US 20090104204A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0104204 A1 Throsby et al. (43) Pub. Date: Apr. 23, 2009

(54) HUMAN BINDING MOLECULES HAVING (30) Foreign Application Priority Data KILLING ACTIVITY AGAINST STAPHYLOCOCC AND USES THEREOF Nov. 16, 2006 (EP) ...... O61242319 Mar. 6, 2007 (EP) ...... O7103.584.4 (76) Inventors: Mark Throsby, Utrach (NL); Publication Classification Cecilia A.W. Geuijen, Moerkapelle (51) Int. Cl Nels Adriaan De Kruif, A 6LX 39/395 (2006.01) e Bilt (NL) C07K 6/12 (2006.01) A6IP3L/04 (2006.01) Correspondence Address: C7H 2L/04 (2006.01) TRASK BRITT CI2N 15/63 (2006.01) P.O. BOX 2.5SO CI2N 5/10 (2006.01) SALT LAKE CITY, UT 84110 (US) CI2P 2L/08 (2006.01) GOIN 33/53 (2006.01) (21) Appl. No.: 12/227,029 (52) U.S. Cl...... 424/142.1; 530/388.15:530/391.3; 536/23.53; 435/320.1; 435/325; 435/69.6; (22) PCT Filed: Jun. 5, 2007 435/7.1 (57) ABSTRACT (86)86) PCT No.:O PCT/EP2007/055527 The present invention provides human binding molecules S371 (c)(1) specifically binding to Staphylococci and having killing activ (2), (4) Date: Nov. 5, 2008 ity against Staphylococci, nucleic acid molecules encoding the human binding molecules, compositions comprising the human binding molecules and methods of identifying or pro Related U.S. Application Data ducing the human binding molecules. The human binding (60) Provisional application No. 60/811,477, filed on Jun. molecules can be used in the diagnosis, prophylaxis and/or 6, 2006. treatment of a condition resulting from Staphylococcus. Patent Application Publication Apr. 23, 2009 Sheet 1 of 6 US 2009/O104204 A1

75

3s 50 O O O) 25 SS

O ------E.------E.------E------E

H N s s Antibody concentration (ug/ml)

FIG. 1 Patent Application Publication Apr. 23, 2009 Sheet 2 of 6 US 2009/O104204 A1

5 O

2 5

Antibody Concentration (ug/ml)

FIG. 2 Patent Application Publication Apr. 23, 2009 Sheet 3 of 6 US 2009/O104204 A1

Antibody Concentration (ug/ml)

FIG. 3 Patent Application Publication Apr. 23, 2009 Sheet 4 of 6 US 2009/O104204 A1

30

3s 20 9 O O O 10 SS t -1------t -E------E.------O H H N N N Q SS Antibody Concentration (ug/ml)

FIG. 4

US 2009/01 04204 A1 Apr. 23, 2009

HUMAN BINDING MOLECULES HAVING therapies or preventative measures for (multi-drug-resistant) KILLING ACTIVITY AGAINST bacterial infections is urgently needed to meet this impending STAPHYLOCOCC AND USES THEREOF healthcare crisis. 0004 Active immunization with vaccines and passive FIELD OF THE INVENTION immunization with immunoglobulins are promising alterna tives to classical small molecule therapy. A few bacterial 0001. The invention relates to medicine. In particular the diseases that once caused widespread illness, disability, and invention relates to the diagnosis, prophylaxis and/or treat death can now be prevented through the use of vaccines. The ment of infection by Staphylococci. vaccines are based on weakened (attenuated) or dead bacte ria, components of the bacterial Surface or on inactivated BACKGROUND OF THE INVENTION toxins. The immune response raised by a vaccine is mainly directed to immunogenic structures, a limited number of pro 0002 Staphylococcus is a genus of gram-positive bacteria teins or Sugar structures on the bacteria that are actively and a member of the micrococcaceae family. Staphylococci processed by the immune system. Since these immunogenic are spherical bacteria that are found primarily on the skin and structures are very specific to the organism, the vaccine needs in the mucous membranes of humans and other warm to comprise the immunogenic components of all variants of blooded animals, and aggregate into Small, grape-like the bacteria against which the vaccine should be protective. clumps. Staphylococci can be divided into two groups, i.e. As a consequence thereof, vaccines are very complex, take coagulase-positive and coagulase-negative staphylococci. long and are expensive to develop. Further complicating the Overall, there are about thirty species of staphylococci. design of vaccines is the phenomenon of antigen replace 0003 Staphylococci can cause a wide variety of diseases ment. This occurs when new strains become prevalent that in humans either through toxin production or invasion. Sta are serologically and thus antigenically distinct from those phylococcus aureus (S. aureus) has been recognized as one of strains covered by the vaccines. The immune status of the the most important and lethal human bacterial pathogens populations at risk for nosocomial infections further compli since the beginning of the previous century. Until the antibi cates vaccine design. These patients are inherently unwell otic era, more than 80% of the patients growing S. aureus and may even be immunocompromised (due to the effect of from their blood died. Through infections caused by coagul immunosuppressive drugs) resulting in delayed or insuffi lase-positive S. aureus were generally known to be poten cient immunity against the infecting pathogens. Furthermore, tially lethal, coagulase-negative staphylococci has been dis except in the case of certain elective procedures, it may not be missed as avirulent skin commensals incapable of causing possible to identify and vaccinate the at risk patients in time to human disease. However, over the past 30 years, coagulase give them Sufficient immune protection from infection. negative staphylococcal infections have emerged as one of 0005 Direct administration of therapeutic immunoglobu the major complications of medical progress. They are cur lins, also referred to as passive immunization, does not rently the pathogens most commonly isolated from infections require an immune response from the patient and therefore of indwelling foreign devices and are the leading cause of gives immediate protection. In addition, passive immuniza nosocomial (hospital-acquired) bacteremias in US hospitals. tion can be directed to bacterial structures that are not immu Staphylococcal infections are commonly treated with antimi nogenic and that are less specific to the organism. Passive crobial agents. However, the ascendancy of staphylococci as immunization against pathogenic organisms has been based pre-eminent nocosomial pathogens also has been associated on immunoglobulins derived from Sera of human or non with a major increase in the proportion of these isolates that human donors. However, blood-derived products have poten are resistant to (multiple) antimicrobial agents. Of the esti tial health risks inherently associated with these products. In mated 2 million hospital infections in the US in 2004, 70% addition, the immunoglobulins can display batch-to-batch was resistant to at least one antibiotic, thereby causing major variation and may be of limited availability in case of sudden medical and consequently economic problems. Ninety per mass exposures. Recombinantly produced antibodies do not cent of the staphylococci strains are penicillin resistant, leav have these disadvantages and thus offer an opportunity to ing only methicillin and Vancomycin to treat the majority of replace immunoglobulins derived from Sera. infections. However, with increasing numbers of reports of 0006 Murine monoclonal antibodies directed against sta methicillin-resistant Staphylococcus aureus (MRSA) chem phylococciare known in the art (see WO 03/059259 and WO ists are faced with the daunting task of generating new anti 03/059260). However, murine antibodies are limited for their biotics with novel modes of action. Despite the urgent need use in vivo due to problems associated with administration of for the development of new antibiotics, the major pharmaceu murine antibodies to humans, such as short serum halflife, an tical companies appear to have lost interest in the antibiotic inability to trigger certain human effector functions and elici market. In 2002, only 5 out of the more than 500 drugs in tation of an unwanted dramatic immune response against the phase II or phase III clinical development were new antibiot murine antibody in a human (HAMA). ics. In the last 6 years only 10 antibiotics have been registered 0007. In WO 03/059259 and WO 03/059260 the attempts and only 2 of those did not exhibit cross-reactivity with exist have been made to overcome the problems associated with the ing drugs (and thus not subject to the same patterns of drug use of fully murine antibodies in humans by preparing chi resistance). This trend has been attributed to several factors: meric antibodies. A disadvantage of these chimeric antibod the cost of new drug development and the relatively small ies is however that they still retain some murine sequences return on investment that infectious disease treatments yield and therefore still elicit an unwanted immune reaction, espe compared to drugs against hypertension, arthritis and lif cially when administered for prolonged periods. estyle drugs e.g. for impotence. Another contributing factoris 0008 WO 2004/043405 relates to polysaccharide vac the increasing difficulty in finding new targets, further driving cines for staphylococcal infections, prepared from poly up development costs. Therefore, investigation into novel N-acetylglucosamine (PNAG) surface polysaccharide from US 2009/01 04204 A1 Apr. 23, 2009

Staphylococci, and the deacetylated form thereof (dPNAG). acid residues, at least 10 contiguous amino acid residues, at WO 2004/043405 also discloses rabbit antiserum to PNAG least 15 contiguous amino acid residues, at least 20 contigu and dPNAG, coupled to Diphteria Toxoid (DTm). ous amino acid residues, at least 25 contiguous amino acid 0009. Although WO 03/059259, WO 03/059260 and WO residues, at least 30 contiguous amino acid residues, at least 20047043405 refer to human antibodies as desired molecules, 35 contiguous amino acid residues, at least 40 contiguous the antibodies actually disclosed and used therein are partly amino acid residues, at least 50 contiguous amino acid resi of murine or completely of rabbit origin, and none of these dues, at least 60 contiguous amino residues, at least 70 con documents actually discloses any human antibodies, nor tiguous amino acid residues, at least 80 contiguous amino sequences thereof. acid residues, at least 90 contiguous amino acid residues, at 0010. In view of their therapeutic benefit in humans, there least 100 contiguous amino acid residues, at least 125 con is thus still a need for human monoclonal antibodies against tiguous amino acid residues, at least 150 contiguous amino Staphylococci. The present invention provides these antibod acid residues, at least 175 contiguous amino acid residues, at ies and their sequences, and shows that they can be used in least 200 contiguous amino acid residues, or at least 250 medicine, in particular for diagnosis, prevention and/or treat contiguous amino acid residues of the amino acid sequence of ment of Staphylococcal infections. the binding molecule. 0018. The term “binding molecule', as used herein DESCRIPTION OF THE FIGURES includes all immunoglobulin classes and Subclasses known in 0011 FIG. 1 shows antibody-mediated phagocytosis of S. the art. Depending on the amino acid sequence of the constant aureus strain Cowan harvested during the log phase of growth domain of their heavy chains, binding molecules can be in the absence of complement with the antibodies CR2430 divided into the five major classes of intact antibodies: IgA, (white dot), CR5132 (black triangle), CR5133 (black dot), Igl), IgE, IgG, and IgM, and several of these may be further and a negative control monoclonal antibody (white square). divided into Subclasses (isotypes), e.g., IgA1, IgA2, IgG1. 0012 FIG. 2 shows antibody-mediated phagocytosis of S. IgG2, IgG3 and IgG4. aureus strain Cowan harvested during the stationary phase of 0019 Antigen-binding fragments include, interalia, Fab, growth in the absence of complement with the antibodies F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining CR2430 (white dot), CR5132 (black triangle), CR5133 region (CDR) fragments, single-chain antibodies (ScPV), (black dot), and a negative control monoclonal antibody bivalent single-chain antibodies, single-chain phageantibod (white square). ies, diabodies, triabodies, tetrabodies, (poly)peptides that 0013 FIG.3 shows antibody-mediated phagocytosis of S. contain at least a fragment of an immunoglobulin that is aureus strain SA125 harvested during the stationary phase of Sufficient to confer specific antigen binding to the (poly) growth in the absence of complement with the antibodies peptide, etc. The above fragments may be produced syntheti CR5132 (black triangle), CR5133 (black dot), and a negative cally or by enzymatic or chemical cleavage of intact immu control monoclonal antibody (white Square). noglobulins or they may be genetically engineered by 0014 FIG. 4 shows antibody-mediated phagocytosis of S. recombinant DNA techniques. The methods of production epidermidis strain SE131 harvested during the stationary are well known in the art and are described, for example, in phase of growth in the absence of complement with the anti Antibodies: A Laboratory Manual, Edited by: E. Harlow and bodies CR5132 (black triangle), CR5133 (black dot), and a D. Lane (1988), Cold Spring Harbor Laboratory, Cold Spring negative control monoclonal antibody (white Square). Harbor, N.Y., which is incorporated herein by reference. A binding molecule or antigen-binding fragment thereof may DESCRIPTION OF THE INVENTION have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another 0.015. Here below follow definitions ofterms as used in the invention. or they may be different. 0020. The binding molecule can be a naked or unconju gated binding molecule but can also be part of an immuno DEFINITIONS conjugate. A naked or unconjugated binding molecule is intended to refer to a binding molecule that is not conjugated, Amino Acid Sequence operatively linked or otherwise physically or functionally 0016. The term "amino acid sequence' as used herein associated with an effector moiety or tag. Such as interalia a refers to naturally occurring or synthetic molecules and to a toxic Substance, a radioactive Substance, a liposome, an peptide, oligopeptide, polypeptide or protein sequence. enzyme. It will be understood that naked or unconjugated Binding Molecule binding molecules do not exclude binding molecules that have been stabilized, multimerized, humanized or in any 0017. As used herein the term “binding molecule' refers other way manipulated, other than by the attachment of an to an intact immunoglobulin including monoclonal antibod effector moiety or tag. Accordingly, all post-translationally ies, such as chimeric, humanized or human monoclonal anti modified naked and unconjugated binding molecules are bodies, or to an antigen-binding and/or variable domain com included herewith, including where the modifications are prising fragment of an immunoglobulin that competes with made in the natural binding molecule-producing cell environ the intact immunoglobulin for specific binding to the binding ment, by a recombinantbinding molecule-producing cell, and partner of the immunoglobulin, e.g. staphylococci. Regard are introduced by the hand of man after initial binding mol less of structure, the antigen-binding fragment binds with the ecule preparation. Of course, the term naked or unconjugated same antigen that is recognized by the intact immunoglobu binding molecule does not exclude the ability of the binding lin. An antigen-binding fragment can comprise a peptide or molecule to form functional associations with effector cells polypeptide comprising an amino acid sequence of at least 2 and/or molecules after administration to the body, as some of contiguous amino acid residues, at least 5 contiguous amino Such interactions are necessary in order to exert a biological US 2009/01 04204 A1 Apr. 23, 2009

effect. The lack of associated effector group or tag is therefore otides and/or amino acids compared to the nucleotide and/or applied in definition to the naked or unconjugated binding amino acid sequences of the parent binding molecule and that molecule in vitro, not in vivo. is still capable of competing for binding to the binding part ner, e.g. staphylococci, with the parent binding molecule. In Biological Sample other words, the modifications in the amino acid and/or nucle otide sequence of the parent binding molecule do not signifi 0021. As used herein, the term “biological sample' cantly affect or alter the binding characteristics of the binding encompasses a variety of Sample types, including blood and molecule encoded by the nucleotide sequence or containing other liquid samples of biological origin, Solid tissue samples the amino acid sequence, i.e. the binding molecule is still able Such as a biopsy specimen or tissue cultures, or cells derived to recognize and bind its target. The functional variant may therefrom and the progeny thereof. The term also includes have conservative sequence modifications including nucle samples that have been manipulated in any way after their otide and amino acid Substitutions, additions and deletions. procurement, Such as by treatment with reagents, solubiliza These modifications can be introduced by standard tech tion, or enrichment for certain components, such as proteins niques known in the art, such as site-directed mutagenesis and or polynucleotides. The term encompasses various kinds of random PCR-mediated mutagenesis, and may comprise natu clinical samples obtained from any species, and also includes ral as well as non-natural nucleotides and amino acids. cells in culture, cell Supernatants and cell lysates. 0026 Conservative amino acid substitutions include the Complementarity Determining Regions (CDR) ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical 0022. The term “complementarity determining regions' properties. Families of amino acid residues having similar as used herein means sequences within the variable regions of side chains have been defined in the art. These families binding molecules, such as immunoglobulins, that usually include amino acids with basic side chains (e.g., lysine, argi contribute to a large extent to the antigenbinding site which is nine, histidine), acidic side chains (e.g., aspartic acid, complementary in shape and charge distribution to the glutamic acid), uncharged polar side chains (e.g., asparagine, epitope recognized on the antigen. The CDR regions can be glutamine, serine, threonine, tyrosine, cysteine, tryptophan), specific for linear epitopes, discontinuous epitopes, or con nonpolar side chains (e.g., glycine, alanine, Valine, leucine, formational epitopes of proteins or protein fragments, either isoleucine, proline, phenylalanine, methionine), beta as present on the protein in its native conformation or, in some branched side chains (e.g., threonine, Valine, isoleucine) and cases, as present on the proteins as denatured, e.g., by solu aromatic side chains (e.g., tyrosine, phenylalanine, tryp bilization in SDS. Epitopes may also consist of posttransla tophan). It will be clear to the skilled artisan that other clas tional modifications of proteins. sifications of amino acid residue families than the one used above can also be employed. Furthermore, a variant may have Deletion non-conservative amino acid Substitutions, e.g., replacement of an amino acid with an amino acid residue having different 0023 The term “deletion', as used herein, denotes a structural or chemical properties. Similar minor variations change in either amino acid or nucleotide sequence in which may also include amino acid deletions or insertions, or both. one or more amino acid or nucleotide residues, respectively, Guidance in determining which amino acid residues may be are absent as compared to the parent, often the naturally substituted, inserted, or deleted without abolishing immuno occurring, molecule. logical activity may be found using computer programs well Expression-Regulating Nucleic Acid Sequence known in the art. 0027. A mutation in a nucleotide sequence can be a single 0024. The term “expression-regulating nucleic acid alteration made at a locus (a point mutation). Such as transi sequence' as used herein refers to polynucleotide sequences tion or transversion mutations, or alternatively, multiple necessary for and/or affecting the expression of an operably nucleotides may be inserted, deleted or changed at a single linked coding sequence in a particular host organism. The locus. In addition, one or more alterations may be made at any expression-regulating nucleic acid sequences, such as inter number of loci within a nucleotide sequence. The mutations alia appropriate transcription initiation, termination, pro may be performed by any suitable method known in the art. moter, enhancer sequences; repressor or activator sequences; efficient RNA processing signals such as splicing and poly Host adenylation signals; sequences that stabilize cytoplasmic 0028. The term “host', as used herein, is intended to refer mRNA sequences that enhance translation efficiency (e.g., to an organism or a cell into which a vector Such as a cloning ribosome binding sites); Sequences that enhance protein sta vector or an expression vector has been introduced. The bility; and when desired, sequences that enhance protein organism or cell can be prokaryotic or eukaryotic. It should be secretion, can be any nucleic acid sequence showing activity understood that this term is intended to refer not only to the in the host organism of choice and can be derived from genes particular Subject organism or cell, but to the progeny of Such encoding proteins, which are either homologous or heterolo an organism or cell as well. Because certain modifications gous to the host organism. The identification and employment may occur in Succeeding generations due to either mutation of expression-regulating sequences is routine to the person or environmental influences. Such progeny may not, in fact, skilled in the art. be identical to the parent organism or cell, but are still Functional Variant included within the scope of the term “host’ as used herein. 0025. The term “functional variant', as used herein, refers Human to a binding molecule that comprises a nucleotide and/or 0029. The term “human', when applied to binding mol amino acid sequence that is altered by one or more nucle ecules as defined herein, refers to molecules that are either US 2009/01 04204 A1 Apr. 23, 2009

directly derived from a human or based upon a human accompany the native nucleic acid molecule in its natural sequence. When a binding molecule is derived from or based host, e.g., ribosomes, polymerases, or genomic sequences on a human sequence and Subsequently modified, it is still to with which it is naturally associated. Moreover, "isolated be considered human as used throughout the specification. In nucleic acid molecules, such as cDNA molecules, can be other words, the term human, when applied to binding mol substantially free of other cellular material, or culture ecules is intended to include binding molecules having vari medium when produced by recombinant techniques, or Sub able and constant regions derived from human germline stantially free of chemical precursors or other chemicals immunoglobulin sequences or based on variable or constant when chemically synthesized. regions occurring in a human or human lymphocyte and modified in Some form. Thus, the human binding molecules Monoclonal Antibody may include amino acid residues not encoded by human 0033. The term “monoclonal antibody” as used herein germline immunoglobulin sequences, comprise Substitutions refers to a preparation of antibody molecules of single and/or deletions (e.g., mutations introduced by for instance molecular composition. A monoclonal antibody displays a random or site-specific mutagenesis in vitro or by Somatic single binding specificity and affinity for a particular epitope. mutation in vivo). “Based on as used herein refers to the situation that a nucleic acid sequence may be exactly copied Accordingly, the term “human monoclonal antibody” refers from a template, or with minor mutations, such as by error to an antibody displaying a single binding specificity which prone PCR methods, or synthetically made matching the has variable and constant regions derived from or based on template exactly or with minor modifications. Semi-synthetic human germline immunoglobulin sequences or derived from molecules based on human sequences are also considered to completely synthetic sequences. The method of preparing the be human as used herein. monoclonal antibody is not relevant.

Insertion Naturally Occurring 0034. The term “naturally occurring as used herein as 0030 The term “insertion', also known as the term “addi applied to an object refers to the fact that an object can be tion', denotes a change in an amino acid or nucleotide found in nature. For example, a polypeptide or polynucleotide sequence resulting in the addition of one or more amino acid sequence that is present in an organism that can be isolated or nucleotide residues, respectively, as compared to the parent from a source in nature and which has not been intentionally Sequence. modified by man in the laboratory is naturally occurring. Intrinsic Activity Nucleic Acid Molecule 0031. The term “intrinsic activity”, when applied to bind ing molecules as defined herein, refers to binding molecules 0035. The term “nucleic acid molecule' as used in the that are capable of binding to certain protein or carbohydrate present invention refers to a polymeric form of nucleotides antigens on the Surface of pathogens such as bacteria and that and includes both sense and anti-sense strands of RNA, can inhibit the ability of the pathogen to grow and divide cDNA, genomic DNA, and synthetic forms and mixed poly normally. Such binding molecules can for example block the mers of the above. A nucleotide refers to a ribonucleotide, entry of specific nutrients required for growth or the transport deoxynucleotide or a modified form of either type of nucle otide. The term also includes single- and double-stranded of toxic waste elements from the bacteria. Through the latter forms of DNA. In addition, a polynucleotide may include action they may also increase the sensitivity of bacteria to the either or both naturally occurring and modified nucleotides action of antibiotic drugs. linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. The nucleic acid molecules Isolated may be modified chemically or biochemically or may contain 0032. The term "isolated, when applied to binding mol non-natural orderivatized nucleotide bases, as will be readily ecules as defined herein, refers to binding molecules that are appreciated by those of skill in the art. Such modifications Substantially free of other proteins or polypeptides, particu include, for example, labels, methylation, Substitution of one larly free of other binding molecules having different anti or more of the naturally occurring nucleotides with an analog, genic specificities, and are also Substantially free of other internucleotide modifications such as uncharged linkages cellular material and/or chemicals. For example, when the (e.g., methyl phosphonates, phosphotriesters, phosphorami binding molecules are recombinantly produced, they are pref dates, carbamates, etc.), charged linkages (e.g., phospho erably substantially free of culture medium, and when the rothioates, phosphorodithioates, etc.), pendent moieties (e.g., binding molecules are produced by chemical synthesis, they polypeptides), intercalators (e.g., acridine, psoralen, etc.), are preferably substantially free of chemical precursors or chelators, alkylators, and modified linkages (e.g., alpha ano other chemicals, i.e., they are separated from chemical pre meric nucleic acids, etc.). The above term is also intended to cursors or other chemicals which are involved in the synthesis include any topological conformation, including single of the protein. The term "isolated when applied to nucleic Stranded, double-stranded, partially duplexed, triplex, hair acid molecules encoding binding molecules as defined pinned, circular and padlocked conformations. Also included herein, is intended to refer to nucleic acid molecules in which are synthetic molecules that mimic polynucleotides in their the nucleotide sequences encoding the binding molecules are ability to bind to a designated sequence via hydrogenbonding free of other nucleotide sequences, particularly nucleotide and other chemical interactions. Such molecules are known in sequences encoding binding molecules that bind binding the art and include, for example, those in which peptide partners other than staphylococci. Furthermore, the term linkages Substitute for phosphate linkages in the backbone of "isolated’ refers to nucleic acid molecules that are substan the molecule. A reference to a nucleic acid sequence encom tially separated from other cellular components that naturally passes its complement unless otherwise specified. Thus, a US 2009/01 04204 A1 Apr. 23, 2009

reference to a nucleic acid molecule having a particular linked immunosorbent assays (ELISA), BIACORE, or other sequence should be understood to encompass its complemen assays known in the art. Binding molecules or fragments tary Strand, with its complementary sequence. The comple thereof that immunospecifically bind to an antigen may be mentary strand is also useful, e.g., for anti-sense therapy, cross-reactive with related antigens. Preferably, binding mol hybridization probes and PCR primers. ecules or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens. Operably Linked 0036. The term “operably linked” refers to two or more Substitutions nucleic acid sequence elements that are usually physically 0040. A “substitution', as used herein, denotes the linked and are in a functional relationship with each other. For replacement of one or more amino acids or nucleotides by instance, a promoter is operably linked to a coding sequence, different amino acids or nucleotides, respectively. if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding Therapeutically Effective Amount sequence should be understood as being “under the control of the promoter. 0041. The term “therapeutically effective amount” refers to an amount of the binding molecule as defined herein that is Opsonic Activity effective for preventing, ameliorating and/or treating a con dition resulting from infection with Staphylococcus. 0037 “Opsonic activity” refers to the ability of an opsonin (generally either a binding molecule, e.g. an antibody, or Treatment serum complement factors) to bind to the Surface of a patho gen either by specific antigenic recognition (in the case of 0042. The term “treatment” refers to therapeutic treatment antibodies) or through the catalytic effect of surface bound as well as prophylactic or preventative measures to cure or molecules (e.g. the increased deposition of C3b as a result of halt or at least retard disease progress. Those in need of Surface bound antibodies). Phagocytosis of opsonized patho treatment include those already inflicted with a condition gens is enhanced due to the specific recognition of receptors resulting from infection with Staphylococcus as well as those on the phagocyte for the opsonin (the Fc receptor in case the in which infection with Staphylococcus is to be prevented. antibodies themselves are the opsonins and the complement Subjects partially or totally recovered from infection with receptor in case complement is the opsonin). Certain bacteria, Staphylococcus might also be in need of treatment. Preven especially encapsulated bacteria that resist phagocytosis due tion encompasses inhibiting or reducing the spread of Staphy to the presence of the capsule, become extremely attractive to lococcus or inhibiting or reducing the onset, development or phagocytes Such as neutrophils and macrophages when progression of one or more of the symptoms associated with coated with an opsonic antibody and their rate of clearance infection with Staphylococcus. from the bloodstream and infected organs is strikingly enhanced. Opsonic activity may be measured in any conven Vector tional manner (e.g. the opsonic phagocytic killing assay). 0043. The term “vector denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for Pharmaceutically Acceptable Excipient introduction into a host where it will be replicated, and in 0038. By “pharmaceutically acceptable excipient' is Some cases expressed. In other words, a vector is capable of meant any inert Substance that is combined with an active transporting a nucleic acid molecule to which it has been molecule Such as a drug, agent, or binding molecule for linked. Cloning as well as expression vectors are contem preparing an agreeable or convenient dosage form. The plated by the term “vector', as used herein. Vectors include, “pharmaceutically acceptable excipient' is an excipient that but are not limited to, plasmids, cosmids, bacterial artificial is non-toxic to recipients at the dosages and concentrations chromosomes (BAC) and yeast artificial chromosomes employed, and is compatible with other ingredients of the (YAC) and vectors derived from bacteriophages or plant or formulation comprising the drug, agent orbinding molecule. animal (including human) viruses. Vectors comprise an origin of replication recognized by the proposed host and in case of Specifically Binding expression vectors, promoter and other regulatory regions 0039. The term “specifically binding, as used herein, in recognized by the host. A vector containing a second nucleic reference to the interaction of a binding molecule, e.g. an acid molecule is introduced into a cell by transformation, antibody, and its binding partner, e.g. an antigen, means that transfection, or by making use of viral entry mechanisms. the interaction is dependent upon the presence of a particular Certain vectors are capable of autonomous replication in a structure, e.g. an antigenic determinant or epitope, on the host into which they are introduced (e.g., vectors having a binding partner. In other words, the antibody preferentially bacterial origin of replication can replicate in bacteria). Other binds or recognizes the binding partner even when the bind vectors can be integrated into the genome of a host upon ing partner is present in a mixture of other molecules or introduction into the host, and thereby are replicated along organisms. The binding may be mediated by covalent or with the host genome. non-covalent interactions or a combination of both. In yet SUMMARY OF THE INVENTION other words, the term "specifically binding' means immuno specifically binding to an antigen or a fragment thereof and 0044) The invention provides human binding molecules not immunospecifically binding to other antigens. A binding capable of specifically binding to staphylococci and exhibit molecule that immunospecifically binds to an antigen may ing killing and/or growth inhibiting activity against Staphy bind to other peptides or polypeptides with lower affinity as lococci. The invention also pertains to nucleic acid molecules determined by, e.g., radioimmunoassays (RIA), enzyme encoding at least the binding region of the human binding US 2009/01 04204 A1 Apr. 23, 2009

