibody is generated from the light chain D and the heavy chain B, designated as modified MAb PB 1.3 (Hg / 1-0).

Example 12

Stable expression of transformed MAb PB1.3 (Hr / Lq) in CHO cells (1) Preparation of vectors (a) pHCMV-1748RH-Y1C-dhfr

This vector is identical to the pHCMV-1748RH-γ1C-neo vector described in Example 11, except that the neo gene was replaced by the dihydrofolate reductase gene linked to a defective SV40 promoter enhancer sequence. To remove the enhancer sequence from the SV40 early promoter, plasmid DNA, pSV2-dhfr (Subramani et al., 1981, Mol. Cell. Biol. 1: 854-864) was digested with SphI and Pvull. , Klenow polymerase and ligated with self to give plasmid pSV2-dhfr-AE A 3.7 kb Eco RI fragment containing the HCMV promoter, the V | - | lys heavy chain variable region and human γ1.

· * <· »» ·

The genomic DNA clone of the constant region I was cut out from the plasmid pHCMVV-lys-γ1-neo by Eco RI digestion. This fragment was ligated into EcoRI-digested plasmid pSV2-dhfr-AE to give plasmid pHCMV-V1 - lys-γ1-dhfr. The genomic clone of the y1 constant region was replaced by a BamHI fragment containing the cDNA clone of the human y1 constant region described in Example 11. The BamHI cleavage site on the 3 'side of the human γ1 constant region is then removed by filling with Klenow polymerase as described in Example 11. The resulting vector is designated pHCMV-1748RH-γC-dhfr. The HindIII-BamHI fragment of this vector containing the unwanted Vnlys can be easily replaced by the V1 of the transformed antibody.

(b) pHCMV-1748RH-γ4-dhfr

This vector is identical to the pHCMV-1748RH-y1C-dhfr vector described above, but the cDNA clone of the human y1 constant region is replaced by a genomic clone of the human y4 constant region. To construct the vector, a 7.0 kb DNA fragment containing the genomic clone of the human? 4 constant region (Brüggeman et al., J. Exp. Med. 166, 1351-1361 (1987)) was cloned into the HindIII site of pUC19. plasmid 428D is obtained. Since the human y4 fragment already contains a BamHI cleavage site near the 3 'end, the subclone is introduced into pUC19 in an orientation that moves this site away from the BamHI cleavage site in the pUC19 polylinker. The 7.0 kb γ4 fragment was excised from 428D by digestion with BamHI and ligated into pHCMV-Vulysneo to give plasmid pHCMV-V1-lys-γ4-neo.

The final plasmid was constructed by triple ligation with a 5.4 kb BamHI-HindIII fragment containing the HCMV enhancer promoter sequence and the pSV2-dhfr-AE sequence, a 0.5 kb base of the transformed human 1748 antibody VH. and a 7.0 kb BamHIBamHI fragment containing the genomic clone of the human γ4 constant region.

(2) Insertion of sequences encoding immunoglobulin

The cDNA encoding the 1748RH heavy chain variable region was cloned into the HCMV-1C-dhfr expression vector to obtain the HCMV-1748RH1-1C-dhfr vector. The cDNA encoding 1748RHB was cloned into the expression vectors HCMV-1C-dhfr and HCMV-4C-dhfr to give plasmids HCMV-1748RHB-1C-dhfr and HCMV-1748RHb4C-dhfr. The nucleotide sequence of the human IgG4 constant region gene and the corresponding amino acid sequence have been reported by Ellison et al., Edison et al., DNA 1: 11-18 (1981). The HCMV-1C-dhfr vector expresses an IgG1 isotype antibody, HCMV-y4C-dhfr expresses an IgG4 isotype antibody when co-expressed with the kappa light chain vector. A schematic map of the dhfr expression vectors is shown in Figures 23 and 24.

CHO dhfr-deficient cells were grown on ocMEM (+ nucleosides, GIBCO / BRL) and 10% fetal calf serum. Plasmid DNA is introduced into COS cells by electroporation using a Gene Pulser instrument. CHO cells were trypsinized and washed once in phosphate buffered saline (PBS). 10 to 10 pg of each heavy chain plasmid DNA and the corresponding light chain plasmid DNA and 1x10 ⁷ cells / ml of cell suspension in PBS are aliquoted into a sterile Gene Pulser cuvette ( 0.4 cm gap). We pass a 1900 volt, 25 microfaradic pulse through the cuvette. After a 10-minute regeneration period at room temperature, the electroporated cells are added to 20 ml of aMEM (+ nucleosides) / 10% FBS. After incubation for 24-48 hours, cells were trypsinized and plated onto 100 mm diameter plates in MEM (84 nucleosides) / 10% dialyzed FBS (to select a plasmid containing dhfr supplemented with 500 pg / ml antibiotic G418 (GIBCO / BRL). for selection of the neo-containing plasmid) The medium is changed every 3-4 days until colonies grow, Individual colonies are isolated by cloning cylinders, propagated, and assayed for IgG production by ELISA.

Three groups of DNA are introduced into CHO dhfr-deficient cells: (1) HCMV1748RHA-γC-dhfr and HCMV-1748RL / KR-neo, which expresses the IgG1 isotype transformed PB1.3 (Ha / L-a) monoclonal antibody; (2) HCMV-1748RHB-γ1Cdhfr and HCMV-1748R1R0-KR-neo, which expresses the IgG1 isotype transformed PB1.3 (/β / Ι-β) monoclonal antibody; and HCMV-1748RHB-γ4C-dhfr and HCMV1748RI_0-KR-neo, which expresses the IgG4 isotype transformed PB1.3 (Hg / Lo) monoclonal antibody.

The CHO cells were propagated and placed in a 10-chamber (6000 cm 2) cell Nunc apparatus. After 72 hours, the medium was harvested and passed through a protein A Sepharose FAst Flow (Pharmacia) column. The column was washed and bovine IgG was eluted at pH 4.5. Humanized antibodies were eluted at pH 3.5, dialyzed in PBS or 20 mM acetate, 0.15 M NaCl, pH 5.5. Antibody concentrations were determined spectrophotometrically and confirmed by ELISA against human IgG. (A purified human IgG4 antibody (kappa, Sigma # 1-4639) was used as a control for the mu MAb PB1.3 (Hb / Lq), IgG4 isotype. Purity was confirmed by SDS-PAGE.

B. Competitive Binding Assay

The relative binding affinity of different versions of the transformed MAb PB1.3 for P-selectin was determined by competitive ELISA. The assay measures the concentration of unlabeled antibody which inhibits 50% of the labeled mu

Binding of MAb PB1.3 to P-selectin.

