MXPA99007584A - Antibody fragment-polymer conjugates and humanized anti-il-8 monoclonal antibodies - Google Patents

Abstract

Humanized anti-IL-8 monoclonal antibodies and variants thereof are described for use in diagnostic applications and in the treatment of inflammatory disorders. Also described is a conjugate formed by an antibody fragment covalently attached to a non-proteinaceous polymer, wherein the apparent size of the conjugate is at least about 500 kD. The conjugate exhibits substantially improved half-life, mean residence time, and/or clearance rate in circulation as compared to the underivatized parental antibody fragment.

Description

CONJUGATES OF ANTIBODY FRAGMENT-POLYMER AND MONOCLONAL ANTIBODIES ANTI-IL-8 HUMANIZED FIELD OF 'THE INVENTION This application relates to the field of antibody fragments derivatized with polymers, and in particular to the use of such derivatization to increase the half-lives in circulation of the antibody-polymer fragment conjugates. This application also relates to humanized anti-interleukin 8 (IL-8) antibodies and to high affinity variants of such antibodies.

BACKGROUND OF THE INVENTION Modification of proteins with pol iA ilengl j. Col ('PEGylation') has the potential to increase microeffects and reduce immunogenicity in. For example, Knauf et al., J. Biol. Chem., 263: 15064-15070 (1988) reported a study of the pharmacodynamic behavior in rats of various species of interleukin-2 modified with polyoxylated glycerol and polyethylene glycol, despite the known advantage REF .: 30916 of PEGylation, PEGylated proteins have not been widely exploited for clinical applications. In the case of antibody fragments, PEGylation has not been shown to extend serum half-life to useful levels. Delgado et al., Br. J. Cancer, 73: 175-182 (1996), Kitamura et al., Cancer Res., 51: 4310-4315 (1991), Kitamura et al., Biochem. Biophys. Res. Comm. , 171: 1387-1394 (1990), and Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994) reported studies that characterize the blood clearance and tissue uptake of certain anti-tumor antigen antibodies. antibody fragments derivatized with low molecular weight PEG (5 kD). Zapata et al., FASEB J., 90 A1479 (1995) reported that low molecular weight PEG (5 or 10 kD) coupled to a sulfhydryl group in the hinge region of a Fab 'fragment reduced clearance compared to ± a Fab 'progenitor molecule. Interleukin 8 (IL-8) is a neutrophilic iotactic qui peptide secreted by a variety of cells in response to inflammatory mediators (for a review see Hebert et al., Cancer Investigation 11 (6): 743 (1993)). IL-8 may play an important role in the pathogenesis of inflammatory disorders, such as adult respiratory distress syndrome (ARDS), septic shock, and multiple organ failure. Immune therapy for such inflammatory disorders may include the treatment of a patient afflicted with anti-IL-8 antibodies. Sticherling et al (J. Im unol. 143: 1628 (1989)) describes the production and characterization of four monoclonal antibodies against IL-8. WO92 / 04372, published March 19, 1992, discloses polyclonal antibodies that react with the site of interaction with the IL-8 receptor and peptide analogs of IL-8, together with the use of such antibodies to prevent an inflammatory response in patients. St. John et al. (Chest 103: 932 (1993)) review irine therapy for ARDS, septic disease, and multiple organ failure, including the potential therapeutic use of anti-IL-8 antibodies. Sekido et al. (Nature 365: 654 (1993)) describes the prevention of damage by pulmonary reperfusion in rabbits by means of a monoclonal antibody against IL-8. Mulligan et al (J. Immunol., 150: 5585 (1993)), describe the protective effects of a murine monoclonal antibody to human IL-8 on inflammatory lung damage in rats. W095 / 23865 (International Application No. PCT / US95 / 02589 published September 8, 1995) demonstrates that anti-IL-8 monoclonal antibodies can be used therapeutically in the treatment of other inflammatory disorders, such as bacterial pneumonias and inflammatory disease of the intestine. Anti-IL-8 antibodies are also useful as reagents for the assay or evaluation of IL-8. For example, Sticherling et al (Arch. Dermatol, Res. 234: 82 (1992)), describe the use of anti-IL-8 monoclonal antibodies as reagents in immunohistochemical studies. Ko et al. (J. Immunol. Methods 149: 227 (1992)) describe the use of anti-IL-8 monoclonal antibodies as reagents in an enzyme-linked immunosorbent assay (LLi A) for 1L-G.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention is a conjugate consisting essentially of one or more fragments of antibodies covalently bound to one or more polymeric molecules, wherein the apparent size of the conjugate is at least about 500 kD. Yet another aspect of the invention is an anti-IL-8 monoclonal antibody or an antibody fragment comprising the regions for determining the complementarity of the amino acid sequence of the light chain polypeptide 6G4.2.5LV11N35E of Figure 45 (SEQ. ID NO: Additional aspects of the invention are a nucleic acid molecule comprising a nucleic acid sequence encoding the anti-Il-8 monoclonal antibody described above or an antibody fragment; an expression vector comprising the nucleic acid molecule operably linked to the control sequences recognized by a host cell transfected with the vector; a host cell transfected with the vector; and a method for producing the dt fragment, antibody, which comprises culturing the host cell under conditions wherein the nucleic acid encoding the antibody fragment is expressed, whereby the antibody fragment is produced, and the antibody fragment from the host cell.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph describing blockade of IL-8-mediated elastase release from neutrophils by anti-IL-8 monoclonal antibody, 5.12.14.

Figure 2 is a graph describing the inhibition of 125I-IL-8 binding to neutrophils by 11-8 unlabelled.

Figure 3 demonstrates that a negative control Fab corresponding to the isotype 'denoted as D5 Fab ") does not inhibit the binding of 125 I-IL8 to human neutrophils.

Figure 4 is a graph describing the inhibition of human neutrophil 125T-tL-8 by the I to chimeric J.J.2.14 with an average ICbu of 1.6 nM.

Figure 5 is a graph describing the inhibition of the binding of 125 I-IL-8 to human neutrophils by the chimeric Fab 6G.4.25 with an average IC 50 of 7.5 nM.

Figure 6 demonstrates the inhibition of neutrophil chemotaxis mediated by human IL-8 by the chimeric Fab 6G4.2.5 and the chimeric Fab 5.12.14.

Figure 7 demonstrates the relative abilities of the chimeric 6G4.2.5 Fab and the chimeric Fab 5.12.14 to inhibit rabbit IL-8 mediated neutrophil chemotaxis.

Figure 8 describes the stimulation of elastase release from human neutrophils by various concentrations of human and rabbit IL-8. The relative degree of elastase release was quantified by measuring the absorbance at 405 nm. The data represent the mean ± SEM of samples in triplicate.

Figure i as a graph describing the ability of the chimeric 6G4.2.5 Fab and the chimeric Fab 5.12.14 to inhibit the release of elastase from human neutrophils, stimulated by human IL-8. The results were normalized to reflect the percentage of elastase release promoted by 100 nM IL-8 alone. The data represent the mean ± SEM of three separate experiments performed on different days with different blood donors. The IC 50 values were calculated by adjusting four parameters.

Figure 10 is a graph describing the relative abilities of the chimeric 6G4.2.5 Fab and the chimeric Fab 5.12.14 to inhibit the release of elastase from human neutrophils, stimulated by rabbit IL-8. The results were normalized to reflect the release rate of elastase promoted by 100 nM IL-8 alone. The data represent the mean + SEM of three separate experiments performed on different days with different blood donors. IC5o values were calculated by adjusting four parameters.

Figures 11A-11J are a group of graphs describing the following parameters in a model of ulcerative colitis in rabbit: Figure HA describes the levels of myeloperoxidase in tissue; Figure 11B describes tissue levels of IL-8; Figure 11C describes the weight of the colon; Figure 1 ID describes gross inflammation; Figure HE describes the edema; Figure 11F describes the degree of necrosis; Figure 11G describes the severity of the necrosis; Figure 11H describes the marginalization of neutrophils; Figure 111 describes the infiltration of neutrophils; and Figure 11J describes the infiltration of mononuclear cells.

Figure 12 is a graph describing the effect of anti-IL-8 monoclonal antibody treatment on the number of neutrophils in bronchoalveolar lavage fluid (BAL) in animals infected with Streptococcus pneumoni ae, Escherichia coli, or Pseudomonas s aerugi nosa Treatment with 6G4.2.5 significantly reduced the number of neutrophils present in the BAL fluid compared to animals treated with the control mouse IgG, isotype (Figure 12).

Guide 13 describes the secu < ucxas? e AD.N (SEQ ID NOS: 1-6) of three primers designed for each of the light and heavy chains. Multiple primers were designed in order to increase the opportunities for primer hybridization and first-strand cDNA synthesis efficiency, for the cloning of light and heavy variable regions of monoclonal antibody 5.12.14.

Figure 14 depicts the DNA sequences (SEQ ID NOS: 7-10) of a forward primer and a reverse primer for the amplification of the light chain variable region of 5.12.14.

Figure 15 describes the DNA sequences (SEQ ID NOS: 11-18) of a forward primer and a reverse primer for the amplification of the heavy chain variable region 5.12.14.

Figure 16 describes the DNA sequence (SEQ ID NO: 19) and the amino acid sequence (SEQ.

ID. NO: 20) of the light chain variable region . 12.14 and the constant light region, murine, partial. CDPs are indicated either by X-ray chromatography (underlined amino acids) or by comparison of Kabat sequences. (amino acids denoted by an asterisk). The important restriction sites are indicated in the italics. The STII signal peptide is amino acids -23 to -l. The variable light region, murine, is amino acids 1 to 109. The partial, murine constant light region is amino acids 110 to 123 (in italics).

Figure 17 describes the DNA sequence (SEQ ID NO: 21) and the amino acid sequence (SEQ.

ID. NO: 22) of the variable region of heavy chain . 12.14 and the murine constant chain region, partial. The CDRs are indicated either by X-ray crystallography (underlined amino acids) or by sequential comparison of Kabat (amino acids denoted by an asterisk). Important restriction sites are indicated in italics. The STII signal peptide is amino acids -23 to -1. The murine variable heavy region is amino acids 1 to 120. The constant, murine, partial heavy region is amino acids 121 to 130.

Fi 'ira 1P describes the sequence of A.DN amplification used to convert the residues of the light and heavy chain constant region, murine, to their human counterparts.

Figure 19 describes the DNA sequence (SEQ ID NO: 27) and the amino acid sequence (SEQ.

ID. NO: 28) for the light chain variable region of 5.12.14 and the light chain constant region of human IgGl. The CDRs are indicated either by X-ray crystallography (underlined amino acids) or by comparison of Kabat sequences (amino acids denoted by an asterisk). The human constant region is denoted in italics. The signal peptide of STII is the amino acids of -23 to -1. The murine variable light region is amino acids 1 to 109. The human constant light region is amino acids 110 to 215.

Figures 20A-20B describe the DNA sequence (SEQ ID NO: 29) and the amino acid sequence (SEQ ID NO: 30) for the heavy chain variable region of 5.12.14 and the chain constant region Heavy of human IgGl. The CDRs are indicated either r.ca or crystallography of X-rays (underlined amino acids) or p ^ r comparison of Kaiíai sequence (amino acids denoted by an asterisk). The human constant region is denoted in italics. The STII signal peptide is amino acids -23 to -1. The murine variable heavy region is amino acids 1 to 120. The human constant heavy region is amino acids 121 to 229.

Figure 21 describes the DNA sequences (SEQ ID NOS: 31-36) of three primers designed for each of the light and heavy chains. The multiple primers were designed in order to increase the opportunities for primer hybridization and the efficiency of the first-strand cDNA synthesis for the cloning of the light and heavy variable regions of the monoclonal antibody 6G4.2.5.

Figure 22 describes the DNA sequences (SEQ ID NOS: 37-40) of a forward primer and a reverse primer for the amplification of the light chain variable region of 6G4.2.5.

Figure 23 describes the DNA sequences (SEQ ID NOS: 41-46) of a forward primer and a reverse primer for the amplification of the heavy chain variable region of 6 < ~ 4 2.5, Figure 24 describes the DNA sequence (SEQ ID NO: 47) and the amino acid sequence (SEQ ID NO: 48) of the light chain variable region 6G4.2.5 and the murine constant, partial light region. The CDRs are indicated either by X-ray crystallography (underlined amino acids) or by Kabat sequence comparison (amino acids denoted by an asterisk). Useful cloning sites are in italics. The STII signal peptide is amino acids -23 to -1. The murine variable light region is amino acids 1 to 114. The murine, partial constant light region is amino acids 115 to 131.

Figure 25 describes the DNA sequence (SEQ ID NO: 49) and the amino acid sequence (SEQ ID NO: 50) of the heavy chain variable region 6G4.2.5 and the constant, murine, partial heavy region . The CDRs are indicated either by X-ray crystallography (underlined amino acids) or by Kabat sequence comparison (amino acids denoted by an asterisk). Useful cloning sites are in italics. The signal peptide of TT? it is the axr i no i -i ^ -23 a1 • 1. The variable heavy metal region is amino acids 1 to 1/7. Heavy, murine, partial heavy region is amino acids 123 to 135.

Figure 26 depicts the DNA sequences (SEQ ID NOS: 51-54) of the primers to convert the murine heavy chain and light chain constant regions to their human counterparts.

Figures 27A-27B describe the DNA sequence (SEQ ID NO: 55) and the amino acid sequence (SEQ ID NO: 56) for the chimeric light chain 6G4.2.5. The CDRs are indicated either by X-ray crystallography (underlined amino acids) or by Kabat sequence comparison (amino acids denoted by an asterisk). The human constant region is denoted in italics. The STII signal peptide is amino acids -23 to -1. The murine variable heavy region is amino acids 1 to 114. The human constant heavy region is amino acids 115 to 220.

Figures 28A-28B describe the sequence of A.DN (SEQ.T.NO:57) and the amino acid sequence (? Ult. ID. NC: b thi, eta x cad au [»osaia 6G4.2.5 chimeric. CDRs are indicated either by X-ray crystallography (underlined amino acids) or by Kabat sequence comparison (amino acids denoted by an asterisk) .The human constant region is denoted in italics.The STII signal peptide is amino acids -23 al -1 The murine variable heavy region is amino acids 1 to 122. The human constant heavy region is amino acids 123 to 231.

Figure 29 depicts an alignment of the amino acid sequence of the light chain variable domain of murine 6G425 (SEQ ID NO: 59), the variable domain of the humanized 6G425 light chain F (ab) -1 (SEQ. NO: 60), and the amino acid sequences of the consensus structure of human light chain (SEQ ID NO: 61), and an amino acid sequence alignment of the murine 6G425 heavy chain variable domain (SEQ. ID NO: 62), the heavy chain variable domain F (ab) -1 of humanized 6G425 (SEQ ID NO: 63), and the heavy chain variable domain of subgroup III of human IgGl (SEQ ID NO: 64), used in the humanization of 6G425 I, the CDRs of ca er? light are "i? r" to os Ll, 1¡¿, L3; ios cj'ils de c e? d pisauc lu, 112 and H3. = y + indicate the CDR sequences as defined by the X-ray crystallographic contacts and the sequential hypervariability, respectively. # indicates a difference between the aligned sequences. The numbering of the waste is according to Kabat and collaborators. The lowercase letters denote the insertion of an amino acid residue in relation to the numbering of the consensus sequence humIII.

Figure 30 is a graph with three panels (A, B and C) that describe the ability of F (ab) -9 (humanized 6G4V11 Fab) to inhibit neutrophil chemotaxis mediated by human wild-type IL-8, human monomeric IL-8 and IL-8 from rhesus monkey, respectively. Panel A presents the inhibition data for F (ab) -9 samples at concentrations of 0.06 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM, for an isotype control antibody (denoted * 4D5" ) of sample at a concentration of 100 nM, and for a control sample without antibody, in the presence of wild-type human IL-8, 2 nM Panel B presents the inhibition data for m? 1? 0ns of F i ah) - 9 a concent t ~ acAnes of 6.25 nM, 12.5 nM, D nM, and 50 nh, for an isotype control antibody sample (denoted * 4D5") at a concentration of 100 nM, and for a control sample without antibody, in the presence of human monomeric IL-8 4 nM (denoted as * BD59"and as * monomeric IL-8"). Panel C presents the inhibition data for F (ab) -9 samples at concentrations of 1 nM, 12.5, 25 nM and 50 nM, for an isotype control antibody sample (denoted * 4D5") at a concentration of 100 nM, and for a control sample without antibody, in the presence of 2 nM rhesus monkey IL-8 In addition, all panels A, B and C each present data for a control sample with buffer without IL-8 (denoted as' Shock absorber ") in the respective inhibition test.

Figure 31A describes the amino acid sequences of the 6G4.2.5V11 light chain of humanized anti-II-8, in an N-terminal fusion with the STII guide peptide (SEQ ID NO: 65), the heavy chain of 6G4.2.5V11 of humanized anti-IL-8 in a fusion N-terminal with the STII guide peptide (SEQ ID NO: 166), and a peptide linker in a C-terminal fusion with the coat protein of the ITI phage M13 gene (SEQ 1L NO: 6 /).

Figure 31B describes the nucleic acid sequence (SEQ ID NO: 68) and the translated amino acid sequence (SEQ ID NO: 65) of the light chain of 6G4.2.5V11 of humanized anti-IL-8, in an N-terminal fusion with the STII guide peptide.

Figure 31C depicts the amino acid sequences of the light chain of 6G4.2.5V19 of humanized anti-IL-8 in an N-terminal fusion with the STII leader peptide (SEQ ID NO: 69), and the heavy chain of 6G4.2.5V19 of humanized anti-IL-8, in an N-terminal fusion with the STII guide peptide (SEQ ID NO: 70).

Figure 32 is a three-dimensional computer model of humanized anti-IL-8 antibody 6G4.2.5V11. Curls or turns of heavy chain CDRs and variable domain regions appear purple, and side chain residues CDR-H3 appear in yellow. The heavy chain constant domain regions appear in red. The turns or curls of light chain CDRs and the variable domain regions appear in white, and the Root Yno of Asn er the osi 0 ^ r 35 of amino acid (N35) in ^ .D Ll appears in verdt. Each region of the light chain constant domain appears in amber.

Figure 33 is a Scatchard plot describing the inhibition of 125I-IL-8 binding to human neutrophils, shown by the intact murine 6G4.2.5 antibody (denoted murine 6G4 mAb), the murine-human chimeric Fab 6G4.2.5 (denoted chimera 6G4), versions 1 and 11 Üe Fab of 6G4.2.5 (denoted VI and VII), and the Fab variant of 6G4.2.5V11N35A (denoted V11N35A).

Figure 34 is a graph with four panels (A, B, C and D) that describe the ability of the 6G4.2.5V11N35A Fab to inhibit neutrophil chemotaxis mediated by human wild-type IL-8, human monomeric IL-8 , Rabbit IL-8, and rhesus monkey IL-8, respectively. Panel A presents the inhibition data for the 6G4.2.5V1135A Fab samples at concentrations of 0.5,], 2, 4, 8, 16 and 33 nM, for an isotype control antibody (denoted '4D5') of sample to a concentration of 33 nM, and for a control sample without antibody (denoted * HuIl-8"), in the presence of wild-type IL-8 hunarn, t 2 nM. Panel B presents the data do? I? iibLuiou jia ia¿ samples dt 'ab ae 6G4.2.5V11N35A at concentrations of 0.5, 1, 2, 4, 8, 16 and 33 nM, for a sample of the mAb 6G4.2.5 intact, at a concentration of 33 nM, for a isotype control antibody sample (denoted as' 4D5") at a concentration of 33 nM, and for a control sample without antibody (denoted * BD59"), in the presence of monomeric IL-8 at concentrations of 2 nM. Panel C presents the inhibition data for Fab samples of 6G4.2.5V1135A at concentrations of 0.5, 1, 2, 4, 8, 16 and 33 nM, for an intact 6G4.2.5 mAb sample at a concentration of 33 nM , for an isotype control antibody sample (denoted '4D5') at a concentration of 33 nM, and for a control sample without antibody (denoted 'Rab IL-8'), in the presence of 2 nM rabbit IL-8. Panel D presents the inhibition data for Fab samples of 6G4.2.5V11N35A at concentrations of 0.5, 1, 2, 4, 8, 16 and 33 nM, for a sample of intact 6G4.2.5 mAb at a concentration of 33 nM, for a sample of isotype control antibody (denoted as D5") at a concentration of 33 nM, and for a control sample without antibody (denoted * Rhe IL-8"), in the presence of II, -R 7 nM. of monkey rherus. In addition, panei s B, C ü ^, is Yin each i? Ü aatot ra control samples of wild type, human IL-8 (denoted "uIL-8") at a concentration of 2 nM in the respective test, and panels A, B, C and D present each data for a control sample of shock absorber without IL-8 (denoted 'Shock absorber') in the respective test.

Figure 35 depicts the amino acid sequences of the humanized anti-IL-8 6G4.2.5V11N35A light chain in an N-terminal fusion with the STII leader peptide (SEQ ID NO: 71), the heavy chain of 6G4. 2.5V11N35A humanized anti-IL-8, in an N-terminal fusion with the STII guide peptide (SEQ ID NO: 66), and the leucine zipper peptide GCN4 (SEQ ID NO: 72). The Ala residue (substituted for the wild type Asn residue) at amino acid position 35 in the light chain of 6G4.2.5V11N35A appears in bold. A putative pepsin cleavage site in the GCN4 leucine zipper sequence is underlined.

Figure 36 describes the DNA sequence (SEQ ID NO: 73) and the amino acid sequence (SEQ ID NO: 71) of the light chain of humanized anti-IL-8 6G4.2.5V11N35A, in an N-terminal position with the Lid guide bl ü -. «. yloiies ae i'etermj na on complementarity Ll, L2 and L3, are underlined.

Figures 37A-37B describe the DNA sequence (SEQ ID NO: 74) and the amino acid sequence (SEQ ID NO: 75) of the heavy chain of humanized anti-IL-8 6G4.2.5V11N35A, in an N-terminal fusion with the STII guide peptide and a C-terminal fusion with the leucine zipper sequence GCN4. The complementarity determination regions Hl, H2 and H3 are underlined.

Figure 38 is a Scatchard plot describing the inhibition of 125I-IL-8 binding to human neutrophils, shown by the Fab of 6G4.2.5V11N35A (denoted Fab), F (ab ') 2 of 6G4.2.5V11N35A (denoted F (ab ') 2), and control of wild-type, human IL-8 (denoted IL-8).

Figure 39 is a graph describing a comparison of the activities of inhibiting chemotaxis of neutrophils mediated by human wild-type IL-8, F (ab ') 2 of 6G .2.5V11N35A and Fab of ^ G4.7.5V11N ^ 5A. The dates < Inhibition presents pdia lac, 6G4 Fab samples. ? .5VI 1 JD (denoted 'Fab N35A') and samples of F (ab ') 2 from 6G4.2.5V11N35A (denoted F (ab ') 2 of N35A) at concentrations of 0.3, 1, 3, 10, 30 and 100 nM, for an isotype control antibody sample (denoted as * 4D5") at a concentration of 100 nM, and for a control sample without antibody, in the presence of human wild-type IL-8, 2 nM In addition, the inhibition data are presented for the control samples of buffer without IL-8 (denoted 'Shock absorber').

Figure 40 is a graph describing the ability of F (ab ') 2 of 6G4.2.5V11N35A to inhibit neutrophil chemotaxis mediated by human monomeric IL-8, rhesus monkey IL-8, and rabbit IL-8. Neutrophil chemotaxis data mediated by human monomeric IL-8 are presented for samples of F (ab ') 2 of 6G4.2.5V11N35A at concentrations of 0.3, 1, 3 and 10 nM, for a sample of isotype control antibody (denoted as * 4D5") at a concentration of 100 nM, and for a control sample without antibody (denoted as 'BD59'), in the presence of human monomeric IL-8 (denoted as 'BD59') at a The 0.5 nM concentration of neutrophil counts measured by rhesus monoclonal IL-L was measured for samples of F (ab ') 2 from 6G .2.5V11N35A at concentrations of 0.3, 1, 3 and 10 nM, and for a control sample without antibody, in the presence of rhesus monkey IL-8 at a concentration of 2 nM.The data of neutrophil chemotaxis mediated by rabbit IL-8 are presented for samples of F (ab ') 2 of 6G4.2.5V11N35A at concentrations of 0.3, 1, 3 and 10 nM, and for a control sample without antibody, in the presence of rabbit IL-8 at a concentration of 2 nM In addition, the inhibition data are presented for a control sample of buffer without IL-8 (denoted as 'Shock absorber') and for a wild-type, human IL-8 at a concentration of 2 nM (denoted as? UIL-8). ").

Figures 41A-41Q describe the nucleic acid sequence (SEQ ID NO: 76) of the F (ab ') 2 vector of p6G4.2.5V11N35A.

Figure 42 depicts the nucleic acid sequences of the stop template primer (SEQ ID NO:) and the NNS scrambling primer (SEQ ID NO:) used for the random mutagenesis of the amino acid position in the CDP -Light cauena variable of anticue i,, or humanized 6G4V11.

Figure 43A is a data table describing the frequencies of different phage visualization clones, obtained from the randomization of the amino acid position in the CDR-Ll variable light chain of the humanized antibody 6G4V11.

Figure 43B contains the graphs of the displacement curves describing the inhibition of the binding of 125I-IL-8 to neutrophils, shown by the Fab's of 6G4V11N35A, 6G4V11N35D, 6G4V11N35E and 6G4V11N35G Figure 44 contains a graph describing the typical kinetics of an anti-IL-8 antibody fragment (f (ab ') 2 of 6G4V11N35A) that binds to IL-8. Figure 44 also contains a data table that provides the equilibrium constant for Fab 6G4V11N35A that binds to IL-8 (speed constants were not determined 'ND'), and equilibrium and velocity constants for F ( abA7 - 6G4V1JN35A and Fab 6G V11N35E sue bind to IL -b.

Figure 45 describes the DNA sequence (SEQ ID NO:) and the amino acid sequence (SEQ.

ID. NO:) of the light chain of 6G4V11N35E in an N-terminal fusion with the STII guide peptide. The complementarity determination regions Ll, L2 and L3 are underlined.

Figure 46 is a graph describing the ability of Fab 6G4V11N35E to inhibit neutrophil chemotaxis mediated by human IL-8 (dark columns) and rabbit 11-8 (light columns). The data is presented for the 6G4V11N35E Fab samples at concentrations of 0.4, 1.2, 3.7, 11 and 33 nM, and for an isotype control antibody sample (4D5) at a concentration of 100 nM, in the presence of human IL-8 2 nM or rabbit IL-8 2 nM. In addition, the inhibition data are presented for a control sample of buffer without IL-8 (denoted 'Shock absorber') and for the control samples of human and rabbit IL-8 (denoted 'IL-8').

In Figure 47 describes the DNA sequence of the strands in - UJICXJ Y. NO:) and in antisense (SEQ ID NO:) of a synthetic nucleotide PvuII-XhoI coding for the amino acids Leu4 to Phe29 of the heavy chain of 6G4V11N35A.

Figures 48A-48T describe the DNA sequence (SEQ ID NO:) of the plasmid p6G4VHN35A.choSD9.

Figure 9 contains the graphs of the displacement curves describing the inhibition of 125I-IL-8 binding to neutrophils, shown by the full length IgG1 forms of the 6G4V11N35A and 6G4V11N35E variants.

Figures 50A-50B are graphs describing the ability of IgGl 6G4V11N35A and IgGl 6G4V11N35E to inhibit neutrophil chemotaxis mediated by human IL-8 (Figure 50A) and rabbit IL-8 (Figure 50B).

Figure 51 contains a graph that shows the typical kinetics of an anti-TL-8 antibody of length e or pLeta (IgG 6G < * V1 iN.s A) that binds to IL-8. Figure 51 also contains a table of data providing the equilibrium and velocity constants for full length IgG2a 6G4.2.5, 6G4V11N35A IgGl and 6G4V11N35E IgGl, which bind to IL-8.

Figure 52 contains graphs of displacement curves describing the results of a competition radioimmunoassay IL-8 / 125I-IL-8 performed with 6G4V11N35A IgGl and full length 6G4V11N35E IgGl.

Figure 53 describes the DNA sequence (SEQ ID NO:) and the amino acid sequence (SEQ.

ID. NO:) of the heavy chain Fab 'of 6G4V11N35A (heavy chain Fab of 6G4V11N35A modified to contain a cysteine residue in the hinge region).

Figures 54A-54C contain graphs of the displacement curves describing the binding of IL-8 and IC50 for the Fab 'molecules of 6G4V11N35A modified with PEG-maleimide.

Figures 0bA-55C are gi whch describe the ability of Fab molecules "to 6G4V11N35A modified with PEG-maleimides to inhibit neutrophil chemotaxis mediated by human IL-8 and rabbit IL-8.

Figures 56A-56C are graphs describing the ability of Fab 'molecules to 6G4V11N35A modified with PEG-maleimides to inhibit the IL-8 mediated release of β-glucuronidase.

Figures 57A-57B contain graphs of displacement curves describing inhibition of 125I-IL-8 binding to neutrophils, shown by 6G4V11N35A Fab'2 molecules modified with PEG-succinimide.

Figures 58A-58B are graphs describing the ability of 6G4V11N35A F (ab ') 2 molecules modified with PEG-succinimide to inhibit neutrophil chemotaxis mediated by human IL-8.

Tables 59A-59E, graphical oon qn-describe the ability of the molecules of F (ab ') 2 of 6G4V11N35A modified with PEG-succinimide to inhibit the human IL-8 mediated release of neutrophil β-glucuronidase.

Figure 60 is a graph that describes the theoretical molecular weight (dashed bars) and the effective size (solid bars) of the molecules of Fab '6G4V11N35A modified with PEG-maleimide, as determined by SEC-HPLC.

Figure 61 is an SDS-PAGE gel that describes the electrophoretic mobility of various molecules of Fab '6G4V11N35A modified with PEG-maleimide.

Figure 62 contains the size exclusion chromatograms (SEC-HPLC) that describe the retention times and effective sizes (hydrodynamic) of various molecules of F (ab ') 2 of 6G4V11N35A.- Figure 63 is a graph that describes the theoretical weight (open columns), the effective size determined? -io by solid SLO iXC IU? I L), and the effective molecular weight determined by SEC-light scattering (shaded columns) for various 6G4V11N35A F (ab ') 2 molecules modified with PEG-succinimide.

Figure 64 is an SDS-PAGE gel describing the electrophoretic mobility of various 6G4V11N35A F (ab ') 2 molecules modified with PEG-succinimide. From left to right, band 1 contains unmodified F (ab ') 2, band 2 contains F (ab') 2 coupled to two 40 kD branched PEG-succinimide molecules (denoted 'Br (2) ~ 40kD (N) -F (ab ') 2"), band 3 contains F (ab') 2 coupled to a branched 40 kD PEG-succinimide molecule (denoted 'Br (1) -40kD- (N) -Fab '2"), band 4 contains a mixture of F (ab') 2 coupled to four linear molecules of PEG-succinimide of 20 kD, and F (ab ') 2 coupled to five linear molecules of PEG-succinimide of 20 kD (denoted 'L (4 + 5) -20kD- (N) -Fab'2"), band 5 contains F (ab') 2 coupled to a linear molecule of PEG-succinimide of 20 kD (denoted 'L (1) -20kD- (N) -Fab '2"), and band 6 contains the standards or hidden weight.

Figure 65 contains graphs comparing serum concentration versus time profiles of various 6G4V11N35A Fab 'molecules modified with PEG-maleimide (upper graph) and various 6G4V11N35A F (ab') 2 molecules modified with PEG-succinimide (bottom graph) in rabbits. In the upper graph, 'bran. (1) 40K (s) Fab '"denotes Fab' of 6G4V11N35A coupled to a branched PEG-maleimide molecule of 40 kD, 'lin. (1) 40K (s) Fab'" denotes Fab 'of 6G4V11N35A coupled to a molecule Linear PEG-Maleimide 40 kD, 'lin. (1) 30K (s) Fab '"denotes Fab' of 6G4V11N35A coupled to a linear molecule of PEG-maleimide of 30 kD, 'lin. (1) 20K (s) Fab'" denotes Fab 'of 6G4V11N35A coupled to a molecule Linear PEG-maleimide 20 kD. In the lower graph, 'bran. (2) 40K (N) Fab '2"denotes F (ab') 2 of 6G4V11N35A coupled to two branched molecules of 40 kD PEG-succinimide, bran. (1) 40K (N) Fab'2" denotes F (ab). ab ') 2 of 6G4V11N35A coupled to a branched PEG-succinimide molecule of 40 kD, and' Fab '2"denotes F (ab') 2 of unmodified 6G4V11N35A In both graphs, * IgG" denotes one equivalent of IgGl Full-length human-to-human kidney Fab non-L-8 ontjO, ueseri lo aae Xj emploi i mas.

Figure 66 contains the graphs comparing the serum concentration profiles versus Fab 'time of 6G4V11N35A coupled to a branched PEG-maleimide molecule of 40 kD (denoted as' bran. (L) 40K (s) Fab' "), the F (ab ') 2 of 6G4V11N35A coupled to a branched PEG-succinimide molecule of 40 kD (denoted as "bran. (1) 40K (N) Fab' 2"), the F (ab ') 2 of 6G4V11N35A not modified (denoted as 'Fab'2'), the Fab 'of 6G4V11N35A (denoted as' Fab '"), and a full-length IgGl equivalent (' denoted as 'IgG') of the human-murine anti-IL chimeric Fab -8 rabbit described in Example F below.

Figure 67 is a graph describing the effect of Fab 'of 6G4V11N35A coupled to a 40 kD PEG-maleimide branched molecule (denoted as' PEG 40 kD') and rabbit anti-rabbit IL-8 6G4 monoclonal antibody. 2.5 (full-length IgG2a) (denoted as '6G4.2.5') on the gross weight of the whole lung in an ARDS rabbit model.

The figure o3 is a gitni.Cc. q. two ibe the Fab 'effect of 6G4V11N35A coupled to a branched 40 kD PEG-maleimide molecule (denoted as' PEG 40 kD') and the rabbit anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (full length IgG2a ) (denoted as '6G4.2.5') on BAL total leukocyte counts (clear columns) and polymorphonuclear cells (dark columns) in the ARDS rabbit model. The data from untreated (non-therapeutic) control animals are denoted as 'Control'.

Figure 69 is a graph describing the effect of FAb 'of 6G4V11N35A coupled to a branched molecule of 40 kD PEG-maleimide (denoted as' PEG 40 kd') and rabbit anti-rabbit IL-8 monoclonal antibody, 6G4 .2.5 (full length IgG2a) (denoted as' 6G4.2.5") on the proportion Pa02 / proportion FÍ02 in the post-treatment of 24 hours (clear columns) and post-treatment of 48 hours (dark columns) in an ARDS rabbit model. The data from untreated (non-therapeutic) control animals are denoted as 'Control'.

DESCRIPTION OF THE PREFERRED MODALITIES I. DEFINITIONS In general, the following words or phrases have the indicated definition when used in the description, examples and claims.

'Polymerase chain reaction "or" PCR "refers to a method or technique in which minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described in US Patent No. 4,683,195 issued on July 28, 1987. In general, sequential information from the ends of the region of interest or beyond, needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The nucleotides d and 5 'end of the two primers may coincide with the ends of the amplified material. The. PCR can be used to amplify specific RNA sequences, specific DNA sequences of the total genomic DNA, and the transcribed cDNA from the cellular RNA tota1. bacteriophage or plasmid sequences, etc. See general llullis et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a nucleic acid known as a nucleic acid. primer, and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid. 'Antibodies' (Abs) and 'immunoglobulins' (Igs) are glycoproteins that have the same structural characteristics. While antibodies show specificity for binding to a specific antigen, immunoglobulins include antibodies and other antibody-like molecules which lack antigen specificity. The polypeptides of the last type are, for example, produced at low levels by the lymphatic system and at levels increased by myelomas. 'Native antibodies and immunoglobulins' are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two heavy chains i'lenlJC i ''. a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has intrachain disulfide bridges, regularly spaced.Each heavy chain has at one end a domain variable (VH) followed by a number of constant domains. "Each light chain has a variable domain at one end (Vi) and a constant domain at its other end.; the constant domain of the light chain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that the particular amino acid residues form an interconnection between the variable domains of light chain and heavy chain (Clothia et al., J. Mol. Biol. 186: 651 (1985); Novotny and Haber, Proc. Nat. Sci. U. S. A., ._ 82: 4592 (3985)). The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among the antibodies and are used in the binding and specificity of the particular antibody to its particular antigen. di Lability c is uniformly distributed throughout the variable domains of antibodies, which is concentrated in three segments called complementarity determination regions (CDRs) or hypervariable regions in both the light chain and the heavy chain variable domains.

The most highly conserved portions of the variable domains are called the structure (FR). The variable domains of the native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form turns or loops that connect, and in some cases that are part of, the sheet structure ß. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from another chain, contribute to the formation of the antigen binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but it does show various effector functions, such as the antigenic activity of the antibody and the antibody-dependent cellular toxicity. Papain digestion of the antibodies produces two identical fragments that bind to the antigen, called 'Fab' fragments, each with a single binding site to the antigen, and a residual 'Fe' fragment, whose name reflects its ability to rapidly crystallize . Treatment with papain produces an F (ab ') 2 fragment that has two antigen binding sites, and is still capable of cross-linking the antigen. 'Fv' is the minimum antibody fragment that contains a complete antigen binding and recognition site.In a two chain Fv species, this region consists of a dimer of a heavy chain variable domain and a light chain dimer in non-covalent, tight or narrow association In a single chain Fv (scFv) species, a variable domain of a heavy chain and a light chain can be covalently linked by a flexible peptide linker such that light and heavy chains can associate in a 'dimeric' structure analogous to that in a homogeneous species of two chains. It is in this configuration that the CDRs or e each variable domain interact to define a binding site for the antigen on the surface of the VH-VL dimer. Collectively, the six CDRs confer specificity of binding to the antigen, to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site. For a review of scFv see Pluckthun, in Th e Ph weapon col ogy of Monocl ona l An tibodi es, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pgs. 269-315 (1994). The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of a few residues at the carboxyl ends of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains have a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cyst between them. Other chemical couplings of antibodies are also known. The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM and several of these can also be divided into subclasses. (isotypes), for example, igGi, IgG2, IgG3, IgG, IgAi, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated a, d, e,?, And μ, respectively. The subunit structures and the three-dimensional configurations of the different classes of immunoglobulins are well known. The term "antibody" is used in the broadest sense and specifically covers the monoclonal antibodies rimóles (including the anti-bodies agonists and ¿n, tagonista) and antibody compositions with poliepitópica specificity. 'Fragment of antibody ", and all grammatical variants thereof, as used herein, are defined as a portion of an intact antibody comprising the antigen binding site or the variable region of the intact antibody, wherein the portion is free of the constant domains of the antibody. heavy chain (eg, CH2, CH3 and CH4, depending on the antibody isotype) of the Fe region of the intact antibody. Examples of antibody fragments include the Fab, Fab ', Fab'-SH, F (ab') 2, and Fv fragments; the diabodies; any antibody fragment which is a polypeptide having a primary structure consisting of an uninterrupted sequence of contiguous amino acid residues (referred to herein as a 'single chain antibody fragment' or 'single chain polypeptide'), including without limitation (1) single chain Fv molecules (scFv) (2) single-chain polypeptides containing only one light chain variable domain, or a fragment thereof, which contains the three CDRs of the light d ^ ca variable domain, without a portion of a picture; ia asoe da and (3 / pol é lid s single chain containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without a portion of associated light chain and the multispecific or multivalent structures formed from the antibody fragments In an antibody fragment comprising one or more heavy chains, the heavy chain (s) may contain any constant domain sequence (eg CH1 in the IgG isotype) found in a non-Fe region of an intact antibody, and / or may contain any hinge region sequence found in an intact antibody, and / or may contain a leucine zipper sequence fused to or located in the sequence of the hinge region or the constant domain sequence of the heavy chain (s) The suitable leucine zipper sequences include the leucine zippers jun and fos shown by Kostelney et al., J. Immunol. , 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the Examples below. Unless specifically indicated to the contrary, the conjugated + "as described and claimed herein, is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragments to one or more polymeric molecules, wherein the heterogeneous molecule is soluble in water, for example, soluble in physiological fluids such as blood, and wherein the heterogeneous molecule is free of any structured aggregate.In the context of the above definition, the term 'structured aggregate' refers to (1) any aggregate of molecules in aqueous solution having a spheroid structure or spheroid shield or shell, such that the heterogeneous molecule is not in a micellar or other emulsion structure, and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insolubilized form, such as a matrix of spheres for chromatography, which does not release the heterogeneous molecule in solution after contact with an aqueous phase. Accordingly, the term "conjugate" as defined herein encompasses the aforementioned heterogeneous molecule in a precipitate, pellet, bioerodible matrix or other solid capable of releasing the heterogeneous molecule in aqueous solution after hydration of the solid. As otherwise indicated, the terms 'polymer', 'polymer molecule', 'non-protein polymer' and 'non-protein polymer molecule' are used interchangeably and are defined as a molecule formed by the covalent bond of two. or more monomers, wherein none of the monomers is contained in the group consisting of alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly) residues. ), histidine (His), isoleucine (lie), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser ), threonine (Thr), valine (Val), tryptophan (Trp), and tyrosine (Tyr). The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, the individual antibodies comprising the population are identical except for possible mutations of origin which may be present in smaller amounts Monoclonal antibodies are highly specific, being directed <; ~ ^ a unique antiquarian site. In addition, in contrast to conventional (polyclonal) preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous since they can be synthesized by culture of hybridomas, not contaminated by other immunoglobulins. The 'monoclonal' modifier identifies the character of the antibody by being obtained from a substantially homogeneous population of antibodies, and should not be considered as the requirement for the production of the antibody by any particular method, for example, the monoclonal antibodies that are going to be used in accordance with the present invention can be used by the hybridoma method first described by Kohler et al., Na ture, 256: 495 (1975), or they can be made by recombinant ADβ methods (see for example, US Pat. No. 4,816,567 to Cabilly et al.) "Monoclonal antibodies" also include clones of antibody fragments that contain the recognition of the tiger and the site of fusion (clones of Fv) on the sides of er-? Lc-cis de an Licucrpo lú icos, aLu a. the techniques described in Clackson et al., Na t ure, 352: 624-628 (1991) and Marks et al., J. Mol. Bi ol. , 222: 581-597 (1991), for example. Monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable domain (including hypervariables) of an anti-IL-8 antibody with a constant domain (eg, "humanized antibodies"), or a light chain with a heavy chain, or a chain from a species with a chain from another species, or fusions with heterologous proteins, regardless of the species of origin or class of immunoglobulin or class designation, as well as antibody fragments (eg , Fab, F (ab ') 2 and Fv), as long as they show the desired biological activity (See, for example, US Patent No. 4,816,567 to Cabilly et al., Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications , pp. 79-97 (Marcel Dekker, Inc., New York, 1987).) Monoclonal antibodies herein specifically include 'chimeric' antibodies (inrruneal obu A n ar) in which unr, norcXón of the string pescd- > / or liyei, e < identical with or homologous to the corresponding sequences in the antibodies derived from a particular species, or belonging to a particular class or subclass of antibody, while the rest of the chain (s) is identical with its homologous to the corresponding sequences in the antibodies derivatives of other species, or belonging to another class or subclass of antibody, as well as fragment of such antibodies, as long as they show the desired biological activity (Cabilly et al., supra; Morrison et al., Proc. Nati. Acad. Sci USA, 81: 6851 (1984)). The "humanized" forms of the non-human (eg, murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2, or other antibody subsequences. which are linked to the antigen) which contain minimal sequence derived from a non-human immunoglobulin For the most part, the humanized antibodies are human immunoglobulins (container antibody) in which the residues from a region of determination of complementarity (CDR ) of the recipient (patient) are relayed by residues from a Cx) R of a non-human species (donor antibody) such as mouse, rat, or rabbit, which has the desired specificity, affinity, and capacity. In some cases, the Fv structural residues of human immunoglobulin are replaced by corresponding non-human residues., the humanized antibodies may comprise residues that are not found in the antibody of the recipient or in the imported CDR or in the structure sequences. These modifications are elaborated to refine and further maximize the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a human immunoglobulin, and all or substantially all regions of FR are those of a consensus sequence of one - >; nmunoglobul.i na human. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fe) typically that of a human immunoglobulin. For additional details see Jones et al., Nat'ire 321: 52? (1 ° 86A * Rei chmann v. Collaborators, Nature 332: 323 (1, 7), and TXosta, lurr, Op. Btruct Biol. 2: 593 (1992). 'Treatment' refers to therapeutic treatment and measures prophylactic or preventive: Those in need of treatment include those already with the disorder, as well as those prone to have the disorder or those in which the disorder is to be prevented. Mammal "for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal in the present is a human. , protein, peptide and polypeptide are used interchangeably to denote an amino acid polymer or a group of two or more amino acid polymers that interact or are linked together.

'Inflammatory disorders' refers to pathological states that result in inflammation, hypothetically caused by chemotaxis of neatrophils.Examples of such disorders include inflammatory skin diseases including psoriasis, responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); ischemic reperfusion; adult respiratory distress syndrome; dermatitis; meningitis; encephalitis; uveitis; autoimmune diseases such as rheumatoid arthritis; Sjorgen syndrome, vasculitis; diseases that involve leukocyte diapedesis; inflammatory disorder of the central nervous system (CNS), multiple organ damage syndrome secondary to septicemia or trauma; alcoholic hepatitis; bacterial pneumonia, diseases mediated by the antigen-antibody complex; inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, and cystic fibrosis; etc. Preferred indications are bacterial pneumonia and inflammatory bowel disease, such as ulcerative colitis. The terms 'hydrodynamic size', 'apparent size', 'apparent molecular weight', 'effective size' and 'effective molecular weight' of a molecule, are used as synonyms in the present and refer to the size of a molecule as determined by comparison to a standard curve produced with the molecular weight standards of the globular protein, in a size exclusion chromatography system, where the standard curve is created by mapping the effective molecular weight of each standard against its elution time, observed in the size exclusion chromatography system Thus, the apparent size of a test molecule is derived by the use of the elution time of the molecule to extrapolate a putative molecular weight from The standard curve Preferably, the molecular weight standards used to create the standard curve are selected such that the apparent size of the test molecule falls within the linear portion of the standard curve.

II. MODES FOR CARRYING OUT THE INVENTION In one part, the invention arises from the surprising and unexpected discovery that antibody-polymer fragment conjugates having an effective and apparent size significantly greater than the antibody-polymer fragment conjugates described in the art, confer an increase in the serum half-life, an increase in mean residence time in circulation (MRT), and / or a decrease in the rate of serum clearance on the non-derivatized antibody fragment, which far exceeds the modest changes in such property or biological properties obtained with the antibody-polymer fragment conjugates, known in the art. The present inventors have determined for the first time that the increase in the effective size of an antibody fragment to at least about 500,000 D, or increasing the effective size of an antibody fragment by at least about 8 times the effective size of the fragment of progenitor antibody, or derivatizing an antibody fragment with a polymer of at least about 20,000 D molecular weight, a molecule with a commercially useful pharmacokinetic profile is produced. The greatly prolonged serum half-life, extended MRT, and / or reduced serum clearance rate of the conjugates of the invention, makes such conjugates viable alternatives to intact antibodies used for the therapeutic treatment of many disease indications. Irrigation in antibodies provides significant advantages over intact antibodies, mainly due to the fact that recombinant antibody fragments can be elaborated in bacterial cell expression systems. Bacterial cell expression systems provide several advantages over expression systems in mammalian cells, including reduced time and cost for research and development, and for the steps of manufacturing a product. In yet another part, the present invention also arises from the humanization of rabbit anti-rabbit IL-8 monoclonal antibody, 6G4.2.5 C6G4.2.5") described in W095 / 23865 (PCT / US95 / 02589 published September 8, 1995), the full disclosure of which is specifically incorporated by reference herein The 6G4.2.5 antibody hybridoma producer was deposited on September 28, 1994 with the North American Collection of Species Crops (American Type Culture Collection) and was given ATCC Accession Number HB 11722 as described in the Examples below In one aspect, the invention provides an identification humanized antibody 6G4.2.5, variant 11 (herein referred to as '6G4.2.5vll'), in which the murine CDRs of 6G4.2.5 are grafted onto a consensus structure for the human light chain? and the subgroup III of the heavy chain of human IgG1, followed by three structural import residues from the variable domain sequence of the progenitor heavy chain of murine 6G4.2.5, within analogous sites in the variab domain the heavy chain sequence of the human template sequence, as described in the Examples below. In yet another aspect, the invention provides variants of the 6G4.2.5vll antibody with certain amino acid substitutions that produce increased affinity for IL-8 and / or that promote greater efficiency in recombinant manufacturing processes. It will be understood that in the context of this Section (II) and all subsections thereof, each reference to 'an antibody fragment' or 'the antibody fragment' contained in a conjugate, will be a reference to one or more antibody fragments in the conjugate (consistent with the definition of the term 'conjugate' described in Section (I) above), except where the number of antibodies in the conjugate is expressly indicated, it will be understood that in the context of this Section (II) and all subsections thereof, any reference to 'a polymer', 'a polymer molecule', 'the polymer', or 'the polymer molecule' contained in a conjugate, will be a reference to one or more polymer molecules in the conjugate (consistent with the definition of the term 'conjugate') ", described in Section (I) above), except where the number of polymer molecules in the conjugate is expressly indicated. 1. CONJUGATES OF ANTIBODY FRAGMENTS-LARGE EFFECTIVE SIZE POLYMER In one aspect, the invention provides an antibody fragment covalently coupled to a polymer to form a conjugate having an effective or apparent size of at least about 500,000 Daltons (D). In yet another aspect, the invention provides an antibody fragment covalently bound to a polymer, to form a conjugate having an apparent size that is at least about 8 times greater than the apparent size of the parent antibody fragment. In yet another aspect, the invention provides an antibody fragment covalently uniao to a polymer of at least about 20,000 D molecular weight (MW). It will be appreciated that the unexpected and surprisingly large increase in the serum half-life of the antibody fragment, the increase in MRT, and / or the decrease in the rate of serum clearance, can be achieved by the use of any type of polymer or any number of polymeric molecules that will provide the conjugate with an effective size of at least about 500,000 D, or by the use of any type of polymer or number of polymeric molecules that will provide the conjugate with an effective size that is at least about 8 times greater than the effective size of the parent antibody fragment, or by the use of any type or number of polymers wherein each polymer molecule is at least about 20,000 D molecular weight. Thus, the invention is not dependent on the use of any particular polymer or molar ratio of polymer to antibody fragment in the conjugate. In addition, the beneficial aspects of the invention extend to the antibody fragments without considering the specificity of antigen. Although variations from antibody to antibody have to be expected, the specificity to the antigen of a given antibody will not substantially impair the extraordinary improvement in the half-life. in serum, the MRT and / or the serum clearance rate for the antibody fragments thereof, which can be obtained by derivatization of the antibody fragments as shown herein. In one embodiment, the conjugate has an effective size of at least about 500,000 D, or at least about 800,000 D, or at least about 900,000 D, or at least about 1,000,000 D, or at least about 1,200,000 D. , or at least about 1'400,000 D, or at least about 1'500,000 D, or at least about 1'800,000 D, or at least about 2'000,000 D, or at least about 2 500,000 D. In another embodiment, the conjugate has an effective size of or about 500,000 D, up to or about 10,000,000 D, or an effective size of or about 500,000 D, up to or about 8,000,000. D, or an effective size of or about 500,000 D up to or about 5,000,000 L, or an effective size of or about 500,000 D up to or about 4,000,000 D, or an effective size of or approximately 500,000 D up to or approximately 3,000,000 D, or an effective size of or approximate 500,000 D up to or about 2'500,000 D, or an effective size of or about 500,000 D up to or about 2'000,000 D, or an effective size of 500,000 D up to or about 1'800,000 D, or an effective size of or about 500,000 D up to or about 1'600,000 D, or an effective size of or about 500,000 D up to or about 1'500,000 D, or an effective size of or about 500,000 D up to or about 1 '000, 000 D. In yet another embodiment, the conjugate has an effective size of or about 800,000 D up to or about 10,000,000, or an effective size of or about 800,000 D up to or about 8,000,000 D, or an effective size of or about 800,000 D, up to or about 5,000,000 D, or an effective size of or about 800,000 D up to or about? '000, 000 D, or an effective size of or about fcü0,000 ü up to or about 3' 000, 000 D, or an effective size of or about 800,000 D up to or about 2 '500,000 D, or an effective size of or about 800,000 D up to or about 2'000,000 D, or an effective size of or about 800,000 D up to or about 1'800,000 D, or an effective size of or about 800,000 D up to or about 1'600, 000 D, or an effective size of or about 800,000 D up to or about 1'500,000 D, or an effective size of or about 800,000 D up to or about 1'000,000 D. In another embodiment, the conjugate has a size effective of or about 900,000 D up to or about 10,000,000 D, or an effective size of or about 900,000 D up to or about 8,000,000 D, or an effective size of or about 900,000 D up to or about 5,000 , 000 D, or an effective size of or about 900,000 D up to or about 4,000,000 D, or an effective size of or about 900,000 D up to or about 3,000,000 D, or an effective size of or about 900,000 JD up to or about 2 '500,000 D, or an effective size of or about 900,000 D up to or about 2'000,000 D, or an effective size of or about 900,000 D up to or about 1' 800,000 D, or an effective size of or about 900,000 D up to about 1'600,000 D, or an effective size of or about 900,000 D up to or about 1'500,000 D. In another embodiment, the conjugate has an effective size of or about 1'000, 000 D up to or about 10'000, 000 D, or an effective size of or about 1'000, 000 D up to or about 8 '000, 000 D, or an effective size of or about 1' 000, 000 D up to or about 5 '000, 000 D, or an effective size of or about 1' 000, 000 D up or about 4,000,000 D, or an effective size of or about 1,000,000 D up to or about 3,000,000 D, or an effective size of or about 1,000,000 D up to or about 2%. 500,000 D, or an effective size of or about 1'000,000 D up to or about 2,000,000 D, or an effective size of or approximately 1,000,000 D up to or about 1'8,000,000 D , or an effective size of or about 1'000,000 D up to or about 1'600,000 D, or an effective size of or about 1'000,000 D up to or about 1'500,000 D.