molecules. The invention further provides for the use of the dii, Shigella flexneri, Shigella sonnei, Pseudomonas aerugi human binding molecules of the invention in the prophylaxis nosa, Pseudomonas mallei, Vibrio cholerae, Vibrio para and/or treatment of a Subject having, or at risk of developing, haemolyticus, Vibrio vulnificus, Vibrio alginolyticus, a Staphylococcus infection. Besides that, the invention per Campylobacter pylori, Helicobacter pylori, Campylobacter tains to the use of the human binding molecules of the inven jejuni, Bacteroides fragilis, Neisseria gonorrhoeae, Neis tion in the diagnosis/detection of Staphylococcus. seria meningitidis, Branhamella catarrhalis, Haemophilus influenzae, Haemophilus ducreyi, Bordetella pertussis, Bru DETAILED DESCRIPTION OF THE INVENTION cella abortus, Brucella abortus, Brucella melitensis, Legionella pneumophila, Treponema pallidum, Treponema 0045. In a first aspect the present invention encompasses carateum, Leptospira interrogans, Leptospira biflexia, Borre binding molecules capable of specifically binding to staphy lia recurrentis, Borrelia burgdorferi, Mycoplasma pneumo lococci. Preferably, the binding molecules are human binding niae, Coxiella burnetii, Clamydia trachomatis, Clamydia molecules. Preferably, the binding molecules of the invention psittaci, Clamydia pneumoniae. The binding molecules of the exhibit killing activity against Staphylococci. In a further invention may be capable of specifically binding to staphy aspect the binding molecules of the invention are capable of lococci and optionally other Gram-positive and/or Gram specifically binding to and/or have killing activity against at negative bacteria that are viable, living and/or infective or that least two different Staphylococcus species. Preferably the are in inactivated/attenuated form. Methods for inactivating/ binding molecules of the invention are capable of specifically attenuating bacteria are well known in the art and include, but binding to and/or have killing activity against at least two, at are not limited to, antibiotic treatment, UV treatment, form least three, at least four, at least five, at least six, at least seven, aldehyde treatment, etc. at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at 0046. The binding molecules of the invention may also be least sixteen, at least seventeen, at least eighteen, at least capable of specifically binding to one or more fragments of nineteen, at least twenty, at least twenty-one, at least twenty staphylococci (and other Gram-positive and/or Gram-nega two, at least twenty-three, at least twenty-four, at least tive bacteria) Such as interalia a preparation of one or more twenty-five, at least twenty-six, at least twenty-seven, at least proteins and/or (poly)peptides derived from Staphylococci or twenty-eight, at least twenty-nine, at least thirty different one or more recombinantly produced Staphylococci proteins Staphylococcus species. Staphylococcus species that the and/or polypeptides. For methods of treatment and/or preven binding molecules of the invention are capable of specifically tion of Staphylococcal infections the binding molecules are binding to and/or have killing activity against are selected preferably capable of specifically binding to surface acces from the group consisting of S. aureus, S. auricularis, S. sible proteins of staphylococci. For diagnostical purposes the capitis, S. Caprae, S. caseolyticus, S. chromogenes, S. cohnii, binding molecules may also be capable of specifically bind S. epidermidis, S. haemolyticus, S. hominis, S. hyicus, S. ing to proteins not present on the Surface of Staphylococci. intermedium, S. lentus, S. lugdunensis, S. saprophyticus, S. The nucleotide and/or amino acid sequence of proteins of Schleiferi, S. Sciuri, S. simulans, S. warneri, and S. xylosus. In various Staphylococcus species and strains can be found in an embodiment the binding molecules of the invention are the GenBank-database, EMBL-database and/or other data capable of specifically binding to and have killing activity bases. It is well within the reach of the skilled person to find against different strains within one Staphylococcus species. Such sequences in the respective databases. In a further embodiment the binding molecules of the inven 0047 Alternatively, binding molecules of the invention tion are capable of specifically binding to and have killing may also be capable of specifically binding to other staphy activity against a Staphylococcus strain in the lag phase, log lococcal molecules including, but not limited to, Surface fac phase, stationary phase and/or death phase. Preferably, they tors that inhibit phagocytic engulfment; factors that enhance specifically bind to and have killing activity against a Staphy their Survival in phagocytes; invasins that lyse eukaryotic cell lococcus strain in the log phase and stationary phase. In membranes; exotoxins that damage host tissues or otherwise another embodiment, the binding molecules of the invention provoke symptoms of disease; polysaccharides; other cell may even be capable of specifically binding to and/or have wall components such as teichoic acid, lipoteichoic acid, killing activity against at least one other Gram-positive bac ribitol, peptidoglycan, pentaglycine oligopeptide, N-acetyl terium and/or Gram-negative bacterium including, but not glucosamine, N-acetylmuramic acid, N-acetylgalac limited to, Group A Streptococci; streptococcus pyrogenes, tosaminuronic acid, N-acetylfucosamine, N-acetylglu Group B streptococci; streptococcus agalactiae, streptococ cosaminuronic acid, N-acetylmannosaminuronic acid, cus milleri, Streptococcus pneumoniae, Viridans strepto O-acetyl, glucosamine, muramic acid, galactosaminuronic cocci; streptococcus mutans, Enterococcus, Enterococcus acid, fucosamine, glucosaminuronic acid, mannosaminu faecalis and Enterococcus faecium, Corynebacterium diph ronic acid and linkage units between any of these compo theriae, Corynebacterium ulcerans, Corynebacterium nentS. pseudotuberculosis, Corynebacterium jeikeium, Corynebac 0048. In another embodiment the binding molecules of the terium xerosis, Corynebacterium pseudodiphtheriticum, invention are capable of specifically binding to a fragment of Bacillus anthracis, Bacillus cereus, Listeria monocytogenes, the above-mentioned proteins and/or other molecules, Clostridium perfringens, Clostridium tetani, Clostridium wherein the fragment at least comprises an antigenic deter botulinum, Clostridium difficile, Mycobacterium tuberculo minant recognized by the binding molecules of the invention. sis, Mycobacterium leprae, Actinomyces israelii, NorCardia An “antigenic determinant’ as used herein is a moiety that is asteroides, Norcardia brasiliensis, Escherichia coli, Proteus capable of binding to a binding molecule of the invention with mirabilis, Proteus vulgaris, Klebsiella pneumoniae, Salmo Sufficiently high affinity to form a detectable antigen-binding nella typhi, Salmonella paratyphi A, B & C. Salmonella molecule complex. enteritidis, Salmonella cholerae-suis, Salmonella virchow, 0049. The binding molecules of the invention can be intact Salmonella typhimurium, Shigella dysenteriae, Shigella boy immunoglobulin molecules such as polyclonal or mono US 2009/01 04204 A1 Apr. 23, 2009 clonal antibodies or the binding molecules can be antigen Manual of Molecular and Clinical Laboratory Immunology, binding fragments including, but not limited to, Fab., F(ab'), 7th Edition. Assays to measure the other mentioned activities F(ab'). Fv, dAb, Fd, complementarity determining region are also known. (CDR) fragments, single-chain antibodies (scFv), bivalent 0055. In a preferred embodiment, the binding molecules single-chain antibodies, single-chain phage antibodies, dia according to the invention comprise at least a CDR3 region, bodies, triabodies, tetrabodies, and (poly)peptides that con preferably a heavy chain CDR3 region, comprising the amino tain at least a fragment of an immunoglobulin that is sufficient acid sequence selected from the group consisting of SEQID to confer specific antigen binding to Staphylococci or a frag NO:9 and SEQID NO:15. The CDR regions of the binding ment thereof. In a preferred embodiment the binding mol molecules of the invention are shown in Table 12. CDR ecules of the invention are human monoclonal antibodies. regions are according to Kabat et al. (1991) as described in Sequences of Proteins of Immunological Interest, US Dept. 0050. The binding molecules of the invention can be used Health and Human Services, NIH, USA (fifth edition). In an in non-isolated or isolated form. Furthermore, the binding embodiment binding molecules may comprise two, three, molecules of the invention can be used alone or in a mixture four, five or even all six CDR regions of the binding mol comprising at least one binding molecule (or variant or frag ecules of the invention. ment thereof) of the invention. In other words, the binding molecules can be used in combination, e.g., as a pharmaceu 0056. In yet another embodiment, the binding molecules tical composition comprising two or more binding molecules according to the invention comprise a heavy chain compris of the invention, variants or fragments thereof. For example, ing the variable heavy chain of the amino acid sequence binding molecules having different, but complementary selected from the group consisting of SEQ ID NO:28 and activities can be combined in a single therapy to achieve a SEQ ID NO:30. In a further embodiment, the binding mol desired prophylactic, therapeutic or diagnostic effect, but ecules according to the invention comprise a light chain com alternatively, binding molecules having identical activities prising the variable light chain of the amino acid sequence can also be combined in a single therapy to achieve a desired selected from the group consisting of SEQ ID NO:34 and prophylactic, therapeutic or diagnostic effect. Optionally, the SEQ ID NO:36. Table 13 specifies the heavy and light chain mixture further comprises at least one other therapeutic agent. variable regions of the binding molecule of the invention. Preferably, the therapeutic agent Such as e.g. an antibiotic is 0057. In another aspect the binding molecules of the useful in the prophylaxis and/or treatment of a staphylococcal invention are capable of specifically binding to one specific infection. Staphylococcus species, preferably one specific Staphylococ cus strain. In other words, they are species- and even strain 0051 Typically, binding molecules according to the specific. Preferably, the binding molecules of the invention invention can bind to their binding partners, i.e. staphylococci exhibit killing activity against the specific Staphylococcus or fragments thereof, with an affinity constant (K-value) that species/strain. In a preferred embodiment the Staphylococcus is lower than 0.2:10M, 1.0*10M, 1.0*10M, 1.0*107 species is S. aureus and the strain is S. aureus strain Cowan. M, preferably lower than 1.0*10 M, more preferably lower The binding molecules of the invention may be capable of than 1.0*10 M, more preferably lower than 1.0*10' M, specifically binding to and exhibit killing activity against the even more preferably lower than 1.0*10' M, and in particu specific Staphylococcus species/strain in any phase, e.g. log lar lower than 1.0*10* M. The affinity constants can vary and/or stationary phase. In a preferred embodiment the bind for antibody isotypes. For example, affinity binding for an ing molecules comprise at least a CDR3 region, preferably a IgM isotype refers to a binding affinity of at least about heavy chain CDR3 region, comprising the amino acid 1.0*107 M. Affinity constants can for instance be measured sequence of SEQID NO:3. The CDR regions of the binding using Surface plasmon resonance, for example using the BIA molecules are shown in Table 12. CDR regions are according CORE system (Pharmacia Biosensor AB, Uppsala, Sweden). to Kabat et al. (1991) as described in Sequences of Proteins of 0052. The binding molecules according to the invention Immunological Interest, US Dept. Health and Human Ser may bind to Staphylococci or a fragment thereof in soluble vices, NIH, USA (fifth edition). In an embodiment binding form such as for instance in a sample or in Suspension or may molecules may comprise two, three, four, five or even all six bind to staphylococci or a fragment thereof bound or attached CDR regions of the binding molecules of the invention. In yet to a carrier or Substrate, e.g., microtiter plates, membranes another embodiment, the binding molecules comprise a and beads, etc. Carriers or Substrates may be made of glass, heavy chain comprising the variable heavy chain of the amino plastic (e.g., polystyrene), polysaccharides, nylon, nitrocel acid sequence of SEQID NO:26. In a further embodiment, lulose, or Teflon, etc. The surface of such supports may be the binding molecules comprise a light chain comprising the Solid or porous and of any convenient shape. variable light chain of the amino acid sequence of SEQ ID 0053. Furthermore, the binding molecules may bind to NO:32. Table 13 specifies the heavy and light chain variable staphylococci in purified/isolated or non-purified/non-iso regions of the binding molecule of the invention. lated form. 0.058 Another aspect of the invention includes functional 0054 The binding molecules of the invention exhibit kill variants of the binding molecules as defined herein. Mol ing activity. Killing activity as meant herein includes, but is ecules are considered to be functional variants of a binding not limited to, opsonic activity or any other activity increas molecule according to the invention, if the variants are ing/augmenting/enhancing phagocytosis and/or phagocytic capable of competing for specifically binding to staphylo killing of bacteria, e.g. staphylococci; intrinsic (killing) activ cocci (or other Gram-positive and/or Gram-negative bacteria) ity, e.g. reduce or inhibit bacterial growth or directly kill or a fragment thereof with the parent human binding mol bacteria; increase the sensitivity of bacteria to antibiotic treat ecules. In other words, when the functional variants are still ment; or any combination thereof. Opsonic activity can for capable of binding to staphylococci or a fragment thereof. instance be measured as described herein. Alternative assays Preferably, the functional variants are capable of competing measuring opsonic activity are described in for instance for specifically binding to the at least two (or more) different US 2009/01 04204 A1 Apr. 23, 2009

Staphylococcus species or fragments thereofthat are specifi ants having killing activity may have a further activity Suit cally bound by the parent human binding molecules. Further able in staphylococcal control. Other activities are mentioned more, molecules are considered to be functional variants of a above. Henceforth, when the term (human) binding molecule binding molecule according to the invention, if they have is used, this also encompasses functional variants of the (hu killing activity against Staphylococci, preferably against the man) binding molecule. at least two (or more) Staphylococcus species against which 0060. The invention provides a panel of useful human the parental binding molecule exhibits killing activity. In monoclonal antibodies that have opsonic phagocytic killing another embodiment the functional variants of a binding mol activity against Staphylococci, said antibodies comprising ecule according to the invention also have killing activity the heavy and light chain variable regions of any one of the against other Gram-positive and/or Gram-negative bacteria. antibodies named CR2430, CR5132, CR5133 CR6166, Functional variants include, but are not limited to, derivatives CR6171, CR6176, CR6187, CR6193, CR6249, CR6273, that are substantially similar in primary structural sequence, CR6389, CR6403, CR6406, CR6410, CR6446, CR6450, but which contain e.g. in vitro or in vivo modifications, CR6452, CR6453, CR6464, CR6471, CR6516, CR6517, chemical and/or biochemical, that are not found in the paren CR6526, CR6528, CR6531, CR6533, CR6536, CR6537, tal binding molecule. Such modifications include inter alia CR6538, CR6540, CR6544, CR6566, or CR6625, or com acetylation, acylation, covalent attachment of a nucleotide or prising variable regions with sequences that are at least 80%, nucleotide derivative, covalent attachment of a lipid or lipid preferably at least 90%, more preferably at least 95%, iden derivative, cross-linking, disulfide bond formation, glycosy tical thereto. Preferably the sequences of the complete anti lation, hydroxylation, methylation, oxidation, pegylation, bodies are at least 80%, more preferably at least 90%, still proteolytic processing, phosphorylation, and the like. more preferably at least 95% identical to the sequences of 0059 Alternatively, functional variants can be binding these antibodies as disclosed herein. The antibodies fell into molecules as defined in the present invention comprising an five distinct groups, based on a target competition assay. amino acid sequence containing Substitutions, insertions, Group A consisted of CR5132, CR5133, CR6187 and deletions or combinations thereof of one or more amino acids CR6453; Group B consisted of CR5140 and CR6171; Group compared to the amino acid sequences of the parent binding C consisted of CR6176: Group D consisted of CR6526; and molecules. Furthermore, functional variants can comprise Group E consisted of the rest of the panel CR6166, CR6193, truncations of the amino acid sequence at either or both the CR6249, CR6273, CR6403, CR6406, CR6410, CR6446, amino or carboxyl termini. Functional variants according to CR6450, CR6452, CR6464, CR6471, CR6516, CR6517, the invention may have the same or different, either higher or CR6528, CR6531, CR6533, CR6536, CR6537, CR6538, lower, binding affinities compared to the parental binding CR6540, CR6544, CR6566, CR6625. Based on the potency, molecule but are still capable of binding to staphylococci or a one antibody from each group was identified as preferred fragment thereof. For instance, functional variants according antibody, and the preferred antibodies are: CR5133, CR6166, to the invention may have increased or decreased binding CR6171, CR6176 and CR6526. These antibodies were all affinities for staphylococci or a fragment thereof compared to shown to bind and have opsonic phagocytic killing activity the parent binding molecules. Preferably, the amino acid against at least two different Staphylococcus species (S. sequences of the variable regions, including, but not limited aureus and S. epidermidis), and against at least 3 different to, framework regions, hyperVariable regions, in particular strains of S. aureus (502, Mn8, Newman). The invention also the CDR3 regions, are modified. Generally, the light chain provides compositions comprising at least 2, at least 3, at least and the heavy chain variable regions comprise three hyper 4, at least 5, or more, of the human monoclonal antibodies of variable regions, comprising three CDRS, and more con the invention. In preferred embodiments, at least 2 of said served regions, the so-called framework regions (FRs). The antibodies in the composition are from different target hyperVariable regions comprise amino acid residues from groups. This has the advantage that different targets on the CDRs and amino acid residues from hypervariable loops. staphylococci are recognized and thus the chances of killing Functional variants intended to fall within the scope of the the bacteria are increased. Of course, higher affinity mutants present invention have at least about 50% to about 99%, or mutants with other advantageous properties can be pre preferably at least about 60% to about 99%, more preferably pared according to routine methods, based on the sequences at least about 70% to about 99%, even more preferably at least of the antibodies as disclosed herein. Such improved antibod about 80% to about 99%, most preferably at least about 90% ies are included within the scope of the present invention, to about 99%, in particular at least about 95% to about 99%, when the variable regions of heavy and light chain are at least and in particular at least about 97% to about 99% amino acid 80%, preferably at least 90%, still more preferably at least sequence homology with the parent human binding mol 95% identical to the sequences of the variable regions of the ecules as defined herein. Computer algorithms such as inter antibodies disclosed herein. alia Gap or Bestfit known to a person skilled in the art can be 0061. In yet a further aspect, the invention includes immu used to optimally alignamino acid sequences to be compared noconjugates, i.e. molecules comprising at least one binding and to define similar or identical amino acid residues. Func molecule as defined herein and further comprising at least one tional variants can be obtained by altering the parent binding tag, Such as inter alia a detectable moiety/agent. Also con molecules or parts thereof by general molecular biology templated in the present invention are mixtures of immuno methods known in the art including, but not limited to, error conjugates according to the invention or mixtures of at least prone PCR, oligonucleotide-directed mutagenesis, site-di one immunoconjugates according to the invention and rected mutagenesis and heavy and/or light chain shuffling. In another molecule. Such as a therapeutic agent or another an embodiment the functional variants of the invention have binding molecule or immunoconjugate. In a further embodi killing activity against Staphylococci. The killing activity ment, the immunoconjugates of the invention may comprise may either be identical, or be higher or lower compared to the more than one tag. These tags can be the same or distinct from parent binding molecules. Furthermore, the functional Vari each other and can be joined/conjugated non-covalently to the US 2009/01 04204 A1 Apr. 23, 2009 binding molecules. The tag(s) can also be joined/conjugated known in the art such as, e.g., recombinantly by constructing directly to the human binding molecules through covalent nucleic acid molecules comprising nucleotide sequences bonding. Alternatively, the tag(s) can be joined/conjugated to encoding the binding molecules in frame with nucleotide the binding molecules by means of one or more linking com sequences encoding the Suitable tag(s) and then expressing pounds. Techniques for conjugating tags to binding mol the nucleic acid molecules. ecules are well known to the skilled artisan. 0065. It is another aspect of the present invention to pro 0062. The tags of the immunoconjugates of the present vide a nucleic acid molecule encoding at least a binding invention may be therapeutic agents, but they can also be molecule, functional variant or immunoconjugate according detectable moieties/agents. Tags Suitable in therapy and/or to the invention. Such nucleic acid molecules can be used as prevention may be toxins or functional parts thereof, antibi intermediates for cloning purposes, e.g. in the process of otics, enzymes, other binding molecules that enhance phago affinity maturation as described above. In a preferred embodi cytosis or immune stimulation. Immunoconjugates compris ment, the nucleic acid molecules are isolated or purified. ing a detectable agent can be used diagnostically to, for 0066. The skilled man will appreciate that functional vari example, assess if a Subject has been infected with a Staphy ants of these nucleic acid molecules are also intended to be a lococcus species or monitor the development or progression part of the present invention. Functional variants are nucleic of a staphylococcal infection as part of a clinical testing acid sequences that can be directly translated, using the stan procedure to, e.g., determine the efficacy of a given treatment dard genetic code, to provide an amino acid sequence identi regimen. However, they may also be used for other detection cal to that translated from the parent nucleic acid molecules. and/or analytical and/or diagnostic purposes. Detectable moi 0067 Preferably, the nucleic acid molecules encode bind eties/agents include, but are not limited to, enzymes, pros ing molecules comprising a CDR3 region, preferably a heavy thetic groups, fluorescent materials, luminescent materials, chain CDR3 region, comprising an amino acid sequence bioluminescent materials, radioactive materials, positron selected from the group consisting of SEQID NO:3, SEQID emitting metals, and non-radioactive paramagnetic metal NO:9 and SEQ ID NO:15. In a further embodiment the ions. The tags used to label the binding molecules for detec nucleic acid molecules encode binding molecules comprising tion and/or analytical and/or diagnostic purposes depend on two, three, four, five or even all six CDR regions of the the specific detection/analysis/diagnosis techniques and/or binding molecules of the invention. methods used such as interalia immunohistochemical stain 0068. In another embodiment, the nucleic acid molecules ing of (tissue) samples, flow cytometric detection, Scanning encode binding molecules comprising a heavy chain com laser cytometric detection, fluorescent immunoassays, prising the variable heavy chain of the amino acid sequence enzyme-linked immunosorbent assays (ELISA's), radioim selected from the group consisting of SEQIDNO:26, SEQID munoassays (RIA's), bioassays (e.g., phagocytosis assays), NO:28 and SEQ ID NO:30. In another embodiment the Western blotting applications, etc. Suitable labels for the nucleic acid molecules encode binding molecules comprising detection/analysis/diagnosis techniques and/or methods a light chain comprising the variable light chain of the amino known in the art are well within the reach of the skilled acid sequence selected from the group consisting of SEQID artisan. NO:32, SEQID NO:34 and SEQID NO:36. 0063. Furthermore, the human binding molecules or 0069. It is another aspect of the invention to provide vec immunoconjugates of the invention can also be attached to tors, i.e. nucleic acid constructs, comprising one or more solid supports, which are particularly useful for in vitro nucleic acid molecules according to the present invention. immunoassays or purification of staphylococci or a fragment Vectors can be derived from plasmids such as interalia F, R1, thereof. Such solid Supports might be porous or nonporous, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages such as planar or non-planar. The binding molecules of the present lambda, lambdoid, M13, Mu, P1, P22, QB, T-even, Todd, T2, invention can be fused to marker sequences, such as a peptide T4, T7, etc.; plant viruses. Vectors can be used for cloning to facilitate purification. Examples include, but are not lim and/or for expression of the binding molecules of the inven ited to, the hexa-histidine tag, the hemagglutinin (HA) tag, tion and might even be used for gene therapy purposes. Vec the myc tag or the flag tag. Alternatively, an antibody can be tors comprising one or more nucleic acid molecules accord conjugated to a second antibody to form an antibody hetero ing to the invention operably linked to one or more conjugate. In another aspect the binding molecules of the expression-regulating nucleic acid molecules are also cov invention may be conjugated/attached to one or more anti ered by the present invention. The choice of the vector is gens. Preferably, these antigens are antigens which are rec dependent on the recombinant procedures followed and the ognized by the immune system of a subject to which the host used. Introduction of vectors in host cells can be effected binding molecule-antigen conjugate is administered. The by interalia calcium phosphate transfection, virus infection, antigens may be identical, but may also differ from each DEAE-dextran mediated transfection, lipofectamin transfec other. Conjugation methods for attaching the antigens and tion or electroporation. Vectors may be autonomously repli binding molecules are well known in the art and include, but cating or may replicate together with the chromosome into are not limited to, the use of cross-linking agents. The binding which they have been integrated. Preferably, the vectors con molecules of the invention will bind to staphylococci and the tain one or more selection markers. The choice of the markers antigens attached to the binding molecules will initiate a may depend on the host cells of choice, although this is not powerful T-cell attack on the conjugate, which will eventually critical to the invention as is well known to persons skilled in lead to the destruction of the staphylococci. the art. They include, but are not limited to, kanamycin, 0.064 Next to producing immunoconjugates chemically neomycin, puromycin, hygromycin, Zeocin, thymidine by conjugating, directly or indirectly, via for instance a linker, kinase gene from Herpes simplex virus (HSV-TK), dihydro the immunoconjugates can be produced as fusion proteins folate reductase gene from mouse (dhfr). Vectors comprising comprising the binding molecules of the invention and a one or more nucleic acid molecules encoding the human Suitable tag. Fusion proteins can be produced by methods binding molecules as described above operably linked to one US 2009/01 04204 A1 Apr. 23, 2009 or more nucleic acid molecules encoding proteins or peptides the invention under conditions conducive to the expression of that can be used to isolate the human binding molecules are the binding molecule, and b) optionally, recovering the also covered by the invention. These proteins or peptides expressed binding molecule. The expressed binding mol include, but are not limited to, glutathione-S-transferase, mal ecules or immunoconjugates can be recovered from the cell tose binding protein, metal-binding polyhistidine, green fluo free extract, but preferably they are recovered from the cul rescent protein, luciferase and beta-galactosidase. ture medium. The above method of producing can also be 0070 Hosts containing one or more copies of the vectors used to make functional variants of the binding molecules mentioned above are an additional subject of the present and/or immunoconjugates of the present invention. Methods invention. Preferably, the hosts are host cells. Host cells to recover proteins, such as binding molecules, from cell free include, but are not limited to, cells of mammalian, plant, extracts or culture medium are well known to the man skilled insect, fungal or bacterial origin. Bacterial cells include, but in the art. Binding molecules, functional variants and/or are not limited to, cells from Gram-positive bacteria or Gram immunoconjugates as obtainable by the above-described negative bacteria such as several species of the genera method are also a part of the present invention. Escherichia, such as E. coli, and Pseudomonas. In the group 0072 Alternatively, next to the expression in hosts, such of fungal cells preferably yeast cells are used. Expression in as host cells, the binding molecules and immunoconjugates of yeast can be achieved by using yeast strains such as interalia the invention can be produced synthetically by conventional Pichia pastoris, Saccharomyces cerevisiae and Hansenula peptide synthesizers or in cell-free translation systems using polymorpha. Furthermore, insect cells such as cells from RNA nucleic acid derived from DNA molecules according to Drosophila and Sf9 can be used as host cells. Besides that, the the invention. Binding molecules and immunoconjugates as host cells can be plant cells such as interalia cells from crop obtainable by the above described synthetic production meth plants such as forestry plants, or cells from plants providing ods or cell-free translation systems are also a part of the food and raw materials such as cereal plants, or medicinal present invention. plants, or cells from ornamentals, or cells from flower bulb (0073. In yet another embodiment, binding molecules of crops. Transformed (transgenic) plants or plant cells are pro the present invention can also be produced in transgenic, duced by known methods, for example, Agrobacterium-me non-human, mammals such as inter alia rabbits, goats or diated gene transfer, transformation of leaf discs, protoplast cows, and secreted into for instance the milk thereof. transformation by polyethylene glycol-induced DNA trans (0074. In yet another alternative embodiment, binding mol fer, electroporation, sonication, microinjection or bolistic ecules according to the present invention, preferably human gene transfer. Additionally, a suitable expression system can binding molecules specifically binding to staphylococci or a be abaculovirus system. Expression systems using mamma fragment thereof, may be generated by transgenic non-human lian cells such as Chinese Hamster Ovary (CHO) cells, COS mammals, such as for instance transgenic mice or rabbits, that cells, BHK cells or Bowes melanoma cells are preferred in the express human immunoglobulin genes. Preferably, the trans present invention. Mammalian cells provide expressed pro genic non-human mammals have a genome comprising a teins with posttranslational modifications that are most simi human heavy chain transgene and a human light chain trans lar to natural molecules of mammalian origin. Since the gene encoding all or a portion of the human binding mol present invention deals with molecules that may have to be ecules as described above. The transgenic non-human mam administered to humans, a completely human expression sys mals can be immunized with a purified or enriched tem would be particularly preferred. Therefore, even more preparation of staphylococci or a fragment thereof. Protocols preferably, the host cells are human cells. Examples of human for immunizing non-human mammals are well established in cells are inter alia HeLa, 911, AT1080, A549, 293 and the art. See Using Antibodies: A Laboratory Manual, Edited HEK293T cells. In preferred embodiments, the human pro by: E. Harlow, D. Lane (1998), Cold Spring Harbor Labora ducer cells comprise at least a functional part of a nucleic acid tory, Cold Spring Harbor, N.Y. and Current Protocols in sequence encoding an adenovirus E1 region in expressible Immunology, Edited by: J. E. Coligan, A. M. Kruisbeek, D. format. In even more preferred embodiments, said host cells H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley are derived from a human retina and immortalized with & Sons Inc., New York, the disclosures of which are incor nucleic acids comprising adenoviral E1 sequences, such as porated herein by reference. Immunization protocols often 911 cells or the cell line deposited at the European Collection include multiple immunizations, either with or without adju of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 vants such as Freund's complete adjuvant and Freund's OJG, Great Britain on 29 Feb. 1996 under number 96022940 incomplete adjuvant, but may also include naked DNA and marketed under the trademark PER.C6(R) (PER.C6 is a immunizations. In another embodiment, the human binding registered trademark of Crucell Holland B.V.). For the pur molecules are produced by B cells or plasma cells derived poses of this application “PER.C6' refers to cells deposited from the transgenic animals. In yet another embodiment, the under number 96022940 orancestors, passages up-stream or human binding molecules are produced by hybridomas, downstream as well as descendants from ancestors of depos which are prepared by fusion of B cells obtained from the ited cells, as well as derivatives of any of the foregoing. above-described transgenic non-human mammals to immor Production of recombinant proteins in host cells can be per talized cells. B cells, plasma cells and hybridomas as obtain formed according to methods well known in the art. The use able from the above-described transgenic non-human mam of the cells marketed under the trademark PER.C6.R as a mals and human binding molecules as obtainable from the production platform for proteins of interest has been above-described transgenic non-human mammals, B cells, described in WO 00/63403 the disclosure of which is incor plasma cells and hybridomas are also a part of the present porated herein by reference in its entirety. invention. (0071. A method of producing a binding molecule accord (0075. In a further aspect, the invention provides a method ing to the invention is an additional part of the invention. The of identifying a binding molecule, such as a human binding method comprises the steps of a) culturing a host according to molecule, e.g. a human monoclonal antibody or fragment US 2009/01 04204 A1 Apr. 23, 2009 thereof, specifically binding to at least two different bacterial cable genetic packages. Alternatively, they may also be organisms or nucleic acid molecules encoding Such binding coupled to a carrier when contact takes place. In another molecules and comprises the steps of: (a) contacting a col embodiment the first and second bacterial organism are from lection of binding molecules on the surface of replicable a different bacterial family, e.g. the first is from a Gram genetic packages with a first bacterial organism under condi negative bacterium and the second is from a Gram-positive tions conducive to binding, (b) selecting at least once for a bacterium. This way, binding molecules capable of specifi replicable genetic package binding to the first bacterial organ cally binding to Gram-positive and Gram-negative bacteria ism, (c) optionally, separating the replicable genetic package can be found. Preferably, the first and second bacterial organ binding to the first bacterial organism from replicable genetic ism are both Gram-positive bacteria. The first and second packages that do not bind to the first bacterial organism, bacterial organism can both be staphylococci. In one embodi contacting the separated replicable genetic packages with a ment the first and second bacterial organism are different second bacterial organism under conditions conducive to strains from the same bacterial species, e.g. a Staphylococcus binding and selecting at least once for a replicable genetic species such as S. aureus or S. epidermidis. This way, species package binding to the second bacterial organism, and (d) specific binding molecules can be found that are capable of separating and recovering the replicable genetic package specifically binding to different strains within one species. In binding to the first and/or second bacterial organism from another embodiment the first and second bacterial organism replicable genetic packages that do not bind to the first and/or are each a member of a different Staphylococcus species, e.g. second bacterial organism. Of course, the above methods the first and second Staphylococcus species are selected from extended with selections on third and further bacterial organ the group consisting of S. aureus and S. epidermidis. This isms are also part of the present invention. way, binding molecules capable of specifically binding to 0076. A replicable genetic package as used herein can be different species within one bacterial genus can be found. prokaryotic or eukaryotic and includes cells, spores, yeasts, Alternatively, first and second bacterial organisms can both bacteria, viruses, (bacterio)phage, ribosomes and polysomes. be enterococci. In one embodiment the first and second bac A preferred replicable genetic package is a phage. The bind terial organism are different strains from the same bacterial ing molecules, such as for instance single chain FVs, are species, e.g. an Enterococcus species such as E. faecalis or E. displayed on the replicable genetic package, i.e. they are faecium. This way, species-specific binding molecules can be attached to a group or molecule located at an exterior Surface found that are capable of specifically binding to different of the replicable genetic package. The replicable genetic strains within one species. In another embodiment the first package is a screenable unit comprising a binding molecule to and second bacterial organism are each a member of a differ be screened linked to a nucleic acid molecule encoding the ent Enterococcus species, e.g. the first and second Enterococ binding molecule. The nucleic acid molecule should be rep cus species are selected from the group consisting of E. faeca licable either in vivo (e.g., as a vector) or in vitro (e.g., by lis and E. faecium. PCR, transcription and translation). In vivo replication can be 0079 Alternatively, the selection step may be performed autonomous (as for a cell), with the assistance of host factors in the presence of a fragment of the bacterial organisms such (as for a virus) or with the assistance of both host and helper as e.g. cell membrane preparations, cell membrane prepara virus (as for a phagemid). Replicable genetic packages dis tions that have been enzymically treated to remove proteins playing a collection of binding molecules is formed by intro (e.g. with protease K), cell membrane preparations that have ducing nucleic acid molecules encoding exogenous binding been enzymically treated to remove carbohydrate moieties molecules to be displayed into the genomes of the replicable (e.g. with periodate), recombinant proteins or polysaccha genetic packages to form fusion proteins with endogenous rides. In yet another embodiment, the selection step may be proteins that are normally expressed from the outer surface of performed in the presence of one or more proteins or (poly) the replicable genetic packages. Expression of the fusion peptides derived from the bacterial organisms, fusion pro proteins, transport to the outer Surface and assembly results in teins comprising these proteins or (poly)peptides, and the display of exogenous binding molecules from the outer Sur like. Extracellularly exposed parts of these proteins can also face of the replicable genetic packages. be used as selection material. The live or inactivated bacterial 0077. The selection step(s) in the method according to the organisms or fragments thereof may be immobilized to a present invention can be performed with bacterial organisms suitable material before use. Alternatively, live or inactivated that are live and still infective or inactivated. Inactivation of bacteria in Suspension are used. In an embodiment the selec bacterial organism may be performed by bacterial inactiva tion can be performed on different materials derived from tion methods well known to the skilled artisan such as inter bacterial organisms. For instance, the first selection round can alia treatment with low pH, i.e. pH 4 for 6 hours to 21 days: be performed on live or inactivated bacterial organisms in treatment with organic solvent/detergent, i.e. addition of Suspension, while the second and third selection round can be organic solvents and detergents (Triton X-100 or Tween-80) performed on recombinant bacterial proteins and polysaccha to the bacterium: UV/light irradiation; gamma-irradiation; rides, respectively. Of course, other combinations are also and treatment with relevant antibiotics. Methods to test, if a contemplated herein. Different bacterial materials can also be bacterial organism is still alive, infective and/or viable or used during one selection/panning step. In a further aspect the partly or completely inactivated are well known to the person invention provides methods wherein the bacterial organisms skilled in the art. The bacterial organisms used in the above used in the selection step(s) are derived from the same or method may be non-isolated, e.g. present in serum and/or different growth phases of the bacteria, e.g. the lag phase, log blood of an infected individual. The bacterial organisms used phase, stationary phase or death phase. This way, phase may also be isolated as discrete colonies after overnight cul specific anti-bacterial binding molecules may be found. For ture at 37°C. on a suitable medium such as sheep blood agar. instance, the first bacterial organism may be a S. aureus in 0078. In an embodiment the first and/or second bacterial stationary phase, while the second bacterial organism is a S. organisms are in Suspension when contacted with the repli aureus in log phase or the first bacterial organism may be a S. US 2009/01 04204 A1 Apr. 23, 2009