2 ml of purified mu MAb PB1.3 (1 mg / ml) were mixed with 100.8 μΙ NHSLC biotin (1 mg / ml, Pierce) and incubated overnight at 4 ° C. A 2.1 mL sample was applied to a NAP 25 column (size exclusion column, Pharmacia). The column was eluted with 0.3 ml PBS fractions. Each fraction was read at 280 nm. The highest OD 280 fractions were pooled. The titer of biotinylated MAb PB1.3 against rsP-selectin is determined to select a concentration that can be used in the competitive binding assay.

A 96-well plate (Microtest III, Falcon # 3912) was coated with 50 μΙ of rsP-selectin (2 μ / / ιτιΙ-Γθ diluted in DPBS) per well, overnight at 4 ° C or for 90 minutes at 37 ° C. The plate is inverted and the remaining liquid is shaken.

The coated plate and an additional blank plate were blocked with 200 μΙ DPBS + 1% bovine serum albumin (BSA, Sigma # A-7888) for 1 hour at room temperature. The blank plate is inverted and the remaining blocking fluid is shaken off. The rsP-selectin-coated was allowed to continue to block during the antibody dilution period. Serial dilution antibody samples (DPBS + 1% BSA) were added to the blank at 50 μΙ / well. The initial concentration of antibodies is 100 μ9 / ιτιΙ. Biotinylated PB1.3 (0.3 μ9 / ΓηΙ-Γθ diluted in DPBS + 1% BSA) was added to the antibody blank on 50 μ lemez / well and pipetted. The rsP-selectin-coated plate is washed three times with DPBS. The antibody sample / biotinylated PB1.3 mixture was pipetted onto a rsPselectin-coated plate (50 μΙ / well) and incubated for one hour at room temperature. The plates are then washed three times with DPBS.

Streptavidin Horseradish Peroxidase Conjugate (Pierce # 21124) diluted 1: 2000 in DPBS1% BSA was added to the plate at 50 μΙ / well and

- 86 - ·: λ '/>

incubate for 1 hour at room temperature. The plate is then washed three times

DPBS.

50 µl / well of tetramethylbenzidine peroxidase substrate / H 2 O 2 (TMB, Kirkegaard and Perry Laboratories # 50-76-00) was added to the plate and the color was allowed to develop for an appropriate time (usually 3-15 minutes) and the reaction was Inject 50 µl of 1 mol / L phosphoric acid. The plates were read at 450 nm

Titles with Multiskan MCC / 340.

Apparent binding affinity is expressed as the concentration of antibody that binds rsP-selectin at half the maximum optical density at 450 nm. The ratio of the concentrations measured at half of the maximum binding of the antibody sample to the corresponding value of the control antibody (unlabeled PB1.3) gives a good estimate of the relative binding affinity of the test antibody for rsP-selectin.

The results of two typical competitive binding experiments are shown in Figures 25 and 26. In the first experiment, twice as much (2.0 pg / ml) of the converted IgG1 isotype PB1.3 (H / \ L / \) monoclonal antibody is required as PB1.3 (1.0 pg / ml) so that the labeled The binding of PB1.3 is inhibited by 50% of the maximum binding. Therefore, this humanized antibody binds rsP-selectin at 50% of PB1.3 binding. Similarly, in the second experiment, PB1.3 / (HbLd) (IgG4 isotype) binds rsPselectin in 50% of the ΡΒ1.3 binding.

Example 13

Enhancement of antibody production in CHO cells by DHFR amplification

CHO cells are seeded into flasks and allowed to confluent before changing media (8 ml for a T-25 cm ² flask or 20 ml for a T75 cm ² flask). After incubation of the cells for 48-72 hours, the medium was harvested and assayed for IgG production by ELISA. The cells were trypsinized and counted. The rate of antibody production is expressed as the amount of antibody produced by one million cells per 24 hours.

The three or six best producing clones are then amplified individually, and the remaining seven best clones are pooled and amplified. Cells were plated at a density of 1 x 10 4 cells / 100 mm plate in selection medium. In the first round, the amplified cells are plated into three groups of plates, which are the MEM (-nucleosides) / 10% dialyzed FBS supplemented with 500 pg / ml G418 and 10, 20 or 50 nM methotrexate (Sigma # A-6770). The cultures are fed every four days. 10-14. On day 1, individual colonies were isolated by cloning cylinders, propagated, and their IgG production assayed by ELISA. Further amplification steps are performed with individual colonies or pools of clones at 5-10 times the initial methotrexate concentration.

Table 5 summarizes the antibody production of CHO cells expressing three humanized versions of PB1.3. An amplified clone expressing the transformed IgG4 isotype PB1.3 (Hg / L0) monoclonal antibody is derived from a pool of clones with expression levels less than 0.2 pg / 10® cells / day and 2 in the first amplification run, 0 pig / ΙΟθ cells / day, and 33.0 pg / 10 ⁶ cells / day in the third amplification run. This clone was selected to produce a greater amount of transformed PB1.3 monoclonal antibody.

Example 14

Bioreactor run

160 g collagen coated microcarrier bead (150-200 microns, JRH

Biosciences # 60142-100) was hydrogenated in 800 ml sterile water for 30 minutes followed by water

discarded. The beads were suspended in 800 ml sterile water and autoclaved for 30 minutes. After cooling to room temperature, the beads are washed twice with sterile serum-free ctMEM (s) and then resuspended in 1 L of complete medium (aMEM (s) / 5% dialyzed FBS).

The CHO-48B4 cell line, which produces the transformed PB1.3 (Hg / Lg) (IgG4 isotype) monoclonal antibody at 33 μρ / 10θ cells / day, is propagated in 36 T-225 cm2 plates with 10% MEM (-nucleosides) / 10%. dialyzed in FBS / 500 nmol / L methotrexate. When the cells mature during growth, they are trypsinized and resuspended in 2 liters of complete medium (without methotrexate) at a concentration of 5.0-8.0x10 ⁴ cells / ml.

pellets, 2 liters of cell suspension, and 2 liters of fresh complete medium were aseptically added to a 10-liter autoclaved container of Proteus 2000 Bioreactor (Wheaton). The air space is continuously rinsed with air + 5% CO2. The cells were allowed to adhere to the microcarriers by programming the Proteus by agitating the cell / microcar suspension at 70 rpm for 1 min and then at 0 rpm for 59 min. After a total of 48 cycles, 5 liters of medium were added to the bioreactor, which was programmed to maintain the slurry at 37 ° C, pH 7, dissolved oxygen at 30% air and continuously at 50 rpm. mixing.

After three days, the bioreactor is programmed to replace the complete medium at a rate of 5 liters / day by attaching a gravity filter to the stripping stub so that the microcarriers remain in the bioreactor. The harvested media was stored at 4 ° C until processing. The bioreactor is run for 5-7 days until accumulated cellular debris causes turbidity in the culture medium (due to degradation by aeration).

