HK1197654B - Compositions and methods related to antibodies to staphylococcal protein a - Google Patents

Description

Compositions and methods related to antibodies to staphylococcal protein a

The invention was made with government support from AI52747 and AI92711 of the National Institute for Allergy and Infectious Diseases (NIAID) and 1-U54-AI-057153 awarded by the national institutes of health. The government has certain rights in the invention.

Priority of united states provisional patent application No. 61/523,751 filed on day 8, 15 of 2011, No. 61/615,083 filed on day 3, 23 of 2012, No. 61/618,417 filed on day 30 of 3, 2012, and No. 61/674,135 filed on day 20 of 7, 2012, all of which are incorporated herein by reference in their entirety.

I. Field of the invention

The present invention relates generally to the fields of immunology, microbiology and pathology. More particularly, it relates to methods and compositions relating to antibodies to bacterial proteins and bacterial peptides for eliciting such antibodies. The protein includes staphylococcal protein a (spa).

II. background of the invention

The number of both community-and hospital-acquired infections has increased in recent years with the increased use of intravascular devices. Hospital-acquired (nosocomial) infections are a leading cause of morbidity and mortality, more specifically, infections of more than two million patients per year in the united states. The most frequent nosocomial infections are urinary tract infections (33% of infections), followed by pneumonia (15.5%), surgical site infections (14.8%) and primary bloodstream infections (13%) (Emorl and Gaynes, 1993).

Staphylococcus aureus (Staphylococcus aureus), coagulase-negative staphylococci (primarily Staphylococcus epidermidis), enterococcus species (enterococcus spp), Escherichia coli (Escherichia coli), and Pseudomonas aeruginosa (Pseudomonas aeruginosa) are major nosocomial pathogens. Although these pathogens cause almost the same number of infections, the severity of the disease they can produce, combined with the frequency of antibiotic resistant isolates, balances this ranking towards staphylococcus aureus (s.aureus) and staphylococcus epidermidis (s.epidermidis) as the most important nosocomial pathogens.

Staphylococci can cause a wide variety of diseases in humans and other animals through toxin production or invasion. Staphylococcal toxins are a common cause of food poisoning because bacteria can grow in improperly stored food.

Staphylococcus epidermidis is a common skin commensal, which is also an important opportunistic pathogen causing infection of damaged medical devices and infection at surgical sites. Medical devices infected with staphylococcus epidermidis include cardiac pacemakers, cerebrospinal fluid shunts, continuous ambulatory peritoneal dialysis catheters, orthopedic devices and artificial heart valves.

Staphylococcus aureus is the most common cause of nosocomial infections with significant morbidity and mortality. It is the cause of several disorders: osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses, and toxic shock syndrome.

Staphylococcus aureus can survive on dry surfaces, increasing the chance of transmission. Any staphylococcus aureus infection can lead to staphylococcal scalded skin syndrome, a skin reaction to exotoxins received into the bloodstream. Staphylococcus aureus can also cause a life-threatening sepsis called sepsis. Methicillin-resistant (Methicillin-resistant) staphylococcus aureus (MRSA) has become a major cause of hospital-acquired infections.

Staphylococcus aureus and staphylococcus epidermidis infections are typically treated with antibiotics, with penicillin (penicillin) being the drug of choice and vancomycin (vancomycin) being used in methicillin-resistant isolates. The percentage of staphylococcus strains that exhibit broad spectrum resistance to antibiotics is increasing, posing a threat to effective antimicrobial therapy. In addition, the recent emergence of vancomycin-resistant staphylococcus aureus strains has caused the following panic: MRSA strains for which no effective therapy is available are beginning to emerge and spread.

An alternative approach to antibiotics in the treatment of staphylococcal infections has been the use of antibodies against staphylococcal antigens in passive immunotherapy. Examples of such passive immunotherapy involve the administration of polyclonal antisera (WO00/15238, WO00/12132) and treatment with monoclonal antibodies to lipoteichoic acid (WO 98/57994).

The first generation of vaccines targeting staphylococcus aureus or exoproteins produced by it have met with limited success (Lee, 1996), but there is still a need to develop additional therapeutic compositions for the treatment of staphylococcal infections.

Disclosure of Invention

Staphylococcus aureus is the most common cause of bacteremia and hospital-acquired infections in the united states. FDA-approved vaccines for preventing diseases caused by staphylococci are currently unavailable.

In some embodiments, there are antibody compositions that inhibit, reduce, and/or prevent staphylococcal infection. In a specific embodiment, there is a polypeptide capable of binding staphylococcal SpA protein. The term polypeptide is understood to mean one or more amino acids or polypeptide chains. For example, the term polypeptide can refer to a single polypeptide chain comprising a heavy chain or a light chain or a heavy and a light chain connected. The term polypeptide may also refer to an immunoglobulin (Ig) monomer, which comprises four polypeptide chains: two heavy chains and two light chains. The term polypeptide may also refer to dimeric, trimeric, tetrameric or pentameric Ig molecules.

In addition, in some embodiments, the SpA-binding polypeptide differs from other SpA antibodies in that it has properties based on or derived from antibodies generated using a variant of SpA, rather than a wild-type protein of SpA, as an antigen. SpA mutants have 1,2, 3, 4,5 or more changes in 1,2, 3, 4, and/or 5 of the A, B, C, D and/or E domains. Furthermore, as discussed herein, these SpA binding polypeptides are capable of specifically binding such SpA variants, including but not limited to KKAA domain variations in all five domains as discussed below.

Some embodiments are directed to recombinant peptides comprising 1,2, 3, 4,5, 6,7, 8,9, 10 or more amino acid segments comprising about, at least, or at most 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids in length, including all values and ranges therebetween, and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid segment of staphylococcal SpA (SEQ ID NO: 1). For example, the amino acid segment from staphylococcal SpA can be from a non-toxic SpA mutant polypeptide (e.g., SpA)_KKAA). PCT publication No. WO2011/005341 and PCT application No. PCT/US11/42845, each incorporated herein by reference, provide a number of non-toxic SpA mutant polypeptides and methods of use thereof. In further aspects, there are antibodies that specifically bind to one or more of these particular amino acid segments.

Embodiments also provide for the use of SpA antibodies in methods and compositions for treating bacterial and/or staphylococcal infections. In some embodiments, the compositions are used in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of bacterial infections, particularly staphylococcal infections. In addition, in some embodiments, there are methods and compositions that can be used to treat (e.g., limit staphylococcal abscess formation and/or persistence in a subject) or prevent bacterial infection.

Some aspects relate to methods of reducing staphylococcal infection or abscess formation comprising administering to a patient having or suspected of having a staphylococcal infection an effective amount of one or more purified polypeptides or proteins that specifically bind to staphylococcal SpA polypeptides. It is contemplated that the polypeptide (or protein) may be referred to as an antibody because it is a polypeptide or protein having an amino acid sequence of one or more CDR regions of an antibody or an amino acid sequence derived from one or more CDR regions of an antibody. In the case of antibodies, any of the embodiments discussed herein may be practiced with respect to a polypeptide or protein, so long as the polypeptide and protein have one or more amino acid regions that are at least 60% identical or homologous over the entire region of the CDRs from the antibody that is capable of specifically binding to a variant of SpA lacking specific Ig-binding activity. The SpA-binding polypeptide can be a purified polyclonal antibody, a purified monoclonal antibody, a recombinant polypeptide, or a fragment thereof. In some aspects, the polypeptide is a humanized antibody, which means that the invariant portion of the antibody has been altered to mimic the constant regions found in human antibodies. Thus, a humanized antibody is expected to be an antibody having the CDR sequences of a non-human antibody (or at least derived from such sequences, i.e., at least 80% identical amino acid sequences).

In some other embodiments, the antibody is a human antibody. In still further aspects, the antibody is a recombinant antibody segment. In some aspects, the monoclonal antibody comprises one or more of 5a10, 8E2, 3a6, 7E2, 3F6, 1F10, 6D11, 3D11, 5a11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8, or 1H7 described in tables 1-2 below and in table 5, incorporated herein by reference. The antibody or polypeptide may be administered at a dose of 0.1, 0.5, 1, 5,10, 50, 100mg or μ g/kg to 5,10, 50, 100, 500mg or μ g/kg. The recombinant antibody segment may be operatively linked to a second recombinant antibody segment. In some aspects, the second recombinant antibody segment binds to a second staphylococcal protein. The method may further comprise administering a second antibody that binds a second staphylococcal protein. In some aspects, the method further comprises administering an antibiotic.

Embodiments relate to monoclonal antibody polypeptides, polypeptides having one or more segments thereof, and polynucleotides encoding same. In some aspects, the polypeptide can comprise all or a portion of the heavy chain variable region and/or the light chain variable region of a SpA-specific antibody. In a further aspect, the polypeptide can comprise an amino acid sequence corresponding to a first Complementarity Determining Region (CDR), a second complementarity determining region, and/or a third complementarity determining region from a light variable chain and/or a heavy variable chain of an antibody, e.g., a SpA-specific antibody. In addition, an antibody or binding polypeptide can have a binding region comprising an amino acid sequence having, at least, or at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology (with conservative amino acid substitutions) (or any range derivable therein) to 1,2, 3, 4,5, or 6 of a CDR sequence discussed herein, including any of SEQ ID NOs 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 51-53, 96-98, 111-113, 116-118, 126-128, 131-133, 16-18, 26-28, 36-38, 46-48, 56-58, 66-68, 76-78, 86-88, 101-103, 106-108, 121-123, 136-138, 141-143. In particular embodiments, antibodies having all or part of one or more CDRs disclosed herein have been humanized in non-CDR regions. In further embodiments, the CDR regions disclosed herein may be altered by 1,2, 3, 4,5, 6,7, or 8 amino acids per CDR, which may be substituted for or performed in addition to humanization. In some embodiments, the alteration may be a deletion or addition of 1,2, or 3 amino acids, or it may be a substitution of any amino acid, which may or may not be an amino acid that is a conserved amino acid.

In some embodiments, the SpA-binding polypeptide or antibody has one, two, three, four, five, six, or seven CDRs that are 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the consensus sequence determined for the CDRs, e.g., as set forth in tables 7-17. It is contemplated that in some embodiments, the SpA-binding polypeptide or antibody has an amino acid sequence corresponding to CDR1, CDR2, and CDR3 of the light chain variable region and CDR1, CDR2, and CDR3 of the heavy chain variable region. As discussed herein, the amino acid sequences corresponding to the CDRs can have a percentage of identity or homology to the CDRs discussed herein. In some embodiments, the consensus sequence is SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, or SEQ ID NO 159. In particular embodiments, the SpA binding polypeptide or antibody has the consensus sequence of a monoclonal antibody from CDR1, CDR2, and/or CDR3 of the light chain variable region. Alternatively or additionally, the SpA-binding polypeptide or antibody has the consensus sequence of a monoclonal antibody from CDR1, CDR2, and/or CDR3 of the heavy chain variable region. It is also contemplated that the SpA binding polypeptide or antibody can have a mixture of CDRs based on a consensus sequence and/or sequence that is identical or homologous to a particular CDR.

In some embodiments, the SpA-binding polypeptide or antibody has one or more consensus sequences for 3F 6. In particular embodiments, the SpA-binding polypeptide or antibody has a consensus sequence of 3F6 from CDR1, CDR2, and/or CDR3 of the light chain variable region. Alternatively or additionally, the SpA-binding polypeptide or antibody has a consensus sequence from 3F6 of CDR1, CDR2, and/or CDR3 of the heavy chain variable region.

In some embodiments, the SpA antibody or binding polypeptide comprises an amino acid sequence that is at least 40% identical to one or more antibody CDR domains from a SpA binding antibody, wherein the polypeptide specifically binds to at least two SpA Ig-binding domains A, B, C, D and E of staphylococcal protein a that lacks non-specific Ig-binding activity. Additional embodiments of this aspect are contemplated below.

In additional embodiments, purified polypeptides that specifically bind to a SpA variant polypeptide lacking specific Ig binding activity are contemplated, wherein the polypeptide pairs at least two and at most five SpAIgG binding Domain A_KKAA、B_KKAA、C_KKAA、D_KKAAAnd E_KKAAHas 0.5 × 10⁹M-¹Or greater association constants. Additional embodiments of this embodiment are contemplated below.

In some aspects, the polypeptide comprises an amino acid sequence QSGPELMKPGASVKISCKAS corresponding to MAb3D11 Variable (VDJ) heavy chainGYSFTSYYMHWVKQSHGKSLEWIGYIDPFNGGTSYNQKFKGKATLTVDKSSSTAYMHLSSLTSEDSAVYYCARYGYDGTFYAMDYWGQGTSVTVSS in whole or in part. CDRs are indicated in bold and underlined. CDRs are regions within an antibody that are complementary in shape to the antigen. Thus, the CDRs determine the affinity and specificity of the protein for a particular antigen. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb3D11, e.g., SEQ ID NO 81, SEQ ID NO 82, and/or SEQ ID NO 83. In further embodiments, the antibody may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence RIVLTQSPAITAASLGQKVTITCSAS corresponding to MAb3D11 Variable (VJ) light chainSSVSYMHWYQQKSGTSPKPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPFTFGSGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb3D11, e.g., SEQ ID NO:86, SEQ ID NO:87, and/or SEQ ID NO: 88. In other embodiments, the polypeptide may have 1,2, or 3 CDRs relative to the CDR,2 and/or 3 amino acid changed CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted or substituted). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb3D11, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb3D 11.

In some aspects, the polypeptide comprises an amino acid sequence corresponding to MAb3F6 Variable (VDJ) heavy chain amino acid sequence EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFSISRDDSQNMLSLQMNNLKTEDTAIYYCVTEHYDYDYYVMDYAll or part of the amino acid sequence of WGQGTSVXSPQ. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb3F6, e.g., SEQ ID NO:51, SEQ ID NO:52, and/or SEQ ID NO: 53. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, alternatively or additionally, the antibody may be a region outside the CDRs and/or variable regionsHumanization of the domains. In some aspects, additionally or alternatively, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence IVLTQSPASLAVSLGQRATISCRAS that corresponds to MAb3F6 Variable (VJ) light chainESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb3F6, e.g., SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO: 58. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb3F6, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb3F 6.

In some aspects, the polypeptide comprises a Variable (VDJ) heavy chain amino acid sequence EVKLVESGGGLVKPGGSLKLSCAAS corresponding to MAb5a10GFAFSNYDMSWVRQTPEKRLEWVATISSGGTYPYYPDSVKGRFTISRDNAKNTLYLQLSSLRSEDTALYYCARGGFLITTRDYYAMDYWGQGTSVTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb5a10, e.g., SEQ ID No. 11, SEQ ID No. 12, and/or SEQ ID No. 13. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence TIVLTQSPAIMSASPGEKVTMTCSAS that corresponds to the Variable (VJ) light chain amino acid sequence of MAb5a10SSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb5a10, e.g., SEQ ID NO 16, SEQ ID NO 17, and/or SEQ ID NO 18. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises at least 60%, 65%, 70%75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of the variable region, i.e.the variable region framework, which is not a CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb5a10, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb5a 10.

In some aspects, the polypeptide comprises an amino acid sequence corresponding to MAb2F2 Variable (VDJ) heavy chain amino acid sequence VKLVESGGDLVKPGGSLKLSCAASRFTFSSYVMSWVRQTPEKRLEWVASIGSGGTTYYPDTVKGRFTISRDNARNILYLQMSSLRSDDTAMYYCTRGRGYGFAWYFDVWGAGTTVTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb2F2, e.g., SEQ ID No. 96, SEQ ID No. 97, and/or SEQ ID No. 98. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence TIVLTQSPAIMSASPGEKVTMTCSAS that corresponds to the Variable (VJ) light chain amino acid sequence of MAb2F2SSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb2F2, e.g., SEQ ID NO 101, SEQ ID NO 102, and/or SEQ ID NO 103. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb2F2, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with the 1,2, 3, 4,5, or 6 CDRs of mAb2F 2.

In some aspects, the polypeptide comprises an amino acid sequence DIVLTQSPASLAVSLGQRATISCRAS that corresponds to MAb4C5 Variable (VJ) light chainESVEYYGASLMQWYQQKSGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPNTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb4C5, such as SEQ ID NO:136, SEQ ID NO:137, and/or SEQ ID NO: 138. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb4C5, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb4C 5.

In some aspects, the polypeptide comprises an amino acid sequence EIVLTQSPAITAASLGQKVTITCSAS that corresponds to the Variable (VJ) light chain amino acid sequence of MAb4D5SSVSYMHWYHQKSGTSPKPWIYETSKLASGVPVRFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPFTFGSGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 variable light weights from MAb4D8The CDRs of the chain, for example SEQ ID NO 141, SEQ ID NO 142 and/or SEQ ID NO 143. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises a Variable (VDJ) heavy chain amino acid sequence EVQLVESGGGLVKPGGSLKLSCAAS corresponding to MAb5a11GFTFSDYYMYWVRQTPEKRLEWVATISDGGTYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARDRDDYDEGPYFDYWGQGTTLTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb5a11, e.g., SEQ ID NO 91, SEQ ID NO 92, and/or SEQ ID NO 93. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence DIVLTQSPASLAVSLGQRATISCRAS that corresponds to the Variable (VJ) light chain amino acid sequence of MAb6B2ESVDYSGASLMQWYQHKPGQPPRLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino groupsTo the carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb6B2, such as SEQ ID NO:121, SEQ ID NO:122, and/or SEQ ID NO: 123. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises a Variable (VDJ) heavy chain amino acid sequence KVQLQQSGAGLVKPGASVKLSCKAS corresponding to MAb8E2GYTFTEYSIHWVKQSSGQGLEWIGWFYPGSGYIKYNEKFKDKATLTADKSSSTVYMEFSRLTSEDSAVYFCARHGYGNYVGYAMDYWGQGTSVTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb8E2, e.g., SEQ ID No. 21, SEQ ID No. 22, and/or SEQ ID No. 23. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence DIQMTQSPASLSASVGETVTITCRAS that corresponds to MAb8E2 Variable (VJ) light chainEIIYSYLAWYQQKQGKSPQLLVYFAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHHYGTPYTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb8E2, e.g., SEQ ID NO:26, SEQ ID NO:27, and/or SEQ ID NO: 28. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb8E2, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with the 1,2, 3, 4,5, or 6 CDRs of mAb8E 2.

In some aspects, the polypeptide comprises a Variable (VDJ) heavy chain amino acid sequence QIQLVQSGPELKKPGETVKISCKAS corresponding to MAb3a6GYNFTDYSMHWVKQAPGKGLKWVGWINTETAESTYADDFKGRFAFSLETSASTAYLQINSLKDEDTATFFCAHFDCWGQGTTLTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide can comprise 1,2, and/or3 CDRs from the variable heavy chain of MAb3A6, e.g., SEQ ID NO 31, SEQ ID NO 32 and/or SEQ ID NO 33. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence DVVMTQISLSLPVTLGDQASISCRAS that corresponds to MAb3a6 Variable (VJ) light chainQSLVHSNGNTYLNWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQITYVPWTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb3a6, e.g., SEQ ID NO:36, SEQ ID NO:37, and/or SEQ ID NO: 38. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb3a6, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb3a 6.

In some aspects, the polypeptide comprises an amino acid sequence QVQLQQSGAELVRPGTSVKVSCKAS corresponding to MAb6D11 Variable (VDJ) heavy chainGNAFTNYLIEWIKQRPGQGLEWIGVINPGSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb6D11, such as SEQ ID NO:71, SEQ ID NO:72, and/or SEQ ID NO: 73. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence HCAHPSPASLAVSLGQRASISCRAS that corresponds to the Variable (VJ) light chain amino acid sequence of MAb6D11ESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPVRFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb6D11, e.g., SEQ ID NO:76, SEQ ID NO:77, and/or SEQ ID NO: 78. In thatIn additional embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb6D11, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb6D 11.

In some aspects, the polypeptide comprises a Variable (VDJ) heavy chain amino acid sequence QVQLQQSGAELVRPGASVKISCKAF corresponding to MAb8D4GSTFTNHHINWVKQRPGQGLDWIGYLNPYNDYTNYNQKFKGKATLTIDKSSSTAYLELSSLTSEDSAVYYCATITFDSQXQ in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb8D4, e.g., SEQ ID NO 111, SEQ ID NO 112, and/or SEQ ID NO 113. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, canAlternatively or additionally, the antibody may be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence KELISSKSEEEKWPGTSVKVSCKAS corresponding to MAb1F10 Variable (VDJ) heavy chainGNAFTNYLIEWIKQRPGQGLEWIGVINPGSGITNYNEKFKGKATLTADKSSNTAYMQLSSLSSDDSAVYFCSGSANWFAYWGQGTLVTVSA in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb1F10, such as SEQ ID NO 61, SEQ ID NO 62, and/or SEQ ID NO 63. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises all or part of an amino acid sequence corresponding to the Variable (VJ) light chain amino acid sequence of MAb1F 10. CDR CSPSPASLAVSLGQRATISCRAS is underlined in boldESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK are provided. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb1F10, e.g., SEQ ID NO:66, SEQ ID NO:67, and/or SEQ ID NO: 68. In further embodiments, the polypeptide may have 1,2 and/or 3 amino groups relative to these 1,2 or 3 CDRsAcid-altered CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substituted). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb1F10, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with 1,2, 3, 4,5, or 6 CDRs of mAb1F 10.