aureus in lag phase, while the second bacterial organism is a has or has had a chronic bacterial infection or an acute bac S. epidermidis in lag phase. Further combinations are well terial infection. Preferably, the human subject has recently within the reach of the skilled artisan. recovered from the bacterial infection. 0080. In a specific embodiment the invention provides a I0083. Alternatively, phage display libraries may be con method as described above wherein, if the first and/or second structed from immunoglobulin variable regions that have Staphylococcus species is a S. aureus Strain, Protein A present been partially assembled in vitro to introduce additional anti on the surface of the S. aureus strain is blocked before the S. body diversity in the library (semi-synthetic libraries). For aureus strain is contacted with replicable genetic packages. example, in vitro assembled variable regions contain Suitable blocking agent may be rabbit serum, purified rabbit stretches of synthetically produced, randomized or partially immunoglobulin, fetal calf serum, pooled human serum randomized DNA in those regions of the molecules that are 0081. In yet a further aspect, the invention provides a important for antibody specificity, e.g. CDR regions. Phage method of obtaining a binding molecule specifically binding antibodies specific for bacteria Such as staphylococci can be to at least two different bacterial organisms or a nucleic acid selected from the library by exposing the bacteria or material molecule encoding such a binding molecule, wherein the thereof to a phage library to allow binding of phages express method comprises the steps of a) performing the above ing antibody fragments specific for the bacteria or material described method of identifying binding molecules, and b) thereof. Non-bound phages are removed by washing and isolating from the recovered replicable genetic package the bound phages eluted for infection of E. coli bacteria and binding molecule and/or the nucleic acid molecule encoding Subsequent propagation. Multiple rounds of selection and the binding molecule. The collection of binding molecules on propagation are usually required to sufficiently enrich for the Surface of replicable genetic packages can be a collection phages binding specifically to the bacteria or material thereof. of scFVs or Fabs. Once a new scEvor Fab has been established If desired, before exposing the phage library to the bacteria or or identified with the above-mentioned method of identifying material thereof the phage library can first be subtracted by binding molecules or nucleic acid molecules encoding the exposing the phage library to non-target material Such as binding molecules, the DNA encoding the schvor Fab can be bacteria of a different family, species and/or strain or bacteria isolated from the bacteria or phages and combined with stan in a different growth phase or material of these bacteria. dard molecular biological techniques to make constructs These subtractor bacteria or material thereof can be bound to encoding bivalent SchvS or complete human immunoglobu a solid phase or can be in Suspension. Phages may also be lins of a desired specificity (e.g. IgG, IgA or IgM). These selected for binding to complex antigens Such as complex constructs can be transfected into suitable cell lines and com mixtures of bacterial proteins or (poly)peptides optionally plete human monoclonal antibodies can be produced (see supplemented with bacterial polysaccharides or other bacte Huls et al., 1999; Boel et al., 2000). rial material. Host cells expressing one or more proteins or 0082. As mentioned before the preferred replicable (poly)peptides of bacteria Such as Staphylococci may also be genetic package is a phage. Phage display methods for iden used for selection purposes. A phage display method using tifying and obtaining (human) binding molecules, e.g. (hu these host cells can be extended and improved by Subtracting man) monoclonal antibodies, are by now well-established non-relevant binders during screening by addition of an methods known by the person skilled in the art. They are e.g. excess of host cells comprising no target molecules or non described in U.S. Pat. No. 5,696,108; Burton and Barbas, target molecules that are similar, but not identical, to the 1994; de Kruifet al., 1995b; and Phage Display: A Laboratory target, and thereby strongly enhance the chance of finding Manual. Edited by: CF Barbas, D R Burton, J K Scott and G relevant binding molecules. Of course, the subtraction may be J Silverman (2001), Cold Spring Harbor Laboratory Press, performed before, during or after the screening with bacterial Cold Spring Harbor, N.Y. All these references are herewith organisms or material thereof. The process is referred to as the incorporated herein in their entirety. For the construction of Mabstract(R) process (Mabstract(R) is a registered trademark of phage display libraries, collections of human monoclonal Crucell Holland B.V., see also U.S. Pat. No. 6,265,150 which antibody heavy and light chain variable region genes are is incorporated herein by reference). expressed on the Surface of bacteriophage, preferably fila I0084. In yet another aspect the invention provides a mentous bacteriophage, particles, in for example single-chain method of obtaining a binding molecule potentially having Fv (scFv) or in Fab format (see de Kruifet al., 1995b). Large killing activity against at least two different bacterial organ libraries of antibody fragment-expressing phages typically isms, wherein the method comprises the steps of (a) perform contain more than 1.0*10 antibody specificities and may be ing the method of obtaining a binding molecule specifically assembled from the immunoglobulin V regions expressed in binding to at least two different bacterial organisms or a the B-lymphocytes of immunized- or non-immunized indi nucleic acid molecule encoding Such a binding molecule as viduals. In a specific embodiment of the invention the phage described above, and (b) verifying if the binding molecule library of binding molecules, preferably scFv phage library, is isolated has killing activity against at least two different bac prepared from RNA isolated from cells obtained from a sub terial organisms. Assays for verifying if a binding molecule ject that has been vaccinated against a bacterium, recently has killing activity Such as opsonic activity are well known in vaccinated against an unrelated pathogen, recently Suffered the art (see for instance Manual of Molecular and Clinical from a chronic or acute bacterial infection, e.g. staphylococ Laboratory Immunology, 7th Edition). In a further embodi cal infection, or from a healthy individual. RNA can be iso ment the binding molecule is also tested for any other activity. lated from inter alia bone marrow or peripheral blood, pref Other useful activities are mentioned above. erably peripheral blood lymphocytes or on isolated B cells or I0085 Inafurther aspect the invention pertains to a binding even on subpopulations of B cells. The subject can be an molecule having killing activity against at least two, prefer animal vaccinated against a bacterium orananimal that has or ably at least three or more, different bacterial organisms. Such has had a bacterial infection. Preferably, the animal is a as e.g. staphylococci, and being obtainable by the methods as human Subject that has been vaccinated against a bacterium or described above. A pharmaceutical composition comprising US 2009/01 04204 A1 Apr. 23, 2009

the binding molecule, the pharmaceutical composition fur sensitivity of bacteria to antibiotic treatment. It is to be under ther comprising at least one pharmaceutically acceptable stood that other combinations are also contemplated herein. excipient is also an aspect of the present invention. Pharma 0090. A pharmaceutical composition according to the ceutically acceptable excipients are well known to the skilled invention can further comprise at least one other therapeutic, person. The pharmaceutical composition according to the prophylactic and/or diagnostic agent. Preferably, the pharma invention may further comprise at least one other therapeutic ceutical composition comprises at least one other prophylac agent. Suitable agents are also well known to the skilled tic and/ortherapeutic agent. Preferably, said further therapeu artisan. tic and/or prophylactic agents are agents capable of I0086. In yet a further aspect, the invention provides com preventing and/or treating a bacterial, e.g. staphylococcal, positions comprising at least one binding molecule preferably infection and/or a condition resulting from Such an infection. a human monoclonal antibody according to the invention, at Therapeutic and/or prophylactic agents include, but are not least one functional variant thereof, at least one immunocon limited to, anti-bacterial agents. Such agents can be binding jugate according to the invention or a combination thereof. In molecules, Small molecules, organic or inorganic com addition to that, the compositions may comprise inter alia pounds, enzymes, polynucleotide sequences, anti-microbial stabilizing molecules, such as albumin or polyethylene gly peptides, etc. Other agents that are currently used to treat col, or salts. Preferably, the salts used are salts that retain the patients infected with bacterial infections such as staphylo desired biological activity of the binding molecules and do coccal infections are antibiotics such as methicillin, 2" and not impart any undesired toxicological effects. If necessary, 3" generation cephalosporins, aminoglycosides, Carbapen the human binding molecules of the invention may be coated ems, Macrollides, Ketolides, Quinolones and miscellaneous in or on a material to protect them from the action of acids or antibiotics Such as daptomycin, lineZolid, nitrofurantoin, other natural or non-natural conditions that may inactivate the quinupristin?dalfopristin, trimethoprim/sulfa, Vancomycin. binding molecules. These can be used in combination with the binding molecules 0087. In yet a further aspect, the invention provides com of the invention. Agents capable of preventing and/or treating positions comprising at least one nucleic acid molecule as an infection with bacteria and/or a condition resulting from defined in the present invention. The compositions may com Such an infection that are in the experimental phase might also prise aqueous solutions such as aqueous Solutions containing be used as other therapeutic and/or prophylactic agents useful salts (e.g., NaCl or salts as described above), detergents (e.g., in the present invention. SDS) and/or other suitable components. 0091. The binding molecules or pharmaceutical composi 0088. Furthermore, the present invention pertains to phar tions of the invention can be tested in suitable animal model maceutical compositions comprising at least one binding systems prior to use in humans. Such animal model systems molecule such as a human monoclonal antibody of the inven include, but are not limited to, murine sepsis and peritonitis tion (or functional fragment or variant thereof), at least one models, rat sepsis and endocarditis models, and rabbit immunoconjugate according to the invention, at least one endocarditis models. composition according to the invention, or combinations 0092 Typically, pharmaceutical compositions must be thereof. The pharmaceutical composition of the invention sterile and stable under the conditions of manufacture and further comprises at least one pharmaceutically acceptable storage. The binding molecules, immunoconjugates, nucleic excipient. acid molecules or compositions of the present invention can 0089. In an embodiment the pharmaceutical compositions be in powder form for reconstitution in the appropriate phar may comprise two or more binding molecules that have kill maceutically acceptable excipient before or at the time of ing activity against a bacterial organism, e.g. a Staphylococ delivery. In the case of sterile powders for the preparation of cus species. In an embodiment, the binding molecules exhibit sterile injectable solutions, the preferred methods of prepa synergistic killing activity, when used in combination. In ration are vacuum drying and freeze-drying (lyophilization) other words, the compositions comprise at least two binding that yield a powder of the active ingredient plus any additional molecules having killing activity, characterized in that the desired ingredient from a previously sterile-filtered solution binding molecules act synergistically in killing a bacterial thereof. organism Such as e.g. a Staphylococcus species. As used 0093. Alternatively, the binding molecules, immunocon herein, the term “synergistic’ means that the combined effect jugates, nucleic acid molecules or compositions of the present of the binding molecules when used in combination is greater invention can be in Solution and the appropriate pharmaceu than their additive effects when used individually. The syn tically acceptable excipient can be added and/or mixed before ergistically acting binding molecules may bind to different or at the time of delivery to provide a unit dosage injectable structures on the same of distinct fragments of the bacterial form. Preferably, the pharmaceutically acceptable excipient organism. In an embodiment the binding molecules acting used in the present invention is Suitable to high drug concen synergistically in killing a bacterial organism may also be tration, can maintain proper fluidity and, if necessary, can capable of killing other bacterial organisms synergistically. A delay absorption. way of calculating synergy is by means of the combination 0094. The choice of the optimal route of administration of index. The concept of the combination index (CI) has been the pharmaceutical compositions will be influenced by sev described by Chou and Talalay, 1984. The two or more bind eral factors including the physico-chemical properties of the ing molecules having synergistic activity have distinct modes active molecules within the compositions, the urgency of the of action. For instance a first binding molecule may have clinical situation and the relationship of the plasma concen opSonizing activity, while the second binding molecule has trations of the active molecules to the desired therapeutic another activity increasing/augmenting/enhancing phagocy effect. For instance, if necessary, the binding molecules of the tosis or a first binding molecule may have intrinsic (killing) invention can be prepared with carriers that will protect them activity, e.g. reduce or inhibit bacterial growth or directly kill against rapid release, such as a controlled release formula bacteria, while the second binding molecule increases the tion, including implants, transdermal patches, and microen US 2009/01 04204 A1 Apr. 23, 2009 capsulated delivery systems. Biodegradable, biocompatible tion may directly inhibit the growth of the bacterium or inhibit polymers can inter alia be used, such as ethylene vinyl virulence factors required for its survival during the infection. acetate, polyanhydrides, polyglycolic acid, collagen, poly 0099. The above-mentioned molecules or compositions orthoesters, and polylactic acid. Furthermore, it may be nec may be employed in conjunction with other molecules useful essary to coat the binding molecules with, or co-administer in diagnosis, prophylaxis and/or treatment. They can be used the binding molecules with, a material or compound that in vitro, ex vivo or in vivo. For instance, the binding mol prevents the inactivation of the human binding molecules. For ecules such as human monoclonal antibodies (or functional example, the binding molecules may be administered to a variants thereof), immunoconjugates, compositions or phar Subject in an appropriate carrier, for example, liposomes or a maceutical compositions of the invention can be co-adminis diluent. tered with a vaccine against the bacterial organism (if avail able). Alternatively, the vaccine may also be administered 0.095 The routes of administration can be divided into two before or after administration of the molecules of the inven main categories, oral and parenteral administration. The pre tion. Instead of a vaccine, anti-bacterial agents can also be ferred administration route is intravenous. employed in conjunction with the binding molecules of the 0096 Oral dosage forms can be formulated inter alia as present invention. Suitable anti-bacterial agents are men tablets, troches, lozenges, aqueous or oily suspensions, dis tioned above. persable powders or granules, emulsions, hard capsules, soft 0100. The molecules are typically formulated in the com gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels, positions and pharmaceutical compositions of the invention or slurries. These formulations can contain pharmaceutically in a therapeutically or diagnostically effective amount. Alter excipients including, but not limited to, inert diluents, granu natively, they may be formulated and administered sepa lating and disintegrating agents, binding agents, lubricating rately. For instance the other molecules such as the anti agents, preservatives, coloring, flavoring or Sweetening bacterial agents may be applied systemically, while the agents, vegetable or mineral oils, wetting agents, and thick binding molecules of the invention may be applied intrathe ening agents. cally or intraventricularly. 0097. The pharmaceutical compositions of the present 0101 Dosage regimens can be adjusted to provide the invention can also be formulated for parenteral administra optimum desired response (e.g., a therapeutic response). A tion. Formulations for parenteral administration can be inter Suitable dosage range may for instance be 0.1-100 mg/kg alia in the form of aqueous or non-aqueous isotonic sterile body weight, preferably 0.5-15 mg/kg body weight. Further non-toxic injection or infusion solutions or suspensions. The more, for example, a single bolus may be administered, sev Solutions or Suspensions may comprise agents that are non eral divided doses may be administered over time or the dose toxic to recipients at the dosages and concentrations may be proportionally reduced or increased as indicated by employed Such as 1,3-butanediol, Ringer's Solution, Hank’s the exigencies of the therapeutic situation. The molecules and Solution, isotonic sodium chloride Solution, oils, fatty acids, compositions according to the present invention are prefer local anesthetic agents, preservatives, buffers, Viscosity or ably sterile. Methods to render these molecules and compo solubility increasing agents, water-soluble antioxidants, oil sitions sterile are well known in the art. The other molecules soluble antioxidants, and metal chelating agents. useful in diagnosis, prophylaxis and/or treatment can be 0098. In a further aspect, the binding molecules such as administered in a similar dosage regimen as proposed for the human monoclonal antibodies (functional fragments and binding molecules of the invention. If the other molecules are variants thereof), immunoconjugates, compositions, or phar administered separately, they may be administered to a maceutical compositions of the invention can be used as a patient prior to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 medicament. So, a method of treatment and/or prevention of minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 4 a bacterial (Gram-positive and/or Gram-negative), e.g. a sta hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 phylococcal, infection using the binding molecules, immu hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, noconjugates, compositions, orpharmaceutical compositions 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6 weeks before), of the invention is another part of the present invention. The concomitantly with, or Subsequent to (e.g., 2 minutes, 5 min above-mentioned molecules can inter alia be used in the utes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 diagnosis, prophylaxis, treatment, or combination thereof, of minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 a bacterial infection. They are suitable for treatment of yet hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 untreated patients suffering from a bacterial infection and hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks patients who have been or are treated for a bacterial infection. or 6 weeks after) the administration of one or more of the They may be used for patients such as hospitalized infants, human binding molecules or pharmaceutical compositions of premature infants, burn victims, elderly patients, immuno the invention. The exact dosing regimen is usually sorted out compromised patients, immunosuppressed patients, patient during clinical trials in human patients. undergoing an invasive procedure, and health care workers. 0102 Human binding molecules and pharmaceutical Each administration may protect against further infection by compositions comprising the human binding molecules are the bacterial organism for up to three or four weeks and/or particularly useful, and often preferred, when to be adminis will retard the onset or progress of the symptoms associated tered to human beings as in vivo therapeutic agents, since with the infection. The binding molecules of the invention recipient immune response to the administered antibody will may also increase the effectiveness of existing antibiotic often be substantially less than that occasioned by adminis treatment by increasing the sensitivity of the bacterium to the tration of a monoclonal murine, chimeric or humanized bind antibiotic, may stimulate the immune system to attack the ing molecule. bacterium in ways other than through opSonization. This acti 0103) In another aspect, the invention concerns the use of Vation may result in long lasting protection to the infection the binding molecules such as killing human monoclonal bacterium. Furthermore, the binding molecules of the inven antibodies (functional fragments and variants thereof), US 2009/01 04204 A1 Apr. 23, 2009 immunoconjugates, nucleic acid molecules, compositions or complex between the human binding molecules and the bac pharmaceutical compositions according to the invention in terial organism or antigenic components thereofthat may be the preparation of a medicament for the diagnosis, prophy present in the sample. The formation of an immunological laxis, treatment, or combination thereof, of a bacterial (Gram complex, if any, indicating the presence of the bacterial positive and/or Gram-negative), e.g. Staphylococcal infec organism in the sample, is then detected and measured by tion. Suitable means. Such methods include, inter alia, homoge 0104. Next to that, kits comprising at least one binding neous and heterogeneous binding immunoassays, such as molecule such as a killing human monoclonal antibody (func radio-immunoassays (RIA), ELISA, immunofluorescence, tional fragments and variants thereof), at least one immuno immunohistochemistry, FACS, BIACORE and Western blot conjugate, at least one nucleic acid molecule, at least one analyses. composition, at least one pharmaceutical composition, at 0107 Preferred assay techniques, especially for large least one vector, at least one host according to the invention or scale clinical screening of patient Sera and blood and blood a combination thereofare also a part of the present invention. derived products are ELISA and Western blot techniques. Optionally, the above-described components of the kits of the ELISA tests are particularly preferred. For use as reagents in invention are packed in suitable containers and labeled for these assays, the binding molecules or immunoconjugates of diagnosis, prophylaxis and/or treatment of the indicated con the invention are conveniently bonded to the inside surface of ditions. The above-mentioned components may be stored in microtiter wells. The binding molecules or immunoconju unit or multi-dose containers as an aqueous, preferably ster gates of the invention may be directly bonded to the microtiter ile, solution or as a lyophilized, preferably sterile, formula well. However, maximum binding of the binding molecules tion for reconstitution. The containers may be formed from a or immunoconjugates of the invention to the wells might be variety of materials such as glass or plastic and may have a accomplished by pre-treating the wells with polylysine prior sterile access port (for example the container may be an to the addition of the binding molecules or immunoconju intravenous Solution bag or a vial having a stopper pierceable gates of the invention. Furthermore, the binding molecules or by a hypodermic injection needle). The kit may further com immunoconjugates of the invention may be covalently prise more containers comprising a pharmaceutically accept attached by known means to the wells. Generally, the binding able buffer. It may further include other materials desirable molecules or immunoconjugates are used between 0.01 to from a commercial and userstandpoint, including other buff 100 ug/ml for coating, although higher as well as lower ers, diluents, filters, needles, Syringes, culture medium for amounts may also be used. Samples are then added to the one or more of the suitable hosts and, possibly, even at least wells coated with the binding molecules or immunoconju one other therapeutic, prophylactic or diagnostic agent. Asso gates of the invention. ciated with the kits can be instructions customarily included 0.108 Furthermore, binding molecules of the invention in commercial packages of therapeutic, prophylactic or diag can be used to identify specific binding structures of a bacte nostic products, that contain information about for example rial organism e.g. a Staphylococcus. The binding structures the indications, usage, dosage, manufacture, administration, can be epitopes on proteins and/or polypeptides. They can be contra-indications and/or warnings concerning the use of linear, but also structural and/or conformational. In one Such therapeutic, prophylactic or diagnostic products. embodiment, the binding structures can be analyzed by 0105. The binding molecules of the invention may also be means of PEPSCAN analysis (see interalia WO 84/03564, used to coat medical devices or polymeric biomaterials. WO93/09872, Slootstra et al., 1996). Alternatively, a random 0106 The invention further pertains to a method of detect peptide library comprising peptides from a protein of a bac ing a bacterial organism (Gram-positive and/or Gram-nega terial organism can be screened for peptides capable of bind tive) in a sample, wherein the method comprises the steps of ing to the binding molecules of the invention. The binding (a) contacting a sample with a diagnostically effective structures/peptides/epitopes found can be used as vaccines amount of a binding molecule (functional fragments and vari and for the diagnosis of bacterial infections. In case fragments ants thereof) or an immunoconjugate according to the inven other than proteins and/or polypeptides are bound by the tion, and (b) determining whether the binding molecule or binding molecules binding structures can be identified by immunoconjugate specifically binds to a molecule of the mass spectrometry, high performance liquid chromatography sample. Preferably, the method is used to detect a Staphylo and nuclear magnetic resonance. coccus in a sample. The sample may be a biological sample 0109. In a further aspect, the invention provides a method including, but not limited to blood, serum, urine, tissue or of screening a binding molecule (or a functional fragment or other biological material from (potentially) infected subjects, variant thereof) for specific binding to the same epitope of a or a non-biological sample such as water, drink, etc. The bacterial organism (Gram-positive and/or Gram-negative), (potentially) infected Subjects may be human Subjects, but e.g. Staphylococcus, as the epitope bound by a human binding also animals that are Suspected as carriers of Such a bacterial molecule of the invention, wherein the method comprises the organism might be tested for the presence of the organism steps of (a) contacting a binding molecule to be screened, a using the human binding molecules or immunoconjugates of binding molecule of the invention and a bacterial organism or the invention. The sample may first be manipulated to make it fragment thereof, (b) measure if the binding molecule to be more suitable for the method of detection. Manipulation screened is capable of competing for specifically binding to means inter alia treating the sample Suspected to contain the bacterial organism or fragment thereof with the binding and/or containing the bacterial organism in Such a way that molecule of the invention. In a further step it may be deter the organism will disintegrate into antigenic components mined, if the screened binding molecules that are capable of Such as proteins, (poly)peptides or other antigenic fragments. competing for specifically binding to the bacterial organism Preferably, the human binding molecules or immunoconju or fragment thereofhave killing activity, e.g. opsonic activity. gates of the invention are contacted with the sample under A binding molecule that is capable of competing for specifi conditions which allow the formation of an immunological cally binding to the bacterial organism or a fragment thereof US 2009/01 04204 A1 Apr. 23, 2009