► · ·· «» · · · ·

The harvested medium was filtered to a mirror and passed through a Millipak-60 0.45 micron filter (Millipore). 20 liters of filtered supernatant were passed through a 2.5 cm x 12 cm protein A Sepharose Fás Flow (Pharmacia) column at a flow rate of 0.55 l / h. After all supernatants were passed through the column, the column was washed with 1.5 L PBS (BioWhittaker, # 17-516Y). The column was eluted with 8 x 10 mL 0.2 M sodium acetate (pH 4.5) followed by 8 x 40 mL 0.2 M glycine, 0.5 M sodium chloride pH 3.5 solution, both into tubes containing 10 ml of 2 M TRIS-HCl, pH 8.0. The single fraction with the highest absorbance at 280 nm was dialyzed into 20 mM acetate, 0.15 M sodium chloride, pH 5.5, and stored at 4 ° C.

200 mg of antibody is recovered daily for 5-7 days. Purification was performed in about 50% recovery. Thus, running a bioreactor can yield 500-700 mg of purified antibody.

Example 15

Effect of mu MAb PB1.3 on tissue injury caused by ischemia reperfusion after rabbit ear replantation

New Zeeland White rabbits are anesthetized with a combination of ketamine and xylazine and then lidocaine is instilled into the base of the ear. The left ear is partially amputated, leaving the central artery, vein and a porchid intact. All nerves are divided to give the ear anesthetic. The ear is then returned and a microvascular clip inserted through the artery to ensure complete ischemia. The rabbits were kept in a room at 23.5 ° C for six hours, then the microvascular clamp was removed and blood was allowed to enter the ear again. Treatment is performed just prior to reperfusion with either mu MAb PB1.3 (2 mg / kg) or saline or a PNB1.6 rodent monoclonal antibody of the same isotype ·· · ··· «« · kg). The volume of the ear is measured daily for 7 days, with water displacement, to quantify tissue edema. On day 7, necrosis is estimated based on the total surface area.

Significant increases in ear volume were observed in animals treated with either saline or PNB1.6 after reperfusion of the ears (Figure 27). Because there was no observable difference between the two control groups, their data were combined. Edema growth was also observed in PB1.3-treated animals, but the extent of growth was significantly less than that observed in the control group at each measurement point. The extent of necrosis (Figure 28), measured at day 7, was also significantly reduced in mu MAb PB1.3-treated animals compared to control animals.

Thus, after reperfusion of ischemia, the transplanted rabbit ear, first responds with an edema followed by extensive tissue destruction. These two manifestations of tissue destruction were significantly and significantly reduced in animals previously treated with mu MAb PB1.3 anti-P-selectin antibody.

16, Example

Effect of mu MAb PB1.3 on vascular potential following canine ischemia and reperfusion of canine skeletal muscle

In this experiment, an ischemic model and reperfusion of isolated canine muscles were used. One aspect of reperfusion injury is a phenomenon that is not referred to as a reperfusion whereby the blood flow through the microcirculation of the previously ischemic tissue is absent or extremely reduced after reperfusion.

·· ··· · · · · ··· · ······

An isolated canine thigh muscle is used as described previously [Carden et al., Circ. Gap. 66: 1436-1444 (1990). Briefly, adult mongrel dogs are anesthetized with sodium pentobarbital. The skin covering the thigh muscle is cut and the connective tissue is removed from the muscle with blunt cuts. The diaphragm is sectioned. The nearby caudal artery and vein are cannulated and each collateral vessel is closed. The femoral artery cannula is connected to a constant flow perfusion pump and the fluid flowing out of the femoral vein is directed to a reperfusion reserve. Arterial and venous pressure in the lateral branches are monitored and blood flow is adjusted to maintain a perfusion pressure of 100 mmHg. Vascular isolation is considered complete when arterial pressure falls below 20 mmHg when the perfusion pump is turned off.

Microvascular potency is estimated by perfusing the isolated thigh muscle with contrast media (Indian ink) at the end of the experimental setup. The computer video image is used to quantify the number of ink-containing microvessels (greater than 10 µm) per muscle strain in histological sections obtained from isolated canine femoral muscle for 4.5 hours of continuous perfusion, 4 hours of ischemia, and 0.5 hours of reperfusion or ischemic reperfusion. were exposed to mu MAb PB1.3 anti-P-selectin antibody (40 pg / ml) in the perfusate.

The ischemia-reperfusion experimental set-up reduces the number of patent microvessels to 36 ± 3% relative to control animals. If mu MAb PB1.3 was also added to the perfusate, the number of patent microvessels after ischemia and reperfusion was 119 ± 18% of the control.

Thus, the mu MAb PB1.3 anti-P-selectin antibody completely prevents the occurrence of non-reflux, which can be determined by the number of patent microvessels in ischemic and reperfused canine femoral muscle.

«··

Example 17

Effect of mu MAb PB1.3 on leukocyte endothelial cell interactions caused by de-granulation of tissue mammalian cells

I. Methods

(a) Intravital microscopy

Male Wistar rats (200-250 grams) were obtained from Harlan Sprague Dawley (Indianapolis) and starved for 12-24 hours prior to surgery. Surgical anesthesia is induced by intramuscular injection of ketamine / rompun / acepromazine. A catheter is inserted into the left cervical vein and a sniffer is made to facilitate spontaneous breathing. The abdomen is washed and shaved, and a cut of about 2 cm in the centerline of the abdominal cavity is made, taking care to avoid bleeding. The rat is placed on a microscope stand specifically designed for intravital microscopy. A well-vascularized posterior loop of the iliac is exposed and the rumen is mounted on an observation stand heated to 37 ° C. During the experiment, the intestinal ripple was passed at 2 ml / min with eyg heated bicarbonate buffered saline (37 ° C, pH 7.4). In addition, plastic foil (Saran-Wrap) previously soaked in alcohol and rinsed in saline is applied to the adjacent areas of the ileum and the caudal areas to keep these areas moist. The microcirculation beds were observed through an intravital microscope (Mikron Instruments, San Diego) equipped with a 10x magnification eyepiece and a 20x magnification lens (water immersion numerical aperture 0.4). A high-resolution video system consisting of a microscope-mounted video camera (Hitachi color CCD), a video timer (American Video Equipment), a video recorder (Sony SVO-9550MD) and a monitor (Sony Trinitron) are used for microscopy. to capture an image on video. For each intestinal rupture, 3 postcapillary venular segments of approximately 180 µm in length are selected for repeated imaging according to the following criteria: (1) A true postcapillary small vessel receiving blood from continuous 15-30 µm diameter capillaries; (2) Rapid blood flow such that individual red blood cells are indistinguishable; (3) Some basic rolling of leukocytes (less than eight leukocytes present in the vascular segment at each time point) but no tight adherent leukocytes. A leukocyte is said to be adherent if it remains immobile for more than 30 seconds.