In a further embodiment, the conjugate has an effective size which is at least about 8 times greater, or at least about 10 times greater, or at least about 12 times greater, or at least about 15 times greater, or at least about 18 times larger, or at least approximately 20 times greater, or at least approximately 25 times greater, or at least approximately 28 times greater, or at least approximately 30 times greater, or at least approximately 40 times greater, than the effective size of the Proger antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 8 times to approximately 100 times greater, or is approximately 8 times to approximately 80 times greater, or is approximately 8 times to approximately 50 times greater, or is approximately 8 times up to approximately 40 times greater, or is approximately 8 times to approximately 30 times greater, or is approximately 8 times to approximately 28 times greater, or is approximately 8 times to approximately 25 times greater, or is approximately 8 times to approximately 20 times greater, or it is about 8 times up to about 18 times greater, or is about 8 times up to about 15 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 12 times to approximately 100 times greater, or is approximately 12 times to approximately 80 times greater, or is approximately 12 times to approximately 50 times greater, or is approximately 12 times up to approximately 40 times greater, or is approximately 12 times to approximately 30 times greater, or is approximately 12 times to approximately 28 times greater, or is approximately 12 times to approximately 25 times greater, or is approximately 12 times to approximately 20 times greater, or it is approximately 12 times to approximately 18 times greater, or is approximately 12 times to approximately 15 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 15 times to approximately 100 times greater, or is approximately 15 times to approximately 80 times greater, or is approximately 15 times to approximately 50 times greater, or is approximately 15 times as much as approximately 40 times greater, or is approximately 15 times to approximately 30 times greater, or is approximately 15 times to approximately 28 times greater, or is approximately 15 times to approximately 25 times greater, or is approximately 15 times to approximately 20 times greater, or it is approximately 15 times up to about 18 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 18 times to approximately 100 times greater, or is approximately 18 times to approximately 80 times greater, or is approximately 18 times to approximately 50 times greater, or is approximately 18 times up to approximately 40 times greater, or is approximately 18 voices up to approximately 30 times greater, or is approximately 18 times or approximately 28 times higher, or is approximately 18 times up to approximately 25 times higher, or is approximately 18 times up to approximately 20 times higher, than the effective size of the parent antibody fragment.

In yet another embodiment, the conjugate has an effective size that is approximately 20 times to approximately 100 times greater, or is approximately 20 times to approximately 80 times greater, or is approximately 20 times to approximately 50 times greater, or is approximately 20 times up to approximately 40 times greater, or is approximately 20 times up to approximately 30 times greater, or is approximately 20 times up to approximately 28 times higher, or is approximately 20 times to approximately 25 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 25 times to approximately 100 times greater, or is approximately 25 times to approximately 80 times greater, or is approximately 25 times to approximately 50 times greater, or is approximately 25 times as much. approximately 40 times greater, or is approximately 25 times to approximately 30 times greater, or is approximately 25 times to approximately 28 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 28 times to approximately 100 times greater, or is approximately 28 times to approximately 80 times greater, or is approximately 28 times to approximately 50 times greater, or is approximately 28 times as much. approximately 40 times greater, or is approximately 28 times to approximately 30 times greater, than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 30 times to approximately 100 times greater, or is approximately 30 times to approximately 80 times greater, or is approximately 30 times to approximately 50 times greater, or is approximately 30 times as much. approximately 40 times greater than the effective size of the parent antibody fragment. In yet another embodiment, the conjugate has an effective size that is approximately 40 times to approximately 100 times greater, or is approximately 40 times to approximately 80 times greater, or is approximately 40 times to approximately 50 times greater, than the effective size of the fragment. of parent antibody. In yet another embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight of at least about 20,000 D. In a further embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight of at least about 30,000 D. In a further embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight of at least about 40,000 D. In another embodiment , the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight that is from or about 20,000 to about 300,000 D, or is from or about 30,000 D up to or about 300,000 D, or is from or approximately 40,000 D up to or approximately 300,000 D. In another form, the conjugate is a an antibody fragment covalently linked to at least one polymer having an effective molecular weight that is from or about 20,000 D up to or about 100,000 D, or is from or about 30,000 D up to or about 100,000 D, or is from or about 40,000 D to or about 100,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight that is from or about 20,000 to about 70,000 D, or is or about 30,000 D to or about 70,000 D, or is from or about 40,000 D up to or about 70,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight that is from or about 20,000 D up to or about 50,000 D, or is from or about 30,000 D up to or about 50,000 D , or is at or about 40,000 D up to or about 50,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently linked to at least one polymer having an effective molecular weight that is at or about 20., 000 D up to or about 40,000 D, or is from or about 30,000 D up to or approximately 40,000 D.

The conjugates of the invention can be made using any suitable technique now known or further developed for the derivatization of antibody fragments with polymers. It will be appreciated that the invention is not limited to conjugates that utilize any particular type of linkage between an antibody fragment and a polymer. The conjugates of the invention include species wherein a polymer is covalently linked to a non-specific site or non-specific sites on the parent antibody fragment, for example, the coupling or polymeric linkage is not directed to a particular region or to an amino acid residue. particular in the parent antibody fragment. In such embodiments, the coupling chemistry can, for example, use non-epsilon air groups free of the lysine residues in the parent antibody, such as the binding or coupling sites for the polymer, wherein such amino groups of the residues of Lysine are randomly derivatized with the polymer. In addition, the conjugates of the invention include species wherein a polymer is covalently linked to a specific site or specific sites on the parent antibody fragment, for example, the polymer coupling is directed to a particular region or to a particular amino residue or residues in the parent antibody fragment. In such embodiments, the coupling chemistry can, for example, use the free sulfhydryl groups of a non-disulfide cysteine residue in the parent antibody fragment. In one embodiment, one or more cysteine residues are engineered at a site or sites selected on the parent antibody fragment for the purpose of providing a specific coupling site or specific coupling sites for the polymer. The polymer can be activated with any functional group that is capable of reacting specifically with the free sulfhydryl thiol group (s) on the parent antibody such as the functional groups maleimide, suifhydyl, Lyoi, thiiflate, tesylate, aziridine, exirano, and -pyridyl. The polymer can be coupled to the parent antibody fragment using any protocol suitable for the chemistry of the selected coupling system, such as the protocols and systems described in Section (II) (I) (b) or Section (T) of the Examples that are described later. In yet another embodiment, the coupling to the polymer is directed to the hinge region of the parent antibody fragment. The site of the hinge region varies according to the isotype of the parent antibody. Typically, the hinge region of the heavy chains of IgG, IgD and IgA types is contained in a proline-rich peptide sequence, which extends between the CH1 and CH2 domains. In a preferred embodiment, a cysteine residue or residues is or is engineered in the hinge region of the parent antibody fragment, for the purpose of coupling the polymer specifically at a site selected from the hinge region. In one aspect, the invention encompasses a conjugate having any molar ratio of polymer to anticuexpo fragment which endows the conjugate with an apparent size in the desired range, as shown herein. The apparent size of the conjugate will depend in part on the size and shape of the polymer used, the size and shape of the size of antibody used, the number of polymer molecules coupled to the antibody fragment, and the location of the site (s). coupling on the antibody fragment. These parameters can be easily identified and maximized to obtain a conjugate with the desired apparent size for any type of antibody fragment, polymer and linker system. In yet another aspect, the invention encompasses a conjugate with a molar ratio of polymer to antibody fragments of no more than about 10: 1., or no more than about 5: 1, or no more than about 4: 1, or just about 3: 1, or no more than about 2: 1, or no more than about 1: 1. In yet another aspect, the invention encompasses a conjugate wherein the antibody fragment is coupled to approximately 10 or fewer polymer molecules, each polymer molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In another embodiment, the conjugate contains an antibody fragment coupled to approximately 5 or fewer polymer molecules, each polymer molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In yet another aspect, the conjugate contains an antibody fragment coupled to about 4 or fewer polymer molecules, each polymer molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In a further embodiment, the conjugate contains an antibody fragment enco With about 3 or less polymer molecules, each polymer molecule has a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In a further embodiment, the conjugate contains a coupled antibody fragment. to about 2 or less polymer molecules, each polymer molecule has a molecular weight of at least about 20,000 L, or at least about 30,000 D, or at least about, or at least about 40,000 D. conjugate containing an antibody fragment coupled to a simple polymer molecule having a molecular weight of at least 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In a further aspect, the invention encompasses a conjugate wherein each polymer molecule in the conjugate has a molecular weight that is from or about 20,000 D up to or approximately 300,000 D, or is from or about 30,000 D up to or about 300,000 D or is from or about 40,000 D up to or about 300,000 D, and wherein the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 molecules polymeric, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules, or no more than about 1 polymeric molecule. In yet another aspect, the invention encompasses a conjugate wherein each polymer molecule in the conjugate has a molecular weight that is from or about 20,000 D up to or about 100,000 D, or is from or about 30,000 D up to or about 100,000 D, or is of or about 40,000 D up to or about 100,000 D, and wherein the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 molecules polymeric, or no more than about 2 polymeric molecules, or no more than 1 polymeric molecule. In yet another aspect, the invention encompasses a conjugate wherein each polymer molecule in the conjugate has a molecular weight that is from or about 20,000 D up to or about 70,000 D, or is from or about 30,000 D up to or about 70,000 D or is from about 40,000 D up to about 70,000 D, and wherein the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymer molecules, or no more than 1 polymer molecule. In another additional aspect, the invention encompasses a conjugate wherein each polymer molecule in the conjugate has a molecular weight that is from or about 20,000 D up to or about 50,000 D, or is from or about 30,000 D up to or about 50,000 D, or is from or about 40,000 D up to or about 50,000 D, and wherein the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymer molecules, or no more than 1 polymer molecule. In yet another aspect, the invention encompasses a conjugate wherein each polymer molecule in the conjugate has a molecular weight that is from or about 20,000 D up to or about 40,000 D, or is from or about 30,000 D up to or about 40,000 D, and in wherein the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules, or no more than about 1 polymer molecule. It is believed that the serum half-life, MRT and the rate of serum clearance of any antibody fragment can be greatly improved by derivatizing the antibody fragment with the polymer, as shown herein. In one embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, scFv and F (ab') 2. In a preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein each polymer molecule in the conjugate is linked to the hinge region of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein each polymer molecule in the conjugate is linked to the hinge region of the antibody fragment, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 molecule-, polymeric, or no more than about 2 polymeric molecules, or no more than 1 polymer molecule. In another preferred embodiment, the conjugate contains an F (ab ') 2 antibody fragment coupled to no more than about 2 polymeric molecules, wherein each polymer molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment. , which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymer molecule, and the polymer is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cy-stein residue in the opposite chain. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, each polymer molecule in the conjugate is at least about 20,000 D molecular weight, or at least about molecular weight of 30,000, or at least about 40,000 D of molecular weight, each polymer molecule in the conjugate is coupled to the hinge region of the antibody fragment, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules, or no more than 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, each polymer molecule in the conjugate is at least about 20,000 D up to or about 300,000 D molecular weight, or is from or about 30,000 D up to or about 300,000 D of molecular weight, or is from or about 40,000 D up to or about 300,000 D of molecular weight, each polymer molecule in the conjugate is coupled to the hinge region of the antibody fragment, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules or no more of 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, each polymer molecule in the conjugate is from or about 20,000 D to or approximately 100,000 D molecular weight, or is from or about 30,000 D up to or about 100,000 D molecular weight, or is from or about 40,000 D up to or about 100,000 D molecular weight, each po limeric molecule in the conjugate is coupled to the hinge region of the antibody, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules. or no more than 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, each polymer molecule in the conjugate is from or about 20,000 D up to or approximately 70,000 D molecular weight, or is from or about 30,000 D up to or about 70,000 D of molecular weight, or is from or about 40,000 D up to or about 70,000 D of molecular weight, each polymer molecule in the conjugate is coupled to the hinge region of the antibody fragment, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules, or no more than 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, each polymer molecule in the conjugate is from or about 20.00 D to or approximately 50,000 D in weight molecular, or is from or about 30,000 D up to or about 50,000 D of molecular weight, or is from or about 40,000 D up to or about 50,000 D of molecular weight, each polymer molecule in the conjugate is coupled to the hinge region of the antibody, and the conjugate contains no more than about 10 polymeric molecules, or no more than about 5 polymeric molecules, or no more than about 4 polymeric molecules, or no more than about 3 polymeric molecules, or no more than about 2 polymeric molecules or no more than 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, each polymer molecule in the conjugate being at or about 20., 000 D up to or about 40,000 D of molecular weight, or is from or about 30,000 D up to or about 40,000 D of molecular weight, each polymer molecule in the conjugate is coupled to the hinge region of the antibody fragment, and the conjugate contains no more than about i polymeric molecules, or no more than about 5 polymer molecules, or no more than about 4 polymer molecules, or no more than about 3 polymer molecules, or no more than about 2 polymer molecules or no more of 1 polymer molecule. In a further embodiment, the conjugate contains an antibody fragment F (ab ') 2 coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is at least about 20,000 D molecular weight, or at least give about 30,000 D of molecular weight, or at least about 40,000 D of molecular weight, and wherein each polymer molecule in the conjugate is coupled to a cysteine residue in the light or heavy chain of the antibody fragment that could ordinarily form the disulfide bridge that binds the light and heavy chains, where the disulfide bridge is avoided by the substitution of another amino acid, as it will be, by the corresponding cysteine residue in the opposite chain. In yet another embodiment, the conjugate contains an F (ab ') 2 antibody fragment coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is from or about 20,000 D up to or about 300,000 D molecular weight or is of or about 30,000 D up to or about 300,000 D of molecular weight, or is of or about 40,000 D up to or about 300,000 D of molecular weight, and wherein each polymer molecule in the conjugate is coupled to a cysteine residue in the heavy or light chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In yet another embodiment, the conjugate contains an antibody fragment F (ab ') 2 coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is from or about 20,000 D up to or about 100,000 D molecular weight , or is from or about 30,000 D up to or about 100,000 D molecular weight, or is from or about 40,000 D up to or about 100,000 D molecular weight, and wherein each polimeric molecule in the conjugate is coupled to a cysteine residue in the light or heavy caaena dtx antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain.

In yet another embodiment, the conjugate contains an F (ab ') 2 antibody fragment coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is from or about 20,000 D up to or about 70,000 D molecular weight or is from about 30,000 D to about 70,000 D of molecular weight, or is from or about 40,000 D up to or about 70,000 D of molecular weight, and wherein each polymer molecule in the conjugate is coupled to a cysteine residue in the heavy or light chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In yet another embodiment, the conjugate contains an antibody dt antibody (ab ') 2 coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is from or about 20,000 D up to or about 50,000 D molecular weight or is of or about 30,000 D up to or about 50,000 D of molecular weight, or is of or about 40,000 D up to or about 50,000 D of molecular weight, and wherein each polymer molecule in the conjugate is coupled to a cysteine residue in the heavy or light chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In yet another embodiment, the conjugate contains an antibody fragment F (ab ') 2 coupled to no more than about 2 polymeric molecules, wherein each polymer molecule in the conjugate is from or about 20,000 D up to or about 40,000 D molecular weight or is of or about 30,000 D up to or about 40,000 D of molecular weight, and wherein each polymer molecule in the conjugate is coupled to a cysteine residue in the light chain or thought of the fragment of the body which could ordinarily form the bridge disulfide that binds light and heavy chains, where the disulfide bridge is avoided by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite chain.

In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is at least about 20,000 D of molecular weight, or at least about 30,000 D of molecular weight, or at least about 40,000 D of molecular weight, wherein the polymer molecule is coupled to a cysteine residue in the light chain or heavy of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is of or about 20,000 D up to or about 300,000 D of molecular weight, or is of or about 30,000 D of molecular weight up to or about 300,000 D of molecular weight, or is from or about 40,000 D up to or about 300,000 D of molecular weight, in where the polymer molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment that could ordinarily form the disulfide bridge that binds the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is of or about 20,000 D up to or about 100,000 D of molecular weight, or is of or about 30,000 D of molecular weight up to or about 100,000 D of molecular weight, or is from or about 40,000 D up to or about 100,000 D of molecular weight, in where the polymer molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is of or approximately 20,000 D up. or about 70,000 D of molecular weight, or is of or about 30,000 D of molecular weight up to or about 70,000 D of molecular weight, or is from or about 40,000 D up to or about 70,000 D of molecular weight, wherein the polymer molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for ei corresponding cysteine residue in the opposite strand. In another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is of or about 20,000 D up to or about 50,000 D of molecular weight, or is of or about 30,000 D of molecular weight up to or about 50,000 D of molecular weight, or is from or about 40,000 D up to or about 50,000 D of molecular weight, in where the polymer molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymer molecule, wherein the polypeptide molecule is of or about 20,000 D up to or about 40,000 D of molecular weight, or is of or about 30,000 D up to or about 40,000 D of molecular weight, wherein the polymer molecule is coupled to a cysteine residue in the light or heavy chain of the fragment of antibody that could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is at least about 20,000 D molecular weight, or at least about 30, 000 D of molecular weight, or at least about 40,000 D of molecular weight, and wherein the polymer molecule is coupled to the hinge region of the antibody fragment. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is assembled to no more than 1 polymeric molecule, wherein the polymeric molecule is at or at least 20,000 D up to or about 300,000 D molecular weight, or is from or about 30,000 D up to or about 300,000 D molecular weight, or is from or about 40,000 D up to or about 300,000 D molecular weight, and in wherein the polymer molecule is coupled to the hinge region of the antibody fragment. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is from or about 20,000 D up to or about 100,000 D molecular weight, or is from or about 30,000 D up to or about 100,000 D molecular weight, or is from or about 40,000 D up to or about 100,000 D molecular weight, and wherein the polymer molecule is coupled to the hinge region of the antibody fragment. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SF, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is from or about 20,000 D up to or about 70,000 D molecular weight, or is from or about 30,000 D up to or about 70,000 D molecular weight, or is from or about 40,000 D up to or about 70,000 D molecular weight, and wherein the polymer molecule is coupled to the hinge region of the antibody fragment. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is from or about 20,000 D up to or about 50,000 D molecular weight, or is from or about 30,000 D up to or about 50,000 D molecular weight, or is from or about 40,000 D up to or about 50,000 D molecular weight, and wherein the polymer molecule is coupled to the hinge region of the antibody fragment. In yet another embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 polymeric molecule, wherein the polymeric molecule is of or approximately 20,000 D up. or about 40,000 D of molecular weight, or is of or about 30,000 D up to or about 40,000 D of molecular weight, and wherein the polymer molecule is coupled to the hinge region of the antibody fragment. Although any type of polymer is contemplated for use in the construction of the conjugates of the invention, including the polymers and chemical link systems described in Section (II) (I) (b) below, polyethylene glycol (PEG) polymers are preferred for use herein. In one embodiment, the conjugate is an antibody fragment covalently linked to at least one PEG having an effective molecular weight of at least about 20,000 D. In another embodiment, the conjugate is an antibody fragment covalently linked to at least one PEG that has an effective molecular weight of at least about 30,000 D. In one embodiment, the conjugate is an antibody fragment covalently linked to at least one PEG having an effective molecular weight of at least about 40,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently coupled to at least one PEG having an effective molecular weight that is from or about 20,000 D up to or about 300,000 D, or is from or about 30,000 D up to or about 300,000 D, or is from or about 40,000 D up to or about 300,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently coupled to at least one PEG e has an effective molecular weight that is from or about 20,000 D up to or about 100,000 D, or is from or about 30,000 D up to or about 100,000 D, or is from or about 40,000 D up to or about 100, 000 D. In another embodiment further, the conjugate is an antibody fragment covalently coupled to at least one PEG having at least one effective molecular weight that is from or about 20,000 D up to or about 70,000 D, or is from or about 30,000 U up to or about 70,000 D , or is from or about 40,000 D up to or about 70,000 D. In yet another embodiment, the conjugate is an antibody fragment covalently coupled to at least one PEG having at least one effective molecular weight that is at or about 20,000 D up to or about 50,000 D, or is from or about 30,000 D up to or about 50,000 D, or is from or about 40,000 D up to or about 50,000 D. In another embodiment s, the conjugate is an antibody fragment covalently coupled to at least one PEG having an effective molecular weight that is from or about 20,000 D up to about 40,000 D, or is from or about 30,000 D up to or about 40,000 D. In another further aspect, the invention encompasses a conjugate with a molar ratio of PEG to the antibody fragment of no more than about 10: 1, or no more than about 5: 1, or no more than about 4: 1, or no more than about 3: 1, or no more than about 2: 1, or no more than 1: 1. In yet another embodiment, the invention encompasses a conjugate wherein the antibody fragment is coupled to approximately 10 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In yet another embodiment, the conjugate contains an antibody fragment coupled to about 5 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In yet another embodiment, the conjugate contains an antibody fragment coupled to approximately 4 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D , or at least about 40,000 D. In a further embodiment, the conjugate contains an antibody fragment coupled approximately to 3 or less PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In a further embodiment, the conjugate contains a coupled antibody fragment. about 2 or less PEG molecules, each PEG molecule having a molecular weight of at least about 20, 000 D, or at least about 30,000 D, or at least about 40,000 D. A conjugate containing an antibody fragment coupled to a single PEG molecule having a molecular weight of at least about 20,000 D is also provided herein. , or at least about 30,000 D, or at least about 40,000 D. In still another aspect, the invention encompasses a conjugate wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to about 300,000 D of molecular weight, or is of or about 30,000 D up to or about 300,000 D of molecular weight, or is of or about 40,000 D up to or about 300,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 molecules. PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule. In yet another aspect, the invention encompasses a conjugate wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 100,000 D molecular weight, or is from or about 30,000 D up to or about 100,000 D of molecular weight, or is of or about 40,000 D up to or about 100,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 molecules of PEG, or no more than about 4 molecules of PEG, or no more than about 3 molecules of PEG, or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In yet another aspect, the invention encompasses a conjugate wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 70,000 D molecular weight, or is from or about 30,000 D up to or about 70,000 D of molecular weight, or is of or about 40,000 D up to or about 70,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 molecules of PEG, or no more than about 4 molecules of P? G, or no more than about 3 molecules of PEG, or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In yet another aspect, the invention encompasses a conjugate wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 50,000 D molecular weight, or is from or about 30,000 D up to or about 50,000 D of molecular weight, or is of or about 40,000 D up to or about 50,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 molecules of PEG, or no more than about 4 molecules of PEG, or no more than about 3 molecules of PEG, or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In yet another aspect, the invention encompasses a conjugate wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 40,000 D molecular weight, or is from or about 30,000 D up to or about 40,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule.

In still another aspect, the invention encompasses a conjugate that contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2, wherein the antibody fragment is coupled to approximately 10 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In another embodiment, the above conjugate contains an antibody fragment coupled at least to 5 or less PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In yet another embodiment, the above conjugate contains an antibody fragment. coupled to approximately 4 or less PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least approximately e 40,000 D. In a further embodiment, the above conjugate contains an antibody fragment coupled to about 3 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In a further embodiment, the above conjugate contains an antibody fragment coupled to about 2 or fewer PEG molecules, each PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D , or at least about 40,000 D. The above conjugate is also provided herein which contains an antibody fragment coupled to a single PEG molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. In still another aspect, the invention encompasses a conjugate that contains a fragment of antibody sele of the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 300,000 D of molecular weight, or is of or about 30,000 D up to or about 300,000 D of molecular weight, or is of or about 40,000 D up to or about 300,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule. In yet another aspect, the invention encompasses a conjugate that contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 100,000 D molecular weight, or is from or about 30,000 D up to or about 100,000 D molecular weight, or is from or about 40,000 D up to or about 100,000 D of molecular weight, and wherein the conjugate contains no more than about 10 molecules of PEG, or no more than about 5 molecules of PEG, or no more than about 4 molecules of PEG, or no more than about 3 molecules of PEG , or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG.

In yet another aspect, the invention encompasses a conjugate that contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 70,000 D molecular weight, or is from or about 30,000 D up to or about 70,000 D molecular weight, or is of or about 40,000 D up to or about 70,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule. In yet another aspect, the invention encompasses a conjugate that contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 50,000 D molecular weight, or is from or about 30,000 D up to or about 50,000 D molecular weight, or is from or about 40,000 D up to or about 50,000 D of molecular weight, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules , or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In still another aspect, the invention encompasses a conjugate that contains an antibody fragment selected from the group consisting of Fab, Fab ', Fab' - SH and F (ab ') 2, wherein the antibody fragment is derivatized with PEG , wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 40,000 D molecular weight, or is from or about 30,000 D up to or approximately 40,000 D molecular weight, and wherein the conjugate contains no more of about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more of 1 PEG molecule.

In a preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D, and wherein each PEG molecule in the conjugate is coupled to the hinge region of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight that is at or about 20,000 D up to about 300,000 D, or is from or about 30,000 D up to or about 300,000 D, or is from or about 40,000 D up to or about 300, or, and where each PEG molecule in the conjugate is coupled to the region of hinge of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight that is at or about 20,000 D up to about 100,000 D, or is from or about 30,000 D up to or about 100,000 D, or is from or about 40,000 D up to or about 100,00 D, and wherein each PEG molecule in the conjugate is coupled to the region of hinge of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight that is at or about 20,000 D up to about 70,000 D, or is from or about 30,000 D up to or about 70,000 D, or is from or about 40,000 D up to or about 70,00 D, and wherein each PEG molecule in the conjugate is coupled to the region of hinge of the antibody fragment. In another piefeixda embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight that is at or about 20,000. D up to about 50,000 D, or is from or about 30,000 D up to or about 50,000 D, or is from or about 40,000 D up to or about 50,00 D, and wherein each PEG molecule in the conjugate is coupled to the region of hinge of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG having a molecular weight that is at or about 20,000 D up to about 40,000 D, or is from or about 30,000 D up to or about 40,000 D, and wherein each PEG molecule in the conjugate is coupled to the hinge region of the antibody fragment. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is at least about 20,000 D of molecular weight, or at least about 30,000 D of molecular weight, or at least about 40,000 D of molecular weight, wherein each PEG molecule in the conjugate molecule is coupled to the hinge region of the antibody fragment, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule. In another preferred modality, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is at or about 20,000 D up to or about 300,000 D of molecular weight, or is from or about 30,000 D up to or about 300,000 D of molecular weight, or is from or about 40,000 D up to or about 300,000 D of molecular weight, wherein each PEG molecule in the The conjugate molecule is coupled to the hinge region of the antibody fragment, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more than about 2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 100,000 D of molecular weight, or is from or about 30,000 D up to or about 100,000 D of molecular weight, or is from or about 40,000 D up to about 100,000 D of molecular weight, wherein each molecule of PEG in the conjugated molecule is coupled to the hinge region of the antibody fragment, and wherein the conjugate contains no more than about 10 molecules of PEG, or no more than about 5 molecules of PEG, or no more than about 4 molecules of PEG. PEG, or no more than about 3 molecules of PEG, or no more than about 7 molecules of PEG, or no more than 1 molecule of? JEG. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 70,000 D of molecular weight, or is from or about 30,000 D up to or about 70,000 D of molecular weight, or is from or about 40,000 D up to or about 70,000 D of molecular weight, wherein each molecule of PEG in the conjugated molecule is coupled to the hinge region of the antibody fragment, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 molecules of PEG, or no more than about 3 molecules of PEG, or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 50,000 D of molecular weight, or is from or about 30,000 D up to or about 50,000 D of molecular weight, or is from or about 40,000 D up to or about 50,000 D of molecular weight, wherein each molecule of PEG in the conjugated molecule is coupled to the hinge region of the antibody fragment, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 molecules of PEG, or no more than about 3 molecules of PEG, or no more than about 2 molecules of PEG, or no more than 1 molecule of PEG. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 40,000 D of molecular weight, or is of or about 30,000 D up to or about 40,000 D of molecular weight, wherein each PEG molecule in the conjugate molecule is coupled to the hinge region of the antibody, and wherein the conjugate contains no more than about 10 PEG molecules, or no more than about 5 PEG molecules, or no more than about 4 PEG molecules, or no more than about 3 PEG molecules, or no more of about 2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibody fragment F (ab ') 2 derivatized with PEG, wherein each PEG molecule in the conjugate is at least about 20,000 D of molecular weight, or at least about 30,000 D of molecular weight, or at least about 40,000 D of molecular weight, wherein the antibody fragment is coupled to no more than about 2 molecules of PEG, and wherein each PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In another preferred embodiment, the conjugate contains an F (ab) 2 antibody fragment derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 300,000 D molecular weight, or is from or about 30,000 D up to or about 300,000 D of molecular weight, or is of or about 40,000 D up to or about 300,000 D of molecular weight, wherein the antibody fragment is coupled to no more than about 2 molecules of PEG, and wherein each molecule of PEG is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, where the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another preferred embodiment, the conjugate contains an antibody fragment F (ab ') 2 derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 100,000 D molecular weight, or is from about 30,000 D up to or about 100,000 D molecular weight, or is from or about 40,000 D up to or about 100,000 D molecular weight, wherein the antibody fragment is coupled to no more than about 2 molecules of PEG, and wherein each PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, where the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another preferred embodiment, the conjugate contains an antibody fragment F (ab ') 2 derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 70,000 D molecular weight, or is of or about 30,000 D up to or about 70,000 D of molecular weight, or is from or about 40,000 D up to or about 70,000 D of molecular weight, wherein the antibody fragment is coupled to no more than about 2 molecules of PEG, and wherein each PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another preferred embodiment, the conjugate contains an antibody fragment F (ab ') 2 derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D to or approximately 50,000 D molecular weight, or is from approximately 30,000 D up. or about 50,000 D of molecular weight, or is of or about 40,000 D up to or about 50,000 D of molecular weight, wherein the antibody fragment is coupled to no more than about 2 molecules of PEG, and wherein each PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is prevented by the substitution of another amino acid such as serine, by the corresponding cysteine residue in the opposite chain. In another preferred modality, the conjugate contains an antibody fragment F (ab ') 2 derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to about 40, OuO D of molecular weight, or is of or about 30,000 D up to or about 40,000 D of molecular weight, wherein the antibody fragment is coupled to no more than about 2 PEG molecules, and wherein each PEG molecule is coupled to a cysteine residue in the light or heavy chain of the fragment of antibody, which could ordinarily form the disulfide bridge that binds the "light and heavy chains," where the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite strand. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is at least about 20,000 D of molecular weight, or at least about 30,000 D of molecular weight, or at least about 40,000 D of molecular weight, wherein the antibody fragment is coupled to no more than one PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily torment the disulfide bridge linking the light and heavy chains, wherein the disulfide bridge is avoided by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand.

In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 300,000 D of molecular weight, or is from or about 30,000 D up to or about 300,000 D of molecular weight, or is from or about 40,000 D up to or about 300,000 D of molecular weight, wherein the fragment of antibody is coupled to no more than about 1 PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge that binds the chains light and heavy, where the disulfide bridge is avoided by the substitution of another amino acid, such as cerin, for the corresponding cysteine residue in the opposite strand. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is of or about 20,000 D up to or about 100,000 D of molecular weight, or is from or about 30,000 D up to or about 100,000 D of molecular weight, or is from or about 40,000 D up to or about 100,000 D of molecular weight, wherein the fragment of antibody is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge that binds the light chains and heavy, wherein the disulfide bridge is prevented by the substitution of another amino acid, such as serine, for the corresponding cysteine residue in the opposite strand. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is dei nuanced with PLG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 70,000 D molecular weight, or is from or about 30,000 D up to or about 70,000 D molecular weight, or is from or about 40,000 D up to or about 70,000 D molecular weight, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light chain 0 heavy of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, where the disulfide bridge is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite strand . In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 50,000 D molecular weight, or is from or about 30,000 D up to or about 50,000 D molecular weight, or is from or about 40,000 D up to or about 50,000 D molecular weight, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light or heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the p? disulfide is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite chain. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab., Fab ', and Fab'-SH, wherein the antibody fragment is derivatized with PEG, wherein each PEG molecule in the conjugate is from or about 20,000 D up to or about 40,000 D molecular weight, or is from or about 30,000 D up to or about 40,000 D of molecular weight, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is coupled to a cysteine residue in the light or heavy chain of the fragment of antibody, which could ordinarily form the disulfide bridge linking the light and heavy chains, wherein the bridge of sulfide is prevented by the substitution of another amino acid, such as serine, by the corresponding cysteine residue in the opposite strand. It will be appreciated that all of the above-described embodiments of the invention utilizing PEG polymers include conjugates wherein the PEG polymers are linear or branched. In a preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is branched and is at least about 40,000 D molecular weight. In a particularly surprising and unexpected finding, the inventors discovered that the above conjugate shows a serum half-life, MRT and serum clearance rate that approximate those of the full-length antibody, as shown in Example X below. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 300,000 D. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'- SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 100,000 D. In another preferred embodiment , the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 70,000 D. In another preferred embodiment, the conjugate contains an antibody fragment selected from the group consisting of Fab, Fab ', and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, and wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 50,000 D. In another preferred embodiment, the invention provides a conjugate containing an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, wherein the PEG molecule is branched and is at least 40,000 D of molecular weight, and the PEG molecule is coupled to the hinge region of the antibody fragment. In another preferred embodiment, the invention provides a conjugate containing an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 300,000 D, and the PEG molecule is coupled to the hinge region of the antibody fragment. In another preferred embodiment, the invention provides a conjugate that contains an antibody fragment selected from the group consisting of FaA Fab 'and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 100,000 D, and the PEG molecule is coupled to the hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugate containing an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 70,000 D, and the PEG molecule is coupled to the hinge region of the antibody fragment. In another preferred embodiment, the invention provides a conjugate containing an antibody fragment selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the antibody fragment is coupled to no more than 1 PEG molecule, wherein the PEG molecule is branched and has a molecular weight that is from or about 40,000 D up to or about 50,000 D, and the PEG molecule is coupled to the hinge region of the antibody fragment. In one aspect, the invention provides any of the conjugates described above wherein the conjugate contains no more than one antibody fragment. Any of the above-described conjugates wherein the conjugate contains one or more antibody fragments linked to one or more polymeric molecules, such as conjugates containing two or more antibody fragments covalently linked together by one or more are also provided herein. polymeric molecules. In one embodiment, a polymer molecule is used to bind together two antibody fragments to form a structure in the form of a dumbbell. Also encompassed herein are those conjugates formed by more than two antibody fragments joined by one or more polymer molecules, to form a rosette or other forms. The antibody fragments in such structures may be the same or a different type of fragment, and may have the same antigen specificity or have different antigen specificities. Such structures can be elaborated by using a polymerized polymerized molecule with multiple functional groups that allow direct coupling, or coupling by means of bi- or multifunctional linkers, of two or more antibody fragments to the polymeric backbone. In yet another aspect, the invention encompasses any of the conjugates described above using an antibody fragment comprising an antigen recognition site that binds to rabbit IL-8 and / or human IL-8. In still another aspect, the invention encompasses any of the conjugates described above using an antibody fragment comprising 6G4.2.5LV / L1N35A or 6G4.2.5LV / L1N35E as defined below. In still another aspect, the invention encompasses any of the conjugates described above using an antibody fragment comprising 6G4.2.5HV11 as defined below. In a further aspect, the invention encompasses any of the conjugates described above using an antibody fragment comprising hu6G4.2LV / LlN35A or hu6G4.2.5LV / L1N35E, as defined below. In a further aspect, the invention encompasses any of the conjugates described above using an antibody fragment comprising hu6G! .2.5HV. Also encompassed herein are any of the conjugates described above, using an antibody fragment comprising 6G4.2.5LV / L1N35A or 6G4.2.5LV / L1N35E and further comprising the CDRs of 6G4.2.5HV as defined below. Also encompassed herein are any of the above-described conjugates that utilize an antibody fragment comprising hu6G4.2.5LV / LlN35A or hu6G4.2.5LV / L1N35E and further comprising hu6G4.2.5HV as defined below. Additionally encompassed herein are any of the conjugates described above, which utilize an antibody fragment comprising 6G4.2.5LV11N35A or 6G4.2.5LV11N35E, as defined below. Any of the conjugates described above is further provided herein, using an antibody fragment comprising 6G4.2.5LV11N35A or 6G4.2.5LV11N35E and further comprising 6G4.2.5HV11 as defined below.

Production of Antibody Fragments Antibody fragments can be produced by any method known in the art. In general, an antibody fragment is derived from an intact parent progenitor. The parent antibody can be generated by obtaining polyclonal serum against the desired antigen, by multiple subcutaneous (se) or intraperitoneal (ip) injections of the antigen and an adjuvant, such as monophosphoryl lipid A (MPL) / trehalose dicrinomycolate (TDM) ) (Ribi Immunochem, Research, Inc., Hamilton, MT), in multiple sites. Two weeks later the animals are reinforced. 7 to 14 days later the animals are bled and the serum is evaluated for the anti-antigen titer. The animals are reinforced up to maximum titles. The sera are harvested from the animals, and the polyclonal antibodies are isolated from the sera by standard immunoglobulin purification methods, such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel filtration, dialysis, or affinity chromatography. antigen. The desired antibody fragments can be generated from the purified polyclonal antibody preparations by conventional enzymatic methods, for example the F (ab ') 2 fragments are produced by pepsin cleavage of the intact antibody, and the Fab fragments are produced by digestion Brief of the intact antibody with papain. Alternatively, the antibody fragments are derived from monoclonal antibodies generated against the desired antigen. Monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Na ture, 256: 495 (1975), or they can be made by recombinant DNA methods (US Patent No. 4, 816, 567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as described above to produce lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used for the immunization. Alternatively, the lymphocytes can be immunized in vi tro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoc l onal Antihodi is: Principi es and Practi ce, pp. 59-103 (Academic tress, 1486)). The hybridoma cells prepared in this way are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, progenitor myeloma cells. For example, if the progenitor myeloma cells lack the enzyme hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of cells deficient in HGPRT. Preferred myeloma cells are those that are efficiently fused, which support stable high-level production of the antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium. Among these, the preferred myeloma cell lines are the murine myeloma lines, such as those derived from the MOP-21 and MC-ll mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and the SP-2 or X63-Ag8-653 cells available from the North American Culture Collection of Fspecies (American Type Culture Collection), Rocxvilie, Maryland JSA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal on An tibody Producti on Techniques and Appli ca ti ons, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cells are being developed is evaluated for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an HIV binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Bi ochem. , 107: 220 (1980). Subsequently, hybridoma cells are identified that produce antibodies of the desired specificity, affinity and / or aclivia, clones can be subcloned by limiting dilution procedures and developed by standard methods (Goding, Monoclonal Antibodi is: Pri ncipi es and Pra c ti ce, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, the D-MEM medium or the RPMI-1640 medium. In addition, the hybridoma cells can be developed as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, the ascites fluid, or the serum by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis. , or affinity chromatography. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (for example, by using oligonucleotide probes that are capable of binding specifically to the genes encoding the heavy and light chains of the monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into the host cells as E cells. col i, simian COS cells, Chinese hamster ovary cells (CHO), or myeloma cells that do not otherwise produce the immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of the DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5_: 256 (1993) and Pluckthun, Immuno1. Revs. , 130: 151 (1992). In a preferred embodiment, the antibody fragment is derived from a humanized antibody. Methods for the humanization of non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced therein, from a non-human source. These non-human amino acid residues are often referred to as 'import' residues, which are typically taken from a 'import' variable domain. It will be appreciated that the variable domain sequence is obtained from any non-human animal phage, shows the Fv clone derived from the library, or from any clone of hybridoma derived antibody, from non-human animal, provided as described herein , can serve as the variable domain of 'importation' used in the construction of the humanized antibodies of the invention Humanization can be essentially performed following the method of Winter et al. (Jones et al., Na ture, 321: 522 (1986 ); Riechmann et al.,? Ature, 332: 323 (1988); Verhoeyen et al., Sci ence, 239: 1534 (1988)), by substituting the CDRs or CDR sequences of non-human animal, eg, rodent, for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly et al., Supra), wherein substantially less than one variable, human, intact domain has been replaced by the corresponding sequence from a non-human species. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non-human, eg, rodent animal antibodies.The choice of human variable domains, either light and heavy, which will be used in the preparation of humanized antibodies, is very important to reduce antigenicity.According to the so-called 'best fit' method, the variable domain sequence of a non-human animal antibody, by example, of rodent, is selected against the complete library of the known sequences of the variable domain h umano The human sequence that is most similar to that of the non-human animal is then accepted as the human structure (FR) for the human antibody (Sims et al, J. Immunol., 151: 2296 (1993); Chothia and Lesk, J. Mol. Bi ol., 196: 901 (1987) .Another method uses a particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.The same structure can be used for several antibodies different humanized systems (Carter et al., Proc. Na ti, Acad.Sci USA, 89: 4285 (1992), Presta et al., J. Immunol., 151: 2623 (1993)) It is also important that the antibodies are humanized with the retention ie the high affinity for the antigen and other favorable biological properties To achieve this goal, according to a preferred method, the humanized antibodies are prepared by a process of analysis of the progenitor sequences and various products s humanized concepts using three-dimensional models of humanized progenitor sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those of skill in the art. Computer programs that illustrate and visually show the probable three-dimensional conformational structures of the selected candidate immunoglobulin sequences are available. The inspection of these visual representations allows the analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of the residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, the FR residues can be selected and combined from the consensus and import sequences, so that the desired antibody characteristic is achieved, such as the increased affinity for the target antigen (s). In general, the CDR residues are directly and more substantially involved in influencing the antigen binding. In addition, antibody fragments for use herein may be derived from human monoclonal antibodies. Human monoclonal antibodies against the antigen of interest can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies have been described, for example, by Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al. Monoclonal on An tibody Production Techniques and Appli ca ti ons, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991). It is now possible to produce transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the gene from the binding region (JH) of the antibody heavy chain in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the arrangement of the human germline immunoglobulin gene in such germline mutant mice will result in the production of human antibodies after challenge with the antigen. See, for example, Jakobovits et al., Proc. Na ti. Aca d. Sci. USA, 90: 2551 (1993); Jakobovits et al., Na t ure, 362: 255 (1993); Bruggermann et al., Year i n Immunol. , 7:33 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348: 552 (1990)) can be used to produce human antibodies and antibody fragments in vi tro, from repertoires of variable domain genes (V) of immunoglobulin, from non-immunized donors. According to this technique, the V domain genes of the antibody are cloned into the structure either in a coat protein gene greater or less than a filamentous bacteriophage, such as M13 or fd, and shown as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in the selection of the gene encoding the antibody that shows those properties. In this way, the phage mimics some of the properties of the B cell. The visual representation of the phage can be performed in a variety of formats; for review see, for example, Johnson et al., Current Opinion in Structural Biology 3: 564 (1993). Several sources of segments of the V gene can be used for the visual representation of the phage. Clackson et al., Nature 352: 624 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed, and antibodies can be isolated for a diverse array of antigens. (including auto-antigens) essentially following the techniques described by Marks and collaborators, J ^ Mol. __. B ^ o.l. 222: 5R1 (1991), or Griffith et al., EMB? J. 12: Y 3 (1993). In a natural immune response, the antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells that show high affinity surface immunoglobulin, are preferably replicated and differentiated during the subsequent challenge with the antigen. This natural process can be imitated by using the technique known as 'chain intermingling' (Marks et al., Bio / Technol 10: 779 (1992).) In this method, the affinity of the 'primary' human antibodies obtained by means of visual representation of the phage, it can be improved by the sequential replacement of the genes of the heavy and light chain V region, with repertoires of variants of natural origin (repertoires) of V domain genes obtained from non-immunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the nM range. A strategy of elaboration of very large repertoires of phage antibodies has been described by Waterhouse et al., Nucí. Ac_ids_Res 21: 2265 (1993). The intermixing of genes can be used to derive human antibodies from non-human, eg, rodent antibodies, where the human antibody has affinities and specificities similar to the initial non-human antibody. According to this method, which is also referred to as an "epitope print", the heavy chain or light chain variable region of a non-human antibody fragment obtained by phage display techniques, as described above, is replaced. with a repertoire of human V domain genes, creating a population of scFv or nonhuman chain / human chain Fab chimeras.The selection with antigen results in the isolation of a scFv or chimeric Fab from nonhuman chain / human chain, wherein the human chain restores the binding site to the destroyed antigen after removal of the corresponding non-human chain in the clone showing the primary phage, for example, the epitope governs (prints) the choice of the human chain partner. the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT / WO 93/06213 published April 1, 19"3). Contrary to the traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which do not have FR or CDR residues of non-human origin. The invention also encompasses the use of bispecific and heteroconjugate antibody fragments having specificities for at least two different antigens. Bispecific and heteroconjugate antibodies can be prepared as full-length antibodies or as antibody fragments (e.g., bispecific antibody fragments of F (ab ') 2). Antibody fragments having more than two valences (eg, trivalent or higher valence antibody fragments) are also contemplated for use herein. Bispecific antibodies, heteroconjugate antibodies, and multivalent antibodies can be prepared as described in Section (II) (3) (C) below. As described above, the DNA coding for the monoclonal antibody or for the antibody fragment of interest can be isolated from its hybridoma or its phage production clone, of origin, and manipulated to create the co-constructions matured by affinity and / or humanized. In addition, known techniques for introducing an amino acid residue or amino acid residues into any desired site on the polypeptide backbone of the antibody fragment can be employed., for example, a cysteine residue placed in the hinge region of the heavy chain, thereby providing a site for the specific coupling of the polymer molecule (s). In one embodiment, the native cysteine residue in the light dinner or in the heavy chain of the antibody fragment, which could ordinarily form the disulfide bridge linking the light and heavy chains, is substituted with another amino acid, such as serine, with the In order to leave the partner cysteine residue in the opposite chain, with a free sulfhydryl for specific coupling of the polymer molecule. After the construction of the clone encoding the desired antibody or antibody fragment, the clone can be used for the recombinant production of the antibody fragment as described in Section (II) (4) below. Finally, the antibody product or antibody fragment can be recovered from the host cell culture and purified as described in Section (II) (4) (F) below. In the case of modalities that use an antibody fragment engineered to lack a cysteine residue that ordinarily forms. the disulfide bridge between the light * and heavy chains, as described above, the preferred recombinant production systems include the bacterial expression and product recovery methods using the low pH osmotic shock method described in the 'Alternative Purification of Fab'-SH ", from Example T below If a full-length antibody is produced, the desired antibody fragment can be obtained from it by subjecting the intact antibody to enzymatic digestion according to known methods, for example , as described in Section (II) (4) (G) below. b. Construction of conjugates Antibody-Polymer Fragment The antibody-polymer fragment conjugates of the invention can be made by quenching the deséalo antibody fragment with an inert polymer. It will be appreciated that any inert polymer that provides the conjugate with the desired apparent size or which has the selected effective molecular weight, as shown herein, is suitable for use in the construction of the antibody-polymer fragment conjugates of the invention. Many inert polymers are suitable for use in pharmaceutical products. See, for example, Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp. 441-451 (1980). In all embodiments of the invention, a non-protein polymer is used. The non-protein polymer is ordinarily a synthetic hydrophilic polymer, for example, a polymer not otherwise found in nature. However, polymers that exist in nature and are produced by recombinant or in vi tro methods are also useful, as are polymers that are isolated from native sources. Hydrophilic polyvinyl polymers fall within the scope of this invention, for example, polyvinyl alcohol and polyvinyl pyrrolidone. Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG); polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and polyoxyethylene and polyoxypropylene block copolymers (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides, which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-acid -manuronic (eg, polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of the acid mucopolysaccharides, for example, hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon. The polymer before crosslinking need not be, but preferably is, soluble in water, but the final conjugate must be soluble in water. Preferably, the conjugate shows a solubility in water of at least about 0.01 mq / ml and more preferably at least about 0.1 mg / ml, and still more preferably at least about 1 mg / ml. In addition, the polymer should not be highly immunogenic in the conjugated form, nor should it possess viscosity that is incompatible with infusion or intravenous injection if the conjugate is intended to be administered by such routes.

In one embodiment, the polymer contains only a single group that is reactive. This helps prevent the cross-linking of protein molecules. However, it is within the scope herein to maximize the reaction conditions to reduce crosslinking, or to purify the reaction products through gel filtration or ion exchange chromatography to recover the substantially homogeneous derivatives. In other embodiments, the polymer contains two or more reactive groups for purposes of binding multiple antibody fragments to the polymer backbone. Again, gel filtration or ion exchange chromatography can be used to recover the desired derivative in substantially homogeneous form. The molecular weight of the polymer can be in the range of up to about 500,000 D, and preferably it is at least about 20, Ouu D, or at least about 30,000 D, or at least about 40,000 D. The molecular weight chosen may depend on the size effective of the conjugate that is going to be achieved, of the nature (e.g., the structure, such as branched or linear) of the polymer, and the degree of derivatization, e.g., the number of polymer molecules per antibody fragment, and the site or sites of polymer coupling on the fragment of antibody. The polymer can be covalently linked to the antibody fragment through a multifunctional crosslinking agent that reacts with the polymer and one or more amino acid residues of the antibody fragment to be ligated. However, it is within the scope of the invention to directly crosslink the polymer by reaction of a derivatized polymer with the antibody fragment, or vice versa. The site of covalent crosslinking on the antibody fragment includes the N-terminal amino group and the epsilon-amino groups found on the lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer EJ can be covalently nested directly to the antibody fragment without the use of multifunctional crosslinking agent (ordinarily bifunctional). Covalent binding to amino acids is achieved by known chemical techniques based on cyanuric chloride, carbonyldiimidazole, reactive aldehyde groups (PEG-alkoxide plus bromoacetaldehyde diethylacetal); PEG plus DMSO and acetic anhydride, or PEG chloride plus "4-hydroxybenzaldehyde phenoxide, activated succinimidyl esters, activated dithiocarbonate PEG, activated PEG with 2, 4, 5-trichlorophenylchloroformate or with P-nitrophenylchloroformate). they are derivatized by the coupling of PEG-amine using carbodiimide.Sulfhydryl groups are derivatized by coupling to PEG substituted with maleimido (for example, alkoxy-PEG-amine plus 4- (N-maleimidomethyl) cyclohexane-1-carboxylate of sulfosuccinimidyl) , as described in International Patent WO 97/10847 published March 27, 1997, or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, AL) Alternatively, free amino groups on the antibody fragment (for example, the epsilon-amino groups on the residues of lysine) can be thioxai with 2-imino-L-iono (Traut's reagent) and then coupled to the derivatives that contain in PEG maleimide, as described in Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994). The polymer will carry a group that is directly reactive with the side chain of amino acids, or the N-terminus or C-terminus of the linked polypeptide, or which is reactive with the multifunctional cross-linking agent. In general, polymers possessing such reactive groups are known for the preparation of immobilized proteins. In order to use such chemistries here, a water-soluble polymer, otherwise derivatized in the same manner as the insoluble polymers used to date for protein immobilization, could be employed. Activation with cyanogen bromide is a particularly useful method for use in the crosslinking of polysaccharides. 'Soluble in water' in reference to the initial polymer means that the polymer or its reactive intermediate used for conjugation is sufficiently soluble in water to participate in a derivative action The degree of substitution with the polymer will vary depending on the number of reactive sites on the antibody fragment, the molecular weight, the hydrophilicity, and other characteristics of the polymer, and the particular derivatization sites of the antibody fragment, chosen In general, the conjugate contains from one to about 10 molecules polymers, but larger numbers of polymer molecules coupled to the antibody fragments of the invention are also contemplated The desired amount of derivatization is easily achieved by the use of an experimental matrix in which the time, temperature and other reaction conditions are varied to change the degree of substitution, after which it is determined mine the level of polymer substitution of the conjugates by size exclusion chromatography or other means known in the art. The polymer, for example PEG, is crosslinked to the antibody fragment by a variety of methods known per se for the covalent modification of proteins with non-protein polymers such as PEG. Some of these methods, however, are not preferred for purposes in the present. The chemistry of cyanuronic chloride leads to many coxa- teral reactions, decreasing the cross-linking of the protein. In addition, it may be particularly likely to lead to the inactivation of proteins containing sulfhydryl groups. The chemistry of carbonyldiimidazole (Beauchamp et al., Anal. Biochem. 131, 25-33 [1983]) requires high pH (> 8.5), which can inactivate proteins. In addition, since the 'PEG activated' intermediate can react with water, a very large molar excess of the 'activated PEG' on the protein is required. The high concentrations of PEG required for the carbonyldiimidazole chemistry also lead to problems in purification, both in gel filtration chromatography and in hydrophilic interaction chromatography, which are adversely affected. In addition, high concentrations of 'activated PEG' can precipitate the protein, a problem that has been previously noted (Davis, US Patent No. 4,179,337). On the other hand, aldehyde chemistry (Royer, US Patent No. 4,002,531) ) is more efficient, since it requires only a 40-fold molar excess of PEG and an incubation of 1 to 2 hours.However, the manganese dioxide suggested by Royer to the preparation of the PEE aldehyde is problematic because of the pronounced tendency of PEG to form complexes with metal-based oxidizing agents "(Harris et al, J. Polym, Sci. Polym, Chem. Ed. 22, 341-52 [1984]). The use of Moffatt oxidation, using DMSO and acetic anhydride, avoids this problem. In addition, the sodium borohydride suggested by Royer should be used at a high pH and has a significant tendency to reduce disulfide bridges. In contrast, sodium cyanoborohydride, which is effective at natural pH and has very little tendency to reduce disulfide bridges, is preferred. In another preferred embodiment, maleimido-activated PEG is used for coupling free thiols on the antibody fragment. PEG polymers functionalized to modify the antibody fragments of the invention are available from Shearwater Polymers, Inc. (Huntsville, AL). Such commercially available PEG derivatives include, but are not limited to, to ino-PEG, amino acid esters of PEG, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG-amino acids, PEG Succinimidyl succinate, PEG-propionate of its cymimidyl ester, succinyl ester or midiyl CoJ. PEG caiboximet bundle, carbon to PEG succinimide, amino acid succinimidyl esters-PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG-vinylsulfone, PEG-maleimide, PEG-ortopyridyldisulfide, heterofunctional PEGs, vinyl derivatives of PEG, silanes of PEG and phospholides of PEG. The reaction conditions for the coupling of these PEG derivatives will vary depending on the protein, the desired degree of PEGylation, and the PEG derivative used. Some factors involved in the choice of PEG derivatives include: the desired point of coupling (such as the R groups of lysine and cysteine), the hydrolytic stability and the reactivity of the derivatives, the stability, toxicity and antigenicity of the link, the adequacy for the analysis, etc. Specific instructions for the use of any particular derivative are available from the manufacturer. The conjugates of this invention are separated from the unreacted starting materials by gel filtration or ion exchange HPLC. The heterologous species of the conjugates are purified by d-; the other in the same way .. The conjugates can also be purified by ion exchange chromatography. The chemistry of many of the electrophilically activated PEGs results in a reduction of the amino group charge of the PEGylated product. In this way, high resolution ion exchange chromatography can be used to separate free and conjugated proteins, and to resolve spaces with different levels of PEGylation. In fact, the resolution of different species (for example, containing one or two PEG residues) is also possible due to the difference in the ionic properties of the unreacted amino acids. In a modality, the species with different levels of PEGylation are resolved according to the methods described in WO 96/34015 (International Application No. PCT / US96 / 05550 published on October 31, 1996). In a preferred embodiment, the conjugate is generated by using the derivatization and purification methods described in Section (T) of the following Examples. In one aspect, the invention provides any of the above-described conjugates formed by sr ^ parte0 composing ts, for example, one or more fragments of a p lici t po co v alentemea joined to one or more polymer molecules, without any foreign matter in the covalent molecular structure of the conjugate. c. Other Derivatives of Conjugates of Large Effective Size In yet another aspect, any of the above-described conjugates can be modified to contain one or more components in addition to the one or more components of antibody fragments and of the polymer component (s), which form the conjugate, wherein the modification does not alter the essential functional property of the conjugate, namely, substantially improved serum half life, MRT and / or a serum clearance rate compared to that of the parent antibody fragment, from which the conjugate is derived. In one embodiment, the invention provides any of the above-described conjugates modified to incorporate one or more non-protein functional groups. For example, the conjugate can be modified to incorporate non-protein markers or reporter molecules, such as radiolabels, including any radioactive substance used in medical treatment or imaging, or used as an effector or tracer function in an animal model, such as the radioisotope levels 99Tc, 90Y, ^ In, 32P, 1C, 125I, 3H, 131I, X1C, 150, 13N, 18F, 35S, 51Cr, 57To, 226Ra, 60Co, 59Fe, 75Se, 152Eu, 67Cu, 217Ci , 11At, 212Pb, 47Sc, 109Pd, 234Th, 40K, and the like, non-radioisotope levels such as 157Gd, 55Mn, 52Tr, 56Fe, etc., fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152 Eu, dansyl, umbelliferone, luciferin, luminal marker, isoluminal marker, an aromatic acridinium ester marker, an imidazole marker, an acridinium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin / avidin, spin labels, stable free radicals, and the like. Conventional methods are available to bind these markers covalently to the polypeptide antibody fragment or to the polymer component of the conjugate. In one aspect, any conjugate of the invention is modified by derivatization of the antibody fragment component with any of the non-protein markers described above, wherein the marker is directly or indirectly (via a coupling agent) attached to the fragment. of antibody, and wherein such derivatization of the antibody fragment does not contribute or introduce any polymeric portion within the molecular structure of the conjugate. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like can be used to label or label the antibody fragment with the fluorescent or chemiluminescent labels described above. See, for example, U.S. Patent No. 3,940,475 (fluorimetry), Morrison, Meth. Enzymol. , 32b, 103 (1974), Svyanen et al., J. Biol. Chem., 284, 3762 (1973), and Bolton and Hunter, Biochem. J., 133, 529 (1973). In the case of the modalities that use rad ers, you can use direct and indirect marking to correct the selected link within the conjugate. As used herein in the context of radiolabeling, the phrases "indirect labeling" and "indirect labeling method" both mean that a chelating agent is covalently bound to the antibody fragment portion or the polymer portion of the conjugate, and at least one radionuclide is inserted into the chelating agent. Preferred chelating agents and radionuclides are described in Srivagtava, S.C. and Mease, RC, 'Progress in Research on Ligands, Nuclides and Techniques for Labeling Monoclonal Antibodies', Nuci, Med. Bio., 18 (6): 589-603 (1991) .A particularly preferred chelating agent is l-1 acid. isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid ('MX-DTPA "). As used herein in the context of radiolabeling, the phrases 'direct labeling' and 'direct labeling method' both mean that a radionuclide is covalently linked directly to the portion of the antibody fragment (typically via an amino acid residue) or to the polymer portion of the conjugate. Preferred radionuclides for use in direct labeling of the conjugate are provided in Srivagtave Mease, supra. Eu one modality. the conjugate is directly labeled with 131I covalently bound to the tyrosine residues. In yet another embodiment, the antibody fragment component of the conjugate is directly or indirectly labeled with any of the previously described radiolabels, wherein such labeling of the antibody fragment does not contribute or introduce any polymeric portion within the molecular structure of the conjugate. d. Therapeutic Compositions and_ Administration of Large Effective Size Conjugates The conjugate of the invention is useful in the treatment of disease indications that are treated with the intact parent antibody. for example, a conjugate derived from an antibody or anti-IL-8 antibody fragment, is useful in the treatment of inflammatory disorders as described in Section (II) (5) (B) below. Therapeutic formulations of the conjugate of the invention can be prepared by using the same procedure described for the formulation of the aneicuepes and fragmontot-anti-IL-8 of the invention in Section (II) (5) (B) below . The conjugate of the invention can be administered in place of the parent antibody for a given indication of disease, by modifying the formulation, dosage, administration protocol, and other aspects of a therapeutic regimen as required by the different pharmacodynamic characteristics. of the conjugate, and is dictated by common medical knowledge and practice. e. Reagent Uses for Conjugates of Large Effective Size The conjugate of the invention also finds application as a reagent in an animal model system for the in vivo study of the biological functions of the antigen recognized by the conjugate. The conjugate could make it possible for the practitioner to inactivate or detect the cognate antigen in circulation or in the tissue, for a much longer period of time than would be possible by the constructions known in the art, while eliminating any interaction of e.g. (which could help the use of an intact antibody) system. In addition, the increased half-life of the conjugate of the invention can be advantageously applied to the induction of tolerance for the non-derivatized antibody fragment in a test animal by using the method of Wie et al., Int. Archs. Allergy Appl. Immunol., 64: 84-99 (1981) for the allergen tolerization, which could allow the practitioner to repeatedly challenge the animal tolerized with the non-derivatized progenitor antibody fragment, without generating an immune response against the parent fragment. 2. ANTIBODY FRAGMENTS AND HUMANIZED MONOCLONAL ANTIBODIES 6G4.2.5 In one embodiment, the invention provides an antibody fragment or a full-length antibody comprising a heavy chain comprising the amino acid sequence of amino acids 1-230 (hereinafter referred to as "6G4.2.5HV11") of the amino acid polypeptide sequence of the humanized anti-IL-8 6G4.2.5V11 heavy chain of Figures 37A-37B (SEQ ID NO: 75) The invention is directed to a fragment of a single chain antibody comprising: 6G4.2.5HV11, with or without any additional amino acid sequence In one embodiment, the invention provides a single chain antibody fragment comprising 6G4.2.5HV11 without any light chain amino acid sequence, associated, for example, with a kind of single strand that constitutes half of a Fab fragment. An antibody or antibody fragment comprising 6G4.2.5HV11 is further provided herein, and further comprising a light chain comprising the sec amino acid sequence of amino acids 1-219 (hereinafter referred to as' 6G4.2.5LV11") of the light chain polypeptide amino acid sequence of humanized anti-IL-8 6G4.2.5V11 of Figure 31B (SEQ. ID. NO: 65). In one embodiment, the invention provides a single chain antibody fragment wherein 6G4.2.5HV11 and 6G4.2.5LV11 are contained in a single chain polypeptide species. In a preferred embodiment, the single chain fragment of the antibody comprises 6G4.2.5HV11 bound to 6G4.2.5LV11 by means of a flexible peptide linker sequence, wherein the chain and light chain domains can be associated in a dimeric structure "analogous to that formed in a Fab double-stranded species., the single chain antibody fragment is a species comprising 6G4.2.5HV11 linked to 6G4.2.5LV11 by a linker that is too short to allow intramolecular pairing of complementary domains, for example, a single chain polypeptide monomer which forms a diabody after dimerization with another monomer. In still another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein one polypeptide chain comprises 6G4.2.5HV11 and a second polypeptide chain comprises 6G4.2.5LV11 and the two polypeptide chains are covalently linked to one or more interchain disulfide bridges. In a preferred embodiment, the double-stranded antibody fragment described above is selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2. The invention also provides an antibody or antibody fragment comprising a heavy chain containing 6G4.2.5HV11 and optionally further comprising a light chain containing ex 6cx.2.0LV11, wherein the heavy chain, and optionally the light chain , is or are fused to an additional portion, such as the constant domain sequence of the additional immunoglobulin. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or full length or partial chain or chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from any animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. (Supra). In a preferred embodiment, the antibody or antibody fragment comprises 6G4.2.5HV11 in a heavy chain that is fused to or contains a leucine zipper sequence. The leucine zipper can increase the affinity and / or production efficiency of the antibody or antibody fragment of interest. Suitable leucine zipper sequences include the iodine and phosphate zippers, as measured by Kostelney et al., J. Immunol. , 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the Examples below. In a preferred embodiment, the antibody or antibody fragment comprises 6G4.2.5HV11 fused at its C-terminus to the leucine zipper GCN4, to produce the amino acid sequence of amino acids 1-275 (hereinafter referred to as * 6G4). .2.5HV11GCN4") of the heavy chain polypeptide amino acid sequence of Figures 37A-37B (SEQ ID NO: 75). 3. VARIANTS OF ANTIBODY FRAGMENTS AND HUMANIZED MONOCLONAL ANTIBODIES 6G4.2.5 The invention further encompasses the humanized anti-IL-8 monoclonal antibody or antibody fragment, comprising the variants of the complementarity determining regions of 6G4.2.5 (CDRs) or the variants of the variable domains of 6G4.2.5vll which show greater affinity for human IL-8 and / or possess properties that produce greater efficiency in the process of recombinant production.

A. VARIANTS OF 6G4.2.5LV In one aspect, the invention provides the humanized anti-IL-8 monoclonal antibodies and the antibody fragments comprising the complementarity determination regions (referred to herein as the "CDGs of 6G4.2.5LV") Ll, L2 and L3 of the amino acid sequence of the 6G4.2.5 light chain variable domain of Figure 24, where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48). a variant of the antibody or humanized antibody fragment 6G4.2.5, comprising a humanized light chain variable domain comprising a variant (hereinafter referred to as a 'variant CDRs of 6G4.2.5LV') of the regions of determination of the comple L, L2 and L3 mentality of the amino acid sequence of the variable light chain domain of 6G4.2.5, of Figure 24 (SEQ 10. NO: -J 8). In one embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of the CDRs of 6G4.2.5LV (hereinafter referred to as' 6G4.2.5LV / L1N35X35") wherein corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID.

NO: 48) with the proviso that any amino acid other than Asn (denoted as 'X35') is replaced by Asn at the position of amino acid 35, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 ( SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) In a preferred embodiment, the invention provides a variant of the humanized antibody 6G4.2.5 or an antibody fragment thereof, comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as' 6G4.2.5LV / L1N35A ") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted for Asn at the position of amino acid 35, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102? E the amino acid sequence os of Figure 24 (SEQ. ID. NO: 48). In still another preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of the CDRs 6G4.2.5LV (hereinafter referred to as '6G4.2.5LV / L1N35E') where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Glu is replaced by Asn at the position of amino acid 35, L2 corresponds to amino acids 55- 61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48). Second aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as' 6G4.2.5LV / L1S26X26") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ. D. NO: 48) with the proviso that any amino acid other than Ser (denoted as 'X26') is replaced by Ser at the position of the amino acid .612 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ. NO: 48). In a preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as 6G4.2.5LV / L1S26A ") wherein Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted for Ser at amino acid position 26, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48). aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as * 6G4.2.5LV / L3H98X98") where Ll corresponds to the amino acids 24-39 of the amino acid sequence of Figure 2 (SEQ ID NO: 48), L2 corresponds to the amino acids 55-6i of the amino acid sequence of Figure 24 (SEQ. ID. NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), with the proviso that any amino acid other than His (denoted as 'Xgs') is substituted by His at the position of amino acid 98. In a preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (referred to herein as 6G4. 2.5LV / L3H98A ") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24. { I KNOW THAT. ID. NO: 48), L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that the amino acid Ala is substituted by His at the position of amino acid 98. In a fourth aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as '6G4.2.5LV / L1S26X26N35X3e') where Ll corresponds to the amino acids 24-C * of the amino acid sequence of Figure 24 (SEQ ID. NO: 48) with the proviso that any amino acid other than Ser (denoted as * X26") is replaced by Ser at the position of amino acid 26 and any amino acid other than Asn (denoted as * X35") is replaced by Asn in the position of amino acid 35, L2 corresponds to amino acids 55-61 of the sequence amino acid of Figure 24 (SEQ. ID. NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48). In a preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G .2.5LV (referred to herein as '6G4.2.5LV / L1S26A, 35A') where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted for Ser at amino acid position 26 and Ala is substituted for Asn in the position of amino acid 35, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) In a fifth aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as' 6G4 .2.5LV / L1N35X35 / 3H98X98") where Ll corresponds to the amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ. ID.

NO: 48) with the proviso that any amino acid other than Asn (denoted as * ° X35") is replaced by Asn at the position of amino acid 35, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24, (SEQ ID NO: 48) with the proviso that any amino acid other than His ( denoted as * Xg8") is replaced by His at the position of amino acid 98. In a preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (referred to as herein as' 6G4.2.5LV / L1N35A / L3H98A ") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted by Asn at amino acid position 35, L2 corresponding to amino acids 05-61 of the amino acid sequence of Figure 24 (SEQ. ID. NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted for His at the position of amino acid 98. " In a sixth aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (hereinafter referred to as' 6G4.2.5LV / LlS26X26 / L3H98X9? ") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that any amino acid other than Ser (denoted as 'X? e') is replaced by Being at the position of amino acid 26, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that any amino acid other than His (denoted com or "Xgs") is substituted for His at the position of amino acid 98. In a preferred embodiment, the invention provides a variant of the antibody or humanized antibody 6G4.2.5 variant comprising a variant of CDRs of 6G4.2.5LV (referred to herein as '6G4.2.5LV / L1S26A / L3H98A') where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ. ID. NO: 48) with the proviso that Ala is substituted for Ser at amino acid position 26, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted by His at the position of amino acid 98. In a seventh aspect, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (referred to herein as' 6G4.2.5LV / L1S26X26, N35X35 / 3H98X98") where Ll corresponds to amino acids 24 -39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that any amino acid other than Ser (denoted as 'X2ß') is replaced by Ser at the position of amino acid 26 and any amino acid different from Asn (denoted as 'X35') is replaced by Psn in the pos Amino acid 35, L2 corresponds to amino acids 55-6Í of the amino acid sequence of Figure 24 (SEQ. ID. NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that any amino acid other than His (denoted as 'X9β') is substituted by His at the position of amino acid 98. In a preferred embodiment, the invention provides a variant of the antibody or fragment of humanized antibody 6G4.2.5 comprising a variant of CDRs of 6G4.2.5LV (referred to herein as' 6G4. 2.5LV / L1S26A, N35A / L3H98A ") where Ll corresponds to amino acids 24-39 of the amino acid sequence of Figure 24 (SEQ ID NO: 48) with the proviso that Ala is substituted for Ser in the position of amino acid 26 and Ala is replaced by Asn at the position of amino acid 35, L2 corresponds to amino acids 55-61 of the amino acid sequence of Figure 24 (SEQ ID NO: 48), and L3 corresponds to amino acids 94-102 of the amino acid sequence of Figure 24 (SEQ. NO: 48) with the proviso that Ala is replaced by His at the position of amino acid 98. The light chain, humanized, variable domains of Already invention, can be constructed by using any of the techniques for the humanization of antibodies, known in the art. Humanization can be essentially done following the method of Winter and collaborators (Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), by substituting CDGs of 6G4.2.5LV or CDRs of a variant of CDGs of 6G4.2.5LV for the corresponding sequences of a human antibody light chain variable domain. Accordingly, such "humanized" derivatives containing the CDRs of 6G4.2.5LV or the CDRs of a variant of CDRs of 6G4.2.5LV, are chimeric (Cabilly et al., Supra.) The humanized light chain variable domain comprising CDRs of 6G4.2.5LV and CDRs of a variant of CDGs of 6G4.2.5LV may also contain some FR residues that are substituted by residues from analogous sites in the light chain variable domain of murine antibody 6G4.2.5 (' 6G4.2.5LV "). The complete amino acid sequence of 6G4.2.5LV is described as amino acids 1-114 of the amino acid sequence of Figure 24 (SEQ ID NO: 48). The invention further provides an antibody or antibody to a humanized antibody comprising a humanized light chain variable domain, comprising the CDRs of 6G4.2.5LV or the CDRs of a variant of CDRs of 6G4.2.5LV as described above, and which further comprises a humanized heavy chain variable domain comprising the complementarity determining regions (CDRs) H1, H2 and H3 of the amino acid sequence of the variable heavy chain domain of 6G4.2.5 (murine monoclonal antibody) of Figure 25 (SEQ ID NO: 50), where H1 corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), where H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and wherein H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). The above described CDRs Hl, H2 and H3 of the heavy chain variable domain of 6G4.2.5 ('6G4.2.5HV ") are collectively referred to as the' CDRs of 6G4.2.5HV '. In yet another embodiment, the invention provides an antibody or humanized antibody fragment comprising a humanized light chain domain, which comprises the LJJLs of 6G4.2.5LV or the CDRs of a CDRs variant of 6G4.2.5LV as described above, and further comprising a humanized heavy chain variable domain comprising a variant (hereinafter referred to as 'CDRs variant of 6G4.2.5HV') of the CDRs of Hl, H2 and H3 of the amino acid sequence of the variable heavy chain domain of 6G4.2.5 (murine monoclonal antibody) of Figure 25 (SEQ ID NO: 50) In a variant of CDGs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / HlS31Z3? "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as' Z3?") be replaced by Ser in the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ.

ID. NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). In a preferred CDG variant of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H1S31A "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ.D.NO: 50) with the condition that Al? is replaced per 1 'the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50).

In a second variant of CDGs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H2S54Z54'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO. : 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), with the proviso that any amino acid other than Ser (denoted as 'Z54') is substituted by Being at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / H2S54A "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO. : c0) with the proviso that Ala is substituted for Ser in I position of amino acid 54, hO corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). In a third variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H3SD100E'), where Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. NO: 50), where H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and where H3 corresponds to amino acids 99-111 of the sequence of amino acids of Figure 25. {SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100. In a fourth variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H3R102K'), where Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ.

NO: 50), where H2 corresponds to the amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and wherein H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg in the position of amino acid 102. In a fifth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H3D106E'), where Hl corresponds to amino acids 26-35 of the amino acid sequence of the Figure 25 (SEQ ID NO: 50), where H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and where H3 corresponds to amino acids 99 -111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106. In a seventh variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / H3D100E, R102K "), wherein Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), wherein H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and where H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is replaced by Arg at the position of amino acid 102. In an eighth variant of CDRs of 6G4.2.5HV (designated in. present as' COI .2.5HV / H3R102K, DIOFOL), e,? where Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), where H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and wherein H3 corresponds to amino acids 99-111 of the amino acid sequence of the Figure 25 (SEQ., ID.

NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102 and Glu is replaced by Asp at the position of amino acid 106. In a ninth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H3D100E, D106E'), where Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), where H2 corresponds to amino acids 50- 66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and wherein H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that Glu is replaced by Asp at the position of amino acid 100 and Glu is replaced by Asp at the position of amino acid 106. In a tenth variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4 7.5FV / H3ÍY 00E, Rl 02K, DI 06E "), where Hl co responds to amino acids 2 -35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), where H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and wherein H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100, Lys is substituted by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a eleventh variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / HlS31Z3? / H2S54Z54"), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that any amino acid other than Ser (denoted as 'Z31') is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO. : 50) with the proviso that any amino acid other than Ser (denoted as * Z5") is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 ( SEQ.ID.O'50;. In a variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H1S31A / H2S54A'), Hl corresponds to amino acids 26-35 of the sequence of amino acids of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso "that Ala is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). In a twelfth variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / HlS31Z3? / H3D100E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25. { I KNOW THAT. ID. NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z3?') Is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEO- TD.NO: 0) with the proviso that Glu is replaced by Asp. in α position of amino acid 100 .. In a variant of CDRs of 6G4.2.5HV (referred to herein as '6G .2.5HV / H1S31A / H3D100E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu s is replaced by Asp at the position of amino acid 100. In a thirteenth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H3R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z3? ") Is replaced by Ser in the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg an the position of the inoea: dc 102. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4. 2.5HV / H1S31A / H3R102K "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the amino acid position 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102. In a fourteenth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H3D106E') , Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z3? ") Is replaced by Ser in the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ciu is replaced by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H1S31A / H3D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso n that Ala is substituted for Ser at amino acid position 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25. { I KNOW THAT. ID. NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106. A Fifteenth variant of CDGs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H3D100E, R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z3?') Is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of the Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SFQ, TD, NO: 50) with the proviso that Glu is substituted for jfor Asp in the position of amino acid 100 and Lys is replaced by Arg at position 102. In a preferred variant of CDRs of 6G4.2.5HV (referred to in the pre as "6G4.2.5HV / H1S31A / H3D100E, R102K"), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is replaced by Arg at the position of amino acid 102. In a sixteenth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H3R102K, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50 ) with the proviso that any amino acid other than Ser (denoted as 'Z3?') is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is substituted by Arg at the position of amino acid 102 and Glu is replaced by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H1S31A / H3R102K, D106E'), Hl corresponds to amino acids 26 -35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is replaced by Ser in the amino position o acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acids 102 and Glu is substituted by Asp at the position of amino acid 106. In a seventeenth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H3D100E, D106E'), Hl corresponds to amino acids 26- 35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as "31") is replaced by S x e x position of the amino acid 31 , H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Glu is replaced by Asp at the position d amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H1S31A / H3D100E, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of the Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Glu is replaced by Asp in the position of amino acid 106. In a eighteenth variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H1S31Z AH3D100E Rl 02K, I 06E'), Hl corresponds to lora amiroa ~? two 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z3?') is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100, Lys is replaced by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (hereinafter referred to as * 6G4.2.5HV / H1S31A / H3D100E, R102K, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), and H3 corresponds to amino acids 99-111 of the sequence of amino acids of Figure 25 (SEQ ID NO: 50) with the proviso that Ciu is replaced by Asp at the position of amino acid 100, L s is replaced by Arg at the position of amino acid 102 and Glu is replaced by Arg at the position of amino acid 106. In a nineteenth variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H2S54Z54 / H3D100E ") Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z5') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is substituted for Asp at the position of amino acid 100. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5 HV / H2S54A / H3D100E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (S EQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Xiguxa 2o (SEQ ID NO: 50) with the condition that Glu is replaced by Asp at the position of amino acid 100. In a twentieth variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H2S54Z54 / H3R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the sequence of amino acids of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99 -111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102. In a preferred variant of CDRs of 6G4.2.5HV (referred to as herein "6G4.2.5HV / H2S54A / H3R102K"), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50- 66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to the 99 amino acids of the amino acid sequence of Figure 05 (SEQ. ID. NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102. In a twenty-first variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H2S54Z54 / H3D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the sequence of amino acids of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99 -111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (referred to as herein as' 6G4.2.5HV / H2S54A / H3D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50- 66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106. In a twenty-second variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H2S54Z54 / H3D100E, R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is substituted by Arg at the position of amino acid 102 In a preferred variant of CDGs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H2S54A / H3D100E, R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 ( SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the cond tion that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is replaced by Arg at the position of amino acid 102.

In a vigesi otercera variant of CDRs of 6G4.2.5HV (denominated in the present as' 6G4.2.5HV / H2S54Z54 / H3R102K, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID. NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z54") is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV ( referred to herein as '6G4.2.5HV / H2S54A / H3R102K, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is replaced by Be at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a twenty-fourth variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / H2S54Z54 / H3D100E, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Glu is replaced by Asp at the position of amino acid 106 In a preferred variant of CDRs of 6G ^ .2.5HV (denoted in _3 present as' 6G4.2.5HV / H2 S54A / H3D100E, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at amino acid position 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Glu is replaced by Asp in the position of amino acid 106. In a twenty-fifth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H2S54Z54 / H3D100E, R102K, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 501, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser ( denoted as 'Z5') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of to amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100, Lys is replaced by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H2S54A / H3D100E, R102K, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. NO: 50), H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at amino acid position 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100, Lys is replaced by Arg at the position of amino acid 102 and Glu is replaced by Asp at the position of amino acid 106. In a twenty-sixth variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H1S31Z31 / H2S54Z5 / H3D100E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50), with the proviso that any amino acid other than Ser (denoted as * Z3Y ') is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is substituted by Asp at the position of amino acid 100. In a preferred variant of CDRs of 6G4.2 .5HV (referred to herein as '6G4.2.5HV / H1S31A / H2S54A / H3D100E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100. In a twenty-seventh variant of CDRs of 6G'i.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z3? ") is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z5') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102. In a preferred variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H1S31A / H2S54A / H3R102K " ), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-? Ll of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102. In a twenty-eighth variant of CDRs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID. NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z3? ") Is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z 4') is replaced by Ser at the position of amino acid 54, and H 3 corresponds to amino acids 99-111 of the sequence of amino acids of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106.

In a preferred variant of CDGs of 6G4.2.5HV (referred to herein as '6G4.2.5HV / H1S31A / H2S54A / H3D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ. ID NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID.

NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 106. In a twenty-ninth variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H1S31Z31 / H2S54Z54 / H3D100E, R102K "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z3?") Is substituted. by Being at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25. { I KNOW THAT. ID. NO: 50) with the proviso that any amino acid other than Ser (denoted as * Z54") is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is substituted by Arg at the position of amino acid 102. In a preferred variant of CDRs of 6G4.2.5HV ( referred to herein as '6G4.2.5HV / H1S31A / H2S54A / H3D100E, R102K'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Lys is replaced by Arg at the position of amino acid 102. In a thirtieth variant of CDRs of 6G4.2.5HV (referred to herein as '6G .2 5HV / HlS31Z3? / H2S54Z54 / H3R102K, Dl 06E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid different from Ser (denoted as 'Z3?') is replaced by Ser at the position of amino acid 31., H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z54') is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. NO: 50) provided that Lys is replaced by Arg at the position of amino acid 102 and Glu s is replaced by Asp at the position of amino acid 106. In a variant of CDRs of 6G4.2.5HV (referred to herein as * 6G4.2.5HV / H1S31A / H2S54A / H3R102K, D106E "), Hl corresponds to amino acids 26- 35 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that Ala is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Lys is replaced by Arg at the position of amino acid 102 and Glu is replaced by Asp at the position of amino acid 106. In a variant variant of CDRs to 6G4.2.5HV (referred to herein as' 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser (denoted as 'Z3i') is substituted. by Being at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid 'differs from Ser (denoted as * Z ") is substituted for Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100 and Glu is substituted by Asp at the position of amino acid 106. In a variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / H1S31A / H2S54A / H3D100E, D106E " ), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) n the condition that Ala is substituted for Ser e the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Ala is replaced by Ser at the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the condition that Glu be replaced by Asp at the position of amino acid 100 and Glu be replaced by Asp at the position of amino acid 106. In a thirty-second variant of CDRs of 6G4.2.5HV (referred to herein as' 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, R102K, D106E "), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid other than Ser ( denoted as * Z3? ") is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that any amino acid different from Ser (denoted as' Z5íJ ") is replaced by Being at the position of the amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ. ID. NO: 50) with the proviso that Glu is replaced by Asp at the position of amino acid 100, Lys is replaced by Arg at the position of amino acid 102 and Glu is substituted by Asp at the position of amino acid 106. In a preferred variant of CDRs of 6G4.2.5HV (hereinafter referred to as '6G4.2.5HV / H1S31A / H2S54A / H3D100E, R102K, D106E'), Hl corresponds to amino acids 26-35 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is substituted for Ser at the position of amino acid 31, H2 corresponds to amino acids 50-66 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Ala is repl by Ser in the position of amino acid 54, and H3 corresponds to amino acids 99-111 of the amino acid sequence of Figure 25 (SEQ ID NO: 50) with the proviso that Glu is repl by Asp at the position of amino acid 100, Lys is substituted by Arg at the position of amino acid 102 and Glu is repl by Asp at the position of amino acid 106. As in the humanization of the light chain variable domain described above, a humanized heavy chain variable domain is constructed by replacing the CDRs of 6G4.2.5HV or the CDRs of a variant of CDRs of 6G4.2.5HV by the corresponding sequences in a human heavy chain variable domain. The humanized heavy chain variable domain comprising the CDRs of 6G4.2.5HV or the CDRs of a variant of CDGs of 6G4.2.5HV may also contain some FR residues that are substituted by residues of analogous sites in the variable chain domain Heavy antibody 6G4.2.5 antibody. The complete amino acid sequence of 6G4.2.5HV is described as amino acids 1-122 of the amino acid sequence of Figure 25 (SEQ ID NO: 50). The choice of human variable domains, whether light and heavy, which are to be used in the preparation of antibodies or humanized antibody fragments, is very important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is selected against the full library of known human variable domain sequences.The human sequence most similar to that of the rodent is then accepted as the FR human structure for the humanized antibody (Sims et al, J. Immuno1: 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196: 901 (1987)). Yet another method uses a particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of light and heavy chains The same structure can be used for several different humanized antibodies (Cárter et al., Proc. Nati Acad. Sci. USA 89: 4285 (1992) Presta et al, J. Im unol. 151: 2623 (1993).) It is also important that the antibodies and antibody fragments of the invention are humanized with high affinity retention for I Human L-8 and other favorable biological properties. To achieve this goal, according to a preferred method, the antibodies or humanized antibody fragments of the invention are prepared by a process of analysis of the progenitor sequences and various conceptual humanized products, using the three-dimensional models of the progenitor and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those of skill in the art. Computer programs are available, which illustrate and show the probable, three-dimensional conformational structures of the selected candidate immunoglobulin sequences. The inspection of these visual representations allows the analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of the residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, the FR residues can be selected and combined from the consensus and progenitor sequences, so that the desired characteristic of the antibody, such as the increased affinity for the target antigen (s), is achieved. Any and all variable domain amino acid sequences of the humanized light chain comprising the 6G4.2.5LV CDRs are collectively referred to herein as 'hu6G4.2.5LV'. Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1N35X35 are collectively referred to herein as 'hu6G4.2.5LV / LlN35X3s' • Any and all variable domain amino acid sequences of the humanized light chain comprising the cDRs of 6G4.2.5LV / L1N35A are collectively referred to herein as' hu6G4.2.5LV / L1N35A. "Any and all domain amino acid sequences variable of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1N35E are collectively referred to herein as 'hu6G4.2.5LV / L1N35E'. Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26X26 are collectively referred to herein as' hu6G4.2.5LV / L1S26X26. "Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26A are collectively referred to herein as' hu6G4.2.5LV / L1S26A. "Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L3H98X98 are collectively referred to herein as 'hu6G4.2.5LV / L3H98X98.' Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L3H98A are collectively referred to herein as' hu6G4.2.5LV / L3H98A. "Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26X26, N35X35 are collectively referred to herein as 'hu6G4.2.5LV / LlS26X26, N35X35' • Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26A, N35A are collectively referred to herein as 'hu6G4.2.5LV / L1S26A, 35A.' Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1N35X35 / 3H98X98 are collectively referred to herein as 'hu6G4.2.5LV / L1N35X35 / 3H98X98'. Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1N35A / L3H98A are collectively referred to herein as' hu6G4.2.5LV / LlN35A / L3H98A. "Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26X26 / 3H98X98 are collectively referred to herein as 'hu6G4.2.5LV / L1S26X26 / L3H98X98'. Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26A / L3H98A are collectively referred to herein as' hu6G4.2.5LV / L1S26A / L3H98A. "Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26X26, N35X35 / 3H98X98 are collectively referred to herein as 'hu6G4.2.5LV / L1S26X26, N35X35 / L3H98X98'. Any and all variable domain amino acid sequences of the humanized light chain comprising the CDRs of 6G4.2.5LV / L1S26A, N35A / L3H98A are collectively referred to herein as 'hu6G4.2.5LV / L1S26A, N35A / L3H98A'. The humanized light chain variable domain amino acid sequences of hu6G4.2.5LV / LlN35X35, hu6G4.2.5LV / L1S26X26, hu6G4.2.5LV / LlS26X26 / 3H98X98, hu6G4.2.5LV / LlS26X26, N35X35, hu6G4.2.5LV / L1N35X35 / L3H98X98, hu6G4.2.5LV / LlS26X26 / L3H98X98, "and hu6G4.2.5LV / LlS26X26, N35X35 / L3H98X98 are collectively referred to herein as' hu6G4.2.5LV / vLl-3X." The variable domain amino acid sequences of Humanized light chain of hu6G4.2.5LV / L1N35A, hu6G4.2.5LV / L1S26A, hu6G4.2.5LV / LlS26A / L3H98A, hu6G4.2.5LV / L1S26A, N35A, hu6G4.2.5LV / LlN35A / L3H98A, hu6G4.2.5LV / L1S26A / L3H98A, hu6G4.2.5LV / L1S26A, N35A / L3H98A are collectively referred to herein as' hu6G4.2.5LV / vLl-3A. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDGs of 6G4.2.5HV are collectively referred to herein as' hu6G4.2.5HV." Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? are collectively referred to herein as 'hu6G4.2.5HV / HlS31Z3?'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A are collectively referred to herein as * hu6G4.2.5HV / H1S31A ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 are collectively referred to herein as' hu6G4.2.5HV / H2S54Z54'A Any and all variable domain amino acid sequences of the humanised heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A are collectively referred to herein as' hu6G4.2.5HV / H2S54A. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3D100E are collectively referred to herein as' hu6G .2.5HV / H3D100E. "Any and all amino acid sequences of the variable domain of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3R102K are collectively referred to herein as 'hu6G4.2.5HV / H3R102K.' Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3D106E are collectively referred to herein as 'hu6G4.2.5HV / H3D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3D100E, R102K are collectively referred to herein as' hu6G4.2.5HV / H3D100E, R102K. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / H3R102K, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3D100E, D106E are collectively referred to herein as' hu6G4.2.5HV / H3D100E, D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H3D100E, R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / H3D100E, R102K, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 are collectively referred to herein as 'hu6G4.2.5HV / HlS31Z3? / H2S54Z54' • Any and all the variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31Z31 / H3D100E are collectively referred to herein as 'hu6G4.2.5HV / H1S31Z31 / H3D100E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31Z31 / H3R102K are collectively referred to herein as 'hu6G4.2.5HV / HlS31Z3? / H3R102K'. and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H3D106E are collectively referred to herein as 'hu6G4.2.5HV / HlS31Z3? / H3D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H3D100E, R102K are collectively referred to herein as 'hu6G4.2.5HV / HlS31Z31 / H3D100E, R102K' Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4, 2.5HV / HlS31Z3? / H3R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / H1S31Z31 / H3R102K, D106E " Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H3D100E, D106E are collectively referred to herein as * hu6G4.2.5HV / HlS31Z3? / H3D100E, D106E "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31Z31 / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / HlS31Z3? / H3D100E, R102K, D106E ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3D100E are collectively referred to herein as' hu6G4.2.5HV / H2S54Z54 / H3D100E. "Any and all variable domain amino acid sequences of the heavy chain humanized comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3R102K are collectively referred to herein as 'hu6G4.2.5HV / H2S54Z54 / H3R102K'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3D106F, are collectively referred to herein as' hu6G4.2.5HV / H2S54Z54 / H3D106E. "Any and all of the variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / H2S54Z54 / H3R102K, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3D100E, D106E are collectively referred to herein as 'hu6G4.2.5HV / H2S54Z54 / H3D100E, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54Z54 / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / H2S54Z54 / H3D100E, R102K , D106E. " Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E are collectively referred to herein as * hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K are collectively referred to herein as' hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K. " Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D106E are collectively referred to herein as' hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D106E Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, R102K are collectively referred to herein as' hu6G4.2.5HV / HlS31Z3? / H2S54Z 4 / H3D100E, R102K ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K, D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, D106E are collectively referred to herein as' hu6G4.2.5 HV / HlS31Z3? / H2S54Z54 / H3D100E, D106E ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / H1S31Z31 / H2S54Z54 / H3D100E, R102K, D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A are collectively referred to herein as' hu6G4.2.5HV / HlS31A / H2S54A ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G .2.5HV / H1S31A / H3D100E are collectively referred to herein as' hu6G4.2.5HV / H1S31A / H3D100E. "Any and all the variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3R102K are collectively referred to herein as hu6G4.2.5HV / HlS31A / H3R102K ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3D106E are collectively referred to herein as' hu6G4.2.5HV / H1S31A / H3D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3D100E, R102K are collectively referred to herein as 'hu6G .2.5HV / H1S31A / H3D100E, R102K'. Any and all variable domain amino acid sequences of the humanised heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / HlS31A / H3R102, D106E.' Any and all variable domain amino acid sequences of the humanised heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3D100E , D106E are collectively referred to herein as 'hu6G4.2.5HV / H1S31A / H3D100E, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / HlS31A / H3D100E, R102K , D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3D100E are collectively referred to herein as' hu6G4.2.5HV / H2S54A / H3D100E." Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3R102K are collectively referred to herein as or 'hu6G4.2.5HV / H2S54A / H3R102K. "Any and all the variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3D106E are collectively referred to herein as 'hu6G4.2.5HV / H2S54A / H3D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / H2S54A / H3R102K, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3D100E, D106E are collectively referred to herein as 'hu6G4.2.5HV / H2S54A / H3D100E, D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H2S54A / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / H2S54A / H3D100E, R102K , D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3D100E are collectively referred to herein as' hu6G4.2.5HV / HlS31A / H2S54A / H3D100E *. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3R102K are collectively referred to herein as' hu6G4.2.5HV / H1S31A / H2S54A / H3R102K. " Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3D106E are collectively referred to herein as 'hu6G4.2.5HV / H1S31A / H2S54A / H3D106E'. Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3D100E, R102K are collectively referred to herein as' hu6G .2.5HV / H1S31A / H2S54A / H3D100E , R102K. " Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / HlS31A / H2S54A / H3R102K , D106E. "Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3D100E, D106E are collectively referred to herein as or hu6G4.2.5HV / HlS31A / H2S54A / H3D100E, D106E ". Any and all variable domain amino acid sequences of the humanized heavy chain comprising the CDRs of 6G4.2.5HV / H1S31A / H2S54A / H3D100E, R102K, D106E are collectively referred to herein as' hu6G4.2.5HV / H1S31A / H2S54A / H3D100E, R102K, D106E. "The amino acid sequences of the humanized heavy chain variable domain of hu6G4.2.5HV / H1S31Z31, hu6G4.2.5HV / H2S54 Z54 hu6G4.2.5HV / H3D100E, hu6G4.2.5HV / H3R102K, hu6G4.2.5HV / H3D106E, hu6G4.2.5HV / H3D100E, RL 02K, hu6G4.2.5HV / H3R102K, D106E, hu6G4.2.5HV / H3D100E, DIO 6E, hu6G4.2.5HV / H3D100E , R102K, D106E, hu6G4.2.5HV / HlS3lZ3? / H2S54Z54, hu6G4.2.5HV / HlS31Z3? / H3D100E, hu6G4.2.5HV / HlS31Z3? / H3R102K, hu6G4.2.5HV / HlS31Z3? / H3D106E, hu6G4.2.5HV / HlS31Z3? / H3D100E, R102K, hu6G4.2.5HV / HlS31Z3? / H3R102K, D106E, hu6G4.2.5HV / HlS31Z31 / H3D100E, D106E, hu6G4.2.5HV / HlS31Z3? / H3D100E, R102K, D106E, hu6G4.2.5HV / H2S54Z54 / H3D100E, hu6G4.2.5HV / H2S54Z54 / H3R102K, hu6G4.2.5HV / H2S54Z5 / H3D106E, hu6G4.2.5HV / H2S54Z54 / H3R102K, D106E, hu6G4.2.5HV / H2S54Z54 / H3D100E, D106E, hu6G4.2.5HV / H2S54Z5 / H3D100E, R102K, D106E, hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D100E, hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3R102K, hu6G4.2.5HV / HlS31Z3? / H2S54Z54 / H3D106E, hu6G4.2.5HV / HlS31Z31 / H2S54Z54 / H3D100E, R102K, hu6G4.2.5HV / HlS31Z3? / H2S5 Z54 / H3R102K, D106E, hu6G4.2.5HV / H1S31Z31 / H2S54Z54 / H3D100E, D106E and hu6G4.2.5HV / H1S31Z31 / H2S54Z54 / H3D100E, R102K, D106E are collectively referred to as in the present as' hu6G4.2 .5HV / VH1-3Z ". The amino acid sequences of the humanized heavy chain variable domain of hu6G4.2.5HV / H1S31A, hu6G4.2.5HV / H2S54A, hu6G4.2.5HV / H3D100E, hu6G4.2.5HV / H3R102K, hu6G4.2.5HV / H3D106E, hu6G4.2.5 HV / H3D100E, Rl 02K, hu6G4.2.5HV / H3R102K, D106E, hu6G4.2.5HV / H3D100E, DIO 6E, hu6G4.2.5HV / H3D100E, R102K, D106E, hu6G4.2.5HV / HlS31A / H2S54A, hu6G4.2.5HV / HlS31A / H3D100E, hu6G4.2.5HV / HlS31A / H3R102K, hu6G4.2.5HV / HlS31A / H3D106E, hu6G4.2.5HV / HlS31A / H3D100E, R102K, hu6G4.2.5HV / HlS31A / H3R102K, D106E, hu6G4.2.5HV / HlS31A / H3D100E, D106E, hu6G4.2.5HV / HlS31A / H3D100E, R102K, D106E, hu6G4.2.5HV / H2S54A / H3D100E, hu6G4.2.5HV / H2S54A / H3R102K, hu6G4.2.5HV / H2S54A / H3D106E, hu6G4.2.5HV / H2S54A / H3R102K, D106E, hu6G4.2.5HV / H2S54A / H3D100E, D106E, hu6G4.2.5HV / H2S54A / H3D100E, R102K, D106E, hu6G4.2.5HV / HlS31A / H2S54A / H3D100E, hu6G4.2.5HV / HlS31A / H2S54A / H3R102K, hu6G4.2 5HV / H1S31A / H2S54A / H3D106E, hu6G4.2.5HV / HlS31A / H2Sb4A / H3D100E, R10K, hu6G4.2.5HV / HlS31A / H2S54A / H3R102K, D106E, hu6G4.2.5HV / HlS31A / H2S54A / H3Dl-00E, D106E, and hu6G4.2.5HV / HlS31 A / H2S54A / H3D1003, R102K, D106E are collectively referred to herein as 'hu6G4.2.5HV / vHl-3A. " The invention provides an antibody or humanized antibody fragment comprising a variable domain of the light chain comprising hu6G4.2.5LV / vLl-3X. In still another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / vLl-3A. In still another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / L1N35X35- In yet another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / L1N35A. In a further embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / LlN35E. The invention further provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / vLl-3X, and further comprising a heavy chain variable domain comprising hu6G4.2.5HV or hu6G4. 2.5HV / vHl-3Z. In yet another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G .2.5LV / vL-3A, and further comprising a heavy chain variable domain comprising hu6G4.2.5 HV or hu6G4.2.5HV / vHl-3Z. In yet another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G .2.5LV / vLl-3A, and further comprising a heavy chain variable domain comprising hu6G4.2.5 HV / vHl-3A. In a further embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / L1N35X35, and further comprising a heavy chain variable domain comprising hu6G4.2. bHV or hu6G4.2.5HV / vHl-3Z. In still another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / N35X35, and further comprising a heavy chain variable domain comprising hu6G4.2.5HV / vHl-3A. In a preferred embodiment, the antibody or antibody fragment comprises a light chain variable domain comprising hu6G4.2.5LV / L1N35X3 and further comprises a humanized heavy chain comprising the amino acid sequence of 6G4.2.5HV11. In a further embodiment, the invention comprises an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / L1N35A, and further comprising a heavy chain variable domain comprising hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z. In another embodiment, the invention provides an antibody or humanized antibody fragment comprising a light chain variable domain comprising hu6G4.2.5LV / N35A, and further comprising a heavy chain variable domain comprising hu6G4.2.5HV / vHl -3A. In yet another embodiment, the antibody or humanized antibody fragment comprises a light chain variable domain comprising hu6G4.2.5LV / L1N35A, and further comprises a heavy chain variable domain comprising hu6G4.2.5HV. In yet another embodiment, the antibody or humanized antibody fragment comprises a light chain variable domain comprising hu6G4.2.5LV / L1N35E, and further comprises a heavy chain variable domain comprising hu6G4.2.5HV. In a preferred embodiment, the antibody or humanized antibody fragment comprises a light chain variable domain comprising hu6G4.2.5LV / L1N35A, and further comprises a humanized heavy chain comprising the amino acid sequence of hu6G4.2.5HV11. In another preferred modality, the antibody or antibody fragment comprises a light chain variable domain comprising hu6G4.2.5LV / L1N35E, and further comprises a humanized heavy chain comprising the amino acid sequence of hu6G4.2.5HV11. The invention encompasses a single chain antibody fragment comprising hu6G4.2.5LV / vLl-3X, with or without any additional amino acid sequence. In one embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5LV / vLl-3X without any amino acid sequence from the variable domain of the associated heavy chain, for example, a single chain species that constitutes the half of a Fv fragment. In still another embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5LV / vLl-3A without any associated heavy chain variable domain amino acid sequence. In still another embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5LV / L1N35X35 without any associated heavy chain variable domain amino acid sequence. In a preferred embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5LV / L1N35A without any associated heavy chain variable domain amino acid sequence. In another preferred embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5LV / L1N35E without any associated heavy chain variable domain amino acid sequence. In one embodiment, the invention provides a single chain antibody fragment wherein hu6G4.2 AL7 / vLl-3X and hu6G4.z.5HV or hu6G4.2.5HV / vHl-3Z are contained in a single chain polypeptide species . In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / vLl-3X linked to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by means of a peptide linker sequence flexible, wherein the heavy chain and light chain variable domains may be associated in a "dimeric" structure analogous to that formed in a double chain Fv species In another embodiment, the single chain fragment is a species comprising hu6G4.2.5LV / vLl-3X linked to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by a linker that is too short to allow intramolecular pairing of the two variable domains, eg, a chain polypeptide monomer simple form a diabody after dimerization with another monomer In another embodiment, the invention provides a single chain antibody fragment wherein hu6G4.2.5LV / vLl-3A and hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z are contained in a species p single chain olipeptide. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / vLl-3A linked to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by means of a peptide linker sequence flexible, where the variable domains of heavy chain and light chain can be associated in a 'dimeric' structure analogous to that formed in a double chain Fv species.

In another embodiment, the single chain antibody fragment is a species comprising hu6G4.2.5LV / vLl-3A linked to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by a linker that is too short to allow the intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer. In another embodiment, the invention provides a single chain antibody fragment wherein hu6G4.2.5LV / vLl-3A and hu6G4.2.5HV / vHl-3A are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / vLl-3A linked to hu6G4.2.5HV / vHl-3A by means of a flexible peptide linker sequence, wherein the variable domains of heavy chain and light chain may be associated in a "dimeric" structure analogous to that formed in a double chain Fv species. In another embodiment, the single chain antibody fragment is a species comprising hu6G4.2.5LV / vLl-3A linked to hu6G4.2.5HV / vHl-3A by a linker that is too short to allow intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer. In still another embodiment, the invention provides a single chain antibody fragment wherein hu6G4.2.5LV / L1N35X35 and hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / LlN35X35 bound to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by means of a flexible peptide linker sequence , wherein the heavy chain and light chain variable domains may be associated in a "dimeric" structure analogous to that formed in a double chain Fv species In another embodiment, the single chain antibody fragment is a species comprising the hu6G4.2.5LV / LlN35X35 bound to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by a linker that is too short to allow intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms one diabody after dimerization with another monomer.

In a further embodiment, the invention provides a single chain antibody fragment wherein hu6G4.2.5LV / L1N35X35 and hu6G4.2.5HV / vHl-3A are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / LlN35X35 linked to hu6G4.2.5HV / vHl-3A by means of a flexible peptide linker sequence, wherein the variable domains of heavy chain and light chain may be associated in a "dimeric" structure analogous to that formed in a double chain Fv species In another embodiment, the single chain fragment of antibody is a species comprising hu6G4.2.5LV / L1N35X35 bound to hu6G .2.5HV / vHl-3A by a linker that is too short to allow intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer. In a further embodiment, the invention provides a single chain antibody fragment wherein the hu6G4.2.5LV / L1N35A and the hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z are contained in a polypeptide species. simple chain. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / LlN35A linked to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by means of a flexible peptide linker sequence, where the heavy chain and light chain variable domains can be associated in a 'dimeric' structure analogous to that formed in a double chain Fv species., the single chain antibody fragment is a species comprising hu6G4.2.5LV / LlN35A bound to hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z by a linker that is too short to allow intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer. Herein also a single chain antibody fragment is provided wherein hu6G4.2.5LV / L1N35E and hu6G4.2.5HV are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / L1N35E linked to hu6G4.2.5HV by means of a flexible peptide linker sequence, wherein the heavy chain variable domains and of light chain may be associated in a "dimeric" structure analogous to that formed in a double-chain Fv species In another embodiment, the single chain antibody fragment is a species comprising hu6G4.2.5LV / L1N35E bound to hu6G4. 2.5HV by a linker that is too short to allow intramolecular pairing of the two variable domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer In a further embodiment, the invention provides a single-chain antibody fragment wherein hu6G4.2.5LV / L1N35A and hu6G4.2.5HV / vHl-3A are contained in a single-chain polypeptide species. d preferred, the single chain antibody fragment is a scFv species comprising hu6G4.2.5LV / HN35A linked to hu6G4.2.5HV / vHl-3A by means of a flexible peptide linker sequence, wherein the chain variable domains heavy and light chain may be associated in a 'dimeric' structure analogous to that formed in a double chain Fv species. In another embodiment, the single chain antibody fragment is a species comprising hu6G4.2.5LV / L1N35A bound to hu6G .2.5HV / vHl-3A by a linker that is too short to allow intramolecular pairing of the two variable domains , for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer. In still another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / vLl-3X and a second polypeptide chain comprises hu6G4.2.5HV or hu6G4.2.5 HV / v Hl-3Z and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In still another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G .2.5LV / vLl-3X and a second polypeptide chain comprises hu6G4.2.5HV / vHl-3A and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises huG4.2.5LV / vLl-3X and a second polypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a further embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprising hu6G4.2.5LV / vLl-3A and a second polypeptide chain comprising hu6G4.2.5HV or hu6G4 .2.5HV / vHl-3Z The two peptide chains are covalently linked by one or more interchain disulfide bonds. In still another embodiment, the present invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / vLl-3A and a second polypeptide chain comprises ei hu6G4. .5HV / vHl-3A and the two polypeptide chains are covalently linked by one or more interchain disulfide bonds. In a preferred embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / vLl-3A and a second polypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptide chains are covalently linked by one or more interchain disulfide bonds or bridges. The invention also encompasses an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35X35 and a second polypeptide chain comprises hu6G4.2.5HV or hu6G4.2.5HV / vHl- 3Z and the two polypeptide chains are covalently linked by one or more interchain disulfide bonds. In still another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35X35 and a second polypeptide chain comprises hu6G4.2.5HV / vHl-3A and the Two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35X35 and a second polypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 and the Two polypeptide chains are covalently linked by one or more interchain disulfide bridges The invention further encompasses an antibody fragment comprising a plurality of polypeptide chains, wherein one polypeptide chain comprises hu6G4.2.5LV / L1N35A and a second polypeptide chain comprises hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges The invention also encompasses an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35E and a second cade A polypeptide comprises hu6G4.2.5HV and the two polypeptide chains are covalently linked by one or more outer chain bridges. In yet another embodiment, the invention provides an antibody fragment that provides a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35A and a second polypeptide chain comprises hu6G4.2.5HV / vHl-3A and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35A and a second polypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In another preferred embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises hu6G4.2.5LV / L1N35E and a second polypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a further embodiment, any of the aforementioned two-chain antibody fragments are selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, and F (ab') 2. In another preferred embodiment, the antibody fragment is selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, and F (ab') 2, wherein the antibody fragment comprises a polypeptide chain comprising hu6G4.2.5LV / L1N35X35 and a second polypeptide chain comprises hu6G4.2.5HV. In another preferred embodiment, the antibody fragment is selected from the group consisting of Fab, Fab ', Fab'-SH, Fv and (F (ab') 2, wherein the antibody fragment comprises a polypeptide chain comprising hu6G4 .2.5LV / L1N35A and a second polypeptide chain comprises hu6G4.2.5HV In a further preferred embodiment, the antibody fragment is selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, and (F ( ab ') 2, wherein the antibody fragment comprises a polypeptide chain comprising hu6G4.2.5LV / L1N35E and a second polypeptide chain comprises 6G4.2.5HV In another, preferred embodiment, the antibody fragment is an F (ab ') 2 comprising a polypeptide chain comprising hu6G4.2.5LV / L1N35A and a second polypeptide chain comprising the amino acid sequence of 6G4.2.5HV11 In a further preferred embodiment, the antibody fragment is an F ( ab ') 2 comprising a polypeptide chain comprising the h u6G4.2.5LV / L1N35E and a second polypeptide chain comprising the amino acid sequence of 6G4.2.5HV11. The invention also provides an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / vLl-3X and optionally further comprising a heavy chain variable domain containing hu6G4.2.5HV or hu6G4 .2.5HV / vHl-3Z, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al.The invention further provides an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / vLl-3X and optionally further comprising a heavy chain variable domain containing the hu6G4.2.5HV / vLl- 3A, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain and / or light chain sequences, to form heavy and / or light chains of full length or partial length chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention further provides an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35X35 and optionally further comprising a heavy chain variable domain containing hu6G4.2.5HV or hu6G4.2.5 HV / v Hl-3Z, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention further provides an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35X35 and optionally further comprising a heavy chain variable domain containing hu6G .2.5HV / vHl-3A, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention also encompasses an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35A and optionally further comprising a heavy chain variable domain containing hu6G4.2.5HV or hu6G4.2.5 HV / v Hl-3Z, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. the invention further provides an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35A and optionally further comprising a heavy chain variable domain containing hu6G4.2.5HV / vHl-3A, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention further encompasses an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35A and optionally further comprising a heavy chain variable domain containing the amino acid sequence of 6G4.2.5HV11, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of which? Isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. . Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kafcat et al. The invention further encompasses an antibody or antibody fragment comprising a light chain variable domain containing hu6G4.2.5LV / L1N35E and optionally further comprising a heavy chain containing the amino acid sequence of 6G4.2.5HV11, wherein the light chain variable domain, and optionally the heavy chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain sequence (s) and / or the light chain to form heavy and / or light chain full or partial length chains or chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from animal or human species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. In another preferred embodiment, the antibody or antibody fragment comprises a light chain variable domain containing hu6G4.2.5LV / vLl-3X, and further comprising hu6G4.2.5HV or hu6G4.2.5HV / vHl-3Z in a heavy chain that is fused to or containing a leucine zipper sequence. The leucine zipper can increase the affinity or production efficiency of the antibody or antibody fragment of interest. Suitable leucine zipper sequences include the leucine jun and fos zippers shown by Kostelney et al., J. Immunol. , 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the examples below.

In particular, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequences of the light chain polypeptide of 6G4.2.5vll anti-IL-8 , humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than Asn (denoted as' X35 ') is replaced by Asn at the position of amino acid 35 (referred to herein as' 6G4.2.5LV11N35X35"). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the 6G4.2.5vll anti-IL light chain polypeptide. -8, humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than Ser (denoted as' X2ß ") is replaced by Ser at the position of amino acid 26 (referred to herein) as' 6G4.2.5LV11S26X26"). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the 6G4.2.5vll anti-IL light chain polypeptide. -8, humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than His (denoted as 'X98') is replaced by His at the position of amino acid 98 (referred to herein) as' 6G4.2.5LV11H98X98"). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the 6G4.2.5vll anti-IL light chain polypeptide. -8, humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than Ser (denoted as 'X2d') is replaced by Ser at the position of amino acid 26 and any different amino acid by Asn (denoted as X35") is substituted for Asn at the position of amino acid 35 (hereinafter referred to as' 6G4.2.5LV11S26X2 / N35X35"). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the humanized 6G4.2.5vll anti-IL-8 light chain polypeptide, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than Asn (denoted as * X3s ") is replaced by Asn at the position of amino acid 35 and any amino acid other than His (denoted as 'X98') is replaced by His at the position of amino acid 98 (referred to herein as' 6G4.2.5LV11N35X35 / H98X98"). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the 6G4.2.5vll anti-IL light chain polypeptide. -8, humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that any other amino acid other than Ser (denoted as 'X26') is replaced by Ser at the position of amino acid 26 and any amino acid other than His (denoted as 'X98') is replaced by His at the position of amino acid 98 (hereinafter referred to as '6G4.2.5LVllS26X26 / H98X9e'). The invention also encompasses an antibody or antibody fragment comprising a light chain that comprises the amino acid sequence of amino acids 1-219 of the amino acid sequence of the humanized anti-IL-8 6G4.2.5vll light chain polypeptide, variant of Figure 31B (SEQ ID No. 65) with the condition that any amino acid other than Ser (denoted as 'X26') is replaced by Ser at the amino acid position 26, any amino acid other than Asn (denoted as * X35") is replaced by Asn at the position of amino acid 35 and any amino acid other than His (denoted as 'X98') is replaced by His at the position of amino acid 98 (referred to as present as' 6G4.2.5LV11S26X26 / N35X35 / H98X98"). In addition, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the polypeptide amino acid sequence of the light chain of humanized anti-IL-8 6G4.2.5vll, variant (SEQ ID No. 71) of Figure 36 (referred to herein as '6G4.2.5LV11N35A'). There is further provided herein an antibody or antibody fragment comprising a light chain comprising amino acid sequence 1-219 of the amino acid sequence of the humanized anti-IL-8 6G4.2.5vll light chain polypeptide, variant (SEQ ID No. 71) of Figure 45 (hereinafter referred to as '6G4.2.5LV11N35E'). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the humanized anti-IL-8 6G4.2.5vll light chain polypeptide, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is replaced by Be at the position of amino acid 26 (hereinafter referred to as * 6G4.2.5LV11S26A "). In yet another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the light chain polypeptide of 6G4.2.bvll anti- Humanized 1L-8, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is replaced by His at the position of amino acid 98 (hereinafter referred to as' 6G4.2.5LV11H98A "). further embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the light chain polypeptide of 6G4.2.5vll anti-IL-8 humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is substituted for Ser at the position of amino acid 26 and Ala is substituted for Asn at the position of amino acid 35 (hereinafter nominated as' 6G4.2.5LV11S26A / N35A "). In still another embodiment, the invention provides an antibody or antibody fragment comprising a light chain comprising the amino acid sequence ie amino acids 1-219 of the amino acid sequence of the 6G4.2.5vll anti-IL light chain polypeptide. -8 humanized, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is replaced by Ser at the position of amino acid 26 and Ala is substituted by His at the position of amino acid 98 (hereinafter referred to as as' 6G4.2.5LV11S26A / H98A ") The invention also encompasses an antibody or antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the light chain polypeptide of Humanized anti-IL-8 6G4.2.5vll, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is substituted for Asn at the position of amino acid 35 and Ala is substituted for His in the position n amino acid 98 (hereinafter referred to as' 6G4.2.5LV11S26A / H98A "). The invention further encompasses an antibody an antibody fragment comprising a light chain comprising the amino acid sequence of amino acids 1-219 of the amino acid sequence of the humanized anti-IL-8 6G4.2.5vll light chain polypeptide, variant of Figure 31B (SEQ ID No. 65) with the proviso that Ala is substituted for Ser at the position of amino acid 26, Ala is substituted for Asn at the position of amino acid 35, and Ala is substituted for His at the position of amino acid 98 (hereinafter referred to as '6G4.2.5LV11S26A / N35A / H98A'). The invention provides a single chain antibody fragment comprising a variant light chain selected from the group consisting of 6G4.2.5LV11N35X35, 6G4.2.5LV11S26X26 6G4.2.5LV11H98X98, 6G4.2.5LV11S26X26 / N35X35, 6G4.2.5LV11N35X35 / H98X98, 6G4.2.5LV11S26X26 / H98X98, and 6G4.2.5LV11S26X26 / N35X35 / H98X98, with or without any additional amino acid sequence. It will be understood that the group consisting of * 6G4.2.5LV11N35X35, 6G4.2.5LV11S26X26, 6G4.2.5LV11H98X98, 6G4.2.5LV11S26X26 / N35X35 / 6G4.2.5LV11N35X35 / H98X98, 6G4.2.5LV11S26X26 / H98X98, and 6G4.2.5LV11S26X26 / N35X35 / H98X98, is collectively referred to herein as the 'group of variants of 6G4.2.5LV11X', and that the individual members of this group are generically referred to herein as a 'variant of 6G4.2.5 LV11X ". In one embodiment, the invention provides a single chain antibody fragment comprising a variant of 6G4.2.5LV11X without any associated heavy chain amino acid sequence, for example a single chain species that constitutes half of a Fab fragment. In a preferred embodiment, the invention provides a variant of 6G4.2.5LV11N35X35 without any associated heavy chain amino acid sequence. The invention encompasses a single chain antibody fragment comprising a variant light chain selected from the group consisting of 6G4.2.5LV11N35A, 6G4.2.5LV11S26A, 6G4.2.5LV11H98A, 6G4.2.5LV11S26A / N35A, 6G4.2.5LV11N35A / H98A 6G4.2.5LV11S26A / H98A, and 6G4.2.5LV11S26A / N35A / H98A, with or without any additional amino acid sequence. It will be understood that the group consisting of 6G4.2.5LV11N35A, 6G4.2.5LV11S26A, 6G4.2.5LV11 H98A, 6G4.2.5LV11S26A / N35A, 6G4.2.5LV11N35A / H98A, 6G4.2.5LV11S26A / H98A, and 6G4.2.5LV11S26A / N35A / H98A is collectively referred to herein as the 'group of variants of 6G4.2.5LV11A', and that the individual members of this group are generically referred to herein as a 'variant of 6G4.2.5LV11A'. In one embodiment, the invention provides a single chain antibody fragment comprising a variant of 6G4.2.5LV11A without any associated heavy chain amino acid sequence, for example, a single chain species that constitutes half of a Fab fragment. In a preferred embodiment, the invention provides 6G4.2.5LV11N35A without any associated heavy chain amino acid sequence. There is further provided herein an antibody or antibody fragment comprising a light chain comprising a variant of 6G4.2.5LV11X, and further comprising a heavy chain comprising a variant of 6G4.2.5LV11. In a preferred embodiment, the invention provides an antibody or antibody fragment comprising a variant of 6G4.2.5LV11N35X35 and further comprising 6G4.2.5LV11. In a preferred embodiment, the invention provides an antibody or antibody fragment comprising 6G4.2.5LV11N35A and further comprising 6G4.2.5HV11. In another preferred embodiment, the invention provides an antibody or antibody fragment comprising 6G4.2.5LV11N35E and further comprising 6G4.2.5HV11. In a further embodiment, the invention provides a single chain antibody fragment wherein a variant of 6G4.2.5LV11X and 6G4.2.5HV11 are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment comprises a variant of 6G4.2.5LV11X linked to 6G4.2.5HV11 by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains can be associated in a 'dimeric' structure analogous to that formed in a two-chain Fab species In yet another embodiment, the single-chain antibody fragment is a species comprising a variant of 6G4.2.5LV11X bound to 6G4.2.5HV11 by a linker which is too short to allow intramolecular pairing of the complementary domains, for example a single chain polypeptide monomer forming a diabody after dimerization with another monomer In yet another embodiment, the invention provides a single chain antibody fragment in where a variant of 6G4.2.5LV11N35X35 and 6G4.2.5HV11 are contained in a single chain polypeptide species., the single chain antibody fragment comprises a variant of 6G .2.5LV11N35X35 bound to 6G4.2.5HV11 by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains may associate in a similar "dimeric" structure to that formed in a two-chain Fab species In yet another embodiment, the single-chain antibody fragment is a species comprising a variant of 6G4.2.5LV11N35X35 bound to 6G4.2.5HV11 by a linker that is too short to allow the intramolecular pairing of the complementary domains, for example, a single chain polypeptide monomer that forms a diabody after dimerization with another monomer.

In a further embodiment, the invention provides a single chain antibody fragment wherein 6G4.2.5LV11N35A and 6G4.2.5HV11 are contained in a single chain polypeptide species. In a preferred embodiment, the single chain antibody fragment comprises 6G4.2.5LV11N35A linked to 6G4.2.5HV11 by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains can be associated in a structure. dimer, "analogous to that formed in a double-stranded Fab species." In yet another embodiment, the single-stranded antibody fragment is a species comprising 6G4.2.5LV11N35A bound to 6G4.2.5HV11 by a linker that is too short to allow intramolecular pairing of the complementary domains, for example, a single chain polypeptide monomer forming a diabody after dimerization with another monomer In a further embodiment, the invention provides a single chain antibody fragment wherein the 6G4 .2.5LV11N35E and 6G4.2.5HV11 are contained in a single chain polypeptide species In a preferred embodiment, the antibody fragment of simple adena comprises 6G4.2.5LV11N35E bound to 6G4.2.5HV11 by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains can associate in a 'dimeric' structure, analogous to that formed in a species Double chain fab. In yet another embodiment, the single-stranded antibody fragment is a species comprising 6G4.2.5LV11N35AE bound to 6G4.2.5HV11 by a linker that is too short to allow intramolecular pairing of complementary domains, eg, a monomer single chain polypeptide forming a diabody after dimerization with another monomer. In still another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein one polypeptide chain comprises a variant of 6G4.2.5LV11X and a second polypeptide chain comprises 6G4.2.5HV11 and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In yet another embodiment, the invention provides an antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises a variant of 6G4.2.5LV11N35X35 and a second polypeptide chain comprising 6G4.2.5HV11 and the two polypeptide chains they are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, any of the above two chain antibody fragments is selected from the group consisting of Fab, Fab ', Fab'-SH, and F (ab') 2. In another preferred embodiment, the two-chain antibody fragment is an F (ab ') 2 wherein one polypeptide chain comprises 6G4.2.5LV11N35A and the second polypeptide chain comprises 6G4.2.5HV11. In a further preferred embodiment, the antibody fragment is a Fab, Fab ', Fab "-SH, or F (ab') 2 wherein one polypeptide chain comprises 6G4.2.5LV11N35E and the second polypeptide chain comprises 6G4.2.5 HV11 A particularly preferred embodiment, the antibody fragment is the leucine zipper species GCN4 of F (ab ') 2 of 6G4V11N35A described in the examples below In another particularly preferred embodiment, the antibody fragment is the leucine GCN4 of F (ab ') 2 of 6G4V11N35E, described in the examples below In another particularly preferred embodiment, the antibody fragment is the Fab of 6G4V11N35E described in the examples below The invention also provides an antibody or fragment of antibody comprising a light chain containing a variant of 6G4.2.5LV11X and optionally further comprising a heavy chain containing 6G4.2.5HV11, wherein the light chain, and optionally the heavy adena, is or are fused to an additional portion, such as an additional immunoglobulin constant domain sequence. The constant domain sequence can be added to the heavy chain and / or light chain sequences, to form heavy or light chain or full length or partial chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al.

The invention also provides an antibody or antibody fragment comprising a light chain containing a variant of 6G4.2.5LVN35X35 and optionally further comprising a heavy chain containing 6G4.2.5HV11, wherein the light chain, and optionally the chain heavy, is or are fused to an additional portion, such as an additional immunoglobulin constant domain sequence. The constant domain sequence can be added to the heavy chain and / or light chain sequences, to form heavy or light chain or full length or partial chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, '. Constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention also provides an antibody or antibody fragment comprising a light chain containing a variant of 6G4.2.5LV11N35A and optionally further comprising a heavy chain containing 6G4.2.5HV11, wherein the light chain, and optionally the chain heavy, is or are fused to an additional portion, such as an additional immunoglobulin constant domain sequence. The constant domain sequence can be added to the heavy chain and / or light chain sequences, to form heavy or light chain or full length or partial chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. The invention also provides an antibody or antibody fragment comprising a light chain containing a variant of 6G4.2.5LV11N35E and optionally further comprising a heavy chain containing 6G4.2.5HV11, wherein the light chain, and optionally the chain heavy, is or are fused to an additional portion, such as an additional immunoglobulin constant domain sequence. The constant domain sequence can be added to the heavy chain and / or light chain sequences, to form heavy or light chain or full length or partial chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. In a preferred embodiment, the antibody or antibody fragment comprises a light chain containing a variant ae 6G4. . bLVilX, and further comprising 6G4.2.5HV11 in a heavy chain that is fused to or containing a leucine zipper sequence. The leucine zipper can increase the affinity or production efficiency of the antibody or antibody fragment of interest. Suitable leucine zipper sequences include the leucine jun and fos zippers shown by Kostelney et al., J. Immunol., 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the following Examples. In another preferred embodiment, the antibody or antibody fragment comprises a light chain containing 6G4.2.5LV11N35A, and further comprising a heavy chain containing 6G4.2.5HV11 fused to the leucine zipper GCN4. In another further preferred embodiment, the antibody or antibody fragment comprises a light chain containing 6G4.2.5LV11N35E, and further comprising a heavy chain containing 6G4.2.5HV11 fused to the leucine zipper GCN4.

B. VARIANTS OF 6G4.2.5HV The invention provides antibodies and humanized antibody fragments comprising the CDRs of a CDR variant of 6G4.2.5HV. The use of a variant of 6G4.2.5HV CDRs in the antibodies or humanized antibody fragments of the invention confers the advantages of higher affinity for IL-8 and / or the improved recombinant manufacturing economy.

A heavy chain variable domain comprising the CDRs of a variant of the CDGs of 6G4.2.5HV can be humanized in conjunction with a light chain comprising the CDRs of 6G4.2.5LV or the CDRs of a variant of the CDRs of 6G4 .2.5LV, essentially as described in Section (II) (2) (A) above. In one embodiment, the invention provides an antibody or humanized antibody fragment, comprising a variant of 6G4.2.5HV CDRs selected from the group consisting of 6G4.2.5HV / HlS31Z3 ?, 6G4.2.5HV / H2S54 Z54, and 6G4.2.5HV / HlS31Z3? / H2S54Z54- In addition, the invention provides an antibody or fragment of humanized antibody, comprising a variant of CDRs of 6G4.2.5HV selected from the group consisting of 6G4.2.5HV / H1S31A, 6G4.2.5 HV / H2S54A, and 6G4.2.5HV / H1S31A / H2S54A. In particular, CDR variants of 6G4.2.5HV can be used to construct a humanized antibody, or antibody comprising hu6G4.2.5HV / vHl-3Z as described in Section (II) (2) (A) above . The invention further provides an antibody or humanized antibody fragment, comprising a heavy chain variable domain which comprises hu6G4.2.5HV / vHl-3Z, and further comprising a light chain variable domain comprising hu6G4.2.5LV or hu6G4.2.5LV / vLl-3X. The invention further encompasses a humanized single-chain antibody fragment, comprising hu6G4.2.5HV / vHl-3Z, with or without any additional amino acid sequence. In one embodiment, the invention provides a single chain antibody fragment comprising hu6G4.2.5HV / vHl-3Z without any heavy chain variable domain amino acid sequence, associated, for example, with a single chain species that constitutes the half of a fragment of Fv. In one embodiment, the invention provides a single chain humanized antibody fragment, wherein hu6G4.2.5HV / vHl-3Z and hu6G4.2.5LV or hu6G4.2.5LV / vLl-3X are contained in a polypeptide species of simple chain. In a preferred embodiment, the single caaena antibody fragment is a scFv species comprising hu6G4.2.5HV / vHl-3Z linked to hu6G4.2.5LV or hu6G4.2.5LV / vLl-3X via a linker sequence peptide, flexible, wherein the heavy chain and light chain variable domains can be associated in a 'dimeric' structure analogous to that formed in a two chain Fv sp. In yet another embodiment, the single chain antibody fragment is a a species comprising hu6G4.2.5HV / vHl-3Z linked to hu6G4.2.5LV or hu6G4.2.5LV / vLl-3X by a linker that is too short to allow intramolecular pairing of the two variable domains, for example a monomer single chain polypeptide forming a diabody after dimerization with another monomer In yet another embodiment, the invention provides a humanized antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain is mprende hu6G4.2.5HV / vHl-3Z and a second polypeptide chain comprises hu6G4.2.5LV or hu6G4.2.5LV / vLl-3X and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, the aforementioned two-chain antibody fragment is selected from the group consisting of Fab, Fab ', Fab'-SH, Fv and F (ab') 2. The invention also provides an antibody or polymerized antibody fragment comprising a variable domain that heavy chain containing the hu6G4.2.5HV / vHl-3Z and optionally further comprising a light chain variable domain containing the hu6G4.2.5LV or the hu6G4.2.5LV / vLl-3X, wherein the heavy chain variable domain, and optionally the light chain variable domain, is or are fused to an additional portion, such as an immunoglobulin constant domain. The constant domain sequence can be added to the heavy chain and / or light chain sequence (s) to form heavy or light chain or partial or full length chains. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD, and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human constant domain sequences can be obtained from Kabat et al. In a preterm embodiment, the antibody or humanized antibody fragment comprises hu6G4.2.5HV / vHl-3Z in a heavy chain that is fused to or containing a leucine zipper sequence. The leucine zipper can increase the affinity or production efficiency of the antibody or antibody fragment of interest. Suitable leucine zipper sequences include the leucine jun and fos zippers shown by Kostelney et al., J. Immunol., 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the following Examples. In addition, the invention provides an antibody or humanized antibody fragment, comprising a heavy chain comprising the amino acid sequence of amino acids 1-230 of the polypeptide amino acid sequence of hu6G4.2.5HVll of Figures 37A-37B (SEQ ID No. 75) with the proviso that Ala is substituted for Ser at the position of amino acid 31 (hereinafter referred to as' 6G4.2.5HV11S31A "). In yet another embodiment, the invention provides an antibody or humanized antibody fragment. comprising a heavy chain comprising the amino acid sequence of amino acids 1-230 of the polypeptide amino acid sequence of 6G4.2.5HV11 of Figures 37A-37B (SEQ ID No. 75) with the proviso that Ala is replaced by Ser at the amino acid position 54 (hereinafter referred to as '6G4.2.5HV11S54A ").

In still another embodiment, the invention provides an antibody or humanized antibody fragment comprising a heavy chain comprising the amino acid sequence of amino acids 1-230 of the polypeptide amino acid sequence of 6G4.2.5HV11 of Figures 37A-37B (SEQ ID No. 75) with the proviso that Ala is substituted for Ser at the position of amino acid 31 and Ala is substituted for Ser at the position of amino acid 54 (hereinafter referred to as' 6G4.2.5HV11S31A / S54A "). A humanized antibody or humanized antibody fragment comprising any of the chain combinations is further provided herein. light and heavy listed in Tables 1 and 2 below.

Table 1 Heavy Chain Light Chain 6G4.2.5HV11S31A 6G4.2.5LV11 6G4.2.5HV11S31A 6G4.2.5LV11N35A 6G4.2.5HV11S31A 6G .2.5LV11S26A 6G4.2.5HV11S31A 6G4.2.5LV11H98A 6G4.2.5HV11S31A 6G4.2.5LV11S26A / N35A 6G4.2.5HV11S31A 6G4.2.5LV11S26A / H98A 6G4.2.5HV11S31A 6G4.2.5LV11N35A / H98A 6G4.2.5HV11S31A 6G4.2.5LV11S26A / N35A / H98A 6G4.2.5HV11S54A 6G4.2.5LV11 6G4.2.5HV11S54A 6G4.2.5LV11N35A 6G4.2.5HV11S54A 6G4.2.5LV11S26A 6G4. 2.5HV11S54A 6G4.2.5LV11H98A Table 2 Heavy Chain Light Chain 6G4.2.5HV11S54A 6G4 2.5LV11S26A / N35A 6G4.2.5FV11S54A 6G4 2.5LV11S26A / H98A 6G4.2.5HV11S54A 6G4 2.5LV11N35A / H98A 6G4.2.5HV11S54A 6G4 2.5LV11S26A / N35A / H98A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11 6G4.2.5HV11S31A / S54A 6G4 2.5LV11N35A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11S26A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11H98A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11S26A / N35A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11S26A / H98A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11N35A / H98A 6G4.2.5HV11S31A / S54A 6G4 2.5LV11S26A / N35A / H98A 6G4.2.5HV11S31A 6G4 2.5LV11 6G4.2.5HV11S31A 6G4 2. 5LV11N35X35 6G4.2.5HV11S31A 6G4.2.5LV11S26X26 6G4.2.5HV11S31A 6G4.2 .5LV11H98X98 6G4.2.5HV11S31A 6G4.2 .5LV11S26X26 / N35X35 6G4.2.5HV11S31A 6G4.2 .5LV11S26X26 / H98X98 6G4.2.5HV11S31A 6G4.2 .5LV11N35X35 / H98X98 6G4.2.5HV11S31A 6G4.2. 5LV11S26X26 / N35X35 / H98X98 6G4.2.5HV11S54A 6G4.2 .5LV11 6G4.2.5HV11S54A 6G4.2 .5LV11N35X35 6G4.2.5HV11S54A 6G4.2 • 5LV11S26X26 6G4.2.5HV11S54A 6G4.2 • 5LV11H98X98 6G4.2.5HV11S54A 6G4.2 .5LV11S26X26 / N35X35 6G4.2.5 HV11S54A 6G4.2 .5LV11N35X35 / H98X98 6G4.2.5HV11S54A 6G4.2. 5LV11S26X2 / N35X35 / H98X98 6G4.2.5HV11S31A / S54A 6G4.2 .5LV11 6G4.2.5HV11S31A / S54A 6G .2 .5LV11N35X35 6G4.2.5HV11S31A / S54A 6G4.2 • 5LV11S26X26 6G4.2.5HV11S31A / S54A 6G4.2 .5LV11H98X98 6G4.2.5HV11S31A / S54A 6G4.2 .5LV11S26X26 / N35X3? 6G4.2.5HV11S31 A / S54A 6G4.2 .5LV11S26X26 / N9 &X98 6G4.2.5HV11S31A / S54A 6G4.2 .5LV11N35X35 / H98X98 6G4.2.5HV11S31A / S54A 6G4.2. 5LV11S26X26 / N35X35 / H98X9B The invention encompasses a single chain humanized antibody fragment comprising a variant heavy chain selected from the group consisting of 6G4.2.5HV11S31A, 6G4.2.5HV11S54A, and 6G4.2.5HV11S31A / S54A, with or without any additional amino acid sequence. It will be understood that the group consisting of 6G4.2.5HV11S31A, 6G4.2.5HV11S54A, and 6G4.2.5HV11S31A / S54A are collectively referred to herein as the 'group of variants of 6G4.2.5HV11A', and that individual members of this group are generally referred to herein as a 'variant of 6G4.2.5HV11A'. In one embodiment, the invention provides a single chain humanized antibody fragment comprising a variant of 6G4.2.5HV11A without any associated light chain amino acid sequence, for example, a single chain species that constitutes half of a Fab fragment. . There is further provided herein an antibody or humanized antibody fragment comprising a heavy chain comprising a variant of 6G .2. ÜHVIIA, and further comprising a light chain comprising a variant of 6G4.2.5LV11A or a variant of 6G4.2.5LV11X. In another modality more, the antibody or humanized antibody fragment comprises any combination of light and heavy chains illustrated in Tables 1 and 2 above. In one embodiment, the invention provides an antibody or humanized antibody fragment comprising a variant of 6G4.2.5LV11A and further comprising 6G4.2.5LV11N35X35. In a preferred embodiment, the invention provides an antibody or humanized antibody fragment comprising a variant of 6G4.2.5HV11A and further comprising 6G4.2.5HV11N35A. In yet another embodiment, the invention provides a single chain humanized antibody fragment wherein a variant of 6G4.2.5HV11A and 6G4.2.5LV11 are contained in a single chain polypeptide species. In yet another embodiment, the invention provides a single chain humanized antibody fragment wherein any pair of light and heavy chains listed in Tables 1 and 2 above is contained in a single chain polypeptide species. In yet another embodiment, the invention provides a fragment of humanized chain-like antibody simpie wherein a variant of 6G4.2.5HV11A and a variant of 6G4.2.5LV11X are contained in a single chain polypeptide species. In still another embodiment, the invention provides a single chain humanized antibody fragment wherein a variant of 6G4.2.5HV11A and a variant of 6G4.2.5LV11N35X35 are contained in a single chain polypeptide species. In a further embodiment, the invention provides a single chain humanized antibody fragment wherein a variant of 6G4.2.5HV11A and the variant of 6G4.2.5LV11N35A are contained in a single chain polypeptide species. In a preferred embodiment, the single chain humanized antibody fragment comprises a variant of 6G4.2.5HV11A bound to a variant of 6G4.2.5LV11X, a variant of 6G .2.5LV11N35X35, a variant of 6G4.2.5LV11N35A, or 6G4. 2.5LV11 by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains can associate in a 'dimeric' structure analogous to that formed in a two-chain Fab species In a further embodiment, the antibody fragment Humanized single chain is a species comprising a variant of 6G4.2.5HV11A bound to a variant of 6G4.2.5LV11X, a variant of 6G4.2.5LV11N35X35, a variant of 6G4.2.5LV11N35A or 6G4.2.5LV11 by a linker that it is too short to allow intramolecular pairing of complementary domains, for example, a single chain polypeptide monomer forming a diabody after dimerization with another monomer. The single chain humanized antibody fragment comprises any pair of light and heavy chains listed in Tables 1 and 2 above, linked by means of a flexible peptide linker sequence, wherein the heavy chain and light chain domains may be associated in a 'dimeric' structure analogous to that formed in a Fab species of two chains. In a further embodiment, the single chain humanized antibody fragment comprises any pair of light and heavy chains listed in the above Tables 1 and 2 linked by a linker that is too short to allow intramolecular pairing of the complementary domains, for example a single chain polypeptide monomer forming a diabody after dimerization with another monomer. In still another embodiment, the invention provides a humanized antibody fragment comprising a plurality of polypeptide chains, wherein a polypeptide chain comprises a variant of 6G4.2.5HV11A and a second polypeptide chain comprises a variant of 6G4.2.5LV11X, a variant of 6G4.2.5LV11N35X35, a variant of 6G4.2.5LV11N35A, or 6G4.2.5LV11, and the two polypeptide chains are covalently linked by one or more interchain disulfide bridges. In a preferred embodiment, the above-mentioned two-chain antibody fragment, is selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab') 2. In a further embodiment, the invention provides a humanized two-chain antibody fragment comprising any pair of light and heavy chains, listed in Tables 1 and 2 above, wherein each chain is contained in a separate molecule. In yet another embodiment, the two-chain antibody fragment comprises any pair of light and heavy chains listed in Tables 1 and 2 above, which are selected from the group consisting of Fab, Fab ', Fab'-SH and F (ab ')2. In a preferred embodiment, the humanized two-chain antibody fragment is an F (ab ') 2 comprising any pair of heavy and light chains listed in Tables 1 and 2 above. In another preferred embodiment, the humanized two-chain antibody fragment is an F (ab ') 2 wherein one polypeptide chain comprises a variant of 6G4.2.5HV11A and the second polypeptide chain comprises 6G4.2.5LV11N35A. The invention also provides an antibody or humanized antibody fragment comprising a heavy chain containing a variant of 6G4.2.5HV11A and optionally further comprising a light chain containing a variant of 6G4.2.5LV11X, a variant of 6G4.2.5LV11N35X35 , a variant of 6G4.2.5LV11N35A, or 6G4.2.5HV11, wherein the heavy chain, and optionally the light chain, is or are fused to an additional portion, such as the additional immunoglobulin constant domain sequence. The constant domain sequence can be added to the heavy chain and / or light chain sequence (s) to form heavy or light chain or full length or partial chain species. It will be appreciated that the constant regions of any isotype can be used for this purpose, including the constant regions of IgG, IgM, IgA, IgD and IgE, and that such constant regions can be obtained from any human or animal species. Preferably, the constant domain sequence is of human origin. Suitable human domain sequences can be obtained from Kabat et al. (Supra). In a preferred embodiment, the antibody or humanized antibody fragment comprises a variant of 6G4.2.5HV11A in a heavy chain that is fused to or contains a leucine zipper sequence. The leucine zipper can increase the affinity or production efficiency of the antibody or antibody fragment of interest. Suitable leucine zipper sequences include the leucine jun and fos zippers shown by Kostelney et al., J. Immunol, 148: 1547-1553 (1992) and the leucine zipper GCN4 described in the Examples below.

Bispecific Antibodies Bispecific antibodies are monoclonal antibodies, preferably numan or humanized, which have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for IL-8, and the other is for any other antigen. For example, bispecific antibodies that bind specifically to an IL-8 and a neurotrophic factor, or two different types of IL-8 polypeptides, are within the scope of the present invention. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two heavy chain-immunoglobulin light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305: 537 (1983)). Due to the randomization of heavy and light immunoglobulin chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually carried out by means of afinidase chromatography steps, is rather problematic, and the yields of the product are low. Similar procedures are described in International Patent WO 93/08829 published May 13, 1993, and in Traunecker et al., EMBO J. 10: 3655 (1991).

According to a different and more preferred method, the variable domains of the antibody with the desired binding specificities (combining sites to the antibody-antigen) are fused to the constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge regions CH2 and CH3. It is preferred to have the first heavy chain constant region (CH1), containing the necessary site for light chain linkage, present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fractions in the modalities when unequal proportions of the three polypeptide chains used in the construction provide the maximum yields. However, it is possible to insert the coding sequences for two or three polypeptide chains into an expression vector, when the production of at least two polypeptide chains in equal proportions results in high yields or when the proportions are not of particular significance. In a preferred embodiment of this method, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a pair of heavy chains-immunoglobulin light chains, hybrid (which provides a second specificity of link) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from the desired nc immunoglobulin chain combinations, such as the presence of an immunoglobulin light chain in only one half of the bispecific molecule, which provides an easy way of separation. For further details of the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Lnzimclogy? 2: 210 (1986). According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from the culture of recombinant cells. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small side chains of amino acids from the interface of the first antibody molecule are replaced with larger side chains (for example tyrosine or tryptophan). Compensatory 'cavities' of identical or similar size to the large side chains are created on the interface of the second antibody molecule by replacement of the amino acid side chains, large ones with smaller ones (eg alanine or threonine.) This provides a mechanism for increasing the performance of the heterodimer over other desired end products such as homodimers Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have been proposed for example to direct cells of the immune system to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (International Patents Nos. WO 91/00360, WO 92/00373 , and European Patent EP 03089). Heteroconjugate antibodies can be used by elaborating any convenient crosslinking methods. Suitable crosslinking agents are well known in the art and are described in U.S. Patent No. 4,676,980, along with a number of crosslinking techniques. The techniques for the generation of bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science, 229: 81 (1985) describe a method wherein intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide. The Fab 'fragments generated are then convexed to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab '-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form spurious bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody molecule F (ab ') 2. Each Fab 'fragment was separately secreted from E. coli and subjected to chemical coupling directed in vitro, to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells that overexpress the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques have also been described for the preparation and isolation of the bispecific antibody fragments, directly from the culture of recombinant cells. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol, 148 (5): 1547-1553 (1992).

The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Nati, Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for the preparation of bispecific antibody fragments.The fragments comprise a variable domain heavy chain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.As a result, the VH and VL domains of a fragment are forced to pair with the complementary VL and VH domains of another fragment, whereby two antigen binding sites are formed.Another strategy for the preparation of bispecific antibody fragments by the use of single chain FV dimers (sFv) has been also reported.

See Gruber et al., J. Immunol., 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., Immunol. 147: 60 (1991). 4. Production of Monoclonal Antibody 6G4.2.5 Humanized Anti-IL-8, Antibody Fragments, and Variants The antibodies and antibody fragments of the invention can be produced using any convenient antibody manufacturing process known in the art. Typically, the antibody or antibody fragment is made using recombinant expression systems. A multiple polypeptide chain antibody or antibody fragment species can be made in a simple host cell expression system, wherein the host cell produces each antibody chain or antibody fragment and assembles the polypeptide chains in a multimeric structure to form the antibody or antibody fragment in vivo, followed by recovery of the antibody or antibody fragment from the host cell. For example, recombinant expression systems suitable for the production of whole antibody or antibody fragment are described in Lucas et al., Nucleic Acids Res., 24: 1774-1779 (1996). Alternatively, the polypeptide chains separated from the desired antibody or antibody fragment can be made into separate expression host cells, separately recovered from the respective host cells, and then in vitro mixtures under conditions that allow the formation of the antibody or antibody fragment. ulti-subunit of interest. For example, U.S. Patent No. 4,816,567 to Cabilly et al., And Carter et al., Bio / Technology, 10: 163-167 (1992) provide methods for the recombinant production of heavy and light chains of antibody in separate expression hosts. , followed by the assembly of the antibody from the separated heavy and light chains, in vitro. The following discussion of recombinant expression methods equally applies to the production of polypeptide species of single-chain antibody and to antibody species and multi-subunit antibody fragments. All recombinant methods for the production of antibody or antibody fragment, provided below will be understood to describe: (1) the manufacture of the single chain antibody species as the desired end product; (2) the manufacture of the antibody species or antibody fragment, multi-subunit, by the production of all the subunits in a simple host cell, the subunit assembly in the host cell, optionally followed by the secretion of the host cell of the host cell. final multi-subunit product towards the culture medium, and the recovery of the multi-subunit final product from the host cell and / or the culture medium; and (3) the manufacture of the antibody or multi-subunit antibody fragment by production of separate host cell subunits (optionally followed by the secretion of the host cell from subunits in the culture medium), the recovery of subunits from the respective host cells and / or culture media, followed by in vitro subunit assembly to form the final multi-subunit product. In the case of an antibody or multi-subunit antibody fragment produced in a single host cell, it will be appreciated that the production of the various subunits can be effected by expression of the nucleic acid sequences encoding multiple polypeptides, carried on a simple vector or by the expression of the nucleic acid sequences encoding the polypeptide, carried on multiple vectors contained in the host cell.

A. Construction of DNA Coding for Humanized 6G4.2.5 Monoclonal Antibodies, Antibody Fragments, and Variants.

After selection of the humanized antibody or antibody fragment of the invention, according to the methods described above, the practitioner can use the genetic code to design ± DNAs encoding the desired antibody or antibody fragment. In one embodiment, the codons preferred by the expression host cell are used in the design of a DNA encoding the antibody or antibody fragment of interest. The DNA encoding the antibody or antibody fragment of interest can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), the full disclosure of which is incorporated by reference herein, such as the triester, phosphite, phosphoramidite and H-phosphonate methods . A variation on the above procedures contemplates the use of gene fusions, where the gene or genes encoding the antibody or the antibody fragment are associated, in the vector, with a gene that codes for another protein or a fragment of another protein. This results in the antibody or antibody fragment that is produced by the host cell as a fusion with another protein. Other protein is often a protein or peptide that can be secreted by the cell, making it possible to isolate and purify the desired protein from the culture medium, and eliminating the need to destroy host cells, which arises when the protein The fusion protein can be expressed intracellularly.It is advantageous to use fusion proteins that are highly expressed.The use of gene fusions, although not essential, can facilitate the expression of heterologous proteins in the cell. coli, as well as the subsequent purification of those gene products (Harris, TJR in Genetic Engineering, Williamson, R., Ed., Academic, London, Vol. 4, p.127 (1983); Uhlen, M. &Mooks , T., Methods Enzymol, 185: 129-143 (1990).) Protein A fusions are frequently used due to the binding of protein A, or more specifically the Z domain of protein A, to IgG p provides an "Affinity Handle" for the purification of the fused protein (Nilsson, B. & Abrahmsen, L. Methods Enzymol. 185: 144-161 (1990)). It has also been shown that many heterologous proteins are degraded when they have been expressed directly in E. coli, but are stable when they are expressed as fusion proteins (Marston, F. A. O., Biochem J. 240: 1 (1986)). The fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which breaks down into a methionine, or hydroxylamine, which breaks between an Asn and Gly. Using the standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids can be inserted just before the 5 'end of the antibody or antibody fragment. Alternatively, proteolytic cleavage of the fusion proteins can be used, which has been recently reviewed (Carter, P. (1990) in Protein Purification: From Molecular Mechanisms to Large Scale Processes, Ladisch, MR, Wilson, R. C , Painton, C. C, and Builder, SE, eds., American Chemical Society Symposium Series No. 427, Ch 13, 181-193). Proteases such as factor Xa, thrombin, subtilisin, and mutants thereof, have been successfully used to cleave fusion proteins. Typically, a peptide linker that is suitable for cleavage by the protease used is inserted between the other "protein (e.g., the Z domain of protein A) and the protein of interest., such as the antibody or anti-IL-8 antibody fragment, humanized. When using the recombinant DNA methodology, the base pairs of the nucleotide coding for the linker are inserted between the genes or gene fragments that code for the other proteins. The proteolytic cleavage of the partially purified fusion protein containing the correct linker can then be carried out on any native fusion protein, or the reduced or denatured fusion protein. Various techniques are also available that can now be employed to produce the humanized antibodies or variant antibody fragments, which code for additions, deletions, or changes in the amino acid sequence of the resulting protein (s), relative to the antibody or fragment of humanized antibody, parent. By way of illustration, with the expression vectors encoding the antibody or humanized antibody fragment at hand, they can be performed on the DNA of the antibody or of the antibody fragment on site-specific mutagenesis (Kinkel et al., Methods Enzymol 204 : 125-139 (1991); Cárter P., and collaborators, Nucí. Acids Res. 13: 4331 (1986); Zoller, M. J. et al., Nucí. Acids Res. 10_6087 (1982)), cassette mutagenesis (Wells, JA et al., Gene 34: 315 (1985)), restriction selection mutagenesis (Wells, JA et al., Philos. Trans., R. Soc. London Ser. A 317, 415 (1986)) or other known techniques. The variant DNA can then be used in place of the parent DNA by insertion into the aforementioned expression vectors. The development of the host bacterium that contains the expression vectors with the mutant DNA allows the production of variant humanized antibodies, or antibody fragments, which can be isolated as described herein.

B • Insertion of DNA into a Cloning Vehicle The DNA encoding the antibody or antibody fragment is inserted into a replicable vector for subsequent cloning (amplification of the DNA) or for expression. Many vectors are available, and the selection of the appropriate vector will depend (1) on whether it will be used for DNA amplification or for DNA expression, (2) the size of the DNA to be inserted into the vector, and (3) of the host cell that is to be transformed with the vector. Each vector comprises various components depending on its function (DNA amplification or DNA expression) and the host cell for which it is compatible). The vector components include in general, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a terminator sequence of the transcription. (i) Component of the Signal Sequence In general, a signal sequence may be a component of the vector, or this may be a part of the antibody DNA or antibody fragment that is inserted into the vector. Preferably, a heterologous signal sequence is selected and fused to the DNA of the antibody or antibody fragment, such that the signal sequence in the corresponding fusion protein is recognized, transported and processed (e.g., cleaved by a signal peptidase) in the protein secretion system of the host cell. In the case of prokaryotic host cells, the signal sequence is selected, for example, from the group of alkaline phosphatase, penicillinase, Ipp, or enterotoxin II guides set to heat. In a preferred embodiment, the STII signal sequence is used as described in the following Examples. For yeast secretion, the native signal sequence can be substituted, for example, by the yeast invertase guide, a factor a guide (including the Saccharomyces factor a and Kl uyveromyces guides), or the phosphatase guide acid, the globamylase guide of C. albi cans, or the signal described in WO 90/13646. In the expression of mammalian cells, mammalian signal sequences as well as viral secretory guides, for example, gD signal of herpes simplex, are available. (ii) Origin of the Replication Component The expression and cloning vectors contain a nucleic acid sequence that makes it possible for the vector to replicate in one or more selected host cells. In general, in the cloning vectors this sequence is one that makes it possible for the vector to replicate independently of the chromosomal DNA of the host, and includes the origins of replication in the sequences of autonomous replication. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the origin of plasmid 2μ is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) and are useful for vectors of cloning in mammalian cells. In general, the origin of the replication component is not necessary for mammalian expression vectors (the SV40 origin can typically be used only because it contains the early promoter). Most expression vectors are shuttle vectors, for example, they are capable of replicating in at least one class of organisms, but they can be transfected in another organism for expression, for example, a vector is cloned. in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even if it is not capable of replicating independently of the chromosome of the host cell.

DNA can also be amplified by insertion into the host's genome. This is easily achieved using the species Ba ci llus as hosts, by including in the vector a DNA sequence that is homologous to a sequence found in Baci l l us genomic DNA. Transfection of the Bacillus with this vector results in homologous recombination with the genome and insertion of the antibody DNA or antibody fragment. (iii) Component of the Selection Gene The expression and cloning vectors must contain a selection gene, also called a selectable marker. This gene codes for a protein necessary for the survival or development of transformed host cells, grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients not available from the complex media, for example the gene coding for the racemase of D-alanine for Bacillus. An example for a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with a heterologous gene express a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the neomycin drugs (Southern et al., J. Molec. Appl. Genet., 1: 327 (1982)), mycophenolic acid (Mulligan et al., Science, 209: 1422 (1980) or hygromycin ( Sugden et al., Mol. Cell, Biol., 5: 410-413 (10985).) The three examples given above employ bacterial genes under eukaryotic control to transfer resistance to the appropriate drug (G418 or neomycin (geneticin), xgpt (acid mycophenolic), and hygromycin, respectively.) Yet another example of selectable markers suitable for mammalian cells are those that make it possible to identify competent cells to pick up the nucleic acid from the antibody or from the antibody fragment, such as dihydrofolate reductase ( DHFR) or thymidine kinase The transformants of mammalian cells are placed under selection pressure to which only the transformants are adapted to survive in vivo. I've got the score. The selection pressure is imposed by culturing the transformants under conditions in which the concentration of the selection agent in the medium is successfully changed, thereby leading to the amplification of the selection gene and the DNA encoding the antibody or antibody fragment. Amplification is the process by which the genes in greatest demand for the production of a critical protein for development, are reiterated in tandem within the chromosomes of successive generations of recombinant cells. The increased amounts of the antibody or antibody fragment are synthesized from the amplified DNA. For example, cells transformed with the DHFR selection gene are first identified by culturing all the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed, is the Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Nati Acad. Sci. USA, 77: 4216 (1980). The transformed cells are then exposed to increased levels of methotrexate.

This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, to multiple copies of another DNA comprising the expression vectors, such as the DNA encoding the antibody or the antibody fragment. This amplification technique can be used with any otherwise suitable host, for example, ATCC No. CCL61 CHO-Kl, regardless of the presence of the endogenous DHFR if, for example, a mutant DHFR gene, which is highly resistant to Mtx. it is employed (European Patent No. LP 117,060). Alternatively, host cells (particularly wild type hosts containing the endogenous DHFR) transformed or co-transformed with DNA sequences encoding the antibody or antibody fragment, for the wild-type DHFR protein, and for another selectable marker such as the aminoglycoside-3'-phosphotransferase (APH) can be selected by "developing the cells in medium containing a selection agent for the selectable marker, such as a glycosidic antibiotic, for example, kanamycin, neomycin, or G418, see US Patent No. 4, 965, 199. A selection gene, suitable for use in yeast is the trpl gene present in the yeast plasmid YRp7. Stinchcomb et al., Nature, 282: 39 (1979); Kings an et al., Gene, 7: 142 (1979); or Tschemper et al., Gene, 10: 157 (1980). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trpl lesion in the kenoma of the yeast host cell then provides an effective environment for the detection of transformation by development in the absence of tryptophan. Similarly, strains of yeast deficient in Leu2. { ATCC 20,622 or 38,626) are contemplated by the known plasmids possessing the Leu2 gene. (iv) Promoter component Expression vectors usually contain a vector that is recognized by the host organism and is operably linked to the nucleic acid of the antibody or antibody fragment. The promoters are untranslated sequences located upstream (5 ') to the start codon of a structural gene (generally within about 100 to 1,000 base pairs) that control the transcription and translation of a particular nucleic acid sequence , such as the sequence encoding the antibody or antibody fragment to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate the increased levels of transcription from the ΔD or their control, in response to some change in culture conditions, for example, the presence or absence of a nutrient or a change in temperature. To this date, a large number of promoters are recognized by a variety of potential host cells.

Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al, Nature, 275: 615 (1978); and Goeddel et al., Nature, 281: 544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980) and European Patent No. EP 36,776) and hybrid promoters such as the tac promoter (de Boer et al., Proc. Nati, Acad. Sci. USA, 80: 21-25 (1983)). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thereby making it possible for a worker skilled in the art to link them operably to the DNA encoding the antibody or the antibody fragment (Siebenlist et al., Cell, 20: 269 (1980)) using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody or antibody fragment. Promotional sequences suitable for use with yeast hosts include promoters for the 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al. contributors, J. Adv. Enzyme Reg., 7: 149 (1968), and Holland, Biochemistry, 17: 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate- decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate-mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucosinase. Other yeast promoters, which are inducible promoters that have the additional advantage of transcription controlled by the conditions of development, are the promoter conditions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, the associated degradative enzymes in the nitrogen metabolism, methotonein, glyceraldehyde-3-iosphate-dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., In European Patent No. EP 73,657A. Yeast enhancers are also advantageously used with yeast promoters. Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription is initiated. Another sequence found at 70 to 80 bases upstream of the start of transcription of many genes is a CXCAAT region where X can be any nucleotide. At the 3 'end most eukaryotic genes are an AATAAA sequence that can be the signal for the addition of the poly-A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into mammalian expression vectors. The transcription of the DNA that codes for the antibody or antibody fragment, driven by the vector, in mammalian host cells can be controlled by promoters obtained from genomes of viruses such as polyoma virus, chickenpox virus (British Patent No. UK 2,211,504 published July 5, 1989). ), adenoviruses (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and more preferably Simian Virus 40 (SV40), from mammalian promoters heterologous, for example, the actin promoter or an immunoglobulin promoter, and heat shock promoters, with the proviso that such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the viral origin of SV40 replication. Fiers et al., Nature, 273: 113 (1978); Mulligan and Berg, Science, 209: 1422-1427 (1980), Pavlakis et al., Proc. Nati Acad. Sci. USA, 78: 7398-7402 (1981). The immediate early promoter of human cytomegalovirus is conveniently obtained as a restriction fragment HindITI E. Greenaway et al., Gene, 18: 355-360 (1982). A system for the expression of DNA in mammalian hosts using the bovine papilloma virus as a vector is described in US Pat. No. 4,419,446. A modification of this system is described in U.S. Patent No. US 4,601,978. See also Gray et al., Nature, 295: 503-508 (1982) on the cDNA expression encoding the immune interferon in monkey cells, Reyes et al., Nature, 297: 598-601 (1982) on expression of the CDNA of human interferon in mouse cells, under the control of a thymidine kinase promoter from the herpes simplex virus, Canaani and Berg, Proc. Nati Acad. Sci. USA, 79: 5166-5170 (1982) on the expression of the human interferon-1 gene in cultured rabbit and mouse cells, and Gorman et al., Proc. Nati Acad. Sci. USA, 79: 6777-6781 (1982) on the expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and NIH-3T3 cells of mouse using the long terminal repeat of the Rous sarcoma virus, as a promoter. (v) Component of the Auirentador Element The transcription of a DNA encoding an antibody or antibody fragment by higher eukaryotic host cells is often increased by inserting an enhancer sequence into the vector. Augmentators are cis-acting elements of DNA, usually about 10-300 base pairs, that act on a promoter to increase its transcription. The augmentators are relatively independent of orientation and position having been found 5 '(Laimins et al., Proc. Nati. Acad. Sci. USA, 78: 993 (1981)) and 3 '(Lusky et al., Mol. Cell Bio., 3: 1108 (1983)) to the transcription unit, within an intron (Banerji et al., Cell, 33: 729 (1983)) as well as well as within the coding sequence itself (Osborne et al., Mol.Cell Bio., 4: 1293 (1984)). Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin-fetoprotein and insulin). Typically, however, someone will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (100-270 base pairs), the enhancer of the cytomegalovirus early promoter, the polyoma enhancer on the late side of the replication origin, and the adenoviral enhancers. See also Yaniv, Nature, 297: 17-18 (1982) on the augmenting elements for the activation of eukaryotic promoters. The enhancer can be spliced into the vector at a 5 'or 3' position to the antibody DNA or antibody fragment, but is preferably located at the 5 'end of the promoter. (vi) Completion of the Transcript Component Expression vectors used in eukaryotic host cells (fungal, yeast, insect, plant, animal, human, or nucleated cells from other multicellular organisms) may also contain sequences necessary for the termination of transcription and for the stabilization of the mRNA. Such sequences are commonly available from the 5 'end and, occasionally, from the 3 rd untranslated regions of the eurarniotic or viral cDNAs or cDNAs. These legions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody or antibody fragment. The 3 'untranslated regions also include the transcription termination sites.

Suitable vectors containing one or more of the above-listed components and the desired coding and control sequences are constructed by standard ligation techniques. The isolated plasmids or DNA fragments are excised, designed, and religated in the desired manner to generate the required plasmids. For the analysis to confirm the correct sequences in the constructed plasmids, mixtures of ligatures are used to transform E. col i K12 strain 294 (ATCC 31, 446) and successful transformants selected for resistance to ampicillin or tetracycline, where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and / or sequenced by the method of Messing et al.

Nucleic Acids Res 9: 309 (1981) or by the method of Maxam et al., Methods in Enzymology, 65: 499 (1980). Particularly useful in the practice of this invention are expression vectors that provide for the transient expression in mammalian cells of the DNA encoding the antibody or antibody fragment. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide. encoded by the expression vector. Other methods, vectors and host cells suitable for the adaptation of the synthesis of the antibody or the antibody fragment in vertebrate recombinant cell culture, are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); Levinson et al., European Patent No. EP 117,060; and European Patent No. EP 117,058. A particularly useful plasmid for the expression in culture of mammalian cells of the IgE peptide antagonist is pRK5 (published European Patent No. 307,247) or pSVl 6B (International Patent PCT published No. WO 91/08291 published on June 13, 1991 ).

C. Selection and Transformation of Host Cells Suitable host cells for the cloning or expression of the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli, Bacilli such as B. subtilis, species of Pseudomonas such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescens. A preferred E. coli cloning host is E. coli 294 (ATCC 31, 446), although other strains are suitable such as E. coli B., E. coli 1776 (ATCC 31,537), and E. coli W3110 (ATCC 27.325). These examples are illustrative rather than limiting. Preferably, the host cell must secrete minimal amounts of the proteolytic enzymes. In a preferred embodiment, the E. coli strain 49D6 is used as the expression host as described in the Examples below. Review articles describing the recombinant production of antibodies in bacterial host cells include Skerra et al., Curr. Opinion in Immunol, 5: 256 (1993) and Pluckthun, Immunol, Revs :, 130: 151 (1992). In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable as hosts for vectors containing the antibody DNA or the antibody fragment. Sa ccharomyces cerevi si a e, or yeast for common bakery, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains, such as S, are commonly available and useful herein. pombe (Beach and Nurse, Nature, 290: 140 (1981)), Kl uyveromyces l a c ti s (Louvencourt et al., J. Bacteriol, 737 (1983)), yarrowi a (European Patent No. 402,226), Pi chi a pa s t ori s (European Patent No. 183,070), Tri ch oderma reesi a (European Patent No. 244.2341, Neurospora crassa (Case et al, Prcc. Nati, Acad. Sci. USA, 76: 5259-5263 (1979)), and Aspergillus hosts such as A. nor dul ans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 (1983); Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al., Proc. Nati Acad. Sci. USA, 81: 1470-1474 (1984)) and A. ni ger (Kelly and Hynes, EMBO J., 4: 475-479 (1985)). Host cells derived from multicellular organisms can also be used in the recombinant production of antibody or antibody fragment. Such host cells are capable of performing complex processing and glycosylation activities. In principle, any culture of higher eukaryotic cells is functional, either from a vertebrate or invertebrate culture. Examples of invertebrate cells include plant cells and insect cells. The numerous strains of baculoviruses and variants and the corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes a egypti (mosquito), Aedes albopi c t us (mosquito), Dros oph i l a mel to n oga s ter (1-fruit fly), and Bombyx mori host cells have also been identified. See for example, Luckow et al., Bio / Technology, 6: 47-55 (1988); Miller and colleagues at Genetic Engineering, Setlow, J.K. et al., 8: 277-279 (Plenum Publishing, 1986), and Maeda et al., Nature, 315: 592-594 (1985). A variety of such viral strains are publicly available, for example, the variant Ll of Au t ographa cal iforni ca NVP and the BM-5 strain of Bomjbyx mori NVP, and such viruses can be used as the virus in the present according to the present invention, particularly for the transfection of Spodoptera frugiperda cells. Plant cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can be used as hosts. Typically, the plant cells are transfected by incubation with certain strains of the bacterium Agroba c t eri um t umefaci ens, which has been previously manipulated to contain the DNA of the antibody or the antibody fragment. During incubation of the plant cell culture with A. t umefa ci ens, the DNA encoding the antibody or antibody fragment is transferred to the host plant cell such that it is transfected, and, under appropriate conditions, will express the DNA of the host cell. antibody or antibody fragment. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopalin-synthase promoter and the polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen, 1: 561 (1982). In addition, DNA segments isolated from the region upstream of the 780 T-DNA gene are capable of activating or increasing the transcription levels of the genes expressed in plants, in plant tissue containing recombinant DNA. See European Patent No. EP 321,196 published June 21, 1989. Vertebrate cell culture is preferred for the recombinant production of full-length antibodies. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of mammalian host cell lines, useful are monkey kidney CV1 lines transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen.

Virol, 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Nati. Acad.

Sci. USA, 77: 4216 (1980)); Mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.

Annals N.Y. Acad. Sci, 283: 44-68 (1982)); cells MRC 5; FS4 cells; and a human hepatoma cell line (Hep G2). Preferred host cells are human embryonic kidney 293 cells and Chinese hamster ovary cells. Myeloma cells that do not otherwise produce the immunoglobulin protein are also useful host cells for the recombinant production of full-length antibodies. The host cells are transfected and preferably transformed with the expression or cloning vectors described above in this invention and cultured in conventional, modified nutritional media, as appropriate for the induction of promoters, selection of transformants, or amplification of genes coding for the desired sequences.

Transfection refers to the capture or taking of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the person of ordinary skill in the art, for example, precipitation with calcium phosphate (CaP04) and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Transformation means the introduction of DNA into an organism, so that DNA is replicable, either as an extrachromosomal element or as a chromosomal integrant. Depending on the host cell used, the transformation is performed using standard techniques appropriate for such cells. The treatment with calcium using calcium chloride, as described in section 1.82 of Sambrook et al., Is generally used for prokaryotes or other cells that contain substantial cell wall barriers. The infection with Agrobacterium um t umefaci ens is used for the transformation of certain plant cells, as described in Shaw et al., Gene, 23: 315 (1983) and International Patent No. WO 89/05859 published on June 29, 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method described in Sections 16.30-16.37 of Sambrook et al., Supra, is preferred. The general aspects of the transformations of the mammalian host cell system have been described by Axel in US Pat. No. 4,399,216 issued August 16, 1983. The transformations within yeast are typically carried out according to the method of Van Solingen et al., J. Bact. , 130: 946 (1977) and Hsiao et al., Proc. Nati Acad. Sci. (USA), 76: 3829 (1979) .- However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or by protoplast fusion can also be used.

D. Culture of Host Cells The prokaryotic cells used to produce the antibody or antibody fragment are cultured in suitable media as generally described in Sambrook et al., Supra.

The mammalian host cells used to produce the antibody or antibody fragment can be cultured in a variety of media. Commercially available media such as Ham's FIO medium (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for the culture of the host cells. In addition, any of the means described in Hamm and Wallace, Meth Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem, 102: 255 (1980), Patents North American Nos. US 4,767,704; 4,657,866; 4,927,762; or 4,560,655; International Patents Nos. WO 90/03430; WO 87/00195; U.S. Patent No. 30,985; or U.S. Patent No. 5,122,469, the descriptions of all of which are incorporated by reference herein, can be used as a medium? of cu1 tive for the host cells. Any of these media can be supplemented as necessary with hormones and / or growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GentamycinRM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolecular range), and glucose or a source of energy equivalent. Any other necessary supplements may be included at appropriate concentrations, which may be known to those of skill in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to one of ordinary skill in the art. Preferred host cells in this description encompass cells in vitro culture, as well as cells that are within a host animal.

E. Detection of Gene Amplification / Expression The amplification and / or expression of the gene can be measured in a sample directly, for example, by conventional Southern staining, northern blotting to quantify mRNA transcription (Thomas, Proc. Nati, Acad. Sci. USA, 77: 5201 -5205 (1980)), stained by spots (DNA analysis), or hybridization in itself, using an appropriately labeled probe, 'based on the sequences provided herein. Various labels, more commonly radioisotopes, particularly 32P, may be employed. However, other techniques can also be employed, such as the use of biotin-modified nucleotides for introduction into a polynucleotide. Biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies that can recognize duplexes or specific pairs, including DNA duplexes, RNA duplexes and duplexes of DNA-RNA hybrids or DNA-protein duplexes, may be employed. The antibodies in turn can be labeled and the assay can be carried out where the duplex is bound to a surface, so that after the formation of the duplex on the surface, the presence of the antibody bound to the duplex can be detected.

The expression of the gene, alternatively, can be measured by immunological methods, such as immunohistochemical staining of the tissue sections, and assay of the cell culture or body fluids, to directly quantify the expression of the gene product. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies, specific to the gene product, where the markers are usually visually detectable, such as enzymatic markers, fluorescent markers, luminescent markers , and similar. A particularly sensitive staining technique, suitable for use in the present invention is described by Hsu et al., Am. J. Clin. Path., 75: 734-738 (1980).

F. Purification of the Anicuis or the Antibody Fragment In the case of a host cell secretion system, the antibody or antibody fragment is recovered from the culture medium. Alternatively, the antibody can be produced intracellularly, or produced in the periplasmic space of a bacterial host cell. If the antibody is produced intracellularly, as a first step, the host cells are lysed, and the resulting particulate waste is removed, for example, by centrifugation or ultrafiltration. Carter and colleagues, Bi o / Technolgy 10: 163-167 (1992) describe a procedure for the isolation of antibodies, which are secreted into the periplasmic space of E. col i. In summary, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) in a period of 30 minutes. The detritus or cellular waste can be removed by centrifugation. Where the antibody is secreted into the medium, the supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the development of adventitious contaminants.

The composition of the antibody prepared from the cells can be "purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography which is the preferred purification technique. Adequacy of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fe domain that is present in the antibody.A protein A can be used to purify antibodies that are based on human heavy chains? 2, or? 4 (Lindmarck et al, J. Immunol, Meh 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human? 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is coupled is more frequently agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXMR resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as ion exchange column fractionation, ethanol precipitation, reverse phase high performance liquid chromatography (HPLC), chromatography on silica, chromatography on heparin, chromatography on Sepharose ™ on an anion exchange resin or cationic (such as a column of polyaspartic acid), chromatofocusing, SDS-PAGE, and precipitation with aluminum sulfate, are also available depending on the antibody to be recovered. After any preliminary purification steps or steps, the mixture comprising the antibody of interest and the contaminants can be subjected to pH-hydrophobic hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low saline concentrations (eg, from about 0 to 0.25 M salt).

G. Production of Antibody Fragments Various techniques have been developed for the production of the humanized antibody fragments of the invention, including the Fab, Fab ', Fab'-SH, or F (ab') 2 fragments. Traditionally, these fragments were derived by means of the proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Bi och emi cal and Bi ophysi cal Meth ods 24: 107-117 (1992) and Brennan et al., Sci ence, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. col i and chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). According to another procedure, the reagents of F (ab ') 2 can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to those of skill in the art.

. Uses of Anti-IL-8 Antibodies A. Diagnostic Uses For diagnostic applications that require the detection or quantification of IL-8, the antibodies or antibody fragments of the invention will typically be labeled with a detectable portion. The detectable portion can be any that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion may be a radioisotope, such as 3 H, 14 C, 3 P. 35 S, or 125 I; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; radioactive isotopic labels, such as, for example, 125 I, 2 P, 14 C, or 3 H; or an enzyme, such as alkaline phosphatase, beta-qalactosidase or horseradish peroxidase. Any method known in the art for separately conjugating the antibody or antibody fragment to the detectable portion can be employed, including those methods described by Hunter et al., Nature 144: 945 (1962); David et al., Biochemistry 13: 1014 (1974); Pain et al., J. Immunol. Meth. 40: 219 (1981); and Nygreen, J. Histochem. and Cytochem. 30: 407 (1982). The antibodies and antibody fragments of the present invention can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. For example, see Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987). Competitive binding assays rely on the ability of a labeled standard (which may be an IL-8 or an immunologically active portion thereof) to compete with the analyte in the test sample (IL-8) to bind to a limited amount of antibody or antibody fragment. The amount of IL-8 in the test sample is inversely proportional to the amount of standard that is bound to bind to the antibodies. To facilitate the determination of the amount of standard that is bound to bind, antibodies or antibody fragments are generally insolubilized before or after competition, so that the standard and the analyte that are bound to the antibodies can be conveniently separated. of the standard and the analyte that remain unattached. The tests or sandwich tests involve the use of two antibodies, each capable of binding to a different antigenic portion, or epitope, of the IL-8 protein to be detected. In a sandwich assay, the analyte in the test sample is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thereby forming an insoluble, three-part complex (US Patent No. 4,376,110). The second antibody can be labeled with a detectable portion (direct sandwich assays) or can be measured using an anti-immunoglobulin antibody that is labeled with a detectable portion (indirect sandwich assay), For example, a type of sandwich assay is an ELISA assay, in which case the detectable portion is an enzyme (eg, horseradish peroxidase). Antibodies and antibody fragments for IL-8 are also useful for the affinity purification of IL-8 from the culture of recombinant cells or from natural sources. For example, these antibodies can be fixed to a solid support by techniques well known in the art, to purify IL-8 from a source such as a culture or tissue supernatant.

B. Therapeutic compositions and_ Administration of Anti-IL-8 Antibody The antibodies and humanized anti-IL-8 antibody fragments of the invention are useful in the treatment of inflammatory disorders, such as adult respiratory distress syndrome (ARDs), hypovolemic shock, ulcerative colitis and rheumatoid arthritis. Therapeutic formulations of humanized anti-IL-8 antibodies and antibody fragments are prepared by storage by mixing the antibody or antibody fragment having the desired degree of purity, with optionally physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, supra), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients or patients, at the doses and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants that include ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or the usina; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). The humanized antibody or IL-8 antibody fragments to be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes, before or after lyophilization or reconstitution. The antibody or humanized anti-IL-8 antibody fragment will ordinarily be stored in lyophilized form or in solution.

The therapeutic compositions of the antibody or humanized anti-IL-8 antibody fragment, are generally placed in a container having a sterile access gate, for example, an intravenous solution bag or a bottle having a stopper pierced by a needle by hypodermic injection. The route of administration of the antibody or humanized anti-IL-8 antibody fragment is according to the known methods, for example, by inhalation, injection or infusion by the intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial route, or intralesional, by enema or suppository, or by sustained release systems as noted below. Preferably, the antibody is administered systemically or at a site of inflammation. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, for example films, or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919, EP 58,881), copolymers of L-glutamic acid and gamma-ethyl-glutamate (Sidman et al., Biopolymers 22: 547 (1983)), poly. (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed, Mater. Res. 15: 167 (1981) and Langer, Chem. Tech. 12:98 (1982)), ethylene vinyl acetate (Langer et al., Supra). ) or the poly-D- (-) -3-hydroxybutyric acid (European Patent EP 133,988). Sustained-release compositions of the antibody or humanized anti-IL-8 antibody fragment also include the antibody or the liposomally entrapped antibody fragment. Liposomes containing an antibody or antibody fragment are prepared by methods known per se: German Patent DE 3,218,121; Epstein et al., Proc. Nati Acad. Sci. U.S.A. 82: 3688 (1985); Hwang et al., Proc. Nati Acad. Sci.

USES. 77: 4030 (1980); European Patent EP 52,322; European Patent EP 36,676; European Patent EP 88,046; European Patent 143,949, European Patent EP 142,611; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and European Patent EP 102,324. Ordinarily, the liposomes are of the small unilamellar type (approximately 200 to 800 Angstroms) in which the lipid content is greater than about 30 mol percent of cholesterol, the selected proportion being adjusted for the most effective therapy with antibody or fragment of antibody. An "effective amount" of the antibody or humanized anti-IL-8 antibody fragment to be used therapeutically will depend, for example, on the therapeutic objectives, the route of administration and the condition of the patient. It is necessary for the therapist to titrate the dose and modify the route of administration, as required, to obtain the optimal therapeutic effect Typically, the clinician will administer the antibody or humanized anti-IL-8 antibody fragment until a dose that reaches The progress of this therapy is easily verified by conventional tests or tests In the treatment and prevention of an inflammatory disorder to an antibody or humanized anti-IL-8 antibody fragment of the invention, the composition of the The antibody will be formulated, dosed and administered in a manner consistent with good medical practice. Context include the particular disorder in question, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the antibody, the particular type of antibody, the method of administration, the administration scheme and other factors known to practicing doctors. The 'therapeutically effective amount' of the antibody, which is to be administered, will be governed by such considerations, and is the minimum amount necessary to prevent, improve, or treat inflammatory disorder, including the treatment of acute or chronic respiratory diseases and the reduction of inflammatory responses. Such amount is preferably below the amount that is toxic to the host, or makes the host significantly more susceptible to infections. As a general proposition, the initial therapeutically effective amount of the antibody fragment or antibody fragment, parenterally per dose, will be in the range of about 0.1 to 50 mg / kg of body weight of the antibody. patient per day, with the typical initial interval of the antibody used which is from 0.3 to 20 mg / kg / day, more preferably 0.3 to 15 mg / kg / day.

As noted above, however, these suggested amounts of antibody or antibody fragment are subject to a fair amount of therapeutic discretion. The key factor in the selection of an appropriate dose and dosing schedule is the result obtained, as indicated above. The antibody or antibody fragment need not be, but is optionally formulated with one or more agents currently used to prevent or treat the inflammatory disorder in question. For example, in rheumatoid arthritis, the antibody must be administered in conjunction with a glucocorticosteroid. The effective amount of the other agents depends on the amount of the antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same doses and routes of administration as used hereinbefore or approximately from 1 to 99% of the doses used to date. The following examples are offered by way of illustration and not by way of limitation. The descriptions of all references cited in the specification, and the descriptions of all citations in such references, are expressly incorporated by reference herein.

EXAMPLES A. GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES HUMAN IL-8 CONTROL Balb / c mice were immunized in each hind paw or intraperitoneally with 10 μg of recombinant human IL-8 (produced as a fusion of (ser-IL-8) 72 with ubiquitin (Hebert et al., J. Immunology 145: 3033-3040 (1990)); IL-8 is commercially available from PeproTech, Inc., Rocky Hill, NJ) resuspended in MPL / TDM (Ribi Immunochem, Research Inc., Hamilton, MT) and boosted twice with the same amount of IJ., - 8 . In these experiments, it is intended that '11 -8"cigniii? Ue (eci IL-8) 72 unless otherwise specified.A final reinforcement of 10 μm of IL-8 was administered 3 days before fusion. The cells of the spleen or cells of the popliteal lymph nodes were fused with the mouse myeloma P3X63Ag8U.l (ATCC CRL1597), a clone of non-secretion of the myeloma P3X63Ag8, using polyethylene glycol at 35% as described above. the fusion, the culture supernatant was selected for the presence of monoclonal antibodies to IL-8 by ELISA The ELISA assay was performed as follows: 96 well Nunc immunoplates (Flow Lab, MacLean, VA) were coated with 50 μg / ml. Well of IL-8 at 2 μg / ml in phosphate buffered saline (PBS) overnight at 4 ° C. The remaining steps were carried out at room temperature.Specific binding sites were blocked with bovine serum albumin. 0.5% (BSA) per 1 hour The plates were then incubated with 50 μl / well of hybridoma culture supernatants from 672 progenitor fusion wells, in development, for 1 hour, followed by incubation with 50 μl / well of 1: 1000 dilution. a reserve solution at 1 mg / .m.! of goat anti-mouse Ig, conjugated to alkaline phosphatase (Tago C?., Foster City, CA) for 1 hour. The level of antibody bound to the enzyme, bound to the plate, was determined by the addition of 100 μl / well of 0.5 mg / ml of r-nitrophenyl phosphate in sodium bicarbonate buffer, pH 9.6. The color reaction was measured at 405 nm with an ELISA plate reader (Titertrek Multiscan, Flow Lab, MacLean, VA). Between each step, the plates were washed three times in PBS containing Tween 20 at 0.05%. Culture supernatants that promoted 4 times more IL-8 binding than the control medium were selected as positive. According to this criterion, 16 of the 672 merging wells of developing parents (2%) were positive. These positive hybridoma cell lines were cloned at least twice by the use of the limiting dilution technique. Seven of the positive hybridomas were further characterized as follows. The isotypes of the monoclonal antibodies were determined by the coating of 96-well Nunc immunoplates (Flow Lab, MacLean, VA) with IL-8 overnight, blocking with BSA, incubating with culture supernatants, sesnid ^ po - the addition of the predetermined amount of goat anti-mouse Ig, conjugated to alkaline phosphatase, specific for isotype (Fischer Biotech, Pittsburgh, PA). The level of conjugated antibodies bound to the plate was determined by the addition of r-nitrophenyl phosphate as described above.

All monoclonal antibodies tested belonged either to the immunoglobulin isotype IgGi or IgG2. The ascites fluid containing these monoclonal antibodies had antibody titers in the range of 10,000 to 100,000, as determined by the reciprocal of the dilution factor that gave 50% of the maximum bond in the ELISA assay. To assess whether these monoclonal antibodies bound to the same epitopes, a competitive binding ELISA assay was performed. At a ratio of biotinylated mAb to the unlabeled mAb of 1: 100, the binding of the biotinylated mAb 5.12.14 was significantly inhibited by its homologous mAb but not by mAb 4.1.3, while the binding of the biotinylated mAb 4.1.3 was inhibited by mAb 4.1.3 but not by mAb 5.12.14. The monoclonal antibody 5.2.3 pp behaved in a manner similar to mAb 4.1.3, while the antioeipos ionocionales 4.8 and 12.3.9 were similar to mAb 5.12.14. Thus, mAb 4.1.3 and mAb 5.2.3 bind to one or more different epitopes than the epitope recognized by monoclonal antibodies 12.3.9, 4.8 and 5.12.14.

Immunoblot spotting analysis was performed to evaluate the reactivity of the antibody to IL-8 immobilized on nitrocellulose paper. The seven antibodies recognized IL-8 immobilized on paper, whereas a mouse IgG antibody, control, did not. The ability of these monoclonal antibodies to capture soluble 125I-IL-8 was evaluated by a radioimmunoprecipitation (RIP) test. Briefly, the 125 I-IL-8 tracer (4 x 104 cpm) was incubated with various dilutions of anti-IL-8 monoclonal antibodies in 0.2 ml of PBS containing 0.5% BSA and 0.05% Tween 20 (buffer assay) for 1 hour at room temperature. One hundred microliters of a predetermined concentration of goat anti-mouse Ig antiserum (Pel-Freez, Rogers, AR) were added, and the mixture was incubated at room temperature for 1 hour. Immune complexes were precipitated by the addition of 0.5 ml of 6% polyethylene glycol (Molecular Weight 8000) maintained at 4 ° C. After centrifugation at 2,000 x g for 20 minutes at 4 ° C, the supernatant was removed by aspiration and the radioactivity remaining in the concentrate or button was counted in a gamma counter. The specific percentage link was calculated as (precipitate cpm - antecedent cpm) / (total cpm - antecedent cpm). Monoclonal antibodies 4.1.3, 5.2.3, 4.8, 5.12.14 and 12.3.9 captured 125I-IL-8 very efficiently, while antibodies 9.2.4 and 8.9.1 were not able to capture 125I-IL- 8 soluble in the RIP test, even though these could bind to the coated IL-8 on the ELISA plates (Table I). The dissociation constants of these monoclonal antibodies are determined using a competitive binding RIP assay. In summary, the competitive inhibition of the binding of each antibody to 125T-lL-8 (20,000 to 40,000 cpm per assay) for various amounts of unlabeled IL-8, was determined by the RIP described above. The dissociation constant (affinity) of each mAb was determined by using the Scatchard plot analysis (Munson et al., Anal. Biochem. 107: 220 (1980)) as propogated in the VersaTerm-PRO computer program (Synergy Software, Reading, PA). The Kd's of these monoclonal antibodies (with the exception of 9.2.4 and 8.9.1) were in the range of 2 x 10"8 to 3 x 10" 10 M. The monoclonal antibody 5.12.14 with a Kd of 3 x 10"10 M showed the highest affinity among all the monoclonal antibodies tested (Table 3).

Table 3. Characterization of the Anti-IL-8 Monoclonal Antibodies Antibody% Specific Link of IL-8 Ka (M) Isotype pl 4. 1.3 58 2 X 10"IgG, 4.3-6.1 . 2.3 34 2 X MT IgGj 5.2-5.6 8.9.1 2 IgG, 6.8-7.6 4. 8 62 3 X 10-8 IgGz. 6.1-7.1 . 12.14 98 3 X 10"10 IgGz, 6.2-7.4 12. 3.9 86 2 X 10 - IgG * 6.5-7.1 To assess the ability of these monoclonal antibodies to neutralize the activity of IL-8, the amount of 125 I-IL-8 bound to human neutrophilies was determined., in the presence of various amounts of culture supernatants, and purified monoclonal antibodies. Neutrophils were prepared by using the Mono-Poly Resolution Medium (M-PRM) (Flow Lab. Inc., McLean, VA). Briefly, fresh heparinized human blood was loaded onto M-PRM at a ratio of blood to the medium, 3.5: 3.0, and centrifuged at 300 x g for 30 minutes at room temperature. Neutrophils enriched in the intermediate layer were collected and washed once in PBS. Such preparation routinely contained more than 95% neutrophils, according to Wright's Giemsa stain. The receptor binding assay was performed as follows. 50 μl of 125 I-IL-8 (5 ng / ml) was incubated with 50 μl of unlabeled IL-8 (100 μg / ml) or monoclonal antibodies in PBS containing 0.1% BSA, for 30 minutes at room temperature. The mixture was then incubated with 100 μl of neutrophils (107 cells / ml) for 15 minutes at 37 ° C. The bound 12SI-IL-8 was separated from the unbound material by loading the mixtures onto 0.4 ml of PBS containing 20% sucrose and 0.1% BSA, and by centrifugation at 300 x g for 15 minutes. The supernatant was removed by aspiration and the radioactivity associated with the button was counted in a gamma counter. Monoclonal antibodies 4.1.3, 5.2.3, 4.8, 5.12.14, t 12.3.9 inhibited more than 85% of the binding of IL-8 to human neutrophils at a molar ratio of 1:25 of IL-8 to mAb . On the other hand, monoclonal antibodies 9.2.4 and 8.9.1 seemed to improve the binding of IL-8 to its receptors on human neutrophils. Since a mouse IgG control also improved the binding of IL-8 on neutrophils, the improvement of the binding of IL-8 to its receptors by mAb 9.2.4 and 8.9.1 appears to be nonspecific. Thus, monoclonal antibodies, 4.1.3, 5.1.3, 4.8, 5.12.14, and 12.3.9 are potential neutralizing monoclonal antibodies, while monoclonal antibodies 8.9.1 and 9.2.4 are non-neutralizing monoclonal antibodies. . The ability of anti-IL-8 antibodies to block neutrophil chemotaxis induced by IL-8 was tested as follows. The chemotaxis of neutrophils induced by IL-8 was determined using a Boyden chamber method (Larsen, et al., Science 243: 1464 (1989)).

One hundred μ "1 d <=> neutrophiles (106 cells / ml) resuspended in Rfp-iJ containing 0. i% Bb'A, were placed in the upper chamber and 29 μl of IL-8 were placed. (20 nM) with or without monoclonal antibodies in the lower chamber The cells were incubated for 1 hour at 37 ° C. The neutrophils that migrated to the lower chamber were stained with Wright's Giemsa stain and counted under the microscope (amplification Approximately 10 different fields per experimental group were examined.The neutralizing monoclonal antibodies 5.12.14 and 4.1.3 blocked almost 70% of the chemotactic activity of the IL-8 neutrophils at a 1:10 ratio of IL- 8 mAb The isoelectric focusing pattern (IEF) of each mAb was determined by applying purified antibodies on an IEF polyacrylamide gel (pH 3-9, Pharmacia) using the Fast gel system (Pharmacia, Piscataway, NJ). The IEF gel was previously treated with Farmalite containing 1% Triton XlOO (Sigma, St. Louis, MO) for 10 minutes before loading the samples. The IEF pattern was visualized by staining with silver according to the manufacturer's instructions. All monoclonal antibodies had different IEF patterns, confirming that these were ee o. gain of different clone. The values of pi for the antibodies are listed in Table 3. All these monoclonal antibodies bound equally well to the forms (ala-IL-8) 77 and (ser-IL-8) 72 of IL -8. Because IL-8 has more than 30% sequential homology with other members of family 4 of platelet factor (PF4) of inflammatory cytokines such as β-TG (Van Damme et al., Eur. J. Biochem. : 337 (1989)); Tanaka et al, FEB 236 (2): 467 (1988)) and PF4 (Deuel et al, Proc. Nati, Acad. Sci. USA 74: 2256 (1977)), these were tested for possible cross-reactivity to β- TG and PF4, as well as another neutrophil activation factor, C5a. No detectable link of any of these proteins was observed, with the exception of mAb 4.1.3, which had a slight cross-activity to β-TG. One of the antibodies, mAb 5.12.14, was also studied to determine if it could block the release measured by IL-8 from elastase by neutrophils. Briefly, human neutrophils were resuspended in Hanks balanced salt solution (Gibco, Grand Island, NY) containing 1.0% BSA, Fraction V (Sigma, St. Louis, MO), 7 mg / ml alpha-D-glucose (Sigma), bicarbonate d-sodic 4.2 (big a) and HLPL3 O ti M, pH 7.1 (JRH Bioscience, Lenexa, KS). A pool of cytochalasin B (Sigma) (5 mg / ml in dimethyl sulfoxide (Sigma) was prepared and stored at 2-8 ° C. Cytochalasin B was added to the neutrophil preparation to produce a final concentration of 5 μg / ml. ml, and incubated for 15 minutes at 37 ° C. Human IL-8 was incubated with mAb 5.12.14 (20 μl), or a negative control antibody, in 1 ml polypropylene tubes (DBM Scientific, San Fernando California) for 30 minutes at 37 ° C. The final assay concentrations of IL-8 were 50 and 500 nM.The monoclonal antibodies were diluted to produce the following proportions (IL-8: Mab): 1:50, 1: 10, 1: 2, 1: 1, and 1: 0.25 Neutrophils treated with cytochalasin B (100 μl / tube) were added and incubated for 2 hours at 25 ° C. The tubes were centrifuged (210 X g, 2- 8 ° C) for 10 minutes, and the supernatants were transferred to 96-well tissue culture plates (30 μl / well). The substrate reserve of elastase, methoxysuccinyl-alanyl-alanyl-propyl 10 mM l-valyl-p-nitroanilide (Calbiochem, La Jolla, California) in DMSO, was prepared and stored at 2-8 ° C. The solution of the elastase substrate (1.2 M substrate, sodium chloride 1.2 14 (Mali inukiodt, Pa? Ir, Kentucky), FEPES 0.12 M, pH 7.2 in distilled water was added (170 μl / well) to the supernatants, and incubated for 0.5 to 2 hours at 37 ° C (until an optical density of the control of 1.0 was reached.) Absorbance was measured at 405 nm (plate reader SLT 340 ATTC, SLT Lab Instruments, Austria).

The results are shown in Figure 1.

At a 1: 1 ratio of IL-8 to mAb 5.12.14, the antibody was able to effectively block the release of elastase from neutrophils. The hybridoma that produces the antibody . 12.14 was deposited on February 15, 1993 with the North American Species Growing Collection (American Type Culture Collection, 12301 Parklawn, Rockville, MD, U.S.A. (ATCC) and was assigned to the ATTC Accession Number HB 11553.

B. GENERATION AND CHARACTERIZATION OF ANTIBODIES MONOCLONABLE AGAINST RABBIT IL-8 Antibodies against rabbit IL-8 were generated essentially in the same process as the human AN + Y-IL-S antibodies using rabbit II-8 as the immunogen .amably provided by C. Broaddus; see also Yoshimura et al J. Immunol. 146: 3483 (1991)). The antibody was characterized as described above for binding to other cytokines coated on ELISA plates; no measurable link was found for MGSA, fMLP, C5a, b-TG, TNF, PF4, or IL-1. The antibody-producing hybridoma 6G4.2.5 was deposited on September 28, 1994 with the North American Species Crop Collection (American Type Culture Collection, 12301 Parklawn, Rockville, MD, USA (ATCC) and assigned ATTC number. Access HB 11722. The recombinant human-urine chimeric Fabs for 5.12.14 and 6G4.2.5 were constructed as described below: A chimeric Fab 6G4.2.5 is compared to a chimeric Fab . 12.14, in detail, later. 1. INHIBITING THE LINK OF IL-8 TO THE HUMAN NEUTRÓFILOS BY FAB 5.12.14 AND FAB 6G4.2.5 The ability of the two chimeric Fabs, the rab of 5.12.14 and ca Lab 6G? 5. to effectively bind to IL-8 and prevent IL-8 from binding to the IL-8 receptors on human neutrophils , was determined by performing a competition binding test, which follows the calculation of the IC50, which is the concentration required to achieve 50% inhibition of the IL-8 binding.

Human neutrophils (5 X 105) were incubated for 1 hour at 4 ° C with 125 I-IL-8 0.5 ° nm, in the presence of various concentrations (0 to 300 nM) of 5.12.14-Fab, 6G4.2.5-Fab, an isotype control (4D5-Fab) or unlabeled IL-8. After incubation, the unbound 125I-IL-8 was removed by centrifugation through a solution of 20% sucrose and 0.1% bovine serum albumin in phosphate buffered saline, and the amount of 125 I-IL-8 bound to the cells was determined by counting the cell buttons in a gamma counter. Figure 2 demonstrates the inhibition of the binding of 125 I-IL-8 to neutrophils by unlabeled IL-8. Figure 3 demonstrates that a negative isotype corresponding to Fab does not inhibit the binding of 125I-IL-8 to human neutrophils. The anti-IL-8 Fabs, Fab 5.12.14 (Figure 4) and Fab 6G4.2.5 (Figure 5) were able to inhibit the binding of 12-I-IL-8 to human neutrophils, with a JC50 averages 1.6 nK and .o nM, respectively. 2. INHIBITION OF THE NEUTRÓFILOS QUIMIOTAXIS MEDIATED BY IL-8, BY 5.12.14 FAB AND 6G4.2.5-FAB Human neutrophils were isolated, counted and resuspended at 5 x 106 cells / ml in Hank's balanced salt solution (abbreviated HBSS, without calcium and magnesium) with 0.1% bovine serum albumin. Neutrophils were labeled by the addition of calcein AM (Molecular Probe, Eugene, OR) at a concentration of 2.0 μM. After a 30 minute incubation at 37 ° C, the cells were washed twice with HBSS-BSA and resuspended at 5 x 10 6 cells / ml. The chemotaxis experiments were carried out in a 96 well chamber of Neuro Probé (Cabin John, MD) model MBB96. The experimental samples (control-only buffer, IL-8 alone or In-0: tcts) were loaded onto a 96-well Polyfiltronics observation plate (Neuro Probé Inc.) placed in a lower chamber. 100 μl of neutrophils labeled with calcein AM were added, to the upper chambers and allowed to migrate through a structured filter of polycarbonate free of PVP, with porosity of 5 micrometers (Neuro Probé Inc.) towards the sample of the lower chamber. The chemotaxis apparatus was then incubated for 40 to 60 minutes at 37 ° C with 5% C02. At the end of the incubation, the neutrophils that remained in the upper chamber were aspirated and the upper chambers were washed three times with PBS. Subsequently, the polycarbonate filter was removed, the cells that did not migrate were wiped with a brush wet with PBS, and the filter was air dried for 15 minutes. The relative number of neutrophils migrating through the filter (Neutrophil migration rate) was determined by measuring the fluorescence intensity of the filter and the fluorescence intensity of the content of the lower chamber, and summing the two values together. The fluorescence intensity was measured with a CytoFlucr 2100 fluorescent plate reader (M ltipore Corp Bedford, M) configured to read a Corning 26 poz.cs plate using the excitation filter at 485-20 nm and an emission filter at 530 -25, with the sensitivity adjusted to 3. The results are shown in Figures 6 and 7. Figure 6 demonstrates the inhibition of neutrophil chemotaxis mediated by human IL-8, by the chimeric Fabs 6G4.2.5 and 5.12.14 . Figure 7 demonstrates the relative abilities of the chimeric Fabs 6G4.2.5 and 5.12.14 to inhibit neutrophil chemotaxis mediated by rabbit IL-8. 3. INHIBITION OF THE RELEASE OF ELASTASE FROM NEUTROFILES MEDIATED BY IL-8, BY VARIOUS CONCENTRATIONS OF THE FABS OF 6G4.2.5 AND 5.12.14 Blood was drawn from healthy male donors, inside heparinized syringes. Neutrophils were isolated by sedimentation with dextran, centrifugation on Lymphocyte Separation Medium (Organon Teknika, Durham, NC), and hypotonic lysis of contaminating red blood cells as described by Berman et al. (J. Cell Biochem. 183 (1993)). The final neutrophil button was either suspended or suspended at a concentration of 1 x 107 cells / ml in the assay buffer, which consisted of the Hanks Balanced Salt Solution (GIBCO, Grand Island, NY) supplemented with 1.0% BSA (fraction V , Sigma, St. Louis, MO), 2 mg / ml glucose, 4.2 mM sodium bicarbonate, and 0.01 M HEPES, pH 7.2.

The neutrophils were stored at 4 ° C for no more than 1 hour. IL-8 (10 μl) was mixed with the anti-IL-8 Fab, an isotype control Fab, or buffer (20 μl) in 1 ml polypropylene tubes and incubated in a 37 ° C water bath for 30 minutes. IL-8 was used at final concentrations in the range of 0.01 to 1000 nM in dose-response studies (Figure 8) and at a final concentration of 100 M in the experiments directed to the effects of the Fabs on the release of elastase ( Figures 9 and 10). The Fab concentrations were in the range of about 20 nM to 300 nM, resulting in molar ratios of Fab: IL-8 from 0.2: 1 to 3: 1. Cytochalasin B (Sigma) was added to the suspension of neutrophils at a concentration of 5 μg / ml (using a stock solution of 5 mg / ml constituted in DMSO), and the cells were incubated for 15 minutes in a water bath. 3 / ° C. The neutrophils treated with cytochalasin B (100 μl) were then added to the IL-8 / Fab mixtures. After an incubation of 3 hours at room temperature, the neutrophils were concentrated by centrifugation (200 x g for 5 minutes), and the aliquots of the cell-free supernatants were transferred to the 96-well plates (30 μl / well). The elastase substrate, methosuccinyl-alanyl-alanyl-propyl-valyl-p-nitroanuide (Calbiochem, La Jolla, CA), was prepared as a stock solution at 10 mM in DMSO and stored at 4 ° C. The working solution of the elastase substrate was prepared just before being used (1.2 mM elastase substrate, 1.2 sodium chloride M, HEPES 0.12, pH 7.2), and 170 μl was added to each well containing the sample. Plates were placed in a tissue culture incubator at 37 ° C for 30 minutes, or until an optical density reading for positive controls reached at least 1.0. Absorbance was measured at 405 nm using an SLT 340 plate reader (SLT Lab Instruments, Austria). The Figure demonstrates the ability of the chimeric Fabs arti-TL-8 to inhibit the release of eiatas from the neumanof loe, stimulated by human IL-8; Figure 10 demonstrates the relative abilities of chimeric anti-IL-8 Fabs to inhibit the release of elastase from human neutrophils stimulated by rabbit IL-8.

C. MOLECULAR CLONING OF THE LIGHT AND HEAVY VARIABLE REGIONS OF THE MURINE MONOCLONAL ANTIBODY 5.12.14 (ANTI-IL-8) Total RNA was isolated from 1 X 108 cells (hybridoma cell line ATCC HB-11722) using the procedure described by Chomczynski and Sacchi (Anal Biochem 162: 156 (1987)). The first-strand cDNA was synthesized by specific priming of the mRNA with the synthetic DNA oligonucleotides designed to hybridize to the regions of the murine RNA coding for the constant region of the kappa light chain or the IgG2a heavy chain (the DNA sequence of these regions are published, in Sequences of Proteins of Immunological Interest, Kabat, EA et al. (1991) NIH Publication 91-3242, V 1-3). Three primers (SEQ ID NOS: 1-6) were designed for each of the light and heavy chains, to increase the opportunities for primer hybridization and the efficiency of first-strand cDNA synthesis (Figure 13). Amplification of first-strand cDNA to double-stranded DNA (ds) was achieved using two sets of synthetic DNA oligonucleotide primers: u forward primer (SEQ ID NOS: 7-9) and a reverse primer (SEQ ID NO: 10) for the amplification of the light chain variable region (Figure 14) and a forward primer (SEQ ID NOS: 11-14) and a reverse primer (SEQ ID NOS: 15-18) for the amplification of the heavy chain variable region (Figure 15). The N-terminal sequence of the first eight amino acids from either the light or heavy chains of 5.12.14 was used to generate a murine, putative DNA sequence corresponding to this region. (A total of 29 amino acids were sequenced from the N-terminus of the light chain and heavy chain variable regions, using the technique of protein isolation by Ed an degradation). This information was used to design the forward amplification primers, which were degenerated in the third position for some codons, to increase the chances of primer hybridization to the natural urxro DNA codons, and also included the unique restriction site, Mlul, for the forward primer of the light chain variable region and the forward primer of the heavy chain variable region, to facilitate ligation to the 3 'end of the STII element in the cloning vector. The reverse amplification primers were designed to anneal with the murine DNA sequence corresponding to a portion of the constant region of the light or heavy chains, near the variable / constant junction. The inverse primer of the variable region of the light chain contained a single BstBI restriction cycle, and the reverse primer of the variable region of the heavy chain contained a unique Apal restriction site for ligation to the 5 'end of either the region light constant of IgGl or constant heavy of IgGl, human, in the vectors, pB13.1 (light chain) and pB14 (heavy chain). The polymerase chain reaction using these groups of primers produced DNA fragments of approximately 400 base pairs. The cDNA coding for the light chain variable region 5.12.14, was cloned into the vector pB13.1, to form pA51214V1, and the heavy chain variable region of 5.12.14 was cloned into the vector pB14, to form pA5l214VH . The ALNc intakes were characterized by DNA sequencing and are presented in the DNA sequence (SEQ ID NO: 19) and the amino acid sequence (SEQ ID NO: 20) of Figure 16 (murine light chain variable region) and in the DNA sequence (SEQ ID NO: 21) and the amino acid sequence (SEQ ID NO: 22) of Figure 17 (murine heavy chain variable region).

D. VECTOR CONSTRUCTION 5.12.14 FAB In the initial construction, pA51214VL, the amino acids between the end of the murine light chain variable sequence of 5.12.14 and the single cloning site, BstBI, in the constant light sequence of human IgGl, were of murine origin corresponding to the first 13 amino acids of the constant region of murine IgGl (Figure 16). Therefore, this plasmid contained a superfluous portion of the murine constant region separating the murine light chain variable region of 5.12.14 and the constant region of human light chain IgGl. This sequence of intervention could alter the amino acid sequence of the chimera and more likely to produce a folded inco-x-positive Fab. This problem was faced by immediately truncating the cDNA clone after A109 and relocating the BstBI site in the variable / constant junction by the polymerase chain reaction. Figure 18 shows the amplification primers used to make these modifications. The forward primer, VL. front (SEQ ID NO: 23), was designed to be coupled to the last five amino acids of the STII signal sequence, including the Mlul cloning site, and the first four amino acids of the murine light chain variable sequence of 5.12.14. The sequence was altered from the original cDNA in the third position of the first two codons DI (T to C) and 12 (C to T) to create a unique EcoRV cloning site which was used for subsequent constructions. The reverse primer, VL.post (SEQ ID NO: 24), was designed to be coupled to the first three amino acids of the constant light sequence of human IgGl and to the last seven amino acids of the light chain variable sequence of 5.12.14, the which included a unique BstBI cloning site. In the BstBI site addition process, the leotidic sequence encoding several amino acids was altered: L106 (TTG to CTT), K107 (AAA to CGA) resulting in a conservative amino acid substitution for arginine, and R108 (CGG a AGA). The PCR product coding for the light chain variable sequence of 5.12.14, modified, was then subcloned into pB13.1 in a two-part ligation. The PCR product of 5.12.14 digested with MluI-BstBI coding for the light chain variable region was ligated into the vector digested with MluI-BstBI, to form the plasmid pA51214VL '. The modified cDNA was characterized by DNA sequencing. The coding sequence for the light chain of 5.12.14 is shown in Figure 19. Likewise, the DNA sequence between the end of the heavy chain variable region and the single cloning site, Apal in the constant domain of Human IgGl heavy chain of pA51214VH, was reconstructed to change the amino acids in this area from murine to human. This was done by the polymerase chain reaction. The amplification of the murine 5.12.14 heavy chain variable sequence was achieved using the primers shown in Figure 18. The forward PCR primer (SEQ ID NO: 25) was designed to collect ioe nucleotides 867-887 in pA51214VH with address upstream of the STII signal sequence and the putative cDNA sequence coding for the heavy chain variable region, and including the single Spel cloning site. The reverse PCR primer (SEQ ID NO: 26) was designed to couple the last four amino acids of the heavy chain variable sequence of 5.12.14 and the first 6 amino acids corresponding to the heavy constant sequence of human IgGl, which also included the single cloning site Apal. The PCR product encoding the modified heavy chain variable sequence of 5.12.14 was then subcloned into the expression plasmid, pMHM24.2.28 in a two part ligature. The vector was digested with Spel-Apal and the PCR product of 5.12.14 digested with Spel-Apal coding for the heavy chain variable region, was ligated into it to form the plasmid pA51214VH '. The modified cDNA was characterized by DNA sequencing. The modification sequence for the heavy chain of 5.12.14 is shown in the DNA sequence (SEQ ID NO: 29) and the amino acid sequence (SEQ ID NO: 30) of Figures 20A-20B. The first expression plasmid, pantilL-8.1, encoding the chimeric Fab of 5.12.14, was prepared by digestion of pA51214VH 'with EcoRV and Bpull02I, to replace the EcoRV-Bpull02I fragment with an EcoRV-Bpull02I fragment coding for the light chain variable region of 5.12.14, murine, of pA51214VL '. The resulting plasmid thus contained the murine-human variable / constant regions of the light and heavy chains of 5.12.14. The preliminary analysis of Fab expression using pantyIL-8.1, showed that the light and heavy chains were produced intracellularly but very little was secreted into the periplasmic space of E. coli To correct this problem, a second expression plasmid was constructed. The second expression plasmid, panti.IL-8.2, was constructed using the plasmid, pmyl87, as the vector. The pantiIl-8.2 plasmid was made by digestion of pmyl87 with MluL and Sphl, and the fragment of Mlul (partial) -Sphl coding for murine-human chimeric Fab of 5.12.14, murine, of pantyIL-8.1, was ligated into East. The resulting plasmid thus contained the murine-human variable / constant regions of the light and heavy chains of 5.12.14. The IL-8.2 plasmid was deposited on February 1, 1995 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, U.S.A. (ATCC) and the ATCC assigned him the accession number ATCC 97056.