with the binding molecule of the invention is another part of EXAMPLES the present invention. In the above-described screening 0111) To illustrate the invention, the following examples method, “specifically binding to the same epitope also con are provided. The examples are not intended to limit the scope templates specific binding to substantially or essentially the of the invention in any way. same epitope as the epitope bound by the a binding molecule of the invention. The capacity to block, or compete with, the Example 1 binding of the binding molecules of the invention to the bacterial organism typically indicates that a binding molecule Construction of scFv Phage Display Libraries Using to be screened binds to an epitope or binding site on the RNA Extracted from Donors Screened for Opsonic bacterial organism that structurally overlaps with the binding Activity site on the bacterial organism that is immunospecifically rec 0112 Samples of blood were taken from donors reporting ognized by the binding molecules of the invention. Alterna a recent gram-positive bacterial infection as well as healthy tively, this can indicate that a binding molecule to be screened adults between 25-50 years of age. Peripheral blood leuko binds to an epitope or binding site which is sufficiently proxi cytes were isolated by centrifugation and the blood serum was mal to the binding site immunospecifically recognized by the saved and frozen at -80° C. Donor serum was screened for binding molecules of the invention to sterically or otherwise opsonic activity using a FACS-based phagocytosis assay inhibit binding of the binding molecules of the invention to (Cantinieaux et al., 1989) and compared to a pool of normal the bacterial organism. healthy donor serum. Sera from donors having a higher 0110. In general, competitive inhibition is measured by phagocytic activity compared to normal serum were chosen means of an assay, wherein an antigen composition, i.e. a to use for the generation of phage display libraries. Total RNA composition comprising a bacterial organism or fragments was prepared from the peripheral blood leukocytes of these thereof, is admixed with reference binding molecules, i.e. the donors using organic phase separation and subsequent etha binding molecules of the invention, and binding molecules to nol precipitation. The obtained RNA was dissolved in be screened. Usually, the binding molecules to be screened RNAse-free water and the concentration was determined by are present in excess. Protocols based upon ELISAs and OD260 nm measurement. Thereafter, the RNA was diluted to Western blotting are suitable for use in such simple competi a concentration of 100 ng/ul. Next, 1 lug of RNA was con tion studies. By using species or isotype secondary antibodies verted into cDNA as follows: To 10 ul total RNA, 13 ul one will be able to detect only the bound reference binding DEPC-treated ultrapure water and 1 Jul random hexamers molecules, the binding of which will be reduced by the pres (500 ng/ul) were added and the obtained mixture was heated ence of a binding molecule to be screened that recognizes at 65° C. for 5 minutes and quickly cooled on wet-ice. Then, Substantially the same epitope. In conducting a binding mol 8 Jul 5x First-Strand buffer, 2 uldNTP (10 mMeach), 2 ul DTT ecule competition study between a reference binding mol (0.1 M), 2 ul RNAse-inhibitor (40 U/ul) and 2 ul ecule and any binding molecule to be screened (irrespective Superscript TMIII MMLV reverse transcriptase (200 U/ul) of species or isotype), one may first label the reference bind were added to the mixture, incubated at room temperature for ing molecule with a detectable label, such as, e.g., biotin, an 5 minutes and incubated for 1 hour at 50° C. The reaction was enzymatic, a radioactive or other label to enable subsequent terminated by heat inactivation, i.e. by incubating the mixture identification. Binding molecules identified by these compe for 15 minutes at 75° C. The obtained cDNA products were tition assays ("competitive binding molecules' or “cross diluted to a final volume of 200 ul with DEPC-treated ultra reactive binding molecules”) include, but are not limited to, pure water. The OD 260 nm of a 50 times diluted solution (in antibodies, antibody fragments and other binding agents that 10 mM Tris buffer) of the dilution of the obtained cDNA bind to an epitope or binding site bound by the reference products was used to determine the cDNA concentration. For binding molecule, i.e. a binding molecule of the invention, as each donor 5 to 10 Jul of the diluted cDNA products were used well as antibodies, antibody fragments and other binding as template for PCR amplification of the immunoglobulin agents that bind to an epitope or binding site sufficiently gamma heavy chain family and kappa or lambda light chain proximal to an epitope bound by the reference binding mol sequences using specific oligonucleotide primers (see Tables ecule for competitive binding between the binding molecules 1-7). In addition, for one donor PCR amplification of the to be screened and the reference binding molecule to occur. immunoglobulin mu heavy chain family and kappa or lambda Preferably, competitive binding molecules of the invention light chain sequences was carried out. PCR reaction mixtures will, when present in excess, inhibit specific binding of a contained, besides the diluted cDNA products, 25 pmol sense reference binding molecule to a selected target species by at primer and 25 pmol anti-sense primer in a final volume of 50 least 10%, preferably by at least 25%, more preferably by at ul of 20 mM Tris-HCl (pH 8.4), 50 mMKC1, 1.5 mMMgCl, least 50%, and most preferably by at least 75%-90% or even 250LM dNTPs and 1.25 units Taq polymerase. In aheated-lid greater. The identification of one or more competitive binding thermal cycler having a temperature of 96° C., the mixtures molecules that bind to about, substantially, essentially or at obtained were quickly melted for 2 minutes, followed by 30 the same epitope as the binding molecules of the invention is cycles of 30 seconds at 96°C., 30 seconds at 55° C. or 60° C. a straightforward technical matter. As the identification of and 60 seconds at 72° C. Finally, the samples were incubated competitive binding molecules is determined in comparison 10 minutes at 72°C. and refrigerated at 4°C. until further use. to a reference binding molecule, i.e. a binding molecule of the 0113. In a first round amplification, each of eighteen light invention, it will be understood that actually determining the chain variable region sense primers (twelve for the lambda epitope to which the reference binding molecule and the light chain (see Table 1: the HuVL1A-Back, HuVL1B-Back competitive binding molecule bind is not in any way required and HuVL1C-Back sense primers were mixed to equimolar in order to identify a competitive binding molecule that binds ity before use, as well as the HuVL9-Back and HuVL10-Back to the same or substantially the same epitope as the reference sense primers) and six for the kappa light chain (see Table 2)) binding molecule. were combined with an anti-sense primer recognizing the US 2009/01 04204 A1 Apr. 23, 2009

C-kappa constant region called HuCK-FOR 5'-ACACTCTC seven products of about 650 base pairs. For one donoran IgM CCCTGTTGAAGCTCTT-3' (SEQ ID NO:37) or C-lambda specific constant region anti-sense primer called HuCIgM constant region HuCL2-FOR 5'-TGAACATTCTG 5'-TGG AAG AGG CAC GTT CTT TTC TTT-3' (SEQ ID TAGGGGCCACTG-3' (SEQ ID NO:38) and HuCL7-FOR NO:42) was used instead of primer HuCIgG. The products 5'-AGAGCATTCTGCAGGGGCCACTG-3 (SEQ ID were purified on agarose gel and isolated from the gel using NO:39) (the HuCL2-FOR and HuCL7-FOR anti-sense prim Qiagen gel-extraction columns. 1/10 of each of the isolated ers were mixed to equimolarity before use), yielding 15 prod products was used in an identical PCR reaction as described ucts of about 650 base pairs. These products were purified on above using eight sense primers, whereby each heavy chain agarose gel and isolated from the gel using Qiagen gel-ex sense primer was combined with one of the four JH-region traction columns. 1/10 of each of the isolated products was specific anti-sense primers (see Table 6: the HuVH1B/7A used in an identical PCR reaction as described above using Back-Sfi and HuVH1 C-Back-Sfi sense primers were mixed eighteen sense primers, whereby each lambda light chain to equimolarity before use). The sense primers used in the sense primer was combined with one of the three Jlambda second round were the same primers as used in the first region specific anti-sense primers and each kappa light chain amplification, but extended with restriction sites (see Table 6) sense primer was combined with one of the five Jkappa to enable directed cloning in the light chain (sub)library vec region specific anti-sense primers (see Table 3: the HuVL1A tor. This resulted in 28 products of approximately 400 base Back-SAL, HuVL1B-Back-SAL and HuVL1C-Back-SAL pairs that were pooled as shown in Table 7 to maintain the sense primers were mixed to equimolarity before use, as well natural distribution of the different J segments and heavy as the HuVL9-Back-SAL and HuVL10-Back-SAL sense chain families within the library and not to over or under primers). The sense primers used in the second amplification represent certain families. The pooled products were purified were the same primers as used in the first amplification, but using Qiagen PCR purification columns. Next, 3 Jug of puri extended with restriction sites (see Table 3) to enable directed fied products was digested with Sfil and XhoI and ligated in cloning in the phage display vector PDV-C06 (SEQ ID the light chain (sub)library vector, which was cut with the NO:40). This resulted in 57 products of approximately 400 same restriction enzymes, using the same ligation procedure base pairs that were pooled as shown in Table 4 to maintain and volumes as described above for the light chain (sub) the natural distribution of the different J segments and light library. Ligation mix purification and Subsequent transforma chain families within the library and not to over or under tion of the resulting definitive library was also performed as represent certain families. The pooled products were purified described above for the light chain (sub)library. All bacteria, using Qiagen PCR purification columns. In the next step, 3 Jug typically ~10, were harvested in 2TY culture medium con of pooled products and 100 ug PDV-C06 vector were digested taining 50 ug/ml amplicillin and 4.5% glucose, mixed with with SalI and Notland purified from gel. Thereafter, a ligation glycerol to 15% (v/v) and frozen in 1.5 ml aliquots at -80°C. was performed overnight at 16° C. as follows. To 500 ng Rescue and selection of each library were performed as PDV-C06 vector either 35, 70 or 140 ng pooled products were described below. The various libraries were named GPB-05 added in a total volume of 50 ul ligation mix containing 50 M01, GPB-05-G01, GPB-05-G02, GPB-05-G03, GPB-05 mM Tris-HCl (pH 7.5), 10 mM MgCl, 10 mM DTT, 1 mM G04 and GPB-05-G05. Two other libraries, RAB-03-G01 and ATP 25 g/ml BSA and 2.5 ul T4 DNA Ligase (400 U/ul). RAB-04-G01, were constructed using a method similar to the The ligation mixes were purified by phenol/chloroform procedure above, as described previously in international extraction, followed by a chloroform extraction and ethanol patent application WO 2005/118644. precipitation, methods well known to the skilled artisan. The DNA obtained was dissolved in 50 pull 10 mM Tris-HCl pH 8.5 Example 2 and per ligation mix 1 or 2 ul was electroporated into 40 ul of TG1 competent E. coli bacteria according to the manufactur Construction of schv Phage Display Libraries Using er's protocol (Stratagene). Transformants were grown over RNA Extracted from Memory B Cells night at 37° C. on 2TY agar supplemented with 50 ug/ml 0115 Peripheral blood was collected from normal healthy ampicillin and 4.5% glucose. Colonies were counted to deter donors, convalescent donors or vaccinated donors by mine the optimal vector to insert ratio. From the ligation mix Venapunction using EDTA anti-coagulation sample tubes. A with the optimal ratio, multiple 1 or 2 ul aliquots were elec blood sample (45 ml) was diluted twice with PBS and 30 ml troporated as above and transformants were grown overnight aliquots were underlayed with 10 ml Ficoll-Hypaque (Phar at 37° C., typically yielding 107 colonies. A (sub)library of macia) and centrifuged at 900xg for 20 minutes at room variable light chain regions was obtained by scraping the temperature without breaks. The supernatant was removed transformants from the agar plates. This (sub)library was carefully to just above the white layer containing the lympho directly used for plasmid DNA preparation using a QiagenTM cytic and thrombocytic fraction. Next, this layer was carefully QIAFilter MAXI prep kit. removed (~10 ml), transferred to a fresh 50 ml tube and 0114 Heavy chain immunoglobulin sequences were washed three times with 40 ml PBS and spun at 400xg for 10 amplified from the same cDNA preparations in a similar two minutes at room temperature to remove thrombocytes. The round PCR procedure and identical reaction parameters as obtained pellet containing lymphocytes was resuspended in described above for the light chain regions with the proviso RPMI medium containing 2% FBS and the cell number was that the primers depicted in Tables 5 and 6 were used. The first determined by cell counting. Approximately 1x10' lympho amplification was performed using a set of eight sense cytes were stained for fluorescent cell sorting using CD24, directed primers (see Table 5: the HuVH1B/7A-Back and CD27 and surface IgM as markers for the isolation of HuVH1 C-Back sense primers were mixed to equimolarity switched and IgM memory B cells. A Becton Dickinson before use) each combined with an IgG specific constant Digital Vantage apparatus set in Yield Mode was used for region anti-sense primer called HuCIgG 5'-GTC CAC CTT physical memory B cell sorting and isolation. Lymphocytes GGT GTT GCT GGG CTT-3' (SEQ ID NO:41) yielding were gated as the small compact population from the FSC/ US 2009/01 04204 A1 Apr. 23, 2009

SSC window. Memory B cells (CD24+/CD27+) were subse (2TY) medium. The obtained bacterial suspension was quently separated from naive B cells (CD24+/CD27-) and divided over two 2TY agar plates supplemented with tetra memory T cells (CD24-/CD27+). In a next step, IgM cyclin, amplicillin and glucose. After overnight incubation of memory B cells (IgM+) were separated from switch memory the plates at 37°C., the colonies were scraped from the plates B cells (IgM-) using IgM expression. In this step IgM and used to prepare an enriched phage library, essentially as memory B cells and switch memory B cells were sorted in described by De Kruif et al. (1995a) and WO 02/103012. separate sample tubes. 1x10 to 1x10° cells of each popula Briefly, scraped bacteria were used to inoculate 2TY medium tion were collected in DMEM/50% FBS and after completion containing amplicillin, tetracycline and glucose and grown at of the sort they were each centrifuged at 400xg for 10 min a temperature of 37° C. to an OD 600 nm of 0.3. CT helper utes. The sorted IgM memory B cells were then used as phages were added and allowed to infect the bacteria after starting material for library construction according to the which the medium was changed to 2TY containing amplicil method described in Example 1, using primer HuCIgM in the lin, tetracycline and kanamycin. Incubation was continued first round amplification of heavy chain immunoglobulin overnight at 30°C. The next day, the bacteria were removed sequences. The various libraries obtained were named MEM from the 2TY medium by centrifugation after which the 05-M01, MEM-05-M02, MEM-05-M03, MEM-05-M04, phages in the medium were precipitated using polyethylene MEM-05-M05, MEM-05-M06, MEM-05-M07, MEM-05 glycol (PEG) 6000/NaCl. Finally, the phages were dissolved M08, MEM-05-M09 and MEM-05-M10. in 2 ml of PBS with 1% bovine serum albumin (BSA), filter sterilized and used for the next round of selection. Example 3 0118. Typically, two rounds of selections were performed before isolation of individual phageantibodies. Selection was Selection of Phages Carrying Single Chain Fv Frag carried out twice on the same strain of bacteria or different ments Specifically Binding to Staphylococci strains were used sequentially (see Table 8 for selection 0116. Antibody fragments were selected using antibody strains). After the second round of selection, individual E. coli phage display libraries, general phage display technology and colonies were used to prepare monoclonal phage antibodies. MAbstract(R) technology, essentially as described in U.S. Pat. Essentially, individual colonies were grown to log-phase in No. 6,265,150 and in WO 98/15833 (both of which are incor 96 well plate format and infected with CT helper phages after porated by reference herein). The antibody phage libraries which phage antibody production was allowed to proceed used were screened donor libraries prepared as described in overnight. The produced phage antibodies were PEG/NaCl Example 1, IgM memory libraries prepared as described in precipitated and filter-sterilized and tested in ELISA and/or Example 2 and a semi-synthetic scFv phage library (JK1994) FACS for binding to Staphylococcus prepared as described which has been described in de Kruif et al., 1995b. The Supra. methods and helper phages as described in WO 02/103012 (incorporated by reference herein) were used in the present Example 4 invention. For identifying phage antibodies recognizing sta Validation of the Staphylococci Specific Single phylococci, phage selection experiments were performed Chain Phage Antibodies using live bacteria in Suspension. The clinical isolates used for selection and screening are described in Table 8. The 0119 Selected single-chain phage antibodies that were isolates are different based on RFLP-typing. obtained in the screens described above were validated in 0117 Bacteria were grown overnight at 37° C. on blood FACS for specific staphylococcal binding activity, i.e. bind agar plates and scraped into RPMI buffer containing 1 mg/ml ing to one or more staphylococcal strain prepared as of Rabbit IgG and 1% BSA at a concentration of 5x10 described Supra but lacking binding to Enterococcus as mea bacteria/ml and incubated for 60 minutes at room tempera Sured by a FACS-based enterococcus binding assay. Phage ture. An aliquot of a phage library (approximately 10" cfu, antibodies were blocked with FACS buffer (20 mM HEPES amplified using CT helper phage (see WO 02/103012)) was buffer pH 7.5, 100 mMNaCl, 1% BSA) for 20 minutes on ice. blocked in blocking buffer (2% ELK in PBS) for 1-2 hours at For each staining 1x10 bacterial cells, scraped from blood room temperature. The blocked phage library was added to agar plates and washed in FACS buffer, were added to each the blocked bacterial Suspension making a total Volume of 1.5 eppendorf tube. The bacteria were blocked with FACS buffer ml and incubated for 2 hours at room temperature in an containing 15% human serum (Biowhittaker) for 30 minutes end-over-end rotor (5 rpm). The Suspension was centrifuged at room temperature. The bacteria were pelleted by centrifu at 6800xg for 3 minutes at room temperature and the super gation at 1700xg for 3 minutes at 4°C. and resuspended with natant was discarded. Bacteria were washed five times with the blocked phage antibodies and incubated for 1.5 hours on RPMI buffer containing 1% BSA and 0.05% v/v Tween-20, ice. The bacteria were then washed with FACS buffer and then five times with RPMI buffer containing 1% BSA to sequentially incubated with murine biotinylated anti-M13 remove unbound phages. Bound phages were eluted from the antibodies (RDI) followed by strepavidin-PE. The cells were antigenby incubation with 1 ml of 0.1 M triethylamine for 10 fixed in buffered 4% formaldehyde and analysed on a FACS minutes at room temperature in an end-over-end rotor (5 caliber. SC05-132 and SC05-133 (both selected from RAB rpm). The entire content of the tube was then mixed with 0.5 03-G01 on strain Cowan in Suspension) showed staining on ml of 1 M Tris-HCl pH 7.5 to neutralize the pH. This mixture all clinical isolates tested indicating that they recognise a was used to infect5 ml of an XL1-Blue E. coli culture that had pan-staphylococcal target. SC02-430 (selected from JK1994 been grown at 37°C. to an OD 600 nm of approximately 0.3. on Strain Cowan in Suspension) showed specific binding to The phages were allowed to infect the XL1-Blue bacteria for the staphylococcal strain Cowan (see Table 9). In further 30 minutes at 37°C. Then, the mixture was centrifuged for 10 selections the single-chain phage antibodies called SCO6 minutes at 3200 g at room temperature and the bacterial 166, SCO6-171, SCO6-176, SCO6-187, SCO6-193, SCO6 pellet was resuspended in 0.5 ml 2-trypton yeast extract 249, SCO6-273, SCO6-389, SCO6-403, SCO6-406, SCO6 US 2009/01 04204 A1 Apr. 23, 2009