(b) Experimental setup

One minute video recordings of selected leukocyte interactions are performed in selected small vessels after complete surgical procedure. All treatments are administered intravenously before the second baseline. 30 minutes after surgery, infusion of 48/80 (Sigma, Cat. No. C4257) into the superfusion buffer was started and continued until the end of the experiment. This results in a concentration of 10 pg / ml in the alimentary canal and is done to induce breast cell degranulation. Small blood vessels were resumed 20 and 30 minutes after the start of 48/80. Leukocyte-endothelial cell interactions are determined at the end of the experiment by studying video tapes. The number of clearly visible leukocytes (i.e., cells that interact with the vessel wall) is determined from a frozen image of the uptake sequence at 0, 15, 30, 45, and 60 seconds, and the average of these three determinations is calculated. Thus, counted leukocytes include rolling cells and those that adhere tightly.

• · * 9 9 99 ·

(c) Statistics

Each experimental group has 5-6 animals and 5-6 small vessels from each animal. Give the mean and the standard error. Significant differences between group means were made by analysis of variance following the Tukey-Kramer HSD test. Differences in pre-treatment and post-treatment blood vessels were examined by paired t-tests. All tests are run with JMP software on a Macintosh llsi machine and the significance is accepted at p <0.05.

II. Results

Topical application of Compound 48/80 results in visible degranulation of more than 90% of the breast cells in the rat gut, which occurs within 3 minutes of treatment. An increase in leukocyte rolling and adhesion is then observed. There is no significant difference in leukocyte accumulation between 20 and 30 minutes after application of 48/80, so the mean of these two times is used in the statistical analysis. Vascular variability is quite high in the model, basal interaction varies from 0 to 4.8 in small blood vessels, and breast cell-induced interaction varies from 2.8 to 25.6 in leukocytes in the PBS-treated group (coefficient of variation, CV, 37%). Animal averages showed much less variability (CV, 16%); as a result, each small blood vessel was treated as a separate experimental unit for statistical reasons.

As shown in Figure 29, intravenous treatment with mu MAb PB1.3 inhibits 90% of breast cell-induced intravascular leukocyte accumulation when compared with the formulation control control group. Non-reactive P6H6 antibody had no effect in this model.

Thus, administration of mu MAb PB1.3 anti-P-selectin antibody to rats completely prevents leukocytes from interacting with the vascular endothelium and subsequently migrating induced by the application of the 48/80 breast cell degranulating agent.

Example 18

Epitope mapping of mu MAb PB1.3

P-selectin is an integral membrane protein found in secretory granulomas of platelets and endothelial cells. Upon cellular activation, P-selectin is rapidly distributed to the plasma membrane. The mature molecule is a multi-domain protein including the lectin region, the EGF region, nine tandem consensus C3b-C4b Regulatory Protein (CRP) repeats, a transmembrane region and a cytoplasmic domain. Descriptions and cloning of P-selectin are described by Johnston et al. Johnston et al., Cell 56: 10331044 (1989). This publication is hereby incorporated by reference in its entirety for all purposes. Figure 30 shows a suggested loop pattern for the P-selectin domain.

P-selectin deletion analysis

To map the epitope specifically bound by mu MAb PB 1.3, mutants of recombinant P-selectin were prepared by deleting consecutive CRP domains (Figure 31). This site-specific mutagenesis was performed by PCR amplification using primers specific for each CRP domain (Table 6). The template used for PCR mutagenesis is a pUC DNA clone of P-selectin which has been truncated and mutated at position 2354 of the DNA sequence (corresponding to the sequence published by P-selectin Johnston et al., Cell 56 , 1033-1044 (1989)]. This template plasmid, pGA1, T, which was first produced by in vitro mutagenesis, lacks the transmembrane domain of P-selectin and the select · · · · · · · ··· ..

cytoplasmic C-terminus, instead encodes the 11 C-terminal amino acids of SV40 large T antigen. This SV40-derived C-terminal is recognized by the monoclonal antibody KT-3 (MacArthur et al., 1984, Journal of Virology 52: 483-491). After PCR amplification, the deletion DNA molecules are purified, ligated, transformed into bacteria, and cloned and sequenced as described below. DNAs having proper binding of CRP to the T antigen signaling sequence were subcloned into pcDNAI expression vector (Invitrogen) and then transfected into COS cells.

PCR mutagenesis was performed on pGMPA1, T. After phosphorylation of the 5 'end, the primers in Table 6 are added at a concentration of 1 pimol / L to each PCR reaction with the lys-1 T antigen sequence. PCR was performed on a Perkin Elmer-Cetus apparatus (30 cycles, 95 ° C for 50 seconds, 50 ° C for 1 minute, and 73 ° C for 4 minutes) with Pfu polymerase (Stratagene) under the conditions recommended by the manufacturer. After purification by agarose gel electrophoresis and elution with Qiaex (Qiagen Corp.), the mutated pUC DNAs were ligated followed by InvF of Escherichia coli. cells (Invitrogen). Cloned DNA carrying the desired mutations is identified by DNA sequence analysis and the Sal I-Hpal fragments containing the mutated Pselectin gene cloned into the Xho I and Xba I sites of the expression vector pcDNAI (Invitrogen). PcDNA clones were transformed into Escherichia coli strain MC1061 (Invitrogen) and purified plasmid DNAs were used for transformation into COS cells.

DEAE-Dextran transfection method

This method is described by M. Kriegler (Kriegler, M .: Gene Transfer and Expression: A Laboratory Manual, WH Freeman and Company (1990)). COS cells were plated at a density of 1x10θ cells / 100 mm plate in DMEM (Biowhittaker), 10% fetal calf serum (FBS). On the second day, plasmid DNA was precipitated with ethanol and then at 20 pg / ml in sterile TE (10 mmol / l). TRIS, pH 8.0, 1 mM EDTA) is suspended. 120 μΙ of DNA is mixed with 300 μΙ of sterile TBS (TRIS-buffered saline, composition: 140 mM NaCl, 5 mM KCl, 1.4 mM Na2HPO4, 25 mM TRIS base, pH 7.5, 1.0 mmol / l of CaCl2, 0.5 mmol / l MgCl ₂₎ and 300 μΙ sterile DEAE dextran (Sigma # D-9885, 1 mg / ml in TBS). The growth medium is aspirated and the monolayer is washed once with PBS and once with TBS. 750 μΙ of the DNA / DEAE dextran / TBS mixture was added to the monolayer. The plate is incubated at room temperature in a lemon box and shaken for 5 minutes for 1 hour. After incubation for one hour, the DNA solution is aspirated and the cells are washed once with TBS and then once with PBS. Cells were incubated in complete medium supplemented with 100 μl of chloroquine (Sigma, # C-6628) at 37 ° C in an atmosphere containing 5% C2. After 4 hours, the medium is replaced with complete medium and the cells are incubated at 37 ° C in an atmosphere containing 5% CC 2. 48 hours after transfection, the cells were fed with serum-free DMEM medium. Twenty-four hours later, the medium was harvested and the cell debris removed by centrifugation (1500 rpm, 5 minutes, in a desktop clinical centrifuge).