E. MOLECULAR CLONING OF THE LIGHT AND REGIONS HEAVY VARIABLES OF THE MONOCLONAL ANTIBODY MURINE 6G4.2.5 Total RNA was isolated from 1 x 108 cells (Hybridoma cell line 6G4.2.5) using the procedure described by Chomczynski and Sacchi (Anal Biochem 162: 156 (1987)). The first-strand cDNA was synthesized by specific priming of the mRNA with the synthetic DNA oligonucleotides designed to hybridize to the regions of the murine RNA that codes for the constant region of the kappa light chain or the heavy chain of IgG2a (the DNA sequence of these regions are published in Sequences of Proteins of Immunological Interest, Kabat et al. (1991) NIH Publication 91-3242, V 1-3). Three primers (SEQ ID NOS: 31-36) were designed for each of the light and heavy chains, to increase the opportunities for primer hybridization and efficiency of the first strand cDNA synthesis (Figure 21). Amplification of first-strand cDNA to double-stranded DNA (ds) was carried out using two sets of synthetic DNA oligonucleotide primers: a forward primer (SEQ ID NOS: 37-39) and a reverse primer (SEQ ID NO; 40) for the amplification of the light chain variable region (Figure 22) and a forward primer (SEQ ID NOS: 41-42) and a reverse primer (SEQ ID NOS: 43-46) for the amplification of the variable region of heavy chain (Figure 23). The N-terminal sequence of the first eight amino acids from either light or heavy chains of 6G4.2.5 was used to generate a murine, putative DNA sequence corresponding to this region. (A total of 29 amino acids were sequenced from the N-terminus of the light chain and heavy chain variable regions, using the protein sequencing technique by Edman degradation). This information was used to design the forward amplification primers which were made degenerate in the third position for some codons, to increase the probabilities of primer hybridization to the murine, natural DNA codons, and also included the i .. single restriction Nsil, for the forward primer of the light chain variable region, and the unique restriction site, Mlul, for the forward primer of the heavy chain variable region, to facilitate ligation to the 3 'end of the STII element in the vector, pchimFab. The reverse amplification primers were designed to anneal with the murine DNA sequence corresponding to a portion of the constant region of the light or heavy chains near the variable / constant junction. The reverse chain primer of the light chain variable region contained a single Muñí restriction site and the heavy chain variable region reverse primer contained a unique Apal restriction site, for ligation to the 5 'end of the light chain regions. constant IgGI or heavy constant of human IgGl, in the vector, pchimFab. The polymerase chain reaction using these primer groups produced DNA fragments of approximately 400 base pairs and were individually cloned into the vector, pchimFab, to form p6g425VL and p6G425VH. The cDNA inserts were characterized by DNA sequencing and are presented in the DNA sequence 'SEQ ID NO: 47) and the amino acid sequence (SLC ID NC: 48) of Figure 24 (murine light chain variable region) and the DNA sequence (SEQ ID NO: 49) and the amino acid sequence (SEQ ID NO: 50) of Figure 25 (murine heavy chain variable region).

F. CONSTRUCTION OF A QUIMÉRICO FAB VECTOR OF 6G4.2.5 In the initial construct, p6G425V1, the amino acids between the end of the murine light chain variable sequence of 6G4.2.5 and the single cloning site, Muñí, in the constant light sequence of human IgG1, were of murine origin. These amino acids must correspond to the amino acid sequence of human IgGl to allow proper folding of the chimeric Fab. Two murine amino acids, D115 and S121, differed dramatically from the amino acids found in the curls of the ß strands of the constant domain of human IgGl, and were converted to the appropriate human amino acid residues, V115 and F121, by site-directed mutagenesis using the primers (SEC ID NOS: 51-54) shown in Figure 26. These specific phaeton mutations confirmed by DNA sequencing and the modified plasmid was named p6G425V1 '. The coding sequence is shown in the DNA sequence (SEQ ID NO: 55) and the amino acid sequence (SEQ ID NO: 56) of Figures 27A-27B.

Similarly, the DNA sequence between the end of the "heavy chain" variable region and the single cloning site, Apal, in the constant domain of the human IgG1 heavy chain of p6G425VH, was reconstructed to change the amino acids in This area from murine to human, this process was facilitated by the discovery of a BstEII site near the end of the heavy chain variable region.This site and the Apal site were used for the addition of a synthetic piece of DNA that codes for The corresponding human IgG amino acid sequence The synthetic oligonucleotides shown in Figure 26 were designed as complements to each other to allow the formation of a 27-base pair DNA double-stranded piece. three parts because the plasmid, p6G425VH contained an additional BstETI site within the vector sequence. Jn fragment of 5309 base pairs of p6G425VH igerido with Mlul-Apal was ligated to a fragment of 388 base pairs that possesses the variable region of heavy chain 6G4.2.5 and a fragment of synthetic DNA of 27 base pairs that codes for the first 6 amino acids of the constant region of IgGl human to form the plasmid p6G425VH '. The insertion of the synthetic piece of DNA was confirmed by DNA sequencing. The coding sequence is shown in the DNA sequence (SEQ ID NO: 57) and the amino acid sequence (SEQ ID NO: 58) of Figures 28A-28B. The expression plasmid, p6G425chim2, encoding the 6G4.2.5 chimeric Fab, was prepared by digestion of p6G425chimVL 'with Mlul and Apal to remove the heavy chain variable region HPC4 STII-murine and replace it with an Mlul-Apal fragment that encodes the variable region of the heavy chain of 6G4.2.5 STII-murine of p6G425chimVH '. The resulting plasmid thus contained the murine-human variable / constant regions of the light and heavy chains of 6G4.2.5. Plasmid p6G425chim2 was deposited on February 10, 1995 with the American Type Culture Collection, 12301 Parklawn isre, Rockville, MP, rj.S.A. (ATCC) and ^ .a A1CC I assigned Accession No. 97055.

G. CONSTRUCTION OF HUMANIZED VERSIONS OF ANTIBODY 6G4.2.5 ANTI-IL-8 The murine cDNA sequence information obtained from the hybridoma cell line, 6G4.2.5, was used to construct recombinant humanized variants of the murine anti-IL-8 antibody. The first humanized variant, F (ab) -I, was elaborated by grafting the synthetic DNA oligonucleotide primers coding for the murine CDRs of the heavy and light chains onto a phagemid vector, pEMXl (Werther et al., J. Immunol, 1_57_: 4986-4995 (1996)), which contains a light chain I of human subgroup 6 and a heavy chain of subgroup III of human IgG (Figure 29). The amino acids corresponding to the structure of the antibody that were pet? Importantly, the maintenance of the recessive conformations for high affinity binding to IL-8 by regions of complementarity determination (CDR) were identified when comparing the molecular models of the variable domains (F (ab (-1) of 6G4.2.5 murine and humanized, using the methods described by Carter et al., PNAS 89: 4285 (1992) and Eigenbrot et al., J. Mol. Biol. 229: 969 (1993). ab) 2-9) were constructed from the information obtained from these models and are presented in the following Table 4. In these variants, the site-directed mutagenesis methods of Kunkel, Proc. Nati, Acad. Sci USA were used. ), 8: 2: 488 (1985) to exchange specific human structural residues with their corresponding murine counterparts 6G4.2.5 Subsequently, the complete coding sequence of each variant was confirmed by DNA sequencing. The expression and purification of each variant of F (ab) was carried out as previously described by Werther et al., Supra, with the exception that the chicken egg white lysozyme was omitted from the purification protocol. The variant antibodies were analyzed by SDS-PAGE, electrospray mass spectroscopy or e, and amino acid analysis.

Table 4 - Variants of 6G425 Humanized IC50C Variant Verdón Template Changes * Purpose 'Mean Deviation N Standard F (ab) -1 version 1 Barter CDR 63.0 12.3 4 F (ab 2 version 2 F (ab) -1 PheH67 Wing packing coa 1 10066..00 17.0 2 CDRH2 F (ab) -3 version 3 F (ab) -1 AigtpiVal packing with 79.8 42.2 CDRs Hl, H2 F (ab ) -4 version 6 F (ab> IleH69e »packaging with 44.7 9.0 CDRH2 F (ab 5 version 7 F (ab l LeuW &Wing packaging with 52.7 31.0 CDRs Hl, H2 F (ab) -6 version 8 F (ab) -1 üeH69¿eu combine 34.6 6.7 LeuH78A¡? F (ab 4 and -5 F (ab 7 version 16 F (ab) -6 LeuH80 to pack with 38.4 9.1 CDRHl Na 'j-8 í (30f Ar ilg /, .mpa ^. '< «m? e ?, n > with 14.t CDRH2 F (ab) -9 version 11 F (ab) -6 Gi HßGln packing with 19.0 5.1 CDRH3 F (ab) 11.4 7.0 13 chimeric11 F (ab) rhu4D5 '> 200μM a Changes in amino acids made in relation to the template used The murine residues are in bold italics and the numbering of the residues is according to Kabat et al. b The purpose for making the changes based on interactions observed in the molecular models of the humanized and murine variable domains. c Concentration in nM of the variant, necessary to inhibit the binding of iodinated IL-8 to human neutrophils in the competitive binding assay. d F (ab) chimeric is an F (ab) possessing the heavy and light chain variable domains, murine, fused to the constant domain kl of light chain, human, and the constant domain I of subgroup III of human heavy chain, respectively . e rhu4D5F (ab) is the same isotype as the F (ab) s of 6G425 humanized, and is a humanized anti-HER2 F (ab) and therefore should not bind to IL-8.

The first humanized variant, F (ab) - 1, was a CDR exchange not to the terada in which all the murine CDR amino acids defined by x-ray crystallography and by sequential hypervariability were transferred to the human structure. . When the purified F (ab) was tested for its ability to inhibit the binding of 1 5IL-8 to human neutrophils, according to the methods described in Section (B) (1) above, a reduction of 5.5 times was evident. in the link affinity, as shown in Table 4 above. Subsequent versions of F (ab) -1 were engineered to conform the three-dimensional structure of the CDR loops or curls to a more favorable conformation for binding to IL-8. The relative affinities of the F (ab) variants, determined from competition binding experiments using human neutrophils, as described in Section (B) (1) above, are presented in Table 4 above. A slight decrease in the binding of xL-8 (< 2 times) was observed for F (ab) -2-3, while only slight increases in the IL-8 bond were noted for F (ab) 3-5 . The variant F (ab) -6 had the highest increase in affinity for IL-8 (approximately 2-fold), showing a binding affinity for IL-8 of 34.6 nM, compared to binding affinity to IL-8. of F (ab) -1, 63 nM. The substitutions of murine Leu in H69 and murine Ala by Leu in H78 are predicted to influence the packing of the Hl and H2 CDRs. Additional structural substitutions using the F (ab) -6 variant as a template were performed to bring the binding affinity closer to that of chimeric F (ab). The binding experiments did not reveal change in affinity for F (ab) -7 (38.4 nM) but a significant improvement in affinity for F (ab) -8/9 of 14 nM, and 19 nM, respectively. By analyzing a computer-generated three-dimensional model of the anti-IL-8 antibody, we hypothesized that the substitution of murine Lys for Arg in H38 in F (ab) -8 influences CDR-H2, while a change in H6 of murine Gln by Glu in F (ab) -9 affects CDR-H3. Examination of the human antibody sequences with respect to amino acid variability revealed that the frequency of Arg in residue H38 is >; 99%, while residue H6 is either Gln approximately 20% or Glu approximately 80% (Kabat et al., Sequences of Proteins of Immunological Interest 5th Ed. (1991)). Therefore, to reduce the likelihood of eliciting an immune response to the antibody, F (ab) -9 over F (ab) -8 was chosen for further affinity maturation studies. The variant F (ab) -9 was also tested for its ability to inhibit chemotaxis mediated by IL-8 (Figure 30). This antibody was able to block the migration of neutrophils induced by human wild-type IL-8, human monomeric IL-8 and Rhesus monkey IL-8 with IC50 = s of approximately 12 nM, 15 nM, and 22 nM, respectively , in IL-8-mediated neutrophil chemotaxis inhibition assays performed as described in Section (B) (2) above. The amino acid sequence for variant F (ab) -8 is provided in Figure 31c. F (ab) -8 was found to block chemotaxis mediated by human and rhesus IL-8 with IC50 = s of 12 nM and 10 nM, respectively, in the inhibition assays of neutrophil chemotaxis mediated by IL-8, performed as described in Section (B) (2) above.

H. CONSTRUCTION OF A GENE FUSION PROTEIN III ANTI-IL-8 FOR THE VISUAL REPRESENTATION OF THE FAGO AND THE ALTAINA EXPLORATION MUTAGENESIS An expression plasmid, pPh6G4, Vll, which codes for a fusion protein (the heavy chain of version 11 of humanized antibody 6G4.2.5 and the coat protein of gene II of phage M13) and the light chain of version 11 of the humanized 6G4.2.5 antibody were assembled to produce a monovalent representation of the anti-IL-8 antibody on phage particles. The construction was performed by digestion of plasmid pFPHX, with EcoRV and Apal to remove the existing irrelevant antibody coding sequence and replace it with an EcoRV-Apal fragment of 1305 base pairs from the plasmid p ^ GAVII, which codes for the antibody 6G4.2.5, version ll, anti-lL-ü. The translated sequence of the humanized 6G4.2.5 heavy chain version 11 (SEQ ID NO: 66), the peptide linker and the coat protein of gene III (SEQ ID NO: 67) is shown in Figure 31A. Plasmid pFPHX is a derivative of phGHam-3 containing an intrastructural amber codon (TAG) between the sequences encoding the DNA of human growth hormone and gene III. When transformed into an amber suppressor strain of E. coli, the codon (TAG) is read as a glutamate that produces a growth hormone (hGH) fusion protein -gen III. Similarly, in a normal strain of E. coli, the codon (TAG) is read as a stop or stop which prevents the complete translational reading of the gene III sequence and thus allows the production of soluble hGH. Plasmid pGHam-3 is described in Methods: A Companion To Methods in Enzymology, 3: 205 (1991). The final product pPh6G4.Vll was used as the template for the alanine scanning mutagenesis of the CDRs and for the construction of randomized CDR libraries of the humanized 6G4.V11 antibody. x- MUTAGÉNESIS ALANINA _DEI.

ANTIBODY HJMANIZADQ 6G4.2.5 VERSION ll The amino acid residues exposed to solvent in the CDRs of the humanized antibody 6G4.2.5 anti-IL-8 version 11 (h5G4Vll) were identified by analysis of a three-dimensional computer-generated model of the IL-8 antibody. In order to determine which amino acids exposed to solvent in the CDRs affect the binding to interleukin 8, each of the amino acids exposed by solvent was individually changed to alanine, creating a panel of mutant antibodies where each mutant contained a substitution of alanine in a simple residue exposed to the solvent. The alanine scanning mutagenesis was performed as described by Leong et al., J. Biol. Chem., 269: 19343 (1994)). The IC 50 's (relative affinities) of wild-type and mutated h6G4Vll antibodies were established using a Phage ELISA Assay described by Cunningham et al. (EMBO J. 13: 2508 81994) and Lee et al. ( Science 270: 1657 (1995)). The test measures the ability of each ant body to bind to IL-? coated on a plate of 9 wells, in the presence of various concentrations of free IL-8 (0.2 to 1 μM) in solution. The first step of the assay requires that the phage concentrations possessed by the wild-type and mutated antibodies be normalized, allowing a comparison of the relative affinities of each antibody. Normalization was achieved by phage titration on the plates coated with IL-8 and establishing its EC o- The 96-well tie plates coated with sulfhydryl (Corning-Costar, Wilmington, MA) were incubated with a 0.1 mg solution / ml of K64C IL-8 (Lysine 64 is replaced with Cysteine to allow the formation of a disulfide bridge between the free thiol group of K64C IL-8 and the sulphydryl-coated plate, which results in the placement of the link to the receptor 11-8 towards the interface of the solution) in phosphate buffered saline (PBS) of pH 6.5, containing 1 mM EDTA for 1 hour at 25 ° C followed by three washes with PBS and a final incubation with a PBS solution containing 1.75 mg / ml of L-cysteine-HCl and 0.1 M NaHC03 to block any reactive sulfhydryl groups , free, on the plate. The plates were washed once more and stored covered at 4 ° C with 200 μl of PBS / well. The phage showing either the reference antibody, h6G4Vll, or the h6G4Vll mutant antibodies were developed and harvested by precipitation with PEG. The phages were resuspended in 500 μl of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and 100 mM sodium chloride, and kept at 4 ° C for no more than 3 hours. An aliquot of each phage was diluted to a quarter in PBS containing 0.05% Tween 20 (BioRad, Richmond, Ca.) And 0.5% RIA grade BSA (Sigma, St, Louis, Mo.) (PBB) and added to plates coated with blocked IL-8 for at least 2 hours at 25 ° C with 50 mg / ml of skimmed milk powder in 25 mM carbonate buffer, pH 9.6. The phages were then serially diluted in 3 gradual steps of descending plaque from well A to well H. Plates were incubated for 1 hour at 25 ° C, followed by 9 rapid washes with PBS containing 0.05% Tween 20 (PBST). The plates were then incubated with a 1: 3200 dilution of anti-phage rabbit antibody and a 1: 1600 dilution of goat anti-rabbit Fc secondary antibody, conjugated to horseradish peroxidase for 15 minutes at 25 ° C, followed by 9 quick washes with PBST. The plates were developed with 80 μl / well of OPD at 1 mg / ml (Sigma, St. Louis, Mo / in Citrate-Phosphate buffer pH 5.0, containing 0.015% H202 for 4 minutes at 25 ° C, and the The reaction was stopped with the addition of 40 μl of 4.5 M H2SO4. The plates were analyzed at an 8492 wavelength in an SLT model 340ATTC plate reader (SLT Lab Instruments) .The individual EC50 = s were determined by analysis of the data using the Kaleidagraph program (Synergy Software, Reading, Pa.) and a 4-parameter adjustment equation Phages maintained at 4 ° C were immediately diluted in PBB to reach a final concentration corresponding to their respective ECD or target 0D4T2 for the competition segment of the experiment, and assortments in a 96-well plate containing serial dilutions to one quarter each of soluble IL-8 in the range of 1 μM in well A and ending with 0.2 μM in well H. Using a 12-channel pipette, transfer They added 100 μl of the phage / IL-8 mixture to a 96-well plate coated with IL-8 and performed as described above. Each sample was made by triplicate-3 columns / sample.

Table_ 5_ - Relative Affinities (IC50) for Pathways 6G4V11 CDR Anti-IL-6, from Alanine Scan Deviation CDR Amino Acid Residue IC50 average (nM) Standard Vil Reference 11. 5 6. 4 CDR-Ll S26 6. 3 2. 9 Q27 10.2 2.4 S28 14.2 5.2 V30 29.1 12.3 H31 580.3 243.0 133 64.2 14.6 N35 3.3 0.7 T36 138.0 nd Y37 NDB nd CDR-L2 K55 24.2 14.9 V56 15.5 3.8 S57 12.4 4.0 N58 17.6 3.7 R59 nd nd CDR-L3 S96 10.8 4.4 T97 70.6 55.2 H98 8.0 1.2 V99 19.6 1.9 CDR-Hl? 28 8.6 3.1 S30 nd nd S31 7.8 2.5 H32 13.3 5.8 Y53 48.2 15.8 CDR-H2 Y50 35.6 13.0 D52 13.3 7.5 S53 6.0 3.4 N54 96.0 5.8 E56 15.8 4.5 T57 8.4 1.6 T58 11.3 1.8 Y59 9.1 3.7 Q61 12.6 6.4 K64 18.5 12.1 CDR-H3 D96 NDB nd Y97 NDB nd R98 36.6 15.3 Y99 199.5 nd N100 278.3 169.4 D102 159.2 44 W103 NDB nd FIO4 NDB nd F105 209.4 72.3 D106 25.3 21.7 Each of ours was done in triplicate / experiment.

NDB ^ No detectable link / nd = value not determined * The numbering of the residue is according to Kabat et al.

The results of the alanine scan are summarized in Table 5 above. Alanine substitutions of many of the mutant antibodies had little or no adverse effect (< 3 times) on the binding affinity for IL-8. The mutants that were found to show no detectable IL-8 (NDB) link presumably contained disruptions in the conformational structure of the antibody, conferred by the structural or hidden crucial amino acids, in the CDR. Based on the results of the examination, CDR-H3 (heavy chain, 3rd CDR) was identified as the dominant binding epitope for the binding to IL-8. The alanine substitutions in this CDR resulted in a decrease of 3 a > 26 times in the link affinity. The amino acids, Y597, Y599 and D602 are of particular interest because it was determined from the computer generated model of the anti-IL-8 antibody, that these residues were exposed to the solvent and that these residues can participate in the hydrogen bond or in charge interactions with IL-8 or other antibody amino acids, which influence either the binding to IL-8 or the conformation of the curl structure of CDR-H3. (See the model described in Figure 32). Unexpected increases in link affinity (1.8> 2.7 fold) were noted for S528 and S531 for CDR-Hl and S553 for CDR-H2. Surprisingly, a significant increase in binding affinity was observed in the alanine mutant N35A located in CDR-Ll (light chain, CDR). A 3 to 6 fold increase in affinity was observed in comparison to the wild-type h6G4Vll antibody. This increase in the binding to IL-8 could be the result of the close proximity of N35A to CDR-H3. The substitution of alanine may have imparted a slight change in the conformation of CDR-Ll, which alters the packaging interaction of the neighboring amino acid residues on CDR-H3, thereby twisting the curl of CDR-H3 to a conformation that facilitates the most appropriate contacts with IL-8. Similarly, N35A can also influence the orientation of the amino acids in CDR-Ll or their interaction directly with IL-8. Unexpected increases in affinity (approximately 2-fold) were also observed for S26 of CDR-Ll and H98 of CDR-L3.

J. CHARACTERIZATION OF THE HUMANIZED ANTIBODY 6G4V11N35A ANTI-IL-8 The soluble antibody 6G4V11N35A Fab was made by transforming an amber non-suppressor strain of E. col i, 34B8, with pPh6G4.Vll and developing the culture in medium with low phosphate content for 24 hours. The periplasmic fraction was collected and passed over a G Hi-Trap protein column (Pharmacia, Piscataway, NJ), followed by a desalination and concentration step. The protein was analyzed by SDS-PAGE, mass spectrometry and amino acid analysis. The protein had the correct size and correct amino acid composition (Figure 35). 6G4V11N35A Fab was tested for its ability to inhibit the binding of 125I-IL-8 to human neutrophils and inhibit chemotaxis of neutrophils mediated by IL-8. as described in Section (B) (1) and (B) (2) above. As shown in Figure 33, the intact murine antibody derived from hybridoma (murine 6G4 monoclonal antibody), the recombinant 6G4 murine-human chimeric Fab, versions 1 and 11 of the recombinant humanized fab, and the 6G4V11N35A Fab were found to inhibit the binding of 125 I-IL -8 to human neutrophils with an average IC50 of 5 nM, 8 nM, 40 nM, 10 nM and 3 nM, respectively. The 6G4V11N35A Fab had at least two times higher affinity than the 6G4.2.5 chimeric fab and an affinity 3 times higher than 6G4V11. As shown in Figure 34, the 6G4V11N35A Fab was found to inhibit neutrophil chemotaxis mediated by IL-8, induced by wild-type and monomeric human IL-8, and by two different animal species of IL-8. , namely, rabbit and rhesus monkey. The irrelevant isotype control Fab (4D5) did not inhibit the migration of neutrophils. The average IC50 values were 3 nM (wild-type IL-8), 1 nM (monomeric IL-8), 5 nM (rabbit IL-8), and 10 nM (rhesus IL-8).

K. CONSTRUCTION OF A LEUCINE ZIPPER OF F (ab ') 2 6G4V11N35A The production of a version of i. { ah '> 2 Fab 6G4V11N35A humanized anti-IL-8 was achieved by constructing a fusion protein with yeast GCN4 leucine zipper. The expression plasmid p6G4VHN35A. F (ab ') 2 was made by digestion of the plasmid? p6G425chim.2. fab2 with Bsal and Apal restriction enzymes to eliminate the DNA sequence encoding the murine-human chimeric Fab of 6G4.2.5 and replacing it with a Bsal-Apal fragment of 2620 base pairs from pPh6G4. V11N35A. The plasmid p6G425chim. fab2 is a derivative of pS1130 which codes for a fusion protein (the leucine zipper GCN4 fused to the anti-CD18 heavy chain) and the light chain of the anti-CD18 antibody. The expression plasmid p6G4VHN35A.F (ab ') 2 was deposited on February 20, 1996 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, U.S.A. (ATTC) and ATCC assigned Accession No. 97890. A pepsin cleavage site in the hinge region of the antibody facilitates removal of the leucine zipper leaving the two immunoglobulin monomers joined by the cysteines generated by the bridges interchain disulfides. The DNA and protein sequence of h6G VI 1N35A. F (ab ') 2 is described in Figures 35-37. An expression host cell was obtained by transforming E. coli 49D6 with p6G4VHN35A. F (ab ') 2 essentially as described in Section (II) (3) (C) above. The transformed host E. coli 49D6 (p6G4VHN35A.F (ab ') 2) was deposited on February 20, 1997 at the ATCC and assigned Accession No. 98332. The transformed host cells were grown in culture, and the F (ab ') 2 product of p6G4VHN35A was harvested from the periplasmic space of the host cells essentially as described in Section (II) (3) (F) above.

I. CHARACTERIZATION OF THE LEUCINE ZIPPER OF F (ab ') 2 6G4V11N35A HUMANIZED The Fab and F (ab ') 2 of 6G4V11N35A were tested for their ability to inhibit the binding of 125I-IL-8 to neutrophils, according to the procedures described in Section (B) (1) above. The displacement curves from a representative binding experiment performed in duplicate are described in Figure 38. The Scatchard analysis of these data shows that the F (ab ') 2 of 6G4V11N35A inhibited the binding of 125I-IL-8 to human neutrophils, with an average IC50 of 0.7 nM (+/- 0.2). This is at least a 7 fold increase in affinity compared to the intact murine hybridoma-derived antibody (average IC50 of 5 nM) and at least a 2.8 fold increase in affinity over the Fab version (average IC50 of 2 nM). ). The F (ab ') 2 of 6G4V11N35A was also tested for its ability to inhibit neutrophil chemotaxis mediated by IL-8, according to the procedures described in Section (B) (2) above. The results of a representative chemotaxis experiment performed in quadruplicate are described in Figure 39. As shown in Figure 39, F (ab ') 2 of 6G4V11N35A inhibited neutrophil chemotaxis mediated by human IL-8. F (ab ') 2 of 6G4V11N35A showed an average IC50 value of 1.5 nM versus 2.7 nM for the 6G4V11N35A Fab, which represents an approximately 2-fold improvement in the ability of the antibody to neutralize the effects of IL-8. The irrelevant isotype control Fab (4D5) did not inhibit the migration of neutrophils. In addition, the F (ab ') 2 antibody of bG4VHN35A retained its ability to inhibit neutrophil chemotaxis mediated by IL-8, by monomeric IL-8, and by two different animal species of IL-8, namely rabbit and of rhesus monkey, in neutrophil chemotaxis experiments, conducted as described above. An individual experiment is shown in Figure 40. The average IC50 values were 1 nM (monomeric IL-8), 4 nM (rabbit IL-8), and 2.0 nM (rhesus monkey IL-8).

M. RANDOM MUTAGENESIS OF THE LIGHT CHAIN AMINO ACID (N35A) IN CDR-LI OF THE HUMANIZED ANTIBODY 6G4V11 A 3 fold increase in IC50 was observed to inhibit the binding of 125I-IL-8 to human neutrophils, when the alanine was replaced by asparagine at position 35 on CDR-Ll (light chain) of the humanized 6G4V11 mAb, as described in Section (I) above. This result can be attributed to an improvement in the contact between the antigen-antibody binding interfaces as a consequence of the replacement of a nonpolar, less bulky side chain (R group) that may have altered the conformation of CDR-1,1 or CDR -H3 neighbor (heavy chain) to become more accessible for antigen coupling. Acceptance of alanine at position 35 of CDR-Ll suggested that this composition contributed to improved affinity and that an evaluation of the re-modeling of CDR loops / regions of antigen binding with other amino acids at this site was ensured . The selection of an affinity-matured version of humanized mAb 6G4.V11 (Kunkel, TA, Proc. Nati, Acad. Sci. USA, 82: 488 81995)) was achieved by random mutagenization of position 35 of CDR-Ll and building an antibody-phage library. The codon for Asparagine (N) at position 35 of CDR-Ll, was targeted for randomization to any of the 20 known amino acids. Initially, a stop template, pPH6G4.Vil-stop, was developed to eliminate the wild-type contaminant-free N35 sequence from the library. This was achieved by performing the site-directed mutagenesis (Muta-Gene Team, Biorad, Richmond, CA) of pPH6G4Vll (described in Section (H) above) to replace the codon (AAC) by N35 with a stop codon (TAA) using the primer SL.97.2 (SEQ ID NO :) (Figure 42). The incorporation of the stop codon was confirmed by DNA sequencing. Subsequently, the single-stranded DNA containing uracil, derived from E. col i CJ236 transformed with the stop template, was used to generate an antibody-phage library following the method described by Lowman (Methods in Molecular Biology, 87 Chapter 25: 1-15 (1997). this library was predicted to produce a collection of antibodies containing one of the 20 amino acids known at the N35 position in CDR-Ll. The amino acid substitutions were achieved by site-directed mutagenesis using the degenerate oligonucleotide primer (SL: 97.3) with the NNS sequence (N = A / G / T / C; S = G / C;) (SEQ ID NO :) (Figure 42) This use of the codon could allow the expression of any of the 20 amino acids, including the codon of amber arrest (TAG) The collection of antibody-phage variants was transfected in E. coli strain XL-1 blue (Stratagene, San Diego, CA) by electroporation and developed at 37 ° C overnight to amplify the library . The selection of the humanized 6G4V11 strong binding Fabs was achieved by panoramic observation of the library on the 96-well plates coated with IL-8 as described in Section (I) above. Before panoramic observation, the phage / library number was normalized to 1.1 x 1013 phages / ml (which produces a maximum reading of OD27o = 1 unit of DO) and the plates coated with IL-8 were incubated with blocking solution (25 mN carbonate buffer containing 50 mg / ml of skimmed milk) for 2 hours before the addition of the phage (each classification used eight plates coated with IL-8 / library). After the blocking and washing steps, each classification began with the addition of 100 μl of antibody-phage (titrated at 1.1 x 1013 phage / ml) to each of the eight wells coated with IL-8, followed by an incubation of 1 hour at 25 ° C. The non-specifically bound antibody-phage was removed by rapid washes with PBS-0.05% Tween 20 (PBS-Tween). For the classification * 1, a high-demand wash (100 μl of PBS-Tween / well for 10 minutes at 25 ° C) was used to capture a small portion of the strong antibody-phage bond bound to the immobilized IL-8. Antibody-phage variants specifically linked to IL-8 were eluted with 100 μl / well of 200 mM Glycine, pH 2.0, for 5 minutes at 25 ° C. The eluted antibody-phage variants from the 8 wells were then combined and neutralized with Tris-HCl pH 8.0 (1/3 of the elution volume). The phage was titled and propagated as described in Section (I) previous. The demand for the washings was successively increased with each round of panning depending on the percentage recovery of the phage at the end of a classification. The washing conditions were as follows: classification # 2 (4 intervals of 15 minutes, total time = 60 minutes) and classification # 3 (any of # 3a: 8 intervals of 15 minutes or # 3b: 12 intervals of 10 minutes; total = 120 minutes). The total number of recovered phage was progressively reduced after each classification, suggesting that no weak linkers or linkers were being selected. The recovery of the negative control (the antibody-phage arrest variant) was constant throughout the panoramic observation (approximately 0.0001 to 0.00001 percent). Eighteen random variables of the # 3 classification were analyzed by DNA sequencing to search for an amino acid consensus at position 35 of C? R-Ll. The data presented in Figure 43A showed that Glycine occupied position 35 in 33% of the sequenced variants. However, after correction for the number of NNS / amino acid codon combinations, the frequency of Glycine was reduced to 16.6%. Glutamic acid was represented with the highest frequency (22%) followed by Aspartic Acid and Glycine (16.6%). The recovery rates of wild type Asparagine and substituted Alanine were only 5.6%. Interestingly, the high frequency of Glycine may suggest that a much wider range of conformations may be allowed for the curl or round of CDR-Ll, which may be attributed to the reduction in steric hindrance of link angle pairing (f-?) as a result of the simple hydrogen atom on the side chain. Conversely, glutamic acid in position 35 can restrict the flexibility of the curl by imposing less freedom of rotation, imposed by the more rigid and voluminously charged polar side chain. The soluble Fab's of the affinity-matured variants (N35G, N35D, N35E and N35A) were elaborated as described in Section (J) above, to evaluate their ability to block the binding to IL-8. As shown in Figure 34B, it was found that variants N35A, N35D, N35E and N35G inhibit the binding of 125I-IL-8 to human neutrophils with an IC5o of approximately 0.2 nM, 0.9 nM, 0.1 nM and 3.0 nM, respectively. All affinity-matured variants showed an improvement in the binding of IL-8 in the range of 3 to 100 times compared to the monoclonal antibody 6G4V11 humanized. The affinity-matured variant, 6G4V11N35E, was 2 times more potent in blocking the binding of IL-8 to human neutrophils than the alanine screening variant, 6G4V11N35A. Kinetic and equilibrium measurements of the 6G4V11N35A and 6G4V11N35E variants were determined using the KinEXAMR automated immunoassay system (Sapidyne Instruments Inc., Idaho City, ID) as described by Blake et al., J. Biol. Chem, 271: 27677 ( nineteen ninety six). The procedure for the preparation of the antigen coated particles was modified as follows: 1 ml of activated agarose spheres (Reacti-Gel 6X; Pierce, Rockford, IL) were coated with antigen in 50 mM Carbonate buffer or 9.6 H, containing 20 μl / ml of human IL-8 and incubated with gentle agitation on a shaker overnight at 25 ° C. The spheres coated with IL-8 were then washed twice with 1 M Tris-HCl, pH 7.5 to inactivate any non-reactive groups on the spheres, and blocked with Superblock (Pierce, Rockford, IL) for 1 hour at 25 ° C to reduce the non-specific link The spheres were resuspended in assay buffer (0.1% bovine serum albumin in PBS) to a final volume of 30 ml. A 550 μl aliquot of the suspension of spheres coated with IL-8 was used each time to package a fresh 4 mm high column in the KinEXA observation cell. The amount of unbound antibody from the antibody-antigen mixtures captured by the spheres coated with IL-8 in the equilibrium and kinetic experiments, was quantified using a secondary antibody, fluorescently labeled. The murine 6G4.2.5 was detected with goat anti-mouse IgG, F (ab ') 2 R-PE AffiniPure, secondary antibody specific to the Fe fragment (Jackson Immuno Research Laboratories, West Grove, PA) and affinity-matured N35A, humanized (Fab and F (ab ') 2) and Fab of N35E were detected with a secondary anti-human donkey IgG antibody (H + L) of F (ab') 2 R-PE AffiniPure (Jackson Imiuno eseerch Laboratories, West Grove, PA); both at a dilution of 1: 1000. Equilibrium measurements were determined by incubating a constant amount of anti-IL-8 antibody (0.005 μg / ml) with various concentrations of human 11-8 (0, 0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625 , 1.25, 2.5 nM). The antibody-antigen mixture was incubated for 2 hours at 25 ° C to allow the molecules to reach equilibrium. Subsequently, each sample was passed over a pack of spheres coated with fresh IL-8, in a KinEXA observation cell at a flow rate of 0.5 ml / minute for a total of 9 minutes / sample. The equilibrium constant (Kd) was calculated using the software (software) provided by Sapidyne Instruments Inc. The rates of association (ka) and dissociation (kd) were determined by co-incubation of a constant amount of antibody and antigen, and by measuring the amount, of un-complexed anti-IL-8, bound to the spheres coated with IL-8, over time. The concentration of antibody used in the kinetic experiments was identical to that used in the equilibrium experiment described above. In general, the amount of human IL-8 used was the concentration derived from the binding curves of the experiment at equilibrium which resulted in 70% inhibition of anti-IL-8 that binds to the beads coated with IL-8. The measurements were taken every 15 minutes to collect approximately nine data points. The ka was calculated using the computer equipment (software) provided by Sapidyne Instruments, Inc. The secondary velocity was determined using the equation: kd = Kd / ka. Figure 44 shows the equilibrium constants (Kd) for the Fab's of the affinity matured variants 6G4V11N35E and 6G4V11N35A that were approximately 54 pM and 114 pM, respectively. The improvement in the affinity of the Fab of 6G4V11N35E for IL-8 can be attributed to a twice faster rate of association (Kon) of 4.7 x 106 for the Fab of 6G4V11N35E versus 2.0 x 166 for the F (ab ') 2 of 6G4V11N35A (The Kd of F (ab ') 2 of 6G4V11N35A and the Fab of 6G4V11N35A are similar). The dissociation rates (K0ff) were not significantly different. Molecular modeling suggests that the substitution of Asparagine with glutumic acid can either affect the interaction of the antibody with IL-8 directly or indirectly, by neutralizing the charge of the neighboring residues R98 (CDR-H3) or K50 (CDR-L2 ) in the CDRs to facilitate contact with IL-8. Another effect may be the formation of a more stable curl conformation for CDR-Ll, which could have facilitated more appropriate contacts of other CDR-Ll curl residues with IL-8. The DNA sequences (SEQ ID NO :) and amino acids (SEQ ID NO :) of p6G4VHN35E. Fab showing the substitution of Asparagine to glutamic acid in the light chain, are presented in Figure 45.

N. CHARACTERIZATION OF THE FAB OF 6G4V11N35E VARIANT ANTI-IL-8, HUMANIZED The affinity matured Fab variant, 6G4V11N35E was tested for its ability to inhibit neutrophil chemotaxis mediated by IL-8 as described in Section (B) (2) above. The reusable 96-well chemotaxis chamber described in Section (B) (2) was replaced with the endotoxin-free disposable chemotaxis chambers containing 5 micron PVP-free polycarbonate filters (ChemoTxl 01-5, Neuro Probé, Inc, Cabin John, MD). As illustrated in Figure 46, the N35E variant effectively blocks neutrophil chemotaxis mediated by IL-8, induced by a 2 nM stimulus from either rabbit or human IL-8. In fact, the level of inhibition at antibody concentrations between 3.7 nM - 33 nM was not significantly different from the buffer control indicating that the N35E variant could completely inhibit this response. ICso's for rabbit and human IL-8 were approximately 2.8 nM and 1.2 nM, respectively. The irrelevant isotype control Fab (4D5) did not inhibit the migration of neutrophils, indicating the results observed for the affinity matured variant, N35E, is specific for 11-8. 0. CONSTRUCTION OF THE LEUCINE ZIPPER 6G4V11N35E F (ab ') 2 HUMANIZED An F (ab ') 2 expression plasmid for 6G4V11N35E was constructed using methods similar to those described in Section (K) above. The expression plasmid p6G4VHN35E.F (ab ') 2, was made by digestion of the p6G4VHN3 A. F (an') 2 plasmid depicted in Section (K) above with the restriction enzymes Apal and Ndel to isolate a fragment of 2805 base pairs coding for the leucine zipper GCN4 of the heavy chain constant domain, and ligating it to an Apal-Ndel fragment of 3758 base pairs of the clone showing the phage PPH6G4V11N35E (which codes for the Fab of 6G4V11N35E) obtained as described in Section (M) previous. The integrity of the entire coding sequence was confirmed by DNA sequencing.

P. CONSTRUCTION OF THE EXPRESSION PLASMIDE OF HUMANIZED IgG 6G4V11N35A, OF COMPLETE LENGTH The full-length IgGi version of the humanized anti-IL-8 6G4V11N35A variant was made using a DHFR-Intron dicistronic expression vector (Lucas et al., Nucleic Acids Res., 24: 1774-1779 (1996)). which contained the full-length recombinant murine-human chimera of the anti-IL-8 monoclonal antibody 6G4.2.5. The expression plasmid coding for the humanized variant 6G4V11N35A was assembled as follows. First, an intermediate plasmid (pSL-3) was developed to release the sequence encoding the variable heavy chain of the humanized anti-IL-8 variant 6G4V11N35A to pRK56G4chim.2Vh - containing the variable heavy region of the chimeric antibody 6G4 .5 anti-IL-8. The vector pRK56G4chim. Vh was digested with PvuII and Apal to remove the heavy chain variable region of the chimeric antibody, and religated it in the 80 base pair synthetic PvuII-XhoI oligonucleotide (coding for Leu4 to Phe29 of 6G4V11N35A) (Figure 47) and a fragment Xhol-Apal of 291 base pairs from p6G4VHN35A.7 possessing the remainder of the variable heavy chain sequence of 6G4V11N35A to create pSL-3. The intermediate plasmid was used in conjunction with two other plasmids, p6G4VHN35A. F (ab ') 2 and p6G425chim2. choSD, to create the mammalian expression plasmid, p6G4VHN35AchoSD.9 (identified as p6G4VHN35A.choSD in a deposit made on December 16, 1997 with the ATCC and assigned Accession No. 209552). This expression construct was assembled into a 4-part ligature using the following DNA fragments: a 5203 base pair Clal-Blpl fragment encoding the regulatory elements of the mammalian expression plasmid (p6G425 chim2. ChoSD), a fragment Clal-Apal of 451 base pairs containing the heavy chain variable region of the humanized 6G4V11N35A antibody (pSL-3), an Apal-EcoRV fragment of 1921 base pairs that possesses the heavy chain constant region of 6G4V11N35A (p6G425chim2. choSD) and a 554 base pair EcoRV-BlpI fragment that codes for the variable and constant regions of light chain of 6G4V11N35A (p6G4Vl 1N35A.F (ab ') 2). The DNA sequence (SEQ ID NO:) of clone p6G4VHN35A. cho. SD.9 was confirmed by DNA sequencing and is presented in Figure 48.

Q. CONSTRUCTION OF HUMANIZED IgG 6G4V11N35E EXPRESSION PLASMIDE, OF COMPLETE LENGTH A mammalian expression vector for the humanized 6G4V11N35E gene was made by bartering or changing the light chain variable region of 6G4V11N35A with 6G4V11N35E as follows: the EcoRV-BlpI fragment of 7566 base pairs (devoid of the 554 base pair fragment coding for the light chain variable region of 6G4VHN35A) from p6G4VHN35A. choSD .9 was ligated to an EcoRV-Blpl fragment of 554 base pairs (coding for the light chain variable region of 6G4V11N35E) from pPH6G4VHN35E .7. The mutation at position N35 of the light chain of p6G4VHN35E. choSD.10 was confirmed by DNA sequencing.

R. CHO CELLULAR LINES STABLE FOR N35A AND N35E VARIANTS For the stable expression of humanized, final IgGl variants (6G4V11N35A and 6G4V11N35E), the DP-12 cells of hamster ovary Chinese (CHO) were transfected with the dicistronic vectors described above (p6G4VHN35A.choSD.9 and p6G4VHN35E. choSI) .10, respectively) designed to coexpress the heavy and light chains (Lucas et al., Nucleic Acid Res. 24: 1774-79 (1996) .The plasmids were introduced into the cells CHO DP12 by means of lipofection and were selected for development in GHT-free medium (Chisholm, V. High efficiency gene transfer in mammalian cells., In: Glover, DM, Hames, BD, DNA Cl oni ng 4. Mammal i an sys t ems Oxford Univ. Press, Oxford pp 1-41 (1996)) Approximately -0 non-amplified clones were randomly selected and re-plated in 96-well plates.The relative specific productivity of each colony was periodically verified using an assay of ELISA to quantify the accumulated full-length human IgG in each well, after 3 days and a fluorescent dye, Calcine AM, as a surrogate marker of number of viable cells per well, based on these data, several clones were chosen. not amplified for subsequent amplification in the presence of increasing concentrations of methotrexate. The individual clones that survive methotrexate 10, 50 and 100 nM were chosen and transferred to 96-well plates for productivity selection. One clone for each antibody (clone # 1933 aIL8.92 NB 28605/12 for 6G4V11N35A, clone # 1934 aIL8.42 NB 28605/14 for 6G4V11N35E), which showed reproducibly high specific productivity, expanded in T flasks and was used to Inoculate a rotary culture. After several passes, the cells adapted to the suspension were used to inoculate production cultures in serum-free medium containing GHT, supplemented with various hormones and protein hydrolysates. The harvested cell culture fluid containing the recombinant humanized anti-IL-8 was purified using protein A-Sepharose CL-4B. The purity after this step was approximately 88%. Subsequent purification to homogeneity was carried out using an ion exchange chromatography step. The production title of the IgG1 6G4V11N35E antibody, humanized, after the first round of amplification and IgG1 6G4V11N35A after the second round of amplification, were 250 mg / L and 150 mg / L, respectively.

S. CHARACTERIZATION OF HUMANIZED IgG 6G4V11N35A / E VARIANTS Full-length, humanized IgG variants of 6G4.2.5 were tested for their ability to inhibit the binding of 125I-IL-8 and to neutralize the activation of human neutrophils; The procedures are described in Sections (B) (1) and (B) (2) above. As shown in Figure 49, the full-length IgG1 forms of the 6G4V11N35A and 6G4V11N35E variants also inhibited the binding of 125-I-IL-8 to human neutrophils, with IC5o 's of approximately 0.3 nM and 0.5 nM, respectively. This represents a 15 to 25 fold improvement in the blocking of the IL-8 binding compared to the full-length murine mAb (IC50 = 7.5 nM). Similarly, the two anti-IL-8 variants showed equivalent neutralization capacity with respect to the inhibition of human neutrophil chemotaxis mediated by IL-8 (Figures 50A-50B). ICso's of IgGl 6G4V11N35A and IgGl 6G4V11N35E for human IL-8 were 4.0 nM and 6.0 nM, respectively, and for rabbit IL-8 were 4.0 nM and 2.0 nM, respectively. Fab isotype control, irrelevant (4D5) did not inhibit the migration of neutrophils. The affinity for IL-8 of these variants relative to mAb 6G4.2.5 was determined using KinExA as described in Section (M). Figure 51 shows the equilibrium constant (Kd) for affinity matured variants, full length IgGl 6G4V11N35E and IgGl 6G4V11N35A that were approximately 49 pM and 88 pM, respectively. The Kd for IgGl 6G4V11N35A was determined directly from the kinetic experiment. As reported with their respective Fabs, this improvement in affinity can be attributed to a two-fold increase in the speed of IgGl 6G4V11N35E (ka - 3.0 x 10") compared to that of IgGl 6G4V11N35A (ka = 8.7 x 105) In addition, these results were confirmed by a competition radioimmunoassay using iodinated human IL-8. 50 pM of 6G4V11N35A IgGl or 6G4V11N35E IgGl was incubated for 2 hours at 25 ° C with 30-50 pM of 125-I-IL- 8 and variant concentrations (0 to 100 nM) of unlabeled IL-8.The antibody-antigen mixture was then incubated for 1 hour at 4 ° C with 10 μl of a 70% suspension of Protein A spheres. (pre-blocked with 0.1% BSA). The spheres were previously shaken in a microcentrifuge and the supernatant was discarded to remove unbound 125-I-IL-8. The amount of 125-I-IL-8 specifically bound to the anti-IL-8 antibodies was determined by counting the concentrates or buttons of protein A in a gamma counter. The approximate Kd values were similar to those determined by KinEXA. The average Kd for 6G4V11N35A IgGl and 6G4V11N35E IgGl were 54 pM (18-90 pM) and 19 pM (5-34 pM), respectively (Figure 52).

T. CONSTRUCTION OF THE HUMANIZED Fab's 6G4V11N35A / E, FOR MODIFICATION BY POLYETHYLENE GLYCOL A Fab 'expression vector for 6G4V11N35A was constructed by digestion of p6G4VHN35A. F (ab ') 2 with the restriction enzymes Apal and Ndel to remove the 2805 base pair fragment encoding the constant domain of human IgGl, fused with yeast GCN4 zipper, and replacing it with the Apal fragment -Ndel of 2683 base pairs from the plasmid pCDNA.18 described in Eigenbrot et al., Proteins: Struct. Funct. Genet., 18049-62 (1994). The Apal-Ndel fragment from pCDNA.18 possesses the coding sequence for the IgG1 human heavy chain constant domain, including the free cysteine from the hinge region that was used to couple the PEG molecule. The 3758 base pair Apal-Ndel fragment (encodes for the light chain variable domain and heavy chain of 6G4V11N35A) isolated from p6G4VHN35A. F (ab ') 2 was ligated to the 2683 base pair Apal-Ndel fragment of pCDNA.18 to create p6G4VHN35A. PEG-1. The integrity of the entire coding sequence was confirmed by DNA sequencing. The translated amino acid sequences and the nucleotides of the heavy chain constant domain with the cysteine at the hinge were shown in Figure 53. A Fab 'expression plasmid for 6G4V11N35E was similarly worked up by digestion of PPH6G4V11N35E (from the Section (O) above) with the restriction enzymes Apal and Ndél to isolate the 3758 base pair Apal-Ndel DNA fragment, which possesses the intact light chain and heavy chain variable domain of 6G4V11N35E, and ligate it to the fragment of Apal-Ndel DNA of 2683 base pairs from p6G4VHN35A.PEG.l to create p6G4VHN35E. PEG-3. The integrity of the entire coding sequence was confirmed by DNA sequencing. The variant of Fab '6G4V11N35A anti-IL-8 was modified with linear methoxy-PEG-maleimide of 20 kD, methoxy-PEG-maleimide linear of 30 kD, methoxy-PEG-maleimide linear of 40 kD, or methoxy-PEG-maleimide branched 40 kD, as described below. All PEG's were commercially obtained from Shearwater Polymers, Inc. to. MATERIALS AND METHODS Purification of Fab '-SH A Fa '-'3H antibody fragment of affinity matured antibody 6G4V11N35A was expressed in E. col i developed at a high cell density in the fermenter as described by Carter et al., Bi o / Technolgy 10, 163-167 (1992). The preparation of the Fab 's-SH fragments was carried out by protecting the Fab'-SH fragments with 4' -4 '-dithiodipyridine (PDS), partially purifying the protected Fab'-PDS fragments, deprotecting the Fab '-PDS with dithiothreitol (DTT) and finally isolating the free Fab'-SH by the use of gel permeation chromatography.

Fab '-SH Protication with PDS The samples of fermentation paste were dissolved in 3 volumes of 20 mM MES, 5 mM EDTA, pH 6. 0 containing 10.7 mg of 4 '-4' -dithiodipyridine per gram of fermentation paste, resulting in a suspension with a pH close to 6.0. The suspension was passed through a homogenizer, followed by the addition of 5% PEI (p / v), pH 6, to homogenize at a final concentration of 0.25%. The mixture was then centrifuged to remove the solids and the clear supernatant was conditioned to a conductivity of less than 3 mS by the addition of cold water.

Partial purification of the Fab'-SH molecule using ion exchange chromatography The conditioned supernatant was loaded onto an ABX column (Baker) in 20 mM Month, pH 6.0. The column was washed with equilibrium buffer, followed by elution of Fab'-SH with a linear gradient of 15 column volumes from 20 mM MES, pH 6.0, to 20 mM MES, 350 mM sodium chloride. The column was checked periodically by absorbance at 280 nm, and the eluate was collected in fractions.

Deprotection of Fab'-SH Antibody Fragments with DTT The pH of the combined ABX was adjusted to 4.0 by the addition of dilute HCl. The solution with the adjusted pH was then deprotected by the addition of DTT to a final concentration of 0.2 mM. The solution was incubated for approximately 30 minutes and then applied to the Sephadex G25 gel filtration column, equilibrated with 15 mM sodium phosphate, 25 M MES, pH 4.0. After elution, the pH of the mixture was raised to pH 5.5 and immediately frozen at -70 ° C for storage or derivatized with PEG-MAL as described below.

Purifi cation to the terna ti va of Fab '-SH Alternatively, the Fab'-SH fragments can be purified using the following procedure. 100 g of the fermentation dough is thawed in the presence of 200 ml of 50 mM acetic acid, pH 2.8, 2 mM EDTA, 1 mM PMSF. After mixing vigorously for 30 minutes at room temperature, the extract is incubated with 100 mg of chicken egg white lysozyme. The DEAE fast-flow resin (approximately 100 ml) is equilibrated with 10 mM MES, pH 5.5, 1 mM EDTA in a sintered glass funnel. The osmotic shock extract containing the Fab'-SH fragment is then filtered through the resin. A column of protein G Sepharose is equilibrated with 10 M MES, pH 5.5, 1 mM EDTA and then loaded with a sample of DEAE flow from side to side. The column is washed, followed by three washes of 4 column volumes with 10 mM MES, pH 5.5, 1 mM EDTA. The Fab'-SH antibody fragment containing a free thiol is eluted from the column with 100 mM acetic acid, pH 2.8, 1 mM EDTA. After elution, the pH of the combination is elevated to pH 5.5. and immediately frozen at -70 ° C for storage or derivatized with PEG-MAL as described below.

Preparation of Fab '-S-PEG The free thiol content of the Fab'-SH preparation obtained as described above was determined by the reaction with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB analysis) according to the Creighton method in Protein Structure : A Practical Approach, Creighton, TE, ed, IRL Press (Oxford, UK: 1990), pp 155-167. The concentration of the free thiol was calculated from the increase on the absorbance at 412 nm, using e4i2 = 14.150 cm "1 M" 1 for the t ionitrobenzoate anion and a Mr = 48.6 * 90 and e28o = l. for the Fab's-0H antibody. The combined Fab'-SH and Sepharose G protein, or the combination of gel permeation and unprotected Fab'-SH, 5 molar equivalents of PEG-MAL, were added and the pH was immediately adjusted to pH 6.5 with NaOH at 10% The Fab '-S-PEG was purified using a cationic change column of 2.5 x 20 cm (Poros 50-HS). The column was equilibrated with a buffer containing 20 M MES, pH 5.5. "The coupling reaction containing the Pegylated antibody fragment was diluted with deionized water to a conductivity of about 2.0 mS.The conditioned coupling reaction was then loaded onto the equilibrated Poros 50 HS column, unreacted PEG-MAL was washed from the column with 2 column volumes of 20 M MES, pH 5.5.The Fab '-S-PEG was eluted from the column using a linear gradient from 0 to 400 mM sodium chloride, in 20 mM MES, pH 5.5, on 15 column volumes Alternatively, a Bakerbond ABX column can be used to purify the Fab '-S-PEG molecule.The molecule is equilibrated with 20 mM MES, pH 6.0 (Shock absorber A) The coupling reaction is diluted with deionized water until the conductivity is equal to that of Shock absorber A (approximately 2.0 mS) and loaded onto the column PEG-MAL unreacted is washed from the column a with 2 column volumes of 20 M MES, pH 6.0. Fab '-S-PEG is eluted from the column using a linear gradient from 0 to 100 mM of amino sulfate [(NH4) 2S04], in 20 mM MES, pH 6.0, on 15 column volumes.

Size Exclusion Chromatography The hydrodynamic or effective size of each molecule was determined using a Pharmacia Superose-6 HR 10/30 column (10 x 300 mm). The mobile phase was 200 mM sodium chloride, 50 mM sodium phosphate pH 6.0. The flow rate was 0.5 ml / minute, and the column was maintained at room temperature. The absorbance at 280 nm was checked periodically where PEG contributed little to the signal. Biorad molecular weight standards containing cyanocobalamin, myoglobin, ovalbumin, IgG, monomer and thyroglobulin dimer, were used to generate a standard curve from which the effective size of the pegylated species was estimated. b. RESULTS Size Exclusion Chromatography The effective size of each modified species was characterized using size exclusion chromatography. The results are shown in Figure 60 below. The theoretical molecular weight of anti-IL-8 Fab fragments modified with PEG 5 kD, 10 kD, 20 kD, 30 kD, 40 kD (linear), 40 kD (branched) or 100,000 kD, are shown together with the molecular weight apparent of the PEGylated fragments obtained by HPLC size exclusion chromatography. When compared to the theoretical molecular weight of the Fab '-S-PEG fragments, the apparent molecular weight (calculated by size exclusion HPLC) increases dramatically as the size of the PEG coupled to the fragments increases. The coupling of a small molecular weight PEG, for example PEG 10,000 D, only increases the theoretical molecular weight of the PEGylated antibody fragment (59,700 D) by 3 times, to an apparent molecular weight of 180,000 D. In contrast, the coupling of a higher molecular weight PEG molecule, for example 100,000 D of PEG to the antibody fragment, increases the theoretical molecular weight of the PEGylated antibody fragment (158,700 D) by 12 times at an apparent molecular weight of 2,000,000 D.

SDS -PAGE In Figure 61, the upper panel shows the size of the anti-IL-8 Fab fragments modified with PEG of molecular weight 5 kD (linear), 10 kD (linear), 20 kD (linear), 30 kD (linear) , 40 kD (linear), 40 kD (branched) or 100 kD (linear) under reduced conditions. The unmodified Fab is shown in band 2 from right to left. The heavy and light chains of the Fab had a molecular weight of approximately 30 kD as determined by PAGE. Each sample of PEGylated fragment yielded two bands: (1) a first band (attributed to the light chain) showing a molecular weight of 30 kD; and (2) a second band (attributed to the heavy chain to which the PEG is specifically coupled in the SH hinge) showing increasing molecular weights of 40, 45, 70, 110, 125, 150 and 300 kD. This result suggested that PEGylation was specifically restricted to the heavy chain of Fab's, while the light chain remained unchanged. The lower panel is reduced n PAGE showing the size of the anti-IL-8 Fab fragments modified with PEG of 5 kD molecular weight (linear), kD (linear), 30 kD (linear), 40 kD (linear), 40 kD (branched), or 100 kD (linear). The fragments PEGylates showed molecular weights of approximately 70 kD, 115 kD, 120 kD, 140 kD, 200 kD and 300 kD. The SDS-PAGE gels confirm that all Fab '-S-PEG molecules were purified to homogeneity and that the molecules differed only with respect to the size of the molecule.

PEG coupled to these.

U. SPECIFIC PENALTY OF AMINA OF FRAGMENTS F (ab ') 2 ANTI-IL-8 PEGylated F (ab ') 2 species were generated by the use of large or branched molecular weight PEGs, in order to achieve a large effective size with minimal protein modification, which may affect activity. The modification involved the chemistry of N-hydroxysuccinamide, which reacts with the primary amides (lysines and the N-terminus). To decrease the probability of modification of the N end, which is in close proximity to the CDR region, a reaction pH of 8 was used, instead of the pH of 7 commonly used. At the pH of 8.0, the amount of reactive species (charged NH3 +) could be considerably higher for the e-NH2 group of the lysines (pKa = 10.3) than for the a-NH2 group (pKa of approximately 7) of the amino terminus. For linear PEGs, a methoxy-succinimidyl derivative of an NHS-PEG was used, due to significantly longer half-life in solution (17 min at 25 ° C a. 8. 0) compared to the NHS esters of the PEG's (which have a half life of 5 to 7 minutes under the above conditions). By using a PEG that is less prone to hydrolysis, a greater degree of modification is achieved with less PEG.

The branched PEGs were used to induce a large increase in the effective size of the antibody fragments. to. MATERIALS All PEG reagents were purchased from Shearwater Polymers and stored at -70 ° C in a desiccator. iv-hydr oxysuccinamide - branched PEG (PEG2-NHS-40 kDa) has a PEG of 20 kDa on each of the two branches, methoxy-succimidyl-propionic acid-PEG (M-SPA-20000) is a molecule of linear PEG with 20 kDa PEG. The protein was recombinantly produced in E. coli and purified as a (Fab) '2 as described in Sections (K) and (O) above. b. METHODS IEX method: A JT Baker HPLC column of Carboxi-sulfona de Poro Ancho (CSX), of 5 microns, of 7.75 x 100 mm, was used for the fractionation of the different pegylated products, taking advantage of the difference in the load according to the the lysines are modified. The column was heated to 40 ° C. A gradient as shown in Table 7 below was used, where Buffer A was 25 mM sodium borate / 25 mM sodium phosphate pH 6.0, and Buffer B was 1 M ammonium sulfate, and C cushion was acetate. 50 mM sodium, pH 5.0.

Table 7 Time% B% C í "luxury xtú (min) 0 10 10 1 .5 20 18 7. 5 1. 5 25 25 7 .5 1 .5 27 70 3. 0 2. 5 29 70 3. 0 2. 30 10 10 2 5 5 10 10 2 5 SEC-HPLC: The effective hydrodynamic size of each molecule was determined using a Pharmacia Superose-6 HR 10/30 column (10 x 300 mm). The mobile phase was 200 mM sodium chloride, 50 mM sodium phosphate, pH 6.0. The flow rate was 0.5 ml / min and the column was maintained at room temperature. The absorbance at 280 nm was checked periodically where PEG contributed little to the signal. Biorad molecular weight standards containing cyanocobalamin, myoglobin, ovalbumin, IgG, monomer and thyroglobulin dimer were used to generate a standard curve from which the effective size of the pegylated species was estimated.

SEC-HPLC-Light Dispersion: For the determination of exact molecular weight, this column was connected to an in-line light scattering detector (Wyatt Minidawn) equipped with three detection angles of 50 °, 90 ° and 135 ° C. A refractive index detector (Wyatt) was also placed online to determine the concentration. All dampers were filtered with 0.1 μ Millipore filters; In addition, a Whatman Anodisc 47 of 0.02 μ was placed in line before the column. The intensity of the scattered light is directly proportional to the molecular weight (M) of the species in dispersion, regardless of the form, according to: M = Ro / K.c where Ro is the Rayleigh ratio, K is an optical constant that is related to the refractive index of the solvent, the wavelength of the incident light, and dn / dc, the differential refractive index between the solvent and the solute with respect to to the change in solute concentration, c. The system was calibrated with toluene (R0 of 1,406 x 10"5 to 632.8 nm) and dn / dc of 0.18, and an extinction coefficient of 1.2 was used, the system had a mass accuracy of approximately 5%.

SDS-PAGE: Tris-Glycine Novex minigels were used at 4-12% together with Tris-glycine buffers supplied by Novex. 10 to 20 μg of protein were applied in each well and the gels were run in a cold box at 150 mV / gel for 45 minutes. The gels were then stained with colloidal Coomassie Blue (Novex) and then washed with water for a few hours and then preserved and dried in drying buffer (Novex).

Preparation of a linear (1) 20kDa- (N) - (Fab ') 2: A 4 mg / ml solution of anti-IL-8 initially formulated in a pH 7.5 buffer, it was dialyzed overnight with a pH 8.0 sodium phosphate buffer. 5 ml of the protein were mixed at a molar ratio of 3: 1. The reaction was carried out in a 15 ml polypropylene Falcon tube and PEG was added while vortexing the sample at low speed for 5 seconds. This was then placed in a nudador for 30 minutes. The degree of modification was evaluated by SDS-PAGE. The complete 5 ml reaction mixture was injected onto the IEX for the removal of any unreacted PEG and purification of slightly or double pegylated species. The above reaction generated a 50% mixture of labeled anti-IL-8 with a single portion. The other 50% of unreacted anti-IL-8 was recycled through the pegylation / purification steps. The combined pegylated product was dialyzed against a pH 5.5 buffer for in vitro assays and PK studies in animals. Endotoxin levels were measured before administration to animals or for cell-based assays. The levels were below 0.5 eu / ml. The fractions were also run on SDS-PAGE to confirm homogeneity. The concentration of the final product was evaluated by absorbance at 280 nm using an extinction coefficient of 1.34, as well as by amino acid analysis.

Preparation of branched (1) 0kDa- (N) - (Fab ') 2: A 4 mg / ml solution of (Fab') 2 anti-IL-8 formulated in a pH 5.5 buffer was dialyzed overnight against a pH 8.0 phosphate buffer. The solid PEG powder was added to 5 ml of protein in two aliquots, to give a final molar ratio of PEG: protein of 6: 1. Each solid aliquot of PEG was added to the protein in a 15 ml polypropylene Falcon tube, while vortexing at low speed for 5 seconds, and then placing the sample on a nudador for 15 minutes. The degree of modification was evaluated by SDS-PAGE using a 4-12% Tris-glycine gel (Novex) and stained with Colloidal Coomasie Blue (Novex). The 5 ml PEG-protein mixture was injected onto the ion exchange column for the removal of any unreacted PEG. The previous reaction generated a mixture of unreacted species (37%), marked with a single portion (45%), double and triple labeled (18%) These were the optimal conditions to obtain the highest recovery of the protein with only one PEG per antibody instead of the higher molecular weight adducts. The unmodified anti-IL-8 was recycled. The pegylated products were separated and fractionated in Falcon tubes and then dialysed against a pH 5.5 buffer for PK assays and studies in animals. The endotoxin levels were below 0.5 eu / ml. The fractions were also run on SDS-PAGE to confirm homogeneity. The concentration of the final product was evaluated by absorbance at 280 nm using an extinction coefficient of 1.34, as well as by amino acid analysis.

Preparation of branched (2) - OKDa- (N) (Fab ') 2: This molecule was * more efficiently elaborated by the addition of three times at 15 minute intervals, from a molar ratio of 3: 1 PEG to the molecule branched (1) -40KDa- (N) - (Fab ') 2 already modified. The molecule was purified on IEX as 50% branched (2) -40KDa- (N) - (Fab ') 2. The unmodified molecule was recycled until approximately 20 mg of protein was isolated for PK studies in animals. The product was characterized by SEC-light scattering and SDS-PAGE. c. RESULTS PEG's significantly increased the hydrodynamic or effective size of the product as determined by gel filtration (SEC-HPLC). Figure 62 shows the SEC profile of the pegylated F (ab ') 2 species with UV detection at 280 nm. The hydrodynamic size of each molecule was estimated by reference to standard molecular weight calibrators. As summarized in Figure 62, the effective size increase of F (ab ') 2 was approximately 7 fold by the addition of a 20 kDa linear PEG molecule, and approximately 11 times by the addition of a branched molecule of PEG. PEG (* Br (l) ") of 40 kDa, and something more with the addition of two branched molecules of PEG (* Br (2)"). Detection by light scattering gave the exact molecular weight of the products and confirmed the degree of modification (Figure 63). The homogeneity of the purified material was shown by SDS-PAGE (Figure 64). The non-derivatized F (ab ') 2 as a 120 kDa species, the linear (1) 20KD- (N) F (ab') 2 migrated as a band at 220 kDa, the Br (1) -40KD (N ) -F (ab ') 2 migrated as a band greater than 400 kDa, and Br (2) -40KD (N) -F (ab') 2 migrated as a larger band around 500 kDa. The proteins appeared somewhat larger than their absolute molecular weight, due to the spherical effect of PEG.

V. CHARACTERIZATION OF ACTI IDAD 1N VITRO OF FRAGMENTS OF Fab 'MODIFIED WITH PEG DE 6G4V11N35A (MALEIMIDA CHEMICAL COUPLING METHOD) Fab 'variants of 6G4V11N35A anti-IL-8 modified with linear PEG molecules of 5-40 kD and a 40 kD branched PEG molecule were tested for their ability to inhibit IL-8 binding and activation of human neutrophils; the procedures were described in Sections (B) (l), (B) (2) and (B) (3) above. The binding curves and the IC50 for the Fab 'molecules of 6G4V11N35A modified with PEG-maleimide, are presented in Figures 54A-54C. The IC50 of the 5 kD pegylated Fab (350 pM) and the average IC50 of the control Fab (366 pM) were not significantly different, suggesting that the addition of PEG with a molecular weight of 5 kD did not link the IL-8 to the Fab 'modified (Figure 54A). However, a decrease in the binding of IL-8 to the pegylated Fab 'molecules of 10 kD and 20 kD was observed as described by the progressively higher IC50 (537 pM) and 732 pM, respectively) compared to IC50. average of the native Fab. These values represent only a minimum loss of link activity (between 1.5 and 2.0 times). A less pronounced difference in the binding of IL-8 was observed for antibodies with linear PEG of 30 kD and 40 kD (Figure 54B). The IC5o's were 524 pM and 1.1 nM, respectively, compared to the value of 802 pM of the Fab control. The Fab 'with 40 kD branched PEG showed the greatest decrease in the binding to IL-8 (2.5 times) in relation to the native Fab (Figure 54C). However, the reduction in the binding of IL-8 by these pegylated Fabs is minimal. The ability of pegylated antibodies to block the activity of human neutrophils mediated by IL-8 was demonstrated using PMN chemotaxis (according to the method described in Section B (2) above) and the release of β-glucuronidase ( according to the method described in Lowman et al., J. Biol. Chem., 271: 14344 (nineteen ninety six)). The IC5o 's for blocking chemotaxis mediated by IL-8 are shown in Figures 55A-55C.

Pegylated Fab 'antibodies with linear PEG of 5-20 kD were able to block chemotaxis mediated by IL-8 within 2 to 3 times of the non-pegylated Fab control (Figure 55A). The difference is not significant because the inherent variation can be up to 2 times for this type of test. However, a significant difference was detected for Fab 'PEGylated antibodies with 30 kD and 40 kD linear PEG, as illustrated by the higher IC50's of linear PEG of 30 kD-Fab' (2.5 nM) and the linear PEG of 40 kD-Fab '(3.7 nM) compared to the control Fab (0.8 nM) (Figure 55B). The ability of the Fab 'molecule with 40 kD branched PEG to block chemotaxis mediated by IL-8 was similar to that of Fab' with 40 kD linear PEG (Figure 55C). At most, the ability of pegylated Fab 'antibodies to block chemotaxis mediated by IL-8 was only reduced by 2 to 3 times. In addition, the release of β-glucuronidase from neutrophil granules was used as another criterion to evaluate the activation of human PMNs mediated by IL-8. Figure 56A (which describes the results obtained with the linear PEGs of 5 kD, 10 kD and 20 kD), Figure 56B (which describes the results obtained with the linear PEGs of 30 kD and 40 kD), and Figure 56C ( which describes the results obtained with the 40 kD branched PEG) show that all pegylated Fab 'antibodies were able to inhibit the IL-8 mediated release of β-glucuronidase, as well as or better than the non-pegylated Fab control. The data collectively show that pegylated Fab 'variants are biologically active and are capable of inhibiting high amounts of exogenous IL-8 in in vitro assays using human neutrophils.

W. CHARACTERIZATION OF IN VITRO ACTIVITY FRAGMENTS OF F (ab ') 2 MODIFIED WITH PEG OF 6G4V11N35A (METHOD OF CHEMICAL COUPLING OF SUCCINIMIDILO) The F (ab ') 2 variant of 6G4V11N35A anti-IL-8 modified with (a) a single linear PEG molecule of 20 kD per F (ab') 2, (b) a single 40 kD branched PEG molecule by F (ab ') 2, (c) with three, four, or five linear PEG molecules of 20 kD with F (ab') 2 (a mixture of: (1) species having three linear PEG molecules of 20 kD by F (ab ') 2; (2) species having four linear PEG molecules of 20 kD per F (ab') 2; and (3) species having five linear PEG molecules of 20 kD per F (ab) ') 2, denoted as * F (ab') 2 with (3,4,5) linear PEG of 20 kD, or (d) with two branched PEG molecules of 40 kD per Fíab ') 2 (denoted as "F (ab ') 2 with (2) branched PEG of 40 KD "), were tested for their ability to inhibit the binding of 125-I-IL-8 and to neutralize the activation of human neutrophils. The procedures used are as described in Sections (B) (l), (B) (2) and (B) (3) above. Link curves for the pegylated F (ab ') 2 variants are shown in Figures 57A-57B. No significant differences were observed between control F (ab ') 2, F (ab') 2 modified with linear 20 kD simple PEG, and F (ab ') 2 modified with branched PEG of 40 kD simple (Figure 57A ). However, F (ab ') 2 variants containing multiple PEG molecules showed a slight (less than 2-fold) reduction in their ability to bind to IL-8. The IC50 's of F (ab') 2 with (3, 4.5) linear PEG of 20 kD and variants of F (ab ') 2 with (2) branched PEG of 40 kD were 437 pM and 510 pM, respectively, compared to 349 pM of F (ab') 2 control (Figure 57B). The ability of these pegylated F (ab ') 2 variants to block neutrophil chemotaxis mediated by IL-8 is presented in Figures 58A-58B. Consistent with the PMN binding data, the F (ab ') 2 variants with only a linear or branched PEG were able to block chemotaxis mediated by IL-b, similar to the control of F (ab') 2 nc pegylated (Figure 58A). The ability of the F (ab ') 2 variant with (2) 40 kD branched PEG to inhibit PMN chemotaxis was identical to the F (ab') 2 control with the mixture of F (ab ') 2 with ( 3,4,5) linear PEG of 20 kD, was able to inhibit within 3 times the control antibody (Figure 58B).

Shown in Figures 59A and 59B are the results of the β-glucuronidase release assay which is a measure of degranulation by human neutrophils stimulated by IL-8. The F (ab ') 2 modified with linear PEG of 20 kD simple and the variants of F (ab') 2 modified with branched PEG of 40 kD simple, were able to inhibit the release of β-glucuronidase as well as the F (ab) ') 2 control (Figure 59A). F (ab ') 2 with (2) 40 kD branched PEG inhibited this response within 2 times of the F (ab') 2 control (Figure 59B). The molecule with linear (3,4,5) PEG of 20 kD was not tested. In total, pegylated anti-IL-8 antibodies F (ab'j2 were biologically active and effectively prevented the binding of IL-8 to human neutrophils and the signaling events that lead to cell activation.

X. PHARMACOKINETIC AND SECURITY STUDY OF EIGHT CONSTRUCTIONS OF PEGILED FRAGMENTS OF F (ab ') 2 AND FAB '(HUMANIZED) ANTI-IL-8 IN RABBITS NORMAL AFTER INTRAVENOUS ADMINISTRATION The aim of this study was to evaluate the effect of pegylation on the pharmacokinetics and safety of six humanized, pegylated anti-IL-8 constructs (pegylated 6G4V11N35A.Fab 'and pegylated 6G4V11N35A.F (ab') 2 as described in Sections (T) and (U) above, relative to non-pegylated fragments in normal rabbits Eight groups of two / three male rabbits received equivalent protein amounts from the constructions of 6G4V11N35A, pegylated Fab 'or 6G4V11N35A. 2) pegylated (2 mg / kg) by means of a single intravenous (IV) bolus dose of an anti-IL-8 construct.The serum samples were collected according to the scheme shown in Table 8 below, and analyzed for anti-IL-8 protein concentrations and antibody formation against anti-IL-8 constructs by ELISA.

Table 8 METHODS Three New Zealand White male rabbits (NZW) or group (with the exception of Group 7, n = 2) received an equivalent amount of the 6G4V11N35A protein (Fab 'or F (ab')?) 2 mg / kg by bolus intravenously in a marginal vein of the ear. The analysis of the amino acid composition and the absorbance at 280 nm using the extinction coefficients of 1.26 for the constructions of 6G4V11N35A. Fab 'and 1.34 for the constructions of 6G4V11N35A F (ab') 2 were performed to determine the protein concentration. The whole blood samples were collected by means of a cannulation in the artery of the ear (ear opposite the dose ear) at the previous time points. Samples were harvested for serum and evaluated for the Fab 'or F (ab') 2 constructs of 6G4V11N35A using an ELISA binding assay to IL-8. The trials were conducted throughout the study as the samples became available. All animals were sacrificed after the last sa toe test, and necropsies were performed on all animals in groups 1, 4-8. Due to the development of antibodies against the 6G4V11N35A constructs, no compartmental pharmacokinetic analysis was conducted on the concentration versus time data only until 168 hours. b. RESULTS In four animals (animals B, P, Q, V), interference to rabbit serum was detected in the ELISA assay (eg, measurable concentrations of anti-IL-8 antibodies to the predose). However, because these values were at negligible levels and the pharmacokinetic analysis was not performed, the data was corrected for this interference. One animal (Animal G, Group 3) was bled before the termination of the study and was excluded from the pharmacokinetic analysis. At 4 hours, the animal showed signs of a shock that was thought not to be related to the drug, since this can occur in rabbits after blood extractions by means of cannulation in the artery of the ear. The average-time concentration profiles of the eight anti-IL-8 constructs of normal rabbits are described in Figure 65, and the pharmacokinetic parameters for the eight constructions are summarized in Table 9 below. Significant antibodies to the anti-IL-8 constructs were present on day 13/14 in all dose groups except Group 1 (Fab 'control).

Table 9. Pharmacokinetic parameters Initial volume of distribution. Volume of distribution in the resting state Maximum observed concentration. Observed time to Cmax. term t? / 2 = half-life associated with the terminal phase of the concentration profile versus time. Area under the concentration versus time curve (extrapolated to infinity). CL = serum clearance MRT = Average residence time.

The initial volume of the distribution approached the plasma volume for Fab 'and F (ab') 2. PEGylation decreased in serum CL of the anti-IL-8 fragments, and extended the terminal half-life and MRT as shown in Table 10 below.

Table 10. Ratio of decrease / increase in clearance, terminal half-life and MRT of pegylated anti-IL-8 fragments For the pegylated anti-IL-8 Fab 'fragments, the CL diminished from 46 to 180 times. The terminal half-life and the MRT were increased from 14 to 35 times and from 53 to 240 times, respectively. For PEGylated anti-IL-8 F (ab ') 2 molecules, CL decreased 15 to 17 fold with PEGylation, and terminal half-life and MRT were increased by more than 5-fold and 13-fold, respectively. Changes in these parameters increased the pegylated Fab 'and F (ab') 2 molecules with the increase in molecular weight of PEG and approached the values of full-length anti-IL-8 (terminal half-life of 74 hours, MRT of 99 hours and CL of 0.47 ml / hr / kg). In the comparison of the branched (1) 40K Fab '(Group 6) and the branched (1) 40K F (ab') 2 (Group 4), unexpected pharmacokinetics were observed. The pegylated fab 'molecule appeared to remain in the serum for longer than the pegylated F (ab') 2 (see Figure 66).

The medium branched CL (1) 40K Fab 'was 0.63 ml / hr / kg, but a higher CL was observed for the branched (l) 40kD F (ab') 2 (CL 0.92 ml / hr / kg). The terminal half-life, likewise, was longer for the Fab 'than for the pegylated F (ab') 2 molecule (110 versus 45 hours). Pharmacokinetic data showed that PEGylation decreased CL and increased the terminal half life and MRT of anti-IL-8 fragments (Fab 'and F (ab') 2) to approximate that of full-length anti-IL-8 . Clearance was decreased with pegylation 46 to 180 times for Fab 'and approximately 16 times for F (ab') 2. The terminal half-life of the anti-IL-8 Fab 'fragment was increased by 14 to 35 times and approximately 5 times for the anti-IL-8 F (ab') 2. MRT, likewise, was extended by 53 to 240 times for the Fab 'and approximately 14 times for the F (ab') 2. The branched (1) 40kD Fab 'had a longer terminal half-life and lower clearance compared to the branched (1) 40kD F (ab') 2.

Y. PROOF OF IN VIVO EFFECTIVENESS OF REAGENTS OF ANTI-IL-8 ANTIBODY IN RABBIT MODEL ISCHEMIA / REPERFUSION AND SYNDROME OF RESPIRATORY INSUFFICIENCY INDUCED WATER BY ACID ASPIRATION (ARDS) The mouse monoclonal antibody 6G4.2.5 anti-IL-8, full-length, the Fab "of 6G4V11N35A with branched PEG of 40 kD and the control antibody (monoclonal antibody 9E3.1F10 anti-HIV gpl20) were tested in a rabbit ARDS model Animals were weighed and anesthetized by intramuscular injection of ina ceta (50 mg / kg body weight), xylazine (5 mg / kg body weight) and acepromazine (0.75 mg / kg body weight). A second dose (20% of the first dose) was administered intramuscularly 15 minutes before the removal of the vascular clamp, and a third dose (60% of the first dose) was administered by tracheotomy. An intraarterial catheter was placed (22G, 2.54 cm (l inch) Angiocath and an intravenous catheter (24G, 2.54 cm (1 inch) Angiocath) in the central artery of the ear and in the vein of the posterior marginal ear for blood sampling (arterial blood gases and CBC) and administration of anti-IL-8 and fluid, res pectively. The anesthetized animals were transferred in a supine position to an operating tray; the abdominal area was shaved and prepared for surgery. By means of a midline laparotomy, the superior mesenteric artery (SMA) was isolated and a microvascular arterial clamp was applied to the aortic origin. Before the temporary closure of the abdomen using a 9 mm coiled clamp (Autoclip, Baxter), 15 mm of normal saline was administered intraperitoneally as a fluid supplement. After 110 minutes of intestinal ischemia, the abdominal incision was reopened and the arterial clamp was freed to allow reperfusion. Before closure, 5 ml of normal saline was administered intraperitoneally for fluid replacement. The laparotomy incision was closed in two layers and the animals were allowed to wake up. After surgery, the animals were placed on a heating pad (38 ° C) and checked continuously for up to 6 hours after reperfusion, and lactated Ringer's solution was administered at 8-12 ml / kg / hr intravenously , as a fluid supplement. At 22-24 hours after reperfusion, a tracheotomy was performed under anesthesia. Normal physiological saline was diluted 1: 3 with water and adjusted to pH 1.5 (adjusted by IN HCL); then 3 ml / kg of body weight were instilled intratracheally. The rectal temperature was maintained at 37 +/- 1 ° C using a homeothermic heat therapy pad (K-Mod II, Baxter). Fluid supplements (LRS) were administered at a rate of 5 ml / kg / hr intravenously. The blood gases were checked periodically every hour. The rabbits were returned to the cage after 6 hours of continuous monitoring. Just before aspiration, the animals were treated with saline, the control monoclonal antibody (IgG 9E3.1F10 anti-HIV gp-120), the murine, full length rabbit anti-IL-8 antibody (murine anti IgG2a) -IL-8 of rabbit, 6g4.2.5) or the Fab 'of pegylated 6G4V11N35A (6G4V11N35A Fab' modified with maleimide-branched PEG of 40 kD as described in Section T above, denoted as 'Fab' 6G4V11N35A with branched PEG of 40 kD "). The data from the animals treated with saline or with the control antibody were combined and presented as 'Control'. Arterial blood gases and A-a P02 gradient measurements were taken daily, and fluid supplementation was performed daily intravenously. The gradient A-a P02 was measured 96 hours after reperfusion. The gradient A-a P02 was calculated as: A-a P02 = [F102 (PB-PH20) - (PaC02 / RQ)] -Pa02 The proportions Pa02 / Fi02 were measured at 24 hours and 48 hours in ambient air and 100% oxygen. After measurement of the final gradient of Aa P02, the animals were anesthetized with Nembutal at 100 mg / kg intravenously, and the animals were sacrificed by transection of the abdominal aorta in order to reduce contamination with red blood cells of the washing fluid. bronchoalveolar (BAL). The lungs were removed en bloc. The whole lungs were weighed and then washed with an intratracheal tube (Hi-Lo tracheal tube, 3 mm), using 30 ml of HBSS and lidocaiaa. The total and differential leukocyte counts in BAL were determined. The lesions / changes were verified by histological examination of each lobe of the right lung of each animal. The gross weight of the lung, total leukocyte and polymorphonuclear cell counts in BAL, and the obtained Pa02 / Fi02 data are described in Figures 67, 68 and 69, respectively. The treatment with the Fab 'of 6G4V11N35A with branched PEG of 40 kD, showed no effect on the biological parameters measured in the model compared to the group' Control. "However, the data do not contradict the pharmacokinetic analysis or the activity analysis. for the Fab 'of 6G4V11N35A with 40 kD branched PEG, presented in Sections (V) and (X) above.In addition, these data do not contradict the Fab' ability of 6G4V11N35A with 40 kD branched PEG, to reach and act on effector disease targets in circulation or other tissues The following biological materials have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): Material ATCC Accession No. Date of Deposit Hybridoma cell line 5.12.14 HB 11553 February 15, 1993 Hybridoma cell line 6G4.2.5 HB 11722 September 28, 1994 paptiIL-8.2, E. coli strain 294 mm 97056 February 10, 1995 p6G425chim2, E. coli strain 294 mm 97055 February 10, 1995 p6G4VllN35A.F (ab * ¡97890 February 20, 1997 E. coli strain 49D6 (p6G4Vl lN35A.F (ab ')?) 98332 February 20, 1997 p6G425Vl lN35A.choSD 209552 December 16, 1997 clone * 1933 aIL8.92 NB 28605/12 CRL-12444 December 11, 1997 clone # 1934 aIL8.42 NB 28605/14 CRL-12445 December 11, 1997 These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures and the Regulations under it (Budapest Treaty). This ensures the maintenance of a viable deposit for 30 years from the date of deposit. These cell lines will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and the ATCC, which ensures the permanent and unrestricted availability of cell lines to the public after the expedition. of the relevant North American patent or after disclosing to the public any US or foreign patent application, whichever comes first, and ensuring the availability of cell lines to someone determined, by the United States Patent and Trademark Commissioner to be authorized for this according to 35 USC Section 122 and the rules of the Commissioner according to this (including 37 CFR Section 1.14 with particular reference to 886 OG 638). The assignee of the patent application has agreed that if the deposited cell lines are lost or destroyed when cultured under suitable conditions, they will be promptly replaced after notification with a specimen of the same cell line. The availability of the deposited cell lines is not considered as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

LIST OF SEQUENCES (i) GENERAL INFORMATION: (i) APPLICANT: Hsei, Vanessa "Koumenis, Iphigenia Leong, Steven R. Presta, Leonard G. Shahrokh, Zahra Zapata, Gerardo A. (ii) TITLE OF THE INVENTION: Conjugates of Antibody-Polymer Fragment and Humanized Anti-IL-8 Monoclonal Antibodies (iii) ) NUMBER OF SEQUENCES: 76 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Genentech Inc. (B) STREET: 1 DNA Way (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA ( F) POSTAL CODE (ZIP): 94080 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: diskette 3.5 inches, 1.44 Mb (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS -DOS (D) SOFTWARE: WinPatin (Genentech) (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: 20-Feb-1998 (C) CLASSIFICATION: (viii) INFORMATION OF THE ATTORNEY / AGENT: (A) NAME: Love, Richard B. (B) REGISTRATION NUMBER: 34,659 (<; - • NUMBER DF, RF, FFR ?: r / X -x ^ or PI Í G, R3G T (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 650 / 225-5530 (B) FAX: 650 / 952- 9881 (2) INFORMATION FOR SEQ ID NO. 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 1: CAGTCCAACT GTTCAGGACG CC 22 (2) INFORMATION FOR SEQ ID NO. 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 2: GTGCTGCTCA TGCTGTAGGT GC 22 (2) INFORMATION FOR SEQ ID NO. 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO . 3: GAAGTTGATG TCTTGTGAGT GGC 23 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 4: GCATCCTAGA GTCACCGAGG AGCC 24 Y) i NFORMAC LON "ARA T / ^, ,,, TGJ NO. ': (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.5: CACTGGCTCA GGGAAATAAC CC 22 (2) INFORMATION FOR SEQ ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 6: GGAGAGCTGG GAAGGTGTGC AC 22 (2) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: "(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 7: ACAAACGCGT ACGCTGACAT CGTCATGACC CAGTC 35 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.

ACAAACGCGT ACGCTGATAT TGTCATGACT CAGTC 35 (2) INFORMATION FOR SEQ ID NO. 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs' &) T7F •,: io, .U-IPK-O (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: ACAAACGCGT ACGCTGACAT CGTCATGACA CAGTC 35 (2) INFORMATION FOR SEQ ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 10: GCTCTTCGAA TGGTGGGAAG ATGGATACAG TTGGTGC 37 (2) INFORMATION FOR SEQ ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. eleven: CGATGGGCCC GGATAGACCG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 12: CGATGGGCCC GGATAGACTG ATGGGGCTGT CGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear x? 1-. , rP '• Oh DE M, .SFV W; SEO ID X '> CGATGGGCCC GGATAGACGG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear. { xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 14: CGATGGGCCC GGATAGACAG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 15: CGATGGGCCC GGATAGACCG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 16: CGATGGGCCC GGATAGACTG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 17:GGATAGACAG ATGGGGCTGT TGTTTTGGC 39 'Y, X-XMA, ON? \ R? THE "Ky"? O] 6 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.18: CGATGGGCCC GGATAGACGG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 369 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 19: GACATTGTCA TGACACAGTC TCAAAAATTC ATGTCCACAT CAGTAGGAGA 50 CAGGGTCAGC GTCACCTGCA AGGCCAGTCA GAATGTGGGT ACTAATGTAG_100_CCTGGTATCA ACAGAAACCA GGGCAATCTC CTAAAGCACT GATTTACTCG 150 TCATCCTACC GGTACAGTGG AGTCCCTGAT CGCTTCACAG GCAGTGGATC 200 TGGGACAGAT TTCACTCTCA CCATCAGCCA TGTGCAGTCT GAAGACTTGG 250 CAGACTATTT CTGTCAGCAA TATAACATCT ATCCTCTCAC GTTCGGTCCT 300 GGGACCAAGC TGGAGTTGAA ACGGGCTGAT GCTGCACCAC CAACTGTATC 350 CATCTTCCCA CCATTCGAA 369 (2) INFORMATION FOR SEQ ID NO. 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 123 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. twenty: Asp He Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly 20 25 30 Thr Asn Va1 Al * Trt JT Gln Hear Lys Pro Glv 'S11 Ser Pro Lye 3b 40 45 Wing Leu He Tyr Being Ser Tyr Arg Tyr Being Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He 65 70 75 Ser His Val Gln Ser Glu Asp Leu Wing Asp Tyr Phe Cys Gln Gln 80 85 90 Tyr Asn He Tyr Pro Leu Thr Phe Gly Pro Gly Thr Lys Leu Glu 95 100 105 Leu Lys Arg Ala Asp Ala Wing Pro Pro Thr Val Ser He Phe Pro 110 115 120 Pro Phe Glu 123 (2) INFORMATION FOR SEQ ID NO. 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. twenty-one: TTCTATTGCT ACAAACGCGT ACGCTGAGGT GCAGCTGGTG GAGTCTGGGG 50 GAGGCTTAGT GCCGCCTGGA GGGTCCCTGA AACTCTCCTG TGCAGCCTCT 100 GGATTCATAT TCAGTAGTTA TGGCATGTCT TGGGTTCGCC AGACTCCAGG 150 CAAGAGCCTG GAGTTGGTCG CAACCATTAA TAATAATGGT GATAGCACCT 200 ATTATCCAGA CAGTGTGAAG GGCCGATTCA CCATCTCCCG AGACAATGCC 250 AAGAACACCC TGTACCTGCA AATGAGCAGT CTGAAGTCTG AGGACACAGC 300 CATGTTTTAC TGTGCAAGAG CCCTCATTAG TTCGGCTACT TGGTTTGGTT 350 ACTGGGGCCA AGGGACTCTG GTCACTGTCT CTGCAGCCAA AACAACAGCC 400 CCATCTGTCT ATCCGGG 417 (2) • INFORMATION FOR SEQ ID NO. 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 130 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION u *. SECU t,?, L \: Shc IJJ No. 22: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Wing Wing Ser Gly Phe He Phe Ser 20 25 30 Ser Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Gly Lys Ser Leu 35 40 45 Glu Leu Val Wing Thr He Asn Asn Asn Gly Asp Ser Thr Tyr Tyr 50 55 60 Pro Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Wing 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Being Ser Leu Lys Ser Glu Asp 80 85 90 Thr Wing Met Phe Tyr Cys Wing Arg Wing Leu He Ser Being Wing Thr 95 100 105 Trp Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Wing 110 115 120 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 125 130 (2) INFORMATION FOR SEQ ID NO. 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 23: ACAAACGCGT ACGCTGATAT CGTCATGACA G 31 (2) INFORMATION FOR SEQ ID NO. 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 24: GCAGCATCAG CTCTTCGAAG CTCCAGCTTG G 31 (21 IN »OPM-'CTON PAR LA I RQ Tt NO 25; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO.

CCACTAGTAC GCAAGTTCAC G 21 (2) INFORMATION FOR SEQ ID NO. 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 26: GATGGGCCCT TGGTGGAGGC TGCAGAGACA GTG 33 (2) INFORMATION FOR SEQ ID NO. 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 714 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 27: ATGAAGAAGA ATATCGCATT TCTTCTTGCA TCTATGTTCG TTTTTTCTAT 50 TGCTACAAAC GCGTACGCTG ATATCGTCAT GACACAGTCT CAAAAATTCA 100 TGTCCACATC AGTAGGAGAC AGGGTCAGCG TCACCTGCAA GGCCAGTCAG 150 AATGTGGGTA CTAATGTAGC CTGGTATCAA CAGAAACCAG GGCAATCTCC 200 TAAAGCACTG ATTTACTCG CATCCTACCG GTACAGTOGA. GTCCCTGATC 250 GCTTCACAGG CAGTGGATCT GGGACAGATT TCACTCTCAC CATCAGCCAT 300 GTGCAGTCTG AAGACTTGGC AGACTATTTC TGTCAGCAAT ATAACATCTA 350 TCCTCTCACG TTCGGTCCTG GGACCAAGCT GGAGCTTCGA AGAGCTGTGG 400 CTGCACCATC TGTCTTCATC TTCCCGCCAT CTGATGAGCA GTTGAAATCT 450 GGAACTGCTT CTGTTGTGTG CCTGCTGAAT AACTTCTATC CCAGAGAGGC 500 CAAAGTACAG TGGAAGÜTGG ATAAeGCCC? CCAATCGGGT AACTCCCAGC JSO AGAGTGTCAC AGAGCAGGAC AGCAAGGACA GCACCTACAG CCTCAGCAGC 600 ACCCTGACGC TGAGCAAAGC AGACTACGAG AAACACAAAG TCTACGCCTG 650 CGAAGTCACC CATCAGGGCC TGAGCTCGCC CGTCACAAAG AGCTTCAACA 700 GGGGAGAGTG TTAA 714 (2) INFORMATION FOR SEQ ID NO. 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 237 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 28: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be He Wing Thr Asn Wing Tyr Wing Asp He Val Met Thr Gln Ser 20 25 30 Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr 35 40 45 Cys Lys Wing Ser Gln Asn Val Gly Thr Asn Val Wing Trp Tyr Gln 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu He Tyr Ser Ser Ser 65 70 75 Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 80 85 90 Gly Thr Asp Phe Thr Leu Thr He Ser His Val Gln Ser Glu Asp 95 100 105 Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn He Tyr Pro Leu Thr 110 115 120 Phe Gly Pro Gly Thr Lys Leu Glu Leu Arg Arg Ala Val Ala Ala 125 130 135 Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 140 145 150 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 155 160 165 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu ßln Ser Gly 170 175 180 Asn Ser Gin Ciu Ser Val Thr Gl? Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu 200 205 210 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 215 220 225 Ser Pro Val Thr Lys Ser * Phe Asn Arg Gly Glu Cys 230 235 237 (2) INFORMATION FOR SEQ ID NO. 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 756 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 29: ATGAAAAAGA ATATCGCATT TCTTCTTGCA TCTATGTTCG TTTTTTCTAT 50 TGCTACAAAC GCGTACGCTG AGGTGCAGCT GGTGGAGTCT GGGGGAGGCT 100 TAGTGCCGCC TGGAGGGTCC CTGAAACTCT CCTGTGCAGC CTCTGGATTC 150 ATATTCAGTA GTTATGGCAT GTCTTGGGTT CGCCAGACTC CAGGCAAGAG 200 CCTGGAGTTG GTCGCAACCA TTAATAATAA TGGTGATAGC ACCTATTATC 250 CAGACAGTGT GAAGGGCCGA TTCACCATCT CCCGAGACAA TGCCAAGAAC 300 ACCCTGTACC TGCAAATGAG CAGTCTGAAG TCTGAGGACA CAGCCATGTT 350 TTACTGTGCA AGAGCCCTCA TTAGTTCGGC TACTTGGTTT GGTTACTGGG 400 GCCAAGGGAC TCTGGTCACT GTCTCTGCAG CCTCCACCAA GGGCCCATCG 450 GTCTTCCCCC TGGCACCCTC CTCCAAGAGC ACCTCTGGGG GCACAGCGGC 500 CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT S50 GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA 600 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGGTGACCG TGCCCTCCAG 650 CAGCTTGGGC ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA 700 ACACCAAGGT GGACAAGAAA GTTGAGCCCA AATCTTGTGA CAAAACTCAC 750 ACATGA 756 (2) INFORMATION FOR SEQ ID NO. 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 251 base pairs (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 30: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be Xle Ala Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser 20 25 30 Gly Gly Gly Leu Val Pro Pro Gly Gly Ser Leu Lys Leu Ser Cys 35 40 45 Wing Wing Ser Gly Phe He Phe Ser Ser Tyr Gly Met Ser Trp Val 50 55 60 Arg Gln Thr Pro Gly Lys Ser Leu Glu Leu Val Wing Thr He Asn 65 70 75 Asn Asn Gly Asp Ser Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg 80 85 90 Phe Thr He Ser Arg Asp Asn Wing Lys Asn Thr Leu Tyr Leu Gln 95 100 105 Met Being Ser Leu Lys Ser Glu Asp Thr Wing Met Phe Tyr Cys Wing 110 115 120 Arg Ala Leu He Ser Ser Ala Thr Trp Phe Gly Tyr Trp Gly Gln 125 130 135 Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser 140 145 150 Val Phe Pro Leu Wing Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 155 160 165 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 170 175 180 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 185 190 195 Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 200 205 210 Val Val Thr Val Pro Ser Ser Be Leu Gly Thr Gln Thr Tyr He 215 220 225 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 230 235 240 V- 1 ír v o TJ S < ?and? Cys Asp t.yr. Thr Hiß Thr .¿45 250 251 (2) INFORMATION FOR SEQ ID NO. 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 31: CAGTCCAACT GTTCAGGACG CC 22 (2) INFORMATION FOR SEQ ID NO. 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 32: GTGCTGCTCA TGCTGTAGGT GC 22 (2) INFORMATION FOR SEQ ID NO. 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 33: GAAGTTGATG TCTTGTGAGT GGC 23 (2) INFORMATION FOR SEQ ID NO. 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 34: GCATCCTAGA GTCACCGAGG AGCC 24 (2) INFORMATION FOR SEQ ID NO. 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 35: CACTGGCTCA GGGAAATAAC CC 22 (2) INFORMATION FOR SEQ ID NO. 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 36: GGAGAGCTGG GAAGGTGTGC AC 22 (2) INFORMATION FOR SEQ ID NO. 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 37: CCAATGCATA CGCTGACATC GTGATGACCC AGACCCC 37 (2) INFORMATION FOR SEQ ID NO. 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 38: CCAATGCATA CGCTGATATT GTGATGACTC AGACTCC 37 (2) INFORMATION FOR SEQ ID NO. 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (c, TYPE DL:? F.RR ^: YES!, - I (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39: CCAATGCATA CGCTGACATC GTGATGACAC AGACACC 37 (2) INFORMATION FOR SEQ ID NO 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs ( B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 40: AGATGTCAAT TGCTCACTGG ATGGTGGGAA GATGG 35 (2) INFORMATION FOR SEQ ID NO. 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 41: CAAACGCGTA CGCTGAGATC CAGCTGCAGC AG 32 (2) INFORMATION FOR SEQ ID NO. 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 42: CAAACGCGTA CGCTGAGATT CAGCTCCAGC AG 32 (2) INFORMATION FOR SEQ ID NO. 43: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) D? SCRItCTJN OF THE SEQUENCE: SLQ iD NO. 43: CGATGGGCCC GGATAGACCG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 44: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 44: CGATGGGCCC GGATAGACTG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 45: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 45: CGATGGGCCC GGATAGACAG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 46: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 46: CGATGGGCCC GGATAGACGG ATGGGGCTGT TGTTTTGGC 39 (2) INFORMATION FOR SEQ ID NO. 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 391 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 47: GATATCGTGA TGACACAGAC ACCACTCTCC CTGCCTGTCA GTCTTGGAGA 50 TCAGGCCTCC ATCTCTTGCA GATCTAGTCA GAGCCTTOTA CACGGTATTG 100 TACCTGCAGA GAAACACCTA TTTACATTGG AGCCAGGCCA GTCTCCAAAG 150 CTCCTGATCT ACAAAGTTTC CAACCGATTT TCTGGGGTCC CAGACAGGTT 200 CAGTGGCAGT GGATCAGGGA CAGATTTCAC ACTCAGGATC AGCAGAGTGG 250 AGGCTGAGGA TCTGGGACTT TATTTCTGCT CTCAAAGTAC ACATGTTCCG 300 CTCACGTTCG GTGCTGGGAC CAAGCTGGAG CTGAAACGGG CTGATGCTGC 350 ACCAACTGTA TCCATCTTCC CACCATCCAG TGAGCAATTG A 391 (2) INFORMATION FOR SEQ ID NO. 48: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 131 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 48: Asp He Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu 1 5 10 15 Gly Asp Gln Wing Being Be Cys Arg Being Ser Gln Being Leu Val 20 25 30 His Gly He Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 35 40 45 Gly Gln Ser Pro Lys Leu Leu He Tyr Lys Val Ser Asn Arg Phe 50 55 60 Ser Gly Val Pro Asp Arg Phe Ser Gly Be Gly Ser Gly Thr Asp 65 70 75 Phe Thr Leu Arg He Ser Arg Val Glu Ala Glu Asp Leu Gly Leu 80 85 90 Tyr Phe Cys Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly Wing 95 100 105 Gly Thr Lys Leu Glu Leu Lys Arg Wing Asp Ala Wing Pro Thr Val 110 115 120 Ser He Phe Pro Pro Ser Ser Glu Gln Leu Lys 125 130 131 (2) INFORMATION FOR SEQ ID NO. 49: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: double (i); X ^ C, TP 1 ip vi (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 49: GAGATTCAGC TGCAGCAGTC TGGACCTGAG CTGATGAAGC CTGGGGCTTC SO AGTGAAGATA TCCTGCAAGG CTTCTGGTTA TTCATTCAGT AGCCACTACA 100 TGCACTGGGT GAAGCAGAGC CATGGAAAGA GCCTTGAGTG GATTGGCTAC 150 ATTGATCCTT CCAATGGTGA AACTACTTAC AACCAGAAAT TCAAGGGCAA 200 GGCCACATTG ACTGTAGACA CATCTTCCAG CACAGCCAAC GTGCATCTCA 250 GCAGCCTGAC ATCTGATGAC TCTGCAGTCT ATTTCTGTGC AAGAGGGGAC 300 TATAGATACA ACGGCGACTG GTTTTTCGAT GTCTGGGGNG NAGGGACCAC 350 GGTCACCGTC TCCTCCGCCA AAACCGACAG CCCCATCGGT CTATCCGGGC 400 CCATC 405 (2) INFORMATION FOR SEQ ID NO. 50: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 135 amino acids (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. fifty Glu He Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly 1 5 10 15 Wing Ser Val Lys He Ser Cys Lys Wing Ser Gly Tyr Ser Phe Ser 20 25 30 Ser His Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu 35 40 45 Glu Trp He Gly Tyr He Asp Pro Be Asn Gly Glu Thr Thr Tyr 50 55 60 Asn Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser 65 70 75 Ser Ser Thr Wing Asn Val His Leu Ser Ser Leu Thr Ser Asp Asp 80 85 90 Be Wing Val Tyr Phe Cys Wing Arg Gly Asp Tyr Arg Tyr Asn Gly 95 100 105 Asp Trp Phe Phe Asp Val Trp Gly Xaa Gly Thr Thr Val Thr Val 110 115 120 ier Ser, ", the t-ys Vh. & p Seo Tro Ti - riy Leu Ser SI and Pro He 125 130 135 (2) INFORMATION FOR SEQ ID NO. 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 51 CTTGGTGGAG GCGGAGGAGA CG 22 (2) INFORMATION FOR SEQ ID NO. 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 pairs of bales (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 52: GAAACGGGCT GTTGCTGCAC CAACTGTATT CATCTTCC 38 (2) INFORMATION FOR SEQ ID NO. 53: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 53: GTCACCGTCT CCTCCGCCTC CACCAAGGGC C 31 (2) INFORMATION FOR SEQ ID NO. 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 54: CTTGGTGGAG GCGGAGGAGA CG 22 (2) INFORMATION FOR SEQ ID NO. 55: (i, CAK? CTF.FUS LCA.q DE L \ SEC? 'EÍ'? T: (A) LENGTH: 729 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 55: ATGAAGAAGA ATATCGCATT TCTTCTTGCA TCTATGTTCG TTTTTTCTAT 50 TGCTACAAAT GCATACGCTG ATATCGTGAT GACACAGACA CCACTCTCCC 100 TGCCTGTCAG TCTTGGAGAT CAGGCCTCCA TCTCTTGCAG ATCTAGTCAG 150 AGCCTTGTAC ACGGTATTGG AAACACCTAT TTACATTGGT ACCTGCAGAA 200 GCCAGGCCAG TCTCCAAAGC TCCTGATCTA CAAAGTTTCC AACCGATTTT 250 CTGGGGTCCC AGACAGGTTC AGTGGCAGTG GATCAGGGAC AGATTTCACA 300 CTCAGGATCA GCAGAGTGGA GGCTGAGGAT CTGGGACTTT ATTTCTGCTC 350 TCAAAGTACA CATGTTCCGC TCACGTTCGG TGCTGGGACC AAGCTGGAGC 400 TGAAACGGGC TGTTGCTGCA CCAACTGTAT TCATCTTCCC ACCATCCAGT 450 GAGCAATTGA AATCTGGAAC TGCCTCTGTT GTGTGCCTGC TGAATAACTT 500 CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC GCCCTCCAAT 550 CGGGTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA GGACAGCACC 600 TACAGCCTCA GCAGCACCCT GACGCTGAGC AAAGCAGACT ACGAGAAACA 650 CAAAGTCTAC GCCTGCGAAG TCACCCATCA GGGCCTGAGC TCGCCCGTCA 700 CAAAGAGCTT CAACAGGGGA GAGTGTTAA 729 (2) INFORMATION FOR SEQ ID NO. 56: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 242 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 56: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be He Wing Thr Asn Wing Tyr Wing Asp He Val Met Thr Gln Thr 20 25 30 Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Be Ser 3 =; 40 45 Cys Arg Ser Ser Gln Ser Leu Val His Gly He Gly Asn Thr Tyr 50 55 60 Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu 65 70 75 He Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe 80 85 90 Be Gly Be Gly Be Gly Thr Asp Phe Thr Leu Arg Be Ser Arg 95 100 105 Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser Thr 110 115 120 His Val Pro Leu Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys 125 130 135 Arg Ala Val Ala Ala Pro Thr Val Phe He Phe Pro Pro Ser Ser 140 145 150 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 155 160 165 Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn 170 175 180 Wing Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 185 190 195 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 200 205 210 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 215 220 225 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 230 235 240 Glu Cys 242 (2) INFORMATION FOR SEQ ID NO. 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 762 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 57: ATGAAAAAGA ATATCGCATT TCTTCTTGCA TCTATGTTCG TTTTTTCTAT 50 Tac, (.. «. '? CACÍTCT ar? AC tcr v T OO TGATGAAGCC TGGGGCTTCA GTGAAGATAT CCTGCAAGGC TTCTGGTTAT 150 TCATTCAGTA GCCACTACAT GCACTGGGTG AAGCAGAGCC ATGGAAAGAG 200 CCTTGAGTGG ATTGGCTACA TTGATCCTTC CAATGGTGAA ACTACTTACA 250 ACCAGAAATT CAAGGGCAAG GCCACATTGA CTGTAGACAC ATCTTCCAGC 300 ACAGCCAACG TGCATCTCAG CAGCCTGACA TCTGATGACT CTGCAGTCTA 350 TTTCTGTGCA AGAGGGGACT ATAGATACAA CGGCGACTGG TTTTTCGATG 400 TCTGGGGCGC AGGGACCACG GTCACCGTCT CCTCCGCCTC CACCAAGGGC 450 CCATCGGTCT TCCCCCTGGC ACCCTCCTCC AAGAGCACCT CTGGGGGCAC 500 AGCGGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA CCGGTGACGG 550 TGTCGTGGAA CTCAGGCGCC CTGACCAGCG GCGTGCACAC CTTCCCGGCT 600 GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACCGTGCC 650 CTCCAGCAGC TTGGGCACCC AGACCTACAT CTGCAACGTG AATCACAAGC 700 CCAGCAACAC CAAGGTGGAC AAGAAAGTTG AGCCCAAATC TTGTGACAAA 750 ACTCACACAT GA 762 (2) INFORMATION FOR SEQ ID NO. 58: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 253 base pairs (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 58: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Being He Wing Thr Asn Wing Tyr Wing Glu He Gln Leu Gln Gln Being 20 25 30 Gly Pro Glu Leu Met Lys Pro Gly Wing Ser Val Lyß He Ser Cys 35 40 45 Lys Wing Ser Gly Tyr Ser Phe Ser Ser His Tyr Met His Trp Val 50 55 60 Lys Gln Ser His Gly Lys Ser Leu Glu Trp He ßly Tyr He Asp 65 70 75 Pro Ser Asn Gly Glu Thr Thr Tyr Asn Gln Lys Phe Lys Gly Lys 80 85 90 Al. '' Hi Le- "'" v? L Aa, - fhi 3rd SJi cei Thr Ala Asn Val Hi ±. 95 100 105 Leu Ser Ser Leu Thr Ser Asp Asp Ser Wing Val Tyr Phe Cys Wing 110 115 120 Arg Gly Asp Tyr Arg Tyr Asn Gly Asp Trp Phe Phe Asp Val Trp 125. 130 135 Gly Wing Gly Thr Thr Val Thr Val Being Ser Wing Being Thr Lys Gly 140 145 150 Pro Ser Val Phß Pro Leu Pro Wing Ser Ser Lys Ser Thr Ser Gly 155 160 165 Gly Thr Ala Ala Leu ßly Cys Leu Val Lys Asp Tyr Phe Pro Glu 170 175 180 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 185 190 195 His Thr Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 200 205 210 Ser Val Val Thr Val Pro Ser Ser Leu Gly Thr ßln Thr 215 220 225 Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 230 235 240 Lys Lys Val ßlu Pro Lys Ser Cys Asp Lys Thr His Thr 245 250 253 (2) INFORMATION FOR SEQ ID NO. 59: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 114 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 59: Asp He Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu 1 5 10 15 ßly Asp ßln Wing Ser He Ser Cys Arg Ser Ser ßln Ser Leu Val 20 25 30 His ßly He ßly Asn Thr Tyr Leu His Trp Tyr Leu ßln Lys Pro 35 40 45 ßly ßln Ser Pro Lys Leu Leu He Tyr Tyr Lys Val Ser Aßn Arg 50 55 60 Phe Ser ßly Val Pro Asp Arg Phe Ser Asp Ser ßly Ser ßly Thr í, b 70 7S Asp Phe Thr Leu Arg Xle Ser Arg Val ßlu Ala ßlu Asp Leu ßly 80 85 90 Leu Tyr Phe Cys Ser ßln Ser Thr His Val Pro Leu Thr Phe ßly 95 100 105 Ala ßly Thr Lys Leu ßlu Leu Lys Arg 110 114 (2) INFORMATION FOR SEQ ID NO. 60: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 114 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 60: Asp He ßln Met Thr ßln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 ßly Asp Arg Val Thr He Thr Cys Arg Ser Ser ßln Ser Leu Val 20 25 30 His ßly He Gly Asn Thr Tyr Leu His Trp Tyr Gln ßln Lys Pro 35 40 45 ßly Lys Ala Pro Lys Leu Leu He Tyr Tyr Lys Val Ser Asn Arg 50 55 60 Phe Ser ßly Val Pro Ser Arg Phe Ser ßly Ser ßly Ser ßly Thr 65 70 75 Asp Phe Thr Leu Thr Xle Ser Ser Leu ßln Pro ßlu Asp Phe Wing 80 85 90 Thr Tyr Tyr Cys Ser ßln Ser Thr His Val Pro Leu Thr Phe ßly 95 100 105 ßln ßly Thr Lys Val ßlu He Lys Arg 110 114 (2) INFORMATION FOR SEQ ID NO. 61: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 109 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 61: Asp He ßln Met Thr ßln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Ast. Ar_! j-h r '1 1 rb,. *',? r, Thr Tie fer 20 25 30 Lys Tyr Leu Wing Trp Tyr Gln Gln Lys Pro ßly Lys Wing Pro Lys 35 40 45 Leu Leu He Tyr Tyr Ser Gly Ser Thr Leu Glu Ser Gly Val Pro 50 55 60 Be Arg Phe Be Gly Ser Gly Be Gly Thr Asp Phe Thr Leu Thr 65 70 75 Be Ser Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys Gln 80 85 90 Gln His Asn ßlu Tyr Pro Leu Thr Phe ßly ßln ßly Thr Lys Val 95 100 105 ßlu He Lys Arg 109 (2) INFORMATION FOR SEQ ID NO. 62: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 117 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 62: Glu Xle ßln Leu ßln ßln Ser ßly Pro ßlu Leu Met Lys Pro ßly 1 5 10 15 Wing Ser Val Lys He Ser Cys Lys Wing Ser ßly and Tyr Ser Phe Ser 20 25 30 Ser His Tyr Met His Trp Val Lys ßln Ser His ßly Lys Ser Leu 35 40 45 ßlu Trp He ßly Tyr He Asp Pro Ser Asn Gly Glu Thr Thr Tyr 50 55 60 Aßn ßln Lys Phe Lys ßly Lys Wing Thr Leu Thr Val Asp Thr Ser 65 70 75 Being Ser Thr Wing Asn Val His Leu Being Ser Leu Thr Being Asp Asp 80 85 90 Being Wing Val Tyr Phe Cys Wing Wing Arg ßly Asp Tyr Arg Tyr Asn 95 100 105 ßly Asp Trp Phe Phe Asp Val Trp ßly Ala ßly Thr 110 115 117 (2) INFORMATION FOR SEQ ID NO. 63: • i) < ? GVTERISTTCA ^ E X SEQUENCE: (A) LENGTH: 117 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 63: Glu Val Gln Leu Val ßlu Ser ßly ßly ßly Leu Val ßln Pro ßly 1 5 10 15 ßly Ser Ser Leu Arg Leu Ser Cys Ala Ala Ser ßly Tyr Ser Phe Ser 20 25 30 Ser His Tyr Met His Trp Val Arg ßln Ala Pro ßly Lys ßly Leu 35 40 45 Glu Trp Val ßly Tyr He Asp Pro Ser Asn ßly Glu Thr Thr Tyr 50 55 60 Asn Gln Lys Phe Lys ßly Arg Phe Thr He Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Wing Wing Arg Gly Asp Tyr Arg Tyr Asn 95 100 105 Gly Asp Trp Phe Phe Asp Val Trp ßly ßln Gly Thr 110 115 117 (2) INFORMATION FOR SEQ ID NO. 64: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 116 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 64: Glu Val ßln Leu Val ßlu Ser Gly ßly Gly Leu Val ßln Pro ßly 1 5 10 15 ßly Ser Leu Arg Leu Ser Cys Ala Wing ßly Phe Ser Phe Thr 20 25 30 ßly His Trp Met Asn Trp Val Arg ßln Ala Pro ßly Lys ßly Leu 35 40 45 ßlu Trp Val ßly Met Met He His Pro Ser Asp Ser ßlu Thr Arg Tyr 50 55 60 Wing Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala ßlu Asp 80 f "9n Thr Ala Val Tyr Tyr Cys Ala Ala Arg ßly He Tyr Phe Tyr ßly 95 100 105 Thr Thr Tyr Phe Asp Tyr Trp ßly ßln ßly Thr 110 115 116 (2) INFORMATION FOR SEQ ID NO. 65: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 242 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 65: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be He Wing Thr Asn Wing Tyr Wing Asp Xle ßln Met Thr ßln Ser 20 25 30 Pro Ser Ser Leu Ser Wing Ser Val ßly Asp Arg Val Thr He Thr 35 40 45 Cys Arg Ser Ser ßln Ser Leu Val His ßly He ßly Asn Thr Tyr 50 55 60 Leu His Trp Tyr ßln ßln Lys Pro ßly Lys Ala Pro Lys Leu Leu 65 70 75 He Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Ser Arg Phe 80 85 90 Ser Gly Ser ßly Ser ßly Thr Asp Phe Thr Leu Thr Be Ser Be 95 100 105 Leu ßln Pro ßlu Asp Phe Wing Thr Tyr Tyr Cys Ser ßln Ser Thr 110 115 120 His Val Pro Leu Thr Phe ßly ßln ßly Thr Lys Val ßlu Xle Lys 125 130 135 Arg Thr Val Ala Ala Pro Ser Val Phe Xle Phe Pro Pro Ser Asp 140 145 150 ßlu ßln Leu Lys Ser ßly Thr Ala Ser Val Val Cys Leu Leu Asn 155 160 165 Asn Phe Tyr Pro Arg ßlu Wing Lys Val ßln Trp Lys Val Asp Asn 170 175 180 Wing Leu ßln Ser ßly Asn Ser ßln ßlu Ser Val Thr ßlu ßln Asp 185 190 195 Ser Lys Atp Ser Thr iyt _ JI Leu S ~ S * t Tht * u rh? Le < ? Ser 200 205 210 Lys Wing Asp Tyr ßlu Lys His Lys Val Tyr Wing Cys ßlu Val Thr 215 220 225 His Gln ßly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg ßly 230 235 240 ßlu Cys 242 (2) INFORMATION FOR SEQ ID NO. 66: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 253 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 66: Met Lys Lys Asn Xle Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be Xle Wing Thr Asn Wing Tyr Wing Glu Val Gln Leu Val ßln Ser 20 25 30 ßly ßly ßly Leu Val ßln Pro ßly ßly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser ßly Tyr Ser Phe Ser Ser His Tyr Met His Trp Val 50 55 60 Arg ßln Ala Pro ßly Lys ßly Leu Glu Trp Val Gly Tyr He Asp 65 70 75 Pro Ser Asn Gly Glu Thr Thr Tyr Asn ßln Lys Phe Lys ßly Arg 80 85 90 Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr Leu ßln 95 100 105 Met Asn Ser Leu Arg Ala ßlu Asp Thr Ala Val Tyr Tyr Cys Ala 110 115 120 Arg ßly Asp Tyr Arg Tyr Asn ßly Asp Trp Phe Phe Asp Val Trp 125 130 135 ßly ßln ßly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys ßly 140 145 150 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser ßly 155 160 165 ßly Thr Ala Ala Leu ßly Cys Leu Val Lys Asp Tyr Phe Pro ßlu 170 175 180 Pro Val Thr Val Ser Trp Asn Ser ßly Ala Leu Thr Ser ßly Val 185 190 195 His Thr Phe Pro Wing Val Leu ßln Ser Ser ßly Leu Tyr Ser Leu 200 205 210 Ser Val Val Thr Val Pro Ser Ser Leu ßly Thr ßln Thr 215 • 220 225 Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 230 235 240 Lys Lys Val ßlu Pro Lys Ser Cys Asp Lys Thr His Thr 245 250 253 (2) INFORMATION FOR SEQ ID NO. 67:, (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 159 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 67:, Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met 1 5 10 15 Wing Asn Wing Asn Lys Gly Wing Met Thr Glu Asn Wing Asp Glu Asn 20 25 30 Ala Leu Gln Ser Asp Ala Lys ßly Lys Leu Asp Ser Val Ala Thr 35 40 45 Asp Tyr ßly Ala Ala He Asp ßly Phe He ßly Asp Val Ser ßly 50 55 60 Leu Ala Asn ßly Asn ßly Ala Thr ßly Asp Phe Ala ßly Ser Ser 65 70 75 Asn Ser ßln Met Ala ßln Val ßly Asp ßly Asp Asn Ser Pro Leu 80 85 90 Met Asn Asn Phe Arg ßln Tyr Leu Pro Ser Leu Pro ßln Ser Val 95 100 105 ßlu Cys Arg Pro Phe Val Phe Ser Ala ßly Lys Pro Tyr ßlu Phe 110 115 120 Be Xle Asp Cys Asp Lys Xle Asn Leu Phe Arg ßly and Val Phe Wing 125 130 135 Phe Leu Leu Tyr Val Wing Thr Phe Met Tyr Val Phe Ser Thr Phe 140 145 150 Ala Asr lio t, eu Arg Asn Lys Glu Per lys (2) INFORMATION FOR SEQ ID NO. 68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 780 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 68: ATGAAAAAßA ATATCßCATT TCTTCTTßCA TCTATßTTCß TTTTTTCTAT 50 TGCTACAAAC GCATACßCTß ATATCCAGAT GACCCAGTCC CCGAGCTCCC 100 TGTCC6CCTC TßTßßGCßAT AGGGTCACCA TCACCTßCAß ßTCAAßTCAA 150 AGCTTAßTAC ATGßTATAGG TAACACßTAT TTACACTGßT ATCAACAßAA 200 ACCAßßAAAA ßCTCCßAAAC TACTGATTTA CAAAGTATCC AATCßATTCT 250 CTßßAGTCCC TTCTCßCTTC TCTGGATCCG GTTCTßßßAC ßßATTTCACT 300 CTßACCATCA ßCAßTCTßCA ßCCAßAAßAC TTCßCAACTT ATTACTßTTC 350 ACAßAßTACT CATßTCCCßC TCACßTTTßß ACAßßßTACC AAßßTGGAGA 400 TCAAACßAAC TßTßßCTßCA CCATCTßTCT TCATCTTCCC ßCCATCTßAT 450 ßAßCAßTTßA AATCTßßAAC TßCTTCTßTT ßTßTßCCTßC T6AATAACTT 500 CTATCCCAßA ßAßßCCAAAß TACAßTßGAA GßTßßATAAC ßCCCTCCAAT 550 CßGGTAACTC CCAGßAßAGT GTCACAGAGC AGGACAßCAA GGACAGCACC 600 TACAßCCTCA ßCAßCACCCT ßACßCTßAßC AAAßCAßACT ACßAßAAACA 650 CAAAßTCTAC ßCCTßCGAAG TCACCCATCA 6GGCCTGAGC TCßCCCßTCA 700 CAAAßAGCTT CAACAGGGGA ßAßTßTTAAß CTßATCCTCT ACßCCGGACß 750 CATCßTßßCC CTAßTACßCA ACTAßTCßTA 780 (2) INFORMATION ?. FOR SEQ ID NO. 69: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 242 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 69: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be Xle Wing Thr Asn Wing Tyr Wing Asp Xle Gln Met Thr ßln Ser 20 25 30 Pro Ser Ser Leu Ser Ala Ser Val ßly Asp Arg Val Thr He Thr 35 40 45 Cys Arg Ser Ser ßln Ser Leu Val His ßly He ßly Asn Thr Tyr 50 55 60 Leu His Trp Tyr ßln ßln Lys Pro ßly Lys Ala Pro Lys Leu Leu 65 70 75 He Tyr Lys Val Ser Asn Arg Phe Ser ßly Val Pro Ser Arg Phe 80 85 90 Be ßly Be ßly Be Gly Thr Asp Phe Thr Leu Thr Be Ser Be 95 100 105 Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys Ser ßln Ser Thr 110 115 120 His Val Pro Leu Thr Phe ßly ßln ßly Thr Lys Val ßlu He Lys 125 130 135 Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp 140 145 150 ßlu ßln Leu Lys Ser ßly Thr Ala Ser Val Val Cys Leu Leu Asn 155 160 165 Asn Phe Tyr Pro Arg ßlu Wing Lys Val ßln Trp Lys Val Asp Asn 170 175 180 Ala Leu ßln Ser Gly Asn Ser Gln ßlu Ser Val Thr ßlu ßln Asp 185 190 195 Being Lys Asp Being Thr Tyr Being Leu Being Being Thr Leu Thr I? U Being 200 205 210 Lys Wing Asp Tyr ßlu Lys His Lys Val Tyr Wing Cys ßlu Val Thr 215 220 225 His ßln ßly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg ßly 230 235 240 Glu Cys 242 (2) INFORMATION FOR SEQ ID NO. 70: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 253 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear,? I) DLSCRiPyíON DF, LA J ,. rfc? '? A: SEQ JL NO. 7J: Met Lys Lys Aßn Xle Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be He Wing Thr Asn Wing Tyr Wing Glu Val Gln Leu Val ßlu Ser 20 25 30 Gly Gly Glu Leu Val Gln Pro Gly Gly Ser Leu Glu Leu Ser Cys 35 40 45 Ala Ala Ser ßly Tyr Ser Phe Ser Ser His Tyr Met His Trp Val 50 55 60 Lys ßln Ala Pro ßly Lys ßly Leu ßlu Trp Val ßly Tyr He Asp 65 70 75 Pro Ser Asn ßly ßlu Thr Thr Tyr Asn ßln Lys Phe Lys ßly Arg 80 85 90 Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Wing Tyr Leu ßln 95 100 105 Met Asn Ser Leu Arg Ala ßlu Asp Thr Wing Val Tyr Tyr Cys Wing 110 115 120 Arg ßly Asp Tyr Arg Tyr Asn ßly Asp Trp Phe Phe Asp Val Trp 125 130 135 ßly ßln ßly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys ßly 140 145 150 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser ßly 155 160 165 ßly Thr Ala Ala Leu ßly Cys Leu Val Lys Asp Tyr Phe Pro ßlu 170 175 180 Pro Val Thr Val Ser Trp Asn Ser ßly Ala Leu Thr Ser ßly Val 185 190 195 His Thr Phe Pro Wing Val Leu ßln Ser Ser ßly Leu Tyr Ser Leu 200 205 210 Being Val Val Thr Val Pro Being Ser Leu ßly Thr ßln Thr 215 220 225 Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 230 235 240 Lys Lys Val ßlu Pro Lys Ser Cys Asp Lys Thr His Thr 245 250 253 (2) INFORMATION FOR SEQ ID NO. 71: (i) CHARACTERISTICS. * - DF THE SEQUENCE: (A) IT GITun: 2 A tract. > < : ic, .- > (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 71: Met Lys Lys Asn Xle Wing Phe Leu Leu Wing Ser Met Phe Val Phe 1 5 10 15 Be Xle Wing Thr Asn Wing Tyr Wing Asp He Gln Met Thr Gln Ser 20 25 30 Pro Ser Ser Leu Ser Wing Val Gly Asp Arg Val Thr He Thr 35 40 45 Cys Arg Ser Ser ßln Ser Leu Val His ßly He ßly Ala Thr Tyr 50 55 60 Leu His Trp Tyr ßln ßln Lys Pro ßly Lys Ala Pro Lys Leu Leu 65 70 75 He Tyr Lys Val Ser Asn Arg Phe Ser ßly Val Pro Ser Arg Phe 80 85 90 Be ßly Be ßly Be Gly Thr Asp Phe Thr Leu Thr Be Ser Be 95 100 105 Leu Gln Pro ßlu Asp Phe Wing Thr Tyr Tyr Cys Ser ßln Ser Thr 110 115 120 His Val Pro Leu Thr Phe ßly ßln ßly Thr Lys Val Glu He Lys 125 130 135 Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp 140 145 150 Glu ßln Leu Lys Ser ßly Thr Ala Ser Val Val Cys Leu Leu Asn 155 160 165 Asn Phe Tyr Pro Arg ßlu Wing Lys Val ßln Trp Lys Val Asp Asn 170 175 180 Ala Leu ßln Ser ßly Asn Ser ßln ßlu Ser Val Thr Glu Gln Asp 185 190 195 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 200 205 210 Lys Wing Asp Tyr ßlu Lys His Lys Val Tyr Wing Cys ßlu Val Thr 215 220 225 His ßln ßly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg ßly 230 235 240 ßlu Cys 242 (2) INFORMATION FOR SEQ ID NO. 72: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 72: Cys Pro Pro Cys Pro Pro Pro ßlu Leu Leu ßly and Gly Arg Met Lys 1 5 10 15 Gln Leu ßlu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His 20 25 30 Leu ßlu Asn ßlu Val Ala Arg Leu Lys Lys Leu Val ßly ßlu Arg 35 40 45 (2) INFORMATION FOR SEQ ID NO. 73: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 780 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID DO NOT. 73: ATGAAAAAGA ATATCGCATT TCTTCTTGCA TCTATßTTCß TTTTTTCTAT 50 TßCTACAAAC ßCATACßCTß ATATCCAGAT GACCCAGTCC CCGAßCTCCC 100 TGTCCßCCTC TGTßGGCGAT AßßßTCACCA TCACCTßCAß ßTCAAßTCAA 150 AßCTTAßTAC ATßßTATAGG TßCTACßTAT TTACACTßßT ATCAACAGAA 200 ACCAGGAAAA ßCTCCßAAAC TACTGATTTA CAAAGTATCC AATC6ATTCT 250 CTßßAßTCCC TTCTCßCTTC TCTßßATCCß ßTTCTßßGAC G6ATTTCACT 300 CTßACCATCA ßCAGTCTGCA ßCCAßAAßAC TTC6CAACTT ATTACTßTTC 350 ACAßAßTACT CATGTCCCGC TCACßTTTßß ACAßßGTACC AAGßTGGAGA 400 TCAAACGAAC TßTGGCTßCA CCATCTßTCT TCATCTTCCC ßCCATCTGAT 450 GAßCAßTTGA AATCTGGAAC TßCTTCTßTT ßTGTGCCTßC TGAATAACTT 500 CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC GCCCTCCAAT 550 CßßßTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA GGACAGCACC 600 TACAGCCTCA GCAGCACCCT GACGCTGAGC AAAßCAßACT ACGAGAAACA 650 •? V > AGT (.lAC ßCCTßCOAAG T'JCCCATCA - -í- <, >, TC-i .. -U t'CA 7? CAAAßAßCTT CAACAßGGGA GAßTßTTAAß CTGATCCTCT ACßCCßßACß 750 CATCßTßßCC CTAßTACßCA ACTAßTCßTA 780 (2) INFORMATION FOR SEQ ID NO. 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 927 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 74: AAAAßßßTAT CTAGAGGTTß AßßTGATTTT ATßAAAAAGA ATATCßCATT 50 TCTTCTTGCA TCTATßTTCG TTTTTTCTAT TGCTACAAAC ßCGTACßCTG 100 AßßTTCAGCT AGTßCAGTCT ßßCßßTGGCC TßßTßCAßCC AßßßßßCTCA 150 CTCCßTTTßT CCTßTßCAGC TTCTßßCTAC TCCTTCTC6A ßTCACTATAT 200 GCACTßßßTC CßTCAßßCCC CßßßTAAßßß CCTßGAATGß ßTTßGATATA 250 TTGATCCTTC CAATßßTßAA ACTACßTATA ATCAAAAßTT CAAGGGCCGT 300 TTCACTTTAT CTCßCßACAA CTCCAAAAAC ACAßCATACC TßCAGATßAA 350 CAßCCTßCßT ßCTßAßßACA CTßCCßTCTA TTACTßTßCA AßAßßßßATT ATCßCTACAA TßßTGACTßß TTCTTCßACß TCTGGßßTCA AßßAACCCTß 450 ßTCACCßTCT CCTCßßCCTC CACCAAßßßC CCATCßßTCT TCCCCCTßßC 500 ACCCTCCTCC AAGAGCACCT CTßßßßßCAC AGCGGCCCTG ßßCTßCCTßß 550 TCAAßßACTA CTTCCCCGAA CCßßTßACßß TßTCßTßßAA CTCAßßCßCC 600 CTßACCAßCß ßCßTßCACAC CTTCCCßßCT ßTCCTACAßT CCTCAßßACT 650 CTACTCCCTC AßCAßCßTßß TßACCßTßCC CTCCAGCAßC TTßßßCACCC 700 AGACCTACAT CTßCAACßTß AATCACAAßC CCAGCAACAC CAAGGTCßAC 750 AAßAAAßTTß AGCCCAAATC TTGT6ACAAA ACTCACACAT ßCCCßCCßTß 800 CCCAßCACCA ßAACTßCTßß GCßßCCßCAT GAAACAGCTA GAßßACAAGß 850 TCGAAßAGCT ACTCTCCAAG AACTACCACC TAGAGAATGA AGTGGCAAGA 900 CTCAAAAAGC TTßTCßGßGA ßCGCTAA 927 < 2) INFORMATION FOR: 'A SSQ ID NO. 75: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 298 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 75: Met Lys Lys Asn He Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Be Xle Wing Thr Asn Wing Tyr Wing Glu Val Gln Leu Val ßln Ser 20 25 30 ßly ßly ßly Leu Val ßln Pro Gly ßly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser ßly Tyr Ser Phe Ser Ser His Tyr Met His Trp Val 50 55 60 Arg Gln Wing Pro Gly Lys Gly Leu Glu Trp Val Gly Tyr He Asp 65 70 75 Pro Ser Asn Gly Glu Thr Thr Tyr Asn Gln Lys Phe Lys ßly Arg 80 85 90 Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr Leu ßln 95 100 105 Met Asn Ser Leu Arg Ala ßlu Asp Thr Ala Val Tyr Tyr Cys Ala 110 115 120 Arg ßly Asp Tyr Arg Tyr Asn ßly Asp Trp Phe Phe Asp Val Trp 125 130 135 ßly Gln Gly Thr Leu Val Thr Val Ser Ser Wing Ser Thr Lys Gly 140 145 150 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser ßly 155 160 165 ßly Thr Ala Ala Leu ßly Cys Leu Val Lys Asp Tyr Phe Pro ßlu 170 175 180 Pro Val Thr Val Ser Trp Asn Ser ßly Ala Leu Thr Ser ßly Val 185 190 195 His Thr Phe Pro Wing Val Leu ßln Ser Ser ßly Leu Tyr Ser Leu 200 205 210 Ser Val Val Thr Val Pro Ser Ser Leu ßly Thr ßln Thr 215 220 225 Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 230 235 240 Lys Lys V * 1 Gxu Pro Lys Ser Cy_s Asp Lys Thr Hxs Thr ..ye Pro 245 250 255 Pro Cys Pro Ala Pro ßlu Leu Leu ßly ßly Arg Met Lys ßln Leu 260 265 270 ßlu Asp Lys Val ßlu ßlu Leu Leu Ser Lys Asn Tyr His Leu ßlu 275 280 285 Asn ßlu Val Ala Arg Leu Lys Lys Leu Val ßly ßlu Arg 290 295 298 (2) INFORMATION FOR SEQ ID NO. 76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6563 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (Xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 76: GAATTCAACT TCTCCATACT TT6GATAAGG AAATACAGAC ATGAAAAATC 50 TCATTßCTßA ßTTßTTATTT AAßCTTßCCC AAAAAßAAGA AGAGTCGAAT 100 GAACTßTßTß CGCAGGTAGA AßCTTTßßAß ATTATCßTCA CTßCAATGCT 150 TCGCAATATß GCGCAAAATG ACCAACAGCß GTTGATTßAT CAGGTAGAGG 200 GßßCßCTßTA CßAßßTAAAß CCCGATßCCA ßCATTCCTßA CßACßATACß 250 ßAßCTßCTßC ßCßATTACßT AAAßAAßTTA TTßAAßCATC CTCßTCAßTA 300 AAAAßTTAAT CTTTTCAACA ßCTßTCATAA AGTTGTCACG ßCCßAGACTT 350 ATAGTCßCTT TßTTTTTATT TTTTAATßTA TTTßTAACTA GAATTCGAGC 400 TCßßTACCCß ßßßATCCTCT CßAGGTTßAß ßTßATTTTAT GAAAAAGAAT 450 ATCßCATTTC TTCTTßCATC TATßTTCßTT TTTTCTATTß CTACAAACßC 500 ATACGCTGAT ATCCAßATGA CCCAGTCCCC GAGCTCCCTß TCCßCCTCTß 550 TßßGCßATAG ßßTCACCATC ACCTßCAßGT CAAGTCAAAß CTTA6TACAT 600 ßßTATAßßTT CTACGTATTT ACACTßßTAT CAACAßAAAC CAGGAAAAßC 650 TCCGAAACTA CTßATTTACA AAGTATCCAA TCßATTCTCT ßßAGTCCCTT 700 CTCßCTTCTC TßßATCCßGT TCTßßßACßß ATTTCACTCT GACCATCAGC 750 AGTCTßCAGC CAGAAßACTT CßCAACTTAT TACTßTTCAC AGAGTACTCA 800 TßTCCCßCTC, r. T vCta r. , nw \ c? A ßGTCGAdATC Afl? Crj? ACTG 850 TßGCTßCACC ATCTßTCTTC ATCTTCCCGC CATCTGATGA GCAßTTßAAA 900 TCTGGAACTG CTTCTßTTßT ßTßCCTßCTß AATAACTTCT ATCCCAGAGA 950 ßßCCAAAßTA CAßTßßAAßß TGGATAACGC CCTCCAATCG GGTAACTCCC 1000 AGGAßAßTßT CACAGAGCAG ßACAßCAAßß ACAßCACCTA CAGCCTCAGC 1050 AßCACCCTßA CßCTßAßCAA AßCAßACTAC GAGAAACACA AAGTCTACßC 1100 CTGCGAAßTC ACCCATCAGß ßCCTGAGCTC ßCCCßTCACA AAßAßCTTCA 1150 ACAßßßGAGA GTGTTAAGCT ßATCCTCTAC ßCCßGACßCA TCßTßßCCCT 1200 AGTACßCAAC TAßTCßTAAA AAGßGTATCT AGAßßTTGAG GTßATTTTAT 1250 ßAAAAAGAAT ATCGCATTTC TTCTTGCATC TATGTTCGTT TTTTCTATTß 1300 CTACAAACßC ßTACßCTßAG GTTCA6CTA6 TßCAßTCTßß CßßTßßCCTß 1350 ßTßCAßCCAß GßßßCTCACT CCGTTTGTCC TßTßCAßCTT CTGGCTACTC 1400 CTTCTCßAGT CACTATATGC ACTßßßTCCß TCAßßCCCCß ßßTAAGGGCC 1450 TßGAATGßßT TßGATATATT GATCCTTCCA ATßßTßAAAC TACßTATAAT 1500 CAAAAßTTCA AGGGCCGTTT CACTTTATCT CßCGACAACT CCAAAAACAC 1550 AGCATACCTß CAGATGAACA GCCTßCßTßC TßAßßACACT ßCCßTCTATT 1600 ACTGTGCAAß AßßßßATTAT CßCTACAATß ßTGACTGßTT CTTCßACßTC 1650 TßßßßTCAAß 6AACCCTGGT CACCßTCTCC TCßGCCTCCA CCAAßßGCCC 1700 ATCßGTCTTC CCCCTßßCAC CCTCCTCCAA ßAßCACCTCT ßßßßßCACAß 1750 CßßCCCTßßß CTßCCTßGTC AAGGACTACT TCCCCßAACC ßßTßACßßTß 1800 TCßTßßAACT CAßßCßCCCT GACCAGCGGC GTßCACACCT TCCCßßCTßT 1850 CCTACA6TCC TCAßßACTCT ACTCCCTCAß CAGCGTßßTß ACCßTßCCCT 1900 CCAßCAßCTT ßGGCACCCAß ACCTACATCT GCAACGTßAA TCACAAßCCC 1950 AßCAACACCA AßßTCßACAA ßAAAGTTGAß CCCAAATCTT ßTßACAAAAC 2000 TCACACATßC CCßCCßTßCC CAßCACCAßA ACTGCTGGGC GGCCGCATßA 2050 AACAßCTAßA ßßACAAßßTC GAAGA6CTAC TCTCCAAßAA CTACCACCTA 2100 GAGAATGAAG TßßCAAßACT CAAAAAßCTT GTCGGGGAGC ßCTAAGCATG 2150 CßACßßCCCT AßAßTCCCTA ACßCTCßßTT ßCCßCCßßßC 6TTTTTTATT 2200 CT-APf TG? T ßTTTCACAGC TTATCATC A T? AGCTTTAA TßCGGTAGTT? 2í > 0 TATCACAGTT AAATTGCTAA CßCAßTCAßG CACCGTGTAT ßAAATCTAAC 2300 AATßCßCTCA TCßTCATCCT CßßCACCßTC ACCCTßßATß CTßTAGßCAT 2350 AßßCTTßßTT ATßCCGGTAC TßCCßßßCCT CTTßCßßßAT ATCßTCCATT 2400 CCGACAGCAT CßCCAßTCAC TATßßCßTßC TßCTAßCßCT ATATßCßTTß 2450 ATßCAATTTC TATßCßCACC CßTTCTCßßA GCACTßTCCß ACCßCTTTßß 2500 CCßCCßCCCA ßTCCTßCTCß CTTC6CTACT TßßAßCCACT ATCßACTACG 2550 CßATCATßßC ßACCACACCC ßTCCTßTßßA TCCTCTAC6C CßßACßCATC 2600 ßTGGCCßßCA TCACCGGCGC CACAGGTGCG GTTGCTGGCG CCTATATCßC 2650 CßACATCACC GATßßßGAAG ATCßßßCTCß CCACTTCßßß CTCATßAGCG 2700 CTTGTTTCGG CßTßßßTATß ßTßGCAGGCC CCßTßßCCßß ßßßACTßTTß 2750 GGCßCCATCT CCTTßCACßC ACCATTCCTT ßCßßCßßCßß TßCTCAACßG 2800 CCTCAACCTA CTACTßßGCT ßCTTCCTAAT ßCAßßAßTCß CATAAGGGAG 2850 AßCßTCßTCC ßATßCCCTTß AGAGCCTTCA ACCCAßTCAß CTCCTTCCßß 2900 TßßßCGCßßG ßCATßACTAT CßTCGCCGCA CTTATGACTß TCTTCTTTAT 2950 CATßCAACTC ßTAGGACAGG TßCCßßCAßC ßCTCTßßßTC ATTTTCßßCß 3000 AßßACCßCTT TCßCTßßAßC ßCGACßATGA TCßßCCTßTC ßCTTßCßßTA 3050 TTCßßAATCT TßCACßCCCT CßCTCAAßCC TTCßTCACTß ßTCCCßCCAC 3100 CAAACßTTTC ßßCGAGAAGC AßßCCATTAT CGCCGßCATß ßCßGCCßACß 3150 CßCTßßßCTA CßTCTTßCTßßßCßTTCßCGA CßCßAßßCTßßATßßCCTTC 3200 CCCATTATßA TTCTTCTC6C TTCCßßCGGC ATCßßGATGC CCGCßTTGCA 3250 ßGCCATßCTG TCCAßßCAGG TAGATGACßA CCATCAßGGA CAGCTTCAAß 3300 ßATCGCTCGC GßCTCTTACC AßCCTAACTT CßATCACTGG ACCGCTGATC 3350 ßTCACßßCGA TTTATGCCßC CTCßßCßAßC ACATßßAACß ßßTTßßCATß 3400 GATTGTAßßC ßCCßCCCTAT ACCTTßTCTG CCTCCCCßCß TTGCGTCßCß 3450 GTGCATGGAG CCßßßCCACC TCGACCTßAA TßßAAßßßßßßß CGßCACCTCß 3500 CTAACGGATT CACCACTCCA AGAATTGGAG CCAATCAATT CTTßCßßAGA 3550 ACTßTßAATß CßCAAACCAA CCCTTßßCAß AACATATCCA TCGCßTCCGC 3600 CATCTCCPOC AGCr ^ c ^ CGC ßGCGCATCTC GßßCAG '"" lT ßßOTCTßGC 3650 CACßßGTßCG CATßATCßTß CTCCTßTCßT TßAßßACCCß ßCTAßßCTGß 3700 CßGGßTTßCC TTACTGGTTA ßCAßAATGAA TCACCGATAC ßCßAßCGAAC 3750 GTßAAßCGAC TGCTßCTßCA AAACßTCTßC ßACCTGAGCA ACAACATßAA 3800 TGGTCTTCßß TTTCCßTßTT TCßTAAAßTC TßßAAACGCß GAAßTCAßCß 3850 CCCTßCACCA TTATßTTCCß GATCTGCATC ßCAßßATGCT GCTGßCTACC 3900 CTßTßßAACA CCTACATCTß TATTAACGAA GCßCTßßCAT TßACCCTGAß 3950 TGATTTTTCT CTGßTCCCßC CßCATCCATA CCßCCAßTTß TTTACCCTCA 4000 CAACGTTCCA GTAACCGGGC ATGTTCATCA TCAGTAACCC GTATCßTGAß 050 CATCCTCTCT CGTTTCATCß ßTATCATTAC CCCCATßAAC AGAAATTCCC 4100 CCTTACACßß AGGCATCAAG TGACCAAACA GGAAAAAACC GCCCTTAACA 4150 TGGCCCGCTT TATCAGAAGC CAGACATTAA CGCTTCTGGA GAAACTCAAC 4200 GAGCTßßACß CGGATGAACA GGCAGACATC TGTGAATCGC TTCACGACCA 4250 CßCTßATßAß CTTTACCßCA ßCTßCCTCßC ßCßTTTCßßT ßATGACGGTß 4300 AAAACCTCTß ACACATßCAß CTCCCGGAGA CGGTCACAGC TTßTCTGTAA 4350 GCGGATGCCG GGAGCAGACA AßCCCßTCAß GGCGCGTCAG CßßßTßTTßß 4400 CßGGTßTCßß GßCßCAßCCA TßACCCAßTC ACGTAGCGAT AGCGGAGTGT 4450 ATACTßßCTT AACTATßCßß CATCAGAßCA GATTßTACTß AGAßTßCACC 4500 ATATßCßßTß TßAAATACCß CACAGATGC6 TAAGGAGAAA ATACCßCATC 550 AßßCßCTCTT CCßCTTCCTC ßCTCACTßAC TCGCTßCGCT CGGTCßTTCß 4600 ßCTGCGßCßA GCßßTATCAG CTCACTCAAA ßßCßßTAATA CGGTTATCCA 4650 CAGAATCAGß ßßATAACßCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA 4700 AAGßCCAGGA ACCßTAAAAA GGCCGCßTTß CTGßCßTTTT TCCATAßßCT 4750 CCßCCCCCCT ßACßAßCATC ACAAAAATCß ACGCTCAAGT CAßAßßTßßC 4800 GAAACCCGAC AßßACTATAA AßATACCAGG CGTTTCCCCC TßßAAGCTCC 4850 CTCGTßCßCT CTCCTßTTCC ßACCCTßCCß CTTACC6GAT ACCTßTCCßC 4900 CTTTCTCCCT TCßßßAAßCß TßßCßCTTTC TCATAßCTCA CßCTßTAßßT 4950 ATCTCAßTTC ßßTßTAßßTC ßTTCßCTCCA AßCTßßßCTCTß TGTßCACßAA 5000 ccccc rvr HJGGCG? CCG C GCOCGTTA TCG? TAAOT ATCG ^ CTT ^ A soro GTCCAACCCG ßTAAGACACß ACTTATCßCC ACTßßCAGCA ßCCACTßßTA 5100 ACAGGATTA6 CAßAGCGAßG TATßTAGGCß ßTßCTACAßA ßTTCTTßAAß 5150 TßßTGGCCTA ACTACGGCTA CACTAGAAGß ACAßTATTTß ßTATCTßCßC 5200 TCTßCTßAAß CCAßTTACCT TCßßAAAAAß AßTTßßTAßC TCTTßATCCß 5250 ßCAAACAAAC CACCGCTGßT AGCßßTßßTT TTTTT6TTT6 CAAßCAßCAß 5300 ATTACßCßCA ßAAAAAAAßß ATCTCAA6AA ßATCCTTTGA TCTTTTCTAC 5350 GGßßTCTßAC ßCTCAßTßGA ACGAAAACTC ACGTTAAßGß ATTTTGßTCA 5400 TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA 5450 AßTTTTAAAT CAATCTAAAß TATATATGAG TAAACTTGGT CTGACAßTTA 5500 CCAATGCTTA ATCAGTGAGß CACCTATCTC AGCGATCTGT CTATTTCßTT 5550 CATCCATAßT TßCCTßACTC CCCßTCGTßT AGATAACTAC GATACGßßAG 5600 ßßCTTACCAT CTßßCCCCAß TßCTßCAATß ATACCßCßAß ACCCACßCTC 5650 ACCßßCTCCA ßATTTATCAß CAATAAACCA ßCCAGCCGßA AßßßCCßAßC 5700 ßCAßAAßTßß TCCTßCAACT TTATCCßCCT CCATCCAGTC TATTAATTGT 5750 TGCCßßßAAß CTAßAGTAAG TAßTTCßCCA ßTTAATAßTT TßCßCAACßT 5800 TßTTGCCATT ßCTßCAßßCA TCßTßßTßTC ACßCTCßTCG TTTGßTATGG 5850 CTTCATTCAß CTCCßGTTCC CAACßATCAA ßßCGAGTTAC ATßATCCCCC 5900 ATßTTßTßCA AAAAAßCGGT TAGCTCCTTC ßßTCCTCCßA TCGTTGTCAß 5950 AAßTAAßTTß ßCCßCAßTßT TATCACTCAT ßßTTATßGCA GCACTßCATA 6000 ATTCTCTTAC TGTCATGCCA TCCßTAAßAT ßCTTTTCTßT ßACTßßTßAß 6050 TACTCAACCA AßTCATTCTß AßAATAGTGT ATßCßGCGAC CßAGTTGCTC 6100 TTGCCCGGCG TCAACACGßß ATAATACCGC GCCACATAßC AßAACTTTAA 6150 AAßTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAG6ATC 6200 TTACCßCTßT TGAßATCCAß TTCßATGTAA CCCACTCGTG CACCCAACTG 6250 ATCTTCAGCA TCTTTTACTT TCACCAßCßT TTCTßßGTGA ßCAAAAACAß 6300 ßAAßGCAAAA TGCCGCAAAA AAßßßAATAA GGGCßACACß GAAATGTTGA 6350 ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGG6TTA 6400 1 CGGGT A ' IG? GCGGATACA TA'TTGA '? TMT? G ^ AA, 'ATAAACAA', 6450 TAGGGGTTCC GC6CACATTT CCCC6AAAA6 TGCCACCTßA CGTCTAAGAA 6500 ACCATTATTA TCATGACATT AACCTATAAA AATAßßCßTA TCACßAGGCC 6550 CTTTCGTCTT CAÁ 6563 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned irwaxam is that which is clear from the present description of the investment. Having described the invention as above, the contents of the following are claimed as property.

Claims (52)

1. A conjugate consisting essentially of one or more antibody fragments covalently bound to one or more non-protein polymer molecules, characterized in that the apparent size of the conjugate is at least about 500 kD.

2. The conjugate according to claim 1, characterized in that the apparent size of the conjugate is at least about 800 kD.

3. The conjugate according to claim 1, characterized in that the apparent size of the conjugate is at least about 1,400 kD.

4. The conjugate according to claim 1, characterized in that the apparent size of the conjugate is at least about 1,800 kD.

5. The conjugate according to claim 1, characterized in that the apparent size of the conjugate is at least 8 times greater than the apparent size of the antibody fragment.

6. The conjugate according to claim 5, characterized in that the apparent size of the conjugate is at least 15 times greater than the apparent size of the antibody fragment.

7. The conjugate according to claim 6, characterized in that the apparent size of the conjugate is at least 25 times greater than the apparent size of the antibody fragment.

8. The conjugate according to claim 1, characterized in that the conjugate contains no more than one antibody fragment, and wherein the antibody fragment is selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, scFv and F (ab ') 2.

9. The conjugate according to claim 8, characterized in that the antibody fragment is F (ab ') 2.

10. The conjugate according to claim 1, characterized in that the antibody fragment is covalently bound to no more than 10 non-protein polymer molecules.

11. The conjugate according to claim 10, characterized in that the antibody fragment is covalently bound to no more than 5 non-protein polymer molecules.

12. The conjugate according to claim 11, characterized in that the antibody fragment is covalently bound to no more than 2 non-protein polymer molecules.

13. The conjugate according to claim 12, characterized in that the antibody fragment is covalently bound to no more than 1 non-protein poiimeric molecule.

14. The conjugate according to claim 12, characterized in that the antibody fragment comprises a heavy chain and a light chain derived from a parent antibody, wherein in the parent antibody the heavy and light chains are covalently linked by a disulfide bridge between a residue of cysteine in the light chain and a cysteine residue in the heavy chain, where in the antibody fragment the cysteine residue in the light or heavy chain is replaced with another amino acid, and the cysteine residue in the opposite chain is covalently bound to a non-protein polymer molecule.

15. The conjugate according to claim 8, characterized in that the antibody fragment is selected from the group consisting of Fab, Fab 'and Fab'-SH.

16. The conjugate according to claim 15, characterized in that the antibody fragment is covalently linked to no more than one non-protein polymer molecule.

17. The conjugate according to claim 16, characterized in that the non-protein polymer molecule in the conjugate is covalently linked to the hinge region of the antibody fragment.

18. The conjugate according to claim 1, characterized in that the non-protein polymer is a polyethylene glycol (PEG).

19. The conjugate according to claim 18, characterized in that the PEG has an average molecular weight of at least 20 kD.

20. The conjugate according to claim 19, characterized in that the PEG has an average molecular weight of at least 40 kD.

21. The conjugate according to claim 20, characterized in that the PEG is a single chain molecule.

22. The conjugate according to claim 20, characterized in that the PEG is a branched chain molecule.

23. The conjugate according to claim 19, characterized in that the conjugate contains no more than one antibody fragment, and wherein the antibody fragment is an F (ab ') 2 and is covalently linked to no more than two PEG molecules.

24. The conjugate according to claim 19, characterized in that the conjugate contains no more than one antibody fragment, and wherein the antibody fragment is selected from the group consisting of Fab, Fab 'and Fab'-SH and is covalently linked to no more than one PEG molecule.

25. The conjugate according to claim 24, characterized in that the PEG molecule is covalently linked to the hinge region of the antibody fragment.

26. The conjugate according to claim 1, characterized in that the antibody fragment has an antigen binding site that binds to human IL-8.

27. The conjugate according to claim 26, characterized in that the conjugate contains no more than one antibody fragment, and wherein the antibody fragment is selected from the group consisting of Fab, Fab 'and Fab'-SH, wherein the fragment The antibody is covalently linked to no more than one non-protein polymer molecule, and wherein the non-protein polymer molecule is a polyethylene glycol having an effective molecular weight of at least 30 kD.

28. The conjugate according to claim 1, characterized in that the antibody fragment is humanized.

29. The conjugate according to claim 1, characterized in that the conjugate contains no more than one antibody fragment.

30. A composition characterized in that it comprises the conjugate according to claim 1 and a carrier.

31. The composition according to claim 30, characterized in that it is sterile.

32. A conjugate formed by one or more antibody fragments covalently linked to one or more non-protein polymeric molecules, characterized in that the apparent size of the conjugate is at least 500 kD, and wherein the molecular structure of the conjugate is free of other material.

33. A conjugate formed by one or more antibody fragments covalently linked to one or more non-protein polymer molecules, characterized in that the apparent size of the conjugate is at least 500 kD, wherein the antibody fragment incorporates a non-protein marker free of any polymer , and where the molecular structure of the conjugate is free of other matter.

34. The conjugate according to claim 33, characterized in that the non-protein label is a radiolabel.

35. A polypeptide, characterized in that it is selected from the group consisting of: (1) a polypeptide which is an antibody or anti-IL-8 monoclonal antibody fragment comprising a light chain amino acid sequence, comprising the regions of complementarity of the amino acid sequence of the light chain polypeptide of Figure 36; and (2) a polypeptide which is an antibody or anti-IL-8 monoclonal antibody fragment comprising a light chain amino acid sequence comprising the regions of complementarity determination of the amino acid sequence of the light chain polypeptide of the Figure 45

36. The polypeptide according to claim 35, characterized in that the light chain amino acid sequence comprises the regions of determination of the complementarity of the amino acid sequence of the light chain polypeptide of Figure 45.

37. The polypeptide according to claim 35, characterized in that it further comprises a heavy chain amino acid sequence, comprising the regions of determination of the complementarity of the amino acid sequence of the heavy chain polypeptide of Figures 37A-37B.

38. The polypeptide according to claim 35, characterized in that the light chain amino acid sequence is selected from the group consisting of: (1) a light chain amino acid sequence comprising amino acids 1-219 of the polypeptide amino acid sequence of light chain of figure 36; and (2) an amino acid sequence of the light chain comprising amino acids 1-219 of the amino acid sequence of the light chain polypeptide of Figure 45.

39. The polypeptide according to claim 38, characterized in that the amino acid sequence of the light chain comprises amino acids 1-219 of the amino acid sequence of the light chain of Figure 45.

40. The polypeptide according to claim 38, characterized in that it further comprises a heavy chain amino acid sequence comprising amino acids 1-230 of the amino acid sequence of the heavy chain polypeptide of Figures 37A-37B.

41. The polypeptide according to claim 40, characterized in that the heavy chain amino acid sequence is fused at its C-terminus to a leucine zipper amino acid sequence.

42. The polypeptide according to claim 41, characterized in that the leucine zipper sequence comprises the amino acids 231-275 of the amino acid sequence of the heavy chain polypeptide of Figures 37A-37B.

43. The polypeptide according to claim 35, characterized in that it is an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, scFv and F (ab') 2.

44. The polypeptide according to claim 38, characterized in that it is an F (ab ') 2 antibody fragment, wherein the antibody fragment comprises a first heavy chain amino acid sequence and a second heavy chain amino acid sequence, comprising each amino acid 1-238 of the amino acid sequence of the heavy chain polypeptide of Figures 37A-37B, and wherein each of the Cys residues at positions 231 and 234 in the first heavy chain amino acid sequence is a disulfide bond with the identical Cys residue in the second amino acid sequence of the heavy chain.

45. The polypeptide according to claim 38, characterized in that it is a Fab 'or Fab'-SH antibody fragment, wherein the antibody fragment comprises a heavy chain amino acid sequence comprising amino acids 1-233 of the amino acid sequence of the heavy chain polypeptide of Figure 53.

46. The polypeptide according to claim 35, characterized in that it is an antibody.

47. A nucleic acid molecule, characterized in that it comprises a nucleic acid sequence encoding the polypeptide according to claim 35.

48. An expression vector, characterized in that it comprises the nucleic acid molecule according to claim 47, operably linked to the control sequences recognized by a host cell transfected with the vector.

49. A host cell, characterized in that it comprises the vector according to claim 48.

50. A method for producing a polypeptide, characterized in that it comprises cultivating the host cell according to claim 49 under conditions wherein the nucleic acid sequence is expressed, whereby the polypeptide is produced, and the polypeptide is recovered from the host cell ..

51. A composition, characterized in that it comprises the polypeptide according to claim 35 and a carrier.

52. The composition according to claim 51, characterized in that it is sterile