410, SCO6-446, SCO6-450, SCO6-452, SCO6-453, SCO6 restriction sites and/or sequences for expression in the IgG 464, SCO6-471, SCO6-516, SCO6-517, SCO6-526, SCO6 expression vectors pSyn-C03-HCY1 (SEQ ID No:43) and 528, SCO6-531, SCO6-533, SCO6-536, SCO6-537, SC06 pSyn-C04-C (SEQ ID No:44). The heavy chain variable 538, SCO6-540, SCO6-544, SCO6-566, SCO6-625 were obtained. These antibodies bound at least one of the clinical region of SC02-430 was cloned into the vector pSyn-C03 isolates tested (see Table 9). SC06-166, SC06-171, SC06-176 HCY1; the light chain variable region of SC02-430 was cloned and SC06-187 were selected from immune libraries, while into the vector pSyn-C04-CV. The VL lambda gene was first the other phageantibodies were selected from IgM memory B amplified using the following oligonucleotides set, 5L-B cell libraries. (SEQID NO:45) and sy3L-A (SEQID NO:46) and the PCR 0120 To test for non-specific reactivity against non-bac product was cloned into vectorpSyn-C04-CV. The nucleotide terial antigens an ELISA assay was used. The complex anti sequence of the construct was verified according to standard gens 5% FBS, 2% ELK and 1% BSA were coated overnight techniques knownto the skilled artisan. The VH gene was first to MaxisorpTM ELISA plates. Selected single-chain phage amplified using the following oligonucleotide set: 5H-F antibodies were incubated for 15 minutes in an equal volume (SEQ ID NO:47) and sy3H-A (SEQ ID NO:48). Thereafter, of PBS containing 1% BSA to obtain blocked phage antibod the PCR product was cloned into vector pSyn-C03-HCY 1 and ies. The plates were emptied, and the blocked single-chain the nucleotide sequence was verified according to standard phage antibodies were added to the wells. Incubation was techniques known to the skilled person in the art. allowed to proceed for two hours at room temperature, the 0.125 Heavy and light chain variable regions of the scFv plates were washed in PBS containing 0.1% V/v Tween-20 called SC05-132, SC05-133, SCO6-166, SCO6-171, SCO6 and bound phage antibodies were detected by means of OD 176, SCO6-187, SCO6-193, SCO6-249, SCO6-273, SCO6 492 nm measurement using an anti-M13 antibody conjugated 389, SCO6-403, SCO6-406, SCO6-410, SCO6-446, SCO6 to peroxidase. As a control, the procedure was performed 450, SCO6-452, SCO6-453, SCO6-464, SCO6-471, SCO6 simultaneously without single-chain phage antibody, with a 516, SCO6-517, SCO6-526, SCO6-528, SCO6-531, SC06 negative control single-chain phageantibody directed against 533, SCO6-536, SCO6-537, SCO6-538, SCO6-540, SC06 West Nile virus envelope protein (SC04-374). As shown in 544, SCO6-566, SCO6-625 were cloned directly by restriction Table 10, the selected phage antibodies called SC02-430, digest for expression in the IgG expression vectors plg-C911 SC05-132 and SC05-133, did not display any detectable HCgammal (SEQ ID NO:49) and plg-C909-Ckappa (SEQ binding to the negative control antigens FBS, ELK and BSA. ID NO:50) or pig-C910-Clambda (SEQ ID NO:115). The heavy chain variable regions of the scFVs called SC05-132, Example 5 SC05-133, SC06-166, SC06-171, SCO6-176, SCO6-187, Characterization of the Staphylococci Specific scFvs SC06-193, SCO6-249, SCO6-273, SCO6-389, SCO6-403, 0121 From the selected specific single-chain phage anti SCO6-406, SCO6-410, SCO6-446, SCO6-450, SCO6-452, body (scFv) clones plasmid DNA was obtained and nucle SCO6-453, SCO6-464, SCO6-471, SCO6-516, SCO6-517, otide sequences were determined according to standard tech SC06-526, SCO6-528, SCO6-531, SCO6-533, SCO6-536, niques. The nucleotide sequences of the Sclvs (including SCO6-537, SCO6-538, SCO6-540, SCO6-544, SCO6-566 and restriction sites for cloning) called SC02-430, SC05-132, and SC06-625 were cloned into the vector plg-C911-HCgammal SC05-133 are shown in SEQID NO:19, SEQID NO:21 and by restriction digest using the enzymes Sfil and XhoI and the SEQ ID NO:23, respectively. The amino acid sequences of light chain variable regions of the schvs called SC05-132, the schvs called SCO2-430, SC05-132 and SC05-133 are SC05-133, SC06-166, SC06-171, SC06-176, SC06-187, shown in SEQID NO:20, SEQID NO:22 and SEQID NO:24, SC06-193, SCO6-249, SCO6-273, SCO6-389, SCO6-403, respectively. SCO6-406, SCO6-410, SCO6-446, SCO6-450, SCO6-452, 0122) The VH and VL gene identity (see Tomlinson I M, SCO6-453, SCO6-464, SCO6-471, SCO6-516, SCO6-517, Williams SC, Ignatovitch O. Corbett SJ, Winter G. VBASE SC06-526, SCO6-528, SCO6-531, SCO6-533, SCO6-536, Sequence Directory. Cambridge United Kingdom: MRC SCO6-537, SCO6-538, SCO6-540, SCO6-544, SCO6-566 and Centre for Protein Engineering (1997)) and the CDR SC06-625 were cloned into the vector plg-C909-Ckappa or sequences of the sclvs Specifically binding staphylococci are pIg-C910-Clambda by restriction digest using the enzymes depicted in Tables 11 and 12, respectively. SalI and NotI. Thereafter the nucleotide sequences were veri 0123. Similar to the single-chain phage antibodies dis fied according to standard techniques known to the person closed above, the nucleotide and amino acid sequence, VL skilled in the art. and VH gene identity and CDR sequences of the single-chain 0.126 The resulting expression plasmids pgG102 phage antibodies called SC06-166, SC06-171, SC06-176, 430C03, pgG105-132C911, pgG105-133C911, pgG106 SC06-187, SC06-193, SC06-249, SC06-273, SC06-389, 166C911, pgG106-171C911, pgG106-176C911, pgG106 SCO6-403, SCO6-406, SCO6-410, SCO6-446, SCO6-450, 187C911, pgG106-193C911, pgG106-249C911, pgG106 SCO6-452, SCO6-453, SCO6-464, SCO6-471, SCO6-516, 273C911, pgG106-389C911, pgG106-403C911, pgG106 SC06-517, SCO6-526, SCO6-528, SCO6-531, SCO6-533, 406C911, pgG106-410C911, pgG106-446C911, pgG106 SC06-536, SCO6-537, SCO6-538, SCO6-540, SCO6-544, 450C911, pgG106-452C911, pgG106-453C911, pgG106 SC06-566 and SCO6-625 were determined (data not shown). 464C911, pgG106-471C911, pgG106-516C911, pgG106 517C911, pgG106-526C911, pgG106-528C911, pgG106 Example 6 531C911, pgG106-533C911, pgG106-536C911, pgG106 Construction of Fully Human Immunoglobulin Mol 537C911, pgG106-538C911, pgG106-540C911, pgG106 ecules (Human Monoclonal Anti-Staphylococci 544C911, pgG106-566C911, and pgG106-625C911 Antibodies) from the Selected Anti-Staphylococci encoding the anti-staphylococci human IgG1 heavy chains Single Chain Fvs and pSyn-C04-V12, pgG105-132C909, pgG105-133C909, 0.124. The heavy and light chain variable region of SC02 pgG106-166C910, pgG106-171C910, pgG106-176C909, 430 was PCR-amplified using oligonucleotides to append pgG106-187C909, pgG106-193C910, pgG106-249C910, US 2009/01 04204 A1 Apr. 23, 2009 20 pgG106-273C910, pgG106-389C910, pgG106-403C910, The amino acid sequences of the light chain of antibodies pgG106-406C910, pgG106-410C910, pgG106-446C910, CR2430, CR5132, CR5133 CR6166, CR6171, CR6176, pgG106-450C910, pgG106-452C909, pgG106-453C909, CR6187, CR6193, CR6249, CR6273, CR6389, CR6403, pgG106–464C910, pgG106-471C910, pgG106-516C909, CR6406, CR6410, CR6446, CR6450, CR6452, CR6453, pgG106-517C910, pgG106-526C910, pgG106-528C910, CR6464, CR6471, CR6516, CR6517, CR6526, CR6528, pgG106-53 1C910, pgG106-533C909, pgG106-536C909, CR6531, CR6533, CR6536, CR6537, CR6538, CR6540, pgG106-537C910, pgG106-538C910, pgG106-540C910, CR6544, CR6566, and CR6625 are shown in SEQID NO:32, pgG106-544C910, pgG106-566C910, pgG106-625C910 SEQ ID NO:34, SEQID NO:36, SEQ ID NO:177, SEQID encoding the anti-staphylococci human Ig light chains were NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID transiently expressed in combination in 293T cells and super NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO.193, SEQ ID NO.195, SEQ ID natants containing human IgG1 antibodies were obtained. NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID The nucleotide sequences of the heavy chains of the antibod NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID ies called CR2430, CR5132, CR5133, CR6166, CR6171, NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID CR6176, CR6187, CR6193, CR6249, CR6273, CR6389, NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID CR6403, CR6406, CR6410, CR6446, CR6450, CR6452, NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID CR6453, CR6464, CR6471, CR6516, CR6517, CR6526, NO:227, SEQIDNO:229, SEQIDNO:231, SEQID NO:233 CR6528, CR6531, CR6533, CR6536, CR6537, CR6538, and SEQID NO:235, respectively. A person skilled in the art CR6540, CR6544, CR6566, and CR6625 are shown in SEQ can determine the variable regions of the heavy and light IDNO:25, SEQID NO:27, SEQID NO:29, SEQID NO:116, chains of the above antibodies and single chain phage anti SEQID NO:118, SEQID NO:120, SEQID NO:122, SEQID bodies by following Kabat et al. (1991) as described in NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID Sequences of Proteins of Immunological Interest, US Dept. NO: 130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID Health and Human Services, NIH, USA (fifth edition). A NO:136, SEQ ID NO: 138, SEQ ID NO:140, SEQ ID person skilled in the art can determine the CDR regions of the NO: 142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID heavy and light chains of the above antibodies and single NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID chain phage antibodies by following Kabat et al. (1991), NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID Chothia and Lesk (1987) or a combination of both. Alterna NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID tively, the variable and CDR regions can be determined using NO:166, SEQIDNO:168, SEQID NO:170, SEQID NO:172 the VBASE database, a database well known to persons and SEQID NO:174, respectively. The amino acid sequences skilled in the art of antibodies. Sequences of the antibodies of of the heavy chains of the antibodies called CR2430. the invention can be compared with immunoglobulin CR5132, CR5133, CR6166, CR6171, CR6176, CR6187, sequences in the VBASE database (see Tomlinson I M. Wil CR6193, CR6249, CR6273, CR6389, CR6403, CR6406, liams SC, Ignatovitch O. Corbett SJ, Winter G. VBASE CR6410, CR6446, CR6450, CR6452, CR6453, CR6464, Sequence Directory. Cambridge United Kingdom: MRC CR6471, CR6516, CR6517, CR6526, CR6528, CR6531, Centre for Protein Engineering (1997)) http://vbase.mrc-cpe. CR6533, CR6536, CR6537, CR6538, CR6540, CR6544, cam.ac.uk/: MRC Centre for Protein Engineering) and on the CR6566, and CR6625 are shown in SEQID NO:26, SEQ ID basis thereof variable regions and CDR regions can be deter NO:28, SEQID NO:30, SEQID NO:117, SEQID NO:119, mined. The variable regions of the some of the antibodies are SEQID NO:121, SEQID NO:123, SEQID NO:125, SEQID given in Table 13. Human anti-staphylococci IgG1 antibodies NO:127, SEQ ID NO.129, SEQ ID NO:131, SEQ ID were validated for their ability to bind to staphylococci by NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID FACS essentially as described for scFvs (see Table 14). The NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID negative control was an anti-West Nile virus antibody NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID (CR4374). Alternatively, batches of greater than 1 mg of each NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID antibody were produced and purified using standard proce NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID dures. NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173 and SEQ ID NO: 175, respectively. The nucleotide sequences of the light Example 7 chain of antibodies CR2430, CR5132, CR5133, CR6166, In Vitro Opsonic Phagocytic Activity of Staphylo CR6171, CR6176, CR6187, CR6193, CR6249, CR6273, coccal Specific IgGs as Measured by FACS CR6389, CR6403, CR6406, CR6410, CR6446, CR6450, CR6452, CR6453, CR6464, CR6471, CR6516, CR6517, I0127. The opsonic activity of anti-staphylococcal IgGs CR6526, CR6528, CR6531, CR6533, CR6536, CR6537, was measured in an opsonophagocytotic (OPA) assay using CR6538, CR6540, CR6544, CR6566, and CR6625 are shown freshly differentiated HL-60 cells. During the OPA assay in SEQID NO:31, SEQID NO:33, SEQID NO:35, SEQID fluorescent bacteria were mixed with differentiated HL-60 NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID cells and serially diluted IgGs. Bacteria were grown to sta NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID tionary or to logarithmic (log) phase prior to labelling. To NO:188, SEQ ID NO.190, SEQ ID NO:192, SEQ ID grow the bacteria to stationary phase different staphylococcal NO:194, SEQ ID NO:196, SEQ ID NO.198, SEQ ID isolates were incubated overnight on sheep blood agar plates NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID at 37°C. The bacteria were resuspended in 5 ml of bicarbon NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID ate buffer (0.1 M NaHCO, pH 8.0), harvested by centrifu NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID gation at 800xg for 10 minutes at room temperature and NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID diluted until a concentration of 2.9x10 bacteria/ml. Bacteria NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID that were grown until logarithmic phase were first cultured NO:230, SEQID NO:232 and SEQID NO:234, respectively. overnight in LB medium at 37° C., then the culture was US 2009/01 04204 A1 Apr. 23, 2009

diluted 10 times and grown for an additional 3 hours in LB that of non-differentiated cells. Samples were assayed with a medium at 37° C. Bacteria were harvested by centrifugation FACSCalibur immunocytometry system (Becton Dickinson at 800xg for 10 minutes and resuspended in bicarbonate and Co., Paramus, N.J.) and were analyzed with CELLOuest buffer washed until a concentration of 2.9x10 bacteria/ml software (version 1.2 for Apple system 7.1: Becton Dickin Fifty microliters of a 5,6-carboxyfluorescein, succinimidyl son). 7,000 gated HL-60 granulocytes were analyzed per ester solution (FAM-SE: Molecular Probes, Eugene, Oreg.); tube. FAM-SE was excited at a wavelength of 488 nm and the 10 mg/ml in dimethyl sulfoxide (Fisher Scientific Co., Fair FAM-SE fluorescence signal of gated viable HL-60 cells was Lawn, N.J.)) was added to 1 ml of 2.9x10 bacteria and the measured for each antibody dilution. IgGs were defined as mixture was incubated for 1 hour at 37°C. without shaking. positive in the phagocytic assay when concentration depen The labeled bacteria were washed three times in 20 ml dent phagocytosis could be observed greater or equal to two opSonophagocytosis buffer (Hanks balanced salt Solution times that of the control IgG. IgGs CR2430, CR5132 and with Ca" and Mg" and 0.2% bovine serum albumin), until CR5133 demonstrated opsonic activity against S. aureus no free dye in the supernatant was observed. FAM-SE-la strain Cowan in both the log (see FIG. 1) and stationary beled bacteria were resuspended in 8 ml OPA buffer and growth phase (see FIG. 2). The three IgGs where more effec stored in aliquots of 500 ulat -20°C. under protection from tive in enhancing phagocytic activity during the log phase of light. growth. IgGs CR5132 and CR5133 enhanced phagocytosis of 0128 HL-60 cells (human promyelocytic leukemia cells; S. aureus strain SA125 compared to the negative control ECACCNO 98070106) were grown in cell densities of 1-9x antibody (see FIG. 3) and antibody CR5133 significantly 10 cells/ml in RPMI 1640 medium containing 2 mM enhanced phagocytic activity of the differentiated HL60 cells L-glutamine supplemented with 10% heat-inactivated fetal against S. epidermidis strain SE131, when compared to the bovine serum (HyClone Laboratories, Logan, Utah) and negative control antibody (see FIG. 4). penicillin/streptomycin. Cells between passage 6 and 35 were used for differentiation. The cells were differentiated into Example 8 granulocytes by culturing in the same medium Supplemented Breadth of Staphylococci Specific IgG1 Binding with 5x107M all-trans-retinoic acid (Sigma), 6x10' M Activity Vitamin-D3 (Sigma) and 30 ng/ml human recombinant G-CSF (R&D). HL-60 cells were harvested by centrifugation 0.130. To determine the extent to which the targets of at 160xg for 10 minutes and washed twice in 15 ml of wash selected human anti-staphylococcal IgG1 antibodies were buffer (Hanks balanced salt solution, without Ca" and Mg", conserved on staphylococci and other gram positive bacteria containing 0.2% bovine serum albumin). The cells were FACS assays were carried out on a extended panel of clinical washed once in opsonophagocytosis buffer, resuspended in 4 bacterial isolates essentially as described before for schvs ml opsonophagocytosis buffer and counted in a hemocytom (see Table 15). From the assay was deducted that CR5132 and eter. The cell concentration was adjusted to 5x10° cells/ml. CR5133 bound to all Strains tested. CR5140 did bind all 0129. The anti-staphylococcal IgGs and a control IgG strains tested with the exception of S. hominis KV111, S. (CR4374) were serially diluted in opsonophagocytosis buffer warneri KV112, S. warneri KV114, S. epidermidis KV115, in a total Volume of 20 Jul to obtain dilutions having an IgG S. haemolyticus KV117, S. warneri vdé5, S. warneri vdé6, S. concentration of 2.50 ug/ml, 1.20 ug/ml, 0.60 ug/ml, 0.30 warneri vd732, S. hominis vd 136, S. hominis Vd 139, and S. ug/ml, 0.15 g/ml, 0.075 ug/ml, 0.0375 ug/ml and 0.019 hominis K136. CR6171 did bind all Strains tested with the ug/ml. Opsonic activity of dilutions was measured in the OPA exception of S. epidermidis KV110, S. hominis KV111, S. assay in a round bottom plate that was blocked with 1% BSA warneri KV112, S. saprophytocis KV113, S. warneri in PBS. As a control, the assay was performed with no IgG. A KV114, S. haemolyticus KV117, S. hominis KV118, S. 15ul aliquot of a bacterial suspension containing 5.4x10° haemolyticus K119, S. warneri vdé5, S. warneri vdé6, S. cells was added to each well of the plate. When a bacterial warneri vd732, S. hominis vd 136, S. hominis Vd 139, and S. Suspension from S. aureus Strain Cowan or S. epidermidis was hominis K136. Finally, CR6453 did bind all strains tested used, the IgG/bacterium suspension was first incubated for 30 with the exception of S. hominis Vd 136 and S. hominis K136. minutes at 37° C. while the plate was horizontally shaking I0131. In addition, using the same FACS based approach (1300 rpm) in a Heidolph titramax 1000. Next, 15 ul of the antibodies from the panel were demonstrated to bind to other differentiated HL-60 cells (total: 75x10 cells) were added to gram-positive bacteria. The antibodies CR5132 and CR6453 each well of the plate and the plate was incubated while were shown to bind Listeria monocytogenes, Bacillus cereus shaking at 37° C. for 30-45 minutes. The final volume in the and Streptococcus group A and CR5132 also bound to Pro well was 50 ul. The reaction was stopped by adding 50 ul of pionibacterium spp. The antibodies CR5133, CR5140 and wash buffer containing 4% v/v formaldehyde. The content in CR6171 were shown to bind Streptococcus group A and each well was resuspended and transferred to polystyrene CR5140 was also shown to bind Enterococcus faecalis (data disposable tubes for flow cytometric analysis. The samples not shown). were stored in the dark at 4°C. until analyzation. The tubes were vortexed for 3 seconds before sampling in the flow Example 9 cytometer. To control the differentiation of the HL-60 cells In Vitro Opsonic Phagocytic Activity of Staphylo the expression of the complement receptor CD11b was mea coccal Specific IgGs Measured by Opsonophago sured. Fc-receptors of differentiated and non-differentiated cytic Killing Assay (OPKA) cells were first blocked with rabbit IgG for 15 minutes on ice and the cells were subsequently labelled with CD11bAPC I0132) To better determine the functional activity of the (BD) for 15 minutes on ice. Cells were considered properly antibody panel an opsonophagocytic assay was conducted to differentiated when the mean fluorescent intensity (MFI) ana quantify the killing activity of anti-staphylococcal human lyzed was at least between 10- to 100-fold higher compared to IgG1 against the Staphylococcus aureus strains 502, Mn8 and US 2009/01 04204 A1 Apr. 23, 2009 22

Newman and Staphylococcus epidermidis strain M187. washing, 100 ul of anti-M13 HRP (1:5000 in 2%(v/v) ELKin Freshly drawn human blood (10 to 30 ml) was mixed with an PBS) was added per well and incubated for 60 minutes at equal volume of dextran-heparin buffer (4.5 g of dextran, room temperature. The wells were washed again and staining Sigma Chemical, St. Louis; 28.4 mg of heparin sodium in 500 was visualized by adding 100 ul OPD-solution to each well. ml of distilled water), and the mixture was incubated at 37°C. Reaction was stopped after 5-10 minutes by adding 50 ul 1 M for 1 hour. The upper layer containing the leukocytes was HSO to each well and OD measured at 492 nm. The experi collected by centrifugation, and hypotonic lysis of the remaining erythrocytes was accomplished by Suspension of ment was repeated twice with the entire panel of antibodies the cell pellet in 1% (w/v) NHC1. The leukocyte population and a control IgG1 CR4374. The results showed that the was subsequently washed in RPMI with 15% (v/v) fetal antibodies fell into five distinct groups. Group A consisted of bovine serum. Trypan blue staining and counting in a CR5132, CR5133, CR6187 and CR6453; Group B consisted hemocytometer were used to determine the concentration of of CR5140 and CR6171; Group C consisted of CR6176: live leukocytes, and the final leukocyte concentration was Group D consisted of CR6526; and Group E consisted of the adjusted to 2x10 cells/ml. The phagocytosis assay was per rest of the panel CR6166, CR6193, CR6249, CR6273, formed in duplicate with or without 100 ul of leukocyte CR6403, CR6406, CR6410, CR6446, CR6450, CR6452, suspension added to 100 ul of bacteria (concentration CR6464, CR6471, CR6516, CR6517, CR6528, CR6531, adjusted spectrophotometrically to 2x107 per ml and con CR6533, CR6536, CR6537, CR6538, CR6540, CR6544, firmed by viable counts), 100 ul of anti-staphylococcal CR6566, CR6625. The binding activity and functional activ human IgG1 diluted in RPMI, and 100 ul of baby rabbit ity of the antibodies was consistent with the grouping. complement. The reaction mixture was incubated on a rotor rack at 37° C. for 90 minutes; samples were taken at time 0 Example 11 and after 90 minutes, diluted in 1% Proteose Peptone (Difco Laboratories, Detroit, Mich.), and plated onto tryptic Soyagar Target Identification of IgG1 in Group A plates. The killing activity (%) of the antibodies was calcu lated as the mean number of CFU surviving in the sample I0135) To determine the binding target of the panel anti containing leukocytes Subtracted from the mean number of bodies, representatives of each of the groups determined CFU surviving in the sample without leukocytes, divided by above (within each group the most potent antibody based on the latter and amplified by 100. The killing activity of the opSonic activity was chosen) was incubated with LTA anti-staphylococcal human IgG1 was tested at two concen extracted from S. aureus in a solid phase ELISA (see Table trations 1250 and 12.5 ng/ml (see Table 16). 17). A solution of 1 ug/ml lipoteichoic acid (Sigma) in PBS 0133. The results show that antibodies CR5132, CR5133, was coated on wells overnight at room temperature. Plates CR6446, CR6453, and CR6566 have more than 20% killing were washed once with PBS and blocked with 400 ul2%(v/v) activity against S. epidermidis strain M187, even at a low ELK in PBS. A serial dilution of each anti-staphylococcal concentration of 12.5 ng/ml. IgG1 Supernatant and negative control Supernatant CR4374 and positive control anti-LTA murine mAb 12248 (Abeam) Example 10 was incubated per well for one hour at room temperature. Wells were washed five times with PBS and 100 ul of anti IgG1 Competition Assay human HRP (1/2000) or anti-mouse HRP (1/2000) diluted in 0134) To establish whether antibodies in the panel com PBSE was added and incubated for one hour at room tem peted for binding to the same target a competition ELISA was perature. Wells were visualized and read as above. The results developed. The S. epidermidis strain SE132 was streaked clearly demonstrate that CR5133 from group A binds onto a blood agar plate and incubated overnight at 37° C. strongly to LTA. The positive control murine monoclonal Colonies were scraped from the plate using 5 ml of 50 mM 12248 showed similar results. In contrast, none of the anti carbonate buffer (8 volumes of 0.2MNaCO, 17 volumes of bodies from the other groups nor the negative control anti 0.2 MNaHCO and 75 volumes of distilled water) and cen body showed significant reactivity with LTA. Antibodies trifuged for 3 minutes at 4000 rpm. The obtained pellet was CR5132 and CR6453 from Group A were consistently shown resuspended in 500 ul of carbonate buffer, centrifuged again to bind LTA, CR6187 however did not show binding reactiv and the pellet was resuspended in 500 ul carbonate buffer. ity to LTA (data not shown). This maybe due to a lower Cell density was determined by measuring OD600 of a dilu affinity of CR6187 compared to the other antibodies in the tion series of the bacteria. The S. epidermidis strain was group. diluted to a density of 5x10 cells/ml and 100 ul (5x10 cells) per well was coated overnight at 4° C. on Nunc-Immuno Example 12 Maxisorp F96 plates. After incubation, the wells were washed three times with PBS and blocked for one hour at room In Vitro Opsonic Phagocytic Activity of Staphylo temperature with 300 ul 2% (v/v) ELK in PBS per well. In coccal Specific IgGs Against Staphylococcus Epider separate tubes 25ul of each Schv-phage maxiprep (produced midis and Staphylococcus aureus Grown Under Dif as above) diluted to subsaturating levels (as determined by ferent Culture Conditions and Measured by ELISA above) was mixed with 25 ul blocking buffer (4% Opsonophagocytic Killing Assay (OPKA) (v/v) ELK/PBS) and 50 ul of IgG1 supernatant diluted to 10 ug/ml in PBS and incubated for 20 minutes on ice. After 0.136 To determine if the bacterial killing activity of the removing the blocking solution, 100 ul of the blocked phages most potent and non-competitive opsonophagocytic anti-sta and IgG1 mixture was added to each well and incubated for phylococcal IgG1 antibodies identified above is affected by one hour at room temperature. The wells were washed three different bacterial growth conditions, the opsonophagocytic times with PBS/0.01% (v/v) Tween and once with PBS. After assay described above was conducted against the Staphyllo US 2009/01 04204 A1 Apr. 23, 2009 23 coccus aureus strain Newman and Staphylococcus epidermi dis strain RP62A grown in different media and under different TABLE 1. conditions. LBA is immune serum taken from an infected patient and served as a positive control. The killing activity of Human lambda chain variable region primers the anti-staphylococcal human IgG1 was tested at five con (sense) . centrations 10,000, 300, 10, 0.3, 0.01 ng/ml or -5,-6.5, -8, Primer -9.5, -11 log g/ml against both staphylococcal strains Primer name nucleotide sequence SEQ NO either grown to mid logarithmic phase (FIG. 5 A, B) or to static phase (FIG. 5 G, H) or in medium consisting of 1% HuVL1A-Back 5'- CAGTCTGTGCTGACT SEO ID NO : 51 glucose (FIG.5 C, D) or 100% human plasma (FIG. 5 E, F). CAGCCACC-3' 0.137 The results show that the antibodies CR5133, HuVL1B-Back 5'- CAGTCTGTGYTGACG SEO NO: 52 CR6166, CR6171, CR6176 and CR6526 have robust CAGCCGCC-3' opSonophagocytic activity against the two staphylococcal strains under all the growth conditions tested. Importantly, HuVL1C-Back 5'- CAGTCTGTCGTGACG SEO ID NO : 53 they were significantly different from the negative control CAGCCGCC-3' antibody CR3009, which showed little or no activity. This HuVL2B-Back 5'-CAGTCTGCCCTGACT SEQ NO:54 Suggests that the targets of the antibody panel are stably CAGCC-3' expressed under a variety of bacterial growth conditions, a factor potentially important for therapeutic application where HuVL3A-Back s" -TCCTATGWGCTGACT SEQ NO:55 the target bacteria may be present in nutrient poor conditions. CAGCCACC-3' HuVL3B-Back 5'-TCTTCTGAGCTGACT SEO ID NO : 56 Example 13 CAGGACCC-3' In Vivo Protective Activity of Staphylococcal Spe- HuVL4B-Back 5'- CAGCYTGTGCTGACT SEO ID NO : 57 cific IgGs in a Lethal Staphylococcus Aureus Chal- CAATC-3' lenge Model HuVL5 - Back 5'- CAGGCTGTGCTGACT SEO NO:58 CAGCCGTC-3' 0138 A bacterial titration experiment in mice is carried out to determine the optimal inoculation dose to produce HuVL 6-Back 5'-AATTTTATGCTGACT SEO ID NO : 59 80%-100% lethality. Animals are inoculated i.p. with S. CAGCCCCA-3' aureus strains Mn3 at doses of 5x10 and 5x10'. Animals are r r observed for 5 days and survival is used as an endpoint. The HuVL7/8-Back Tito GACY SEQ ID NO: 60 dose that results in 0% survival after 5 days is chosen as the challenge dose for further experiments. HuVL9-Back 5'- CWGCCTGTGCTGACT SEO ID NO : 61 0.139. Using the dose determined above for the bacterial CAGCCMCC-3' inoculum, a set of challenge experiments is conducted to assess the protective activity of the panel of Staphylococcal HuVL1O-Back 5'- CAGGCAGGGCTGACT SEO NO: 62 binding mAb (CR5133, CR6166, CR6171, CR6176 and CAG-3 CR6526) that have demonstrated in vitroopsonic phagocytic activity. For each experiment, purified mAbs (one isotype control IgG1 and 5 test IgG1) are injected i.p. (0.5-1 ml in TABLE 2 PBS), at a dose of 15 mg/kg. 5 mAb are tested against S. aureus Min8. Human kappa chain variable region primers 0140. After 24 hours animals are inoculated i.p. with the S. SeeSee aureus strain at the inoculation dose determined above. Primer Immediately prior to inoculation, a small amount of blood Primer name nucleotide sequence SEQ NO (50-100 ml) is collected (using the tail cut method) to measure circulating antibody levels. The blood is kept at room tem- HuVK1B-Back SATCAGTGAcco SEO ID NO: 63 perature between 30 min and 2 hours, to allow the blood to clot, then centrifuged at 4°C. for 5 min. The serum is removed HuVK2-Back 5'-GATGTTGTGATGACTC SEO NO: 64 and stored at -20°C. A human IgG1 ELISA is performed on AGTCTCC-3' all blood samples prior to inoculation and after sacrifice. HuVK2B2 5'-GATATTGTGATGACCC SEO NO: 65 Animals with no measurable antibody in their blood prior to AGACTCC-3' inoculation are excluded from further analysis. 0141 Mice are observed daily for five days and sacrificed HuVK3B-Back SartorgaCRC SEQ NO: 66 when showing signs of severe distress. Survival is scored in each group at the end of five days. To validate each experi- HuVK5-Back 5' - GAAACGACACTCACGC SEQ NO: 67 ment there must be less than 20% survival in the negative AGTCTCC-3' control IgG1 group. Further experiments are carried out in the HuVK6-Back 5' - GAAATTGTGCTGACTC SEO NO: 68 model described above where the antibodies are titrated at AGTCTCC-3' half-log doses from 10 mg/kg to determine their protective potency in vivo.