Inclusive ELISA for analysis of secreted P-selectin mutant proteins

A 96-well Costar plate was coated with KT-3 antibody (produced in ascites and purified with Protein G Sepharose (Pharmacia)) at 100 µg / well at 40 µg / ml at 4 ° C overnight. The plate is washed three times with DPBS and blocked with 250 μΙ / well DPBS + 1% BSA for 1 hour at room temperature. The plate was washed three times with DPBS and incubated with COS cell supernatant at 100 μΙ / well for 2 hours at room temperature. After washing the plate three times with DPBS, the bound P-selectin is detected by: or 100 μΙ / well of (a) 1 ng / ml rabbit anti-P-selectin polyclonal Ig, or (b) biotinylated mu MAb PB1.3, or (c) biotinylated P6H6, DPBS / 1. Diluted in% BSA for 1 hour at room temperature. After washing the plate three times with DPBS, the antibody, rabbit antibody, was detected using 100 μΙ 1: 1000 diluted HRP conjugated goat anti-rabbit antibody (BioRad) in KBS in DPBS / 1% BSA. Tissue culture from 3 cell lines blocked with culture medium (10%) for 30 minutes at room temperature. The biotinylated antibodies were detected in 100 μΙ / well HRP-streptavidin (Pierce) diluted 1: 1000 (30 min, room temperature). The bound HRP is incubated with TMB and inoculated with 1 M phosphoric acid. Figure 32 shows the signal without background from the supernatant of COS cells that had undergone false transfection. The False column is supernatant from cells transfected with the full-length form of P-selectin lacking the SV40 T antigen tag and therefore cannot be detected in this KT-3 capture ELISA. The X axis determines the number of CRPs expressed by the various mutant P-selectin cDNAs, from all 9 CRPs in column 2 to one CRP in column 10. The results show that although P-selectin mutants lacking C-terminal CRP can be detected by rabbit polyclonal Ig, these molecules do not react with PB1.3. This suggests that the PB1.3 binding site is located in the fifth CRP repeat domain.

To confirm the results obtained from epitope mapping, we extend the deletion analysis to cut only the fifth CRP domain, the

Leaving the C-terminal sequences of P-selectin intact (Figure 30). PCR amplifiers were designed by deleting amino acid residues 407-468 of P-selectin (Table 6), using the same procedure as described above.

Set supernatants were analyzed directly by ELISA using mu MAb PB1.3 and P6H6 antibodies (Figure 33). Although mu MAb PB1.3 binds to intact Pselectin, it is unable to bind to the deletion molecule. P6H6 binds equally to both molecules.

Analysis of synthetic peptides

The mu MAb PB1.3 epitope is further characterized by synthesizing peptides that cover the 62 amino acid regions of the fifth CRP of P-selectin (Figure 34) and then detecting mu MAb PB1.3 binding by ELISA. The fifth CRP covers amino acids 407-468, according to the nomenclature of Johnston et al. The peptide sequences are as follows: amino acids 968.01, 408-426; 968.02, 418-436os; 968.04, 408-433 amino acids; 968.05, which is an oxidized version of 968.04 .; 968.06, 428-447; and 968.08, 448-467. Each peptide was dissolved in dimethylsulfoxide (DMS) at a concentration of 2 mg / ml and covered with a mad 96-well microtiter plate, dissolved in 1 pg in 100 μΙ PBS and dried overnight at 37 ° C. Coated cells were washed with PBS and blocked with 250 μΙ / well in PBS / 1% BSA at room temperature for 30 minutes. The wells were washed with PBS and incubated with PB1.3 diluted to 1 pg / ml in PBS / 1% BSA at room temperature at 100 μΙ / well. The bound mu MAb PB 1.3 antibody was detected with HRP-conjugated goat anti-mouse antibody reagent and TMB substrate as described above. Figure 34 shows a comparison of the reactivity of the peptides PB1.3. The C-terminal peptide, 968.08, strongly reacts with PB1.3. As a result, the epitope recognized by PB1.3 is mapped to residues 448-467 of the fifth CRP of human P-selectin.

For purposes of clarity, the invention is described in these examples and in the preceding description. However, it is obvious that certain changes and • · «

Modifications may be made without departing from the scope of the appended claims. All publications and patent applications cited above are hereby incorporated by reference in their entirety for the same purposes as if each were to be mentioned individually.

101 Table 1

Alignment of amino acid sequences leading to the design of the transformed human PB1.3 heavy chain variable region

Jacket # You are FR CDR Mouse 1747 Mouse IF human I human 21 / 28'CL RH \ / a Note 1747 1 1 HC1 E E Q PCA E Near H 1, the likely role of anti- qén meq 2 2 I I THE V V V THE An unusual amino acid, Near H1, the antigen is likely to play a role meqkötésben 3 3 I Q Q Q Q Q 4 4 1 L L L L L 5 5 1 Q Q V V V 6 6 1 Q Q Q Q Q 7 7 1 1 S S S S S 8 8 1 1 G G G G G 9 9 1 1 P P THE THE THE 10 10 1 1 E E E E E 11 11 l 1 L L V V V 12 12 1 1 V V K K K 13 13 1 1 E K K K K 14 14 1 1 P P P P P 15 15 1 1 G G G G G 16 16 1 1 THE THE THE THE THE 17 17 1 1 S S S S S 18 18 1 1 V V V V V 19 19 1 1 K K K K K 20 20 1 1 V L V V V 21 21 1 1 S S s s s 22 22 1 1 C c c c c 23 23 1 J K K K K K