In some aspects, the polypeptide comprises all or part of an amino acid sequence corresponding to the Variable (VJ) light chain amino acid sequence of MAb4C 1. CDR VLTQSPASLAVSLGQRATISCRAS is underlined in boldESVEYSGASLMQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK are provided. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb4C1, e.g., SEQ ID NO:106, SEQ ID NO:107, and/or SEQ ID NO: 108. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspectsAlternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence FFGVSLGQRASISCRAS that corresponds to the Variable (VJ) light chain of MAb2B8ESVEYSGASLIQWYQHKPGQPPKLLIYAASNVESGVPVRFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb2B8, e.g., SEQ ID NO:126, SEQ ID NO:127, and/or SEQ ID NO: 128. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence corresponding to MAb2C3 Variable (VDJ) heavy chain amino acid sequence EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYDMSWVRQTPEKRLEWVATISSGGTYPYYPDSVKGRFTISRDNAENTLYLQLSSLRSEDTALYYCARGGFLITTRDYYAMDYWGQGTSVTVSS in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb2C3, e.g., SEQ ID NO:131, SEQ ID NO:132, and/or SEQ ID NO: 133. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In another implementationIn the protocol, alternatively or additionally, the antibody may be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence corresponding to MAb7E2 Variable (VDJ) heavy chain amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTDYSVHWVKQAPGKGLKWMAWINTATGEPTFADDFKGRFAFSLETSARTAYLQINNLKNEDTATYFCAPQLTGPFAYWGHGTLVTVSA in whole or in part. CDRs are indicated in bold and underlined. CDRs are regions within an antibody that are complementary in shape to the antigen. Thus, the CDRs determine the affinity and specificity of the protein for a particular antigen. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable heavy chain of MAb7E2, e.g., SEQ ID NO:41, SEQ ID NO:42, and/or SEQ ID NO: 43. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In some aspects, the polypeptide comprises an amino acid sequence DIQMTQSPASLSASVGETVTITCRAS that corresponds to MAb7E2 Variable (VJ) light chainENIHNYLAWYQQKQGKSPQLLVYNAKTLTDGVPSRFSGSGSGTQFSLKINSLQAGDFGSYYCQHSWSIPYTFGGGTRLQIRR in whole or in part. CDRs are indicated in bold and underlined. From amino to carboxy terminus, the CDRs are CDR1, CDR2, and CDR 3. In some aspects, the polypeptide may comprise 1,2, and/or 3 CDRs from the variable light chain of MAb7E2, e.g., SEQ ID NO 46,47 and/or 48. In further embodiments, the polypeptide may have CDRs with 1,2, and/or 3 amino acid changes relative to these 1,2, or 3 CDRs (1 or 2 amino acids added, 1 or 2 amino acids deleted, or substitutions). In further embodiments, the antibody may alternatively or additionally be humanized in regions outside the CDRs and/or variable regions. In some aspects, alternatively or additionally, the polypeptide comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to the amino acid sequence of the variable region, i.e., the variable region framework, of the non-CDR sequence.

In other embodiments, the polypeptide or protein comprises 1,2, 3, 4,5, or 6 CDRs from one or both of the light and heavy variable regions of mAb7E2, and 1,2, 3, 4,5, or 6 CDRs may have 1,2, and/or 3 amino acid changes relative to these CDRs. In some embodiments, all or part of the antibody sequence outside of the variable region has been humanized. The protein may comprise one or more polypeptides. In some aspects, a protein may contain one or two polypeptides similar to a heavy chain polypeptide and/or 1 or 2 polypeptides similar to a light chain polypeptide. In further embodiments, the polypeptide may be a single chain antibody or other antibody discussed herein, so long as it has at least 70% sequence identity or homology with the 1,2, 3, 4,5, or 6 CDRs of mAb7E 2.

In still further aspects, the polypeptide of an embodiment comprises one or more amino acid segments in any of the amino acid sequences disclosed herein. For example, a polypeptide can comprise 1,2, 3, 4,5, 6,7, 8,9, 10, or more amino acid segments comprising about, at least, or at most 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 96, 95, 98, 100, 106. 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 amino acids, including all values and ranges therebetween, and the amino acid segment is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences disclosed herein. In some aspects, the amino segment is selected from one of the amino acid sequences of SpA-binding antibodies as provided in table 5.

In still further aspects, the polypeptide of the embodiments comprises an amino acid segment of any of the amino acid sequences disclosed herein, wherein the segment is 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 93, 97, 98, 99, 98, 97, 98, 99, and combinations thereof in any of the sequences provided herein 100. 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 amino acids and beginning at amino acids 4,5, 6,7, 8,9, 11, 16, 17, 18, 11, 16, 17, 18, 17, 18, 21, 13, 21, 22. 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 129, 149, 130, 135, 147, 135, 134, 142, 136, 143, 142, 143, 142, 146, 143, 146, 142, 146, 142, 150. 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 amino acids. In some aspects, the amino segment or portion thereof is selected from one of the amino acid sequences of SpA binding antibodies as provided in table 5.

In still further aspects, the polypeptides of the embodiments comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) of the same amino acid segment as the V, VJ, VDJ, D, DJ, J or CDR domains of the SpA binding antibody (as provided in table 5). For example, a polypeptide may comprise 1,2, or 3 amino acid segments that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to CDR1, 2, and/or 3 of an SpA binding antibody as provided in table 5.

In further aspects, the nucleic acid molecule of an embodiment comprises one or more nucleic acid segments of any of the nucleic acid sequences disclosed herein. For example, a nucleic acid molecule can comprise 1,2, 3, 4,5, 6,7, 8,9, 10, or more nucleic acid segments comprising about, at least, or at most 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 95, 98, 100, 106. 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 217, 215, 220, 217, 220, 227, 229, 230, 223, 225, 224, 225, 224, 223, 225, 224, 230, 223, 225, 224, 225, 234, 224, 225, 224, 225, 223, 225, 224, 225, 235. 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000, or more nucleic acids, including all values and ranges therebetween, and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) of the nucleic acid segment is identical to any of the nucleic acid sequences disclosed herein. In some aspects, the nucleic acid segment is selected from one of the nucleic acid sequences encoding a portion of a SpA binding antibody as provided in table 5.

In still further aspects, the nucleic acid molecules of embodiments comprise a nucleic acid segment that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) identical to a sequence encoding a V, VJ, VDJ, D, DJ, J, or CDR domain of an SpA binding antibody as provided in table 5. For example, a nucleic acid molecule may comprise 1,2 or 3 nucleic acid segments that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) identical to the sequences encoding the CDRs 1,2 and/or 3 of the SpA binding antibodies as provided in table 5.

In still further aspects, some embodiments provide a hybridoma cell line that produces a monoclonal antibody. In some embodiments, the hybridoma cell line is a line that produces a 5a10, 8E2, 3a6, 7E2, 3F6, 1F10, 6D11, 3D11, 5a11, 1B10, 4C1, 2F2, 8D4, 7D11, 2C3, 4C5, 6B2, 4D5, 2B8, or 1H7 monoclonal antibody, one or more of which may be deposited. In another aspect, 1,2, and/or 3 CDRs from the light chain variable region and/or the heavy chain variable region of the MAb may be comprised in a humanized antibody or a variant thereof. In other embodiments, bispecific antibodies are contemplated in which the binding polypeptide is capable of binding at least two different antigens.

Some aspects relate to a method of treating a subject having or suspected of having a staphylococcal infection comprising administering to a patient having or suspected of having a staphylococcal infection an effective amount of a purified antibody or binding polypeptide that specifically binds staphylococcal protein a.

In another aspect, the method relates to a method of treating a subject at risk for staphylococcal infection comprising administering to a patient at risk for staphylococcal infection an effective amount of an antibody or binding polypeptide that binds to a staphylococcal protein a polypeptide prior to infection by staphylococci.

Antibodies or binding polypeptides contemplated for use in these embodiments include those capable of reducing bacterial load, increasing survival, reducing bacterial abscesses, administering protective immunity, reducing the number of days of antibiotic use, reducing the risk of sepsis or sepsis, reducing the risk of shock, or providing some other protective effect.

Certain embodiments are directed to antibody or binding polypeptide compositions comprising an isolated and/or recombinant antibody or polypeptide that specifically binds a peptide segment as described above. In some aspects, an antibody or polypeptide has a sequence in which, at least, or at most 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) is identical to all or part of any of the monoclonal antibodies provided herein. In still further aspects, an isolated and/or recombinant antibody or polypeptide has, has at least, or has at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a contiguous amino acid from any sequence or combination of these sequences provided herein.

In further embodiments, there are pharmaceutical compositions comprising one or more polypeptides or antibodies or antibody fragments discussed herein. Such compositions may or may not contain additional active ingredients.

In some embodiments, there is a pharmaceutical composition consisting essentially of a polypeptide comprising one or more of the antibody fragments discussed herein. It is contemplated that the composition may contain inactive ingredients.

Some aspects relate to nucleic acid molecules encoding the heavy chain variable region and/or the light chain variable region of an antibody that specifically binds SpA or a non-toxic variant of SpA.

Other aspects relate to pharmaceutical compositions comprising an effective antibacterial amount of an antibody that specifically binds to the above-described peptide and a pharmaceutically acceptable carrier.

The term "providing" is used according to its ordinary meaning to mean "supplying or supplying for use". In some embodiments, the protein is provided directly by administering a composition comprising an antibody or fragment thereof described herein.

The subject will typically have (e.g., be diagnosed as having a persistent staphylococcal infection), will be suspected of having, or is at risk for having a staphylococcal infection. The compositions comprise an effective amount of a SpA-binding polypeptide to achieve the intended purpose-treatment or protection against staphylococcal infection. The term "binding polypeptide" refers to a polypeptide that specifically binds to a target molecule, e.g., the binding of an antibody to an antigen. The binding polypeptide may, but need not, be derived from an immunoglobulin gene or a fragment of an immunoglobulin gene. More specifically, an effective amount means the amount of active ingredient necessary to provide resistance to, reduction of, or remission from infection. In a more specific aspect, the effective amount prevents, alleviates or alleviates a symptom of the disease or infection, or prolongs the survival of the subject being treated. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any formulation used in the methods described herein, an effective amount or dose can first be estimated by in vitro assays, cell culture assays, and/or animal model assays. For example, the dosage may be formulated in animal models to achieve the desired response. This information can be used to more accurately determine useful doses for humans.

The composition may comprise an antibody or cell that binds SpA. The antibody may be an antibody fragment, a humanized antibody, a monoclonal antibody, a single chain antibody, or the like. In some aspects, the SpA antibody is elicited by providing a SpA peptide or antigen or epitope that results in the production of an antibody that binds SpA in a subject. The SpA antibody is typically formulated into a pharmaceutically acceptable composition. The SpA antibody composition can further comprise at least 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 antibodies to more staphylococcal antigens or immunogenic fragments thereof. Staphylococcal antigens include, but are not limited to, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, IsdA, IsdB, SdrC, SdrD, SdrE, ClfA, ClfB, Coa, Hla (e.g., the H35 mutant), IsdC, SasF, vWbp, SpA and variants thereof (see U.S. provisional application No. 61/166,432 filed on 4/3 of 2009; 61/170,779 filed on 4/20 of 2009; and 61/103,196 filed on 10/6 of 2009; each of which is incorporated herein by reference in its entirety), 52kDa vitronectin binding protein (WO01/60852), Aaa (GenBank CAC80837), Aap (GenBank accession No. AJ249487), Ant (GenBank accession No. NP-372518), autolysin aminoglycoside, autolysin amidase, collagen binding protein (US), collagen binding protein (E.B), EF6240214, fibronectin (EPB), fibronectin binding protein (EPS), fibronectin binding protein (EPB) (EPF894041), fibronectin binding protein (EPB) (EPH 8941) binding protein (EPB), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, immunodominant ABC transporter, IsaA/PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO00/12689), SdrG/Fig (WO00/12689), SdrH (WO00/12689), SEA exotoxin (WO00/02523), SEB exotoxin (WO00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), Ssa, SSP-1, vitronectin-2 and/or PCT connexin binding protein (see WO 2007/392007, WO 2006/392006, WO2006/032472, each of which is incorporated herein by reference. The staphylococcal antigen or immunogenic fragment or segment can be administered simultaneously with the SpA antibody. The staphylococcal antigen or immunogenic fragment and SpA antibody may be administered in the same or different compositions and simultaneously or non-simultaneously.

The SpA antibody composition can further comprise at least 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 antibodies, antibody fragments, or antibody subfragments of a further staphylococcal antigen or immunogenic fragment thereof. Staphylococcal antigens to which such antibodies, antibody fragments or subfragments are directed include, but are not limited to, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, IsdA, IsdB, SdrC, SdrD, SdrE, ClfA, ClfB, Coa, Hla (e.g., the H35 mutant), IsdC, SasF, vWbp, SpA and variants thereof (see U.S. provisional application No. 61/166,432 filed on 4/3 of 2009; 61/170,779 filed on 4/20 of 2009; and 61/103,196 filed on 6 of 2009; each of which is incorporated herein by reference in its entirety), 52kDa vitronectin binding protein (WO01/60852), Aaa (GenBank CAC80837), Aap (GenBank accession No. AJ249487), Ant (GenBank accession No. NP-372518), autolysin aminoglycoside, autolysin amidase, collagen binding protein (EfA), collagen binding protein (EfB), Epstein Barn binding protein (E.B) (EPFb 8841), fibrin binding protein (EPB) (EPFbpA) binding protein 8841, Ab;) and variants thereof, Fibronectin binding protein (US5840846), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, immunodominant ABC transporter, IsaA/PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue (US5648240), MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SdrF (WO00/12689), SdrG/Fig (WO00/12689), SdrH (WO00/12689), SEA exotoxin (WO00/02523), SEB exotoxin (WO00/02523), SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (US5,801,234), SsaA, SSP-1, SSP-2, and/or vitronectin binding protein (see PCT publications WO2007/113222, WO2007/113223, WO2006/032472, WO2006/032475, WO2006/032500, each of which is incorporated herein by reference in its entirety). Antibodies, antibody fragments, or antibody subfragments to other staphylococcal antigens or immunogenic fragments thereof may be administered simultaneously with the SpA antibody. Antibodies, antibody fragments or antibody subfragments to other staphylococcal antigens or immunogenic fragments thereof may be administered to the SpA antibodies in the same or different compositions and at the same or different times.

As used herein, the term "modulating" encompasses the meaning of the word "inhibiting". "modulation" of activity is a decrease in activity. As used herein, the term "modulator" refers to a compound that affects the function of a staphylococcus bacterium, including the enhancement, inhibition, down-regulation, or repression of a protein, nucleic acid, gene, or organism, or the like.

Embodiments include compositions with or without bacteria. The composition may or may not comprise attenuated or viable or intact staphylococcal bacteria. In some aspects, the composition comprises a non-staphylococcal bacterium or a bacterium that does not contain a staphylococcal bacterium. In some aspects, the bacterial composition comprises an isolated or recombinantly expressed SpA antibody or a nucleic acid encoding same. In still further aspects, the SpA antibody multimerizes, e.g., dimerizes, trimerizes, tetramers, and the like.

In some aspects, the peptide or antigen or epitope can be presented as a multimer of 1,2, 3, 4,5, 6,7, 8,9, 10 or more peptide segments or peptide mimetics.

The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material or culture medium (when produced by recombinant DNA techniques) or chemical precursors or other chemicals (when chemically synthesized) of its origin. Furthermore, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, a compound may not simply be considered "isolated" when it is attached to a column or embedded in an agarose gel. In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that is not naturally occurring in fragment form and/or is not generally in a functional state.

The composition, e.g., an antibody, peptide, antigen, or immunogen, may be conjugated or linked, covalently or non-covalently, to other moieties, e.g., adjuvants, proteins, peptides, carriers, fluorescent moieties, or labels. The terms "conjugation" or "immunoconjugates" are used broadly to define the effective association of one moiety with another agent, and are not intended to refer to any type of effective association only, and are not particularly limited to chemical "conjugation". Recombinant fusion proteins are specifically contemplated.

The term "SpA antibody" refers to a polypeptide that binds to SpA protein from staphylococcus bacteria.

In further aspects, the composition can be administered to the subject more than once, and can be administered 1,2, 3, 4,5, 6,7, 8,9, 10, 15, 20, or more times. Administration of the compositions includes, but is not limited to, oral, parenteral, subcutaneous, and intravenous administration, or various combinations thereof, including inhalation or aspiration.

The compositions are typically administered to human subjects, but administration to other animals capable of providing therapeutic benefit against staphylococcal bacteria is contemplated, particularly cattle, horses, goats, sheep and other livestock, i.e. mammals. In a further aspect, the staphylococcus bacterium is staphylococcus aureus. In still further aspects, the methods and compositions may be used to prevent, reduce or treat infection of a tissue or gland, such as a mammary gland infection, particularly mastitis, and other infections. Other methods include, but are not limited to, prophylactically reducing bacterial load in subjects who do not show signs of infection, particularly those subjects suspected of or at risk of colonization by the target bacteria, such as patients at or at risk of infection or susceptible to infection during hospitalization, treatment and/or rehabilitation.

Still further embodiments include a method for providing a protective or therapeutic composition against staphylococcus bacteria to a subject, comprising administering to the subject an effective amount of a composition comprising (i) an SpA antibody; or (ii) a nucleic acid molecule encoding therefor; or (iii) administering an SpA antibody with any combination or permutation of the bacterial proteins described herein.

The embodiments of the examples section are to be understood as embodiments applicable to all aspects of the invention, including compositions and methods.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly stated to refer only to alternatives, or alternatives are mutually exclusive, although the disclosure supports the definition of alternatives and "and/or". It is also contemplated that anything listed using the term "or" may also be specifically excluded

Throughout this application, the term "about" is used to indicate that a numerical value includes the standard deviation of error for the device or method used to determine the value.

In accordance with long-standing patent law, the words and phrases used in the claims or specification, and "comprising" and "including" if not limited to a number, mean one or more unless specifically stated otherwise.

As used in this specification and claims, the words "comprise," "have," "include," or "contain" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description.

It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

So that the manner in which the above recited features, advantages and objects of the present invention, as well as others which will become apparent and can be understood in detail, are attained and can be understood in detail, certain embodiments of the invention are illustrated in the appended drawings. Which form a part of the specification. It is to be noted, however, that the appended drawings illustrate some embodiments of the invention and are therefore not to be considered limiting of its scope.

FIG. 1: SpA_KKAASpecific monoclonal antibodies (mAb) protect mice from MRSA infection. By using isotype controls (IgG)_2a) Or 20 mg/kg^-1SpA of_KKAAOf mAb (3F6)Groups of animals (n =10) were immunized by intraperitoneal injection 24 hours after immunization with 5 × 10⁶CFU staphylococcus aureus MW2 challenged animals. (A) At 15 days post challenge, animals were euthanized to count the number of staphylococcal load in the kidneys. (B) Serum samples from infected 15-day mice were analyzed for staphylococcal antigen matrix (ClfA, aggregation factor A; ClfB, aggregation factor B; FnBPA, fibronectin binding protein A; FnBPB, fibronectin binding protein B; IsdA, iron surface determinant A; IsdB, iron surface determinant B; SdrD, serine-aspartate repeat protein D; SpAKKAA, non-toxic staphylococcal protein A; Coa, coagulase; EsxA, Ess [ ESAT-6 (early secretory antigen target 6kDa)]Secretion system]An extracellular matrix A; EsxB, Ess [ ESAT-6 (early secretory antigen target 6kDa)]Secretion system]Antibodies to extracellular matrix B, Hla, α -hemolysin, LukD, leukocidin D, vWbp, von Willebrand binding protein numerical values represent fold increases (for IgG) in samples from mAb3F 6-treated animals relative to serum samples from isotype control animals_2aN =7 for 3F6, n = 8). Data are mean and error bars represent ± SEM. The results in A-B represent two independent analyses.

FIG. 2: affinity of protein a specific monoclonal antibodies. Monoclonal antibodies were incubated with increasing concentrations (0-4M) of ammonium thiocyanate to perturb (A) IgG₁Monoclonal antibody of the same type, (B) IgG_2aMonoclonal antibodies of the same type and (C) IgG_2bAntigen-antibody specific interactions in monoclonal antibodies of the same type. Data are mean and error bars represent ± SEM. Results in A-C represent three independent analyses.

FIG. 3: SpA_KKAASpecific mAb binding to wild type protein A. (A) Examination of immobilized wild-type protein A (SpA) and isotype control antibody (IgG)₁、IgG_2aOr IgG_2b) Or SpA_KKAA-binding ELISA of specific mabs (5a10, 3F6 and 3D 11). (B) Horse Radish Peroxidase (HRP) conjugated SpA_KKAASpecific mAbs (5A10-HRP, 3F6-HRP and 3D11-HRP) with immobilized SpA_KKAAIn plate reader experiments, in which SpA_KKAAFirst use the sameType control antibody (IgG)₁、IgG_2aOr IgG_2b) Or three different SpA_KKAASpecific mabs (5a10, 3F6 and 3D11) were cultured to evaluate the possibility of competitive inhibition of antibodies binding to the same or closely related sites (n = 3). Measuring OD_405nmAnd is expressed as SpA_KKAAInteractions with HRP-conjugated SpA-specific mabs were normalized. Data are mean and error bars represent ± SEM. The data in panels a and B represent three independent analyses. Asterisks indicate statistical significance (P)<0.05)。

FIG. 4: SpA_KKAAThe mAb prevents the association of staphylococcal protein A with immunoglobulins. (A) Isotype control antibody or SpA_KKAAmAb used to perturb human IgG to proteins immobilized on ELISA plates (wild type SpA, or lack of binding to Fc γ (SpA)_KK) Or Fab (SpA)_AA) A variant of (d) in a cell. Values were normalized to the interaction of protein a without antibody with human IgG (n = 4). (B) Staphylococci grew to mid log phase and were cultured using isotype control antibody or mAb3F6, followed by 2. mu.g of wild-type Sbi_1-4After cultivation, α -SpA was used after affinity purification_KKAAMeasurement of Sbi by immunoblotting with rabbit antibodies_1-4And (4) consumption. Values are expressed as Sbi without antibody_1-4Sedimentation (no Ab) was normalized. (C) Subjecting the affinity-purified S_pA(200_μg) 85 for injection_μg(5m_g·k_g ^-1) Of mAb3F6 the animals were euthanized at the indicated time points to affinity purified α -SpA_KKAARabbit antibodies the amount of SpA in circulating blood was measured by immunoblotting (n =3 per time point). Values were normalized to the total amount of SpA injected at 0 min. Data are mean and error bars represent ± SEM. The results in A-C represent two independent analyses. Asterisks indicate statistical significance (P)<0.05)。

FIG. 5: SpA_KKAAmAb promotes opsonophagocytic killing of Staphylococcus aureus in blood of mice and humans. (A) Lepirudin (Lepirudin) anticoagulated mouse blood in isotype mouse antibody control or SpA_KKAA-mAb(2_μg·ml^-1) In the presence of (2) with 5 × 10⁵CFU staphylococcus aureus Newman was incubated for 30 minutes and survival measurements were taken (n = 3). (B) Lepirudin anticoagulated human whole blood in isotype mouse antibody control or SpA_KKAA-mAb(10μg·ml^-1) In the presence of (2) with 5 × 10⁶CFU staphylococcus aureus MW2 was incubated for 120 min and survival measurements were performed (n = 3). (C-H) at 60 min of incubation of Staphylococcus in anticoagulated human blood, extracellular Staphylococcus clusters were detected in samples incubated with mouse isotype antibody control (grey arrow), while in SpA_KKAAStaphylococci were found in neutrophils (black arrows) in mAb-cultured samples. Data are mean and error bars represent ± SEM. Results in A-H represent three independent analyses. Asterisks indicate statistical significance (P)<0.05)。

FIG. 6: a protein a-specific immune response was generated by mAb3F 6. Measurement of protein A variants (SpA ) that have received 20. mu.g by ELISA_KK、SpA_AA、SpA_KKAAAnd PBS) and 85 μ g of mAb3F6 (an IgG2a antibody) or isotype control thereof (n =5 per group). The titers were normalized to their isotype control standards. Data are mean and error bars represent ± SEM. Results represent two independent analyses.