US 2009/01 04204 A1 Apr. 23, 2009 26

TABLE 6- continued TABLE 7-continued Human IgG heavy c hain variable region primers Percentage of the different heavy chain products in the final mixture. extended with Sfil/NooI restriction sites (sense) and human IgG heavy chain J-region Sense primer Antisense primer Product Percentage prlmers extended with XhoI/BstEII restriction HuJH4.5-FOR-Xho H73 O.6% sites (anti-sense) . HuJH6-FOR-Xho H74 O.2%

Primer Primer name nucleotide sequence SEQ ID NO TABLE 8 HuJH3 - FOR-Xho 5'-GAGTCATTCTCGACT SEO ID NO : 112 CGAGACGGTGACCATTGT Staphylococcal clinical isolates used for selection and screening of anti CCC-3' staphylococcal single-chain (scFV) phage antibodies. ID Strain Hospital Code Site of Isolation HuJH4/5 - FOR-Xho 5'-GAGTCATTCTCGACT SEO ID NO : 113 CGAGACGGTGACCAGGGT Cowan S. auretis NA NA TCC-3 SA099 Satiretts D3 Anterior Nares SA100 S. attreus D8 Anterior Nares HuJH6 - FOR-Xho 5'-GAGTCATTCTCGACT SEO ID NO : 114 SA 8. t B Air N. geograac CGTGGT SA103 S. attreus D16 Anterior Nares SA104 Satiretts D17 Anterior Nares SA105 S. attreus D18 Anterior Nares SA108 S. attreus D2O Anterior Nares SA109 Satiretts D21 Anterior Nares TABLE 7 SA110 S. attreus D23 Anterior Nares SA111 S. attreus D26 Anterior Nares Percentage of the different heavy chain products in the final mixture. SA112 S. attreus D34 Anterior Nares SA113 S. attreus D43 Anterior Nares Sense primer Antisense primer Product Percentage SA114 Satiretts D44 Anterior Nares SA115 S. attreus Kv2 Renal Dialysis HuVH1B 7A-Back-Sf HuJH1.2-FOR-XhoIB H1.J1 2.5% SA116 S. airetts Kv3 Renal Dialysis HuVH1C-Back-Sf HuJH3-FOR-Xho H12 2.5% SA117 S. attreus KyS Blood HuJH4S-FOR-Xho H1.J3 15.0% SA118 S. attreus Kv6 Blood HuJH6-FOR-Xho H14 S.0% SA119 S. attreus Ky7 Blood HuVH2B-Back-Sf HJH1 2-FOR-XhoIB H21 O.2% SA120 S. attreus Kw8 Wound HuJH3-FOR-Xho H22 O.2% SA121 S. attreus Kw'9 Wound HuJH4S-FOR-Xho H23 1.2% SA122 S. attreus Kv11. Wound HuJH6-FOR-Xho H24 O.4% SA123 S. attreus Kv24 CSF HuVH3A-Back-Sf HJH1 2-FOR-XhoIB H31 2.5% SA124 S. attreus Ky2S CSF HuJH3-FOR-Xho H32 2.5% SA125 S. attreus Kv27 Lung Pleura HuJH4S-FOR-Xho H33 15.0% SA126 S. attreus Kv28 Lung Pleura HuJH6-FOR-Xho H34 S.0% SA127 Satiretts Kv30 Pericardiac HuVH3C-Back-Sf HuJH1 2-FOR-XhoIB H4J1 2.5% SA128 S. attreus Kv31 oint HuJH3-FOR-Xho H42 2.5% SA129 S. attreus Kv32 oint HuJH4S-FOR-Xho H43 15.0% SE130 S. epidermidis 587/29 Blood HuJH6-FOR-Xho H44 S.0% SE131 S. epidermidis 688,35 Blood HuVH4B-Back-Sf HJH1 2-FOR-XhoIB HS1 O.2% SE132 S. epidermidis 724,42 Blood HuJH3-FOR-Xho HS2 O.2% SE133 S. epidermidis 587 (Kv11.0) Unknown HuJH4S-FOR-Xho HSJ3 1.2% SE134 S. epidermidis V48 (Kv115) Unknown HuJH6-FOR-Xho HSJ4 O.4% SE135 S. epidermidis 354 (Kv118) Unknown HuVH4C-Back-Sf HJH1 2-FOR-XhoIB H61 2.0% SE136 S. epidermidis V16 Renal Dialysis HuJH3-FOR-Xho H62 2.0% SE137 S. epidermidis V29 Renal Dialysis HuJH4S-FOR-Xho H63 12.0% SE138 S. epidermidis V33 Renal Dialysis HuJH6-FOR-Xho H64 4.0% SE139 S. epidermidis V65 Renal Dialysis HuVH6A-Back-Sf HJH1 2-FOR-XhoIB HAJ1 O.1% SE140 S. epidermidis V75 Renal Dialysis HuJH3-FOR-Xho H72 O.1%

TABLE 9 Staphylococcal specific binding activity of single-chain (scFv) phage antibodies as measured by FACS. Name phage Staphylococcal Strains (90 positive antibody Cowan SA102 SA103 SA12O SA124 SA125 SE130 SA131 SA132

SCO2-430 89.0 ND 3O.O 13.0 ND ND ND ND ND SCOS-132 21.9 ND 82.7 86.5 ND 84.2 ND ND ND SCOS-133 48.2 ND 77.9 83.4 ND 76.2 ND ND ND sc06-166 31.2 51.4 48.1 ND 58.4 59.0 22.0 S3.3 43.2 ScO6-171 32.1 69.7 67.4 ND 71.7 71.2 S.O 39.3 29.2 US 2009/01 04204 A1 Apr. 23, 2009 27

TABLE 9-continued Staphylococcal specific binding activity of single-chain (ScFw) phage antibodies as measured by FACS. Name phage Staphylococcal Strains (90 positive antibody Cowan SA102 SA103 SA120 SA124 SA125 SE130 SA131 SA132

ScO6-176 30.1 11.7 30.1 ND 29.9 27.2 1.9 27.6 15.1 ScO6-187 24.5 72.5 65.5 ND 67.8 63.8 36.6 31.4 43.7 ScO6-193 12.O 27.7 37.2 ND S.O.3 56.2 2.9 7.0 8.9 sc06-249 10.4 ND ND ND ND ND ND ND 7.6 ScO6-273 S.1 10.1 33.2 ND 36.9 44.0 2.2 2.4 8.0 ScO6-389 7.3 12.9 35.7 ND 46.4 44.2 3.O 4.4 2.3 scO6-403 6.3 8.8 7.7 ND 10.4 11.5 0.7 5.4 2.7 scO6-406 6.8 14.7 28.5 ND 36.7 48.3 5.3 4.4 8.0 sc06-410 13.3 ND ND ND ND ND ND ND 8.1 scO6-446 9.5 16.9 4.6 ND 14.3 26.8 1.O 7.3 2.0 ScO6-450 46.7 61.1 58.4 ND 63.9 55.1 1.3 4.0 6.4 ScO6-452 9.6 ND ND ND ND ND 1.2 8.5 2.5 ScO6-453 410 26.2 33.6 ND 56.7 59.3 36.O 55.8 42.O scO6-464 20.4 33.2 9.6 ND 45.2 47.2 6.2 25.7 7.2 ScO6-471 2.1 53.5 46.0 ND 64.4 62.8 0.4 0.7 1.O ScO6-516 12.2 ND ND ND ND ND 3.7 22.3 1O.O ScO6-517 26.5 21.6 7.7 ND 24.4 24.9 12.4 4.3 13.8 ScO6-526 8.5 8.1 3.4 ND 15.7 16.3 3.6 6.7 6.3 ScO6-528 29.9 19.6 O.1 ND 31.3 28.4 15.5 7.6 24.3 ScO6-531 10.4 10.2 O.2 ND 15.6 12.O O.8 5.3 1.7 ScO6-533 15.7 3.9 8.6 ND 15.8 8.3 ND 6.0 O.8 ScO6-536 14.5 9.8 2.6 ND 20.1 10.9 2.O 7.5 3.1 ScO6-537 38.0 5.5 O.O ND 9.2 22.4 2.6 23.5 8.3 ScO6-538 14.3 6.2 9.6 ND 7.9 16.4 0.4 9.1 2.1 ScO6-540 9.3 7.3 O.S ND 22.7 23.4 O6 6.4 1.7 sc06-544 22.6 8.5 2.1 ND 7.6 17.2 1.6 13.8 11.7 ScO6-566 8.00 13.5 22.6 ND 37.1 39.4 1.O 13.4 1.7 ScO6-625 9.00 8.OO 5.4 ND 21.4 24.2 O.9 8.OO 1.9 Neg. Ctrl 13.2 1.5 2.5 ND 5.8 20.8 O.9 1.4 O.S

ND not determined

TABLE 10 TABLE 1 1-continued Non-specific binding activity of staphylococci reactive single-chain scFV) phage antibodies measured by ELISA at 492 nm. Data of the Staphylococcus specific single-chain Fvs. Negative controls SEQID NO SEQID NO of Name phage ELISA (OD492 mm Name of nucl. amino acid antibody BSA (1%) FBS (5%) ELK (2%) ScFw Sequence sequence VH-locus VL-locus

SCO2-430 O.04 O.04 O.OS SCOS-133 23 24 VH3 (3-11) VkIII (A27) SCOS-132 O.04 O.04 O.04 SCOS-133 O.04 O.04 O.04 (Vh 1-120; No phage O.04 O.04 O.04 VI 137-244) antibody Negative control O.04 O.O6 O16 between brackets the amino acids making up the heavy chain variable region (VH) and the light chain variable region (VL) is shown

TABLE 11 TABLE 12 Data of the Staphylococcus specific single-chain Fvs. Data of the CDR regions of the Staphylococcus Specific single-chain FVS. SEQID NO SEQ ID NO of Name of nucl. amino acid HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 ScFw Sequence sequence VH-locus VL-locus HCDR1 (SEQ (SEQ (SEQ (SEQ (SEQ (SEQID ID ID ID ID ID SCO2-430 19 2O VH4 (4-31) V12 (2b2) Name scFw NO:) NO:) NO:) NO:) NO:) NO:) (Vh 1-118; VI 134-242) SCO2-430 1 2 3 4 5 6 SCOS-132 21 22 VH3 (3-07) VkI (L12) SCOS-132 7 8 9 10 11 12 (Vh 1-118; SCOS-133 13 14 15 16 17 18 V1135-242) US 2009/01 04204 A1 Apr. 23, 2009

TABLE 13 TABLE 13-continued Data of the Staphylococcus specific IgGs. Data of the Staphviococcus specific IgGs. SEQ SEQ ID NO of SEQID NO of ID NO of SEQID NO of SEQID NO of amino acid: SEQID NO of amino acid* Name SEQIDnucl. sequence NO of aminosequence acid SEQIDnucl. sequence NO of sequenceamino acid light Name nucl. sequence Sequence nucl. sequence sequence light IgG h hai h hai li hai hai IgG heavy chain heavy chain light chain chain 9. eavy chain eavy chain ight chain Cl8ll CRS133 29 30 35 36 CR2430 25 26 31 32 (Vh 1-120) (VI 1-110) (Vh 1-118) (VI 1-109) CRS132 27 28 33 34 between brackets the amino acids making up the heavy chain variable (Vh 1-118) (VI 1-110) region (VH) and the light chain variable region (VL) is shown

TABLE 1.4 Staphylococcal specific binding activity of IgG1 molecules as measured by FACS. Name phage Staphylococcal strains (MFI antibody Cowan SA102 SA103 SA124 SA125 SE130 SA131 SA132

CR2430 2814 ND ND ND ND ND ND ND CRS132 1924 9.7 9.3 20.1 13.7 222.5 141.5 128.5 CRS133 285.8 ND ND ND ND 229.9 2O3.3 252.6 Neg. Ctrl 3.6 3.2 3.0 3.3 3.5 2.5 3.1 2.7

ND not determined

TABLE 1.5 Staphylococcal binding activity of IgG1 antibodies as measured by FACS. Isolation sitef IgG1 binding activity (MFI

Strain resistance Name Ctrl. CRS132 CRS133 CRS140 CR6171 CR6453

S. airetts CAPDND KVO1 4.OS 1064 8SO 756 2 S64 S. airetts CAPDND KVO2 16.63 919 558 433 47 552 S. airetts CAPDND KVO3 36.3 949 583 358 64 668 S. airetts CAPDND KVO4 1164 1.123 629 S46 97 752 S. airetts Blood ND KVOS 12.33 S64 652 447 34.2 525 S. airetts Blood ND KVO6 10.41 634 526 386 42.2 439 S. airetts Blood ND KVO7 21.04 881 705 441 68.4 614 S. airetts Wound ND KVO9 23.83 754 483 305 34.7 515 S. airetts Wound ND KV11 16.12 363 28O 226 O6.7 362 S. airetts Wound ND KV12 27.55 571 381 224 27.4 457 S. airetts Blood ND KV13 23.19 S76 403 278 4.1.8 503 S. airetts NAND Newman 8.01 655 430 384 53.1 387 S. airetts CAPDND KV1S 22.1 674 311 232 99.8 481 S. airetts CAPDND KV16 9.09 458 291 248 97.9 334 S. airetts CAPDND KV17 8.4 226 1845 1611 57.4 54.5 S. airetts CAPDND KV18 13.91 269 2O3 1662 62.4 58.7 S. airetts Blood ND KV19 2.66 190.9 1946 2O3 44.6 83.3 S. airetts Blood ND KV2O S.12 311 298 251 64.9 95 S. airetts Blood ND KV21 3.67 353 266 290 73.9 40 S. airetts Liquor/ND KV24 4:28 320.2 242 223 69.9 O2 S. airetts Liquor/ND KV25 3.37 269 219 1885 53.3 05.5 S. airetts Liquor/ND KV26 1O.O3 217 183.7 1629 38.6 86.4 S. airetts PleuraND KV27 4.03 348 235 239 S2.9 29.4 S. airetts PleuraND KV28 6.98 217.4 1846 2O3 46.7 74.1 S. airetts PleuraND KV29 2.99 1834 1826 147.9 38.5 10.2 S. airetts Pericard ND KV30 3.55 357 358 372 77.7 52.1 S. airetts JointND KV31 4.89 200 192.3 178.7 38.1 O6.5 S. airetts JointND KV33 5.88 222 232 177 58.5 744 S. airetts Wound ND KV34 7.45 286 199 1608 59.6 83.5 S. airetts Wound ND KV35 4.02 237 213 232 70.2 90.9 S. airetts Wound ND KV36 3.44 285 247 229 76.4 218 S. airetts Wound ND KV37 4.OS 217 215 212 42.6 25.5 S. airetts ND, MRSA KV38 6.1 920 642 1923 20.4 683 US 2009/01 04204 A1 Apr. 23, 2009 29

TABLE 15-continued Staphylococcal binding activity of IgG1 antibodies as measured by FACS. Isolation sitef IgG1 binding activity (MFI

Strain resistance Name Ctrl. CRS132 CRS133 CRS140 CR6171 CR6453

S. airetts ND, MRSA KV39 6.06 953 657 615 173 604 S. airetts ND, MRSA KV41 6.8 1038 854 732 226 739 S. airetts ND, MRSA KV42 12.41 1340 950 678 221 973 S. airetts ND, MRSA KV43 S.S.S 1084 711 48O 129.6 772 S. airetts Enterotoxin-f KV46 18.38 1144 607 247 79 776 ND S. airetts enterotoxin-f KV47 8.58. 809 513 353 102.1 436 ND S. airetts Blood pediatrict KV48 S.29 306 271 210 34.5 153 ND S. airetts Blood pediatrict KV49 6.53 747 S62 522 99.7 388 ND S. airetts Blood pediatric? KV50 15.86 939 539 397 117.8 864 ND S. airetts Blood pediatric? KV51 10.2S 818 68O 510 111.9 410 ND S. airetts NAND MW2 9.15 1080 1021 774 210 818 S. airetts NAND COL 19.62 471 S42 192 61.7 339 S. epidermi-dis NAND KV110 9.01 438 1221 499 7.04 1210 S. hominis NAND KV111 4.57 16.91 39.1 4.11 4.01 13.43 S. warneri NAND KV112 2.95 126.4 11.7 5.44 4.39 105.6 S. saprof. NAND KV113 6.35 186.2 17.34 136.6 9.16 118.8 S. warneri NAND KV114 8.67. 292 303 8.63 9.17 113.4 S. epidermi-dis NAND KV115 12.58 886 1577 11.76 90.2 369 S. haemolyti-cus NAND KV117 7.23 111.8 79.5 9.89 6.44 79.9 S. hominis NAND KV118 11 1334 2085 97.8 9.02 17SO S. haemolyti-cus NAND K119 16.71 816 888 103.9 11.71 371 S. warneri NAND wd.65 8.24 419 1922 S.O8 4.78 73.4 S. warneri NAND wd66 5.77 237 104.9 6.23 5.57 80.5 S. warneri NAND wd732 7.82 285 289 7.62 4.32 100.6 S. warneri NAND K7O6 4.21 214 225 14.62 10.3 68.7 S. hominis NAND wd136 4S4 25.4 815 7.37 4.13 6.4 S. hominis NAND wd139 5.64 90.3 211 5.47 4.4 133.7 S. hominis NAND K136 6.48 25.3 842 10.57 6.83 6.02

TABLE 16 TABLE 16-continued Staphylococcal killing activity of IgG1 Staphylococcal killing activity of IgG1 M abits 8S st by OPKA. 9. antibodies as measured by OPKA. Mean staphylococcal killing activity (%) Mean staphylococcal killing activity (%) Strain Strain 502 Min8 Newman M187 ng/ml 502 Min8 Newman M187 ng/ml IgG1 antibody 1250 12.5 1250 12.5 1250 12.5 1250 12.5 CRS132 83.9 43.2 85.0 37.3 70.4 47.5 80.9 64.O IgG1 antibody 1250 12.5 1250 12.5 1250 12.5 1250 12.5 CRS133 92.1 62.S 84.S 46.4 72.4 53.1 78.1 54.9 CR6166 71.6 35.1 S2.1 S.S. 64.8 35.1. 19.3 3.3 CR6516 85.9 49.4 68.1 36.1 59.9 23.2 8.5 3.9 CR6171 81.9 40.1 88.8 S2.7 62.8 39.9 29.O 14.7 CR6517 79.4 36.1 59.8 18.4 54.8 21.5 S.8 S.1 CR6176 78.4 38.2 70.7 31.9 74.3 SS.8 31.9 11.0 CR6526 88.8 S5.3 S1.1. 16.7 S6.S. 23.7 35.2 9.4 CR61.87 78.1 47.1 70.3 39.0 47.3 24.7 S.9 3.7 CR6528 89.6 47.0 49.0 16.4 55.7 27.O 64 1.8 CR6193 61.O 37.6 81.1 44.1 61.5 28.5 6.O -0.8 CR 6531 77.5 35.6 61.2 37.5 62.1 23.O 7.9 -0.7 CR6249 82.2 30.3 90.4 46.5 51.6 26.4 4.O 1.2 CR 6533 73.6 38.4 53.6 28.9 67.2 37.8 7.1 3.3 CR6273 91S 58.2 64.0 9.1 58.8 39.9 14.8 4.7 CR 6536 91.1 59.6 46.3 17.5 69.1 48.3 46 -1.4 CR6403 85.4 35.9 62.1 21.7 59.8 35.6 22.7 7.6 CR6537 70.3 28.9 69.1 21S 60.4 23.3 2.5 3.9 St. s 3. . 3. 3. i. CR 6538 64.9 22.6 63.9 15.2 66.3 35.2 3.3 2.0 CR6446 695 41.3 54.6 536 64.1 41.2 59. 48.6 CR 6540 92.6 53.O 63.9 16.4 61.1 38.2 8.9 4.4 CR6450 76.3 21.9 67.O 284 60.6 35.4 2.0 -0.7 CR 6544 79.8 28.8 S9.3 22.S 62.3 25.4 3.2 2.0 CR6452 83.9 30.6 91.6 41.3 S7.S. 36.O 7.9 2.6 CR6566 20.9 14.2 21.3 8.7 6.3 -1.6 54.3 30.4 CR6453 85.9 46.0 67.O 21.O 74.1 49.7 83.2 57.5 CR 6625 20.2 9.7 8.6 -0.8 51.O 23.3 43.8 19.1 CR6464 85.9 36.7 SSS 11.4 S7.2 30.7 6.8 1.4 Neg. Ctrl ND IND IND IND 4.O ND 4.5 O.O CR6471 96.O 68.2 44.2 7.1 62.6 34.7 8.0 O.O US 2009/01 04204 A1 Apr. 23, 2009 30

new cytofluorometric method using FITC and paraformal TABLE 17 dehyde. J Immunol. Methods 121:203-208. 0145 Chothia and Lesk (1987), Canonical structures for LTA binding activity of IgG1 antibodies as measured by ELISA. the hypervariable regions of immunoglobulins. J. Mol. ELISA binding to LTA (OD492 mm Biol. 196:901-917. 0146 Chou, TC and PTalalay (1984), Quantitative analy IgG1 10 3 1 O.3 O.1 O.O3 O.O1 sis of dose-effect relationships: the combined effects of CRS133 3.3 2.58 2.093 1.429 O.631 O.356 0.171 multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. CR61.66 0.052 O.OS1 O.OS1 O.049 O.OS4 O.OS2 O.O49 22:27-55. CR6171 O.133 0.127 0.121 O.116 0.091 O.O73 0.06S CR6176 O.O48 0.053 O.OS O.046 (0.046 (O.062 O. 111 0147 De Kruif J. Terstappen L. Boel E and Logtenberg T CR6526 O.O49 O.OS3 O.OS O.049 0.048 0.053 0.052 (1995a), Rapid selection of cell subpopulation-specific CR4374 0.093 0.099 0.084 O.O73 O.O7 O.O7 O.069 human monoclonal antibodies from a synthetic phageanti 12248 2.574 2.297 2.054 1.457 0.799 0.402 0.26 body library. Proc. Natl. Acad. Sci. USA 92:3938. PBS O.113 O.124 O.O98 O.O94 O.09 O.108 O.O94 0148 De Kruif J. Boel E and Logtenberg T (1995b), Selection and application of human single-chain FV anti body fragments from a semi-synthetic phage antibody dis REFERENCES play library with designed CDR3 regions. J. Mol. Biol. 0142 Boel E, Verlaan S, Poppelier MJ, Westerdaal NA. 248:97-105. Van Strijp J A and Logtenberg T (2000), Functional human 0149 Huls G, Heijnen I J. Cuomo E. van der Linden J. monoclonal antibodies of all isotypes constructed from Boel E. van de Winkel J and Logtenberg T (1999), Antitu phage display library-derived single-chain FV antibody mor immune effector mechanisms recruited by phage dis fragments. J. Immunol. Methods 239:153-166. play-derived fully human IgG1 and IgA1 monoclonal anti 0143 Burton DR and Barbas CF (1994), Human antibod bodies. Cancer Res. 59:5778-5784. ies from combinatorial libraries. Adv. Immunol. 57:191 (O150 Slootstra J. W. Puijk W. C. Ligtvoet GJ, Langeveld J 28O. P. Meloen R H (1996), Structural aspects of antibody 0144 Cantinieaux B, Hariga C, Courtoy P. Hupin J and antigen interaction revealed through Small random peptide Fondu P (1989) Staphylococcus aureus phagocytosis. A libraries. Mol. Divers. 1:87-96.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 235

SEO ID NO 1 LENGTH: 7 TYPE PRT ORGANISM: Homo sapiens <4 OO SEQUENCE: 1 Ser Gly Gly Tyr Tyr Trp Ser 1. 5

SEO ID NO 2 LENGTH: 16 TYPE PRT ORGANISM: Homo sapiens <4 OO SEQUENCE: 2 Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser Lieu Lys Ser 1. 5 1O 15

SEO ID NO 3 LENGTH: 8 TYPE PRT ORGANISM: Homo sapiens

<4 OO SEQUENCE: 3 Thr Val Met Asn Ser Phe Phe Asp 1. 5

<210 SEQ ID NO 4 <211 LENGTH: 14 US 2009/01 04204 A1 Apr. 23, 2009 31

- Continued

&212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 4 Thr Gly. Thir Ser Ser Asp Val Gly Ser Tyr Asn Lieu Val Ser 1. 5 1O

<210 SEQ ID NO 5 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 5 Glu Val Ser Lys Arg Pro Ser 1. 5

<210 SEQ ID NO 6 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 6 Cys Ser Tyr Ala Gly Ser Ser Trp Val 1. 5

<210 SEQ ID NO 7 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 7 Asn Tyr Trp Met Thr 1. 5

<210 SEQ ID NO 8 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 8 Asn. Ile Asin Arg Asp Gly Ser Asp Llys Tyr His Val Asp Ser Val Glu 1. 5 1O 15 Gly

<210 SEQ ID NO 9 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 9 Gly Gly Arg Thr Thr Ser Trp Tyr Trp Arg Asn 1. 5 1O

<210 SEQ ID NO 10 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 10 Arg Ala Ser Glin Ser Ile Ser Ser Trp Lieu Ala 1. 5 1O US 2009/01 04204 A1 Apr. 23, 2009 32

- Continued

<210 SEQ ID NO 11 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 11 Lys Ala Ser Ser Lieu. Glu Ser 1. 5

<210 SEQ ID NO 12 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 12 Gln Glin Tyr Asn Ser Tyr Pro Leu. Thr 1. 5

<210 SEQ ID NO 13 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 13 Asp Tyr Tyr Met Thr 1. 5

<210 SEQ ID NO 14 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 14 His Ile Ser Gly Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Arg 1. 5 1O 15 Gly

<210 SEQ ID NO 15 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 15 Gly Gly Arg Ala Thr Ser Tyr Tyr Trp Val His 1. 5 1O

<210 SEQ ID NO 16 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 16 Arg Ala Ser Glin Ser Val Ser Gly Tyr Lieu. Gly 1. 5 1O

<210 SEQ ID NO 17 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 17 US 2009/01 04204 A1 Apr. 23, 2009 33

- Continued Gly Ala Ser Ser Arg Ala Thr 1. 5

<210 SEQ ID NO 18 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 18 Gln Glin Tyr Gly Ser Ser Pro Leu. Thr 1. 5

<210 SEQ ID NO 19 <211 LENGTH: 726 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: SCO2-430

<4 OO SEQUENCE: 19 taggtgcagc tigcaggagtic gggcc cagga citggtgaagc Ctt cacagaC cctgtcc ct c 6 O acctgcactg. tct Ctggtgg Ct c catcagc agtggtggitt act actggag Ctggat.ccgg 12 O