102 1. Continuing Table

Jacket # You are FR CDR Mouse Mouse human I human 21 / 28'CL RH \ / a Note 1747 1747 IF 24 24 I 1 THE THE THE THE THE 25 25 1 1 S S S S S 26 26 1 1 G G G G G * 27 27 1 1 Y Y Y Y Y * 28 28 1 1 29 29 1 F F F F F * 30 30 FR1 T T T T * T 31 31 CDR1 N D S S N * 32 32 I Y Y Y Y Y * 33 33 I l V Y THE THE V 34 34 1 M M I M M * 35 35 1 I H N S H H 35A 1 1 35B CDR1 36 36 FR2 w w w w w 37 37 I 1 V V V V V 38 38 1 1 K K R R R 39 39 1 1 Q Q Q Q Q 40 40 1 1 K S THE THE THE 41 41 1 1 P P P P P 42 42 1 1 G G G G G 43 43 1 1 Q K Q Q Q 44 44 1 1 G S G R R 45 45 1 1 L L L L L 46 46 1 1 E E E E E 47 47 1 1 W W W W W 48 48 1 1 I I M M M 49 49 FR2 G G G G G 50 50 CDR2 F D W W W 51 51 1 I I I I I 52 52 t N N N N N

103 1. Continuing Table

Jacket # You are FR CDR Mouse 1747 Mouse IF human I human 21 / 28'CL RH _V a 1747 Comment 52A 53 I 1 P P P THE P * 52B 1 1 52C 1 l 53 54 1 1 s G G G s * 54 55 1 N N N N N * 55 56 1 D G G G D * 56 57 1 G G D N G 57 58 1 P T T T P 58 59 1 1 K s N K K 59 60 1 Y Y Y T Y 60 61 1 N N THE s N 61 62 1 E Q Q Q E 62 63 1 1 R K K K R 63 64 l 1 F F F F F 64 65 1 K K Q Q K 65 66 CDR2 N G G G N 66 67 FR3 K K R R R 67 68 1 í THE THE V V V 68 69 1 ! T T T T T 69 70 1 1 L L L L L 70 71 1 1 T T T T T 71 72 1 1 s V THE R s * H2 canonical structure- ra, can support H2 72 73 1 1 D D D D D 73 74 1 ] K K T T T 74 75 1 I S S s s s 75 76 1 J S S T THE THE 76 77 1 J S S s S S 77 78 1 1 T T T T T 78 79 1 1 THE THE THE THE THE 79 80 1 Y Y Y Y Y

• ·

1. Continue Table

Jacket # You are FR CDR Mouse 1747 Mouse IIA human I human 21 / 28'CL RHya Note 1747 80 81 1 1 M M M M M 81 82 1 1 E Q E E E 82 83 1 1 L L L L L 82A 84 1 1 S S S S S 82B 85 1 S S S S S 82C 86 1 L L L L L 83 87 1 T T R R R 84 88 1 s s S S S 85 89 1 1 E E E E E 86 90 1 V D D D D 87 91 1 s S T T T 88 92 1 THE THE THE THE THE 89 93 1 1 V V V V V 90 94 1 1 Y Y Y Y Y 91 95 1 F Y Y Y Y 92 96 1 C C C C C 93 97 1 1 THE THE THE THE THE 94 98 FR3 R R R R R * 95 99 CDR3 THE G THE G THE 96 100 1 1 R X P R 97 101 1 1 P Y G P 98 102 1 1 G Y Y G 99 103 1 1 F S G G F 100 104 1 1 D S S D 100A t 1 S G 100B 1 1 Y G 100C 1 1 M G 100D 1 1 X C 100E 1 1 THE Y 100F 1 1 X R Y 100G 1 I X G Y

105 1. Continuing Table

Coat # FR or Mouse Mouse Human Human RH \ / a Note

CDR 1747 HA I_21 / 28'CL 1747

100H | YDG _:

1001 105! W Y Y S_W

100J 106! Y A - G_Y

10QK 107 l_F F F S_F

101 108! D D D N_D

102 109 CDR3 V Y Y_Y_V

103 110 FR4 WWW W_W

104 111 i G G G G G 105 112! THE Q Q Q Q 106 113! G G G G G 107 114! T T T T T 108 115! T T L L L 109 116! V V V V V 110 117! T T T T T 111 118! V V V V V 112 119! s s S s S 113 120 FR4 s s S s S

Table 2

The transformed human PB1.3 light chain leads to the design of the variable region

alignment of amino acid sequences Jacket # FR or Mouse Mouse q V human q-l human DEN RHV _L Note 1747 CDR 1747 1 1 FR1 D D D D D 2 2 I 1 L L L L L 3 3 1 1 V Q Q Q Q 4 4 1 1 M M M M M 5 5 1 1 T T T T T 6 6 1 1 Q Q Q Q Q 7 7 1 1 S S S S S 8 8 1 Q P P P P

• · ·

106 2. Continuing Table

Jacket # You are FR CDR Mouse 1747 Mouse q V human q-l human DEN RHV _l Note 1747 9 9 I 1 K S S S S 10 10 I F S S T T 11 11 1 1 M L L L L 12 12 1 1 S S S S S 13 13 1 1 T THE THE THE THE 14 14 1 s S S S S 15 15 1 1 V L V V V 16 16 1 G G G G G 17 17 l D D D D D 18 18 1 1 R R R R R 19 19 1 1 V V V V V 20 20 1 l S T T T T 21 21 1 1 V I I I V supports L1.1, can destroy the maq 22 22 1 1 T T T T T 23 23 FR1 c c c C c 24 24 CDR1 K R R R K 25 25 I 1 THE THE THE T THE* 26 26 1 1 S S S S S * 27 27 1 1 Q Q Q Q Q * 27A 28 1 1 N D S S N 27B 1 1 L 27C 1 1 V 27D 1 1 27E 1 1 27F 1 I 28 29 1 1 V D s I V * 29 30 1 1 THE I I s THE* 30 31 1 1 T s s R * T 31 32 l i N N N / S W N *

• · ··

- 107 2, Continuing Table

Jacket You are # FR Mouse Mouse human human rhv _l Comment CDR 1747 q V Q-L DEN 1747 32 33 | V Y Y L V * 33 34 | V L L THE V * 34 CDR1 N THE 35 35 FR2 w W W w w 36 36! Y Y Y Y Y 37 37! Q Q Q Q Q 38 38 i Q Q Q Q Q 39 39 ί R K K K K 40 40 i P P P P P 41 41 | G G G G G 42 42 I Q Q K E E 43 43! S S THE THE THE 44 44! P P P P P 45 45 i K K K K K 46 46 | THE L L L THE can support L2, L you can change L2 orientations 47 47 | L L L L L 48 48! I I I I I * 49 49 FR2 Y Y Y Y Y 50 50 CDR2 T Y THE G * T 51 51 ' THE THE THE THE THE 52 52 i S S S S S * 53 53! Y R S N Y 54 54! R L L L R 55 55 I F H E E F 56 56 CDR2 S S S S S 57 57 FR3 G G G G G 58 58! V V V V V 59 59! P P P P P

• · • «· · ·

- 108 ···· • · · · * · ··· ··· • · «

Table 2

Jacket # You are FR CDR Mouse 1747 Mouse q V human q-l human DEN RHV _l 1747 Comment 60 60 I E S S S E for group 54, the In L2, on the surface and can bind antiqene 61 61 I 1 R R R R R 62 62 1 1 F F F F F 63 63 1 1 S S S S S 64 64 1 1 G G G G G 65 65 1 1 S S S S S 66 66 1 1 G G G G G 67 67 1 1 S S S S S 68 68 1 1 G G G G G 69 69 1 1 T T T T T 70 70 1 1 D D D E D charge interaction can be with group 24 in L1 71 71 1 1 F Y F F F 72 72 1 1 T S T T T 73 73 1 1 L L L L L 74 74 1 1 T T T T T 75 75 1 1 I I I I I 76 76 I 1 T s s S s 77 77 1 1 N N s S s 78 78 1 1 V L L L L 79 79 1 1 Q E Q Q Q 80 80 1 « S Q P S S 81 81 1 1 E E E D D 82 82 1 ! D D D D D 83 83 1 1 L I F F F 84 84 1 1 THE THE THE THE THE 85 85 1 I D T T T T 86 86 1 1 Y Y Y Y Y 87 87 1 1 F F Y Y Y

109 Table 2.

Jacket # You are FR CDR Mouse 1747 Mouse q V human q-l human DEN RHV _l Note 1747 88 88 FR3 C C C C C 89 89 CDR3 Q Q Q Q Q 90 90 1 Q Q Q Q Q * 91 91 1 Y G Y Y Y * 92 92 1 N N N D N * 93 93 1 1 N T S S N * 94 94 1 Y L L F Y * 95 95 1 P P P P P * 95A 1 I E 95B 1 95C 1 95D 1 t 95E 1 95F 1 96 96 1 Y R w Y Y * 97 97 CDR3 T T T T T 98 98 FR4 F F F F F 99 99 1 G G G G G 100 100 1 1 G G Q Q Q 101 101 1 1 G G G G G 102 102 l 1 T T T T T 103 103 1 1 K K K K K 104 104 1 1 V L V L L 105 105 1 1 E E E E E 106 106 1 1 I I I I I 106A I 1 107 107 FR4 Q K K K K

110 Table 3

Discrepancies in the amino acid sequence of the heavy chain variable domains of the three transformed versions of PB 1.3

Construction 1 amino acid 2 73 1748RH _A Glu below Thr 1748RHb Gin With Thr 1748RH _C Glu below Lys

Table 4

Discrepancies in the amino acid sequence of the four light chain variable domains of PB1.3

Construction 60 amino acid 73 1748RL _A Glu asp 1748RL _B Ser asp 1748RLq Glu Glu 1748RL _D Ser Glu

111 Table 5.

Antibody production rate of the amplified CHO cell line producing three different humanized PB 1.3 (CY1748) versions (microqram / 106 cells / day)

Ellenanyaq Non-amplified 1st round 2nd round 3rd round 1448-IgG1 0.36 2.1 8.2 29.6 0.36 2.1 13.7 21.8 0.36 0.90 9.9 19.7 1748B-IgG1 0.10 1.6 23.9 0.24 1.5 21.1 <0.2 7.3 18.6 0.10 1.6 17.8 1748B-IgG4 <0.3 <0.2 33.0 <0.3 <2.0 26.9 0.75 2.6 23.2 <0.3 3.0 20.8 0.80 6.5 20.8 <0.3 <2.0 19.4 0.78 2.5 18.6 0.78 8.8 17.8

112 Table 6.

PCR amplifiers used for the construction of human P-selectin CRP deletion mutants

DNA sequence

The given number of CRP

PCR 3'-amplimer

TTTCACAGCTCTGCATGC 5'-3 '

8-9 5'-TGCTATGCCTTTGCAGGT-3 '7-9 5'-CTTGATGGCTTCACACAT-3'

6-9 5'-GGGAATGGCTTGGCATTC-3 '5-9 5'-CTGCAAAGCTTGACAGAC-3'

4-9 5'-CGAAATAGCCTCACAGGT-3 '3-9 5'-CTGCACAGCTTTACACAC-3' 2-9 5'-CTGGGCAGCTAAACACTG-3 '

The amino acid sequence used to predict the mutant to be predicted

CRP Broad Craven TIQEA LK .. CKGIA TIQEA LK .. CEAIK TIQEA LK., CQAIP TIQEA LK. CQALQ TIQEA LK .. CEAIS TIQEA LK .. CKAVQ TIQEA LK .. CLAAQ TIQEA LK ..

5'-amplimer, lys-1

AAACCTCCCACACCCCCT 5'-3 '

The two amplimers used to excise only CRP #: 5'-amplimer 5'-TGCACACCTTTGCTAAGC-3 '3'-amplimer 5'-CTGCAAAGCTTGACAGAC-3'

Claims (56)

PATENT CLAIMS Claims 1. A blocking P-selectin antibody that competitively inhibits the binding of an antibody secreted by a cell line deposited at ATCC HB11041 to P-selectin based on a competitive inhibition assay. The blocking P-selectin antibody of claim 1, which inhibits the binding of neutrophils to P-selectin in the absence of Ca ⁺⁺ . The blocking P-selectin antibody of claim 1, which inhibits the binding of neutrophils to P-selectin in the presence of the CQNRYTDLVAIQNKNE peptide. The blocking P-selectin antibody of claim 1, which is an IgG immunoglobulin. The blocking P-selectin antibody of claim 1, which is a rodent antibody. The blocking P-selectin antibody of claim 1, produced by an ATCC accession number HB11041. The antibody of claim 1, which is a Fab, Fab ', F (ab') 2 'Fabc or Fv fragment. 8. A method of detecting P-selectin, comprising the steps of: administering the antibody of claim 1 to a patient or tissue sample thereof; and detecting complexes resulting from specific binding between the antibody and the P-selectin present in the sample. 9. A method of treating an immune system disorder, wherein the patient suffering from the disorder is a P-selectin blocking agent according to claim 1. A therapeutically effective amount of a pharmaceutical composition comprising 114 antibodies and a pharmaceutically acceptable carrier. 10. The method of claim 9, wherein the blocking Pselectin antibody is IgG. 11. The method of claim 9, wherein the blocking Pselectin antibody is of murine origin. 12. The method of claim 9, wherein the blocking Pselectin antibody is produced by cell line ATCC Accession No. HB11041. 13. The method of claim 9, wherein the disease is due to acute lung injury. 14. The method of claim 9, wherein the disease is due to ischemia-reperfusion. 15. The method of claim 9, wherein the blocking Pselectin antibody is administered intravenously. 16. A method of treating a condition of the immune system, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the blocking P-selectin antibody of claim 1 and a pharmaceutically acceptable carrier. A pharmaceutically acceptable composition comprising a pharmaceutically acceptable carrier and the blocking P-selectin antibody of claim 1. The pharmaceutical composition of claim 17, wherein the blocking Pselectin antibody is secreted by cell line ATCC HB11041. 19. The pharmaceutical composition of claim 17, wherein the antibody is of murine origin. 20. A composition comprising the cell line ATCC HB11041. 115 21. A humanized immunoglobulin comprising a humanized heavy chain and a humanized light chain: (1) the humanized light chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having the amino acid sequence derived from the corresponding complementarity determining regions of the murine PB1.3 immunoglobulin light chain and comprising a variable region framework of a human kappa light chain variable region. a region from a framework sequence; and (2) the humanized heavy chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having the amino acid sequence derived from the corresponding complementarity determining regions of the murine PB1.3 immunoglobulin heavy chain, and comprising a variable region framework of a human kappa heavy chain. a variable region from a framework sequence; where the humanized immunoglobulin specifically binds to P-selectin, its lower affinity for binding is about 10 ⁷ mol / l and its upper limit is about five times the binding affinity for mouse PB1.3 immunoglobulin. 22. The humanized immunoglobulin of claim 21, wherein the human kappa light chain variable region framework sequence is altered at at least one position, the change being made at positions L21, L46, L60 and L70; an amino acid at an equivalent position in the framework sequence of the murine PB1.3 immunoglobulin light chain variable region; and altering the human heavy chain variable region framework sequence at at least one position, such change being at positions H1, H2, H71 and H73; an amino acid at an equivalent position in the framework sequence of the murine PB1.3 immunoglobulin heavy chain variable region. 116 »··· ♦ a _w • ♦ · * · * ·· ··· • * · ff« •• ff «« ·· · «· _judgment » The humanized immunoglobulin of claim 22, wherein the humanized heavy chain variable region framework sequence is a 21 / 28'CL heavy chain variable region framework sequence. The humanized immunoglobulin of claim 23, wherein the humanized light chain variable region framework is a DEN light chain variable region framework sequence. The humanized immunoglobulin of claim 24, wherein the humanized light chain variable region framework sequence is substituted at at least two positions from the first group. The humanized immunoglobulin of claim 25, wherein the humanized heavy chain variable region framework sequence is substituted at at least two positions from the second group. 27. The humanized immunoglobulin of claim 21, wherein the humanized light chain comprises the amino acid sequence (SEQ ID NO: 17) of the 1748RLA mature light chain variable region. The humanized immunoglobulin of claim 21, wherein the humanized light chain comprises the amino acid sequence of SEQ ID NO: 1748RLB (see Figure 15). 29. The humanized immunoglobulin of claim 21, wherein the humanized light chain comprises the amino acid sequence (SEQ ID NO: 17) of the 1748RLD mature light chain variable region. 30. The humanized immunoglobulin of claim 21, wherein the humanized light chain comprises the amino acid sequence (SEQ ID NO: 17) of the 1748RLD mature light chain variable region. 117 ·· ••• F · «• *» »• · ♦ ·« ·· • · · · · · · · · · 31. The humanized immunoglobulin of claim 21, wherein the humanized heavy chain comprises the amino acid sequence of the 1748RHA mature heavy chain variable region (SEQ ID NO: 11). 32. The humanized immunoglobulin of claim 21, wherein the humanized heavy chain comprises the amino acid sequence of SEQ ID NO: 1748RHB (see Figure 12). 33. The humanized immunoglobulin of claim 21, wherein the humanized heavy chain comprises the amino acid sequence of the 1748RHC mature heavy chain variable region (SEQ ID NO: 13). 34. The humanized immunoglobulin of claim 31, wherein the humanized heavy chain comprises the amino acid sequence of the 1748RHB mature heavy chain variable region (SEQ ID NO: 12). 35. The humanized immunoglobulin of claim 21 which is a Fab, Fab ', F (ab') 2> Fabc or Fv fragment. 36. The humanized immunoglobulin of claim 34, further comprising a constant region domain. 37. The humanized immunoglobulin of claim 36, wherein the constant region domain has an effector function. 38. The humanized immunoglobulin of claim 34, wherein the constant region lacks an effector function. 39. The humanized immunoglobulin of claim 37, wherein the effector function is capable of complement fixation or antibody-dependent cellular toxicity. 40. The nucleic acid encoding the heavy chain of the humanized immunoglobulin of claim 21. 41. A nucleic acid encoding the humanized immunoglobulin light chain of claim 21. 118 »·» · i · »'• · · ♦ · ··· · Μ • · · ·« »·· #« (· · «· ι» A pharmaceutical composition comprising a humanized antibody of claims 42-34 and a pharmaceutically acceptable carrier. 43. A pharmaceutical composition comprising the humanized antibody of claim 21 and a pharmaceutically acceptable carrier. 44. The pharmaceutical composition of claim 43, wherein the humanized immunoglobulin is a Fab fragment. 45. A method for detecting P-selectin, comprising the steps of: adding the humanized antibody of claim 34 to a patient or tissue sample thereof; and detecting specific binding complexes between the immunoglobulin and the P-selectin present in the sample. 46. A method of treating a patient suffering from a disorder of the immune system comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to claim 43. 47. The method of claim 46, wherein the disease is due to acute lung injury. 48. The method of claim 46, wherein the disease is due to ischemia-reperfusion injury. 49. The method of claim 48, wherein the patient is suffering from ischemic epidermal, myocardial, renal, cerebral, spleen, liver, spinal cord, visceral, lung, body or whole body. 50. A method of treating a patient suffering from a condition of the immune system comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition according to claim 43. 119 - 51. A stable cell line comprising: a nucleic acid segment encoding the heavy chain of the humanized immunoglobulin of claim 34 operably linked to a promoter that permits expression of the heavy chain; a second nucleic acid segment encoding the humanized immunoglobulin light chain of claim 34 operably linked to a second promoter that permits expression of the light chain; the stable cell line is capable of producing the humanized antibody of claim 34. 52. The cell line of claim 51, which is capable of producing 30 pigs / 10θ cells / day from the humanized antibody. 53. The cell line CHO-48B4 of claim 52 which is deposited under ATCC CRL 11596. 54. A purified P-selectin polypeptide consisting of up to 100 amino acids comprising at least five contiguous amino acids from positions 448 and 467 of the amino acid sequence shown in Figure 34 (SEQ ID NO: 1). 55. The purified polypeptide of claim 54, comprising up to 100 amino acids, the up to 100 amino acids having at least five contiguous amino acids at positions 448 and 467 of the amino acid sequence shown in Figure 34. 56. The purified polypeptide of claim 55, comprising essentially the contiguous amino acid sequence (SEQ ID NO: 44) of positions 448 to 467 of the amino acid sequence shown in Figure 34.