FIG. 7: interaction of human immunoglobulin fragments with protein a variants. Analysis of immobilized protein A variants by ELISA (wild type SpA, SpA)_KK、SpA_AAOr SpA_KKAA) With human immunoglobulins (hIgG) and their Fc or F (ab)₂The fragments were associated and normalized to SpA interaction with human IgG. Statistical significance of SpA variants and SpA with Each ligand (human IgG, Fc or F (ab))₂Fragment, n =4) were compared. Data are mean and error bars represent ± SEM. Results represent three independent analyses. Asterisks indicate statistical significance (P)<0.05)。

FIGS. 8A-B: SpA_KKAAAnd (5) carrying out mAb CDR comparison. Alignment using ClustalW2Amino acid sequences of CDRs (complementarity determining regions) obtained from hybridoma cell line immunoglobulin genes. Asterisks indicate positions with single, fully conserved residues. (colon) indicates strongly similar properties-scoring in the Gonnet PAM250 matrix>0.5 conservation between groups. (period) indicates weakly similar properties-score in Gonnet PAM250 matrix = in Gonnet PAM250 matrix<0.5 conservation between groups. mAb ranking based on CFU reduction in the murine kidney abscess model is shown as a superscript prior to mAb identification. Indicating a mouse IgG isotype. AVFPMILW-small (small + hydrophobic (including aromatic-Y)), DE-acidic, RK-basic-H, STYHCNGQ-hydroxy + mercapto + amine + G.

FIG. 9: the SpA monoclonal antibody (SpA27) failed to elicit protective immunity in mice. (A) Examination of SpA-mAb (SpA27) and SpA_KKAAmAb (3F6) with immobilized wild-type protein A (SpA) and absence via Fc γ (SpA)_KK)、Fab(SpA_AA) Or Fc gamma and Fab (SpA)_KKAA) (ii) an association ELISA of immunoglobulin-binding variants of (1) (n = 3). (B) By using 5 or 50m_g·k_g ^-13F6, 5m_g·k_g ^-1Groups of animals (n =9-15) were immunized with intraperitoneal injections of Spa27, or a mimic (PBS) twenty-four hours after immunization, with 5 × 10⁶CFU staphylococcus aureus USA300 to challenge animals. (A) Four days after challenge, animals were euthanized to count the number of staphylococcal load in the kidneys.

FIGS. 10A-E: mab358a76.1 specifically recognized the E domain of staphylococcal protein a. ELISA examination of (A) mAbs358A76.1 and (B)3F6 with immobilized non-toxic protein A variants (SpA)_KKAA) And each immunoglobulin binding domain (E)_KKAA、D_KKAA、A_KKAA、B_KKAAAnd C_KKAA) And is derived from E_KKAAAssociation of synthetic linear peptides of three helices (H1, H2, H3, H1+2, H2+3) of immunoglobulin binding domains (igbds). (C) Alignment of the amino acid sequences of the five igbds of protein a revealed amino acid residues in the E domain that differ from the conserved amino acid residues of the remaining four igbds (dashed box). Amino acid residues substituted in non-toxic protein AIdentified by a grey frame. (D) Amino acid sequence homology levels are compared using ClustalW, and numbers represent the percentage of amino acid homology between immunoglobulin binding domains. (E) Horse Radish Peroxidase (HRP) -conjugated mAbs (358A76.1-HRP and 3F6-HRP) with SpA immobilized in an ELISA plate_KKAAIn a plate reader experiment, in which SpA_KKAAFirst, isotype control antibody (IgG) was used_2a) Or mabs (358a76.1 and 3F6) to determine competitive inhibition of antibodies binding to the same or closely related sites. Record OD_405nmThe numerical value is expressed as SpA_KKAAInteractions with HRP-conjugated SpA-specific mabs were normalized.

FIGS. 11A-B: SpA monoclonal antibody 358a76.1 failed to elicit protective immunity in mice. (A) By using 5m_g·k_g ^-1Mimic of (I)_gG_2aIsotype control mAb), mab358a76.1 or mAb3F6 were immunized by intraperitoneal injection into a cohort of animals (n =10) twenty-four hours post immunization with 5 × 10⁶CFU s s.aureus USA300 challenged animals via intravenous inoculation. Four days after challenge, animals were euthanized to count the number of staphylococcal load in the kidneys. (B) Anticoagulated mouse blood was assayed in IgG2a isotype control mAb, mAb358A76.1 or mAb3F6 (10)_μg·ml^-1) In the presence of (2) with 5 × 10⁵CFU staphylococcus aureus USA300(LAC) for 30 minutes; staphylococcal survival was measured. (C) Isotype control antibodies, mAb358a76.1 or mAb3F6 were used to perturb the binding of human IgG to wild-type protein a (spa) immobilized on ELISA plates. Values were normalized to the interaction of protein a with human IgG in the absence of antibody.

Detailed Description

Staphylococcus aureus is a commensal of human skin and nostrils, and is a major cause of blood, skin and soft tissue infections (Klevens et al, 2007). The recent dramatic increase in staphylococcal disease mortality has been due to the spread of methicillin-resistant staphylococcus aureus (MRSA) strains, which are generally not susceptible to antibiotics (Kennedy et al, 2008). In a large retrospective study, the incidence of MRSA infection was 4.6% of all admissions in the united states (Klevens et al, 2007). Annual healthcare costs for 94300 infected individuals in the united states exceed $ 24 billion (Klevens et al, 2007). Current MRSR infections have triggered the public health crisis that must be addressed by the development of prophylactic vaccines (Boucher and Corey, 2008). To date, no FDA-approved vaccine for the prevention of staphylococcus aureus is available.

The inventors herein describe staphylococcal protein a binding antibodies and antigen binding determinants thereof. In particular, a collection of monoclonal antibodies have been made using SpA mutant proteins that lack both toxicity (Fc γ interaction) and superantigen activity against B cells (Fab interaction). Many antibodies were found to interact with SpA with high affinity and specificity. Importantly, the antibody is able to counteract the molecular mechanisms of staphylococcal protein a. In addition, the antibodies reduce bacterial load and abscess formation following challenge with virulent staphylococcus aureus when administered to animals. These antibodies may also enhance the host immune response following staphylococcal infection because these molecules can block the immunosuppressive effects of SpA. Thus, the SpA binding molecules of the embodiments provide a novel and effective way to treat or prevent staphylococcal disease.

SPA polypeptides

Some aspects of the embodiments relate to SpA polypeptides, such as wild-type SpA provided herein as SEQ ID No. 1. However, in some aspects, embodiments relate to mutant or variant SpA polypeptides, such as polypeptides that lack B cell superantigen activity and/or non-specific immunoglobulin binding activity (i.e., bind Ig that is independent of the CDR sequences of Ig). In particular, some embodiments relate to polypeptides that specifically bind to SpA polypeptides that lack B cell superantigen activity and/or non-specific immunoglobulin binding activity (e.g., polypeptides comprising antibody CDR domains).

The N-terminal part of protein A is composed of four or five immunoglobulin binding domains (IgBD A-E) of 56-61 amino acid residues; the SpA variants used in accordance with embodiments may be, for example, full-length SpA variants comprising variant A, B, C, D and/or the E domain. In some aspects, the SpA variant comprises or consists of an amino acid sequence 80%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 7. In other embodiments, the variant SpA comprises a segment of SpA. The SpA segment can comprise at least or at most 1,2, 3, 4,5, or more IgG binding domains. The IgG domain may be at least or at most 1,2, 3, 4,5 or more variant A, B, C, D or E domains. In some aspects, the SpA variant comprises at least or at most 1,2, 3, 4,5, or more variant a domains. In another aspect, the SpA variant comprises at least or at most 1,2, 3, 4,5 or more variant B domains. In still further aspects, the SpA variant comprises at least or at most 1,2, 3, 4,5, or more variant C domains. In yet a further aspect, the SpA variant comprises at least or at most 1,2, 3, 4,5 or more variant D domains. In some aspects, the SpA variant comprises at least or at most 1,2, 3, 4,5, or more variant E domains. In another aspect, the SpA variant comprises a combination of A, B, C, D and E domains in various combinations and permutations. The combination may include all or part of a SpA signal peptide segment, a SpA region X segment, and/or a SpA class signal segment. In other aspects, the SpA variant does not include a SpA signal peptide segment, a SpA region X segment, and/or a SpA class signal segment. In some aspects, the variant a domain comprises substitutions at positions 7,8, 34, and/or 35 of SEQ ID No. 4. In another aspect, the variant B domain comprises substitutions at positions 7,8, 34, and/or 35 of SEQ ID NO 6. In still further aspects, the variant C domain comprises substitutions at positions 7,8, 34, and/or 35 of SEQ ID No. 5. In some aspects, the variant D domain comprises substitutions at positions 9, 10, 36, and/or 37 of SEQ ID No. 2. In another aspect, the variant E domain comprises substitutions at positions 6,7, 33, and/or 34 of SEQ ID NO 3.

In some aspects, a SpA domain D variant, or equivalent thereof, can be comprised at positions 9 and 36 of SEQ ID No. 2; positions 9 and 37; 9 and 10 bits; 36 and 37 bits; 10 and 36 bits; positions 10 and 37; 9. 36 and 37 bits; 10. 36 and 37, 9, 10 and 36; or mutations at positions 9, 10 and 37. In another aspect, similar mutations may be included in one or more of domains A, B, C or E.

In further aspects, the amino acid glutamine (Q) at position 9 of SEQ ID NO:2 (or its analogous amino acid in other SpA domains) can be substituted with alanine (a), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y). In some aspects, the glutamine at position 9 may be replaced with arginine (R). In another aspect, glutamine at position 9 of SEQ ID NO. 2 or an equivalent thereof may be replaced with lysine or glycine. Any 1,2, 3, 4,5, 6,7, 8,9, 10 or more substitutions may be explicitly excluded.

In another aspect, the amino acid glutamine (Q) at position 10 of SEQ ID NO. 2 (or its analogous amino acids in other SpA domains) can be substituted with alanine (A), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y). In some aspects, the glutamine at position 10 may be replaced with arginine (R). In another aspect, glutamine at position 10 of SEQ ID NO. 2 or an equivalent thereof may be replaced with lysine or glycine. Any 1,2, 3, 4,5, 6,7, 8,9, 10 or more substitutions may be explicitly excluded.

In some aspects, aspartic acid (D) at position 36 of SEQ ID NO:2 (or its analogous amino acid in other SpA domains) can be substituted with alanine (a), asparagine (N), arginine (R), cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y). In some aspects, the aspartic acid at position 36 may be replaced with glutamic acid (E). In some aspects, the aspartic acid at position 36 of SEQ ID NO. 2 or an equivalent thereof can be replaced with an alanine or a serine. Any 1,2, 3, 4,5, 6,7, 8,9, 10 or more substitutions may be explicitly excluded.

In another aspect, aspartic acid (D) at position 37 of SEQ ID NO. 2 (or its analogous amino acid in other SpA domains) can be substituted with alanine (A), asparagine (N), arginine (R), cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y). In some aspects, the aspartic acid at position 37 can be replaced with glutamic acid (E). In some aspects, the aspartic acid at position 37 of SEQ ID NO. 2 or an equivalent thereof can be replaced with an alanine or a serine. Any 1,2, 3, 4,5, 6,7, 8,9, 10 or more substitutions may be explicitly excluded.

In a specific embodiment, the amino group at position 9 of SEQ ID NO:2 (or similar amino acids in the additional SpA domain) is substituted with alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S) or valine (V). In some aspects, the amino acid at position 9 of SEQ ID NO. 2 is substituted with glycine. In another aspect, the amino acid at position 9 of SEQ ID NO. 2 is substituted with a lysine.

In a specific embodiment, the amino group at position 10 of SEQ ID NO:2 (or similar amino acids in the additional SpA domain) is substituted with alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S) or valine (V). In some aspects, the amino acid at position 10 of SEQ ID NO. 2 is substituted with glycine. In another aspect, the amino acid at position 10 of SEQ ID NO. 2 is substituted with a lysine.

In a specific embodiment, the amino group at position 36 of SEQ ID NO:2 (or similar amino acids in the other SpA domain) is substituted with alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S) or valine (V). In some aspects, the amino acid at position 36 of SEQ ID NO. 2 is substituted with serine. In another aspect, the amino acid at position 36 of SEQ ID NO. 2 is substituted with alanine.

In a specific embodiment, the amino group at position 37 of SEQ ID NO:2 (or similar amino acids in the additional SpA domain) is substituted with alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S) or valine (V). In some aspects, the amino acid at position 37 of SEQ ID NO. 2 is substituted with serine. In another aspect, the amino acid at position 37 of SEQ ID NO. 2 is substituted with alanine.

In some aspects, the SpA variant comprises (a) one or more amino acid substitutions that disrupt or reduce binding to IgG Fc in the IgG Fc binding domain of SpA domain A, B, C, D and/or E, and (b) V A, B, C, D in SpA domain and/or E_H3 disruption or reduction of binding in the binding Domain to V_H3, or a substitution of one or more amino acid substitutions of the binding. In still further aspects, the amino acid sequence of the SpA variant comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% (including all values and ranges therebetween) identical to the amino acid sequence of SEQ ID NOs 2-6.

In another aspect, the SpA variant comprises (a) one or more amino acid substitutions that disrupt or reduce binding to IgG Fc at corresponding amino acid positions in the IgG Fc binding domain of SpA domain D or in other IgG domains, and (b) V at SpA domain D_H3 in the Binder Domain or at the corresponding amino acid position in the other IgG Domain_H3, or one or more amino acid substitutions of the binding. In some aspects, amino acid residues F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and/or K35(SEQ ID NO:2, QQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNES) of the IgG Fc binding subdomain of domain D are modified or replaced. In some aspects, V for domain D_H3 amino acid residues of the binding subdomain Q26, G29, F30, S33, D36, D37, Q40, N43 and/or E47(SEQ ID NO:2) modified or substituted so as to react with Fc or V_H3, the binding is weakened. In further aspects, respective modifications or substitutions can be made in respective locations of domains A, B, C and/or E. The corresponding positions are defined by aligning the domain D amino acid sequence with one or more amino acid sequences from other IgG binding domains of SpA. In some aspects, the amino acid substitution can be any of the other 20 amino acids. In another aspect, conservative amino acid substitutions may be specifically excluded from possible amino acid substitutions. In other aspects, only non-conservative substitutions are included. In any case, any substitution or combination of substitutions that reduce binding of the domain such that SpA toxicity is significantly reduced is contemplated. The significance of reduced binding refers to variants that produce minimal to no toxicity when introduced into a subject and can be evaluated using the in vitro methods described herein.

In some embodiments, the variant SpA comprises at least or at most 1,2, 3, 4,5, 6,7, 8,9, 10 or more variant SpA domain D peptides. In some aspects, 1,2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more amino acid residues of the variant SpA are substituted or modified-including, but not limited to, amino acids F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and/or K35(SEQ ID NO:2) of the IgG Fc binding domain of domain D and V35 of domain D_H3 binding subdomain amino acid residues Q26, G29, F30, S33, D36, D37, Q40, N43 and/or E47(SEQ ID NO: 2). In one aspect, the glutamic acid residue at position 9 and/or 10 of SEQ ID NO 2 (or corresponding positions in other domains) is mutated. In another aspect, the aspartic acid residues 36 and/or 37 of SEQ ID NO 2 (or corresponding positions in other domains) are mutated. In another aspect, glutamic acid 9 and 10 and aspartic acid residues 36 and 37 are mutated. Purified non-toxic SpA or SpA-D mutants/variants described herein are no longer capable of significantly binding (i.e., demonstrate attenuated or disrupted binding affinity) Fc γ or f (ab)₂V_H3, nor does it stimulate B cell apoptosis.

It is contemplated that variants of SpA may also include the same variations in domains A, B, C and/or E as those in domain D described above. In some embodiments, the SpA binding polypeptide or antibody can bind to a SpA variant having a KKAA variation described herein in each of domains A, B, C, D and E. In other embodiments, the same SpA-binding polypeptide or antibody may also bind to variants with GGSS variations in each domain rather than KKAA. In addition, in some embodiments, SpA-binding polypeptides or antibodies may bind to variant Sbi antigens that are altered in one or more of their domains as in SpA. An example of this is shown in figure 4.

Furthermore, it is contemplated that the SpA binding polypeptides or antibodies described herein may be capable of competing with SpA in binding to the Fc or Fab regions of immunoglobulins, or may prevent SpA disruption of immunoglobulin function. It is also contemplated that the SpA binding polypeptides or antibodies described herein may or may be capable of disrupting SpA disruption of immunoglobulin function or SpA binding to the Fc or Fab regions of an immunoglobulin. In some embodiments, this property enables therapeutic compounds to be used to treat infections. In addition, the method involves a SpA-binding polypeptide or antibody that is capable of counteracting SpA disruption of immunoglobulin function or binding to SpA of the Fc or Fab region of an immunoglobulin.

The non-toxin producing protein a variants can be used as subunit vaccines, eliciting humoral immune responses and conferring protective immunity against challenge with s. Immunization with the SpA-D variants resulted in an increase in protein A-specific antibodies compared to wild-type full-length protein A or wild-type SpA-domain D. Other SpA variants and methods of using the same are provided in PCT publication No. WO2011/005341 and PCT application No. PCT/US11/42845, both of which are incorporated herein by reference.

Compositions of proteins

Alternative variants typically contain exchanges of one amino acid with another at one or more positions within the protein, and may be designed to modify one or more properties of the polypeptide, with or without loss of other function or property. Substitutions may be conservative, i.e., an amino acid is replaced with an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamic acid to asparagine; glutamate to aspartate; glycine to proline; histidine to aspartic acid or glutamic acid; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitutions may be non-conservative, such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the replacement of a residue with a chemically different residue, such as the replacement of a non-polar or uncharged amino acid with a polar or charged amino acid, and vice versa.

The protein may be recombinant, or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is also contemplated that bacteria containing such variants can be practiced in compositions and methods. The protein then does not need to be isolated.

The term "functionally equivalent codons" is used herein to refer to codons encoding the same amino acid, e.g., six codons for arginine or serine, and also to codons encoding biologically equivalent amino acids (see table below).

Cipher sub-table

It will also be appreciated that the amino acid and nucleic acid sequences may comprise additional residues, for example additional N-terminal or C-terminal amino acids, respectively, or 5 'or 3' sequences, but still be substantially as hereinbefore described in one of the sequences disclosed herein, provided that the sequence meets the above criteria, including maintenance of biological protein activity when protein expression is involved. The addition of terminal sequences applies in particular to nucleic acid sequences which may, for example, comprise various non-coding sequences flanking either the 5 'or 3' portion of the coding region.

The following is a discussion based on changing the amino acids of a protein to create an equivalent or even improved second generation molecule. For example, some amino acids may be replaced with other amino acids in a protein structure without an observable loss of binding capacity to interact with a structure, such as an antigen binding region of an antibody or a binding site on a substrate molecule. Because it is the interactive capacity and nature of proteins that define the biological functional activity of proteins, some amino acid substitutions can be made in the protein sequence and in the underlying DNA coding sequence, and still produce proteins with similar properties. The inventors therefore contemplate that various changes may be made in the DNA sequence of a gene without observable loss of its biological efficacy or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the amino acid hydropathic index in conferring interactive biological functions on proteins is well understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophilic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It is also understood in the art that substitutions of similar amino acids can be made efficiently based on hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, illustrates that the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still produce a biologically and immunologically equivalent protein.

As outlined above, amino acid substitutions are typically based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamic acid and aspartic acid; and valine, leucine and isoleucine.

It is contemplated that in the composition, there is between about 0.001mg and about 10mg of total polypeptide, peptide and/or protein per ml. Thus, the concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0mg/ml or more (or any range derivable therein). Wherein about, at least about, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, (ii) or, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% can be antibodies that bind SpA, and can be used in combination with other staphylococcal proteins or protein binding antibodies described herein.

A. Polypeptides and polypeptide production

Embodiments relate to polypeptides, peptides and proteins and immunogenic fragments thereof for use in various aspects described herein. For example, specific antibodies are tested for or used to counteract or inhibit staphylococcal infection, or are used in counteracting or inhibiting staphylococcal infection. In particular embodiments, all or a portion of the proteins described herein may also be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be administered according to known protocols. See, e.g., Stewart and Young, (1984); tam et al, (1983); merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference. Alternatively, recombinant DNA techniques may be employed, wherein the nucleotide sequence encoding the peptide or polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression.

One embodiment includes the use of gene transfer into cells, including microorganisms, for the production and/or presentation of proteins. The gene for the protein of interest can be transferred into a suitable host cell, and the cell can then be cultured under suitable conditions. Nucleic acids encoding virtually any polypeptide may be employed. The generation of the recombinant expression vector and the elements contained therein are discussed herein. Alternatively, the protein to be produced may be an endogenous protein that is normally synthesized by the cells used for protein production.

In a particular aspect, an immunogenic SpA fragment comprises substantially all of the extracellular domain of a protein that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 97% to 99% identical, including all values and ranges therebetween, to a selected sequence over the length of the fragment sequence.

Fusion proteins consisting of staphylococcal proteins or immunogenic fragments of staphylococcal proteins (e.g. SpA) are also included in the immunogenic composition. Alternatively, embodiments also include individual fusion proteins of staphylococcal proteins or immunogenic fragments thereof as fusion proteins with heterologous sequences such as T cell epitopes or providers of purification markers, for example: beta-galactosidase, glutathione-S-transferase, Green Fluorescent Protein (GFP), epitope tags such as FLAG, myc tags, polyhistidine, or viral surface proteins such as influenza hemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM 197.

B. Antibodies and antibody-like molecules

In some aspects, one or more antibodies or antibody-like molecules (e.g., polypeptides comprising antibody CDR domains) can be obtained or produced that are specific for SpA. These antibodies can be used in various diagnostic and therapeutic applications as described herein.

As used herein, the term "antibody" is intended to refer broadly to any immunological binding agent, such as IgG, IgM, IgA, IgD, and IgE, as well as polypeptides comprising CDR domains of antibodies that retain antigen binding activity. Thus, the term "antibody" is used to refer to any antibody-like molecule having antigen binding regions and includes antibody fragments, such as Fab ', Fab, F (ab')₂Single Domain Antibodies (DAB), Fv, scFv (single chain Fv) and polypeptides with antibody CDRs, scaffold domains displaying CDRs (e.g., anticalins) or nanobodies. For example, the nanobody may be an antigen-specific VHH (e.g. a recombinant VHH) from camelid IgG2 or IgG3, or a CDR display framework from such camelid igs. Techniques for making and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing Antibodies are also well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

Embodiments are also contemplated that use "minibodies" or "minibodies". Mini antibodies are sFv polypeptide chains that comprise an oligomerization domain separated from the sFv at their C-terminus by a hinge region. Pack et al (1992). The oligomerization domain comprises a self-associated alpha-helix that can be further stabilized by additional disulfide bonds, such as a leucine zipper. The oligomerization domain is designed to be adapted for transmembrane vector folding, a process thought to contribute to folding of polypeptides into functional binding proteins in vivo. Typically, the mini-antibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al (1992); cumber et al (1992).

Antibody-like binding to the mimetic peptide is also contemplated in embodiments. Liu et al (2003) describe an "antibody-like binding mimetic peptide" (ABiP), which is an antibody that is a reduced antibody and has the advantages of longer serum half-life and less cumbersome synthetic methods.

Alternative scaffolds, e.g., CDRs, for the antigen binding peptides are also available and can be used to generate SpA binding molecules according to embodiments. In general, the skilled person knows how to determine the type of protein scaffold onto which to graft at least one CDR produced by the original antibody. More particularly, it is known that this type of stent to be selected must meet the following maximum number criterion (Skerra, 2000): good phylogenetic conservation; known three-dimensional structures (e.g., by crystallography, NMR spectroscopy, or any other technique known to those skilled in the art); small size; little or no post-transcriptional modification; and/or ease of production, expression and purification.

The origin of such protein scaffolds may be, but is not limited to, a structure selected from the group consisting of: fibronectin and preferably fibronectin type III domain 10, lipocalin, anticalin (Skerra, 2001), protein Z produced by domain B of protein a of staphylococcus aureus, thioredoxin a or proteins with repeated motifs such as "ankyrin repeats" (Kohl et al, 2003), "armadillo repeats", "leucine rich repeats" and "triangular tetrapeptide repeats". For example, anticalin or lipocalin derivatives are a class of binding proteins with affinity and specificity for various target molecules and can be used as SpA binding molecules. Such proteins are described in U.S. patent publication nos. 20100285564, 20060058510, 20060088908, 20050106660 and PCT publication No. WO2006/056464, which are incorporated herein by reference.

Protein Inhibitors (PINs) derived from toxins, such as those from scorpions, insects, plants, molluscs, etc., scaffolds and neuronal NO synthase may also be used in some aspects.

Monoclonal antibodies (mabs) have been identified as having several advantages, such as reproducibility and large-scale production. Embodiments include monoclonal antibodies of human, mouse, monkey, rat, hamster, rabbit, and chicken origin.

"humanized" antibodies are also contemplated, as are chimeric antibodies derived from mouse, rat, or other species, bispecific antibodies, recombinant and engineered antibodies and fragments thereof, bearing domains of human constant and/or variable regions. As used herein, the term "humanized" immunoglobulin refers to an immunoglobulin comprising human framework regions and one or more CDRs from a non-human (typically mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. To describe the antibodies of some embodiments, the strength of binding of an antibody molecule to an epitope, referred to as affinity, can be measured. The affinity of an antibody can be measured by the association constant (K)_a) Or dissociation constant (K)_d) To be determined. An antibody considered useful in some embodiments may have about, at least about, or up to about 10⁶、10⁷、10⁸、10⁹Or 10¹⁰M or any range of association constants derivable therein. Similarly, in some embodiments, the antibody may have about, at least about, or up to about 10^-6、10^-7、10^-8、10^-9Or 10^-10M or any range of dissociation constants derivable therein. These values are reported for the antibodies discussed herein, and the same assay can be used to evaluate the binding properties of such antibodies.

In some embodiments, the polypeptide that specifically binds SpA is capable of counteracting protein a and/or promoting opsonophagocytic killing of staphylococci. In addition, in some embodiments, the polypeptides used may provide protective immunity against s. It is contemplated that mab358a76.1 may be excluded from these embodiments.

1. Method for producing antibodies

Methods for producing antibodies (e.g., monoclonal antibodies and/or monoclonal antibodies) are known in the art. Briefly, polyclonal antibodies are prepared by immunizing an animal according to an embodiment with a SpA polypeptide (e.g., non-toxic SpA) or portion thereof and collecting antisera from the immunized animal.

A wide range of animal species can be used to produce antisera. Typically, the animal used for the production of antisera is a rabbit, mouse, rat, hamster, guinea pig or goat. The choice of animal may be determined by the ease of handling, cost, or desired amount of serum, as is known to those skilled in the art. It will be appreciated that antibodies can also be produced transgenically by the production of mammals or plants transgenic for the immunoglobulin heavy and light chain sequences of interest and from which the antibodies are produced in recoverable form. For transgenic production in mammals, antibodies can be produced and recovered in the milk of goats, cattle and other mammals. See, for example, U.S. patent nos. 5,827,690, 5,756,687, 5,750,172 and 5,741,957.

As is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, protoplasms, synthetic compositions, or vectors encoding such adjuvants.

Adjuvants that may be used according to embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI in a 2% squalene/tween 80 emulsion containing three components extracted from the bacteria MPL, Trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS) is also expected. Even MHC antigens may be used. Exemplary adjuvants may include Freund's complete adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and/or aluminum hydroxide adjuvant.

In addition to adjuvants, it is also desirable to co-administer Biological Response Modifiers (BRMs), which have been shown to upregulate T-cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CiM; 1200mg/d) (Smith/Kline, PA); low doses of cyclophosphamide (CYP; 300mg/m2) (Johnson/Mead, NJ), cytokines, such as interferon, IL-2 or IL-12, or genes encoding proteins involved in immune-accessory functions, such as B-7.

The amount of immunogenic composition used in the production of antibodies varies depending on the nature of the immunogen and the animal used for immunization. The immunogen may be administered using a variety of routes including, but not limited to, subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal routes. Antibody production can be monitored by sampling the blood of the immunized animal at various points after immunization.

A second booster dose (such as that provided in an injection) may also be administered. The process of advancing and titrating is repeated until the appropriate titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal may be bled, serum isolated and stored, and/or the animal may be used to produce mabs.

For the production of rabbit polyclonal antibodies, blood may be drawn from the animal by ear vessels or by cardiac puncture. The removed blood is allowed to clot and then centrifuged to separate serum components from whole blood and blood clots. The serum may be used for various applications, or the desired antibody fraction may be purified by well-known methods, e.g. affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using e.g. protein a or protein G chromatography, etc.

Mabs can be readily prepared by using well-known techniques, such as those exemplified in U.S. patent No. 4,196,265, which is incorporated herein by reference. Generally, the technology involves immunizing a suitable animal with a selected immunogenic composition, e.g., a purified or partially purified protein, polypeptide, peptide, or domain, which is a wild-type or mutated composition. The immunizing composition is administered in a manner effective to stimulate the antibody-producing cells.

The process for producing monoclonal antibodies (mabs) generally begins along the same route as the process for making polyclonal antibodies. In some embodiments, rodents, such as mice and rats, are used to produce monoclonal antibodies. In some embodiments, rabbit, sheep or frog cells are used to produce monoclonal antibodies. The use of rats is well known and may provide several advantages (Goding, 1986, pages 60 to 61). Mice (e.g., BALB/c mice) are routinely used and typically give a high percentage of stable fusions.

The animals are typically injected with antigen as described above. The antigen may be mixed with an adjuvant, such as Freund's complete or incomplete adjuvant. Boosting administration with the same antigen or with DNA encoding the antigen can occur at intervals of about two weeks. As discussed in the examples, the antigen may be altered compared to the antigen sequence found in nature. In some embodiments, variant or altered protein a peptides or polypeptides are employed to generate antibodies. In some embodiments, a variant of SpA has 1,2, 3, 4,5, 6,7, or 8 changes in 1,2, 3, 4, or all 5 of the A, B, C, D or E domains of SpA.

After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in MAb generation protocols. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from peripheral blood samples. Typically spleen cells are a rich source of antibody producing cells in the plasmablast stage. Generally, peripheral blood cells can be easily obtained because peripheral blood is easily available.

In some embodiments, a group of animals will be immunized, the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe, typically, the spleen from the immunized mouse will contain about 5 × 10⁷To 2 × 10⁸And (4) lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with immortal myeloma cells, which are typically cells of the same species as the immunized animal. Myeloma cell lines suitable for use in hybridoma-producing fusion processes are preferably non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growth in specific selective media that support the growth of only the desired fused cells (hybridomas).

Any of a number of myeloma cells may be used, as known to those skilled in the art (Goding, pages 65 to 66, 1986; Campbell, pages 75 to 83, 1984). For example, when the animal being immunized is a mouse, P3X63/Ag8, X63Ag8.653, NS1/1.Ag41, Sp210Ag14, FO, NSO/U, MPC11, MPC11X45GTG1.7, and S194/5XX0 Bul; for rats, r210.rcy3, y3ag1.2.3, IR983F and 4B 210; u266, GM1500GRG2, LICR LON HMy2, and UC7296 were all useful for human cell fusion. See Yoo et al (2002), for a discussion of myeloma expression systems.

One murine myeloma cell is the NS-1 myeloma cell line (also known as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human gene mutant cell depository (Human Genetic mutant) by requiring cell line accession number GM 3573. Another mouse myeloma cell line that may be used is the 8 azaguanine resistant mouse myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen cells or lymph node cells and myeloma cells generally involve mixing the cells with myeloma cells in a 2:1 ratio, although the ratio can vary from about 20:1 to about 1:1, respectively, in the presence of agents (chemical or electrical) that promote cell membrane fusion. Fusion methods using Sendai (Sendai) virus have been described by Kohler and Milstein (1975; 1976), and those using ethylene glycol (PEG), e.g., 37% (v/v) PEG, have been described by Gefter et al (1977). It is also suitable to use an electrically induced fusion method (Goding, pages 71-74, 1986).

The fusion process is typically performed at a low frequency, about 1 × 10^-6To 1 × 10^-8Viable hybrids were generated. However, this does not pose a problem because viable, fused hybrids are differentiated from parent, unfused cells (particularly unfused cells that generally continue to divide indefinitely) by culturing in selective media. The selective medium is typically a medium containing an agent that blocks de novo synthesis of nucleotides in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of purines and pyrimidines, whereas azaserine blocks purine synthesis only. When aminopterin and methotrexate are used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine.

The selection medium was HAT. Only cells that are capable of manipulating the nucleotide salvage pathway are able to survive in HAT medium. Myeloma cells are deficient in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT), which cannot survive. B cells can manipulate this pathway, but they have a limited life span in culture and typically die within about two weeks. Thus, the only cells that can survive in selective media are those hybrids formed from myeloma and B cells.

This culture provides a population of hybridomas from which a particular hybridoma is selected. Generally, selection of hybridomas is performed as follows: cells were cultured by monoclonal dilution in microtiter plates and supernatants of individual clones were then tested (after about two or three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mabs. Cell lines can be used for MAb production in two basic ways. First, a sample of the hybridoma can be injected (usually into the peritoneal cavity) into a tissue-compatible animal type (e.g., a syngeneic mouse) that is used to provide somatic and myeloma cells for the initial fusion. Optionally, the animal is rendered susceptible to a hydrocarbon, particularly an oil, such as pristane (tetramethylpentadecane), prior to injection. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fused cell hybrids. The body fluids of the animal, such as serum or ascites fluid, may then be withdrawn to provide a high concentration of MAb. Secondly, individual cell lines can be cultured in vitro, where the MAb is naturally secreted into the culture medium from which high concentrations of MAb can be easily obtained.

In addition, expression of antibodies (or other portions thereof) from the producer cell line can be enhanced using a number of known techniques. For example, glutamine synthetase and DHFR gene expression systems are common methods for enhancing expression under certain conditions. High expression cell clones can be determined using conventional techniques, such as limiting dilution cloning and microdroplet (Microdrop) techniques. The GS system is discussed in whole or in part in european patent nos. 0216846, 0256055 and 0323997 and in european patent application No. 89303964.4.

MAb produced by either means can be further purified, if desired, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Fragments of monoclonal antibodies can be obtained from monoclonal antibodies produced as follows: by methods involving digestion with enzymes such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments may be synthesized using an automated peptide synthesizer.

It is also contemplated that monoclonal antibodies can be produced using molecular cloning methods. In one embodiment, combinatorial immunoglobulin phagemid libraries are made from RNA isolated from the spleen of immunized animals, and phagemids expressing appropriate antibodies are selected by panning using antigen-expressing cells and control cells. The advantage of this approach over traditional hybridoma technology is that approximately 104-fold more antibody can be produced and screened in a single round, and new specificities are generated from the combination of H and L chains, which further increases the chances of finding a suitable antibody.

Another embodiment relates to the production of antibodies, for example as found in U.S. patent No. 6,091,001, which describes a method of producing cells expressing antibodies from the genomic sequence of cells comprising modified immunoglobulin loci using Cre-mediated site-specific recombination. The method involves first transfecting the antibody-producing cell with a homologous targeting vector comprising a lipoxygenase site and a targeting sequence homologous to a first DNA sequence adjacent to an immunoglobulin locus region of the genomic sequence, which immunoglobulin locus region is to be converted to a modified region, so that the first lipoxygenase site is inserted into the genomic sequence via site-specific homologous recombination. The cells are then transfected with a lipoxygenase targeting vector comprising a second lipoxygenase site suitable for Cre-mediated recombination with the intact lipoxygenase site and a modification sequence to convert the immunoglobulin locus region into a modified region. The conversion is performed by allowing the lipoxygenase site to interact with Cre in vivo, such that the modifying sequence is inserted into the genomic sequence via Cre-mediated site-specific recombination of the lipoxygenase site.

Alternatively, monoclonal antibody fragments can be synthesized using automated peptide synthesizers or by expression of full-length genes or gene fragments in e.

It is also contemplated that monoclonal antibodies may be further screened and optimized for properties related to specificity, affinity, half-life, immunogenicity, binding association, binding dissociation or overall functional properties with respect to treatment as an infection. Thus, it is contemplated that a monoclonal antibody may have 1,2, 3, 4,5, 6 or more changes in the amino acid sequence of 1,2, 3, 4,5, 6 or 6 CDRs of monoclonal antibodies 5a10, 8E2, 3a6, 3F6, 1F10, 6D11, 3D11, 5a11, 1B10 or 4C 1. It is contemplated that the amino acid in position 1,2, 3, 4,5, 6,7, 8,9 or 10 of the VJ region or VDJ region of the light or heavy variable region of monoclonal antibodies 5a10, 8E2, 3A6, 3F6, 1F10, 6D11, 3D11, 5a11, 1B10 or 4C1, CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 may have insertions, deletions or substitutions with conservative or non-conservative amino acids. Such amino acids that may be substituted or constitute substitutions are disclosed above.

In some embodiments, fragments of an intact antibody may perform the function of binding antigen. Examples of binding fragments are (i) Fab fragments consisting of VL, VH, CL and CHl domains; (ii) an Fd fragment consisting of VH and CHl domains; (iii) an Fv domain consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH or VL domains (Ward, 1989; McCafferty et al, 1990; Holt et al, 2003); (v) an isolated CDR region; (vi) a F (ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv) in which a VH domain and a VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al, 1988; Huston et al, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; Holliger et al, 1993). Fv, scFv or diabody molecules can be stabilized by incorporating a disulfide bridge connecting the VH and VL domains (Reiter et al, 1996). Mini-antibodies comprising a scFv linked to a CH3 domain can also be made (Hu et al, 1996). The citation in this paragraph is incorporated by reference.

Antibodies also include bispecific antibodies. Bispecific or bifunctional antibodies form a second generation monoclonal antibody in which two different variable regions are combined in the same molecule (Holliger, P.&Winter, G.1999cancer and dmetastasis rev.18:411-419, 1999). Their use in both the diagnostic and therapeutic fields is demonstrated by their ability to target several molecules or complement new effector functions on the surface of tumor cells. When bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be manufactured in various ways (Holliger et al, PNAS U)SA90:6444-6448, 1993), for example chemically or by hybrid hybridomas, or may be any of the bispecific antibody fragments described above. These antibodies can be obtained by chemical methods (Glennie et al, 1987J. Immunol.139, 2367-. Examples of bispecific antibodies include BiTE^TMThose of the art, where the binding domains of two antibodies with different specificities can be used and directly linked via a short flexible peptide. This combines two antibodies on a short single peptide chain. Diabodies and scFvs can be constructed using only the variable domains without the Fc region, possibly reducing the effect of the anti-idiotypic reaction. The citation in this paragraph is incorporated by reference in its entirety.

Bispecific antibodies can be constructed as intact IgG, bispecific Fab '2, Fab' PEG, diabodies, or bispecific scFv. In addition, two bispecific antibodies can be linked to form a tetravalent antibody using conventional methods known in the art.

Bispecific diabodies, as opposed to bispecific whole antibodies, can also be particularly useful because they can be easily constructed and expressed in E.coli. Diabodies (and many other polypeptides, such as antibody fragments) with suitable binding specificities can be readily selected using phage display from libraries (WO 94/13804). If one arm of a diabody is to be kept constant, e.g. with specificity for SpA, a library can be made in which the other arm is altered, and an antibody of the appropriate specificity selected. Bispecific whole antibodies can be made by alternative engineering methods as described in Ridgeway et al, (Protein Eng., 9: 616-.

C. Antibody and polypeptide conjugates

Embodiments provide antibodies and antibody-like molecules to SpA protein, polypeptides and peptides linked to at least one agent to form antibody conjugates or payloads. To enhance the efficacy of an antibody molecule as a diagnostic or therapeutic agent, at least one desired molecule or moiety is typically linked or covalently bound or complexed. Such a molecule or moiety may be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having a desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibodies, radiolabeled nucleotides, and the like. In contrast, a reporter is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands such as biotin.

Some examples of antibody conjugates are those in which the antibody is linked to a detectable label. A "detectable label" is a compound and/or element that can be detected due to its particular functional and/or chemical properties, the use of which enables the antibodies to which they are attached to be detected and/or further quantified if desired.

Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics are generally divided into two categories, those used for in vitro diagnostics, such as those used in various immunoassays, and/or those used in vivo diagnostic protocols, commonly referred to as "antibody direct imaging". Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies (see, e.g., U.S. patent nos. 5,021,236, 4,938,948, and 4,472,509, each of which is incorporated herein by reference). The imaging moiety used may be paramagnetic particles, radioisotopes, fluorescent dyes, NMR detectable substances, X-ray imaging.

In the case of paramagnetic ions, mention may be made by way of example of ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to lanthanum (III), gold (III), lead (II), and bismuth (III) among others.

In the case of radioisotopes for therapeutic and/or diagnostic applications, astatine may be used²¹¹Carbon, carbon¹⁴Chromium (III)⁵¹Chlorine, chlorine³⁶Cobalt, cobalt⁵⁷Cobalt, cobalt⁵⁸Copper, copper⁶⁷、Eu¹⁵²Gallium, gallium⁶⁷Hydrogen, hydrogen³Iodine, iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹³¹Indium, indium¹¹¹Iron, iron⁵⁹Phosphorus, phosphorus³²Rhenium¹⁸⁶Rhenium¹⁸⁸Selenium, selenium⁷⁵Sulfur, sulfur³⁵Technetium, technetium^99mAnd/or yttrium⁹⁰。¹²⁵I is generally used in some embodiments, technetium^99mAnd/or indium¹¹¹But also because of its low power and suitability for remote detection. Radiolabeled monoclonal antibodies may be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and/or potassium iodide and a chemical oxidant, such as sodium hypochlorite, or an enzymatic oxidant, such as lactoperoxidase. Monoclonal antibodies can be used with technetium by a ligand exchange process^99mLabelling, for example by reducing pertechnetate with a stannous salt solution, chelating the reduced technetium to a Sephadex column and applying the antibody to the column. Alternatively, direct labelling techniques may be used, for example by culturing pertechnetate, reducing agents such as SnCl₂Buffer solutions such as sodium potassium phosphate solution and antibodies. An intermediate functional group commonly used to bind radioisotopes present as metal ions to antibodies is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Included among the fluorescent labels contemplated for use as conjugates are Alexa350, Alexa430, AMCA, BODIPY630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green488, Oregon Green500, Oregon Green514, Pacific Blue, REG, rhodamine Green, rhodamine Red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine/or Texas Red, and the like.

Antibody conjugates include those intended for use primarily in vitro, wherein the antibody is linked to a second binding ligand and/or an enzyme (enzyme label) that produces a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. Preferred second binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Pat. nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241; each of which is incorporated herein by reference.

Another known method of site-specifically attaching molecules to antibodies involves the reaction of antibodies with hapten-based affinity labels. Basically, hapten-based affinity labels react with amino acids at the antigen binding site, thereby disrupting the site and preventing specific antigen reactions. However, this may not be advantageous because it results in loss of antigen binding by the antibody conjugate.

Molecules containing azido groups can also be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low intensity uv light (Potter & Haley, 1983). In particular, 2-azido and 8-azido analogs of purine nucleotides have been used as site-directed optical probes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al, 1985). 2-azido and 8-azido nucleotides have also been used to determine the position of the nucleotide binding domain of purified proteins (Khation et al, 1989; King et al, 1989; and Dholakia et al, 1989) and can be used as antibody binding agents.

Several methods for attaching or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods involve the use of metal chelating complexes that employ organic chelators, such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriamine tetraacetic acid, N-chloro-p-toluenesulfonamide, and/or tetrachloro-3, 6-diphenylglycoluril-3 attached to antibodies (U.S. patent nos. 4,472,509 and 4,938,948, each of which is incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent, such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these coupling agents or by reaction with isocyanates. In U.S. patent No. 4,938,948, imaging of breast tumors is accomplished using monoclonal antibodies, and a detectable imaging moiety is conjugated to the antibody using a linking agent such as methyl p-hydroxybenzoate or N-succinimidyl 3- (4-hydroxyphenyl) propionate.

In some embodiments, it is contemplated that the immunoglobulins are derivatized by selectively introducing thiol groups in the Fc region of the immunoglobulin using reaction conditions that do not alter the binding site of the antibody. Antibody conjugates produced according to this method are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector molecules or reporter molecules coupled to carbohydrate residues in the Fc region has also been disclosed in the literature (O' Shannessy et al, 1987). This approach has been reported to produce antibodies that are promising in diagnostic and therapeutic terms, which are currently under clinical evaluation.

In some embodiments, antibodies against SpA are attached to semiconductor nanocrystals, such as those described in U.S. patent nos. 6,048,616, 5,990,479, 5,690,807, 5,505,928, 5,262,357 (which are all incorporated herein in their entirety), and PCT publication No. 99/26299 (published 5/27 of 1999). In particular, exemplary materials for use as semiconductor nanocrystals in biological and chemical analysis include, but are not limited to, those described above, including group II-VI, III-V, and IV semiconductors, such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge, and Si, and ternary or quaternary mixtures thereof. Methods for attaching semiconductor nanocrystals to antibodies are described in U.S. patent nos. 6,630,307 and 6,274,323.

Nucleic acids

In some embodiments, there are recombinant polynucleotides encoding the proteins, polypeptides, or peptides described herein. Contemplated polynucleotide sequences include those encoding an antibody to SpA, or a SpA-binding portion thereof.

As used herein, the term "polynucleotide" refers to a nucleic acid molecule that is recombinant or has been isolated from total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids having 100 or fewer residues in length), recombinant vectors, including, for example, plasmids, cosmids, phagemids, viruses, and the like. In some aspects, a polynucleotide comprises regulatory sequences substantially isolated from the coding sequence of a gene or protein in which it naturally occurs. The polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may, but need not, be present within the polynucleotide.

In this regard, the terms "gene," "polynucleotide," or "nucleic acid" are used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequence required for appropriate transcription, post-translational modification, or localization). As will be understood by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or may be suitable for expression of proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may comprise a contiguous nucleic acid sequence encoding all or part of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that contains changes with slightly different amino acid sequences but encodes the same or substantially similar protein (see above).

In particular embodiments, there are isolated nucleic acid segments and recombinant vectors comprising a nucleic acid sequence encoding a polypeptide (e.g., an antibody or fragment thereof) that binds SpA. The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, which generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a replication product of such a molecule.

Regardless of the length of the coding sequence itself, the nucleic acid segment may be used in combination with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that its overall length may vary significantly. It is thus contemplated that accounting fragments of almost any length may be employed, with the overall length preferably being limited by ease of preparation and use in contemplated recombinant nucleic acid protocols. In some cases, the nucleic acid sequence may encode the polypeptide sequence with additional heterologous coding sequences, e.g., to provide purification, transport, secretion, post-translational modification of the polypeptide, or to provide a therapeutic benefit, e.g., targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not identical to the modified polypeptide.

In some embodiments, there are polypeptide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more (including all values and ranges therebetween) sequence identity compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In some aspects, an isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and greater identity over the entire length of the sequence to an amino acid sequence described herein; or comprising a nucleotide sequence complementary to said isolated polynucleotide.

A. Carrier

The polypeptide may be encoded by a nucleic acid molecule. The nucleic acid molecule may be in the form of a nucleic acid vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence may be inserted for introduction into a cell, where it may be replicated and expressed. A nucleic acid sequence may be "heterologous," meaning that it is in an environment different from the cell into which it is introduced into the vector, or different from the nucleic acid into which it is incorporated, comprising a sequence that is homologous to a sequence in the cell or in the nucleic acid, but not in a location within the host cell or nucleic acid in which it is not normally found. Vectors include DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the art will be able to construct vectors by standard recombinant techniques (e.g., Sambrook et al, 2001; Ausubel et al, 1996, both of which are incorporated herein by reference). The vector may be used in a host cell to produce an antibody that binds SpA.

The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of a gene product capable of being transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various "control sequences" which refer to nucleic acid sequences required for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. Vectors and expression vectors may contain nucleic acid sequences having other functions in addition to control sequences that govern transcription and transfer, and are described herein.

A "promoter" is a control sequence. Promoters are generally regions of a nucleic acid sequence that control the initiation and rate of transcription. It may contain genetic factors where regulatory proteins and molecules can bind, for example, RNA polymerase and other transcription factors. The phrases "operatively placed," "operatively linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and/or orientation with respect to the nucleic acid sequence to control transcriptional initiation and expression of the sequence. Promoters may or may not be used with "enhancers" which refer to cis-acting regulatory sequences involved in the activation of transcription of a nucleic acid sequence.

The particular promoter used to control expression of the polynucleotide encoding the peptide or protein is not considered critical, so long as it is capable of expressing the polynucleotide in the targeted cell, preferably a bacterial cell. When targeting human cells, the polynucleotide coding region is preferably placed adjacent to and under the control of a promoter that is capable of being expressed in human cells. In general, such promoters may include bacterial, human or viral promoters.

Effective translation of a coding sequence may also require specific initiation signals. These signals include the ATG initiation codon or adjacent sequences. It may be desirable to provide exogenous transcriptional control signals, including an ATG initiation codon. One skilled in the art would be readily able to determine this and provide the necessary signals.

The vector may contain a Multiple Cloning Site (MCS), which is a region of nucleic acid containing multiple restriction enzyme sites, any of which may be used with standard recombinant techniques to digest the vector. (see Carbonelli et al, 1999, Levenson et al, 1998 and Cocea, 1997, incorporated herein by reference.)

Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splice sites to ensure proper processing of the transcript for protein expression. (see Chandler et al, 1997, incorporated herein by reference.)

The carrier or construct will typically contain at least one termination signal. A "termination signal" or "terminator" is comprised of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in some embodiments, a termination signal is contemplated that terminates production of the RNA transcript. The terminator may be necessary in vitro to achieve the desired level of messenger. In eukaryotic systems, the terminator region may also comprise specific DNA sequences that allow site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized exogenous polymerase to add a stretch of about 200A residues (polyA) to the 3' end of the transcript. RNA molecules modified with the polyA tail appear to be more stable and may be translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator comprises a signal for RNA cleavage, more preferably that the terminator signal promotes polyadenylation of the messenger.

In expression, particularly eukaryotic expression, a polyadenylation signal will typically be included to produce appropriate polyadenylation of the transcript.

For propagation of the vector in a host cell, it may contain one or more origins of replication (often referred to as "ori"), which are specific nucleic acid sequences at the start positions of replication. Alternatively, when the host cell is yeast, an Autonomously Replicating Sequence (ARS) is employed.

B. Host cell

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. All of these terms also include their progeny, which are any and all of the progeny. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can and have been used as recipients for vectors or viruses. A host cell can be "transfected" or "transformed," which refers to a process by which a foreign nucleic acid, e.g., a recombinant protein coding sequence, is transferred or introduced into the host cell. Transformed cells include primary test cells and their progeny.

Some vectors may employ control sequences that allow them to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One skilled in the art will further understand the conditions under which all of the above host cells are cultured to maintain them and allow the vector to replicate. It is also understood and appreciated that conditions will allow for large scale production of the vector and the production of the nucleic acid encoded by the vector and its cognate polypeptide, protein or peptide.

C. Expression system

There are a number of expression systems comprising at least part or all of the above compositions. Prokaryotic-and/or eukaryotic-based systems may be employed for embodiments to produce nucleic acid sequences or their homologous polypeptides, proteins, and peptides. Many such systems are commercially available or readily available.

The insect cell/baculovirus system can produce high levels of protein expression of heterologous nucleic acid segments, such as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both of which are incorporated herein by reference, and the system can be derived, for example, fromBy name2.0 purchased and derived fromBACPACK by name^TMBaculovirus expression systems are commercially available.

In addition to the disclosed expression systems, other examples of expression systems includeCOMPLETE CONTROL (C)^TMAn inducible mammalian expression system, which relates to the synthetic ecdysone inducible receptor or its pET expression system, an E.coli expression system. Another example of an inducible expression system can be derived fromObtaining, which carries T-REX^TM(tetracycline regulated expression) system, an inducible mammalian expression system using the full-length CMV promoter.Also provided is a yeast expression system, called the Pichia methanolica (Pichia methanolica) expression system, designed for high level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One skilled in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

D. Method for gene transfer

Suitable methods for nucleic acid delivery to effectively express the composition are believed to include virtually any method by which nucleic acids (e.g., DNA, including viral and non-viral vectors) can be introduced into cells, tissues, or organisms, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, for example by injection (U.S. Pat. nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and VanDer Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct acoustic loading (Fechheimer et al, 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al, 1989; Kato et al, 1991); by microprojectile bombardment (PCT publication Nos. WO94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al, 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); transformation mediated by Agrobacterium (Agrobacterium) (U.S. Pat. nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al, 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); mediated DNA uptake by desiccation/inhibition (Potrykus et al, 1985). Organelles, cells, tissues or organisms can be transformed permanently or temporarily by application of techniques such as these.

Method of treatment

As discussed above, compositions and methods using these compositions can treat subjects (e.g., limit bacterial burden or abscess formation or persistence) having, suspected of having, or at risk of having an infection or a related disease, particularly those associated with staphylococci. One use of the composition is to prevent nosocomial infections by vaccinating the subject prior to hospitalization.

As used herein, the phrase "immune response" or its equivalent "immunological response" refers to an immunological response (antibody-mediated), a cellular response (mediated by antigen-specific T cells or their secretory products), or both an immunological and a cellular response in a recipient patient to a protein, peptide, or polypeptide of the invention. The treatment or therapy may be an active immune response induced by administration of the immunogen or a passive therapy resulting from administration of the antibody, antibody-containing material, or primed T cells.

As used herein, "passive immunity" refers to any immunity that is administered to a subject by the application of an immune effector (e.g., a polypeptide that binds SpA protein) that includes a cellular mediator or a protein mediator. The antibody compositions may be used in passive immunization for the prevention or treatment of infections caused by organisms carrying antigens recognized by the antibodies. Antibody compositions can comprise antibodies or polypeptides comprising antibody CDR domains that bind various antigens, which can be associated with various organisms. The antibody component may be a polyclonal antiserum. In some aspects, the antibody is affinity purified from an animal or a second subject that has been challenged with the antigen. Alternatively, a mixture of antibodies, which are a mixture of monoclonal and/or polyclonal antibodies to an antigen present in the same, related or different microorganism or organism, such as a gram positive bacterium, a gram negative bacterium, including but not limited to a staphylococcal bacterium, may be used.

Passive immunization may be administered to a patient or subject by administering to the patient an immunoglobulin (Ig) or fragment thereof and/or other immune factors obtained from a donor or other non-patient source with known immune reactivity. In other aspects, a composition of antigens can be administered to a subject, who then serves as a source or donor of globulins that are produced ("hyperimmunoglobulins") in response to an attack from the composition that contain antibodies to staphylococci or other organisms. The subject so treated will donate plasma from which the hyperimmune globulin is then obtained via conventional plasma separation methods and administered to another subject to confer resistance to or treat a staphylococcal infection. Hyperimmune globulin is particularly useful for individuals who lack immunity, for individuals who undergo an invasive procedure, or for situations where the time is not allowed for the individual to produce its autoantibodies in response to vaccination. For exemplary methods and compositions related to passive immunization, see U.S. patents 6,936,258, 6,770,278, 6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018, each of which is incorporated by reference in its entirety.

For the purposes of the present specification and appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen that is responsive to or recognized by B cells and/or T cells. B cell epitopes can be formed from both contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are generally retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are generally treated with denaturing solventsIt is lost in time. Epitopes generally comprise at least 3, more usually at least 5 or from 8 to 10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an Epitope include those described in Epitope Mapping Protocols (1996). T cells recognize a contiguous epitope of about 9 amino acids of CD8 cells or about 13 to 15 amino acids of CD4 cells. T cells recognizing the epitope can be identified by an in vitro assay that measures antigen-dependent proliferation, such as that triggered by activated T cells in response to the epitope³H-thymidine incorporation (Burke et al, 1994), through antigen-determined killing (T lymphocyte assay for cytotoxicity, Tigges et al, 1996), or through cytokine secretion.

The presence of a cell-mediated immunological response can be determined by a proliferation assay (CD4(+) T cells) or CTL (cytotoxic T lymphocytes) assay. The corresponding contributions of the humoral and cellular responses to the protective or therapeutic effect of the immunogen can be distinguished by isolating IgG and T cells, respectively, from the immunocompromised animal and measuring the protective or therapeutic effect in a second subject. As used herein and in the claims, the terms "antibody" or "immunoglobulin" may be used interchangeably.

Optionally, the antibody, or preferably the immunological portion of the antibody, may be chemically conjugated to, or expressed as, a fusion protein with other proteins. For the purposes of the present specification and claims, all such fused proteins are included in the definition of antibody or immunological portion of an antibody.

In one embodiment, the method comprises treatment of a disease or condition caused by a staphylococcal pathogen. In some aspects, embodiments include methods of treating a staphylococcal infection, for example a hospital-acquired nosocomial infection. In some embodiments, the treatment is administered in the presence of a staphylococcal antigen. Further, in some embodiments, the treatment includes administering other agents commonly used against bacterial infections, such as one or more antibiotics.

The therapeutic composition is administered in a manner compatible with the dosage form and in an amount that will be therapeutically effective. The amount administered depends on the subject to be treated. The precise amount of active ingredient to be administered depends on the judgment of the practitioner. Suitable regimens for initial application and synergy are also variable, but typically are initial application followed by subsequent application.

The manner of application may vary widely. Any conventional method for administering a polypeptide therapeutic is applicable. These are considered to include oral use, parenteral, by injection, etc. on a solid physiologically acceptable carrier or in a physiologically acceptable dispersion. The dosage of the composition will depend on the route of administration and will vary depending on the size and health of the subject.

In some examples, it is desirable to have multiple administrations of the composition, e.g., 2,3, 4,5, 6, or more administrations. Administration can be at 1,2, 3, 4,5, 6,7, 8 to 5,6, 7,8, 9, 10, 11, 12 twelve week intervals, including all ranges therebetween.

A. Antibodies and passive immunization

Some aspects relate to methods of producing antibodies for preventing or treating staphylococcal infection comprising the steps of immunizing a recipient with a vaccine and isolating antibodies from the recipient or producing recombinant antibodies. Another aspect is an antibody prepared by these methods and used to treat or prevent staphylococcal infection. Another aspect is a pharmaceutical composition comprising an antibody that specifically binds SpA and a pharmaceutically acceptable carrier, which can be used in the manufacture of a medicament for treating or preventing staphylococcal disease. Another aspect is a method for treating or preventing a staphylococcal infection comprising administering to a patient an effective amount of a pharmaceutical formulation.

The inoculum for polyclonal antibody production is generally prepared by dispersing an antigenic composition (e.g., a peptide or antigen or epitope of SpA or a common portion thereof) in a physiologically tolerable diluent, such as physiological saline or other adjuvant suitable for human use, to form an aqueous composition. An immunostimulatory amount of the inoculum is administered to the mammal, and the vaccinated mammal is maintained for a period of time sufficient for the antigen composition to induce protective antibodies. Antibodies can be isolated to the desired extent by well-known techniques, such as affinity chromatography (Harlow and Lane, Antibodies: A laboratory Manual 1988). The antibody may comprise an antiserum preparation from various commonly used animals, such as goats, primates, donkeys, pigs, horses, guinea pigs, rats or humans. Animals were bled and serum restored.

The antibody may comprise a whole antibody, an antibody fragment, or a subfragment. The antibody may be a whole immunoglobulin of any class (e.g., IgG, IgM, IgA, IgD, or IgE), a chimeric antibody, a human antibody, a humanized antibody, or a hybrid antibody with bispecific to two or more antigens. They may also be fragments (e.g., F (ab ')2, Fab', Fab, Fv, etc., including hybrid fragments). Antibodies also include natural, synthetic, or genetically engineered proteins that behave like antibodies by binding to a specific antigen with sufficient affinity.

The vaccine may be administered to a recipient, which then serves as a source of antibodies that are produced in response to challenge from a specific vaccine. The subject so treated will donate plasma from which antibodies will be obtained via conventional plasma separation methods. The isolated antibody will be administered to the same or a different subject to confer resistance to or treat a staphylococcal infection. The antibodies are particularly useful for treating or preventing staphylococcal disease in infants, in immunocompromised individuals, or in situations where treatment is required but the individual has not had time to mount a response to vaccination.

Further aspects are pharmaceutical compositions comprising two or more antibodies or monoclonal antibodies (or fragments thereof; preferably human or humanized) reactive with at least two components of an immunogenic composition, which can be used for the treatment or prevention of infections caused by gram-positive bacteria, preferably staphylococci, more preferably staphylococcus aureus or staphylococcus epidermidis.

B. Combination therapy

The compositions and related methods, particularly the administration of antibodies that bind to SpA or peptides or their consensus peptides to patients/subjects, can also be used in combination with the administration of traditional therapies. These include, but are not limited to, administration of antibiotics such as streptomycin, ciprofloxacin, doxycycline, gentamicin, chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin, tetracycline, or a combination of various antibiotics.

In one aspect, the contemplated treatment is used with an antimicrobial treatment. Alternatively, the treatment may precede or follow treatment with other agents at time intervals ranging from minutes to weeks. In embodiments, when the other agent and/or protein or polynucleotide is administered separately, it will generally be ensured that no significant period of time passes between the times of delivery, such that the therapeutic composition will always be able to exert a beneficial combined effect on the subject. In such cases, it is contemplated that the two modalities may be administered within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. In some cases, it is desirable to significantly extend the period for administration, however, with days (2, 3, 4,5, 6, or 7) or weeks (1, 2,3, 4,5, 6,7, or 8) passing between the respective administrations.

Various combinations of treatments may be employed, for example, antibiotic treatment is "a" and antibody treatment including antibodies that bind SpA or peptides or their consensus peptides is "B":

administration of the antibody composition to a patient/subject will follow the general protocol for administering such compounds, taking into account the toxicity of the composition, if any. It is contemplated that the processing cycle may be repeated if desired. It is also contemplated that various standard treatments, such as hydrotherapy, may be administered in combination with the treatment.

C. General pharmaceutical compositions

In some embodiments, the pharmaceutical composition is administered to a subject. Various aspects can involve administering an effective amount of the composition to a subject. In some embodiments, an antibody that binds SpA or a peptide or a consensus peptide thereof may be administered to a patient to prevent or treat infection by one or more bacteria from the genus staphylococcus. Alternatively, expression vectors encoding one or more such antibodies or polypeptides or peptides may be administered to a patient as a prophylactic treatment. In addition, such compositions may be administered in combination with an antibiotic. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce side effects, allergic reactions, or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, may also be incorporated into the composition.

The active compounds can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous or even intraperitoneal routes. Generally, such compositions may be prepared as liquid solutions or suspensions; solid forms suitable for addition of liquids to prepare solutions or suspensions prior to injection may also be prepared; and may also emulsify the formulation.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations comprising sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The protein composition may be formulated in a neutral or salt form. Pharmaceutically acceptable salts, including the acid addition salts (formed using the free amino groups of the protein), are formed using inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

The pharmaceutical compositions may comprise a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained by: for example by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by the following method: the active compound is combined in the required amount with the various other ingredients enumerated above, in a suitable solvent, followed by filter sterilization or an equivalent process, if desired. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients selected from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Administration of the compositions will generally be via a variety of conventional routes. This includes, but is not limited to, oral, nasal or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In some embodiments, the vaccine composition may be inhaled (e.g., U.S. patent 6,651,655, which is specifically incorporated by reference). Such compositions will typically be administered in a pharmaceutically acceptable composition comprising a physiologically acceptable carrier, buffer or other excipient.

An effective amount of the therapeutic or prophylactic composition is determined based on the intended goal. The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a composition calculated to produce the desired response described above in connection with its administration, i.e., the appropriate route and regimen. The amount to be administered depends on the desired protection, both on the number of treatments and the unit dose.

The precise amount of the composition will also depend on the judgment of the practitioner and will be unique to each individual. Factors that affect dosage include the physical and clinical state of the subject, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and efficacy, stability, and toxicity of the particular composition.

After formulation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically or prophylactically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above.

V. examples

The following examples are given for the purpose of illustrating various embodiments and are not intended to limit the invention in any way. Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples of the present invention, together with the methods described herein, presently represent preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Those skilled in the art will envision other uses and variations therein that are within the spirit of the invention as defined by the scope of the claims.

Example 1

Monoclonal antibody of staphylococcus aureus protein A

SpA_KKAAmAb protection of mice from diseases caused by staphylococci. BALB/c mice use purified SpA using a prime-boost protocol_KKAAImmunisation and antigen specific IgG responses were quantified by ELISA. Animals were euthanized and their spleen cells were fused with myeloma cells. The resulting hybridomas are screened to produce antigen-specific mabs. Initially, protein a-specific mabs were screened using functional assays and a murine infection model (table 1). After the primary screen, three mabs (5a10, 3F6, and 3D11) were selected for further characterization, as these antibodies showed the best immunoprotection in each isotype group (table 1). BALB/c mice were immunized with affinity purified mAbs (5 m)_g·k_g ^-1Body weight) and by mixing 1 × 10⁷CFU staphylococcus aureus Newman, a methicillin-sensitive clinical isolate (MSSA) (Baba et al, 2007) was injected into the periorbital sinus of the right eye for challenge. Four days after challenge the ability of staphylococci to grow abscesses in kidney tissue was examined by histopathological section (table 1). In control mice (with 5 mg. kg)^-1Isotype control mAb) yielded 5.02log in homogenized kidney tissue₁₀CFU·g^-1(IgG₁)、4.64log₁₀CFU·g^-1(IgG_2a) And 5.24log₁₀CFU·g^-1(IgG_2b) Average staphylococcal load (table 1). Animals receiving the protein A-specific mAb showed a reduction in staphylococcal load compared to control treated with isotype mAb [2.80log₁₀CFU·g^-1(5A10)、2.28log₁₀CFU·g^-1(3F6) And 2.72log₁₀CFU·g^-1(3D11)]And a reduction in abscess formation (table 1). Notably, not all SpA's are_KKAAMabs all protected against disease caused by staphylococci (table 1), although these antibodies bound with considerable affinity to their antigens (see e.g. 3a6 and 6D11 in table 3).

TABLE 1 SpA_KKAAThe monoclonal antibody of (3) is passively immunized against mice

SpA_KKAAmAbb protects mice from MRSA challenge. With mAb5A10, 3F6, 3D11(5mg kg)^-1) Or a combination of these three mAbs (15mg kg)^-1) A group of BALB/c mice was immunized and challenged with strain MW2, a community-acquired virulent MRSA isolate (Baba et al, 2002). Animals receiving any of the three mabs (5a10, 3F6, 3D11) carried a reduced bacterial load and fewer staphylococcal abscesses in kidney tissue compared to isotype mAb treated controls (table 2). Using a mixture of these three mAbs (15mg kg)^-1) Immunized animals showed even greater reduction in staphylococcal load (2.03 log reduction)₁₀CFU·g^-1；P<0.0002) and reduction of abscess formation (vaccine versus mock, P)<0.0004). The enhanced protection is likely due to an increased concentration of mAb administered (15mg kg)^-1Relative to 5 mg/kg^-1). We conclude this hypothesis because these three antibodies, although recognizing similar structural features, do not appear to occupy the same binding site on SpA (see below). In addition, increasing the concentration of only one of the three mabs (3F6) produced the same effect: enhanced protection against diseases caused by staphylococci (see below).

In addition to providing immediate protection against staphylococcal challenge, SpA provides immediate protection_KKAAThe specific mAb also counteracts B-cell superantivity of SpAProbiotics (Goodyea and Silverman, 2003), thereby enabling the infected host to mount an antibody response to a number of different staphylococcal antigens (Kim et al, 2010 a). To test this possibility, mAb3F6 or its IgG was used_2aIsotype control (20 mg. kg)^-1) BALB/c mice were passively immunized and then challenged intravenously with Staphylococcus aureus MW 2. Fifteen days after challenge, animals were euthanized and examined for staphylococcal load in organ tissues (fig. 1A). Mice immunized with mAb3F6 carried a reduced staphylococcal load (4.77 log reduction)₁₀CFU·g^-1P =0.0013) and reduced number of abscesses [ from 10.14(± 2.08) (IgG)_2a) To 3.00(± 1.00) (3F6), P = 0.0065; FIG. 1A]. Blood samples drawn 15 days after challenge were examined for serum IgG reactivity against fourteen staphylococcal antigens: coa, ClfA, ClfB, EsxA, EsxB, FnBPA, FnBPB, Hla, IsdA, IsdB, LukD, SdrD, SpA_KKAAAnd vWbp (detent et al, 2012), these fourteen staphylococcal antigens are considered as protective antigens for vaccine development. SpA actively inoculated as before_KKAAAs observed in the animals of (a), mice immunized with mAb3F6 developed higher serum IgG titers against several different staphylococcal antigens (fig. 1B) (Kim et al, 2010 a). In particular, IgG levels to Coa, ClfA, EsxA, EsxB, FnBPB, Hla, IsdA, LukD, SdrD and vWbp were increased in serum samples from animals immunized with mAb3F6 compared to the control group. However, there was no increase in serum IgG to IsdB (staphylococcal hemoglobin carrier (Mazmanian et al, 2003)) (fig. 1B). After mAb3F6 passive transfer, to SpA_KKAAThe IgG titer of (1) was maintained for fifteen days (FIG. 1B).

During infection by staphylococci, soluble SpA is expected to be recognized by mAb3F6 to form Immune Complexes (IC) which are then phagocytosed by immune cells. The phagocytosed SpA is then treated with proteases in phagolysosomes and peptide fragments are presented to T and B cells to produce polyclonal antibodies. As a validation experiment, groups of animals received affinity purified recombinant protein A variants [ SpA, SpA ] on days 0 and 11 in the presence of mAb3F6 or its isotype control_KK、SpA_AA、SpA_KKAAAnd Simulant (PBS)]A mixture of (a). On day 21, animals were euthanized and their ability to elicit different species of SpA-specific antibodies was measured by ELISA. All animals not treated with mAb failed to produce SpA-specific antibody responses (figure 6). In addition, B cell superantigens (SpA and SpA) were received_KK(ii) a See below)) failed to produce SpA-specific IgG even in the presence of mAb3F6₁And IgG_2aAntibodies (fig. 6). However, with SpA variants lacking B cell superantigen activity (SpA)_AAAnd SpA_KKAA(ii) a See below)) the treated mice were able to produce significant amounts of IgG₁(FIG. 6). Although estimated amount of soluble protein A during infection (every 10 th)⁷CFU, 5-10 ng) was much lower than the dose of affinity purified protein a injected into animals in these experiments, but the data in fig. 6 indicate the potential role of SpA-specific T/B cells in counteracting B cell superantigen activity. Taken together, the inventors speculate that active vaccination SpA_KKAA(Kim et al, 2010a), passive immunization of mice infected with s.aureus (see below) without a neutralizing mAb can increase the significant levels of protein a-specific antibodies.

TABLE 2 SpA_KKAAmAb immunization protects mice from MRSA challenge

mAb Spa27 failed to recognize Spa_KKAAAnd therefore do not elicit protective immunity in mice. Spa27 is a commercially available protein a-specific monoclonal antibody (Sigma) that has been used for the detection of staphylococcal protein a over the last two decades (Perry et al, 2002). The Spa27 hybridoma was obtained from a mouse that had been immunized with wild-type staphylococcal protein a purified from s.aureus strain Cowan I (Sjoquist et al, 1972). Previous work showed that wild-type protein A elicits clonal expansion and shrinkage of B cell populations (Forsgren et al, 1976; Goodyear et al, 2003), thereby preventing protein A-specific immune responses in mice (Goodyear et al, 2004), and wild-type protein A elicitsType A protein contains Fc gamma and Fab V_H3 (Graille et al, 2000; Stahlenheim et al, 1970). Therefore, we wanted to know whether Spa27 recognizes wild-type protein a as an antigen. To solve this problem, we used ELISA using purified recombinant protein a (SpA), or its lack of specific binding to Fc γ (SpA)_KK)、V_H3 Fab Domain (SpA)_AA) Or both (SpA)_KKAA) (iii) variants of (iii) (fig. 7). The data show that Spa27 binds strongly to wild-type Spa and Spa_KKBut not bound to SpA_AAOr SpA_KKAA(FIG. 9B). Spa27 is a mouse IgG₁Isotype antibodies, which explain their inability to bind protein a via Fc γ (Kronvall et al, 1970). Spa27 and Spa_AAOr SpA_KKAAThe weak association between them may be due to the seemingly least likely SpA27 requiring residues D36/D37 in each of the five igbds to recognize antigen, or the more likely SpA27 for us to bind SpA via its Fab domain, assuming that the antibody belongs to V_H3 or a related species.

For pairwise comparison, we performed mAb3F6 or Spa27(5 mg. kg)^-1) The biological function of Spa27 was examined by injection into the peritoneal cavity of BALB/c mice. These animals were then challenged with a community-acquired virulent MRSA strain (Diep et al, 2006), staphylococcus aureus USA300(LAC), which is prevalent in the united states. On day 4 post challenge, animals were euthanized and bacterial load in the kidneys of infected animals was determined (fig. 9B). Animals receiving mAb3F6 carried reduced bacterial load (1.38 log reduction) compared to mock (PBS) treatment₁₀CFU·g^-1P = 0.0011). In contrast to the protective immunity elicited by 3F6, mAb Spa27 failed to reduce bacterial load in the kidneys of infected animals (a 0.20log reduction)₁₀CFU·g^-1P = 0.2111). These data indicate that mAb Spa27 does not provide protection against disease caused by staphylococci. In addition, the experiments of Spa27 demonstrated that immunization of mice with wild-type protein a might not elicit monoclonal antibodies that can pass through the immunomodulatory properties of protein a binding this molecule as an antigen.

Recognition by mAbsSpA_KKAA. By SpA_KKAAMicrotiter plates were coated and the affinity constant (K) of the purified mAb was determined by ELISA_a=[mAb·Ag]/[mAb]×[Ag]). mAb3F6 showed the highest affinity (K)_a22.97×10⁹M^-1) And secondly mAb5A10 (K)_a8.47×10⁹M^-1) And mAb3D11 (K)_a3.93×10⁹M^-1Table 3). Each of the five IgBDs is examined individually (E)_KKAA、D_KKAA、A_KKAA、B_KKAAAnd C_KKAA) Or checking for inclusion of IgBD E_KKAAAntibody binding of the peptides of helices 1,2 or 3 and helices 1+2 and 2+3 of the domain (table 3). mAb5A10 and mAb3F6 to SpA_KKAAThe same affinity binds all five igbds. mAb5a10 was unable to bind to the helical peptide, whereas mAb3F6 showed weak affinity for the helical 1+2 peptide. mAb3D11 binding to B_KKAAAnd C_KKAAWeakly bound to A_KKAACannot be bound to E_KKAAAnd D_KKAA. In conclusion, SpA, which gave the highest level of protection against staphylococcal disease in mice_KKAAMabs bind to some or all of the five igbds, but not to peptides that contain only one or two of the three helices of igbds. These data indicate that the protective mAb recognizes conformational epitopes of the three helical bundles of each IgBD.

To examine whether the affinity of the mAb plays a significant role in immune protection, ELISA was performed with increasing concentrations of the chaotropic agent ammonium thiocyanate (figure 2). The measured avidity of mAb3F6 was significantly higher than that of mAb5a10 and mAb3D11 (fig. 2). Notably, 3D11 exhibited a relatively low affinity, probably due to its specific interaction with only two of the five igbds (fig. 2 and table 3). We can therefore conclude that the avidity of mabs may not be a major determinant of their immune protection against mice.

U.S. patent application publications US2008/0118937 and US2010/0047252 describe a murine hybridoma cell line obtained from a mouse immunized with wild-type protein a. The corresponding antibody mab358a76.1.1 was reported to associate with wild-type protein a; however, the molecular nature of this association has not been revealed. To further explore the properties of B cells in response to wild-type protein a, mab358a76.1.1 was isolated and its functional attributes studied. Unlike the SpAKKAA-mAb, the 358a76.1 antibody only binds to the E domain of SpA, and is unable to bind to any of the other four igbds (D, A, B and C). In addition, mab358a76.1 neither counteracted protein a nor promoted opsonophagocytic killing of staphylococci, and passive transfer of mab358a76.1 failed to protect mice from disease caused by staphylococcus aureus.

Monoclonal antibody 358a76.1 only weakly binds to the E domain of SpA. To determine the affinity constant (K) for mAb358A76.1 binding to protein A_a=[mAb·Ag]/[mAb]×[Ag]) By SpA_KKAAIgBD (E)_KKAA、D_KKAA、A_KKAA、B_KKAAOr C_KKAA) And comprises E_KKAASynthetic peptides for each helix (H1, H2, and H3) or for both helices (H1+2 and H2+3) of the three helix bundle coat the microtiter plates. mAb3F6 is derived from SpA_KKAAThe resulting mouse monoclonal antibody to full-length SpA_KKAA(K_a22.97×10⁹M^-1) And each of five IgBD_a12.41-27.46×10⁹M^-1) All have high affinity. mAb358A76.1 vs. SpA compared to mAb3F6_KKAAShow much weaker affinity (K)_a1.00×10⁹M^-1FIGS. 10A-B). Notably, mab358a76.1 only binds to E_KKAA(K_a0.21×10⁹M^-1) And cannot bind to any of the other four IgBD (D)_KKAA、A_KKAA、B_KKAAOr C_KKAATable 6). In addition, the inclusion of E could not be identified by mAb358A76.1_KKAAAny of the synthetic peptides of one or both helices (H1, H2, H3, H1+2, and H2+3) of IgBD. In comparison, mAb3F6 showed weak affinity for helix 1+2 peptide (table 6). Alignment of the amino acid sequences of all five igbds all show that the E domain is the least similar domain (Sjodahl, 1977). However, the importance of dissimilarity of the E domain, like the other four igbds, in association with human and animal immunoglobulins has not been noted (Moks et al,

1986) (FIGS. 10C-D). mab358a76.1 can specifically bind to conformational epitopes of the non-conserved amino acids of helices 1 and 3 that contain the E domain (fig. 10C-D).

To check whether mAbs 3F6 and 358A76.1 share SpA_KKAA(E_KKAA) The epitope binding site of (A), we used an increased concentration of IgG_2aIsotype control, mab358a76.1 or mAb3F6 performed competition ELISAs. At 30_μg·ml^-1Isotype control antibody (I) at concentration_gG_2a) Did not interfere with HRP-conjugated mAb358A76.1 or mAb3F6 binding to SpA_KKAA(FIG. 10E). As expected, the mAb358A76.1 competed with the HRP-conjugated mAb358A76.1 for binding to SpA_KKAAHowever, it did not interfere with binding of HRP-conjugated mAb3F6 (fig. 10E). Notably, mAb3F6 was able to completely block HRP-conjugated mab358a76.1 from associating to SpA_KKAA(96.4% inhibition, fig. 10E), whereas mab358a76.1 produced only 88.0% inhibition (mAb3F6 versus mab358a76.1, P = 0.0007).

Monoclonal antibody 358a76.1 did not elicit protective immunity in mice. 5 mg/kg^-1mAb358A76.1 or mAb3F6 was injected into the peritoneal cavity of a group of BALB/c mice. Passively immunized animals were challenged with virulent MRSA strains associated with the community prevalent in the United states (Diep et al, 2006; Kennedy et al, 2008) Staphylococcus aureus USA300 (LAC). Animals receiving mAb3F6 carried reduced bacterial load in kidney tissue (1.26 log reduction) compared to controls treated with IgG2a isotype mAb₁₀CFU·g^-1(ii) a P =0.0021, fig. 11A). Interestingly, animals receiving mab358a76.1 showed only a small reduction in bacterial load, which failed to achieve statistical significance (0.42 log reduction)₁₀CFU·g^-1(ii) a P =0.0948, fig. 11A). Passive transfer of mAb3F6 produced increased protection against CA-MRSA strain USA300 in immunized mice (0.84 log reduction) compared to mAb358A76.1₁₀CFU·g^-1(ii) a P =0.0011, fig. 11A).

Opsonophagocytosis to kill invading microorganisms is a key defense strategy for infected hosts and also represents a key defense against protective immunity for many different bacterial vaccinesIn combination (Robbins et al, 1987; Robbins et al, 1990). By testing opsonophagocytic killing in fresh mouse blood, we sought whether mab358a76.1 could promote opsonophagocytic killing of MRSA strain USA 300. Simply, at 10. mu.g.ml^-1Anticoagulated blood obtained from 6-week-old BALB/c mice first tested was cultured with Staphylococcus aureus USA300 in the presence or absence of mAb358A76.1, mAb3F6 or mAb IgG2a isotype control. Blood samples were lysed, plated on agar medium and staphylococcal load calculated. The mab358a76.1 and IgG2a controls failed to activate opsonophagocytic killing of staphylococci compared to mAb3F6 which reduced staphylococcal load by 49% in blood (mAb3F6 vs PBS, P<0.0001; mAb3F6 relative to 358a76.1, P = 0.0007; fig. 11B).

During infection, protein a, together with immunoglobulins, traps and decorates the surface of staphylococci. By associating with the Fc γ domain of immunoglobulins, SpA prevents complement activation, Fc receptor involvement, and opsonization of staphylococci by phagocytes. In addition, the SpA molecules released from the bacterial surface crosslink V_HType 3B cell receptors activate lymphocytes, eventually leading to their apoptosis and impeding the development of an adaptive immune response to staphylococci. We used ELISA to determine if mab358a76.1 could counteract the immunoglobulin binding activity of protein a. As a control, the concentration was 6. mu.g/ml^-1And 30. mu.g.ml^-1Next, mAb3F6 counteracted the ability of protein a to bind human IgG, while the IgG2a isotype control mAb did not (fig. 11C). In addition, the concentration was 6. mu.g/ml^-1Or 30. mu.g/ml^-1At concentrations of (a), mab358a76.1 did not prevent human IgG from associating to protein a (fig. 11C).

Table 6mAb358A76 and mAb3F6 binding to SpA_KKAAAnd association constant of fragments thereof

^aCross-coating with antigen (for SpA)_KKAA20nM, 100nM for IgG binding domain) ELISA plates serially diluted with affinity purified antibody (100. mu.g.ml)^-1) To utilize(GraphPad Software, Inc.) to calculate the association constants. To investigate the binding of antibodies to the protein A antigen, we used each of the five immunoglobulin-binding domains (IgBD) in protein A [ E (residues 1-56), D (residues 57-117), A (residues 118-]SpA with four amino acids replaced_KKAAVariants (residues 1-291 of mature SpA carrying six N-terminal histidine residues). In each IgBD, glutamine is lysine (Q) at positions 9 and 10 (amino acid residue of IgBD-E)⁹K，Q¹⁰K) Instead, aspartic acids 36 and 37 are replaced by alanine (D)³⁶A，D³⁷A) In that respect Introducing the same substitution across each IgBD: E_KKAA、D_KKAA、A_KKAA、B_KKAAAnd C_KKAA(all expressed with six N-terminal histidine tags and purified). SpA_KKAAAnd each IgBD was purified from the e.coli extract by affinity chromatography. Peptides H1, H2, H3, H1+2, and H2+3 were synthesized on a peptide synthesizer and purified by HPLC. The peptide comprises E_KKAAIgBD(SpA_KKAAResidues 1 to 56: NH (NH)₂-AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPK-COOH) helix 1 of three helix bundles (H1: NH)₂-AQHDEAKKNAFYQVLNMPNLNA-COOH)、2(H2:NH₂-NMPNLNADQRNGFIQSLKAAPSQ-COOH)、3(H3:NH₂-AAPSQSANVLGEAQKLNDSQAPK-COOH)、1+2(H1+2:NH₂-AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQ-COOH), or 2+3(H2+3: NH)₂-NMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPK-COOH).^bSymbol<Indicating that the measurement results are too low to allow determination of the association constant.

mAb3F6 bound Sbi. The secretory protein Sbi of Staphylococcus aureusFive different domains are composed (Zhang et al, 1998). The two N-terminal domains (1 and 2) are homologous to IgBD from SpA (Zhang et al, 1999). Domains 3 and 4 associate with complement component C3 and factor H, and it has been proposed that the C-terminal domain retains some of the secreted Sbi molecules within the staphylococcal envelope by binding to lipoteichoic acid (Burman et al, 2008; Smith et al, 2012). Domains 1 and 2 bind to the Fc γ portion of immunoglobulins (Atkins et al, 2008); this activity, consistent with the C3 and factor H binding properties of domains 3 and 4, promotes useless consumption of fluid complement components (Haupt et al, 2008). Sbi does not appear to produce B cell superantigen activity because its two igbds (domains 1 and 2) lack two typical aspartic acid residues at positions 36 and 37 (Graille et al, 2000; Lim et al, 2011). Recombinant protein His-Sbi comprising IgBD and complement binding domain_1-4Human IgG was retained in affinity chromatography experiments. His-Sbi_1-4/KKAAIs a conserved glutamine residue (Q) in domains 1 and 2^51,52And Q^103,104) That is, the predicted Fc gamma binding site of Sbi IgBD is replaced by lysine (K) and arginine (R) of the complement binding domain²³¹) And aspartic acid (D)²³⁸) Variants with residues replaced by alanine (a) (Haupt et al, 2008). His-Sbi_1-4/KKAAHuman IgG was not retained during the performance of affinity chromatography. His-Sbi when examined by ELISA_1-4Binds to mouse as well as human IgG and to the Fc and Fab domains of human IgG, while His-Sbi_1-4/KKAAThis is not the case. mAb5A10 and mAb3D11 did not bind to His-Sbi_1-4/KKAAHowever, 3F6 binds to the protein. Thus, mAb3F6 could either counteract Sbi or remove secreted Sbi from circulation, thereby preventing the consumption of complement factor C3 by staphylococci.

Using SpA_KKAABinding site competition experiments for mAbs. The ELISA study showed that three mabs 5a10, 3F6, and 3D11 bound to wild-type SpA with similar affinities (fig. 3A). IgG compared to 5A10₁The control antibody showed a small affinity for SpA. In addition, IgG compared to the affinity of mAb3D11_2bThe affinity of the control antibody decreased. IgG compared to 3F6_2aThe control antibody bound SpA with slightly reduced affinity. In the use of horseradish peroxidase conjugated mAb (5A)10-HRP, 3F6-HRP, and 3D11-HRP), the isotype control antibody did not prevent HRP-conjugated mAb from binding to SpA (fig. 3B). Addition of equimolar amounts of each mAb reduced binding of the corresponding HRP-conjugate (fig. 3B). mAb3D11 did not prevent HRP-5a10 or HRP-3F6 from associating with SpA, however mAb5a10 and mAb3F6 prevented HRP-3D11 from binding to SpA. mAb3F6 resulted in a slight decrease in binding of HRP-5a10 to SpA (fig. 3B). Finally, mAb5a10 was a weak competitor for 3F6-HRP binding to SpA (fig. 3B). These data indicate that the binding sites of the three mabs on the surface of the SpA triple helix bundle may be close to each other or even partially overlapping (table 3).

SpA_KKAAThe mAb prevents the immunoglobulin from associating with protein A. Same kind of V_HMouse antibodies of 3-related families (e.g. 7183, J606 and S107) bind SpA via their Fab portion, while those of the other VH families (J558, Q52, Sm7, VH10, VH11 and VH12) do not bind SpA (Cary et al, 1999). SpA_KKAAThe amino acid sequence of the Complementarity Determining Regions (CDRs) of a specific mAb is determined by sequencing cDNA derived from hybridoma transcripts. The data show that mAb5A10 belongs to the same species V_HFamily 37183; the Fab domain likely showed SpA affinity (table 4). mAb3F6 and mAb3D11 are members of the VH10 and J558 families, respectively (table 4); the association of the Fab domains of these antibody families with SpA is unknown.

TABLE 4 amino acid sequence of CDR region of monoclonal antibody

Wild type SpA and its variant SpA_KKAA、SpA_KKAnd SpA_AAPurified and used in ELISA binding studies with human IgG. As expected, SpA binds to IgG or its Fc γ and F (ab)₂Fragment of and SpA_KKAANot (fig. 7). SpA_KKVariants (10 glutamine residues all replaced by lysine) have a diminished ability to bind to Fc gamma fragments, but bind to F (ab)₂The capacity of the fragment was not impaired, whereas SpA_AAVariants (10 aspartic acid residues all replaced by alanine) bind to Fc γ, but not F (ab)₂(FIG. 7). All three mabs (5a10, 3F6, and 3D11) prevented human IgG from binding to SpA in a manner that exceeded the competition of isotype control mabs (fig. 4A). All three antibodies prevented human IgG from binding to SpA_KK(Fab binding) or SpA_AA(Fab binding) (FIG. 4A). Thus, SpA_KKAASpecific mabs prevent non-immunological association of SpA with immunoglobulins. Based on these data, we speculate that protein a-specific mabs interact with conformational epitopes comprising structural elements involved in helix 2, Fc γ and Fab interactions of SpA of IgBD.

If mAb3F6 binds to wild type SpA as an antigen on the surface of staphylococci, its Fc γ domain should be available to recognize complement or Fc receptors on the surface of immune cells. To validate this prediction, 3F6, its isotype control, and affinity purified Sbi were used_1-4And (5) culturing the staphylococcus aureus. Antibody-mediated co-precipitation resulted in soluble Sbi in the supernatant_1-4Reduction, which was analyzed as a measure of the availability of Fc γ sites on the surface of the bacteria. Incubation of staphylococci with control mAbs that can only associate with SpA in a non-immunological manner resulted in soluble Sbi_1-4Moderate reduction (fig. 4B). In comparison, incubation of staphylococci with mAb3F6 resulted in soluble Sbi_1-4Reduction, indicating that mAb3F6 bound SpA antigen on the bacterial surface while presenting its Fc γ domain with Sbi_1-4Association (fig. 4B).

To test whether mAb3F6 binding to SpA did occur in vivo, BALB/c mice were immunized with mAb3F6 or isotype control antibody. After injection of purified SpA into the peritoneal cavity, its abundance in circulation was assessed by taking a blood sample at 30 minutes. Injection of mAb3F6 treated animals resulted in accelerated clearance of SpA from the bloodstream compared to animals treated with control mAb (fig. 4C). It is postulated that immune recognition of SpA by mAb3F6 provides its Fc γ domain to mediate Fc-receptor mediated removal of antigen-antibody complexes from the bloodstream.

SpA_KKAAmAb to promote the opsonization of staphylococci in human and mouse bloodPhagocytosis and killing. The induction of an adaptive immune response that promotes opsonophagocytic killing of pathogens is a common goal of vaccine development and licensing (Robbins et al, 1996). This has not been achieved for s.aureus because this pathogen is protected from opsonizing antibodies by SpA and Sbi molecules exposed and secreted from its surface (Kim et al, 2011). To test the SpA_KKAAWhether mAb can promote opsonophagocytosis, using a test of bacterial kill in fresh blood developed by RebeccaLancefield (Lancefield, 1928). In 2_μg·ml^{- 1}Lepirudin anticoagulated blood from 6-week old BALB/c mice that were first tested was cultured with MSSA strain Newman in the presence or absence of mAb5a10, mAb3F6, and mAb3D11 or their isotype controls. Blood samples were lysed, plated on agar medium and staphylococcal load calculated (figure 5A). All three mabs caused opsonophagocytic killing of staphylococci, ranging from 37% (3D11, P =0.0025) to 33% (3F6, P =0.0478) and 16% (5a10, P =0.0280) of inoculum. As a test for opsonophagocytic killing of staphylococci in human blood, the inventors recruited healthy human volunteers and examined their sera for SpA _KKAAAntibody specificity. As previously reported, none of the volunteers carried serum antibodies against protein a (data not shown) (Kim et al, 2010 a). At 10. mu.g/ml^-1Anticoagulated human fresh blood samples were cultured with MSSA strain USA400(MW2) in the presence or absence of mAb5a10, mAb3F6 and mAb3D11 or their isotype controls (fig. 5B). All three mabs caused opsonophagocytic killing of staphylococci, ranging from 52% (3D11, P =0.0002) to 44% (3F6, P =0.0001) and 34% (5a10, P =0.0035) of the inoculum. Blood samples were spread on glass slides, stained with Giemsa and analyzed by microscopy. Blood samples cultured in the presence of mAb5a10, mAb3F6, and mAb3D11 carried staphylococci associated with neutrophils, i.e. they could be associated with these leukocytes or located intracellularly (fig. 5C-E). Blood samples cultured with isotype control mAb carried clusters of extracellular staphylococci (red arrows) and staphylococci associated with leukocytes (blue arrows) (fig. 5F-H).

Discussion of the related Art

Monoclonal antibodies offer unique opportunities for studying the biological properties of the humoral adaptive immune response of microbial surface products, showing the molecular properties of microbial immune evasion and protective immunity (Fischetti, 1989). For example, group A streptococcal M protein, a key virulence factor and the alpha-helical coiled-coil surface protein (Phillips et al, 1981), confers resistance to opsonophagocytic clearance, which can be overcome by a humoral adaptive immune response during infection (Lancefield, 1962; Scott et al, 1986). mAbs that bind to the alpha-helical coiled coil of the M protein do not induce opsonophagocytic killing of group A streptococci, however, they are achieved by mAbs directed against the N-terminal random coil domain (Jones and Fischetti, 1988; Jones et al, 1986). The N-terminal domain of the M protein is highly variable between clinical isolates, representing the molecular basis for type-specific immunity (Hollingshead et al, 1987; Lancefield, 1962).

Like streptococcal M protein, protein a also functions as a protective antigen for staphylococcus aureus (Stranger-Jones et al, 2006). Virtually all clinical isolates of S.aureus express protein A, however the amino acid sequence of IgBD is very conserved (McCarth)_yAnd Lindsa_y,2010). Infection by staphylococci in mice or humans does not elicit a protein a-specific humoral immune response (Kim et al, 2010a), which is explained by the B-cell superantigen activity of the molecule (Silverman and Goodyear, 2006). Using protein A variants, especially SpA_KKAAMolecular immunization elicits a humoral immune response in mice and rabbits; these antibodies cross-react with wild-type protein a and provide protection against staphylococcal disease in mice (Kim et al, 2010 a). When cultured in the blood of anticoagulated mice, the polyclonal rabbit antibody subjected to affinity purification can block B cell superantigen activity of wild-type protein A in the mice and enhance opsonophagocytic capability of neutrophils in the mice. In addition, mutants of Staphylococcus aureus lacking the structural gene of protein A (spa) exhibit significant pathogenic defects and are also permissiveThere is a number of developments in humoral immune responses to a number of different staphylococcal antigens as well as the development of protective immunity (Cheng et al, 2009; Kim et al, 2011). Thus, antibodies that counteract the immune-modulating properties of SpA may not only provide protection against acute staphylococcal infection, but may also enable the development of protective immune responses to other staphylococcal antigens and prevent recurrent staphylococcus aureus infections. The inventors combated SpA by raising mAb_KKAATo validate the prediction. All monoclonal antibodies that elicit protective immunity in mice recognize conformational epitopes of protein a and interact with their triple helix folds of IgBD. Importantly, SpA_KKAAThese monoclonal antibodies with strong affinity and cross-reactivity to multiple or all igbds also recognize wild-type protein a. When tested in vitro, mabs, specifically mAb5a10, mAb3F6, and mAb3D11, prevented protein a from associating with the Fc γ and Fab domains of immunoglobulins and triggered opsonophagocytic killing of staphylococcus aureus by phagocytic cells in mouse and human blood. When injected into the peritoneal cavity of mice, mabs elicited effective immunoprotection against both MSSA and MRSA staphylococcus aureus isolates. In addition, SpA_KKAAmAb mediated in vivo neutralization of SpA stimulates humoral immune response against several different S.aureus antigens, supporting SpA_KKAAHypothesis that antibodies inhibit the B-cell superantigen activity of staphylococci.

Notably, the magnitude of the antibody response to staphylococcal antigen in passively immunized mice was compared to that of SpA_KKAAThe immune response elicited in actively immunized mice was much lower. The main difference between active and passive immunization protocols is the development of antigen-specific T/B cell populations that control the host immune response, which is the result of the active immunization protocol. Warranting further investigation to confirm that protein A-specific T/B cells are improving appropriate systemic immune responses such as T_H1/17 whether or not the supplementation of Staphylococcus aureus infection by functional phagocytes mediated is critical (Spellberg et al, 2012).

Previous work demonstrated protein A vs V in mice_HOf type 3B cell receptorsSuperantigen activity (Goodyear et al, 2003). Notably, only 5-10% of mouse B cells are V_H3-cloned and sensitive to protein a superantigens (Silverman et al, 2006). However, protein A mutant staphylococci show a major defect in the causative agent of abscess formation in a mouse model for this disease (Cheng et al, 2009; Kim et al, 2011). Human V compared to mouse_HClone 3B cells contained up to 50% of the total B cell population (Berberian et al, 1993, Huang et al, 1992), suggesting that the effect of protein a superantigen activity during infection by staphylococci is likely to be greater than the human B cell population (Silverman et al, 2006). If so, protein A-mediated B cell activation can trigger V_HThe biased use of 3B cell clones and the development of non-physiological B cell populations. Finally, staphylococcal protein A expects V_H3 Positive B cells and V_HHuman hosts of type 3 antibodies. A similar protocol applies to the HIV envelope glycoprotein gp120, which is also similar to V_H3 clonal B cell interactions leading to V in AIDS patients_HClonal deletion of 3B cells (antibody genes) (Berberian et al, 1993; Berberian et al, 1991). These events are likely to be key factors in the prevention of counteracting antibody responses during HIV and staphylococcus aureus infections (Kim et al, 2012 b).

Work by others has attempted to isolate monoclonal antibodies against protein a. One such antibody, SPA27(Sigma, st. louis, MO), is classified as mouse IgG1, an Fc_γClasses of antibodies whose domains do not interact with protein A (Kronvall et al, 1970). However, even antibodies of the IgG1 subtype can bind to protein a by pseudo-immune association, provided that these antibodies carry VH 3-type Fab domains (Car)_yEt al, 1999; sasso et al, 1989). We have recently developed reagents that can distinguish between these possibilities. For example, wild type protein a (spa) binds immunoglobulins through Fc γ and VH 3-type Fab domains (Silverman et al, 2006). SpA_KKAssociated only with the Fab domain of VH 3-type antibodies, whereas SpA_AABinds only to the Fc γ domain of IgG, but not to the Fab domain of VH 3-type immunoglobulins (Kim et al, 2012 a). Using these reagents, we observed binding of SPA27 toWild type SpA and SpA_KKBut not bound to Sp_AAAOr SpA_KKAA(Kim et al, 2012 a). Therefore, SPA27 isolated from hybridomas after immunization of mice with wild-type protein a from staphylococcus aureus Cowan1 did not specifically recognize protein a (Kim et al, 2012 a). Instead, SPA27 is bound by protein a (Kim et al, 2012 a). As can be expected from these observations, SPA27 was unable to counteract the IgG or IgM binding activity of protein a and did not provide protection in mice later challenged by s.aureus infection (Kim et al, 2012 a).

We wanted to know if it is a general phenomenon that the host immune system is unable to produce counteracting antibodies during s.aureus infection or after immunization with wild-type protein a (Kim et al, 2012a, Kim et al, 2012 b). To test this model, we purified mab358a76.1, an antibody isolated after immunization of mice with wild-type protein a from staphylococcus aureus Cowan1 (Sjoquist et al, 1972) (U.S. patents US2008/0118937a1 and US2010/0047252a 1). Unlike mABSPA27, mAB358A76.1 is related to SpA and SpA_KK、Sp_AAAAnd SpA_KKAAAll showed immunoreactivity (Kim et al, 2012 a). We observed that the specific binding site for mAb358A76.1 was restricted to E_KKAADomain, and the antibody cannot recognize D_KKAADomain, A_KKAADomain, B_KKAADomain and C_KKAAA domain. At E_KKAABased on the amino acid dissimilarity with the other four igbds, we hypothesized that mab358a76.1 recognizes conformational epitopes on the surface of the E-IgBD domain. The association between mab358a76.1 and the E domain of protein a did not counteract the other four igbds (D, A, B and C). Unexpectedly, passive transfer of mab358a76.1 to naive mice did not protect against challenge with staphylococcus aureus and did not trigger opsonophagocytic killing of staphylococcus in the blood. Based on these observations, we propose to use only non-toxin-producing protein A such as SpA_KKAAImmunization can lead to the development of antibodies that counteract all of the IgBD of protein a and confer protection against s.

The general hypothesis that the cancellation of Fc γ and Fab binding activity of SpA for the data presented herein represents a correlation to protective immunity to staphylococcus aureus provides evidence: such antibodies are expected to trigger opsonophagocytic killing of pathogens in the blood and elicit antibodies that counteract the secreted pathogenic factors of staphylococci (Mazmanian et al, 1999).

Sequence analysis

Wild-type staphylococcus aureus protein a interacts with human IgG through 2 non-antigenic binding sites. The first is with the Fc constant region and the second is with the Fab heavy chain of human VH3 race (Fab binding also occurs with IgA and IgM). Thus, a mAb belonging to the mouse race corresponding to human VH3 is likely to have three possible and possibly competitive binding affinities to the wild-type antigen, one for Fc, one for Fab, and the third for antigen-specific binding mediated by CDRs. CDR sequence analysis was performed on hybridoma cell lines producing SpAKKAA-specific monoclonal antibodies. Each individual antibody comprises two antigen recognition sites (Fab fragments), wherein each Fab portion comprises three CDRs in the light chain and three CDRs in the heavy chain. Mabs identified as conferring protection against s.aureus infection included 5a10, 3F6, 3D11, 5a11, 1B10, and 4C 1.

3F6, which provides important protection, presented the most distinctive sequence among a series of SpAkkaa mAbs. While having the same light chain CDR sequences as 1F10 and 6D11, 3F6 has a unique heavy chain CDR sequence that differs from 1F10 and 6D 11. Finally, 1F10 and 6D11 shared common light chain CDR sequences and heavy chain CDR sequences, indicating that they are cognate mabs. Neither the 1F10 antibody nor the 6D11 antibody produced significant immunoprotection in mice. The three mabs (5a10, 3F6, and 2F2) that gave the most promising protection and that had been further characterized are shown in bold.

Three major groups of light chain sequences were identified and joined by similar sequences. The first group comprised 3F6, 1F10, 6D11, 4C1, 6B2, 2B8, and 4C5, all of which shared a common light chain CDR sequence, except for one amino acid difference in 6B2 and two amino acid differences in 4C 5. Despite sharing only the light chain sequence, these mabs still gave rise to multiple protective effects, suggesting that specific differences in the heavy chain sequence may significantly affect the functional effects of these mabs. The second group shares a set of light chain CDR sequences (different heavy chain CDR sequences) and comprises 5a10 and 2F2, including one of the primary protective antibodies, 5a 10.

The percent identity of the corresponding CDRs for all antibodies was calculated and shown in matrix form in 7-15. Antibodies having greater than 40% sequence identity of each CDR relative to each CDR of 3F6, 5a10, or 3D11 are summarized in table 16. The consensus sequences for each set of CDRs having greater than 40% identity to the corresponding CDRs of 3F6 are shown in table 17.

Example 2

Materials and methods

Bacterial strains and growth conditions. Staphylococcus aureus strains Newman and MW2 were grown in Tryptic Soy Broth (TSB) at 37 ℃. Coli strains DH 5a and BL21(DE3) were grown in Luria-Bertani (LB) broth containing 100. mu.g ml-1 ampicillin at 37 ℃.

A monoclonal antibody. Preparation of mouse monoclonal antibody by conventional method (And c.milstein.1975). Briefly, 100. mu.g of purified SpA emulsified with Complete Freund's Adjuvant (CFA, DIFCO)1:1 by intraperitoneal injection_KKAABALB/c mice (8 weeks old, female, Jackson laboratory) were immunized. On days 21 and 42, mice were boosted by intraperitoneal injection of 100 μ g of the same antigen emulsified with Complete Freund's Adjuvant (CFA, DIFCO)1: 1. On days 31 and 52, blood and serum samples of mice were collected and screened for specific antibodies by ELISA. Seventy-nine days after the initial immunization, the antigen-immunoreactivity shown to be strong by ELISA was boosted with 25. mu.g of the same antigenA mouse. Three days later, splenocytes were harvested and fused with the mouse myeloma cell line SP2/mIL-6 (interleukin 6 secreting derivative of SP2/0 myeloma cell line). Supernatants from the resulting hybridomas were screened by ELISA, and antigen-specific clones were further subcloned by limiting dilution to generate monoclonal antibody-secreting hybridomas elicited by single cells. Antibodies were purified from the cell line culture supernatants used. Spa27 monoclonal antibody was purchased from Sigma. Hybridoma cell line 358A76.1.1(ATCC accession number PTA-7938) was purchased from the American Type culture Collection and expanded at the Fitch monoclonal antibody factory (University of Chicago).

And (4) purifying the recombinant protein. Synthesis of a peptide from SpA-E by CPC Scientific Inc (Sunnyvale, USA)_KKAAA polypeptide of the amino acid sequence of a domain. The lyophilized peptide samples were dissolved with distilled water or dimethyl sulfoxide (DMSO), then divided into equal portions and frozen at-80 ℃. The use of wild-type SpA and SpA has been described previously_KKAAThe plasmid of (Kim et al, 2010 a). Synthesis of DNA for SpA Synthesis by Integrated DNA Technologies, Inc (USA)_KK(Q in each of the five IgBDs⁹K，Q¹⁰K is replaced), SpA_AA(D in each of the five IgBDs³⁶A、D³⁷A is replaced), SpA_KKAAThe respective IgBD (E, D, A, B and C). SpA_KKAACloning of the PCR product of the variant into the production of N-terminal His₆-in the pET15b vector of the labeled recombinant protein. Sbi_1-4The coding sequence of (A) was PCR amplified with two primers 5'-AAAAAAGCTAGCTGGTCTCATCCTCAATTTGAGAAGACGCAACAAACTTCAACTAAG-3' (SEQ ID NO:8) and 5'-AAAAAACTCGAGTTTCCAGAATGATAATAAATTAC-3' (SEQ ID NO:9) from the Newman chromosomal DNA of Staphylococcus aureus with an engineered N-terminal Strep tag (WSHPQFEK (SEQ ID NO: 10)). Sbi_1-4Cloning of the PCR product of (1) into the production of C-terminal His with an engineered N-terminal Strep tag (WSHPQFEK (SEQ ID NO:10))₆-in the pET24b vector of the labeled recombinant protein. All plasmids were converted to BL21 for affinity purification (DE 3). An overnight culture of the recombinant E.coli strain was diluted 1:100 into fresh medium and grown at 37 ℃ to A₆₀₀0.5Bacterial cells were pelleted by centrifugation, suspended in column buffer (50mM Tris-HCl (pH7.5), 150mM NaCl) and disrupted with a French press at 14000 psi.the membrane and insoluble components were cleared from the lysate by ultracentrifugation at 40000 × g.Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was performed on the protein in the cleared lysate.the protein was eluted in column buffer containing successively higher concentrations of imidazole (100-500 mM.) the protein concentration was determined by a dicumyl-benzoic acid (BCA) assay (Thermo Scientific).

Enzyme-linked immunosorbent assay. To determine SpA-specific serum IgG, affinity-purified SpA was used_KKAAELISA plates (NUNC Maxisorp) were coated at 1. mu.g.ml^-1Overnight in 0.1M carbonate buffer (pH9.5 at 4 ℃). The next day, plates were blocked, incubated with hyperimmune serum and developed with OptEIA reagents (BD Biosciences). To determine the binding affinity of the SpA-specific mAbs, the individual immunoglobulin binding domains or sequences purified by affinity were derived from SpA-E_KKAASynthetic peptides of sequence (H1, H2, H3, H1+3, and H2+3) coated ELISA plates. The plates were coated with peptide overnight at a concentration of 100nM in 0.1M carbonate buffer (pH9.5, at 4 ℃). The next day, plates were blocked with 1% BSA solution in PBS-T and incubated with different concentrations of SpA-specific mAb. To determine the avidity of specific mabs, increasing concentrations (0-4M) of ammonium thiocyanate were used to interfere with antibody-antigen interactions. For the SpA and Sbi binding assays, affinity purified SpA and Sbi were applied at 1. mu.g/ml in 0.1M carbonate buffer (pH9.5 at 4 ℃ C.)^-1Coating on ELISA plate overnight. The next day, the plate was blocked and peroxidase-conjugated human IgG, Fc and F (ab) were used₂(Jackson Laboratory) dilution or isotype control antibody and SpA_KKAA-specific mAb dilution for incubation; the assay was developed using OptEIA reagents. To measure the inhibition of the immunological association between human IgG and SpA, 20. mu.g.ml were used before ligand binding^-1Isotype control antibody or SpA_KKAASpecific mAb plates. For competition assays, the assay was performed at 4 ℃ with 0.1M carbonate buffer (pH 9)5) 10 ng/ml^-1SpA_KKAAPlates were coated overnight. The next day, the plates were blocked and 30. mu.g/ml were used^-1Isotype control antibody or SpA_KKAASpecific mAb culture followed by 100-200ng ml^-1At final concentrations, incubated with HRP-conjugated SpA-specific mAbs (Innova biosciences) or human IgG.

Mouse kidney abscess model. Affinity purified antibody in PBS at 5, 15, 20 or 50 mg-kg^-1Concentrations of experimental animal body weight were injected into the peritoneal cavity of BALB/c mice (6 weeks old, female, Charles River Laboratories) and challenged with Staphylococcus aureus 4-24 hours later. An overnight culture of S.aureus strains was diluted 1:100 into fresh TSB and grown at 37 ℃ for 2 hours. Staphylococci were pelleted, washed and suspended in PBS to the desired bacterial concentration. The inoculum was quantified by spreading an aliquot of the sample on TSA and counting the colonies formed after incubation. By intraperitoneal injection of 100 mg/ml per kg body weight^-1Ketamine and 20 mg/ml^-1BALB/c mice were anesthetized with xylazine by mixing 1 × 10⁷Newman or 5 × 10 of CFU Staphylococcus aureus⁶Mice were infected with CFU s aureus USA300(LAC) or USA400(MW2) injected into the periorbital sinus of the right eye. On day 4 or day 15 post challenge, by CO₂The mice were killed by inhalation. Both kidneys were removed and staphylococcal load in one organ was analyzed by homogenizing kidney tissue with PBS, 0.1% Triton X-100. Serial dilutions of the homogenate were spread on TSA and cultured until colonies formed. The remaining organs were examined by histopathological section. Briefly, kidneys were placed in 10% formalin for 24 hours at room temperature. Tissues were embedded in hematoxylin-eosin stained thin paraffin discs and examined by light microscopy to calculate abscessed lesions. Immune serum samples collected at day 15 post infection were examined by immunoblotting against 14 affinity purified staphylococcal antigens immobilized on nitrocellulose membranes at 2 μ g. The signal strength was quantified as described previously (Kim et al, 2010 b). According to the Institutional Biosafety Committee (IBC) and Institutional Animal Care and UseCommittee (IA) in compliance with the university of ChicagoCUC) guidelines for the reviewed and approved protocol all mouse experiments were performed.

Staphylococci survive in the blood. Puncture through the heart and use 10. mu.g.ml^-1Lepirudin inhibits clotting whole blood was collected from BALB/c mice. At 2. mu.g.ml^-1In the presence of mAb 50. mu.l 5 × 10⁵CFU·ml^-1Staphylococcus aureus Newman was mixed with 950. mu.l of mouse blood. Samples were incubated at 37 ℃ with slow rotation for 30 minutes, then incubated with 1% saponin/PBS on ice. For human blood studies, at 10. mu.g.ml^-1In the presence of mAb 50. mu.l 5 × 10⁶CFU·ml^-1Staphylococcus aureus MW2 was mixed with 950. mu.l of freshly drawn human blood. The tubes were incubated at 37 ℃ for 120 minutes with slow rotation. Aliquots were incubated with 1% saponin/PBS on ice to lyse blood cells. Dilutions of staphylococci were plated on agar for colony formation. Experiments with blood from human volunteers were performed with an Institutional Review Board (IRB) of Chicago university that had been reviewed, approved, and supervised.

SpA-specific serum IgG. In the presence of 85 u g mAb3F6 or its isotype control, in day 0 and day 11 will be 20 u g affinity purification of SpA variants injected into BALB/c mice in the intraperitoneal. On day 21, whole blood was collected from BALB/c mice to obtain hyperimmune serum.

SpA abundance in the circulation was measured. 200 μ g of affinity purified wild type SpA was injected intraperitoneally into passively immunized BALB/c mice. At the indicated time intervals, 10. mu.g.ml are used^-1Lepidiudin anticoagulant whole blood was collected from BALB/c mice all samples and 1% saponin/PBS were kept on ice for 10 minutes, then the solubilized samples were diluted with 1:10PBS and mixed with 1:1 SDS-PAGE sample buffer, the samples were boiled at 90 ℃ for 5 minutes, then subjected to SDS-PAGE gel electrophoresis, the samples were transferred to PDVF and subjected to affinity purified rabbit α -SpA_KKAAAntibodies were analyzed by immunoblotting.

Sbi consumption assay. An overnight culture of Staphylococcus aureus Newman 1:100 was diluted into fresh TSB and grown for 2 hoursIn combination with pre-cooled TSB, A600 was adjusted to 0.4(1 × 10)⁸CFU·ml^-1). Cells were washed and 100. mu.l of isotype control or mAb3F6 at 100. mu.g.ml^-1After incubation, the staphylococci were washed with precooled TSB and incubated with 2. mu.g of affinity purified wild type Sbi for 1 hour at 4 ℃ the staphylococci were pelleted by centrifugation at 13000 × g for 1 minute, the supernatant removed and mixed with sample buffer (1: 1). the sample was boiled at 90 ℃ for 5 minutes and then subjected to SDS-PAGE gel electrophoresis, the sample was electro-transferred to PDVF membrane and incubated with affinity purified rabbit α -SpA_KKAAAntibodies were analyzed by immunoblotting.

Sequencing of monoclonal antibodies total RNA samples from hybridoma cells were isolated using a standardized protocol 1.4 × 10, simply cultured in DMEM-10 medium with 10% FBS⁷Individual hybridoma cells were washed with PBS, pelleted by centrifugation and lysed in trizol (invitrogen), the samples were mixed with 20% chloroform, incubated at room temperature for three minutes, and centrifuged at 10,000 × g for fifteen minutes at 4 ℃, RNA in the aqueous layer was removed and washed with 70% isopropanol, RNA was pelleted by centrifugation and washed with 75% diethyl coke-carbonate (DEPC) -ethanol, the pellets were dried and RNA was lysed in DEPC.

And (5) carrying out statistical analysis. Bacterial burden and number of abscesses in experimental animal models for staphylococcus aureus infection were analyzed using a two-tailed Mann-Whitney test to measure statistical significance. Unpaired two-tailed Student's t-test was performed to analyze the statistical significance of ELISA data, immunoblot signals, and in vivo blood survival data. All data were analyzed by Prism (GraphPad Software, Inc.) and P values less than 0.05 were considered significant.

Reference to the literature

The following references are specifically incorporated by reference herein in order to provide exemplary methodology or other details supplementary to those set forth herein.

U.S. Pat. No. 3,817,837

U.S. Pat. No. 3,850,752

U.S. Pat. No. 3,939,350

U.S. Pat. No. 3,996,345

U.S. Pat. No. 4,196,265

U.S. Pat. No. 4,275,149

U.S. Pat. No. 4,277,437

U.S. Pat. No. 4,338,298

U.S. Pat. No. 4,366,241

U.S. Pat. No. 4,472,509

U.S. Pat. No. 4,472,509

U.S. Pat. No. 4,554,101

U.S. Pat. No. 4,684,611

U.S. Pat. No. 4,748,018

U.S. Pat. No. 4,879,236

U.S. Pat. No. 4,938,948

U.S. Pat. No. 4,938,948

U.S. Pat. No. 4,952,500

U.S. Pat. No. 5,021,236

U.S. Pat. No. 5,196,066

U.S. Pat. No. 5,262,357

U.S. Pat. No. 5,302,523

U.S. Pat. No. 5,310,687

U.S. Pat. No. 5,322,783

U.S. Pat. No. 5,384,253

U.S. Pat. No. 5,464,765

U.S. Pat. No. 5,505,928

U.S. Pat. No. 5,512,282

U.S. Pat. No. 5,538,877

U.S. Pat. No. 5,538,880

U.S. Pat. No. 5,548,066

U.S. Pat. No. 5,550,318

U.S. Pat. No. 5,563,055

U.S. Pat. No. 5,580,859

U.S. Pat. No. 5,589,466

U.S. Pat. No. 5,591,616

U.S. Pat. No. 5,610,042

U.S. Pat. No. 5,648,240

U.S. Pat. No. 5,656,610

U.S. Pat. No. 5,690,807

U.S. Pat. No. 5,702,932

U.S. Pat. No. 5,736,524

U.S. Pat. No. 5,741,957

U.S. Pat. No. 5,750,172

U.S. Pat. No. 5,756,687

U.S. Pat. No. 5,780,448

U.S. Pat. No. 5,789,215

U.S. Pat. No. 5,801,234

U.S. Pat. No. 5,827,690

U.S. Pat. No. 5,840,846

U.S. Pat. No. 5,871,986

U.S. Pat. No. 5,945,100

U.S. Pat. No. 5,981,274

U.S. Pat. No. 5,990,479

U.S. Pat. No. 5,994,624

U.S. Pat. No. 6,008,341

U.S. Pat. No. 6,048,616

U.S. Pat. No. 6,091,001

U.S. Pat. No. 6,274,323

U.S. Pat. No. 6,288,214

U.S. Pat. No. 6,630,307

U.S. Pat. No. 6,651,655

U.S. Pat. No. 6,756,361

U.S. Pat. No. 6,770,278

U.S. Pat. No. 6,793,923

U.S. Pat. No. 6,936,258

U.S. Pat. No. 61/103,196

U.S. Pat. No. 61/166,432

U.S. Pat. No. 61/170,779

U.S. patent publication 2002/0169288

U.S. patent publication 20050106660

U.S. patent publication 20060058510

U.S. patent publication 20060088908

U.S. patent publication 20100285564

Atherton et al, biol. of Reproduction,32:155-171,1985.

Atkins et al, mol. Immunol.,45:1600-1611,2008.

Ausubel et al, In Current Protocols In Molecular Biology, John, Wiley & Sons, Inc, New York,1996.

Baba et al, J.Bacteriol.,190:300-310,2007.

Baba et al, Lancet 359:1819-1827,2002.

Barany and Merrifield in The Peptides, Gross and Meienhofer (Eds.), Academic Press, NY,1-284,1979.

Berberian et al, Blood78: 175-.

Berberian et al, Science261: 1588-.

Boucher and Corey, clin. infect. dis, 46(5) S344-349,2008.

Burke et al, J.Inf.Dis.,170:1110-1119,1994.

Burman et al, J.biol.chem.,283: 17579-.

Campbell, in Monoclonal Antibody Technology, Laboratory technologies in biochemistry and Molecular Biology, Burden and Von Knippenberg (Eds.), Elseview, Amsterdam,13:71-74/75-83,1984.

Carbonelli et al, FEMS Microbiol. Lett.,177(1):75-82,1999.

Cary et al, mol. Immunol.,36:769-776,1999.

Chandler et al, Proc. Natl. Acad. Sci. USA,94(8): 3596-.

Chen and Okayama, mol.cell biol.,7(8):2745-2752,1987.

Cheng et al, FASEB J.,23: 3393-.

Cocea,Biotechniques,23(5):814-816,1997.

Cumber et al, J.immunology,149B:120-126,1992.

de Bono et al, J.mol.biol.,342(1): 131-.

Dedent et al, Semin. Immunopathol, 34: 317-.

Dholakia et al, J.biol.chem.,264,20638-20642,1989.

Deep et al, Lancet367:731-739,2006.

Emorl and Gaynes, Clin. Microbiol. Rev.,6:428-442,1993.

Epitope mapping protocols in Methods in Molecular Biology, Vol.66, Morris (Ed.),1996,

european patent 0216846

European patent 0256055

European patent 0323997

European patent application 89303964.4

Fechheimer et al, Proc Natl.Acad.Sci.USA,84: 8463-.

Fischetti,Clin.Microbiol.Rev.,2:285-314,1989.

Forsgren et al, Eur.J.Immunol.6:207-213,1976.

Fraley et al, Proc.Natl.Acad.Sci.USA,76: 3348-.

Gefter et al, solar Cell Gene, 3: 231-.

Goding, in Monoclonal Antibodies: Principles and Practice,2d ed., Academic Press, Orlando, Fl, pages 60-61,71-74, 1986.

Goding, in Monoclonal Antibodies: Principles and Practice,2d ed., Academic Press, Orlando, Fl,65, page 66, 1986.

Goodyear and Silverman, j.exp.med.,197:1125-1139,2003.

Goodyear and Silverman Proc. Natl. Acad. Sci. USA101: 11392-.

Gopal,Mol.Cell Biol.,5:1188-1190,1985.

Graham and Van Der Eb, Virology,52:456-467,1973.

Graille, et al, Proc. Nat. Acad. Sci. USA97:5399-5404,2000.

Harland and Weintraub, J.cell biol.,101(3):1094-1099,1985.

Harlow et al, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Chapter8,1988.

Haupp et al, PloS Patholog, 4: e1000250,2008.

Hollingbeam et al, infection. Immun.,55:3237-3239,1987.

Huang et al, J.Clin.invest.89:1331-1343,1992.

Jones and Fischetti, J.Exp.Med.,167:1114-1123,1988.

Jones et al, J.exp.Med.,164:1226-1238,1986.

Kaeppler et al, Plant Cell Rep.,8: 415-418, 1990.

Kaneda et al, Science,243: 375-.

Kato et al, J.biol.chem.,266:3361-3364,1991.

Kennedy et al, Proc. Natl. Acad. Sci., USA,105(4):1327-1332,2008. Khaton et al, Ann. of Neurology,26, 210-.

Kim et al, FASEB J.,25: 3605-E3612, 2011.

Kim et al, J.Exp.Med.,207: 1863-one 1870,2010a.

Kim et al, Vaccine,28:6382-6392,2010b.

Kim et al, curr, Opin, Microbiol.15:92-99,2012b.

Kim et al, feed.Immun.80 EPub head of press.2012a

King et al, J.biol.chem.,269,10210-10218,1989.

Klevens et al, JAMA,298: 1763-.

Kohl et al, Proc. Natl. Acad. Sci., USA,100(4):1700-1705,2003.

Kohler and Milstein, Eur.J.Immunol.,6:511-519,1976.

Kohler and Milstein, Nature,256: 495-.

Kronvall et al, J.Immunol.105:1115-1123.1970

Kyte and Doolittle, J.mol.biol.,157(1):105-132,1982.

Lancefield,J.Exp.Med.,47:91-103,1928.

Lancefield,J.Immunol.,89:307-313,1962.

Lee,Trends Microbiol.,4(4):162-166,1996.

Levenson et al, hum. Gene ther.,9(8) 1233-1236,1998.

Liu et al, Cell mol. biol.,49(2):209-216,2003.

Mazmanian et al, science 285:760-763,1999.

McCarthy and Lindsay, BMC Microbiology,10:173,2010.

Merrifield,Science,232(4748):341-347,1986.

Moks et al, Eur.J.biochem.156:3577-3588,1986.

Nicolau and Sene, Biochim. Biophys. acta,721:185-190,1982.

Nicolau et al, Methods Enzymol, 149:157-176,1987.

Nimmerjahn and ravatch, nat. rev. immunol.,8(1):34-47,2008.

Omirulleh et al, Plant mol.biol.,21(3):415-28,1993.

O' Shannessy et al, J.Immun.meth, 99,153-161,1987.

Owens and Haley, J.biol.chem.,259:14843-14848,1987.

Pack et al, biochem, 31: 1579-.

Perry et al, J Biol chem.277:16241-16248,2002.

PCT application PCT/US11/42845

PCT application WO00/02523

PCT application WO00/12132

PCT application WO00/12689

PCT application WO00/15238

PCT application WO01/60852

PCT application WO2006/032472

PCT application WO2006/032475

PCT application WO2006/032500

PCT application WO2007/113222

PCT application WO2007/113223

PCT application WO2011/005341

PCT application WO94/09699

PCT application WO95/06128

PCT application WO98/57994

PCT publication WO2006/056464

PCT publication WO99/26299

Phillips et al, Proc.Natl.Acad.Sci., USA,78:4689-4693,1981.

Potrykus et al, mol.Gen.Genet.,199(2):169-177,1985.

Potter and Haley,Methods Enzymol,91:613-633,1983.

Rippe et al, mol.cell biol.,10:689-695,1990.

Robbins et al, Peditar. Infect. Dis. J.6:791-794,1987.

Robbins et al, J.Infect.Dis.161:821-832,1990.

Robbins et al, adv. exp. Med. biol.,397:169-182,1996.

Sambrook et al, in Molecular cloning, Cold Spring Horbor laboratory Press, Cold Spring Horbor, NY, 2001.

Sasso et al, J.Immunol.142: 2778-.

Scott et al, J.Exp.Med.,164:1641-1651,1986.

Silverman and Goodyear, nat. Rev. Immunol.,6:465-475,2006.

Et al, Eur.J.biochem.73:343-351,1977.Et al, Eur.J.biochem.29:572-578,1972.

Skerra,J.Biotechnol.,74(4):257-75,2001.

Skerra,J.Mol.Recogn.,13:167-187,2000.

Smith et al, mol. Microbiol.,83:789-804,2012.

Spellberg et al, Semin Immunopathol.34:335-348,2012.

Stahlenheim et al, J.Immunol.103: 467-.

Stewart and Young, in Solid Phase Peptide Synthesis,2d.ed., Pierce chemical Co., 1984.

Stranger-Jones et al, Proc. Natl. Acad. Sci., USA,103: 169442-16947, 2006.

Tam et al, j.am.chem.soc.,105:6442,1983.

Tigges et al, J.Immunol.,156(10): 3901-.

Ton-That et al, Proc. Natl. Acad. Sci., USA,96: 12424-.

Wong et al, Gene,10:87-94,1980.

Yoo et al, J.Immunol.methods,261(1-2):1-20,2002.

Zhang et al, Microbiology,144: 985-.

Zhang et al, Microbiology,145: 177-.

Claims (46)

1. Use of an effective amount of a purified SpA binding polypeptide capable of specifically binding at least two SpAIgG binding domains A, B, C, D and E of a SpA variant lacking non-specific Ig binding activity, wherein said purified polypeptide comprises six CDR amino acid regions from the VH domain and the VL domain of a 3F6 monoclonal antibody, wherein the VH domain of said 3F6 monoclonal antibody comprises three CDR amino acid regions of sequences SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, respectively, and the VL domain of said 3F6 monoclonal antibody comprises three CDR amino acid regions of sequences SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, respectively, for the manufacture of a medicament for treating a staphylococcal infection in a patient having or at risk of staphylococcal infection.

2. The use of claim 1, wherein the purified SpA-binding polypeptide binds to at least two and up to five SpAIgG-binding domain A_KKAA、B_KKAA、C_KKAA、D_KKAAAnd E_KKAAAnd (4) combining.

3. The use of claim 2, wherein the purified SpA binding polypeptide is directed to at least two and up to five SpA IgG binding domains a_KKAA、B_KKAA、C_KKAA、D_KKAAAnd E_KKAAHas 0.5 × 10⁹M^-1Or greater association constants.

4. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is capable of reducing staphylococcal load in the patient.

5. The use of any one of claims 1 to 3, wherein the SpA-binding polypeptide is capable of mediating opsonophagocytic killing of Staphylococcus aureus.

6. The use of any one of claims 1 to 3, wherein the SpA-binding polypeptide is capable of disrupting the binding of human IgG to wild-type SpA.

7. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is a humanized antibody comprising CDR amino sequences from a 3F6 monoclonal antibody.

8. The use of any one of claims 1 to 3, wherein treating a staphylococcal infection comprises reducing abscess formation or reducing bacterial load in a patient.

9. The use of any one of claims 1-3, wherein the SpA polypeptide lacking non-specific Ig-binding activity is SpA_KKAA。

10. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide competes for binding to SpA with the 3F6 monoclonal antibody_KKAAA polypeptide.

11. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 0.5 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1The association constant between.

12. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 1.0 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1The association constant between.

13. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 2.0 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1The association constant between.

14. The use of any one of claims 1-3, further comprising administering an effective amount of two or more purified SpA-binding polypeptides.

15. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is a recombinant polypeptide.

16. The use of claim 15, wherein the purified SpA binding polypeptide is a humanized antibody.

17. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is a human antibody.

18. The use of any one of claims 1 to 3, wherein the purified SpA-binding polypeptide comprises a VH domain and a VL domain from a 3F6 monoclonal antibody.

19. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide comprises a VH domain from a 3F6 monoclonal antibody.

20. The use of any one of claims 1 to 3, wherein the purified SpA-binding polypeptide comprises a VL domain from a 3F6 monoclonal antibody.

21. The use of claim 15, wherein the recombinant polypeptide comprises a scaffold from a polypeptide selected from the group consisting of immunoglobulin, fibronectin, lipocalin, or staphylococcus aureus protein Z.

22. The use of claim 15, wherein the recombinant purified SpA binding polypeptide is operably linked to a second purified SpA binding polypeptide.

23. The use of claim 22, wherein the second purified SpA binding polypeptide binds a second staphylococcal protein.

24. The use of any one of claims 1 to 3, further comprising administering a second purified SpA-binding polypeptide that binds a second staphylococcal protein.

25. The use of any one of claims 1 to 3, further comprising administering an antibiotic or staphylococcal vaccine composition.

26. The use of any one of claims 1-3, wherein the purified SpA-binding polypeptide is administered at a dose of 0.1mg/kg to 500 mg/kg.

27. A purified polypeptide comprising six CDR amino acid regions from the VH domain and the VL domain of a 3F6 monoclonal antibody, wherein the polypeptide is capable of specifically binding to at least two SpA IgG binding domains A, B, C, D and E of a staphylococcal protein a (SpA) polypeptide variant lacking non-specific Ig binding activity, wherein the VH domain of the 3F6 monoclonal antibody comprises three CDR amino acid regions of sequences SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, respectively, and the VL domain of the 3F6 monoclonal antibody comprises three CDR amino acid regions of sequences SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, respectively.

28. The polypeptide of claim 27, wherein the SpA polypeptide lacking non-specific Ig-binding activity is SpA_KKAA。

29. The polypeptide of claim 28, wherein the purified polypeptide competes for binding to SpA with the 3F6 monoclonal antibody_KKAAA polypeptide.

30. The polypeptide of any one of claims 28-29, wherein the purified polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 0.5 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1Often in association with each otherAnd (4) counting.

31. The polypeptide of any one of claims 28-29, wherein the purified polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 1.0 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1The association constant between.

32. The polypeptide of any one of claims 28-29, wherein the purified polypeptide is for SpA_KKAAThe polypeptide has a molecular weight at 2.0 × 10 as measured by ELISA⁹M^-1And 100 × 10⁹M^-1The association constant between.

33. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide is a humanized monoclonal antibody.

34. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide is a human antibody.

35. The polypeptide of any one of claims 27-29, wherein the purified polypeptide is recombinant.

36. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide comprises the VH domain and VL domain of a 3F6 monoclonal antibody.

37. The polypeptide of claim 27, wherein the purified polypeptide comprises the VH domain of the 3F6 monoclonal antibody.

38. The polypeptide of claim 27, wherein the purified polypeptide comprises the VL domain of the 3F6 monoclonal antibody.

39. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide comprises a scaffold from a polypeptide selected from the group consisting of an immunoglobulin, fibronectin, lipocalin, or staphylococcus aureus protein Z.

40. The polypeptide of any one of claims 27-29, wherein the purified polypeptide is operably linked to a recombinant polypeptide that specifically binds to a second staphylococcal protein.

41. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide is a 3F6 monoclonal antibody.

42. The polypeptide of any one of claims 27 to 29, wherein the purified polypeptide is an antibody comprising (a) a heavy chain and (b) a light chain, wherein the heavy chain comprises the VH region and a human hinge region, CH1 region, CH2 region, and CH3 region from an IgG1, IgG2, IgG3, or IgG4 subtype; the light chain comprises the VL region, and either human kappa CL or human lambda CL.

43. A pharmaceutical composition comprising the purified polypeptide of any one of claims 27-42.

44. The pharmaceutical composition of claim 43, comprising a single unit dose of the purified polypeptide in a sealed container.

45. The pharmaceutical composition of claim 43, comprising at least one second antibacterial agent.

46. The pharmaceutical composition of claim 45, wherein the second antibacterial agent is an antibiotic, a staphylococcal vaccine composition or a polypeptide that specifically binds to a second staphylococcal protein.