Cagc.ccc.cag ggaagggact ggagtggatt ggg tacatct attacagtgg gag caccitac 18O tacaactic git coct caagag togagttaccatat cagtag acacgt.ctaa gaaccagttc 24 O t ccctgaagc tigagct ctgt gactg.ccg.cg gacacgg.ccg togt attact g togcaaagacg 3OO gttatgaatt C9ttctittga Ctgggggcaa ggit accctgg to accgt Ct c gagtggtgga 360 ggcggttcag gcggaggtgg Ctctggcggit ggcggat.cgg alaattgagct cacgcagcc.g 42O

C cct cogtgt Ctgggit ct co tacagtic at cac catct cctgcactgg alaccagcagt 48O gatgttggga gttata acct ttct Cotgg taccalacago acccaggcaa agcc.cccaaa 54 O Ct catgattt atgaggltdag taagcggc cc ticaggggttt Ctaatcgctt Ctctggct cc 6OO aagttctggca acacggcctic cctgacaatc. tctgggcticc aggctgagga Caggctgat 660 tatt actgct gct catatgc agg tagtagc tigggtgttcg gcggagggac Caagctgacc 72 O gtcc ta 726

<210 SEQ ID NO 2 O <211 LENGTH: 242 &212> TYPE: PRT <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: SCO2-430

<4 OO SEQUENCE: 2O Glin Val Glin Leu Gln Glu Ser Gly Pro Gly Lieu Val Llys Pro Ser Glin 1. 5 1O 15 Thr Lieu Ser Lieu. Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 2O 25 3O Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Lieu. Glu 35 4 O 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser SO 55 6 O Lieu Lys Ser Arg Val Thir Ile Ser Val Asp Thir Ser Lys Asn Glin Phe 65 70 7s 8O Ser Lieu Lys Lieu. Ser Ser Val Thir Ala Ala Asp Thr Ala Val Tyr Tyr US 2009/01 04204 A1 Apr. 23, 2009 34

- Continued

85 90 95 Cys Ala Lys Thr Val Met Asn Ser Phe Phe Asp Trp Gly Glin Gly Thr 1OO 105 11 O Lieu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 12 O 125 Gly Gly Gly Gly Ser Glu Ile Glu Lieu. Thr Gln Pro Pro Ser Val Ser 13 O 135 14 O Gly Ser Pro Gly Glin Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser 145 150 155 160 Asp Val Gly Ser Tyr Asn Lieu Val Ser Trp Tyr Glin Gln His Pro Gly 1.65 17O 17s Lys Ala Pro Llys Lieu Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly 18O 185 19 O Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn. Thir Ala Ser Lieu 195 2OO 2O5 Thir Ile Ser Gly Lieu. Glin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys 21 O 215 22O Ser Tyr Ala Gly Ser Ser Trp Val Phe Gly Gly Gly Thr Lys Lieu. Thr 225 23 O 235 24 O

Wall Lieu

<210 SEQ ID NO 21 <211 LENGTH: 726 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: SCOS-132

<4 OO SEQUENCE: 21 gaggtgctgg agtctggggg aggcttggtc. Cagc.cgggggggtCCCtgag actgtcCt9t 6 O t cagacitctg gattct cott taataact at tatgacct gggtc.cgc.ca ggctic.cgggg 12 O aaggggctgg agtgggtggc caa.catalaat Cagatggala gtgacaagta C catgtagac 18O tctgtggagg gcc.gatt cac catct coaga gacaact coa agaact cact atacctgcaa. 24 O atgaacaa.cc tagagc.cga cacgcggcg gtatatttitt gtgcgagagg C9gc.cggact 3OO actagotggt attggaga aa citgggggcag ggaac cctgg to accgt.ctic gagcigtacg 360 ggcggttcag gcggaaccgg cagcggcact ggcgggt ca C9gacat coa gatgaccCag 42O tctic ct tcca ccctgtctgc atctgtagga gacagagtca ccatcacttig ccgggc.cagt 48O cagagt atta gtagctggitt ggcctggitat cagcagaaac Cagggaaagc ccctaagctic 54 O

Ctgatctata aggcgt.ctag tittagaaagt ggggtcc cat Caaggttcag C9gcagtgga 6OO tctgggacag aatt cact ct cac catcago agcctgcago citgatgattt togcaactitat 660 tact.gc.caac agtataatag ttacCCCCtc actitt.cggcg gagggaccala gctggagat C 72 O aaacgt. 726

<210 SEQ ID NO 22 <211 LENGTH: 242 &212> TYPE: PRT <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: SCOS-132

<4 OO SEQUENCE: 22 US 2009/01 04204 A1 Apr. 23, 2009 35

- Continued

Glu Wall Luell Glu Ser Gly Gly Gly Lieu Wall Glin Pro Gly Gly Ser Luell 1O 15

Arg Luell Ser Cys Ser Asp Ser Gly Phe Ser Phe Asn Asn Tyr Trp Met 25

Thir Trp Wall Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Wall Ala Asn 35 4 O 45

Ile Asn Arg Asp Gly Ser Asp Llys Tyr His Val Asp Ser Wall Glu Gly SO 55 6 O

Arg Phe Thir Ile Ser Arg Asp Asn Ser Lys Asn Ser Lell Tyr Lieu. Glin 65 70 7s 8O

Met Asn Asn Lieu. Arg Ala Asp Asp Ala Ala Wall Phe Ala Arg 85 90 95

Gly Gly Arg Thir Thr Ser Trp Tyr Trp Arg Asn Trp Gly Glin Gly Thr 105 11 O

Lell Wall Thir Val Ser Ser Gly Thr Gly Gly Ser Gly Gly Thir Gly Ser 115 12 O 125

Gly Thir Gly Gly Ser Thr Asp Ile Gln Met Thr Glin Ser Pro Ser Thr 13 O 135 14 O

Lell Ser Ala Ser Val Gly Asp Arg Wall. Thir Ile Thir Arg Ala Ser 145 150 155 160

Glin Ser Ile Ser Ser Trp Lieu Ala Trp Tyr Glin Glin Pro Gly Lys 1.65 17O 17s

Ala Pro Lieu. Lieu. Ile Tyr Lys Ala Ser Ser Lell Glu Ser Gly Val 18O 185 19 O

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thir Lieu. Thir 195 2O5

Ile Ser Ser Lieu. Glin Pro Asp Asp Phe Ala Thr Tyr Glin Glin 21 O 215 22O

Tyr Asn Ser Tyr Pro Leu. Thr Phe Gly Gly Gly Thir Luell Glu Ile 225 23 O 235 24 O Lys Arg

SEQ ID NO 23 LENGTH: 732 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: SCO5-13 3

SEQUENCE: 23 gaggtgcagc tiggtggagac tgggggaggc ttggit Caagc CtggagggtC cctgagactic 6 O t cct gcticag cct ctagatt Cagct tcagg gac tact aca tgacgtggat cc.gc.caggct 12 O cCagggalagg ggc.cggaatg ggttt cacac atalagtggca gtggcagtac gatt tactac 18O gCagacitctg tgagggg.ccg att Caccatc. tcc agggaca acgc.caagag ctic ctitgitat 24 O

Ctgcaaatgg atagoctaca ggc.cgacgac acggc.cgitat attactgtgc gagagggggt 3OO cgc.gc.cacca gttact actg ggtcc actgg ggc.ccgggaa c cctdgt cac cgt.ct cagc 360 ggtacggg.cg gttcaggcgg alaccggcagc ggCactggcg aattgttgttg acgcagt citc cagccacc ct gtc.tttgtct cCaggggaaa gagccaccct CtcCtgcagg gccagt caga gtgttagcgg c tact taggc tgg taccaac agaalacctgg c caggct coc 54 O US 2009/01 04204 A1 Apr. 23, 2009 36

- Continued aggctic ct catctatggtgc atc.ca.gcagg gcc actggca toccaga cag gttcagtggc 6OO agtgggtctg ggacagacitt cactict cacc atcagc.cggc tiggagcctga agattittgca 660 gtgt attact gtcagoagta tigtagctica cc.gct cactt toggcggagg gaccalagctg 72 O gagatcaaac git 732

<210 SEQ ID NO 24 <211 LENGTH: 244 &212> TYPE: PRT <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: SCOS-133

<4 OO SEQUENCE: 24 Glu Val Glin Lieu Val Glu Thr Gly Gly Gly Lieu Val Llys Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ser Ala Ser Arg Phe Ser Phe Arg Asp Tyr 2O 25 3O Tyr Met Thr Trp Ile Arg Glin Ala Pro Gly Lys Gly Pro Glu Trp Val 35 4 O 45 Ser His Ile Ser Gly Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val SO 55 6 O Arg Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Ser Ser Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asp Ser Lieu. Glin Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Thir Ser Tyr Tyr Trp Val His Trp Gly Pro 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser Gly Thr Gly Gly Ser Gly Gly Thr 115 12 O 125 Gly Ser Gly Thr Gly Gly Ser Thr Glu Ile Val Lieu. Thr Glin Ser Pro 13 O 135 14 O Ala Thr Lieu. Ser Lieu. Ser Pro Gly Glu Arg Ala Thr Lieu. Ser Cys Arg 145 150 155 160 Ala Ser Glin Ser Val Ser Gly Tyr Lieu. Gly Trp Tyr Glin Gln Llys Pro 1.65 17O 17s Gly Glin Ala Pro Arg Lieu. Lieu. Ile Tyr Gly Ala Ser Ser Arg Ala Thr 18O 185 19 O Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 2OO 2O5 Lieu. Thir Ile Ser Arg Lieu. Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 21 O 215 22O Gln Glin Tyr Gly Ser Ser Pro Leu. Thr Phe Gly Gly Gly Thr Lys Lieu. 225 23 O 235 24 O Glu Ile Lys Arg

<210 SEQ ID NO 25 <211 LENGTH: 1344 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1344)

<4 OO SEQUENCE: 25

US 2009/01 04204 A1 Apr. 23, 2009 38

- Continued agc gtg Ct c acc gtg ctg. cac cag gac tog ctgaac ggc aag gag tac 96.O Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O aag to aag gtg agc aac aag gcc ctg cct gcc ccc atc gag aag acc OO8 Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 atc agc aag gCC aag ggc cag ccc cqg gag ccc cag gtg tac acc Ctg O56 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O

CCC cc C agc cgg gag gag atg acc aag aac cag gtg tcc ct c acc tit 104 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365

Ctg gtg aag ggc titc tac CCC agc gac at C goc gtg gag tig gag agc 152 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O aac ggc cag ccc gag aac aac tac aag acc acc C cc cct gtg ctg. gac 2OO Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO agc gaC ggc agc titc titc Ctg tac agc aag ctic acc gtg gaC aag agc 248 Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 cgg togg cag cag ggc aac gtg tt C agc tigc agc gtg atg cac gag gCC 296 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O ctg. cac aac cac tac acc cag aag agc ctg agc Ctg agc ccc ggc aag 344 Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44 O 445

<210 SEQ ID NO 26 <211 LENGTH: 448 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 26 Glin Val Glin Leu Gln Glu Ser Gly Pro Gly Lieu Val Llys Pro Ser Glin 1. 5 1O 15 Thr Lieu Ser Lieu. Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 2O 25 3O Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Lieu. Glu 35 4 O 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser SO 55 6 O Lieu Lys Ser Arg Val Thir Ile Ser Val Asp Thir Ser Lys Asn Glin Phe 65 70 7s 8O Ser Lieu Lys Lieu. Ser Ser Val Thir Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Lys Thr Val Met Asn Ser Phe Phe Asp Trp Gly Glin Gly Thr 1OO 105 11 O Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125 Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s US 2009/01 04204 A1 Apr. 23, 2009 39

- Continued

Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5 Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr 21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44 O 445

<210 SEQ ID NO 27 <211 LENGTH: 1344 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1344)

<4 OO SEQUENCE: 27 gag gtg Ctg gag tict gig gaggc titg gtC Cag CC9 gig gag to C Ctg 48 Glu Val Lieu. Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly Ser Lieu. 1. 5 1O 15 aga citg to c tdt to a gac tot gga titc. tcc titt aat aac tat togg atg 96 Arg Lieu. Ser Cys Ser Asp Ser Gly Phe Ser Phe Asn Asn Tyr Trp Met 2O 25 3O acc tig gtC C9C Cag gct CC9 g aag gig citg gag tig gtg gcc aac 144 Thir Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ala Asn 35 4 O 45 ata aat ca gat gga agt gac aag tac cat gta gac tot gtg gag ggc 192

US 2009/01 04204 A1 Apr. 23, 2009 41

- Continued Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365

Ctg gtg aag ggc titc tac CCC agc gac at C goc gtg gag tig gag agc 152 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O aac ggc cag ccc gag aac aac tac aag acc acc C cc cct gtg ctg. gac 2OO Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO agc gaC ggc agc titc titc Ctg tac agc aag ctic acc gtg gaC aag agc 248 Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 cgg togg cag cag ggc aac gtg tt C agc tigc agc gtg atg cac gag gCC 296 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O

Ctg cac aac cac tac acc cag aag agc ctg agc Ctg agc ccc ggc aag 344 Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44 O 445

<210 SEQ ID NO 28 <211 LENGTH: 448 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 28 Glu Val Lieu. Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly Ser Lieu. 1. 5 1O 15 Arg Lieu. Ser Cys Ser Asp Ser Gly Phe Ser Phe Asn Asn Tyr Trp Met 2O 25 3O Thir Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ala Asn 35 4 O 45 Ile Asin Arg Asp Gly Ser Asp Llys Tyr His Val Asp Ser Val Glu Gly SO 55 6 O Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Ser Lieu. Tyr Lieu. Glin 65 70 7s 8O Met Asn. Asn Lieu. Arg Ala Asp Asp Ala Ala Val Tyr Phe Cys Ala Arg 85 90 95 Gly Gly Arg Thr Thr Ser Trp Tyr Trp Arg Asn Trp Gly Glin Gly Thr 1OO 105 11 O Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 O 125 Lieu Ala Pro Ser Ser Lys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly 13 O 135 14 O Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val Lieu. Glin 1.65 17O 17s Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 18O 185 19 O Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser 195 2OO 2O5 Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr 21 O 215 22O His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser 225 23 O 235 24 O US 2009/01 04204 A1 Apr. 23, 2009 42

- Continued Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 26 O 265 27 O Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 27s 28O 285 Lys Thr Llys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 29 O 295 3 OO Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 3. OS 310 315 32O Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thr 3.25 330 335 Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 34 O 345 35. O Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 355 360 365 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 37 O 375 38O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 385 390 395 4 OO Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Lys Lieu. Thr Val Asp Llys Ser 4 OS 41O 415 Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 42O 425 43 O Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44 O 445

<210 SEQ ID NO 29 <211 LENGTH: 1350 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1350)

<4 OO SEQUENCE: 29 gag gtg cag Ctg gtg gag act gig gaggc titg gtC aag cct giga gig 48 Glu Val Glin Lieu Val Glu Thr Gly Gly Gly Lieu Val Llys Pro Gly Gly 1. 5 1O 15 tcc ctd aga ct c ticc togc tica gcc tot aga titc agc titc agg gac tac 96 Ser Lieu. Arg Lieu. Ser Cys Ser Ala Ser Arg Phe Ser Phe Arg Asp Tyr 2O 25 3O tac atg acg tdg atc cqC cag gct coa ggg aag ggg cc.g. gala tig gtt 144 Tyr Met Thr Trp Ile Arg Glin Ala Pro Gly Lys Gly Pro Glu Trp Val 35 4 O 45 tca cac at a agt ggc agt ggc agt acg att tac tac gca gac tot gtg 192 Ser His Ile Ser Gly Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val SO 55 6 O agg ggc cga ttic acc atc. tcc agg gac aac goc aag agc ticc titg tat 24 O Arg Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Ser Ser Lieu. Tyr 65 70 7s 8O

Ctg caa atg gat agc cta cag gcc gac gac acg gcc gta tat tac tit 288 Lieu. Glin Met Asp Ser Lieu. Glin Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga ggg ggit cqc gcc acc agit tac tac tdg gtc. cac togggc cc.g 336 Ala Arg Gly Gly Arg Ala Thir Ser Tyr Tyr Trp Val His Trp Gly Pro

US 2009/01 04204 A1 Apr. 23, 2009 44

- Continued

4 OS 41O 415 aag agc cgg tog Cag cag ggc aac gtg tt C agc tigc agc gtg atg cac 1296 Lys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His 42O 425 43 O gag gCC Ctg cac aac Cac tac acc cag aag agc Ctg agc ctg agc ccc 1344 Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 44 O 445 ggc aag 1350 Gly Lys 450

<210 SEQ ID NO 3 O <211 LENGTH: 450 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 30 Glu Val Glin Lieu Val Glu Thr Gly Gly Gly Lieu Val Llys Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ser Ala Ser Arg Phe Ser Phe Arg Asp Tyr 2O 25 3O Tyr Met Thr Trp Ile Arg Glin Ala Pro Gly Lys Gly Pro Glu Trp Val 35 4 O 45 Ser His Ile Ser Gly Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val SO 55 6 O Arg Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Ser Ser Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asp Ser Lieu. Glin Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Ala Thir Ser Tyr Tyr Trp Val His Trp Gly Pro 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 12 O 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 13 O 135 14 O Lieu. Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val 1.65 17O 17s Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro 18O 185 19 O Ser Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Lys 195 2OO 2O5 Pro Ser Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp 21 O 215 22O Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly 225 23 O 235 24 O Pro Ser Val Phe Lieu. Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 26 O 265 27 O Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 27s 28O 285 US 2009/01 04204 A1 Apr. 23, 2009 45

- Continued Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr Tyr Arg 29 O 295 3 OO Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys 3. OS 310 315 32O Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile Glu 3.25 330 335 Llys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr 34 O 345 35. O Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. 355 360 365 Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 37 O 375 38O Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 4 OO Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp 4 OS 41O 415 Lys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His 42O 425 43 O Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 44 O 445 Gly Lys 450

<210 SEQ ID NO 31 <211 LENGTH: 660 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) . . (660)

<4 OO SEQUENCE: 31 Cag to C gcc ctg acc cag C cc cqc to a gtg tct ggg tdt CCt gga cag 48 Gln Ser Ala Lieu. Thr Glin Pro Arg Ser Val Ser Gly Ser Pro Gly Glin 1. 5 1O 15 tcg at C acc at c toc to act gga acc agc agt gat gtt ggg agt tat 96 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 2O 25 3O aac citt gtc. tcc togg tac caa cag cac cca ggc aaa goc ccc aaa ct c 144 Asn Lieu Val Ser Trp Tyr Glin Gln His Pro Gly Lys Ala Pro Llys Lieu 35 4 O 45 atg att tat gag gtc. agt aag cig ccc to a ggg gtt t ct aat cqc ttic 192 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe SO 55 6 O tct ggc ticc aag tot ggc aac acg gcc toc ctd aca atc. tct ggg ct c 24 O Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Lieu. Thir Ile Ser Gly Lieu. 65 70 7s 8O

Cag gct gag gac gag gct gat tat tac tec tec tica tat gca ggit agt 288 Glin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90 95 agc tigg gtg tt C gga act ggc acc aag gtg acc gtg Ctg aag ctt acc 336 Ser Trp Val Phe Gly Thr Gly Thr Llys Val Thr Val Lieu Lys Lieu. Thr 1OO 105 11 O gtg Ctg ggc cag C cc aag gcc gct coc agc gtg acc Ctg tt C ccc ccc 384 Val Lieu. Gly Glin Pro Lys Ala Ala Pro Ser Val Thr Lieu Phe Pro Pro 115 12 O 125 US 2009/01 04204 A1 Apr. 23, 2009 46

- Continued tcc to C gag gag Ctg cag gcc aac aag gCC acc Ctg gtg tdc ct C at C 432 Ser Ser Glu Glu Lieu. Glin Ala Asn Lys Ala Thr Lieu Val Cys Lieu. Ile 13 O 135 14 O agc gaC tt C tac Cct ggc gcc gtg acc gtg gCC tig aag gCC gaC agc 48O Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser 145 150 155 160 agc ccc gtg aag gcc ggc gtg gag acc acc acc C cc agc aag cag agc 528 Ser Pro Val Lys Ala Gly Val Glu Thir Thr Thr Pro Ser Lys Glin Ser 1.65 17O 17s aac aac aag tac gcc gcc agc agc tac ctg agc ctic acc ccc gag cag 576 Asn Asn Llys Tyr Ala Ala Ser Ser Tyr Lieu. Ser Lieu. Thr Pro Glu Glin 18O 185 19 O tgg aag agc cac cq9 agc tac agc tigc cag gtg acc cac gag ggc agc 624 Trp Llys Ser His Arg Ser Tyr Ser Cys Glin Val Thr His Glu Gly Ser 195 2OO 2O5 acc gtg gag aag acc gtg gcc ccc acc gag tec agc 660 Thr Val Glu Lys Thr Val Ala Pro Thr Glu. Cys Ser 21 O 215 22O

<210 SEQ ID NO 32 <211 LENGTH: 220 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 32 Gln Ser Ala Lieu. Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Glin 1. 5 1O 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 2O 25 3O Asn Lieu Val Ser Trp Tyr Glin Gln His Pro Gly Lys Ala Pro Llys Lieu 35 4 O 45 Met Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe SO 55 6 O Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Lieu. Thir Ile Ser Gly Lieu. 65 70 7s 8O Glin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90 95 Ser Trp Val Phe Gly Thr Gly Thr Llys Val Thr Val Lieu Lys Lieu. Thr 1OO 105 11 O Val Lieu. Gly Glin Pro Lys Ala Ala Pro Ser Val Thr Lieu Phe Pro Pro 115 12 O 125 Ser Ser Glu Glu Lieu. Glin Ala Asn Lys Ala Thr Lieu Val Cys Lieu. Ile 13 O 135 14 O Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser 145 150 155 160 Ser Pro Val Lys Ala Gly Val Glu Thir Thr Thr Pro Ser Lys Glin Ser 1.65 17O 17s Asn Asn Llys Tyr Ala Ala Ser Ser Tyr Lieu. Ser Lieu. Thr Pro Glu Glin 18O 185 19 O Trp Llys Ser His Arg Ser Tyr Ser Cys Glin Val Thr His Glu Gly Ser 195 2OO 2O5 Thr Val Glu Lys Thr Val Ala Pro Thr Glu. Cys Ser 21 O 215 22O

<210 SEQ ID NO 33

US 2009/01 04204 A1 Apr. 23, 2009 48

- Continued Val Gly Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Ser Ile Ser 2O 25 3O Ser Trp Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys Lieu. 35 4 O 45 Lieu. Ile Tyr Lys Ala Ser Ser Lieu. Glu Ser Gly Val Pro Ser Arg Phe SO 55 6 O Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Lieu. Thir Ile Ser Ser Lieu. 65 70 7s 8O Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Tyr Asn Ser Tyr 85 90 95 Pro Lieu. Thir Phe Gly Gly Gly. Thir Lys Lieu. Glu Ile Lys Arg Ala Ala 1OO 105 11 O Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 12 O 125 Gly. Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu 13 O 135 14 O Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lieu. 1.65 17O 17s Ser Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val 18O 185 19 O Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val Thir Lys 195 2OO 2O5 Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210 SEQ ID NO 35 <211 LENGTH: 645 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) . . (645)

<4 OO SEQUENCE: 35 tcq acg gaa att gtg ttg acg cag tot coa goc acc ctd tot ttgtct 48 Ser Thr Glu Ilie Wall Lieu. Thir Glin Ser Pro Ala Thir Lieu. Ser Lieu. Ser 1. 5 1O 15 cca ggg gala aga gcc acc Ct c to C to agg gcc agt cag agt gtt agc 96 Pro Gly Glu Arg Ala Thr Lieu. Ser Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3O ggc tac tta ggc tigg tac Caa cag aaa cct ggC Cag gct coc agg ct c 144 Gly Tyr Lieu. Gly Trp Tyr Glin Glin Llys Pro Gly Glin Ala Pro Arg Lieu. 35 4 O 45

Ctc at C tat ggit gca t cc agc agg gcc act ggc atc cca gac agg tt C 192 Lieu. Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe SO 55 6 O agt ggc agt ggg tot ggg aca gac ttic act citc acc atc agc cgg Ctg 24 O Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Arg Lieu. 65 70 7s 8O gag cct gala gat titt gca gtg tat tac tt cag cag tat ggit agc tica 288 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin Tyr Gly Ser Ser 85 90 95 ccg Ct c act tt C ggc gga ggg acc aag ctg gag atc aaa cit gcg gCC 336 Pro Lieu. Thir Phe Gly Gly Gly. Thir Lys Lieu. Glu Ile Lys Arg Ala Ala US 2009/01 04204 A1 Apr. 23, 2009 49

- Continued

1OO 105 11 O gca CCC agc gtg titc atc ttic ccc ccc to C gac gag cag ctgaag agc 384 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 12 O 125 ggc acc gcc agc gtg gtg to ctg. Ctgaac aac titc tac ccc cqg gag 432 Gly. Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu 13 O 135 14 O gcc aag gtg cag tog aag gtg gaC aac gcc ctg. Cag agc ggc aac agc 48O Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser 145 150 155 160

Cag gag agc gtg acc gag cag gac agc aag gac toc acc tac agc ctg 528 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lieu. 1.65 17O 17s agc agc acc ct c acc Ctg agc aag gcc gac tac gag aag cac aag gtg 576 Ser Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val 18O 185 19 O tac gcc tic gag gtg acc cac cag ggc ctg agc agc ccc gtg acc aag 624 Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val Thir Lys 195 2OO 2O5 agc titc aac cqg ggc gag tt 645 Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210 SEQ ID NO 36 <211 LENGTH: 215 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 36

Ser Thr Glu Ilie Wall Lieu. Thir Glin Ser Pro Ala Thir Lieu. Ser Lieu. Ser 1. 5 1O 15 Pro Gly Glu Arg Ala Thr Lieu. Ser Cys Arg Ala Ser Glin Ser Val Ser 2O 25 3O Gly Tyr Lieu. Gly Trp Tyr Glin Glin Llys Pro Gly Glin Ala Pro Arg Lieu. 35 4 O 45 Lieu. Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe SO 55 6 O Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Arg Lieu. 65 70 7s 8O Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin Tyr Gly Ser Ser 85 90 95 Pro Lieu. Thir Phe Gly Gly Gly. Thir Lys Lieu. Glu Ile Lys Arg Ala Ala 1OO 105 11 O Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 12 O 125 Gly. Thir Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe Tyr Pro Arg Glu 13 O 135 14 O Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin Ser Gly Asn. Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lieu. 1.65 17O 17s Ser Ser Thr Lieu. Thir Lieu. Ser Lys Ala Asp Tyr Glu Lys His Llys Val 18O 185 19 O Tyr Ala Cys Glu Val Thr His Glin Gly Leu Ser Ser Pro Val Thir Lys 195 2OO 2O5 US 2009/01 04204 A1 Apr. 23, 2009 50

- Continued

Ser Phe Asn Arg Gly Glu. Cys 21 O 215

<210 SEQ ID NO 37 <211 LENGTH: 24 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Anti-sense primer HuCK-FOR <4 OO SEQUENCE: 37 acact citc cc ctdttgaagc tictt 24

<210 SEQ ID NO 38 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Anti-sense primer HuCL2-FOR <4 OO SEQUENCE: 38 tgaacattct gtaggggc.ca Ctg 23

<210 SEQ ID NO 39 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Anti-sense primer HuCL7- FOR <4 OO SEQUENCE: 39 agagcattct gcaggggc.ca Ctg 23

<210 SEQ ID NO 4 O <211 LENGTH: 4941 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Wector PDW- CO6

<4 OO SEQUENCE: 40 aagcttgcat gcaaatticta titt Caaggag acagt cataa tgaaatacct attgcctacg 6 O gcagcc.gctggattgttatt act coggcc Cagc.cggc.ca tgg.ccgaggt gtttgactaa 12 O tggggcgc.gc ct Cagggaac cctggtcacc gtc.tc.gagcg gtacggg.cgg ttcaggcgga 18O accggcagcg gCactggcgg gtcgacggaa attgttgctica cacagt citcc agccaccctg 24 O tctttgtc.t.c Caggggaaag agccaccctic t ccticaggg Ccagt cagag tgttagcagc 3OO tact taggct ggtaccaa.ca gaaac Ctggc caggctic cca ggctic ct cat ctatatgca 360 tccaac aggg C cactggcat CCC agc.cagg ttcagtggca gtgggtctgg gacagacttic actic to acca totagoagc ct agagcctgaa gattittgcag titt attactg t cagcagcgt. agcaactggc Ctcc.ggctitt cgg.cggaggg accalaggtgg agatcaaacg tgcggcc.gca 54 O catcatcatc accatcacgg ggcc.gcat at accatattg aaatgaaccg Cctgggcaaa gggg.ccgcat agactgttga aagttgttta gcaaaacctic atacagaaaa tt catt tact 660 aacgtctgga aagacgacaa aactittagat cgttacgcta act atgaggg Ctgtctgtgg 72 O aatgct acag gcgttgttggit ttgtactggit gacgaaactic agtgttacgg tacatgggitt

US 2009/01 04204 A1 Apr. 23, 2009 59

- Continued agtgct catc attggaaaac gttct tcggg gcgaaaactic ticaaggat.ct taccgctgtt 6 OOO gagatccagt togatgtaac ccact cqtgc acccaactga t ctitcagcat ctitt tactitt 6 O6 O

Caccagcgtt totgggtgag caaaaac agg aaggcaaaat gcc.gcaaaaa agggaataag 612 O gg.cgacacgg aaatgttgaa tact catact citt cotttitt caat attatt gaagcattta 618O t cagggittat tdt ct catga gcggatacat atttgaatgt atttagaaaa ataaacaaat 624 O aggggttc.cg cgcacatttic ccc.gaaaagt gcc acctgac gtc 6283

<210 SEQ ID NO 45 <211 LENGTH: 52 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide 5L-B

<4 OO SEQUENCE: 45 acctgtct cq agttitt coat ggcticagt cc gcc ct gaccc agcc.ccgctic ag 52

<210 SEQ ID NO 46 <211 LENGTH: 43 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide sy3L-A <4 OO SEQUENCE: 46 Ccagcacggit aagctt cagc acggt cacct ttgc.cagt t cc 43

<210 SEQ ID NO 47 <211 LENGTH: 50 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide 5H-F <4 OO SEQUENCE: 47 acctgtcttgaattct coat ggcc.caggtg cagctgcagg agt ccggcc.c SO

<210 SEQ ID NO 48 <211 LENGTH: 47 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Oligonucleotide sy3H-A

<4 OO SEQUENCE: 48 gccCttggtg Ctagdgctgg agacggtcac Cagggtgc.cc tdgcc cc 47

<210 SEQ ID NO 49 <211 LENGTH: 105.15 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Vector pig- C911 -HCGamma1 &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1326) . . (5076) &223> OTHER INFORMATION: Stuffer

<4 OO SEQUENCE: 49 tcgacggatc gggagatcto C catc.ccct atggtgcact ct cagtacaa tictgctctga 6 O

US 2009/01 04204 A1 Apr. 23, 2009 69

- Continued

<211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1C-Back

<4 OO SEQUENCE: 53

Cagt ctgtcg tacgcagcc gcc 23

<210 SEQ ID NO 54 <211 LENGTH: 2O &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L2B-Back

<4 OO SEQUENCE: 54 cagtctg.ccc tacticagoc

<210 SEQ ID NO 55 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L3A-Back

<4 OO SEQUENCE: 55 toctatowgc tigacticagoc acc 23

<210 SEQ ID NO 56 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L3B-Back

<4 OO SEQUENCE: 56 tottctgagc tigacticagga ccc 23

<210 SEQ ID NO 57 <211 LENGTH: 2O &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L4B-Back

<4 OO SEQUENCE: 57 cagoytgtgc tigact caatc

<210 SEQ ID NO 58 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJLS-Back

<4 OO SEQUENCE: 58

Caggctgtgc tigacticagoc gtc 23

<210 SEQ ID NO 59 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: US 2009/01 04204 A1 Apr. 23, 2009 70

- Continued

<223> OTHER INFORMATION: Primer Huw L6-Back

<4 OO SEQUENCE: 59 aattittatgc tigacticagoc cca 23

<210 SEQ ID NO 60 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuVL7/8-Back <4 OO SEQUENCE: 60

Cagrictgtgg tacycagga gcc 23

<210 SEQ ID NO 61 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L9-Back

<4 OO SEQUENCE: 61 cwgcctgtgc tigacticagoc mcc 23

<210 SEQ ID NO 62 <211 LENGTH: 18 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1O-Back

<4 OO SEQUENCE: 62

Caggcagggc tigacticag 18

<210 SEQ ID NO 63 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK1B-Back

<4 OO SEQUENCE: 63 gacatc.ca.gw tdacccagtic ticc 23

<210 SEQ ID NO 64 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK2-Back

<4 OO SEQUENCE: 64 gatgttgttga tigacticagtic ticc 23

<210 SEQ ID NO 65 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK2B2

<4 OO SEQUENCE: 65 US 2009/01 04204 A1 Apr. 23, 2009 71

- Continued gatattgttga tigacccagac toc 23

<210 SEQ ID NO 66 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK3B-Back

<4 OO SEQUENCE: 66 gaaattgttgw tdacrcagtic ticc 23

<210 SEQ ID NO 67 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJKS-Back

<4 OO SEQUENCE: 67 gaaacgacac toacgcagtic ticc 23

<210 SEQ ID NO 68 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK6-Back

<4 OO SEQUENCE: 68 gaaattgttgc tigacticagtic ticc 23

<210 SEQ ID NO 69 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K1B-Back-SAL

<4 OO SEQUENCE: 69 tgagcacaca ggtcgacgga catcCagWitg acc cagt cto c 41

<210 SEQ ID NO 70 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K2-Back-SAL

<4 OO SEQUENCE: 7 O tgagcacaca ggtcgacgga tigttgttgatg act cagt cto c 41

<210 SEQ ID NO 71 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K2B2- SAL

<4 OO SEQUENCE: 71 tgagcacaca ggtcgacgga tattgttgatg acccagactic C 41

<210 SEQ ID NO 72 US 2009/01 04204 A1 Apr. 23, 2009 72

- Continued

<211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K3B-Back-SAL

<4 OO SEQUENCE: 72 tgagcacaca ggtcgacgga aattgttgWitg acrcagt cto c 41

<210 SEQ ID NO 73 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K5-Back-SAL

<4 OO SEQUENCE: 73 tgagcacaca ggtcgacgga aacga cactic acgcagt cto c 41

<210 SEQ ID NO 74 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw K6-Back-SAL

<4 OO SEQUENCE: 74 tgag cacaca ggit cacgga aattgttgctg act cagt citc C 41

<210 SEQ ID NO 75 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK1-FOR-NOT

<4 OO SEQUENCE: 75 gagt cattct cqacttgcgg cc.gcacgttt gattt coacc ttggit coc 48

<210 SEQ ID NO 76 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK2- FOR-NOT

<4 OO SEQUENCE: 76 gagt cattct cacttgcgg cc.gcacgttt gat ct coagc titggit coc 48

<210 SEO ID NO 77 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJK3 - FOR-NOT

<4 OO SEQUENCE: 77 gagt cattct cqacttgcgg cc.gcacgttt gatat coact ttggit coc 48

<210 SEQ ID NO 78 <211 LENGTH: 47 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: US 2009/01 04204 A1 Apr. 23, 2009 73

- Continued

&223> OTHER INFORMATION: Primer HuJK4 - FOR-NOT

<4 OO SEQUENCE: 78 gagt cattct cqacttgcgg ccgacgtttg atctocacct tdgtc. cc 47

<210 SEQ ID NO 79 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJKS-FOR-NOT

<4 OO SEQUENCE: 79 gagt cattct cqacttgcgg cc.gcacgttt aatct coagt cqtgtc.cc 48

<210 SEQ ID NO 8O <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1A-Back-SAL

<4 OO SEQUENCE: 80 tgagcacaca ggtcgacgca gtctgtgctg act cago cac C 41

<210 SEQ ID NO 81 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1B-Back-SAL

<4 OO SEQUENCE: 81 tgagcacaca ggtcgacgca gtctgtgyitg acgcago.cgc C 41

<210 SEQ ID NO 82 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1C-Back-SAL

<4 OO SEQUENCE: 82 tgagcacaca ggtcgacgca gtctgtcgtg acgcago.cgc C 41

<210 SEQ ID NO 83 <211 LENGTH: 38 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L2B-Back-SAL

<4 OO SEQUENCE: 83 tgagcacaca ggtcgacgca gtctg.ccctg act cago C 38

<210 SEQ ID NO 84 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L3A-Back-SAL

<4 OO SEQUENCE: 84 US 2009/01 04204 A1 Apr. 23, 2009 74

- Continued tgagcacaca ggtcgacgt.c ctatgWgctg act cago cac C 41

<210 SEQ ID NO 85 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L3B-Back-SAL

<4 OO SEQUENCE: 85 tgagcacaca ggtcgacgt.c ttctgagctg act caggacc C 41

<210 SEQ ID NO 86 <211 LENGTH: 38 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L4B-Back-SAL

<4 OO SEQUENCE: 86 tgagcacaca ggtcgacgca gCytgtgctg act caatc 38

<210 SEQ ID NO 87 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L5-Back-SAL

<4 OO SEQUENCE: 87 tgagcacaca ggtcgacgca ggctgtgctg act cago.cgt C 41

<210 SEQ ID NO 88 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L6-Back-SAL

<4 OO SEQUENCE: 88 tgagcacaca ggtcgacgaa ttt tatgctg act cago Ccc a 41

<210 SEQ ID NO 89 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuVL7/8-Back-SAL

<4 OO SEQUENCE: 89 tgagcacaca ggtcgacgca grotgtggtg acycaggagc C 41

<210 SEQ ID NO 90 <211 LENGTH: 41 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L9-Back-SAL

<4 OO SEQUENCE: 9 O tgagcacaca ggtcgacgcW gcc titgctg act cago Cmc C 41

<210 SEQ ID NO 91 US 2009/01 04204 A1 Apr. 23, 2009 75

- Continued

<211 LENGTH: 36 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer Huw L1O-Back-SAL

<4 OO SEQUENCE: 91 tgagcacaca ggtcgacgca ggcagggctg act cag 36

<210 SEQ ID NO 92 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJIL1-FOR-NOT

<4 OO SEQUENCE: 92 gagt cattct cacttgcgg cc.gcacctag gacggtgacc ttggit coc 48

<210 SEQ ID NO 93 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuJIL2/3 - FOR-NOT <4 OO SEQUENCE: 93 gagt catt ct cacttgcgg cc.gcacctag gacggtcago ttggit coc 48

<210 SEQ ID NO 94 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJIF-FOR-NOT

<4 OO SEQUENCE: 94 gagt cattct cacttgcgg cc.gcaccgag gacggtcago toggtgcc 48

<210 SEQ ID NO 95 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuVH1B/7A-Back

<4 OO SEQUENCE: 95 Cagrtgcagc tiggtgcartc td 23

<210 SEQ ID NO 96 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH1C-Back

<4 OO SEQUENCE: 96 Sagg to cago togtrc agtic tig 23

<210 SEQ ID NO 97 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: US 2009/01 04204 A1 Apr. 23, 2009 76

- Continued

&223> OTHER INFORMATION: Primer HuJH2B-Back

<4 OO SEQUENCE: 97 Cagrtc acct talaggagtic tig 23

<210 SEQ ID NO 98 <211 LENGTH: 18 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuwH3A-Back

<4 OO SEQUENCE: 98 gaggtgcagc tiggtggag 18

<210 SEQ ID NO 99 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH3C-Back

<4 OO SEQUENCE: 99 gaggtgcagc tiggtggagWC ygg 23

<210 SEQ ID NO 100 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH4B-Back

<4 OO SEQUENCE: 1.OO Caggtgcagc tacagcagtgggg 23

<210 SEQ ID NO 101 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH4C-Back

<4 OO SEQUENCE: 101 Cagstgcagc tigcaggagtic Sgg 23

<210 SEQ ID NO 102 <211 LENGTH: 23 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuwH6A-Back

<4 OO SEQUENCE: 102 Cagg tacagc tigcagcagtic agg 23

<210 SEQ ID NO 103 <211 LENGTH: 56 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuVH1B/7A-Back-Sfi

<4 OO SEQUENCE: 103

US 2009/01 04204 A1 Apr. 23, 2009 78

- Continued

<211 LENGTH: 56 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuwH6A-Back-Sfi

<4 OO SEQUENCE: 110 gtcCtc.gcaa citgcggcc.ca gcc.ggc.catg gcc Cagg tac agctgcagca gtcagg 56

<210 SEQ ID NO 111 <211 LENGTH: 36 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuJH1/2-FOR-XhoIB <4 OO SEQUENCE: 111 gagt cattct cactic gaga crgtgaccag ggtgcc 36

<210 SEQ ID NO 112 <211 LENGTH: 36 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH3 - FOR-Xho

<4 OO SEQUENCE: 112 gagt catt ct cactic gaga C9gtgacc at tdt CCC 36

<210 SEQ ID NO 113 <211 LENGTH: 36 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Primer HuJH4/5 - FOR-Xho

<4 OO SEQUENCE: 113 gagt cattct cactic gaga C9gtgaccag ggttcC 36

<210 SEQ ID NO 114 <211 LENGTH: 36 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer HuJH6 - FOR-Xho

<4 OO SEQUENCE: 114 gagt cattct cactic gaga C9gtgaccgt ggit coc 36

<210 SEQ ID NO 115 <211 LENGTH: 8792 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Vector plig- C910 - Clambda &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1330) . . (3869) &223> OTHER INFORMATION: Stuffer

<4 OO SEQUENCE: 115 tcgacggatc gggagatcto C catc.ccct atggtgcact ct cagtacaa tictgctctga 6 O tgcc.gcatag ttaa.gc.cagt atctgctic cc ticttgttgttgttggaggtog Ctgagtagt 12 O

US 2009/01 04204 A1 Apr. 23, 2009 84

- Continued aag gag tac aag tic aag gtg agc aac aag gCC Ctg cct gcc cc C at C OO8 Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile 3.25 330 335 gag aag acc at C agc aag gcc aag ggC cag ccc cqg gag ccc cag gtg O56 Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 34 O 345 35. O tac acc ctg. CCC ccc agc cqg gag gag atg acc aag aac Cag gtg to C 104 Tyr Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser 355 360 365

Ct c acc tit ctg gtg aag ggc titc tac cc C agc gac atc gcc gtg gag 152 Lieu. Thir Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 37 O 375 38O tgg gag agc aac ggc cag C cc gag aac aac tac aag acc acc ccc cct 2OO Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 4 OO gtg Ctg gac agc gac ggc agc titc ttic ctg tac agc aag ct c acc gtg 248 Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 4 OS 41O 415 gac aag agc cgg tog Cag cag ggc aac gtg titc agc tigc agc gtg atg 296 Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 42O 425 43 O cac gag gCC ctg. cac aac Cac tac acc cag aag agc ctg agc ctg agc 344 His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 435 44 O 445

CCC ggc aag 353 Pro Gly Lys 450

<210 SEQ ID NO 117 <211 LENGTH: 451 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 117 Glin Val Glin Lieu Val Glin Ser Gly Ala Glu Val Llys Llys Pro Gly Glu 1. 5 1O 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 2O 25 3O Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Lieu. Glu Trp Met 35 4 O 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe SO 55 6 O Gln Gly Glin Val Thr Ile Ser Ala Asp Llys Ser Ile Ser Thr Ala Tyr 65 70 7s 8O Lieu Gln Trp Ser Ser Lieu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Ala Ser Ile Val Gly Ala Thr His Phe Asp Tyr Trp Gly 1OO 105 11 O Gln Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 12 O 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 13 O 135 14 O Ala Leu Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala US 2009/01 04204 A1 Apr. 23, 2009 85

- Continued

1.65 17O 17s Val Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val 18O 185 19 O Pro Ser Ser Ser Lieu. Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His 195 2OO 2O5 Llys Pro Ser Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys 21 O 215 22O Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly 225 23 O 235 24 O Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 26 O 265 27 O Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 27s 28O 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 29 O 295 3 OO Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly 3. OS 310 315 32O Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile 3.25 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 34 O 345 35. O Tyr Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser 355 360 365 Lieu. Thir Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 37 O 375 38O Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 4 OO Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 4 OS 41O 415 Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 42O 425 43 O His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 435 44 O 445 Pro Gly Lys 450

<210 SEQ ID NO 118 <211 LENGTH: 1353 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1353)

<4 OO SEQUENCE: 118 gag gtg cag Ctg gtg gag act gig ga gtC gcg gtC Cag cct gig agg 48 Glu Val Glin Lieu Val Glu Thr Gly Gly Val Ala Val Glin Pro Gly Arg 1. 5 1O 15 tcc ctd aga ct c ticc tdt gcg gog tot gga titc agt titc aga gat tat 96 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Asp Tyr 2O 25 3O ggc atg cac td gtC C9C Cag gct gca gC aag gigg Ctg gag tig gtg 144

US 2009/01 04204 A1 Apr. 23, 2009 87

- Continued Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 34 O 345 35. O tac acc ctg. CCC ccc agc cqg gag gag atg acc aag aac Cag gtg to C 104 Tyr Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser 355 360 365

Ct c acc tit ctg gtg aag ggc titc tac cc C agc gac atc gcc gtg gag 152 Lieu. Thir Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 37 O 375 38O tgg gag agc aac ggc cag C cc gag aac aac tac aag acc acc ccc cct 2OO Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 4 OO gtg Ctg gac agc gac ggc agc titc ttic ctg tac agc aag ct c acc gtg 248 Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 4 OS 41O 415 gac aag agc cgg tog Cag cag ggc aac gtg titc agc tigc agc gtg atg 296 Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 42O 425 43 O cac gag gCC ctg. cac aac Cac tac acc cag aag agc ctg agc ctg agc 344 His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 435 44 O 445

CCC ggc aag 353 Pro Gly Lys 450

<210 SEQ ID NO 119 <211 LENGTH: 451 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 119 Glu Val Glin Lieu Val Glu Thr Gly Gly Val Ala Val Glin Pro Gly Arg 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Asp Tyr 2O 25 3O Gly Met His Trp Val Arg Glin Ala Ala Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Phe Ile Trp Pro His Gly Val Asn Arg Phe Tyr Ala Asp Ser Met SO 55 6 O Glu Gly Arg Phe Thir Ile Ser Arg Asp Asp Ser Lys Asn Met Lieu. Tyr 65 70 7s 8O Lieu. Glu Met Asn. Asn Lieu. Arg Thr Glu Asp Thr Ala Lieu. Tyr Tyr Cys 85 90 95 Thir Arg Asp Glin Asp Tyr Val Pro Arg Llys Tyr Phe Asp Lieu. Trp Gly 1OO 105 11 O Arg Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 12 O 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 13 O 135 14 O Ala Leu Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala 1.65 17O 17s Val Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val 18O 185 19 O Pro Ser Ser Ser Lieu. Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His US 2009/01 04204 A1 Apr. 23, 2009 88

- Continued

195 2OO 2O5 Llys Pro Ser Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys 21 O 215 22O Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly 225 23 O 235 24 O Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 26 O 265 27 O Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 27s 28O 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 29 O 295 3 OO Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly 3. OS 310 315 32O Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile 3.25 330 335 Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 34 O 345 35. O Tyr Thr Lieu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Glin Val Ser 355 360 365 Lieu. Thir Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu. 37 O 375 38O Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 4 OO Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 4 OS 41O 415 Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 42O 425 43 O His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 435 44 O 445 Pro Gly Lys 450

<210 SEQ ID NO 120 &2 11s LENGTH: 1377 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1377)

<4 OO SEQUENCE: 120 Cag gtg cag ctg. Cag gag ticg ggc ccg aga ctg gtg aag cct tcg gag 48 Glin Val Glin Lieu. Glin Glu Ser Gly Pro Arg Lieu Val Llys Pro Ser Glu 1. 5 1O 15 acc ctd to c ct c act tcc aat gtc. tct gat gac toc atc acg agt tat 96 Thr Lieu Ser Lieu. Thr Cys Asn Val Ser Asp Asp Ser Ile Thr Ser Tyr 2O 25 3O ggit tac tat tig ggc tigg atc. c9c cag ccc cca ggg gag gCa ctg gag 144 Gly Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Glu Ala Leu Glu 35 4 O 45 tgg att ggc aat gtc titt tac agt ggc atg got tat tac aac cc.g. tcc 192 Trp Ile Gly Asn Val Phe Tyr Ser Gly Met Ala Tyr Tyr Asn Pro Ser SO 55 6 O

US 2009/01 04204 A1 Apr. 23, 2009 90

- Continued gag atg acc aag aac Cag gtg to C Ct c acc tdt Ctg gtg aag ggc titc 152 Glu Met Thr Lys Asn Glin Val Ser Lieu. Thr Cys Lieu Val Lys Gly Phe 37 O 375 38O tac cc C agc gac atc gcc gtg gag togg gag agc aac ggc cag ccc gag 2OO Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Glin Pro Glu 385 390 395 4 OO aac aac tac aag acc acc CCC cct gtg ctg gac agc gac ggc agc titc 248 Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp Ser Asp Gly Ser Phe 4 OS 41O 415 ttic ctg tac agc aag Ct c acc gtg gac aag agc cq9 tdg cag cag ggc 296 Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp Llys Ser Arg Trp Glin Glin Gly 42O 425 43 O aac gtg tt C agc tigc agc gtg atg cac gag gCC Ctg cac aac cac tac 344 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lieu. His Asn His Tyr 435 44 O 445 acc cag aag agc Ctg agc Ctg agc ccc ggc aag 377 Thr Gln Lys Ser Lieu. Ser Leu Ser Pro Gly Lys 450 45.5

<210 SEQ ID NO 121 <211 LENGTH: 459 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 121 Glin Val Glin Lieu. Glin Glu Ser Gly Pro Arg Lieu Val Llys Pro Ser Glu 1. 5 1O 15 Thr Lieu Ser Lieu. Thr Cys Asn Val Ser Asp Asp Ser Ile Thr Ser Tyr 2O 25 3O Gly Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Glu Ala Leu Glu 35 4 O 45 Trp Ile Gly Asn Val Phe Tyr Ser Gly Met Ala Tyr Tyr Asn Pro Ser SO 55 6 O Lieu Lys Ser Arg Val Thir Ile Lieu. Ile Asp Thir Ser Llys Lys Glin Phe 65 70 7s 8O Ser Lieu. Arg Lieu. Asn. Ser Val Thir Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Val Pro Phe Leu Met Phe Arg Val Lys Ile Val Glin Gly 1OO 105 11 O Thr Gly Ala Phe Asp Ile Trp Gly Glin Gly Thr Met Val Thr Val Ser 115 12 O 125 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 13 O 135 14 O Llys Ser Thir Ser Gly Gly Thr Ala Ala Lieu. Gly Cys Lieu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lieu. Thr 1.65 17O 17s Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Lieu. Tyr 18O 185 19 O Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Lieu. Gly Thr Glin 195 2OO 2O5 Thr Tyr Ile Cys Asn Val Asn His Llys Pro Ser Asn Thr Llys Val Asp 21 O 215 22O Lys Arg Val Glu Pro Llys Ser Cys Asp Llys Thr His Thr Cys Pro Pro US 2009/01 04204 A1 Apr. 23, 2009 91

- Continued

225 23 O 235 24 O Cys Pro Ala Pro Glu Lieu. Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255 Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg Thr Pro Glu Val Thr 26 O 265 27 O Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Llys Phe Asn 27s 28O 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Llys Pro Arg 29 O 295 3 OO Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lieu. Thr Val 3. OS 310 315 32O Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr Lys Cys Llys Val Ser 3.25 330 335 Asn Lys Ala Lieu Pro Ala Pro Ile Glu Lys Thir Ile Ser Lys Ala Lys 34 O 345 35. O Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu Pro Pro Ser Arg Glu 355 360 365 Glu Met Thr Lys Asn Glin Val Ser Lieu. Thr Cys Lieu Val Lys Gly Phe 37 O 375 38O Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Glin Pro Glu 385 390 395 4 OO Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp Ser Asp Gly Ser Phe 4 OS 41O 415 Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp Llys Ser Arg Trp Glin Glin Gly 42O 425 43 O Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lieu. His Asn His Tyr 435 44 O 445 Thr Gln Lys Ser Lieu. Ser Leu Ser Pro Gly Lys 450 45.5

<210 SEQ ID NO 122 <211 LENGTH: 1350 &212> TYPE: DNA <213> ORGANISM: Homo sapiens &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (1350)

<4 OO SEQUENCE: 122 gag gtg cag Ctg gtg gag tict 999 giga gaC ttg gta Cag cc.g. gigg gig 48 Glu Val Glin Lieu Val Glu Ser Gly Gly Asp Lieu Val Glin Pro Gly Gly 1. 5 1O 15 tcc ctd cqa ct c ticc tdt gta ggc tict gga titc acc titt ggc cqc tat 96 Ser Lieu. Arg Lieu Ser Cys Val Gly Ser Gly Phe Thr Phe Gly Arg Tyr 2O 25 3O gcc atg agt td gtC C9C Cag gct CC a gigg aag gigg Ctg gag tig gtC 144 Ala Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 gcq tot att aac aat aat giga aat coa tac tac gca gac toc gtg aag 192 Ala Ser Ile Asn. Asn. Asn Gly Asn Pro Tyr Tyr Ala Asp Ser Val Lys SO 55 6 O ggc cqa titc acc atc. tcc gca gac aat tcc aag agc aca gtt tat ctd 24 O Gly Arg Phe Thr Ile Ser Ala Asp Asn Ser Lys Ser Thr Val Tyr Lieu. 65 70 7s 8O caa atgaat agc Ctg aga gcc gala gac acg gCC atg tat tac tit gcg 288

US 2009/01 04204 A1 Apr. 23, 2009 93

- Continued Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 4 OO

Ctg gac agc gaC ggc agc titc ttic ctg tac agc aag Ct c acc gtg gac 1248 Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val Asp 4 OS 41O 415 aag agc cgg tog Cag cag ggc aac gtg tt C agc tigc agc gtg atg cac 1296 Lys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His 42O 425 43 O gag gCC Ctg cac aac Cac tac acc cag aag agc Ctg agc ctg agc ccc 1344 Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 44 O 445 ggc aag 1350 Gly Lys 450

<210 SEQ ID NO 123 <211 LENGTH: 450 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 123 Glu Val Glin Lieu Val Glu Ser Gly Gly Asp Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Val Gly Ser Gly Phe Thr Phe Gly Arg Tyr 2O 25 3O Ala Met Ser Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Ser Ile Asn. Asn. Asn Gly Asn Pro Tyr Tyr Ala Asp Ser Val Lys SO 55 6 O Gly Arg Phe Thr Ile Ser Ala Asp Asn Ser Lys Ser Thr Val Tyr Lieu. 65 70 7s 8O Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thir Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Asp His Tyr Ser Ser Gly Trp Pro Ala Phe Asp His Trp Gly Glin 1OO 105 11 O Gly Thr Lieu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 12 O 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 13 O 135 14 O Lieu. Gly Cys Lieu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Lieu. Thir Ser Gly Val His Thr Phe Pro Ala Val 1.65 17O 17s Lieu. Glin Ser Ser Gly Lieu. Tyr Ser Leu Ser Ser Val Val Thr Val Pro 18O 185 19 O Ser Ser Ser Leu Gly Thr Glin Thr Tyr Ile Cys Asn Val Asn His Lys 195 2OO 2O5 Pro Ser Asn. Thir Lys Val Asp Lys Arg Val Glu Pro Llys Ser Cys Asp 21 O 215 22O Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly Gly 225 23 O 235 24 O Pro Ser Val Phe Lieu. Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu