WO2014027697A1

WO2014027697A1 - Anti-pneumococcal surface protein (psp) monoclonal antibody

Info

Publication number: WO2014027697A1
Application number: PCT/JP2013/072041
Authority: WO
Inventors: Takayuki Ohta; Yutaka Kanda; Sascha KRISTIAN; Tomoyuki Tahara; Tsuguo Kubota
Original assignee: Kyowa Hakko Kirin Co., Ltd.
Priority date: 2012-08-13
Filing date: 2013-08-13
Publication date: 2014-02-20

Abstract

The present invention relates to bacteriology and antibody drug development. More specifically, the present invention relates to monoclonal antibody against Streptococcus pneumoniae surface protein (Psp) which has both preventive (protective) and therapeutic efficacy against Streptococcus pneumonia-induced infectious diseases including pneumonia

Description

DESCRIPTION

Anti-pneumococcal surface protein (Psp) monoclonal antibody Technical Field

The embodiments described herein relate to bacteriology and antibody drug development. More specifically, the various embodiments relate to monoclonal antibody against Streptococcus pneumoniae surface protein A (PspA) which has both preventive (protective) and therapeutic efficacy against Streptococcus pneumonia-induced infectious diseases including pneumonia.

Background Art

Streptococcus pneumoniae (S. pneumoniae) is a well known human pathogen and a major etiologic agent for pneumonia, meningitis, otitis media as well as sepsis, among primarily young children, older adults and immuno-compromised patients.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of invasive bacterial infection (NPL1). This pathogen was previously known as Diplococcus pneumoniae because it is typically seen clinically in the form of diplococci or short bacterial chains. Antibodies to a capsular polysaccharide may provide protection against pneumococci expressing the same capsular serotype. Nowadays over 90 different serotypes are identified and reported. Currently available pneumococcal vaccines contain a mixture of capsular polysaccharide of multiple serotypes. For example, one pneumococcal vaccine (Pneumovax 23) contains capsular polysaccharide from twenty-three commonly found serotypes including serotype 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The most recently developed type of vaccine contains capsular polysaccharide from seven to thirteen serotypes (serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) that are conjugated to a protein molecule (Prevnar 13). A seven- valent conjugate vaccine (PCV7, Prevnar; having serotype 4, 6B, 9V, 14, 18C, 19F, and 23F) was Approved for clinical use and launched in the USA, 2000, and has reduced the incidence of invasive pneumococcal diseases in children and in adults. On the other hand, such vaccine prevailing among the world has revealed the population of non-responder to the vaccine drugs and led to the emergence of non-vaccine serotypes in community and hospital. Moreover Pneumovax 23 has not been indicated in children less than 2 years of age. Children in this age group respond poorly to the capsular types contained in this polysaccharide vaccine. Further, Pichichero et al. discovered and reported multidrug-resistant strains of S. pneumoniae including Legacy strain which is resistant to all Food and Drug Administration (FDA)-approved antimicrobial drugs and to 8 different non-FDA-approved antimicrobial drugs used to treat acute otitis media (AOM) in children (NPL2). However, any passive immunotherapy including monoclonal antibody drugs against Streptococcus pneumonia has not been developed in clinical yet.

Pneumococcal infections are controlled by host neutrophils, which kill this pathogen via opsonophagocytosis (OPA or OPH), a process that requires opsonization of bacteria by the complement system (NPL3, NPL4, NPL5). Activation of this system results in the covalent deposition of complement component 3 (C3, C3b, C3d) onto bacterial surfaces (NPL6). On gram-negative bacteria, this can lead to direct complement- mediated lysis of cells, while gram-positives are resistant to lysis due to their thick peptidoglycan layer. However, C3, C3b and C3d can also interact with complement receptors on neutrophils such as Mac-1 (CD1 lb/CD18) to promote phagocytosis.

Deposition of C3 onto pneumococci can result from activation of either the classical or alternative pathways (NPL7). Activation of the classical pathway can be directed to bacterial surfaces with the aid of antibodies, while the alternative pathway stochastically activates complement on bacterial surfaces. In addition, members of the leukocyte immunoglobulin (Ig) G Fc receptor (FcgammaR) family play a key role in antibody- mediated phagocytosis and can either enhance antigen presentation or down-modulate immune responses (NPL8). Once opsonized, bacteria can be recognized by surface receptors on neutrophils, monocytes or macrophages and ingested by phagocytosis. Once internalized, S. pneumoniae is efficiently killed in the phagolysosome (NPL9).

Pneumococci resist opsonization by complement due to their surface capsular

polysaccharide (NPL10), which masks underlying structures and activates complement poorly. In addition to capsule, the pneumococcus has surface proteins that directly interact with serum components to evade complement and subsequent phagocytosis (NPL1 1, NPL12).

Streptococcus pneumoniae surface protein A (PspA) is a surface protein of S. pneumoniae found in every characterized pneumococcal strain. Its size is strain-dependent and varies from ~67 to 99 kDa. PspA proteins are classified into 3 families and 6 clades. It is attached to pneumococci through non-covalent interactions of the C-terminal repeat region with the terminal choline residues of the teichoic or lipoteichoic acids present on the pneumococcal cell wall and classified as a choline-binding protein. The PspA molecule is built from four distinct domains which include the antigenic N-terminal part (alpha-helical coiled-coil domain) followed by a highly flexible, tether-like proline-rich region, a repeat region which is responsible for the attachment to the choline residues (choline-binding domain), and a C-terminal hydrophobic tail. The N-terminal moiety likely protrudes outside of the capsule, interacts with many of antibodies reactive to PspA, and has been described as the functional part of this protein. This part is electronegative and has already been implicated in PspA's anti-complementary properties (resistance to complement) which prevent the host complement system from attaching to S. pneumoniae. Additionally, Hyams et al. reported that the resistance of TIGR4 strain to complement-mediated immunity can vary with the capsular serotype independently of other genetic differences between S. pneumonia strains (NPL13). Moreover the N-terminal domain plays a role to suppress the bactericidal activity of lactoferrin (NPL14). This part of PspA is essential for full pneumococcal virulence. The proline-rich region of PspA likely serves as a flexible tether anchoring PspA to the cell wall through the choline-binding domain. This region, present in all reported PspA and almost all PspC (pneumococcal surface protein C) molecules, consists of irregular repeats marked by the presence of a proline residue every two or three amino acids. The most common other amino acids are alanine and lysine. The most common sequence motif is PAPAP, which is also conserved in many different kinds of mammalian proteins including human proteins having proline-rich coiled-coil domains. Around 56% of PspA and 77% of PspC protein molecules are interrupted by a highly conserved block of amino acids termed the non-proline block (NPB) (NPL15). The NPB, mainly present within the proline-rich region, contains 33 amino acids, none of which are prolines.

PspA has been shown to elicit antibodies protective against pneumococcal infection and to be necessary for full pneumococcal virulence in mice. Crain et al.

reported that monoclonal antibodies were raised against PspA of Streptococcus

pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough Streptococcus pneumoniae strain R36A. The mouse monoclonal antibodies produced by two hybridoma clones, Xi64 (IgM) and Xil26 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39 (NPL16). However, Waltman et al. reported that only 14% of 499 pneumococcal isolates reacted with one or both of the monoclonal antibodies Xi64 and Xil26 (NPL17). Because of the antigenic variation in PspA, it seemed likely that many of the strains failing to react with Xi64 or Xil26 might express PspA molecules antigenically distinct from those detected by these antibodies. Crain produced five additional mouse monoclonal antibodies specific for PspA including clone SR4W4

(IgG2a), 2A4 (IgM), 1A4 (IgM), 7D2 (IgG2a) and XiR278 (IgGl). The panel of the seven monoclonal antibodies was used to study PspA in 57 strains of pneumococci. The frequency of strains reactive with each individual antibody was as follows: Xi64, 19%; ΧΠ26, 35%; SR4W4, 49%; 2A4, 31%; 1A4, 39%; 7D2, 25%; and XiR278, 42%. Four of the 57 strains, the type 6 strains DBL1 , BG25-9, BG5-8A, and the type 3 strain BG6380, failed to react with any of the seven monoclonal antibodies. According to the pattern of reactivity of the 57 strains with the seven monoclonal antibodies, it was revealed that there were 31 different molecule types of PspA protein among the 57 isolates (NPL18). Daniels showed that the passive immunization of clone 2A4, whose epitope is within N-terminal alpha helical domain, protected the death of septic mice infected with WU2 strain but did not protected the mice infected with BG12730 strain (NPL19). Kolberg et al. newly established 16 different monoclonal antibodies specific to PspA by immunizing BALB/c mice with heat-treated clinical isolates of S. pneumoniae. This study showed that some of the monoclonal antibodies bound to PspA epitopes expressed by a low number of strains whereas other antibodies bound to broadly distributed epitopes, that is to say, the three monoclonal antibodies in the narrow reacting group, 143F-2 (IgA), 149B-3 (IgG2a) and 162C-9 (IgGl), bound to epitopes found in 21 to 25% of the strains in a dot blot analysis whereas the four antibodies in the broad reacting group, 164A-2 (IgGl), 169H-6 (IgGl), 170E-1 1 (IgG2a) and 183F-6 (IgM) detected 57 to 77% of the analyzed strains (NPL20). Unfortunately, the clone 183F-6, which reacted with a high number (77%) of pneumococci by dot blotting, showed relatively weak binding in flow cytometric analysis even with the isolate that was used as immunogen (strain 16/89). The discrepancy between the two methods can possibly indicate that a low number of epitopes is exposed on live exponential phase grown pneumococci. The epitope of 183F-6 has not been fully determined yet (NPL21). At University of Alabama at Birmingham, in 1994 McDaniel et al. reported that the protective epitopes of PspA is located within N-terminal alpha-helical domain (NPL22). After sixteen years from the report, Daniels et al. established three different anti-PspA murine monoclonal antibodies, PR-1A4.7 (IgGl), PR-5C4.6 (IgG2a) and PR-6A5.12 (IgGl), by immunizing the recombinant peptide fragment of amino acids 288 to 588 of strain Rxl PspA comprising the proline-rich region, non-proline block and choline-binding domain. In their sepsis models in mice, both PR-1A4.7 and PR-6A5.12 showed the preventive activity against the infection with the strain WU2 but did not show the activity against the strain BG12730 at all. On the other hand, PR-5C4.6 exhibited the activity against BG12730 but did not show the activity against WU2 at all. An epitope mapping study revealed that epitopes of clone PR-1A4.7 and PR-6A5.12 as well as K67 were assumed to be within NPB and that clone PR-5C4.6 may bind to the sequence PKPEQ in the proline-rich region except NPB. Moreover it was also possible that the epitope of PR- 5C4.6 was a conformational epitope that requires the presence of both the NPB and the C- terminal proline-rich sequence. Regarding the theoretical coverage of these three monoclonal antibodies, about 56% of S. pneumoniae strains express the PspA molecule with NPB, and 46% of strains express the PspA molecule with an exact copy of PKPEQ in the proline-rich region (NPL23). For the all of circumstances stated above, any therapeutic epitope within PspA molecule, which is highly conserved (e.g. over 80%) among S.

pneumoniae strains has not been fully determined yet. Also, none of prior art to establish anti-PspA monoclonal antibody having both preventive and therapeutic efficacy against S. pneumoniae has ever been found in publication.

Citation List

Non Patent Literature

[NPL1] O'Brien et al., 2009, Lancet 374, 893-902

[NPL2] JAMA 2007;298: 1772-1778

[NPL3] Dalia et al, 2010 Infect. Immun S, 2108-21 16.;

[NPL4] Lysenko et al., 2007 PLoS Pathog. 3, el 18.;

[NPL5] Matthias et al., 2008 J. Immunol. 180, 6246-6254

[NPL6] Lambris et al., 2008 Nat. Rev. Microbiol. 6, 132-142

[NPL7] Brouwer et al, 2008 J. Immunol. 180, 4124-4132

[NPL8] J Infect Dis. 2003 Jun 1 ;187(1 1): 1686-93

[NPL9] Standish and Weiser, 2009 J. Immunol. 183, 2602-2609

[NPL10] Hyams et al., 2010 Infect. Immun. 78, 704-715

[NPL11] Dalia et al., 2010 Infect. Immun S, 2108-21 16.;

[NPL12] Jarva et al., 2003 Mol. Immunol. 40, 95-107

[NPL13] Infection and Immunity 2010;78:716-725

[NPL14] Infection and Immunity 2004;72:5031-5040

[NPL15] J. Bacteriol 1992;174:601-609

\N? 16] J Exp Med. 1984;160(2):386-397

[NPL17] Microb. Pathog. 1988;5: 159-167

[NPL18] Infection and Immunity 1990;58:3293-3299

[NPL19] Infection and Immunity 2010;78:2163-2172

[NPL20] FEMS Immunol and Medical Microbiol 2001 ;31 : 175-180

[NPL21] FEMS Immunol and Medical Microbiol 2003;39:265-273

[NPL22] Microbial Pathogenesis 17, 323 (1994)

[NPL23] Infection and Immunity 2010;78:2163-2172 Summary of Invention

Technical Problem

The embodiments relating to bacteriology and antibody drug development are desired. More specifically, the various embodiments relating to monoclonal antibody against Streptococcus pneumoniae surface protein (Psp) which has both preventive (protective) and therapeutic efficacy against Streptococcus pneumonia-induced infectious diseases including pneumonia are desired. Solution to Problem

The present invention provides antibodies or antigen-binding fragments thereof specifically directed against peumococcal surface protein (Psp) such as PspA and PspC. Optionally, the antibody is isolated from a hybridoma generated from an immunized animal or from a B cell from a human donor. Exemplary monoclonal antibodies include 139G3, 140G1, 140G1 1, 140H1 and its variants described herein. Alternatively, the antibody is an antibody that binds to the identical or overlapped epitope as that ofl39G3, 140G1, 140G1 1 or 140H1. The antibodies respectively referred to herein are anti-PspA antibodies. The anti-PspA antibodies have one or more of the following mechanism(s): a) bind to an epitope in PspA or PspC polypeptide of a Streptococcus pneumoniae strain; b) bind to Streptococcus pneumoniae cells; c) show a complement-dependent cytotoxicity against Streptococcus pneumoniae cells, c) show an opsonophagocytic killing activity against S. pneumoniae cells.

The epitope that the said anti-PspA antibody binds to is a linear or non-linear epitope of a PspA polypeptide. The non-linear epitopes of the present invention are included as conformational structures of PspA or PspC which is presented on cell surface of Streptococcus pneumoniae. Any epitopes are included in the present invention as presenting in the PspA protein, for example, the epitopes of the antibody in the present invention are included as an epitope which is existed within at least one amino acid sequence selected from TPAPAPKPEQPA, KPAPAPQP and

DDQQAEEDYARRSEEEYNRLPQQQPPKAE of a Proline-rich domain of PspA. Further the epitope of the antibody in the present invention comprising at least one peptide selected from TPAPAPKPEQPA, KPAPAPQP and

DDQQAEEDYARRSEEEYNRLPQQQPPKAE of a Proline-rich domain are exemplified. Preferably, the epitope is conserved between PspA and PspC. More preferably, the epitope is located within the portion of the proline-rich region inserted between alpha helical coiled-coil domain and non-proline block (NPB) of a PspA polypeptide. Most preferably, the epitope wholly or partially includes the amino acid sequence KPAPAPQP.

An antibody or the antigen-binding fragment thereof of the present invention preferably includes the antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein in the VH domain and the VL domain each complementarity-determining region (CDR) includes the following amino acid sequences: VH CDR1 : SEQ ID NO: l ; VH CDR2: SEQ ID NO:2; VH CDR3: SEQ ID NO:3; VL CDR1 : SEQ ID NO:4; VL CDR2: SEQ ID NO:5; VL CDR3: SEQ ID NO:6. The antibody or antigen-binding fragment binds to PspA.

Alternatively, or in addition, an antibody, or antigen-binding fragment thereof , comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the three heavy chain CDRs include an amino acid sequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs: l, 2, 3, and a light chain with three CDRs that include an amino acid sequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs:4, 5, 6. The antibody or antigen-binding fragment binds to PspA.

In another aspect, the invention provides a composition including an anti-PspA antibody according to the invention. The composition is optionally a pharmaceutical composition including any one of the anti-PspA antibodies or antigen-binding fragments described herein and a pharmaceutical carrier. In various aspects, the composition further includes an anti-bacterial drug, a bacterial growth inhibitor, a bacterial toxin inhibitor, a bacterial phagocytosis mediator, or a bacterial attachment inhibitor. The anti-bacterial drug is for example an enzymatic inhibitor or a modulator of the bacterial membrane. The enzymatic inhibitor is for example cephem, etc. In a further aspect, the composition further includes a second anti-bacterial antibody.

In another aspect, the invention provides methods of treating infectious disease comprising administering the anti-PspA antibodies or antigen-binding fragments described herein to a subject prior to, and/or after exposure to a Streptococcus pneumoniae strain. In other words, the anti-PspA antibody of the invention is used to prevent or treat the infection with S. pneumoniae. The anti-PspA antibody is administered at a dose sufficient to promote bacterial clearance.

Also included in the invention is a method for determining the presence of a S. pneumoniae infection in a patient, by contacting a biological sample obtained from the patient with an anti-PspA antibody or antigen-binding fragment described herein; detecting an amount of the antibody that binds to the biological sample; and comparing the amount of antibody that binds to the biological sample to a control value. The invention further provides a diagnostic kit comprising an anti-PspA antibody or antigen-binding fragment described herein.

The present invention also includes a process for producing the antibody for Psp such as PspA and PspC. The method of process for producing the antibody preferably includes the process for producing the monoclonal antibody or antigen-binding fragment thereof recognizes one epitope comprising a peptide selected from TPAPAP PEQPA, KPAPAPQP and DDQQAEEDYARRSEEEYNRLPQQQPPKAE of a Proline-rich domain, and binds to Psp.

Advantageous Effects of Invention

Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. Brief Description of Drawings

FIG. 1 shows the design of the expression construct of recombinant PspA proteins with a 6x histidine-tag.

FIG. 2 shows the summarized result of epitope mapping for the four anti-PspA monoclonal antibodies. The upper scheme shows the position of the four PspA

polypeptides SI to S4 within the PspA peptide from BAA-658 strain. The lower scheme shows, (1) the amino acid sequence of the five PR domain partial sequences PR1 to PR4,

(2) the result of the peptide-binding ELISA experiments in the bottom of the right side, and

(3) the binding site of the four anti-PspA monoclonal antibodies including 139G3, 140G1, 140G1 1, and 140H1 is depicted in each PspA sequence.

FIG. 3 shows the sensorgrams from surface plasmon resonance analysis for the four anti-PspA monoclonal antibodies including 139G3, 140G1, 140G11, and 140H1 towards PspA D39 by using BIAcore 3000. In each sensorgram, Y-axis indicates the resonance unit and X-axis indicates the time after injection in each binding experiment.

FIG. 4 shows the sensorgrams from surface plasmon resonance analysis for the four anti-PspA monoclonal antibodies including 139G3, 140G1, 140G1 1, and 140H1 towards PspA BAA-658 by using BIAcore 3000. In each sensorgram, Y-axis indicates the resonance unit and X-axis indicates the time after injection in each binding experiment.

FIG. 5 shows the sensorgrams from surface plasmon resonance analysis for the four anti-PspA monoclonal antibodies including 139G3, 140G1, 140G1 1, and 140H1 towards PspA TIGR4 by using BIAcore 3000. In each sensorgram, Y-axis indicates the resonance unit and X-axis indicates the time after injection in each binding experiment.

FIG. 6 shows the summary of the coverage analysis by using flow cytometry to 28 Streptococcus pneumoniae wild-type and 3 mutant strains for the ten anti-PspA monoclonal antibodies. The binding of 10 mouse anti-PspA mAbs to a panel of 28 S. pneumoniae strains was determined. Live, exponential phase bacteria were incubated with buffer only, control or mouse anti-PspA mAbs. Primary antibody bound to the bacteria was detected with phycoerythrin-labeled anti-mouse IgG secondary antibodies and flow cytometry. Moreover, the binding of the mAbs to three genetically engineered PspA- deficient mutants was determined to assess whether candidate antibodies can cross-react with other pneumococcal antigens.

FIG. 7 shows the results from the complement-deposition analysis in mouse serum by using flow cytometry to 5 Streptococcus pneumoniae strains for the four anti- PspA monoclonal antibodies including 139G3, 140csGl, 140G11, and HOcsHl (mouse IgG2a). Exponential phase pneumococci were washed in HBSS, 5% BSA, and then incubated in 0.2 mL HBSS, 3.75% BSA at 37°C at 600 rpm with or without isotype or anti- PspA mAbs and mouse serum at concentrations that were optimized for each pneumococcal strain. After 30 min, cells were washed with ice-cold PBS, 0.5% BSA, and then resuspended in 100 μL PBS, 0.5% BSA, containing 2 μg/mL fluorescein-labeled anti- mouse C3 antibody. After 30-60 min incubation at 4°C without mixing, bacteria were washed in ice-cold PBS, 0.5% BSA. Subsequently, cells were fixed with formalin and subjected to flow cytometry after additional washing. The FL-1 fluorescence intensity of the cells was measured with a BD FACSCalibur™. The histograms show overlays of the FL-1 data for bacteria that were subjected to negative control (tinted; gray lines) or the respective anti-PspA (black lines) mAbs. A shift in FL-1 intensity correlated with deposition of C3b on the bacteria. Please note that 139G3 did not bind to ATCC-6305. The arrowheads in the histograms indicate the detected complement C3-deposition.

FIG. 8 shows the summary of the complement-deposition analysis in mouse serum by using 9 Streptococcus pneumoniae strains for the four anti-PspA monoclonal antibodies including 139G3, 140csGl, 140G1 1, and 140csHl (mouse IgG2a).

FIG. 9 shows the results from the opsonophagocytic killing (OPK) experiments with rabbit complement and polymorphonuclear neutrophile (PMN)-like HL- 60 cells to a Streptococcus pneumoniae strain PJ-1324 for the four anti-PspA monoclonal antibodies including 139G3, 140csGl, 140G1 1, and 140csHl (mouse IgG2a). ~2xl0⁴ colony forming units (CFU) of exponential phase PJ-1324 were pre-opsonized in 10% baby rabbit complement for 60 min in the presence of 1 μg/mL of the indicated mouse IgG2a control or anti-PspA antibodies. Then, 10⁶ differentiated PMN-like HL-60 cells in HBSS, 10 mM glucose, or vehicle alone were added to the bacteria. At the indicated time points, samples were taken to enumerate the number of surviving bacteria. Samples were run in triplicate or quadruplicate and mean CFU values/mL + SD of one representative experiment of two performed are shown (***, p<0.0005 anti-PspA vs. isotype antibody at given time points, unpaired, two-tailed t-test).

FIGs. 10A, 10B and IOC show the summary of mouse in vivo efficacy against a Streptococcus pneumoniae strain BAA-658 for the four anti-PspA monoclonal antibodies including 139G3, 140csGl, 140G1 1, and 140csHl (mouse IgG2a). 4-6 h before intraperitoneal (i.p.), intranasal (i.n.), or intravenous (i.v.) infection with the indicated S. pneumoniae strains, female CD-I or Swiss- Webster mice were pretreated i.p. with the indicated amounts of mAbs in PBS. In most experiments, heparinized tail vein blood was collected and bacterial colony forming unit (CFU) enumerated (Left panels). For the blood data, the results of one to two independent experiments are shown (*, p<0.05; CFU comparisons of isotype vs. anti-PspA mAb treatment with unpaired t-test). Survival of the mice was followed for 13-14 days. The combined survival results of two to three independent experiments are shown in the graphs (*, p<0.05; **, p<0.005; ***, p<0.0005, survival curve comparisons of isotype vs. anti-PspA antibodies with Mantel-Cox test). FIGs. 11 A, 1 IB and 1 1C show the summary of mouse in vivo efficacy against a Streptococcus pneumoniae strain WU2 for the four anti-PspA monoclonal antibodies including 139G3, HOcsGl, 140G11, and 140csHl . 4-6 h before intraperitoneal (i.p.), intranasal (i.n.), or intravenous (i.v.) infection with the indicated S. pneumoniae strains, female CD-I or Swiss- Webster mice were pretreated i.p. with the indicated amounts of mAbs in PBS. In most experiments, heparinized tail vein blood was collected and bacterial CFU enumerated (Left panels). For the blood data, the results of one to two independent experiments are shown (*, p<0.05; CFU comparisons of isotype vs. anti-PspA mAb treatment with unpaired t-test). Survival of the mice was followed for 13-14 days. The combined survival results of two to three independent experiments are shown in the graphs (*, p<0.05; **, p<0.005; ***, p<0.0005, survival curve comparisons of isotype vs. anti- PspA antibodies with Mantel-Cox test).

FIGs. 12 A, 12B and 12C show the summary of mouse in vivo efficacy against a. Streptococcus pneumoniae strain PJ-1324 for the four anti-PspA monoclonal antibodies including 139G3, HOcsGl, 140G11, and 140csHl . Survival of the mice was followed for 13 days. The combined survival results of two to three independent experiments are shown in the graphs (**, p<0.005; ***, p<0.0005, survival curve comparisons of isotype vs. anti-PspA antibodies with Mantel-Cox test).

FIGs. 13 A, 13B and 13C show the summary of mouse in vivo efficacy against a Streptococcus pneumoniae strain NCTC-11905 for the four anti-PspA monoclonal antibodies including 139G3, HOcsGl, 140G1 1, and HOcsHl . 4-6 h before intraperitoneal (i.p.), intranasal (i.n.), or intravenous (i.v.) infection with the indicated S. pneumoniae strains, female CD-I or Swiss- Webster mice were pretreated i.p. with the indicated amounts of mAbs in PBS. In most experiments, heparinized tail vein blood was collected and bacterial CFU enumerated (Left panels). For the blood data, the results of one to two independent experiments are shown (*, p<0.05; CFU comparisons of isotype vs. anti-PspA mAb treatment with unpaired t-test). Survival of the mice was followed for 13-14 days. The combined survival results of two to three independent experiments are shown in the graphs (*, p<0.05; **, p<0.005; ***, p<0.0005, survival curve comparisons of isotype vs. anti-PspA antibodies with Mantel-Cox test).

FIG 14 shows the summary of the five mouse in vivo studies. Degrees of antibody activity: +++: strong protection observed, >66.7% of mice survived 13-15-day observation period; ++: survival proportion >33.3%, but <66.7%; +, survival proportion <33.3%, but statistically significant protection compared to isotype antibody treated mice; neg: no protection compared to negative control group; CFU, no significant protection in terms of survival, but significantly reduced bacterial numbers in tail vein blood observed 24 h after infection compared to negative control antibody treated mice; n.t.: not tested, since antibody does not bind to respective S. pneumoniae strain. The bacterial doses were chosen as such that all isotype treated mice would succumb to infection which was the case in all experiments with the exception of the ATCC-6301 model where 2/20 mice survived. The results of two to four experiments were combined for each animal model to calculate the degree of anti-PspA antibody protection. Please note that CD-I mice were used for all but the WU2 passive immunization model in which Swiss Webster mice were used.

FIGs. 15A and 15B show the reduction of lung CFU by HOcsHl in S.

pneumoniae mouse lung infection model using PJ-1324. Six hours after intratracheal infection with 0.75-2.7 x 10⁶ CFU of S. pneumoniae PJ-1324 in 50 μΐ, PBS, CD-I mice were treated intraperitoneally with 200 xL PBS containing 200 μg isotype IgG2a mAb C44 (open circles) or HOcsHl (closed circles). Twenty- four hours after infection, CFU numbers in (A.) lungs and (B.) tail vein blood of the animals was determined. The combined results of three independent experiments are shown. The detection limit of 100 CFU is indicated by dotted line. *, p<0.04, C44 vs. HOcsHl treated mice, unpaired t-test.

FIG. 16 shows the survival improvement by HOcsHl in a S. pneumoniae mouse lung infection model using PJ-1324. Twenty-four hours after intratracheal infection with 0.75-2.7 x 10⁶ CFU of S. pneumoniae PJ-1324 in 50 PBS, CD-I mice were treated intraperitoneally with 200 iL PBS containing 200 μg isotype IgG2a mAb C44 (open circles) or HOcsHl (closed circles). Mice were treated 24 h after infection with C44 (open circles; n= 12 total) or HOcsHl (closed circles; n= 13 total). The survival of the mice was followed for 15 days after infection. The combined results of two independent

experiments are shown in the graph. **, p<0.004, C44 vs. HOcsHl survival curve, Mantel-Cox test.

FIGs. 17A and 17B show the survival improvement by combination therapy of HOcsHl and ceftriaxone in a S. pneumoniae mouse passive immunization model using PJ- 1324. Twenty-four hours after intraperitoneal (i.p.) infection with ~2.1-3.2x 10⁴ CFU of S. pneumoniae PJ-1324, which equals around 21- to 32-fold the LD100, in 200 μΐ, PBS, CD- 1 mice were treated i.p. with 200 μί PBS alone (vehicle), or 200 μΐ, PBS containing 100 μg isotype IgG2a mAb C44 or HOcsHl + 1 mg ceftriaxone (-50 mg/kg). Survival of the mice was followed for 15 days after infection (14 days after treatment). The combined results of three independent experiments are shown in the graph. *, p<0.05, survival curve comparison with Mantel-Cox test. A) indicates antibody treatment, and B) indicates combination therapy of antibody and ceftriaxone.

FIG. 18 shows Activity of anti-PspA mAb HOcsHl in a mouse passive immunization model with a multi-drug resistant pneumococcal strain BAA-658. ~30 min after intraperitoneal (i.p.) infection with 3.2-6.5 x 10⁷ exponential phase CFU of the erythromycin (ERM)-resistant pneumococcal strain BAA-658, groups of 4-5 female CD-I mice were treated i.p. with 0.2 ml of PBS containing 300 microgram of mouse isotype IgG2a or anti-PspA mAb 140csHl . Then, mice were treated intragastrically with either 200 PBS, 1.5% ethanol (vehicle) or 200 xL PBS, 1.5% ethanol, 1.5 mg/niL ERM (Dose: 15 mg/kg). The survival of the mice was monitored for 8 days. The results of three independent experiments were combined for the graph shown. Mantel-Cox test was applied to calculate if mortality curves are statistically significantly different from each other. ***, pO.0001 vehicle+140csHl vs. ERM+140csHl ; n.s., not significant, vehicle+isotype mAb vs. ERM+isotype mAb.

FIG. 19 shows the MFI data of the increased activity of human IgGl/IgG3 versions of mouse/human chimeric 140H1 in complement deposition assays with human serum using a Streptococcus pneumoniae strain WU2. 10 CFU/mL of highly

encapsulated S. pneumoniae WU2 were incubated for 15 min in 2.5% unabsorbed human serum pool, in the presence or absence of 5 μg/mL of control or chimeric anti-PspA antibodies. Subsequently, C3b deposition was detected with a FITC-labeled anti-C3 antibody. Samples were run in triplicate and the FL-1 intensity of the cells was measured by flow cytometry. Average Mean Fluorescence Intensity (MFI) values + SD of one representative experiment of two performed with similar results are shown in the graph. ***, p<0.0005, vs. chimeric Fuc+ and Fuc- hlgGl versions of 140H1, unpaired Student's t-test).

FIGs. 20A, 20B and 20C show the histogram data of the increased activity of human IgGl/IgG3 versions of mouse/human chimeric 140H1 in complement deposition assays with human serum using a Streptococcus pneumoniae strain WU2. 10 CFU/mL of highly encapsulated S. pneumoniae WU2 were incubated for 15 min in 2.5% unabsorbed human serum pool, in the presence or absence of 5 microgram/mL of control or chimeric anti-PspA antibodies. Subsequently, C3b deposition was detected with a FITC-labeled anti-C3 antibody. Samples were run in triplicate and the FL-1 intensity of the cells was measured by flow cytometry. The histogram shows the FL-1 data for 10,000 bacterial particles for one representative sample.

FIGs. 21 A to 21H show the histogram data of the increased activity of human IgGl/IgG3 versions of mouse/human chimeric 140H1 in complement deposition assays with human serum using two Streptococcus pneumoniae strains BAA-658 and PJ-1324. 10 CFU/mL of highly encapsulated S. pneumoniae strains were incubated for 15 min in 2.5% unabsorbed human serum pool, in the presence or absence of 5 microgram/mL of control or chimeric anti-PspA antibodies. Subsequently, C3b deposition was detected with a FITC-labeled anti-C3 antibody. Samples were run in triplicate and the FL-1 intensity of the cells was measured by flow cytometry. Experiments with S. pneumoniae BAA-658 (FIGs. 21A-21D) and PJ-1324 (FIGs. 21E-21H) were conducted as above, but with 40% absorbed human plasma as complement source and 20,000 analyzed particles per sample.

FIG. 22 shows the summary of the increased activity of human IgGl/IgG3 versions of mouse/human chimeric 140H1 and 140G1 in complement deposition assays with human serum using three Streptococcus pneumoniae strains WU2, BAA-658 and PJ- 1324.

FIG. 23 shows Increased activity of non-fucosylated mouse/human chimeric 140H1 in opsonophagocytosis assays (OP As) against D39 strain with human phagocytes. In 96-well plates, 2.5 x 10⁶ fluorescein-labeled pneumococcal particles were incubated for 30 min with 5 x 10⁵ purified human blood PMN in 200 μΐ HBSS/PBS buffer and 10 μg/mL or isotype control or chimeric anti-PspA versions. Subsequently, the samples were washed and 200 live PMN/sample analyzed by fluorescence microscopy for phagocytosis of pneumococci. Ethidium bromide at an endconcentration of 0.25 mg/mL was added to the samples to be able to differentiate between intra- and extracellular pneumococci. Samples were run in quadruplicate and average values + SD of one representative experiment of at least two performed are shown in the graphs. **, p<0.005; ***, p<0.0005 vs. chimeric Fuc+ IgGl version of 140H1, unpaired Student's t-test.

FIG. 24 shows increased activity of non-fucosylated mouse/human chimeric 140G1 in opsonophagocytosis assays (OP As) against D39 strain with human phagocytes. In 96-well plates, 2.5 x 10⁶ fluorescein-labeled pneumococcal particles were incubated for 30 min with 5 x 10⁵ purified human blood PMN in 200 μΐ, HBSS/PBS buffer and 2.5 μg/mL or isotype control or chimeric anti-PspA versions. Subsequently, the samples were washed and 200 live PMN/sample analyzed by fluorescence microscopy for phagocytosis of pneumococci. Ethidium bromide at an endconcentration of 0.25 mg/mL was added to the samples to be able to differentiate between intra- and extracellular pneumococci.

Samples were run in quadruplicate and average values + SD of one representative experiment of at least two performed are shown in the graphs. **, p<0.005; ***, p<0.0005 vs. chimeric Fuc+ IgGl version of 140G1, unpaired Student's t-test.

FIGs. 25 A to 25D show binding of chimeric and humanized 140H1 antibody variants to live pneumococci. Live, exponential phase cells of the indicated S. pneumoniae strains were washed, and then incubated for 1 h with purified isotype human IgGl or the indicated chimeric or humanized anti-PspA antibodies at 4°C with shaking. Then, cells were washed and bound primary antibody detected with PE-labeled anti-human IgG. Subsequently, the FL-2 mean fluorescence intensities (MFI) of > 20,000 bacterial particles per sample were measured after excitation with a 488 nm laser with a FACSCalibur™.

Samples were run in duplicate and average MFI values for each data point are shown in the graphs. FIG. 26 shows activity of chimeric and humanized 140H1 antibody variants in opsonophagocytosis assays with human phagocytes. In 96-well polypropylene round bottom plates, 2.5 x 10⁶ fluorescein-labeled pneumococcal particles were incubated for 15 min with 5 x 10⁵ purified human blood polymorphonuclear neutrophils (PMN) in 200 HBSS, 10% PBS containing 100 μg/mL human anti-DNP antibody +/- 10 μg/mL of the indicated non-fucosylated (Fuc-) chimeric negative control or chimeric or humanized anti- PspA human IgGl (hlgGl) antibody versions. Subsequently, the samples were washed once. Ethidium bromide at an endconcentration of 0.25 mg/mL was added to the samples to stain extracellular bacteria to differentiate between intra- and extracellular pneumococci by fluorescence microscopy using a triple dichroic filter for simultaneous visualization of green and red fluorescence. Samples were run in hexuplicate and at least 100 live

PMN/sample analyzed for phagocytosis of pneumococci. Average values + SD are shown in the graph. ***, pO.0001, comparison with negative control antibody by unpaired two- tailed t-test.

Description of Embodiments

The present invention relates to the following (1) to (21):

(1) A monoclonal antibody or antigen-binding fragment thereof which recognizes one epitope comprising a peptide selected from TPAPAPKPEQPA, KPAPAPQP and

DDQQAEED YAPvRSEEE YNPvLPQQQPPKAE of a Proline-rich domain, and binds to pneumococcal surface protein (hereinafter described as Psp).

(2) The antibody or antigen-binding fragment thereof according to (1), wherein Psp is at least one Psp selected from PspA and PspC.

(3) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody binds to Streptococcus pneumoniae which expresses PspA.

(4) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody binds to PspA polypeptide derived from Streptococcus pneumoniae strain selected from D39 and BAA-658 at the affinity less than 10 nM.

(5) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody binds to Streptococcus pneumoniae strains consisting of ATCC-6301, ATCC-

49619, NCTC-1 1888, BAA-658, ATCC-700675, NCTC-1 1886, ATCC-700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC-11905, NCTC-1 1906, ATCC-700673, NCTC-1 1897, BAA- 612 and ATCC-700671.

(6) The antibody or antigen-binding fragment thereof according to (1), which binds to Streptococcus pneumoniae strains consisting of ATCC-6301, NCTC-1 1910,

ATCC-49136, ATCC49619, NCTC-1 1888, EF3030, BAA-658, ATCC-700675, ATCC- 6305, WU2, NCTC-7978, D39, NCTC-1 1886, BAA-475, BAA-340, ATCC-700905, PJ- 1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-1 1906, ATCC-700673, NCTC-11897, BAA-612, ATCC-49150, ATCC-700671 and DS2341-94.

(7) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody comprises complementarity determining regions (hereinafter, described as CDR) 1 to 3 of heavy chain variable region (hereinafter described as VH) comprising amino acid sequences of SEQ ID NOs: l to 3, respectively, and CDRs 1 to 3 of light chain variable region (hereinafter described as VL) comprising amino acid sequences of SEQ ID NOs:4 to 6, respectively.

(8) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody comprises VH comprising amino acid sequence consist of SEQ ID NO: 7 and VL comprising amino acid sequence consist of SEQ ID NO: 8.

(9) The antibody or antigen-binding fragment thereof according to (1), wherein the antibody is any recombinant antibodies selected from a chimeric antibody, humanized antibody and human antibody.

(10) A humanized antibody or antigen-binding fragment thereof according to (9), is selected from (a) and (b) as followed,

(a) wherein VH of the humanized antibody comprises an amino acid sequence in which at least one substitution selected from substitutions of Val at position 2 with He, Ser at position 9 with Pro, Val at position 20 with lie, Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with He, Val at position 93 with Thr, Tyr at position 95 with Phe, and Ala at position 97 with Gly is introduced in the amino acid sequence represented by SEQ ID NO: 10; and

wherein VL of the humanized antibody comprises an amino acid sequence in which at least one substitution selected from substitutions of He at position 2 with Thr, Leu at position 15 with Val, Ala at position 19 with Val, He at position 21 with Met, Pro at position 49 with Ser, and Leu at position 84 with Val is introduced in the amino acid sequence represented by SEQ ID NO: 12, or

(b) wherein VH of the humanized antibody comprises the amino acid sequence represented by any one of SEQ ID NOs:10, 14, and 16, and

wherein VL of the humanized antibody comprises the amino acid sequence represented by SEQ ID NOs: 12, 18, and 20.

(11) A method for detecting Psp polypeptide or Psp-expressed on Streptococcus pneumoniae comprising a use of the antibody or antigen-binding fragment thereof according to (1). (12) A method for treating a Streptococcus pneumoniae-induced infectious disease of a subject, comprising administration of the antibody or antigen-binding fragment thereof according to (1) to the subject.

(13) The method for treating the infectious diseases according to (12), wherein the infectious disease is selected from pneumonia, sepsis, septic shock, otitis media, pericarditis, peritonitis, bronchitis, bacteremia and meningitis.

(14) A method for treating the infectious diseases according to (12), wherein the infectious disease is caused by at least one of Streptococcus pneumoniae strains being resistant to complement activation.

(15) A method for treating the infectious diseases according to (12), wherein the infectious disease is caused by at least one of Streptococcus pneumoniae strains being resistant to multi-drugs.

(16) A method for treating the infectious diseases according to (12), wherein the administration of the antibody is combined with at least one antibiotic.

(17) A method for treating a Streptococcus pneumoniae-m^' d ced infectious disease of a subject described in (16), wherein the antibiotic is selected from cephem antibiotic and macrolide antibiotic.

(18) A nucleotide encoding amino acid sequence of the antibody and antigen- binding fragment thereof according to (1).

(19) A recombinant vector which comprises the DNA according to (18).

(20) A transformant obtainable by introducing the recombinant vector according to claim (19) into a host cell.

(21) A process for producing the antibody or the antibody fragment thereof according to (1), comprising culturing the transformant described in (20) in a medium to form and accumulate the antibody or the antibody fragment thereof described in (1) in the culture, and collecting the antibody or the antibody fragment thereof from the culture.

The present invention can provide a monoclonal antibody or an antigen- binding fragment thereof, which specifically recognizes a portion of proline-rich region of Streptococcus pneumoniae surface protein (Psp) and binds to the region with a high affinity, and also exhibits a high phagocytotic activity and/or a high complement- deposition activity against Streptococcus pneumoniae; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which comprises the DNA; a transformant obtainable by introducing the vector; a process for producing the antibody or the antigen-binding fragment thereof using the hybridoma or the transformant; and a therapeutic agent and a diagnostic agent using the antibody or the antigen-binding fragment thereof. In the present invention, pneumococcal surface protein (hereinafter described as Psp) includes PspA and PspC. PspA of the present invention includes a polypeptide comprising the amino acid sequence represented by NCBI reference sequence

NP_344663.1 (TIGR4) or YP_815641.1 (D39); a polypeptide comprising an amino acid sequence in which one or more amino acid residue(s) is/are deleted, substituted or added in the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) and having the activity of PspA; a polypeptide comprising an amino acid sequence having at least 60% homology, preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology, with the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) and having the activity of PspA; and the like. The amino acid sequence of PspA polypeptide from D39 is identical to that of strain Rxl [Infect. Immu. 68:5889-5900 (2000)].

The polypeptide comprising an amino acid sequence in which one or more amino acid residue(s) is/are deleted, substituted and/or added in the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) can be obtained, for example, by introducing a site-specific mutation into DNA encoding a polypeptide comprising the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) by site-specific mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press

(1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), or Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] or the like. The number of amino acid residues which are deleted, substituted or added is not particularly limited, and the number is preferably, 1 to dozens, such as 1 to 20, and more preferably 1 to several, such as 1 to 5.

As a gene encoding PspA, the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39) may be exemplified. As the gene encoding PspA, the gene encoding PspA of the present invention also included a gene containing a DNA comprising a nucleotide sequence having deletion(s), substitution(s) or addition(s) of one or more nucleotides in the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39) and also encoding a polypeptide having the function of PspA; a gene containing a DNA consisting of a nucleotide sequence having at least 60% or higher homology, preferably 80% or higher homology, and more preferably 95% or higher homology, with the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39), and also encoding a polypeptide having the function of PspA; a gene consisting of a DNA which hybridizes with a DNA having the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39) under stringent conditions and also containing a DNA that encodes a polypeptide having the function of PspA; and the like.

In the present invention, the DNA which hybridizes under stringent conditions refers to a DNA which is obtained by colony hybridization, plaque hybridization, Southern blot hybridization, DNA microarray or the like using a DNA having the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39) as a probe. A specific example of such DNA is a hybridized colony- or plaque derived DNA which can be identified by performing hybridization at 65°C in the presence of 0.7 to 1.0 mol/L sodium chloride using a filter or slide glass with the PCR product or oligo DNA having immobilized thereon, and then washing the filter or slide glass at 65°C with a 0.1 to 2-fold concentration SSC solution (1-fold concentration SSC solution: 150 mmol L sodium chloride and 15 mmol/L sodium citrate). Hybridization can be carried out according to the methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Lab. Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987- 1997); DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)] and the like. Specifically, the DNA capable of hybridization includes DNA having at least 60% or more homology, preferably 80% or more homology, more preferably 90% or more homology, and most preferably 95% or more homology to the nucleotide sequence represented by NCBI gene ID 929896 (TIGR4) or 4441373 (D39).

In the nucleotide sequence of the gene encoding a protein of a prokaryote, genetic polymorphism is often recognized. The PspA gene used in the present invention also includes a gene in which small modification is generated in the nucleotide sequence by such polymorphism as the gene used in the present invention.

The number of the homology in the present invention may be a number calculated by using a homology search program known by the skilled person, unless otherwise indicated. Regarding the nucleotide sequence, the number may be calculated by using BLAST [J. Mol. Biol, 2]_5, 403 (1990)] with a default parameter or the like, and regarding the amino acid sequence, the number may be calculated by using BLAST2

[Nucleic Acids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997) with a default parameter; http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] or the like.

As the default parameter, G (cost to open gap) is 5 for the nucleotide sequence and 11 for the amino acid sequence; -E (cost to extend gap) is 2 for the nucleotide sequence and 1 for the amino acid sequence; -q (penalty for nucleotide mismatch) is -3; -r (reward for nucleotide match) is 1; -e (expect value) is 10; -W (wordsize) is 1 1 residues for the nucleotide sequence and 3 residues for the amino acid sequence; -y [dropoff (X) for blast extensions in bits] is 20 for blastn and 7 for a program other than blastn; -X (X dropoff value for gapped alignment in bits) is 15; and -Z (final X dropoff value for gapped alignment in bits) is 50 for blastn and 25 for a program other than blastn

(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

The polypeptide comprising a partial sequence of the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) can be prepared according to a method known by the skilled person. For example, it can be prepared by deleting a part of DNA encoding the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) and culturing a transformant into which an expression vector containing the DNA is introduced. Also, based on the polypeptide or DNA prepared by using the above method, a polypeptide comprising an amino acid sequence in which one or more amino acid(s) is/are deleted, substituted or added in a partial sequence of the amino acid sequence represented by NCBI reference sequence NP 344663.1 (TIGR4) or YP_815641.1 (D39) can be prepared in the same manner as described above. In addition, the polypeptide comprising a partial sequence of the amino acid sequence represented by NCBI reference sequence

NP 344663.1 (TIGR4) or YP 815641.1 (D39); or a polypeptide comprising an amino acid sequence in which one or more amino acid(s) is/are deleted, substituted or added in a partial sequence of the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) can be produced by a chemical synthesis method such as fluorenylmethoxycarbonyl (Fmoc) method or t-butyloxycarbonyl (tBoc) method.

The function of PspA in the present invention means that PspA is one of the important cell wall-anchored virulence factors of S. pneumoniae, and inhibits complement activation and deposition, and inhibits phagocytosis by host immune cells, and impairs bactericidal activity of plasma protein such as lactoferrin. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human Factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (slgA) via PspC. PspC is classified in 11 groups based on variations of the gene.

Binding of the antibody or antigen-binding fragment of the present invention to the proline-rich region of PspA can be confirmed by a method in which the binding ability of a cell expressing a specified antigen and an antibody for the specific antigen can be examined, for example, by a radioimmunoassay using a solid phase sandwich method or the like, or a conventionally known immunological detecting method for a cell expressing PspA using an enzyme immuno assay (ELISA) method, preferably a fluorescent cell staining method or the like. Examples include a fluorescent antibody staining method using the FMAT8100HTS system (manufactured by Applied Biosystem), [Cancer Immunol. Immunother. , 36, 373 (1993)], a fluorescent cell staining method using a flow cytometry, a surface plasmon resonance using the Biacore system (manufactured by GE Healthcare) and the like. In addition, it can also be confirmed by a combination of conventionally known immunological detecting methods [Monoclonal Antibodies- Principles and Practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] and the like.

In the present invention, as a cell expressing PspA includes any cells which so long as express PspA. Examples include a cell of Streptococcus pneumoniae, and a recombinant cell which is produced by using gene recombinant techniques.

The cell which infects in the human body includes a cell expressing PspA in the body of a patient suffering from infectious disease, at-a-risk patient in ventilation, surgical operation, organ transplantation, trauma, and burn.

Specific examples of the cell obtained by using gene recombination technique includes a cell expressing PspA, which is prepared by introducing an expression vector comprising cDNA encoding PspA into a bacterial cell, etc., and the like.

Examples of the antibody of the present invention include a monoclonal antibody or an antigen-binding fragment thereof against Streptococcus PspA which has a dissociation constant (hereinafter, referred to as "KD") less than 10 x 10^"9M of the antibody to PspA, binds to an proline-rich region of PspA with high affinity and has high antibody- dependent phagocytotic activity.

Examples of the antibody of the present invention include a monoclonal antibody or an antigen-binding fragment thereof against Streptococcus PspA which has a dissociation constant "KD" of the antibody of 10 x 10^"9M or less to the antigen and has a high antibody dependent phagocytotic activity and a high complement-deposition activity. Examples of the antibody of the present invention also include an antibody or an antigen- binding fragment which has complement deposition activity on a strain of Streptococcus pneumonia expressing PspA.

The monoclonal antibody of the present invention includes an antibody produced by a hybridoma and a recombinant antibody produced by a transformant transformed with an expression vector containing a gene encoding the antibody.

The monoclonal antibody is an antibody secreted by a single clone antibody- producing cell, and recognizes only one epitope (also called antigen determinant) and has uniform amino acid sequence (primary structure).

In the present invention, the monoclonal antibody has a structure comprising a heterotetramer consisting of two H chains and two L chains. A H chain comprises a H chain variable region (hereinafter referred to as "VH") and a H chain constant region (hereinafter referred to as "CH"); and a L chain comprises a L chain variable region (hereinafter referred to as "VL") and a L chain constant region (hereinafter referred to as "CL"). In addition, CH comprises four domains: CHI domain, hinge domain, CH2 domain and CH3 domain. Furthermore, a domain consisting of CH2 domain and CH3 domain together is defined as "Fc region", "Fc domain" or simply "Fc" of an antibody.

Examples of the epitope include a single amino acid sequence, a three- dimensional structure formed by an amino acid sequence, and the like, which a monoclonal antibody recognizes and binds to. Examples of the epitope of the monoclonal antibody of the present invention include preferably a portion of proline-rich region in PspA

polypeptide such as TP APAPKPEQPA, KPAPAPQP and

DDQQAEEDYARRSEEEY RLPQQQPPKAE, further preferably a portion of proline- rich region in PspA polypeptide which is inserted between N-terminal alpha-helical coiled- coil domain and C-terminal non-proline block (NPB), and an epitope corresponding to position 340 to 347 of the amino acid sequence represented NCBI reference sequence YP_815641.1 (D39) or to position 450 to 457 of the amino acid sequence represented NCBI reference sequence NP_344663.1 (TIGR4).

The antibody of the present invention can bind to at least one of Psp selected from PspA and PspC. Accordingly the antibody can bind to Streptococcus pneumoniae in which at least one of Psp selected from PspA and PspC is expressed. Preferably the antibody of the present invention can bind to PspA and PspC, and bind to both of them expressed Streptococcus pneumoniae. These antibodies can be effective for broad spectrum of bacteria expressed by PspA and PspC.

In the present invention, an antibody which binds to PspA with high affinity is an antibody which has enough affinity for a therapeutic antibody, preferably an antibody which binds to PspA derived from Streptococcus pneumoniae strain selected from BAA- 658, D39, and TIGR4, with dissociation constant ¾ value less than 10 x 10^"9M, preferably less than 1 x 10^"9M, in terms of affinity.

Affinity is measured by kinetic analysis, and for example, can be measured by using a Biacore T100 and Biacore 3000 (manufactured by GE Healthcare Bio-Sciences), Octet (manufactured by ForteBio) or the like.

In the present invention, the term "dissociation is slow" means that a value of a dissociation rate constant kd of an antibody calculated by Biacore T100, Biacore 3000 or Octet has a smaller value. The smaller dissociation rate constant represents that an antibody does not easily dissociate from an antigen-expressing cell. By increasing an amount of an antibody binding to the cell surface to thereby extend the effective time of the antibody, then a high medicinal efficacy can be expected. A dissociation rate constant kd is measured, for example, using a Biacore T100, Biacore 3000, or Octet and can be calculated by software attached to the apparatus, Biacore T100 evaluation software (manufactured by Biacore), or the like.

The antibody of the present invention includes an antibody which binds to at least one of Streptococcus pneumoniae strains consisting of ATCC-6301, NCTC-1 1910, ATCC-49136, ATCC-49619, NCTC-1 1888, EF3030, BAA-658, ATCC-700675, ATCC- 6305, WU2, NCTC-7978, D39, NCTC-11886, BAA-475, BAA-340, ATCC-700905, PJ- 1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-11906, ATCC-700673, NCTC-1 1897, BAA-612, ATCC-49150, ATCC-700671 and DS2341-94.

Preferably, an antibody which can bind to at least Streptococcus pneumoniae strains consisting of ATCC-6301, ATCC49619, NCTC-11888, BAA-658, ATCC-700675, NCTC-11886, ATCC-700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC- 1 1906, ATCC-700673, NCTC-1 1897, BAA-612 and ATCC-700671, is exemplified.

Further preferably an antibody which can binds to Streptococcus pneumoniae strains consisting of ATCC-6301, NCTC-1 1910, ATCC-49136, ATCC49619, NCTC- 11888, EF3030, BAA-658, ATCC-700675, ATCC-6305, WU2, NCTC-7978, D39, NCTC- 1 1886, BAA-475, BAA-340, ATCC-700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC- 1 1905, NCTC-1 1906, ATCC-700673, NCTC-1 1897, BAA-612, ATCC-49150, ATCC- 700671 and DS2341 -94, is exemplified.

In the present invention, the term "antibody-dependent phagocytotic activity" refers to an activity which leads to the phagocytosis to a target bacterial cell by such a manner that an antibody bound to an antigen on the target cell binds to an Fc receptor of an immune cell through an Fc region of the antibody, consequently resulting in the activation of the immune cell (neutrophil, macrophage, etc.).

An Fc receptor (hereinafter referred to as "FcR") is a receptor which binds to Fc region of an antibody and leads to various effector activities by binding to the antibody. An FcR corresponds to a subclass of an antibody, and IgG, IgE, IgA and IgM specifically binds to FcyR, FcsR, Fc R and FcμR, respectively. Moreover, an FcyR has subtypes of FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) and these have isoforms of FcyRIA, FcyRIB, FcyRIC, FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA and FcyRIIIB, respectively. The above different FcyRs exist on different cells {Annu. Rev. Immunol , 9:457-492 (1991)). In human, FcyRI specifically expresses on a macrophage and FcyRIIA specifically expresses on a neutrophil, macrophage and FcyRIIIB specifically expresses on a neutrophil and FcyRIIIA expresses on a monocyte, a Natural Killer cell (NK cell) and some part of T cell. Binding of an antibody through FcyRI, FcyRIIA, FcyRIIIB, leads to an immune cell- dependent phagocytotic activity.

In the present invention, the term "complement deposition activity" refers to an activity which leads to the complement C3b deposition on a target bacterial cell by such a manner that an antibody bound to an antigen on the target cell activates a series of cascades (complement activation pathways) containing complement-related protein groups in blood. In addition, protein fragments generated by the activation of a complement can induce the migration, phagocytosis and activation of immune cells.

Examples of the antibody of the present invention include a monoclonal antibody or an antigen-binding fragment which binds to the proline-rich (PR) region of PspA, especially to the proline-rich region inserted between N-terminal alpha-helical coiled-coil domain and C-terminal non-proline block (NPB).

Further, among the antibodies of the present invention, examples of an antibody which recognizes proline-rich region include a monoclonal antibody in which a heavy chain constant region (hereinafter referred to as VH) of the antibody comprises the amino acid sequences of CDRs 1 to 3 represented by SEQ ID NOs: l to 3, respectively, and a light chain constant region (hereinafter referred to as VL) of the antibody comprises the amino acid sequences of CDRs 1 to 3 represented by SEQ ID NOs:4 to 6, respectively, and the like.

Moreover, specific examples of the monoclonal antibody of the present invention include a monoclonal antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO: 7 and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:8 and the like.

In addition, examples of the monoclonal antibody of the present invention include a monoclonal antibody which competes with the above monoclonal antibody in the binding of the proline-rich region of PspA, a monoclonal antibody which binds to the same epitope as an epitope in a proline-rich region of PspA to which the above monoclonal antibody binds.

The hybridoma can be prepared, for example, by preparing the above

Streptococcus pneumoniae cell or recombinant PspA polypeptide as an antigen

(immunogen), inducing an antibody-producing cell having antigen specificity from an animal immunized with the antigen, and fusing the antigen-producing cell with a myeloma cell. The anti-PspA monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma cell into an animal to cause ascites tumor in the animal and separating and purifying the culture or the ascites.

The animal immunized with an antigen may be any animal, so long as a hybridoma can be prepared, and mouse, rat, hamster, chicken, rabbit, or the like is suitably used. Also, the antibody of the present invention includes an antibody produced by a hybridoma obtained by fusion of the cell having antibody-producing activity can be obtained from such an animal, and immune in vitro with a myeloma cell. In the present invention, the recombinant antibody includes an antibody produced by gene recombination, such as a human chimeric antibody, a humanized antibody (complementarity determining region (hereinafter referred to as CDR)-grafted antibody), a human antibody and an antigen-binding fragment thereof. Among the recombinant antibodies, one having characters of a monoclonal antibody, low

immunogenecity and prolonged half-life in blood is preferable as a therapeutic agent. Examples of the recombinant antibody include an antibody in which the above monoclonal antibody of the present invention is modified by gene recombination technology.

CH of the recombinant antibody of the present invention is preferably of human origin and includes CHI domain, hinge domain, CH2 domain and CH3 domain.

Fc region of the recombinant antibody of the present invention may include one or more amino acid modification, so long as it has binding activity to FcyRs.

The human chimeric antibody is an antibody comprising a heavy chain variable region VH and a light chain variable region VL of an antibody of a non-human animal and CH and CL of a human antibody. Specifically, the human chimeric antibody of the present invention can be produced by obtaining cDNAs encoding VH and VL from a hybridoma which produces a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region, inserting each of them into an expression vector for animal cell comprising DN As encoding CH and CL of human antibody to thereby construct a vector for expression of human chimeric antibody, and then introducing the vector into an animal cell to express the antibody.

As the CH of the human chimeric antibody, any CH can be used, so long as it belongs to human immunoglobulin (hereinafter referred to as "hlg"), and those belonging to the hlgG class are preferred, and any one of the subclasses belonging to the hlgG class, such as hlgGl, hIgG2, gG3 and hIgG4, can be used. Additionally an engineered constant region of human IgGl/IgG3 chimeric isotypes can be also used [Cancer Res. 2008 May 15;68(10):3863-72.]. As the CL of the human chimeric antibody, any CL can be used, so long as it belongs to the hlg class, and those belonging to κ class or λ class can be used.

Specific examples of the human chimeric antibody of the present invention include a human chimeric antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO: 7 and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:8, and the like.

In addition, examples of the chimeric antibody of the present invention include a chimeric antibody which competes with the above chimeric antibody in the binding of the proline-rich region of PspA and a chimeric antibody which binds to the same epitope as an epitope in a proline-rich region of PspA to which the above chimeric antibody binds. A humanized antibody is an antibody in which amino acid sequences of CDRs of VH and VL of an antibody derived from a non-human animal are grafted into appropriate positions of VH and VL of a human antibody. The humanized antibody of the present invention can be produced by constructing cDNAs encoding a V region in which the amino acid sequences of CDRs of VH and VL of an antibody derived from a non- human animal produced by a hybridoma which produces a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region are grafted into frame work region (hereinafter referred to as "FR") of VH and VL of any human antibody, inserting each of them into a vector for expression of animal cell comprising genes encoding CH and CL of a human antibody to thereby construct a vector for expression of humanized antibody, and introducing it into an animal cell to thereby express and produce the humanized antibody.

As the CH of the humanized antibody, any CH can be used, so long as it belongs to the hlg class, and those of the hlgG class are preferred and any one of the subclasses belonging to the hlgG class, such as hlgGl, hIgG2, hIgG3 and hIgG4 can be used. Additionally an engineered constant region of human IgGl/IgG3 chimeric isotypes can be also used [Cancer Res. 2008 May 15;68(10):3863-72.]. As the CL of the humanized antibody, any CL can be used, so long as it belongs to the hlg class, and those belonging to the κ class or λ class can be used.

Examples of the humanized antibody of the present invention include a humanized antibody wherein CDRs 1 to 3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs: l to 3, respectively, and CDRs 1 to 3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:4 to 6.

Moreover, specific examples of the humanized antibody of the present invention include the following humanized antibodies:

with regard to the amino acid sequence of VH of the antibody, a humanized antibody wherein VH of the antibody has an amino acid sequence in which Val at position 2, Ser at position 9, Val at position 20, Arg at position 38, Gin at position 39, Glu at position 46, Met at position 48, Phe at position 68, Val at position 93, Tyr at position 95 with Phe, and Ala at position 97 in the amino acid sequence represented by SEQ ID NO: 10 are substituted with other amino acid residues,

preferably, a humanized antibody wherein VH of the antibody has an amino acid sequence in which Ser at position 9, Arg at position 38, Gin at position 39, Glu at position 46, Met at position 48, Phe at position 68, Val at position 93, Tyr at position 95 with Phe, and Ala at position 97 in the amino acid sequence represented by SEQ ID NO: 10 are substituted with other amino acid residues. The amino acid sequence of VH of the antibody obtained by the above amino acid modifications include an amino acid sequence in which at least one modification selected from among amino acid modifications for substituting Val at position 2 with He, Ser at position 9 with Pro, Val at position 20 with He, Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with lie, Val at position 93 with Thr, Tyr at position 95 with Phe, and Ala at position 97 with Gly is introduced in the amino acid sequence represented by SEQ ID NO: 10.

A specific example of the amino acid sequence of VH in which eleven modifications are introduced in the amino acid sequence represented by SEQ ID NO: 10 include the following amino acid sequences:

An amino acid sequence in which substitutions of Val at position 2 with lie, Ser at position 9 with Pro, Val at position 20 with He, Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with He, Val at position 93 with Thr, Tyr at position 95 with Phe, and Ala at position 97 with Gly are introduced, and the like.

A specific example of the amino acid sequence of VH in which nine modifications are introduced in the amino acid sequence represented by SEQ ID NO: 10 include the following amino acid sequences:

An amino acid sequence in which substitutions of Ser at position 9 with Pro,

Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with lie, Val at position 93 with Thr, Tyr at position 95 with Phe, and Ala at position 97 with Gly are introduced, and the like.

With regard to the amino acid sequence of VL of the antibody, a humanized antibody wherein VL of the antibody has an amino acid sequence in which He at position 2, Leu at position 15, Ala at position 19, He at position 21, Pro at position 49, and Leu at position 84 in the amino acid sequence represented by SEQ ID NO: 12 are substituted with other amino acid residues,

preferably, a humanized antibody wherein VL of the antibody has an amino acid sequence in which He at position 2, Ala at position 19, He at position 21 , and Pro at position 49 in the amino acid sequence represented by SEQ ID NO: 12 are substituted with other amino acid residues.

A specific example of the amino acid sequence of VL in which six modifications are introduced in the amino acid sequence represented by SEQ ID NO: 12 include the following amino acid sequences: an amino acid sequence in which substitutions of lie at position 2 with Thr, Leu at position 15 with Val, Ala at position 19 with Val, He at position 21 with Met, Pro at position 49 with Ser, and Leu at position 84 with Val are introduced, and the like.

A specific example of the amino acid sequence of VL in which four modifications are introduced in the amino acid sequence represented by SEQ ID NO: 12 include the following amino acid sequences:

an amino acid sequence in which substitutions of lie at position 2 with Thr, Ala at position 19 with Val, He at position 21 with Met, and Pro at position 49 with Ser are introduced, and the like.

Specific example of the humanized antibody of the present invention includes a humanized antibody in which VH comprises the amino acid sequence represented by one selected from SEQ ID NOs: 10, 14, and 16, and/or VL comprises the amino acid sequence represented by one selected from SEQ ID NOs: 12, 18 and 20. Furthermore, specific example of the humanized antibody of the present invention specifically include:

a humanized antibody in which H chain of variable region comprises the amino acid sequence represented by SEQ ID NO: 10 and/or L chain of variable region comprises the amino acid sequence represented by SEQ ID NO: 12;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 14 and/or VL comprises the amino acid sequence represented by SEQ ID NO: 12;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 16 and/or VL comprises the amino acid sequence represented by SEQ ID NO: 12;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 10 and/or VL comprises the amino acid sequence represented by SEQ ID NO: 18;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 14 and/or VL comprises the amino acid sequence represented by SEQ ID NO: 18;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 16 and/or VL comprises the amino acid sequence represented by SEQ ID NO: 18;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 10 and/or VL comprises the amino acid sequence represented by SEQ ID NO:20; a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 14 and/or VL comprises the amino acid sequence represented by SEQ ID NO:20;

a humanized antibody in which VH comprises the amino acid sequence represented by SEQ ID NO: 16 and/or VL comprises the amino acid sequence represented by SEQ ID NO:20; and the like.

In addition, examples of the humanized antibody of the present invention include a humanized antibody which competes with the above humanized antibody in the binding of the proline-rich region of PspA and a humanized antibody which binds to the same epitope as an epitope in a proline-rich region of PspA to which the above humanized antibody binds.

A human antibody is originally an antibody naturally existing in the human body, and it also includes an antibody obtained from a human antibody phage library or a human antibody-producing transgenic animal, which is prepared based on the recent advanced techniques in genetic engineering, cell engineering and developmental engineering.

The antibody existing in the human body can be prepared, for example by isolating a human peripheral blood lymphocyte, immortalizing it by infecting with EB virus or the like and then cloning it to thereby obtain lymphocytes capable of producing the antibody, culturing the lymphocytes thus obtained, and purifying the antibody from the supernatant of the culture.

The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting a gene encoding an antibody prepared from a human B cell into a phage gene. A phage expressing an antibody fragment having the desired antigen binding activity can be recovered from the library, using its activity to bind to an antigen-immobilized substrate as the index. The antibody fragment can be converted further into a human antibody molecule comprising two full H chains and two full L chains by genetic engineering techniques.

A human antibody-producing transgenic animal is an animal in which a human antibody gene is integrated into cells. Specifically, a human antibody-producing transgenic animal can be prepared by introducing a gene encoding a human antibody into a mouse ES cell, grafting the ES cell into an early stage embryo of other mouse and then developing it. A human antibody is prepared from the human antibody-producing transgenic non-human animal by obtaining a human antibody-producing hybridoma by a hybridoma preparation method usually carried out in non-human mammals, culturing the obtained hybridoma and forming and accumulating the human antibody in the supernatant of the culture. In the amino acid sequence constituting the above antibody or antibody fragment, a monoclonal antibody or antibody fragment thereof in which one or more amino acids are deleted, substituted, inserted or added, having activity similar to the above antibody or antibody fragment is also included in the monoclonal antibody or antibody fragment of the present invention.

The number of amino acids which are deleted, substituted, inserted and/or added is one or more, and is not specifically limited, but it is within the range where deletion, substitution or addition is possible by known methods such as the site-directed mutagenesis [Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1 89), Current protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Set, USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)] or the like. For example, the number is 1 to dozens, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.

The expression "one or more amino acids are deleted, substituted, inserted or added" in the amino acid sequence of the above antibody means the followings. That is, it means there is deletion, substitution, insertion or addition of one or plural amino acids at optional positions in the same sequence and one or plural amino acid sequences. Also, the deletion, substitution, insertion or addition may occur at the same time and the amino acid which is substituted, inserted or added may be either a natural type or a non-natural type. The natural type amino acid includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Preferable examples of mutually substitutable amino acids are shown below.

The amino acids in the same group are mutually substitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

Group C: asparagine, glutamine

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid

Group E: proline, 3-hydroxyproline, 4-hydroxyproline

Group F: serine, threonine, homoserine

Group G: phenylalanine, tyrosine The antibody fragment of the present invention includes Fab, F(ab')₂, Fab', scFv, diabody, dsFv, a peptide comprising CDR and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating an IgG antibody molecule with a protease, papain (cleaved at an amino acid residue at position 224 of the H chain), are bound together through a disulfide bond.

An F(ab')₂ is an antibody fragment having a molecular weight of about 100,000 and antigen binding activity and comprising two Fab regions which are bound in the hinge position obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG, with an enzyme, pepsin. The F(ab')₂ of the present invention can be produced by treating a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region with a protease, pepsin. Also, the F(ab')₂ can be also produced by binding Fab' described below via a thioether bond or a disulfide bond.

An Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cleaving a disulfide bond at the hinge region of the above F(ab')₂. The Fab' of the present invention can be produced by F(ab')₂ which specifically recognizes PspA and binds to the proline-rich region, with a reducing agent, such as dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote to express the Fab'.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH and one chain VL are linked using an appropriate peptide linker (hereinafter referred to as "P") and is an antibody fragment having antigen binding activity. The scFv of the present invention can be produced by obtaining cDNAs encoding VH and VL of a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment wherein scFv is dimerized, is an antibody fragment having divalent antigen binding activity. In the divalent antigen binding activity, two antigens may be the same or different. The diabody of the present invention can be produced by obtaining cDNAs encoding VH and VL of a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region, constructing DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 or less residues, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acid residue of each of VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on a three-dimensional structure estimation of the antibody in accordance with a known methods [Protein Engineering, 7, 697 (1994)]. The dsFv of the present invention can be produced by obtaining cDNAs encoding VH and VL of a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region, constructing DNA encoding dsFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv.

A peptide comprising CDR is constituted by including one or more regions of CDRs of VH or VL. Peptide comprising plural CDRs can be bound directly or via an appropriate peptide linker. The peptide comprising CDR of the present invention can be produced by constructing DNA encoding CDRs of VH and VL of a monoclonal antibody which specifically recognizes PspA and binds to the proline-rich region, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the peptide. The peptide comprising CDR can also be produced by a chemical synthesis method such as Fmoc method or tBoc method.

The monoclonal antibody of the present invention includes an antibody conjugate in which a monoclonal antibody or an antibody fragment thereof which specifically recognizes a three-dimensional structure of an proline-rich region of PspA and binds to the proline-rich region is chemically or genetically bound to a radioisotope, an agent having a low molecular weight, an agent having a high molecular weight, a protein, a therapeutic antibody or the like.

The antibody conjugate of the present invention can be produced by chemically conjugating a radioisotope, an agent having a low molecular weight, an agent having a high molecular weight, a protein, a therapeutic antibody or the like to the N-terminal side or C-terminal side of an H chain or an L chain of the monoclonal antibody or the antibody fragment thereof, an appropriate substituent or side chain of the antibody or the antibody fragment, a sugar chain in the antibody or the antibody fragment or the like, which specifically recognizes a three-dimensional structure of an proline-rich region of PspA and binds to the proline-rich region in the present invention [Antibody Engineering Handbook, published by Chijin Shokan (1994)]. Also, the antibody conjugate can be genetically produced by linking a DNA encoding the monoclonal antibody or the antibody fragment thereof which specifically recognizes three-dimensional structure of an proline-rich region of PspA and binds to the proline-rich region in the present invention to other DNA encoding a protein or a therapeutic antibody to be conjugated, inserting the DNA into a vector for expression, and introducing the expression vector into an appropriate host cell.

The radioisotope includes ¹³¹1, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ⁹⁹Tc, ⁷⁷Lu, ^{i n}In, ^I88Re, ²¹¹At,

213 *

Bi, and the like. The radioisotope can directly be conjugated with the antibody by Chloramine-T method. Also, a substance chelating the radioisotope can be conjugated with the antibody. The chelating agent includes 1 -isothiocyanate benzyl-3- methyldiethylene-triaminepentaacetic acid (MX-DTPA) and the like.

The agent having a low molecular weight includes an anti-bacterial agent such as an antibiotic penicillin, cephalosporins, macrolides (such as erythromycin), tetracycline, clindamycin, quinolones, vancomycin, beta-lactam antibiotics (cephalosporins), fluoroquinolones such as levofloxacin and moxifloxacin and derivatives thereof.

The method for conjugating the agent having low molecular weight with the antibody includes a method in which the agent and an amino group of the antibody are conjugated through glutaraldehyde, a method in which an amino group of the agent and a carboxyl group of the antibody are conjugated through water-soluble carbodiimide, and the like.

The agent having a high molecular weight includes polyethylene glycol (hereinafter referred to as "PEG"), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropylmethacrylamide, and the like. By binding these compounds having a high molecular weight to an antibody or antibody fragment, the following effects are expected: (1) improvement of stability against various chemical, physical or biological factors, (2) remarkable prolongation of half life in blood, (3) disappearance of immunogenicity, suppression of antibody production, and the like [Bioconjugate Drug, Hirokawa Shoten (1993)]. For example, the method for binding PEG to an antibody includes a method in which an antibody is allowed to react with a PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. The PEG- modifying reagent includes a modifying agent of ε-amino group of lysine (Japanese Published Unexamined Patent Application No. 178926/86), a modifying agent of a carboxyl group of aspartic acid and glutamic acid (Japanese Published Unexamined Patent Application No. 23587/81), a modifying agent of a guanidino group of arginine (Japanese Published Unexamined Patent Application No. 1 17920/90) and the like. The immunostimulator may be any natural products known as immunoadjuvants. Examples of an agent enhancing immunogen include β(1— »3)glucan (lentinan, schizophyllan), a-galactosylceramide and the like.

The protein includes a cytokine or a growth factor which activates a immunocompetent cell, such as NK cell, macrophage or neutrophil, a toxic protein, and the like.

Examples of the cytokine or the growth factor include interferon (hereinafter referred to as "INF")-a, INF-β, I F-γ, interleukin (hereinafter referred to as "IL")-2, IL- 12, IL-15, IL-18, IL-21, IL-23, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF) and the like. The toxic protein includes ricin, diphtheria toxin, ONTAK and the like, and also includes a toxic protein wherein mutation is introduced into a protein in order to control the toxicity.

A fusion antibody with a protein or therapeutic antibody can be produced by linking a cDNA encoding a monoclonal antibody or an antibody fragment to a cDNA encoding the protein, constructing a DNA encoding the fusion antibody, inserting the DNA into an expression vector for prokaryote or eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the fusion antibody.

In the case where the above antibody conjugate is used for the detecting method, method for quantitative determination, detection reagent, reagent for quantitative determination or diagnostic agent in the present invention, examples of the agent to which a monoclonal antibody or an antibody fragment thereof of the present invention which specifically recognizes a proline-rich region of PspA and binds to the proline-rich region is bound includes a label used in routine immunological detecting or measuring method. The label includes enzymes such as alkaline phosphatase, peroxidase and luciferase, luminescent materials such as acridinium ester and lophine, fluorescent materials such as fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (RITC), and the like.

In addition, the present invention relates to a therapeutic agent for a disease relating to a Streptococcus pneumoniae bacterial cell which comprises a monoclonal antibody which specifically recognizes a proline-rich region of PspA and also binds to the proline-rich region, or an antigen-binding fragment thereof as an active ingredient.

The Streptococcus pneumoniae bacterial cell-associated disease may be any disease so long as it is an infectious disease relating to Streptococcus pneumoniae, and examples include pneumonia, sepsis, septic shock, bacteremia, otitis media, bronchitis, pericarditis, peritonitis and bacterial meningitis. Further more specific diseases related to Streptococcus pneumonia treated by antibodies of the present invention includes pneumococcal pneumonia, pneumococcal sepsis, pneumococcal septic shock,

pneumococcal bacteremia, pneumococcal otitis media, pneumococcal meningitis, pneumococcal pericarditis , pneumococcal peritonitis, pneumococcal bronchitis, and so on.

Further, in the present invention, Streptococcus pneumoniae strains which are resistant to complement-dependent cototoxicity (CDC) caused by an antibody and deposition of serum complement through the CDC mechanism, and Streptococcus pneumoniae strains which are resistant to at least one of antibiotics as cephem, macrolide, and so on, are exemplified as a target of antibodies of the present invention in therapy for infectious diseases.

Examples of the Streptococcus pneumoniae strain are ATCC-6301, NCTC- 1 1910, ATCC-49136, ATCC49619, NCTC-1 1888, EF3030, BAA-658, ATCC-700675, ATCC-6305, WU2, NCTC-7978, D39, NCTC-1 1886, BAA-475, BAA-340, ATCC- 700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-1 1906, ATCC-700673, NCTC-11897, BAA-612, ATCC-49150, ATCC-700671, and DS2341-94, and the like.

The therapeutic agent in the present invention includes a therapeutic agent comprising the above monoclonal antibody or an antigen-binding fragment of the present invention as an active ingredient.

The therapeutic agent comprising the antibody or antibody fragment thereof, or conjugate thereof of the present invention may comprise only the antibody or antibody fragment thereof, or conjugate thereof as an active ingredient. It is generally preferred that the therapeutic agent is prepared as a pharmaceutical preparation produced by an appropriate method well known in the technical field of pharmaceutics, and by mixing it with one or more pharmaceutically acceptable carriers.

The antibodies of the present invention can prevent or inhibit bacterial infection and have exhibited therapeutic effect after bacterial infection. Namely these antibody can inhibit an infection process of Psp expressed Streptococcus pneumoniae, growth of Psp expressed Streptococcus pneumoniae in patient body, decrease of colony forming unit (CFU) derived from infectious patient, as a result treat the infectious diseases.

In the present invention, a therapeutic method for infectious diseases includes single administration of the antibody and further combining with at least one antibiotic selected from cephem antibiotics and macrolide antibiotics as erythromycin.

It is preferred to administer the therapeutic agent by the route that is most effective for the treatment. Examples include oral administration and parenteral administration, such as buccal, tracheal, rectal, subcutaneous, intramuscular or intravenous administration is preferred.

The therapeutic agent may be in the form of spray, capsules, tablets, granules, powder, syrup, emulsion, suppository, injection, ointment, tape, and the like. Although the dose or the frequency of administration varies depending on the objective therapeutic effect, administration method, treating period, age, body weight and the like, it is usually 10 μg/kg to 100 mg/kg per day and per person (child or adult).

Further, the present invention relates to a method for immunologically detecting or measuring PspA, an agent for immunologically detecting or measuring PspA, a method for immunologically detecting or measuring Streptococcus pneumoniae cell, an agent for immunologically detecting or measuring a Streptococcus pneumoniae cell, and an agent for diagnosing a disease relating to a Streptococcus pneumoniae cell, comprising a monoclonal antibody or an antibody fragment thereof which specifically recognizes a proline-rich region of PspA and binds to the proline-rich region, as an active ingredient.

As a method for detection or determination of the amount of PspA in the present invention, any known method may be included. For example, an immunological detecting or measuring method may be exemplified.

An immunological detecting or measuring method is a method in which an antibody amount or an antigen amount is detected or determined using a labeled antigen or antibody. Examples of the immunological detecting or measuring method are radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting method, physico-chemical means and the like.

The above disease relating to Streptococcus pneumoniae can be diagnosed by detecting or measuring a Streptococcus pneumoniae cell by using the monoclonal antibody or antigen-binding fragment of the present invention.

For the detection of the Streptococcus pneumoniae cell, known immunological detecting methods can be used, and an immunoprecipitation method, a fluorescent cell staining method, an immune tissue staining method and the like are preferably used. In addition, a fluorescent antibody staining method using FMAT 8100 HTS system (Applied Biosystem) and the like can be used.

In the present invention, the living body sample to be used for detecting or measuring PspA is not particularly limited, so long as it has a possibility of containing the polypeptide, such as tissue cells, blood, blood plasma, serum, pancreatic fluid, urine, fecal matter, tissue fluid or culture fluid.

The diagnostic agent containing the monoclonal antibody or an antigen-binding fragment thereof, or conjugate thereof may further contain a reagent for carrying out an antigen-antibody reaction or a reagent for detection of the reaction depending on the desired diagnostic method. The reagent for carrying out the antigen-antibody reaction includes a buffer, a salt, and the like. The reagent for detection includes a reagent generally used for the immunological detecting or measuring method, such as labeled secondary antibody which recognizes the monoclonal antibody, antibody fragment thereof or conjugates thereof and substrate corresponding to the labeling.

A process for producing the antibody of the present invention, a method for treating the disease and a method for diagnosing the disease are specifically described below.

1. Preparation method of monoclonal antibody

(1) Preparation of antigen

PspA polypeptide as an antigen can be obtained by introducing an expression vector comprising cDNA encoding a full length of PspA or a partial length thereof is introduced into Escherichia coli, or the like. In addition, PspA can be purified from various Streptococcus pneumoniae cells. Furthermore, a synthetic peptide having a partial sequence of the Psp A can be prepared by a chemical synthesis method such as Fmoc method or tBoc method and used as an antigen.

PspA used in the present invention can be produced, for example, by expressing a DNA encoding PspA in a host cell using a method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) or the like according to the following method.

Firstly, a recombinant vector is prepared by inserting a full length cDNA comprising the region encoding PspA into downstream of a promoter of an appropriate expression vector. At this time, if necessary, a DNA fragment having an appropriate length containing a region encoding the polypeptide based on the full length cDNA, and the DNA fragment may be used instead of the above full length cDNA. Next, a transformant producing PspA can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.

The expression vector includes vectors which can replicate autonomously in the host cell to be used or vectors which can be integrated into a chromosome comprising an appropriate promoter at such a position that the DNA encoding the polypeptide can be transcribed.

The host cell may be any one, so long as it can express the objective gene. Examples include a microorganism which belongs to the genera Escherichia, such as Escherichia coli, and the like.

When a prokaryote such as Escherichia coli is used as the host cell, it is preferred that the recombinant vector used in the present invention is autonomously replicable in the prokaryote and comprises a promoter, a ribosome binding sequence, the DNA comprising the portion encoding PspA and a transcription termination sequence. The recombinant vector is not necessary to have a transcription termination sequence, but a transcription termination sequence is preferably set just below the structural gene. The recombinant vector may further comprise a gene regulating the promoter.

Also, the above recombinant vector is preferably a plasmid in which the space between Shine-Dalgarno sequence (also referred to as SD sequence), which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 nucleotides).

Furthermore, the nucleotide sequence of the DNA encoding PspA can be substituted with another base so as to be a suitable codon for expressing in a host cell, thereby improve the productivity of the objective PspA.

Any expression vector can be used, so long as it can function in the host cell to be used. Examples of the expression vector includes pBTrp2, pBTacl, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8

(manufactured by QIAGEN), pKYPIO (Japanese Published Unexamined Patent

Application No. 1 10600/83), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from

Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Published Unexamined Patent Application No. 221091/85], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Published Unexamined Patent Application No. 221091/85], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUBl 10, pTP5, pC194, pEG400 [J. BacterioL, 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by

Novagen), pMEl 8SFL3 and the like.

Any promoter can be used, so long as it can function in the host cell to be used. Examples include promoters derived from Escherichia coli, phage and the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter and T7 promoter. Also, artificially designed and modified promoters, such as a promoter in which two Ptrp are linked in tandem, tac promoter, lacT7 promoter and letl promoter, can be used.

Examples of the host cell include Escherichia coli XL 1 -Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MCI 000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W31 10, Escherichia coli NY49, Escherichia coli DH5a and the like. Any introduction method of the recombinant vector can be used, so long as it is a method for introducing DNA into the host cell, and examples include a method using a calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 11 (1979)].

PspA can be produced by culturing the transformant derived from a microorganism, or the like having a recombinant vector comprising the DNA encoding PspA in a medium to form and accumulate PspA in the culture, and recovering it from the culture. The method for culturing the transformant in the medium is carried out according to the usual method used in culturing of hosts.

When a microorganism transformed with a recombinant vector containing an inducible promoter is cultured, an inducer can be added to the medium, if necessary. For example, isopropyl- -D-thiogalactopyranoside or the like can be added to the medium when a microorganism transformed with a recombinant vector using lac promoter is cultured; or indoleacrylic acid or the like can be added thereto when a microorganism transformed with a recombinant vector using trp promoter is cultured.

Regarding the expression method of the gene encoding PspA, in addition to direct expression, secretory production, fusion protein expression and the like can be carried out according to the method described in Molecular Cloning, A Laboratory

Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

The process for producing PspA includes a method of intracellular expression in a host cell, a method of proline-rich secretion from a host cell, a method of producing on a host cell membrane outer envelope, and the like. The appropriate method can be selected by changing the host cell used and the structure of the PspA produced.

When the PspA is produced in a host cell or on a host cell membrane outer envelope, PspA can be positively secreted proline-richly in accordance with the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], the methods described in Japanese Published Unexamined Patent Application No. 336963/93 and WO 94/23021, and the like.

The resulting PspA can be isolated and purified, for example, as follows.

When PspA is intracellularly expressed in a dissolved state, the cells after culturing are recovered by centrifugation, suspended in an aqueous buffer and then disrupted using ultrasonicator, French press, Manton Gaulin homogenizer, dynomill or the like to obtain a cell-free extract. The cell-free extract is centrifuged to obtain a

supernatant, and a purified preparation can be obtained by subjecting the supernatant to a general enzyme isolation and purification techniques such as solvent extraction; salting out with ammonium sulfate etc. ; desalting; precipitation with an organic solvent; anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical); cation exchange

chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia); hydrophobic chromatography using a resin such as butyl-Sepharose or phenyl-Sepharose; gel filtration using a molecular sieve; affinity chromatography; chromatofocusing;

electrophoresis such as isoelectric focusing; and the like which may be used alone or in combination.

When PspA is expressed intracellularly by forming an inclusion body, the cells are recovered, disrupted and centrifuged in the same manner, and the inclusion body of PspA are recovered as a precipitation fraction. The recovered inclusion body of the protein is solubilized with a protein denaturing agent. The protein is made into a normal three- dimensional structure by diluting or dialyzing the solubilized solution, and then a purified preparation of PspA is obtained by the same isolation purification method as above.

Also, PspA used in the present invention can be produced by a chemical synthesis method, such as Fmoc method or tBoc method. Also, it can be chemically synthesized using a peptide synthesizer manufactured by Advanced ChemTech, Perkin- Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive,

Shimadzu Corporation, or the like. (2) Immunization of animal and preparation of antibody-producing cell for fusion

A mouse, rat or hamster 3 to 20 weeks old is immunized with the antigen prepared in the above (1), and antibody-producing cells are collected from the spleen, lymph node or peripheral blood of the animal.

The immunization is carried out by administering the antigen to the animal through subcutaneous, intravenous or intraperitoneal injection together with an appropriate adjuvant (for example, complete Freund's adjuvant, combination of aluminum hydroxide gel with pertussis vaccine, or the like). When the antigen is a partial peptide, a conjugate is produced with a carrier protein such as BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin) or the like, which is used as the antigen.

The administration of the antigen is carried out 5 to 10 times every one week or every two weeks after the first administration. On the 3rd to 7th day after each

administration, a blood sample is collected from the fundus of the eye, the reactivity of the serum with the antigen is tested, for example, by enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or the like. An animal showing a sufficient antibody titer in their sera against the antigen used for the

immunization is used as the supply source of antibody-producing cells for fusion. Three to seven days after final administration of the antigen, tissue containing the antibody-producing cells such as the spleen from the immunized animal is excised to collect the antibody-producing cells. When the spleen cells are used, the spleen is cut out and loosened, followed by centrifugation. Then, antibody-producing cells for fusion are obtained by removing erythrocytes.

(3) Preparation of myeloma cell

An established cell line obtained from mouse is used as myeloma cells.

Examples include 8-azaguanine-resistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-Ul (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NSl/l-Ag41 (NS-1) [European ! Immunology, 6, 51 1 (1976)], SP2/0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3- X63-Ag8 (X63) [Nature, 256, 495 (1975)] and the like.

The myeloma cells are subcultured in a normal medium [a medium in which glutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine are added to RPMI1640 medium] and they are subcultured in the normal medium 3 or 4 days before cell fusion to ensure the cell number of 2x 10 or more on the day for fusion.

(4) Cell fusion and preparation of hybridoma for producing monoclonal antibody

The antibody-producing cells for fusion obtained by the above (2) and myeloma cells obtained by the above (3) were sufficiently washed with a minimum essential medium (MEM) or PBS (1.83 g of disodium hydrogen phosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2) and mixed to give a ratio of the antibody-producing cells: the myeloma cells = 5 to 10: 1, followed by centrifugation. Then, the supernatant is discarded. The precipitated cell group is sufficiently loosened. After loosening the precipitated cell, the mixture of polyethylene glycol- 1000 (PEG- 1000), MEM and dimethylsulfoxide is added to the cell under stirring at 37°C. In addition, 1 to 2 mL of MEM medium is added several times every one or two minutes, and MEM is added to give a total amount of 50 mL. After centrifugation, the supernatant is discarded. After the cells are gently loosen, the cells are gently suspended in HAT medium [a medium in which hypoxanthine, thymidine and aminopterin is added to the normal medium]. The suspension is cultured in a 5% C0₂ incubator for 7 to 14 days at 37°C.

After the culturing, a portion of the culture supernatant is sampled and a hybridoma which is reactive to an antigen containing PspA and is not reactive to an antigen not containing PspA is selected by binding assay as described below. Then, cloning is carried out twice by a limiting dilution method [Firstly, HT medium (HAT medium from which aminopterin is removed) is used, and secondly, the normal medium is used], and a hybridoma which shows a stably high antibody titer is selected as the monoclonal antibody-producing hybridoma. (5) Preparation of purified monoclonal antibody

The hybridoma cells producing a monoclonal antibody obtained by the above (4) are administered by intraperitoneal injection into 8- to 10-week-old mice or nude mice treated with 0.5 niL of pristane (2,6,10,14-tetramethylpentadecane (pristane) is

intraperitoneally administered, followed by feeding for 2 weeks). The hybridoma develops ascites tumor in 10 to 21 days. The ascitic fluid is collected from the mice, centrifuged to remove solids, subjected to salting out with 40 to 50% ammonium sulfate and then precipitated by caprylic acid, passed through a DEAE-Sepharose column, a protein A column or a gel filtration column to collect an IgG or IgM fraction as a purified

monoclonal antibody.

Furthermore, a monoclonal antibody-producing hybridoma obtained by the above (4) is cultured in RPMI1640 medium containing FBS or the like and the supernatant is removed by centrifugation. The precipitated cells are suspended in Hybridoma SFM medium containing 5% DIGO GF21 and cultured for 3 to 7 days. The purified monoclonal antibody can be obtained by centrifusing the obtained cell suspension, followed by purifying the resulting supernatant with Protein A column or Protein G column to collect the IgG fractions.

The subclass of the antibody can be determined using a subclass typing kit by enzyme immunoassay. The amount of the protein can be determined by the Lowry method or from the absorbance at 280 nm.

(6) Selection of monoclonal antibody

Selection of monoclonal antibody is carried out by the following binding assay using an enzyme immunoassay method and kinetic analysis with Biacore. (6-a) Binding assay

As the antigen, a gene-introduced cell or a recombinant protein obtained by introducing an expression vector containing a cDNA encoding PspA obtained in (1) into Escherichia coli, yeast, an insect cell, an animal cell or the like, or a purified polypeptide or partial peptide obtained from a human tissue is used. When the antigen is a partial peptide, a conjugate is prepared with a carrier protein such as BSA or KLH and is used.

After making these antigens into a solid layer by dispensing in a 96-well plate, a substance to be tested such as serum, a culture supernatant of a hybridoma or a purified monoclonal antibody is dispensed therein as the primary antibody and allowed to react. After thoroughly washing with PBS or PBS-Tween, an anti- immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent material, a radiation compound or the like is dispensed therein as the secondary antibody and allowed to react. After thoroughly washing with PBS-Tween, the reaction is carried out in response to the label of the secondary antibody to select a monoclonal antibody which specifically reacts with the antigen.

The antibody which competes with the anti-PspA monoclonal antibody of the present invention can be prepared by adding an antibody to be tested to the above- mentioned binding assay system and carrying out reaction. That is, a monoclonal antibody which competes with the thus obtained monoclonal antibody for its binding to the proline- rich region of PspA can be prepared by carrying out a screening of an antibody by which the binding of the monoclonal antibody is inhibited when the antibody to be tested is added.

Furthermore, an antibody which binds to an epitope which is the same as the epitope recognized by the monoclonal antibody which binds to the proline-rich region of PspA of the present invention can be obtained by identifying the epitope of the antibody obtained in the above binding assay, and preparing a partial synthetic peptide, a synthetic peptide mimicking the conformational structure of the epitope or the like, followed by immunization.

(6-b) Kinetic analysis with Biacore

The kinetics between an antigen and a test substance is measured using Biacore T100 and then the obtained results are analyzed using analysis software accompanied with the apparatus. After anti-IgG mouse antibody is immobilized onto to the a CM5 sensor chip by an amine coupling method, a test substance such as culture supernatant of a hybridoma, a purified antibody is allowed to flow, bind at an appropriate amount, and further flow an antigen at plural known concentrations, followed by measuring the binding and dissociation. Using the obtained data and the software accompanied with the apparatus, the kinetics analysis is carried out using the 1 : 1 binding model to obtain necessary parameters. Otherwise, after PspA is immobilized onto the sensor chip by an amino coupling method, a purified monoclonal antibody is allowed to flow at plural known concentrations followed by measuring the binding and dissociation. Using the obtained data and the software accompanied with the apparatus, the kinetics analysis is carried out using bivalent analyte model to obtain necessary parameters. 2. Preparation of recombinant antibody

As production examples of recombinant antibodies, processes for producing a human chimeric antibody and a humanized antibody are shown below. ( 1 ) Construction of vector for expression of recombinant antibody

A vector for expression of recombinant antibody is an expression vector for animal cell into which DNAs encoding CH and CL of a human antibody have been inserted, and is constructed by cloning each of DNAs encoding CH and CL of a human antibody into an expression vector for animal cell.

The C region of a human antibody may be CH and CL of any human antibody.

Examples include CH belonging to γΐ subclass, CL belonging to κ class, and the like. As the DNAs encoding CH and CL of a human antibody, the cDNA may be generally used and a chromosomal DNA comprising an exon and an intron can be also used. As the expression vector for animal cell, any expression vector can be used, so long as a gene encoding the C region of a human antibody can be inserted thereinto and expressed therein. Examples include pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., KM , 1307 (1987)], pHSG274 [Gene, 21, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSGlbd2-4 [Cytotechnol, 4, 173 (1990)], pSElUKlSedl-3 [Cytotechnol, 13, 79 (1993)] and the like. Examples of a promoter and enhancer used for an expression vector for animal cell include an SV40 early promoter [J. Biochem., 101, 1307 (1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 4_i, 479 (1985)] and enhancer [Cell, 33, 717 (1983)] and the like.

The vector for expression of recombinant antibody may be either of a type in which a gene encoding an antibody H chain and a gene encoding an antibody L chain exist on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a vector for expression of recombinant antibody, easiness of introduction into animal cells, and balance between the expression amounts of antibody H and L chains in animal cells, a tandem type of the vector for expression of recombinant antibody is more preferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandem type of the vector for expression of recombinant antibody include pKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and the like. (2) Obtaining of cDNA encoding V region of antibody derived from non-human animal and analysis of amino acid sequence

Obtaining of cDNAs encoding VH and VL of a non-human animal antibody and analysis of amino acid sequence are carried out as follows.

mRNA is extracted from hybridoma cells producing an antibody derived from a non-human animal to synthesize cDNA. The synthesized cDNA is cloned into a vector such as a phage or a plasmid, to prepare a cDNA library. Each of a recombinant phage or recombinant plasmid containing cDNA encoding VH or VL is isolated from the library using DNA encoding a part of the C region or V region of a mouse antibody as the probe. The full length of the nucleotide sequences of VH and VL of a mouse antibody derived from a non-human animal of interest on the recombinant phage or recombinant plasmid are determined, and the full length of the amino acid sequences of VH and VL are deduced from the nucleotide sequences, respectively.

Examples of the non-human animal for preparing a hybridoma cell which produces a non-human antibody include mouse, rat, hamster, rabbit or the like. Any animals can be used so long as a hybridoma cell can be produced therefrom.

Examples of the method for preparing total RNA from a hybridoma cell include a guanidine thiocyanate-cesium trifluoroacetate method [Methods in EnzymoL, 154, 3 (1987)], the use of a kit such as RNA easy kit (manufactured by Qiagen) and the like.

Examples of the method for preparing mRNA from total RNA include an oligo (dT) immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], a method using a kit such as Oligo-dT30 <Super> mRNA Purification Kit (manufactured by Takara Bio) and the like. Also, examples of a kit for preparing mRNA from a hybridoma cell include Fast

Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia) and the like.

Examples of the method for synthesizing cDNA and preparing a cDNA library include known methods [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab. Press (1989); Current Protocols in Molecular Biology, Supplement 1 , John Wiley & Sons (1987-1997)]; a method using a kit such as Super Script Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by GIBCO BRL), ZAP-cDNA Kit

(manufactured by Stratagene), etc. ; and the like.

The vector into which the synthesized cDNA using mRNA extracted from a hybridoma cell as the template is inserted for preparing a cDNA library may be any vector, so long as the cDNA can be inserted. Examples include ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], zapII (manufactured by Stratagene), λgtlO and Xgtl 1 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech), ExCell and pT7T3 18U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], and the like.

Any Escherichia coli for introducing the cDNA library constructed by a phage or plasmid vector may be used, so long as the cDNA library can be introduced, expressed and maintained. Examples include XLl-Blue MRF' [Strategies, 5, 81 (1992)], C600

[Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222: 778 (1983)], NM522 [J. Mol Biol, 166, 1 (1983)], K802 [J. Mol. Biol, 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], and the like.

A colony hybridization or plaque hybridization method using an isotope- or fluorescence-labeled probe may be used for selecting cDNA clones encoding VH and VL of a non-human antibody or the like from the cDNA library [Molecular Cloning, A

Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].

Also, the cDNAs encoding VH and VL can be prepared through polymerase chain reaction (hereinafter referred to as "PCR"; Molecular Cloning, A Laboratory

Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)) by preparing primers and using cDNA prepared from mRNA or a cDNA library as the template.

The nucleotide sequence of the cDNA can be determined by digesting the cDNA selected with appropriate restriction enzymes and the like, cloning the fragments into a plasmid such as pBluescript SK(-) (manufactured by Stratagene), carrying out the reaction by a usually used nucleotide analyzing method. For example, a nucleotide analyze is carried out by using an automatic nucleotide sequence analyzer such as ABI PRISM3700 (manufactured by PE Biosystems) and A.L.F. DNA sequencer (manufactured by Pharmacia) after a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)].

Whether the obtained cDNAs encode the full amino acid sequences of VL and VL of the antibody containing a secretory signal sequence can be confirmed by estimating the full length of the amino acid sequences of VH and VL from the determined nucleotide sequence and comparing them with the full length of the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept.

Health and Human Services (1991)]. The length of the secretory signal sequence and N- terminal amino acid sequence can be deduced by comparing the full length of the amino acid sequences of VH and VL of the antibody comprising a secretory signal sequence with full length of the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the subgroup to which they belong can also be known. Furthermore, the amino acid sequence of each of CDRs of VH and VL can be found by comparing the obtained amino acid sequences with amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

Moreover, the novelty of the full length of the amino acid sequence of VH and VL can be examined by carrying out a homology search with sequences in any database, for example, SWISS-PROT, PIR-Protein or the like using the obtained full length of the amino acid sequences of VH and VL, for example, according to the BLAST method [J Mol. Biol , 215, 403 (1990)] or the like.

(3) Construction of vector for expression of human chimeric antibody

cDNA encoding each of VH and VL of antibody of non-human animal is cloned in the upstream of genes encoding CH or CL of human antibody of vector for expression of recombinant antibody mentioned in the above (1) to thereby construct a vector for expression of human chimeric antibody.

For example, in order to ligate cDNA comprising a nucleotide sequence of 3'- terminal of VH or VL of antibody of non-human animal and a nucleotide sequence of 5'- terminal of CH or CL of human antibody, each cDNA encoding VH and VL of antibody of non-human animal is prepared so as to encodes appropriate amino acids encoded by a nucleotide sequence of a linkage portion and designed to have an appropriate recognition sequence of a restriction enzyme. The obtained cDNAs encoding VH and VL of antibody are respectively cloned so that each of them is expressed in an appropriate form in the upstream of gene encoding CH or CL of human antibody of the vector for expression of humanized antibody mentioned in the above (1) to construct a vector for expression of human chimeric antibody.

In addition, cDNA encoding VH or VL of a non-human animal antibody is amplified by PCR using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends and each of them is cloned to the vector for expression of recombinant antibody obtained in the above (1).

(4) Construction of cDNA encoding V region of humanized antibody

cDNAs encoding VH or VL of a humanized antibody can be obtained as follows.

Amino acid sequences of framework region (hereinafter referred to as "FR") in VH or VL of a human antibody to which amino acid sequences of CDRs in VH or VL of an antibody derived from a non-human animal antibody are transplanted are respectively selected. Any amino acid sequences of FR of a human antibody can be used, so long as they are derived from human. Examples include amino acid sequences of FRs of human antibodies registered in database such as Protein Data Bank or the like, and amino acid sequences common to subgroups of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the like. In order to inhibit the decrease in the binding activity of the antibody, amino acid sequences having high homology (at least 60% or more) with the amino acid sequence of FR in VH or VL of the original antibody is selected.

Then, amino acid sequences of CDRs of the original antibody are grafted to the selected amino acid sequence of FR in VH or VL of the human antibody, respectively, to design each amino acid sequence of VH or VL of a humanized antibody. The designed amino acid sequences are converted to DNA sequences by considering the frequency of codon usage found in nucleotide sequences of genes of antibodies [Sequence of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the DNA sequence encoding the amino acid sequence of VH or VL of a humanized antibody is designed.

Based on the designed nucleotide sequences, several synthetic DNAs having a length of about 100 nucleotides are synthesized, and PCR is carried out using them. In this case, it is preferred that 6 synthetic DNAs per each of the H chain and the L chain are designed in view of the reaction efficiency of PCR and the lengths of DNAs which can be synthesized.

Furthermore, the cDNA encoding VH or VL of a humanized antibody can be easily cloned into the vector for expression of humanized antibody constructed in (1) by introducing the recognition sequence of an appropriate restriction enzyme to the 5' terminal of the synthetic DNAs existing on the both ends.

Otherwise, it can be carried out using a synthetic DNA as one DNA encoding each of the full-length H chain and the full-length L chain based on the designed DNA sequence.

After the PCR, an amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene) or the like, and the nucleotide sequence is determined according to a method similar to the method described in (2) to obtain a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL of a desired humanized antibody.

(5) Modification of amino acid sequence of V region of humanized antibody

It is known that when a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non-human animal into FRs of VH and VL of a human antibody, its antigen binding activity is lower than that of the original antibody derived from a non-human animal [BIO/TECHNOLOGY, 9, 266 (1991)]. In humanized antibodies, among the amino acid sequences of FRs in VH and VL of a human antibody, an amino acid residue which directly relates to binding to an antigen, an amino acid residue which interacts with an amino acid residue in CDR, and an amino acid residue which maintains the three-dimensional structure of an antibody and indirectly relates to binding to an antigen are identified and modified to an amino acid residue which is found in the original non-humanized antibody to thereby increase the antigen binding activity which has been decreased.

In order to identify the amino acid residues relating to the antigen binding activity in FR, the three-dimensional structure of an antibody is constructed and analyzed by X-ray crystallography [J. Mol. Biol , 1 12, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501 (1994)] or the like. In addition, various attempts must be currently be necessary, for example, several modified antibodies of each antibody are produced and the correlation between each of the modified antibodies and its antibody binding activity is examined.

The modification of the amino acid sequence of FR in VH and VL of a human antibody can be accomplished using various synthetic DNA for modification according to PCR as described in (4). With regard to the amplified product obtained by the PCR, the nucleotide sequence is determined according to the method as described in (2) so that whether the objective modification has been carried out is confirmed.

(6) Construction of vector for expression of humanized antibody

A vector for expression of humanized antibody can be constructed by cloning each cDNA encoding VH or VL of a constructed recombinant antibody into upstream of each gene encoding CH or CL of the human antibody in the vector for expression of recombinant antibody as described in (1).

For example, when recognizing sequences of an appropriate restriction enzymes are introduced to the 5'-terminal of synthetic DNAs positioned at both ends among synthetic DNAs used in the construction of VH or VL of the humanized antibody in (4) and (5), cloning can be carried out so that they are expressed in an appropriate form in the upstream of each gene encoding CH or CL of the human antibody in the vector for expression of a humanized antibody as described in (1).

(7) Transient expression of recombinant antibody

In order to efficiently evaluate the antigen binding activity of various humanized antibodies produced, the recombinant antibodies can be expressed transiently using the vector for expression of humanized antibody as described in (3) and (6) or the modified expression vector thereof.

Any cell can be used as a host cell, so long as the host cell can express a recombinant antibody. Generally, COS-7 cell (ATCC CRL1651) is used in view of its high expression amount [Methods in Nucleic Acids Res., CRC Press, 283 (1991)].

Examples of the method for introducing the expression vector into COS-7 cell include a DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press, 283 (1991)], a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.

After introduction of the expression vector, the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant can be determined by the enzyme immunoassay [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] and the like.

(8) Obtaining transformant which stably expresses recombinant antibody and preparation of recombinant antibody

A transformant which stably expresses a recombinant antibody can be obtained by introducing the vector for expression of recombinant antibody described in (3) and (6) into an appropriate host cell.

Examples of the method for introducing the expression vector into a host cell include electroporation [Japanese Published Unexamined Patent Application

No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.

As the host cell into which a vector for expression of a recombinant antibody is introduced, any cell can be used, so long as it is a host cell which can produce the recombinant antibody. Examples include CHO-K1 (ATCC CCL-61), DUkXBl 1 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat NO.: 11619), rat myeloma cell YB2/3HL.P2.G1 1.16Ag.20 (also referred to as YB2/0), mouse myeloma cell NSO, mouse myeloma cell SP2/0-Agl4 (ATCC No. CRL1581), mouse P3X63-Ag8.653 cell (ATCC No. CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "dhfr") is defective [Proc. Natl. Acad. Sci. U.S.A. , 77, 4216 (1980)], lection resistance-acquired Lecl 3 [Somatic Cell and Molecular genetics, 12, 55 (1986)], CHO cell in which l,6-fucosyltransaferse gene is defected (WO 2005/35586, WO 02/31140), rat YB2/3HL.P2.G1 1.16Ag.20 cell (ATCC No. CRL1662), and the like.

In addition, host cells in which activity of a protein such as an enzyme relating to synthesis of an intracellular sugar nucleotide, GDP-fucose, a protein such as an enzyme relating to the modification of a sugar chain in which 1 -position of fucose is bound to 6- position of N-acetylglucosamine in the reducing end through -bond in a complex type N- glycoside-linked sugar chain, or a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body are introduced is decreased or deleted, preferably CHO cell in which al,6-fucosyltransferase gene is defected as described in WO05/35586, WO02/31 140 or the like, can also be used.

After introduction of the expression vector, transformants which express a recombinant antibody stably are selected by culturing in a medium for animal cell culture containing an agent such as G418 sulfate (hereinafter referred to as "G418") or the like (Japanese Published Unexamined Patent Application No. 257891/90).

Examples of the medium for animal cell culture include RPMI1640 medium

(manufactured by Invitrogen), GIT medium (manufactured by Nihon Pharmaceutical), EX- CELLS 01 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium (manufactured by Invitrogen), media obtained by adding various additives such as fetal calf serum (hereinafter referred to as "FCS") to these media, and the like. The recombinant antibody can be produced and accumulated in a culture supernatant by culturing the selected transformants in a medium. The expression amount and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like. Also, in the transformant, the expression amount of the recombinant antibody can be increased by using DHFR amplification system or the like according to the method disclosed in Japanese Published Unexamined Patent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant of the transformant by using a Staphyrococcus Protein A column [Monoclonal Antibodies- Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. For example, the recombinant antibody can be purified by a combination of gel filtration, ion-exchange chromatography, ultrafiltration and the like.

The molecular weight of the H chain or the L chain of the purified recombinant antibody or the antibody molecule as a whole is determined by polyacrylamide gel electrophoresis (hereinafter referred to as "SDS-PAGE") [Nature, 227, 680 (1970)], Western blotting [Monoclonal Antibodies-Principles and practice, Third edition,

Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], and the like.

3. Activity evaluation of the monoclonal antibody or antibody fragment

The activity of the purified monoclonal antibody or antibody fragment of the present invention can be evaluated in the following manner. The binding activity to Streptococcus pneumoniae cell is evaluated by the binding assay described in the above 1 -(6-a) and a surface plasmon resonance method using such as the Biacore system described in the above (6-b). Furthermore, it can be measured by fluorescent antibody technique [Cancer Immunol. Immunother., 36, 373 (1993)], a surface plasmon resonance method using such as BIAcore system or the like. Furthermore, it can be measured by fluorescent antibody technique [Cancer Immunol. Immunother. , 36, 373 (1993)].

In addition, complement-deposition activity or antibody dependent phagocytotic activity against an antigen positive bacterial cell is evaluated by a known method [Vaccine. 201 1 Feb 24;29( 10): 1929-34., PLoS One. 201 l ;6(10):e24581].

4. Method of controlling effector activity of antibody

As a method for controlling an. effector activity of the anti-Psp A monoclonal antibody of the present invention, a method for controlling an amount of fucose

(hereinafter, referred to also as "core fucose") which is bound in a- 1,6 linkage to N- acetylglucosamine (GlcNAc) present in a reducing end of a complex type N-linked sugar chain which is bound to asparagine (Asn) at position 297 of an Fc region of an antibody (WO2005/035586, WO2002/31 140, and WO00/61739), a method for controlling an effector activity of a monoclonal antibody by modifying amino acid group(s) of an Fc region of the antibody, and the like are known. The effector activity of the anti-PspA monoclonal antibody of the present invention can be controlled by using any of the methods.

The "effector activity" means an antibody-dependent activity which is induced via an Fc region of an antibody. As the effector activity, an antibody dependent cytotoxicity (ADCC), an antibody-dependent phagocytotic activity, a complement- dependent cytotoxicity (CDC), a complement-deposition activity, and the like are known.

By controlling a content of core fucose of a complex type N-linked sugar chain of Fc of an antibody, an effector activity of the antibody can be increased or decreased. According to a method for lowering a content of fucose which is bound to a complex type N-linked sugar chain bound to Fc of the antibody, an antibody to which fucose is not bound can be obtained by the expression of an antibody using a CHO cell which is deficient in a gene encoding al,6-fucosyltransferase. The antibody to which fucose is not bound has a high antibody-dependent phagocytotic activity. On the other hand, according to a method for increasing a content of fucose which is bound to a complex type N-linked sugar chain bound to Fc of an antibody, an antibody to which fucose is bound can be obtained by the expression of an antibody using a host cell into which a gene encoding al ,6-fucosyltransferase is introduced. The antibody to which fucose is bound has a lower antibody-dependent phagocytotic activity than the antibody to which fucose is not bound. In the present invention, an antibody which has no core-fucose is defined as non- fucosylated antibody, afucosylated antibody or Potelligent® antibody in case.

Further, by modifying amino acid residue(s) in an Fc region of an antibody, the antibody-dependent phagocytotic activity or complement deposition activity can be increased or decreased. The antibody-dependent phagocytotic activity can be controlled by increasing or decreasing the binding activity to FcyR due to the modification(s) of amino acid residue(s) in an Fc region. In addition, the complement deposition activity can be controlled by increasing or decreasing the binding activity of complement due to the modification(s) of amino acid residue(s) in an Fc region. For example, the binding activity to an antibody can be increased by using the amino acid sequence of the Fc region described in US2007/0148165. Further, the antibody-dependent phagocytotic activity or complement deposition activity can be increased or decreased by modifying the amino acid as described in US Patent Nos. 6,737,056, or 7,297,775 or WO2005/070963, and some scientific papers [Mol Cancer Ther. 2008 Aug;7(8):2517-27., MAbs. 2010 Mar- Apr;2(2): 181-9].

In the present invention, it is particularly defined as Complegent® or fucosylated Fc-engineered version (IgGl/IgG3) that the antibody which has a domain exchanged Fc between human IgGl and IgG3. The antibody has an enhanced CDC activity by both of glycoengineering and Fc engineering.

In the present invention, it is particularly defined as AccretaMAb® or afucosylated Fc-engineered version (IgGl/IgG3) that the antibody which has a domain exchanged Fc between human IgGl and IgG3, and is afucoslated. The antibody has an enhanced ADCC activity and an enhanced CDC activity by both of glycoengineering and Fc engineering.

Furthermore, an antibody in which the effector activity is controlled can be obtained by combining the above methods; the method for controlling a sugar chain and the method for modifying amino acid(s) in an Fc region.

5. Method for treating disease using the anti-PspA monoclonal antibody or antibody fragment of the present invention

A monoclonal antibody which recognizes a native structure of PspA and binds to the proline-rich region, or an antibody fragment thereof of the present invention can be used for treating a disease relating to Streptococcus pneumoniae.

Examples of a route of administration include oral administration and parenteral administration, such as buccal, tracheal, rectal, subcutaneous, intramuscular or intravenous administration. In the case of an antibody or peptide formulation, intravenous administration is preferred. Examples of the dosage form includes sprays, capsules, tablets, powder, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

The pharmaceutical preparation suitable for oral administration includes emulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using, as additives, water; sugars such as sucrose, sorbitol and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; antiseptics such as p-hydroxybenzoic acid esters; flavors such as strawberry flavor and peppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using, as additives, excipients such as lactose, glucose, sucrose and mannitol; disintegrating agents such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropylcellulose and gelatin; surfactants such as fatty acid ester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administration includes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, a glucose solution or a mixture of both thereof.

Suppositories can be prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

Sprays can be prepared using the antibody or antibody fragment as such or using it together with a carrier which does not stimulate the buccal or airway mucous membrane of the patient and can facilitate absorption of the compound by dispersing it as fine particles. The carrier includes lactose, glycerol and the like. It is possible to produce pharmaceutical preparations such as aerosols and dry powders.

In addition, the components exemplified as additives for oral preparations can also be added to the parenteral preparations.

6. Method for diagnosing disease using the anti-PspA monoclonal antibody or antibody fragment of the present invention

A disease relating to Streptococcus pneumoniae can be diagnosed by detecting or determining PspA or a bacterial cell expressing PspA using the monoclonal antibody or antigen-binding fragment of the present invention. A diagnosis of infectious disease, one of the diseases relating to Streptococcus pneumoniae, can be carried out by, for example, the detection or measurement of

Streptococcus pneumoniae as follows.

The diagnosis of infectious disease can be carried out by detecting PspA expressing on the bacterial cell in a patient's body by an immunological method such as a flow cytometer or immunoblotting.

An immunological method is a method in which an antibody amount or an antigen amount is detected or determined using a labeled antigen or antibody. Examples of the immunological method include radioactive substance-labeled immunoantibody method, enzyme immunoassay, fluorescent immunoassay, luminescent immunoassay, Western blotting method, physico-chemical means and the like.

Examples of the radioactive substance-labeled immunoantibody method include a method, in which the antibody or antibody fragment of the present invention is allowed to react with an antigen, a cell expressing an antigen or the like, then anti- immunoglobulin antibody subjected to a radioactive labeling or a binding fragment thereof is allowed to react therewith, followed by determination using a scintillation counter or the like.

Examples of the enzyme immunoassay include a method, in which the antibody or antibody fragment of the present invention is allowed to react with an antigen, a cell expressing an antigen or the like, then an anti-immunoglobulin antibody or an binding fragment thereof subjected to antibody labeling is allowed to react therewith and the colored pigment is measured by a spectrophotometer, and, for example, sandwich ELISA may be used. As a label used in the enzyme immunoassay, any known enzyme label {Enzyme Immunoassay, published by Igaku Shoin, 1987) can be used as described already. Examples include alkaline phosphatase labeling, peroxidase labeling, luciferase labeling, biotin labeling and the like.

Sandwich ELISA is a method in which an antibody is bound to a solid phase, antigen to be detected or measured is trapped and another antibody is allowed to react with the trapped antigen. In the ELISA, two kinds of antibody which recognizes the antigen to be detected or measured or the antibody fragment thereof in which antigen recognizing site is different are prepared and the first antibody or antibody fragments is previously adsorbed on a plate (such as a 96-well plate) and the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. The plate to which the above antibody is adsorbed is allowed to react with the cell separated from living body or disrupted cell suspension thereof, tissue or disintegrated solution thereof, cultured cells, serum, pleural effusion, ascites, eye solution or the like, then allowed to react with a labeled monoclonal antibody or an antibody fragment and a detection reaction corresponding to the labeled substance is carried out. The antigen concentration in the sample to be tested can be calculated from a calibration curve prepared by a stepwise dilution of antigen of known concentration. As antibody used for sandwich ELISA, any of polyclonal antibody and monoclonal antibody may be used or antibody fragments such as Fab, Fab' and F(ab)₂ may be used. As a combination of two kinds of antibodies used in sandwich ELISA, a combination of monoclonal antibodies or antibody fragments recognizing different epitopes may be used or a combination of polyclonal antibody with monoclonal antibody or antibody fragments may be used.

A fluorescent immunoassay includes a method described in the literatures [Monoclonal Antibodies - Principles and practice, Third Edition, Academic Press (1996); Manual for Monoclonal Antibody Experiments, Kodansha Scientific (1987)] and the like. As a label for the fluorescent immunoassay, any of known fluorescent labels [Fluorescent Immunoassay, by Akira Kawao, Soft Science (1983)] may be used as described already. Examples of the label include FITC, RITC and the like.

The luminescent immunoassay can be carried out using the methods described in the literature [Bioluminescence and Chemical Luminescence, Rinsho Kensa, 42, Hirokawa Shoten (1998)] and the like. As a label used for luminescent immunoassay, any of known luminescent labels can be included. Examples include acridinium ester, lophine or the like may be used.

Western blotting is a method in which an antigen or a cell expressing an antigen is fractionated by SDS-polyacrylamide gel electrophoresis [Antibodies-A

Laboratory Manual (Cold Spring Harbor Laboratory, 1988)], the gel is blotted onto PVDF membrane or nitrocellulose membrane, the membrane is allowed to react with antigen- recognizing antibody or antibody fragment, further allowed to react with an anti-mouse IgG antibody or antibody fragment which is labeled with a fluorescent substance such as FITC, an enzyme label such as peroxidase, a biotin labeling, or the like, and the label is visualized to confirm the reaction. An example thereof is described below. Cells or tissues in which a polypeptide having the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or YP_815641.1 (D39) is expressed are dissolved in a solution and, under reducing conditions, 0.1 to 30 μg as a protein amount per lane is electrophoresed by an SDS-PAGE method. The electrophoresed protein is transferred to a PVDF membrane and allowed to react with PBS containing 1 to 10% of BSA (hereinafter referred to as "BSA-PBS") at room temperature for 30 minutes for blocking. Here, the monoclonal antibody of the present invention is allowed to react therewith, washed with PBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as "Tween-PBS") and allowed to react with goat anti-mouse IgG labeled with peroxidase at room temperature for 2 hours. It is washed with Tween-PBS and a band to which the monoclonal antibody is bound is detected using ECL Western Blotting Detection Reagents (manufactured by Amersham) or the like to thereby detect a polypeptide having the amino acid sequence represented by NCBI reference sequence NP_344663.1 (TIGR4) or

YP 815641.1 (D39). As an antibody used for the detection in Western blotting, an antibody which can be bound to a polypeptide having no three-dimensional structure of a natural type is used.

The physicochemical method is specifically carried out by reacting PspA as the antigen with the antibody or antibody fragment of the present invention to form an aggregate, and detecting this aggregate. Other examples of the physicochemical methods include a capillary method, a one-dimensional immunodiffusion method, an

immunoturbidimetry, a latex immunoturbidimetry [Handbook of Clinical Test Methods, Kanehara Shuppan (1988)] and the like. For example, in a latex immunodiffusion method, a carrier such as polystyrene latex having a particle size of about of 0.1 to 1 μηι sensitized with antibody or antigen may be used and when an antigen-antibody reaction is carried out using the corresponding antigen or antibody, scattered light in the reaction solution increases while transmitted light decreases. When such a change is detected as absorbance or integral sphere turbidity, it is now possible to measure antigen concentration, etc. in the sample to be tested.

For the detection of the bacterial cell expressing PspA, known immunological detection methods can be used, and an immunoprecipitation method, an immuno cell staining method, an immune tissue staining method, a fluorescent antibody staining method and the like are preferably used.

An immunoprecipitation method is a method in which a cell expressing PspA is allowed to react with the monoclonal antibody or antibody fragment of the present invention and then a carrier having specific binding ability to immunoglobulin such as protein G-Sepharose is added so that an antigen-antibody complex is precipitated. Also, the following method can be carried out.

The above-described antibody or antibody fragment of the present invention is solid-phased on a 96-well plate for ELISA and then blocked with BSA-PBS. When the antibody is in a non-purified state such as a culture supernatant of hybridoma cell, anti- mouse immunoglobulin or rat immunoglobulin or protein A or Protein G or the like is previously adsorbed on a 96-well plate for ELISA and blocked with BSA-PBS and a culture supernatant of hybridoma cell is dispensed thereto for binding. After BSA-PBS is discarded and the residue is sufficiently washed with PBS, reaction is carried out with a dissolved solution of cells or tissues expressing PspA. An immune precipitate is extracted from the well-washed plate with a sample buffer for SDS-PAGE and detected by the above-described Western blotting. An immune cell staining method or an immune tissue staining method are a method where cells or tissues in which antigen is expressed are treated, if necessary, with a surfactant, methanol or the like to make an antibody easily permeate to the cells or tissues, then the monoclonal antibody of the present invention is allowed to react therewith, then further allowed to react with an anti-immunoglobulin antibody or binding fragment thereof subjected to fluorescent labeling such as FITC, enzyme label such as peroxidase or biotin labeling and the label is visualized and observed under a microscope. In addition, cells of tissues can be detected by an immunofluorescent staining method where cells are allowed to react with a fluorescence-labeled antibody and analyzed by a flow cytometer

[Monoclonal Antibodies - Principles and practice, Third Edition, Academic Press (1996), Manual for Experiments of Monoclonal Antibodies, Kodansha Scientific (1987)] in which cells are allowed to react with a fluorescence-labeled antibody and analyzed by a flow cytometer. Particularly, the monoclonal antibody or antibody fragment of the present invention which binds to an proline-rich region of the PspA can detect a cell expressing the polypeptide maintaining a natural structure.

In addition, in the case of using FMAT8100HTS system (manufactured by Applied Biosystems) and the like among fluorescent antibody staining methods, the antigen quantity or antibody quantity can be measured without separating the formed antibody-antigen complex and the free antibody or antigen which is not concerned in the formation of the antibody-antigen complex.

The present invention is described below by Examples; however, the present invention is not limited to the following Examples^'.

Example 1 : Establishment of anti-PspA monoclonal antibodies

(1) Construction of C-terminal His-tagged PspA protein expression vectors

A vector for bacterial expression of a fragment of the Streptococcus pneumoniae bacteria protein PspA with an added carboxy-terminal (C-term) six histidines (hereafter called "6xHis-tag" or "His-tag") was constructed by insertion of a pspA gene fragment coding for the amino-terminal (N-term) region of the PspA protein including alpha-helical and proline-rich domains from Streptococcus pneumoniae bacteria strains D39 (Accession NO.: CP000410) and TIGR4 (accession NO.:AE005672.1) into plasmid pET20b(+) (manufactured by Novagen). Insertion of the pspA gene was performed such that the pET20b(+) plasmid would append an amino-terminal (N-term) 22 amino acid signal peptide sequence from pectate lyase B (pelB), and the C-terminal amino acid sequence (-Leu-GIu-His-His-His-His-His-His) (FIG. 1). The pelB signal peptide induces post-translational transport of the attached protein to the periplasm of the bacteria, followed by subsequent removal of the peptide from the protein by enzymatic cleavage. RNA was isolated from Streptococcus pneumoniae strains D39 and TIGR4 using RiboPure™ Bacteria Kit (manufactured by Ambion) as per the manufacturer's instructions. The RNA was used as template for first strand cDNA synthesis via reverse transcription (RT) with SuperScript™II (manufactured by Invitrogen) using primer as per the manufacturer's instructions. The PspA gene fragment was amplified from first strand cDNA by using polymerase chain reaction (PCR) with specific primers. To prepare the D39 pspA gene fragment for insertion into the expression vector, PCR was performed on the previously amplified cDNA with KOD Hot Start polymerase (manufactured by

Novagen), applying the manufacturer's recommended conditions and standard methods. The amplified DNA was fractionated by eletrophoresis on a 1% agarose TAE gel, and the band migrating at the expected molecular weight was excised and purified using a

QIAquick Gel Extraction kit (manufactured by QIAGEN). The isolated gene fragment was subsequently digested with restriction enzymes Ncol and Xhol (manufactured by New England Biolabs) using manufacturer's recommended conditions. The bacterial expression vector pET20b(+) (manufactured by Novagen) was digested in the same manner. Both DNAs were purified using a QIAquick Gel Extraction kit, then ligated together using Mighty Mix DNA ligation kit (manufactured by TaKaRa) according to the manufacturer's instructions. The ligated DNA mixture was transformed into DH5a-TlR E. coli bacteria strain (manufactured by Invitrogen Corp.) using the manufacturer's protocol. Plasmid DNAs were isolated (QIAprep Spin Miniprep kit, manufactured by QIAgen) from several transformed bacteria colonies that grew on ampicillin-containing LB-Miller agar plates. A plasmid containing the D39 PspA gene fragment of interest was identified by NcollXhol double restriction digest, and confirmed by Sanger DNA sequencing (performed by

Genewiz Inc.). The completed plasmid was named pET-20b(+)-PspA-D39 and the nucleotide sequence of the expression construct is SEQ ID NO:21. The same method was used to construct a vector for the expression of the homologous region of the pspA gene fragment from TIGR4 strain. The completed plasmid was named pET-20b(+)-PspA- TIGR4 and the nucleotide sequence of the expression construct is SEQ ID NO:22. (2) Production of His-tagged PspA proteins

Plasmids containing cDNA coding for various forms and strains of PspA protein were transformed into chemically competent E. coli strain BL21(DE3)

(manufactured by Stratagene) as per the manufacturer's instructions, and selected on LB- Miller agar plates containing ampicillin. A colony was tested for expression by growing in LB-Miller medium + ampicillin at 37°C with 250 rpm shaking to an optical density at 600 nM (OD₆₀o) between 0.6-1.0 units. Expression of the protein was induced by addition of isopropyl β-D-l-thiogalactopyranoside (IPTG) (manufactured by BioPioneer Inc.) to 1 mM, and the cultures were as before for 3-4 hours. Expression of the desired protein was confirmed by protein immunoblot (western blot) technique. First, each sample was fractionated by standard Laemmli sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) method. 200 μΐ, of IPTG-induced or non-induced bacterial culture was centrifuged to pellet the bacteria, media was aspirated and the cells were lysed using 2x Laemmli reducing sample buffer, vortexing and 5 minute 95°C incubation.

Debris was removed by centrifugation, and the soluble fraction was loaded on a 4-20% tris-glycine polyacrylamide gel (manufactured by Invitrogen). Fractionated proteins were transferred from the gel onto blotting membrane (manufactured by Invitrogen) by electrophoresis. The blots were blocked with 5% dry skim milk (manufactured by BD Biosciences) in tris buffered saline with 0.05% Tween 20 (referred to herein as "PBS-T") (manufactured by SIGMA). Blots were probed with peroxidase-conjugated primary antibodies anti-His (manufactured by Clontech) to detect the respectively tagged proteins.

To produce recombinant PspA-6xHis proteins in bacteria, the expression vector pET-20b(+)-PspA-D39 or pET-20b(+)-PspA-TIGR4 was transformed into BL21 (DE3) competent cells and bacterial cultures were induced with lmM IPTG (isopropyl-beta-D- thiogalactopyranoside). The amino acid sequence of the expression construct in pET- 20b(+)-PspA-D39 is SEQ ID NO:39, and the amino acid sequence of the expression construct in pET-20b(+)-PspA-TIGR4 is SEQ ID NO:40. Cells were harvested by centrifugation for protein purification. Bacterial cells were lysed with microfluidizer (model M10L, Microfluidics, Inc.) and separated from the lysate by centrifugation. The obtained lysate was further clarified and sterilized using a 0.22-μπι pore size vacuum filter unit (Millipore). D39 and TIGR4 versions of recombinant PspA with C-terminal HisTag® were purified from the obtained clarified bacterial lysate by metal chelate affinity chromatography with Ni Sepharose 6 Fast Flow resin (GE Healthcare) according to the manufacturer instructions. PspA was eluted from the column with 200 mM imidazole, and subsequently dialyzed against 10 mM Tris-HCl buffer, pH 7.5, 0.1M NaCl. Protein concentration was determined by DC Lowry protein assay (Bio-Rad) using BSA standard (Pierce Biotechnology) in the same buffer.

(3) Immunization and hybridoma screening

Recombinant PspA with His-tag, D39 (Family 1) and TIGR4 (Family 2) were used as immunogens. BALB/c mice (n = 110, 60 for D39 and 50 for TIGR4) were immunised intraperitoneally with 50 μg protein in 0.25 ml saline mixed with 0.25 ml Freund's incomplete adjuvant, followed by a booster injection 2 weeks later with the same mixture. After a rest period 1 month one booster injection of 50 μg proteins in saline was given intravenously 4 days before fusion of spleen cells from the immunised mouse with Sp2/0 myeloma cells by standard methods [Microbiology 143, 55-61 (1997)]. Hybridoma supernatants were screened by both ELISA against the immunogen and flow cytometry against WU2 and EF3030 strains. Hybridoma cells were cloned by limiting dilution and the isotyping of mAbs in hybridoma supernatant fluids was performed by ELISA. Overall 9916 wells (4664 from D39 and 5252 from TIGR4) were screened and finally the following 10 monoclonal antibody clones were established (5 from D39 and 5 from

TIGR4), 139F3, 139G3, 13913, 13916, 13918, 140G1, 140G5, 140G6, 140G1 1, and, 140H1.

The binding activity of the 10 clones was evaluated in flow cytometric analysis using Streptococcus pneumoniae strain panel which consists of twenty-eight Streptococcus pneumoniae strains including ATCC-6301 , NCTC- 1 1910, ATCC-49136, ATCC49619, NCTC-11888, EF3030, BAA-658, ATCC-700675, ATCC-6305, WU2, NCTC-7978, D39, NCTC-1 1886, BAA-475, BAA-340, ATCC-700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-11906, ATCC-700673, NCTC-1 1897, BAA-612, ATCC-49150, ATCC-700671, ATCC-6303, and DS2341-94. The following 4 monoclonal antibody clones were selected in terms of broader reactivity to the strain panel; clone 140G1 (mlgGl, kappa), 140G11 (mIgG2a, kappa), 140H1 (mlgGl, kappa) were from D39 (Family 1 PspA), and clone 139G3 (mIgG2a, kappa) from TIGR4 (Family 2 PspA).

Example 2: Production of recombinant anti-PspA antibodies

(1) Isolation of PspA antibody genes 139G3, 140G1, 140G11 and 140H1

The cDNA coding for the rearranged immunoglobulin heavy chain variable domains (herein referred to as "VH") (this includes the signal peptide + variable gene region (V) + diversity gene region (D) + joining gene region (J)), and immunoglobulin light chain variable domains (herein referred to as "VL") (this includes the signal peptide + variable gene region (V) + joining gene region (J)) from select hybridoma cell lines were extracted using 5'-SMART-RACE-PCR (Switching Mechanism at 5' End of RNA

Template-Rapid Amplification of cDNA Ends-Polymerase Chain Reaction) and Sanger- sequenced. This was performed on hybridoma clones 139G3, 140G1, 140G1 1 and 140H1 , which produce murine immunoglobulin antibodies 139G3, 140G1 , 140G1 1 or 140H1 respectively.

To do this, cultured hybridoma cells were collected by centrifugation. Total RNA was purified from these cells using RNeasy kit (manufactured by QIAGEN Inc.) following the manufacturer's instructions. Using the RNA as a template, SMART RACE cDNA Amplification Kit (manufactured by Clontech Co.) with the reverse transcriptase SuperScript™II (manufactured by Invitrogen) were used, as per the manufacturer's written instructions, for 5'SMART-RACE-PCR to amplify the cDNA that encodes the VH and VL regions of the immunoglobulin genes. Briefly, first strand cDNA was prepared by reverse transcriptase from 2 μg of RNA. This cDNA was used as a template for PCR amplification of the VH or VL regions plus a short part of the constant region of heavy or light chains. PCR was performed as per the SMART-RACE kit manufacturer's instructions with KOD Hot Start DNA polymerase (manufactured by Novagen).

The thermal cycling program was 1 cycle of 94°C x 4 min: 35 cycles of: 94°C x 30 sec, 55°c 30 sec, 68°C x 1.5 min. followed by an extension at 72°C x 7 min.

Amplified DNA fragments were isolated by agarose gel electrophoresis, and purified by QIAquick Gel Extraction Kit (manufactured by Qiagen). Purified DNA fragments of VH and VL were separately integrated into PCR Bluntll-TOPO plasmid using the Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen) as per the manufacturer's instructions. Each plasmid was transformed into DH5a-TlR E. coli, and plated on kanamycin-containing LB-Miller agar plates. Plasmids were isolated from bacterial colonies grown in LB-Miller medium with kanamycin grown overnight, using QIAprep Spin Miniprep kit (manufactured by QIAgen). Plasmids were screened by EcoRI restriction digest and sequenced by S anger-sequencing (performed by GENEWIZ Inc.). Nucleotide sequences of each insert in the construct plasmids were analyzed using the nucleotide-alignment software program Sequencher (manufactured by Gene Codes Corp.). Based on consensus alignment of multiple plasmid clones obtained from each antibody, cDNA sequences were identified for the VH and VL regions of the heavy and light chains respectively. This was performed for all four hybridoma clones: 139G3 VH (nucleotide SEQ ID NO:28, amino acid SEQ ID NO.:61)( amino acid residues from at the position 1 to 19 are signal sequence), 139G3 VL (nucleotide SEQ ID NO:29, amino acid SEQ ID NO:46) (amino acid residues from at the position 1 to 20 are signal sequence); 140H1 VH (nucleotide SEQ ID NO:30, amino acid SEQ ID NO:47) ( amino acid residues from at the position 1 to 19 are signal sequence), 140H1 VL (nucleotide SEQ ID NO.:31, amino acid SEQ ID NO:48) ( amino acid residues from at the position 1 to 20 are signal sequence); 140G1 VH (nucleotide SEQ ID NO:32, amino acid SEQ ID NO:49) ( amino acid residues from at the position 1 to 19 are signal sequence), 140G1 VL (nucleotide SEQ ID NO:33, amino acid SEQ ID NO:50) ( amino acid residues from at the position 1 to 20 are signal sequence); 140G11 VH (nucleotide SEQ ID NO:34, amino acid SEQ ID NO:51) ( amino acid residues from at the position 1 to 19 are signal sequence), 140G11 VL (nucleotide SEQ ID NO:35, amino acid SEQ ID NO:52) ( amino acid residues from at the position 1 to 20 are signal sequence). Antibody VH and VL domains were analyzed for germline V, D and J genes using the IMGT/V-quest program {Brocket, Lefranc et al. 2008). Antibody complementarity determining regions (CDRs) were defined using the Kabat definition. (2) Creation vectors of expression of class switched antibodies 140csGl and HOcsHl

Vectors for the expression of "class-switched" recombinant mouse IgG2a,kappa-isotype versions of antibodies 140G1 or 140H1 (herein called "140csGl" or "HOcsHl" respectively) were created by in-frame fusion of a gene fragment of each antibody clone VH domain to a gene fragment coding for mouse IgG2a constant heavy (CH) immunoglobulin (nucleotide SEQ ID NO:36, amino acid SEQ ID NO:53).

Subsequently, the full length gene for the mouse light chain was amplified by PCR and inserted in the vector containing the corresponding class-switched heavy chain gene. (3) Creation of mouse/chimeric engineered human IgGl/IgG3 Fc antibodies from 140G1 and 140H1

The mouse/human chimeric antibodies (human IgGl, kappa) from 139G3, 140G1 and 140H1 were prepared following the method of Shitara et al. [Cancer Immunol Immunother. 1993 Jun;36(6):373-80]. The nucleotide sequence of the constant region in human IgGl heavy chain is SEQ ID NO:37. The amino acid sequence of the constant region in human IgGl heavy chain is SEQ ID NO:54.

Next, the engineered IgGl/IgG3 Fc is the 1 13F variant (Natsume et al. Cancer Res. 2008 May 15;68(10):3863-72) with a single amino acid substitution N392K (EU numbering) (herein referred to as "IgGl/IgG3 Fc" or IgGl/IgG3" or "1 13F (N392K)"). Briefly described, IgGl/IgG3 chimeric Fes were previously produced by shuffling constant domains from human IgGl and IgG3 using standard recombinant DNA techniques

(Natsume et al. Cancer Res. 2008 May 15;68(10):3863-72). The resulting chimeric antibodies were screened for various desired characteristics, enhanced complement- dependent cytotoxicity (CDC) and retention of IgGl -like protein A binding being of first- order importance. Variant 1 13F consists of IgG3 sequence for CH2 and CH3, though amino acid 422, replacing the homologous domain in human IgGl to form a "chimeric" Fc. The variant used here contained an additional single amino acid substitution which reverted IgG3-type amino acid N392 to IgGl-type K392. The nucleotide sequence of the constant region in the 1 13F (N392K) heavy chain is SEQ ID NO:38. The amino acid sequence of the constant region in the 113F (N392K) heavy chain is SEQ ID NO:55.

Vectors were created for the expression of 140G1 and 140H1 mouse/human chimeric IgGl/IgG3 Fc antibodies by replacing the human IgGl constant domain-coding gene fragments in the previously constructed expression vectors for expression of mouse/human IgGl chimeric 140G1 or 104H1 antibodies, with a gene fragment coding for human IgGl/IgG3 constant domains. The mouse/human kappa chimeric light chain cDNA was unaltered in the vectors. (4) Production of recombinant antibody from 293 -F cells

Suspension cultures of FreeStyle™293 cells (herein called "293-F") (manufactured by Invitrogen) were maintained in FreeStyle™293 Expression Medium (manufactured Invitrogen) while shaking at -90-110 rpm/min in an 8% C0₂ humidified incubator at 37°C. Cells were transfected with the desired expression vector DNA based on pKANTEX93 (Mol. Immunol.(2000), 37:1035-1046) using FreeStyle™MAX transfection reagent (manufactured by Invitrogen) following manufacturer's standard method outlined in product manual (the ratio of DNA to FreeStyle™MAX reagent used was 1 :1, weight to volume). Transfectants were subsequently incubated under normal growth conditions for six days. Supernatant was clarified by centrifugation followed by 0.22 μπ filtration (manufactured by Millipore). This material was then used directly for assays, or the antibody was purified.

(5) Production of chimeric human IgGl and IgGl/IgG3-Fc recombinant antibodies from CHO-S cells

Suspension cultures of FreeStyle™CHO-S cells (herein called "CHO-S") (manufactured by Invitrogen) were maintained in FreeStyle™CHO Expression Medium (manufactured by Gibco) while shaking at -90-1 10 rpm/min in an 8% C0₂ humidified incubator at 37°C. Cells were transfected with expression vector DNA containing genes coding for the desired antibody (either standard Fc or IgGl/IgG3-type Fc) using

FreeStyle™MAX transfection reagent (manufactured by Invitrogen) following

manufacturer's standard method (the ratio of DNA to FreeStyle™MAX reagent used was 1 : 1, weight to volume). Transfectants were incubated under normal growth conditions for six days. Supernatant was clarified by centrifugation followed by 0.22 μπι filtration (manufactured by Millipore). This material was then used directly in assays, or was further processed for purified antibody.

(6) Production of non-fucosylated chimeric human IgGl and non-fucosyalted chimeric human IgGl/IgG3-Fc recombinant antibodies

To express antibody without fucose carbohydrate modification on the Fc, an alpha-(l ,6)-fucosyltransferase-double-knockout CHO cell line Ms704 {Biotechnol Bioeng. 2004 Sep 5;87(5):614-22), was transiently transfected using identical methods to those described for CHO-S cells. Plasmids containing cDNA for antibodies with standard human Fc that were transfected in CHO Ms704 cells produced non-fucosylated IgGl-type antibody. Plasmids containing cDNA for antibodies with human IgGl/IgG3-Fc that were transfected into CHO Ms704 cells produced non-fucosylated IgGl/IgG3 Fc-type antibody. (7) Obtaining of purified chimeric antibody

After culturing each transformant, cell suspensions were recovered and centrifuged at 3000 rpm and 4°C for 20 minutes to recover the culture supematants. Then, the culture supematants were sterilized using a 0.22-μηι pore size vacuum filter unit (Millipore). All versions of the anti-PspA chimeric antibodies were purified from the obtained culture supematants using a Protein A MabSelect Sure resin (GE Healthcare) according to the manufacturer instructions. Supematants were first concentrated using a small-scale Sartorius tangential flow system (Sartorius-Stedim Biotech SA). The concentrated supematants were filtered with a 0.22 μηι vacuum filter unit (Millipore) and loaded onto a Protein A column (see above) of appropriate capacity for the amount of human antibody in the medium. The column was washed thoroughly with 20 column volumes of PBS and the antibody was eluted with 0.1 M Gly-HCl, pH 3.6 and neutralized with 1 M Tris-HCl, pH 8.0. The fractions were analyzed by SDS-PAGE and the positive fractions were pooled and concentrated with a centrifugal concentrator (Vivaspin, 50,000 MWCO, Sartorius-Stedim Biotech SA). Concentrated antibody was buffer exchanged into PBS or other appropriate buffer via dialysis. Finally, the antibody was filter sterilized using syringe filters with 0.22 μηι pore diameters and the antibody concentration was determined by the Lowry assay with DC Lowry kit (Bio-Rad). Example 3: Epitope domain mapping using overlapping bacterial expressed PspA fragments

(1) Preparation for MBP-tagged recombinant PspA fragments

The following plasmids were constructed, using the same molecular biology scheme, to express short overlapping fragments of PspA peptides (depicted in FIG. 2) with identical N-term MBP-tags from the above mentioned three S. pneumoniae strains: strain BAA-658 FL (nucleotide SEQ ID NO:23, amino acid SEQ ID NO.:41), SI (nucleotide SEQ ID NO:24, amino acid SEQ ID NO:42), S2 (nucleotide SEQ ID NO:25, amino acid SEQ ID NO:43), S3 (nucleotide SEQ ID NO:26, amino acid SEQ ID NO:44), S4

(nucleotide SEQ ID NO:27, amino acid SEQ ID NO:45).

To narrow the region within PspA that may contain the epitopes of the various antibodies, overlapping recombinant fragments of PspA from strain BAA658 were produced and used in ELISAs with the anti-PspA antibodies. PspA (BAA658) was selected because it was the shorted protein to which all four antibodies bound.

To do this, E. coli bacteria strain BL21(DE3) (manufactured by Invitrogen) were transformed with plasmids expressing various MBP-tagged PspA-fusion peptide fragments, or control plasmid expressing MBP-non-PspA fusion peptide, MBP-B6R, and selected on ampicillin-containing LB-Miller agar plates. LB-Miller liquid bacterial cultures were grown to OD₆₀o -0.6-1.0, then induced to express recombinant peptides with ImM IPTG. After a 3 hr incubation with IPTG, bacteria were harvested from 500 of culture by centrifugation. The cell pellet was lysed by addition of 50 μΐ, of IX PBS followed by three freeze-thaw cycles (1 min ethanol-dry ice bath, 1 min 37°C bath followed by vortexing). 1.2 mL of IX PBS were mixed with the lysate, cleared of insoluble debris by centrifugation. 50 of the cleared lysate was coated in wells of 96- well clear MaxiSorp™ ELISA plates (manufactured by Nunc), which were then incubated over night at 4°C. Plates were washed three times with 300 μΐ. TBST per well, then blocked with 200 iL per well SuperBlock® (TBS) Dry Blend blocking buffer (Thermo Scientific) for 30 min at room temperature. Primary antibodies 139G3, 140G1 , 140H1 , 140G1 1 and rabbit-anti-MBP (manufactured by New England Biolabs) antibodies were diluted in lx TBST (0.05 % Tween-20 in Tris Buffered Saline, pH 8 (Sigma)) to 1 μg/mL, 50 μΐ. was added to respective antigen coated wells in duplicate, and incubated for 1 hour at 37°C. Each plate was washed as described previously, and 50 μΐ, of the secondary antibody (Peroxidase-conjugated donkey-anti -mouse IgG (H+L) (manufactured by Jackson Immunoresearch) or peroxidase-conjugated goat-anti-rabbit IgG) (manufactured by Jackson Immunoresearch)), diluted 1 :5,000 in 1% SuperBlock®/TBST, was added to each well. After 1 hr 37°C incubation, each plate was washed as described previously, and visualized with 100 μΐ, peroxidase-reactive colorimetric substrate (TMB+ Substrate- chromogen (DAKO)), and fixed with 50 μΐ. 2.0 N sulfuric acid (LabChem, Inc.) after 5-10 min. Plates were read at OD₄₅₀ using a VersaMax microplate reader (Molecular Devices). Wells with higher ODs were considered to contain primary antibodies with affinity for the PspA protein coated in that well.

As the result, all antibodies bound to full BAA-658 peptide. Anti-PspA antibodies 139G3 and 140H1 bound to both of partial peptides of BAA-658 (S3) and (4), the other anti-PspA antibodies 140G1 and 140G1 1 bound to only the partial peptide of BAA-658 (S4) (the left of the bottom in FIG. 2). Therefore it is found that 139G3 and 140H1 antibodies bind to the epitope being present in the proline-rich domain of PspA, and 140G1 and 140G1 1 antibodies bind to the epitope being present in the non-proline block (NPB) included in the proline-rich domain of PspA.

(2) Epitope mapping of 139G3 and 140H1 with synthetic overlapping peptides

Five short overlapping synthetic peptides covering the 37 amino acid long overlapping region of proteins MBP-BAA658(S3) and MBP-BAA658(S4), that corresponds to PspA (BAA658) amino acids 281 -317, were synthesized with N-terminal biotin-7-aminohexanoyl (biotin-Ahx) and C-terminal amidation modifications

(manufactured by GenScript): BAA658-PR1 (SEQ ID NO:56), BAA658-PR1.2 (SEQ ID NO.:57), BAA658-PR2 (SEQ ID NO:58), BAA658-PR3 (SEQ ID NO:59), and, BAA658- PR4 (SEQ ID NO:60) (depicted in the bottom of FIG. 2). All lypohylized peptides, except BAA658-PR2 were, reconstituted directly in Milli-Q quality H₂0. Peptide BAA658-PR2 was reconstituted in 100% DMSO, then diluted in Milli-Q pure water to a final

concentration of 30% DMSO, 2.4 mg/mL peptide. To perform the ELISA, pre-blocked (SuperBlock®) NeutrAvidin 96-well, 8-well strip plates (Pierce) were washed three times with 200 μΐ of TBST, pH 8.0 wash buffer. 50 μΐ of biotinylated BAA658 PspA peptides at 1 μg/mL in coating buffer (10% SuperBlock® in TBST) were added to wells and incubated at 37°C for 1-2 hours. Wells were washed 3 times with 300 μΐ, TBST pH 8.0. 50 μΐ, of 1 μg/mL primary mouse-anti-PspA antibodies 139G3, 140H1 , 140G1 and 140G1 1 were added and incubated at 37°C for 1-2 hours. In this experiment, antibody 140G1 1 is a negative control because it is known to not bind this region of BAA658 PspA. Wells were washed as before. 50 μΐ, of peroxidase-conjugated secondary antibody donkey-anti-mouse IgG(H+L) (manufactured by Jackson Immunoresearch), diluted 1 :5,000 in 10% SuperBlock/TBST pH 8.0, was added to the wells and incubated for 1 hour at 37°C. Wells were washed as before. Colorimetric visualization was performed by adding 100 μΐ, peroxidase-reactive colorimetric substrate (TMB+ Substrate-chromogen (DA O)), and fixed with 50 \xh 2.0 N sulfuric acid (LabChem, Inc.) after 5-10 minutes. Plates were read at OD₄₅₀ using a VersaMax microplate reader (Molecular Devices). Wells with higher ODs were considered to contain primary antibodies with affinity for the PspA peptide coated in that well.

The reverse of this experiment was also performed with the same results.

Briefly, anti-PspA antibodies were captured on an ELISA plate with anti-mouse IgG Fc antibodies, followed by capture of peptides and detection with strepavidin-peroxidase. To do this, goat-anti-mouse IgG (Fc-specific) antibody (manufactured by Jackson

Immunoresearch) diluted in 50 mM carbonate-bicarbonate buffer pH 9.6 (manufactured by SIGMA) was coated on MaxiSorp 96-well ELISA plates, 50 μΐ, per well, overnight at 4°C. Wells were washed as described above. Wells were then blocked with 200 μΕ

SuperBlock® for 30 min at room temperature. Mouse anti-PspA antibodies 139G3, 140H1 or 140G1 1 were captured by adding 50 μΐ, of each at 5 μg/mL in 10% SuperBlock®/TBST pH 8.0. After 1 hr 37°C incubation, the wells were washed as before. The biotinylated PspA(BAA658) peptides described above were added to various wells in 50 μΐ of 10% SuperBlock®/TBST pH 8.0 at a concentration of ~6 μg/mL. These were incubated at 37°C for 1 hour, then wells were washed as before. The presence of the PspA peptides was determined by detection of the conjugated biotin with peroxidase-conjugate streptavidin (Jackson Immunoresearch) diluted 1 :5,000 in 10% SuperBlock®/TBST pH 8.0, and incubated at 37°C for 1 hour. After a final wash, as described above, the assay was visualized by adding 100 μΐ, peroxidase-reactive colorimetric substrate (TMB+ Substrate-chromogen (DAKO)), and fixed with 50 μΐ, 2.0 N sulfuric acid (LabChem, Inc.) after 5-10 minutes. Plates were read at OD₄₅₀ using a VersaMax microplate reader (Molecular Devices). A summary of epitope mapping results can be seen in FIG. 2.

As results, anti-PspA antibody 139G3 bound to the synthetic peptides of

BAA658-PR1.2 and BAA658-PR2, and 140H1 bound to the synthetic peptides of BAA658-PR3 and BAA658-PR4, on the other hand, anti-PspA antibody 140G1 and 140G1 1 didn't bind to any peptides in the experiment (the left of the bottom in FIG. 2). Therefore it is found that 139G3 binds to the epitope comprising/being present in

TPAPAPKPEQPA of the proline-rich domain of PspA and 140H1 binds to the epitope comprising/being present in KPAPAPQP of the proline-rich domain of PspA.

Example 4: Biacore-based evaluation of binding activity of anti-PspA mouse antibodies to recombinant PspA-D39, PspA-BAA658, and PspA-TIGR4.

In order to kinetically analyze the binding activity of anti-PspA mouse antibodies 139G3, 140G1, 140G1 1, and 140H1 to recombinant PspA proteins, the binding activity was measured by surface plasmon resonance method (SPR). All of the following manipulations were carried out using a Biacore 3000 (manufactured by GE Healthcare Bio-Sciences). The three different recombinant PspA proteins were immobilized on each different CM5 sensor chip (manufactured by GE Healthcare Bio-Sciences) by an amine coupling method in accordance with the protocols attached thereto. In particular, the kinetic assay was carried out by immobilizing on the chip different amounts of

recombinant proteins, in order to achieve low, medium, and high protein immobilization level. Thereafter, the anti-PspA antibodies diluted from a high concentration in six steps (from 40 to 1.25 nM, or from 20 to 0.625 nM) were allowed to run at a flow rate of 30 μΐνη ίη onto the chip, and the sensorgram corresponding to each concentration was obtained (FIGs. 3, 4, 5). The analysis was carried out using a bivalent binding model, using the analysis software attached to the apparatus, Biacore 3000 Evaluation software (manufactured by Biacore), thereby calculating an association rate constant kal and a dissociation rate constant kdl, as well as ka2 and kd2 of respective antibodies for the different recombinant PspA proteins.

As a result of using a bivalent binding model, an association rate constant kal, a dissociation rate constant kdl, and a dissociation constant KDI (kdl /kal), as well as an association rate constant ka2, a dissociation rate constant kd2, and a dissociation constant KQ₂ (kd/ka), of individual antibodies thus obtained are given in Table 1 to 3. Among the four antibodies, only 140H1 showed a clear and strong binding (KDI is less than 100 nM) to all of the three tested ligands including PspA-D39, PspA-BAA658, and PspA-TIGR4. Although the clone 140H1 was established from the immunization using PspA-D39 (Family 1), the clone can bind to PspA-TIGR4 (Family 2).

Table 1 : Summar of SPR ex eriments usin Ps A-D39 as the li and

Table 2: Summar of SPR ex eriments usin Ps A-BAA658 as the li and.

Table 3 : Summar of SPR ex eriments usin Ps A-TIGR4 as the li and.

Example 5: Binding spectrum analysis of mouse anti-PspA mAbs 140G1 , 140G1 1, 140H1 and 139G3 using 28 S. pneumoniae (Sp) wild-type strain and three PspA-deficient mutant strain.

To identify anti-PspA mAbs with broad binding spectrum, the reactivity of 10 mouse anti-PspA mAb antibody candidates to a panel of 28 Sp strains was assessed by flow cytometry. Notably, the pneumococcal strain selection used for the binding spectrum analyses contained representatives of the most prevalent serotypes currently observed in invasive pneumococcal disease, including multi-drug resistant strains, and all clinically relevant PspA clades (Clades 1-5)

Live, exponential phase bacteria were incubated with buffer only, control or mouse anti-PspA mAbs. Primary antibody bound to the bacteria was detected with phycoerythrin-labeled anti-mouse IgG secondary antibodies and flow cytometry using a Becton Dickinson FACSCalibur™ instrument. Moreover, the binding of the mAbs to three genetically engineered PspA-deficient mutants was determined to assess whether candidate antibodies can cross-react with other pneumococcal antigens.

Three mouse anti-PspA IgG antibodies with a broad binding spectrum to the strain collection have been generated: 140G1 (mouse IgGl), 140G1 1 (mouse IgG2a) and 140H1 (mouse IgGl) bound to >93% of the strains. Candidate 139G3 (mouse IgG2a) bound to 75% of the strains.

- 140G1 : 93% spectrum (bound to 26 of the 28 tested Sp strains)

- 140G11 : 93% spectrum (bound to 26/28 strains)

- 140H1 : 96% spectrum (bound to 27/28 strains) - 139G3 : 75% spectrum (bound to 21 /28 strains)

A list of the Sp strains used for the analysis and the binding pattern of the four lead candidates can be seen in FIG. 6. 140G1 and 140G11 showed an identical binding pattern. All of the four antibodies bound to at least one of the three tested PspA-deficient mutant strains, indicated that the antibodies could bind not only to PspA, but could also cross-react with non-PspA proteins such as PspC, a characteristic that is expected to enhance antibody efficacy. It is know that PspA expressed strains as D39, TIGR4 and so on, also express PspC (Ogunniyi et al, INFECTION AND IMMUNITY, Apr. 2007, p. 1843-1851 ; Kerr et al, INFECTION AND IMMUNITY, Sept. 2006, p. 5319-5324; Li et al, INFECTION AND IMMUNITY, Dec. 2007, p. 5877-5885.). PspA deficient would be caused by some mechanisms as insertion, frame-shift, deletion, and substitution. In the above results, it is clearly shown that the antibodies of the present invention can bind to PspA deficient strains in which PspA is deficient by some mechanisms and in which PspC is also expressed. Therefore, these antibodies can be effective for broad spectrum of bacteria.

Examples 6: Anti-PspA mAbs show activity in complement deposition assays (CD As) with 5^*. pneumoniae strains representing PspA clades 1-5.

Complement is known to be critical for the human host defense during invasive pneumococcal disease. The classical pathway of complement activation is initiated by antibody-mediated antigen recognition leading to the deposition of C3b-iC3b on S.

pneumoniae resulting in complement receptor mediated uptake and destruction of the bacteria by phagocytes. Complement deposition assays (CD As) and opsonophagocytic killing (OPK) experiments with anti-pneumococcal antibodies and hyper-immune sera have been proposed as surrogates of protection assays that can predict the protective efficacy of anti-S¹. pneumoniae antibodies and vaccines, respectively.

As mentioned elsewhere in this document, 140G1 and 140H1 are mouse IgGl mAbs, whereas 139G3 and 140G1 1 are IgG2a molecules. IgG affinity to Fc receptors on phagocytes is specific to the IgG subclass. Along this line, mouse IgG2a, the counterpart of human IgGl, is more potent than mouse IgGl in binding Clq and thus activating the classical complement pathway. To facilitate an appropriate comparison of the in vitro and in vivo activities of the anti-PspA mAbs, mouse IgG2a (class switched) versions of 140G1 and 140H1 were prepared, which were named 140csGl and 140csHl .

CD As with mouse serum and nine S. pneumoniae strains representing PspA clades 1-5 were conducted to determine if the four mouse anti-PspA mAbs confer C3b deposition on live pneumococci. ~10 exponential phase pneumococcal colony forming units (CFU) were incubated in 0.2 niL HBSS, 3.75% bovine serum albumin (BSA) at 37°C at 600 rpm horizontal shaking with or without mouse anti-PspA mAbs and commercially available BALB/c or CD-I mouse serum (Innovative Research) at concentrations that were optimized for each bacterial strain. After 30 min, cells were washed with ice-cold PBS, 0.5% BSA, and resuspended in 100 μΐ, refrigerated PBS, 0.5% BSA, containing 2 μg/mL fluorescein-labeled anti-mouse or anti-human C3 antibodies (Cedarlane Laboratories USA Inc.). After 30-60 min incubation at 4°C, bacteria were washed in ice-cold PBS, 0.5% BSA. After one additional washing step, the cells were subjected to flow cytometric analysis. The FL- 1 fluorescence intensity of the cells was measured with a BD

FACSCalibur™ and analyzed with FlowJo software. The histograms show overlays of the FL-1 data for bacteria that were subjected to negative control (tinted; gray lines) or the respective anti-PspA (black lines) mAbs. A shift in FL-1 intensity correlated with deposition of C3b on the bacteria. 139G3 did not bind to ATCC-6305.

The anti-PspA mAbs mediated C3b deposition in a strain specific manner.

Typical CD A results for one strain each for PspA clades 1-5 are shown as histograms in FIG. 7 and all CDA results are summarizes the results of CD As with the nine

pneumococcal strains in FIG. 8. High activity was observed for all mAbs in assays with ATCC-49619 (PspA clade 1), BAA-658 (Clade 1), WU2 (Clade 2), PJ-1324 (Clade 3), and NCTC-1 1905 (Clade 4). HOcsGl, 140G1 1, and 140csHl also mediated strong C3 deposition on D39 (Clade 2). Only 140csHl was able to confer significant C3 deposition on ATCC-6305 (Clade 2). In TIGR4 (Clade 3) experiments 139G3 and 140csHl showed higher activity than 140G1 1 and HOcsGl . All antibodies were able to confer C3 deposition on ATCC-700673 (Clade 5). 140csHl was the only antibody for which CDA activity was observed for all nine tested strains.

Example 7: Activity of mouse anti-PspA mAbs HOcsGl, 140G11, 140csHl and 139G3 in opsonophagocytic killing (OPK) experiments.

To further test the therapeutic potential of the mouse anti-PspA mAb candidates, OPK assays with differentiated, polymorphonuclear neutrophil (PMN)-like HL-60 cells were performed. PMNs are critical first-line innate immune host defense effector cells that sense and destroy invading microbial organism. Along this line, the ability of anti-pneumococcal antibodies to confer killing of S. pneumoniae by PMN is a second surrogate of protection that, besides CD As, is thought to predict in vivo efficacy of antibodies.

The serotype 6B S. pneumoniae strain PJ-1324 was grown to the exponential phase in THY, washed in PBS. Then, ~2xl0⁴ colony forming units (CFU) of PJ-1324 were pre-opsonized in 10% baby rabbit complement for 60 min in the presence of 1 μg/mL mouse IgG2a control or anti-PspA mAbs. Then, 10⁶ differentiated HL-60 cells in HBSS, 10 mM glucose, or vehicle alone were added to the bacteria. At the indicated time points, samples were, serially diluted and plated on agar plates to enumerate the number of surviving colony forming units (CFU). Samples were run in triplicate or quadruplicate.

The four anti-PspA mAbs showed a strong colony forming unit (CFU) reduction of S. pneumoniae PJ-1324 that was dependent on effector cells (FIG. 9). At 60 min, 139G3 and HOcsHl showed a stronger OPK activity than 140csGl and 140G11.

Taken the results outlined in Examples 5-7 together, HOcsHl antibody showed superior coverage, CD A and OPK activity.

Example 8: Activity of anti-Psp A mAbs 140csGl, 140G1 1, HOcsHl and 139G3 in mouse passive immunization experiments of pneumococcal sepsis when given before infection (prophylactically).

Mouse passive immunization experiments were conducted with 140csGl,

140G1 1, and HOcsHl, and 139G3 to assess whether broad binding spectrum and in vitro activity of anti-PspA antibodies translates into a wide range of in vivo efficacy. Such data would validate the potential of PspA as a target for the treatment of severe pneumococcal disease by mAbs.

The activities of the four anti-PspA candidates alone or in combination with each other were compared in up to 13 mouse septicemia models in which the antibodies were applied prophylactically. For the experiments, exponential phase bacteria were washed twice in PBS and then either injected intraperitoneally (i.p.) or intravenously (i.v.) into unanesthetized mice, or intranasally (i.n.) or intratracheally (i.t.) into mice

anesthetized by 2, 2, 2-Tribromoethanol in PBS as vehicle. For the i.n. instillation, 40 μΐ, bacterial suspensions were dropped into the nostrils; for i.t. instillation, 50 μΐ, bacteria were delivered through aspiration by occluding the trachea of the mice with the ball of a sterile animal feeding needle using a Hamilton syringe. 100-300 μg isotype control IgG2a mAb or anti-PspA mAbs in 200 μΐ, PBS were given i.p. 4-6 h before infection. In most experiments, tail vein blood was collected 1-3 days after infection to enumerate the bacterial burden in the blood. Survival of the mice was followed for 13-15 days. All experiments were conducted at least twice to confirm results. To calculate statistical significance, survival curves were compared with Mantel-Cox Test and results of two or more experiments were combined for most of the animal models.

Anti-PspA mAb activity was tested in up to 13 mouse passive immunization models in which the mAbs were applied prophylactically. Results with S. pneumoniae strains representing typical PspA clades 1-5 are shown in FIGs. lOA-H. The four anti-PspA mAbs displayed statistically significant efficacy in the S. pneumoniae BAA-658 (PspA family 1, clade 1) intraperitoneal (i.p.) infection model with 139G3 having the strongest activity (FIGs. 1 OA- IOC). In two of three experiments performed, the bacterial burden in blood was determined 24 h after infection; mice treated with anti-PspA mAbs had lower median blood CFU numbers compared to isotype treated animals, but the data did not reach statistical significance.

140csGl, 140G1 1 and 140csHl protected mice from mortality in a mouse intranasal challenge model with highly encapsulated S. pneumoniae WU2 (PspA family 1 , clade 2); 24 h after infection, no statistically significant differences in blood CFU were observed (FIGs. 1 lA-1 1C). 139G3 was not tested in this model due to lack of binding to WU2.

All four anti-PspA mAbs had activity in a S. pneumoniae PJ-1324 (PspA family 2, clade 3) i.p. infection model with 139G3 and HOcsHl conferring 100% survival; 24 h after infection, no bacteria were detectable in their blood of most of the anti-PspA mAb treated mice, whereas 80% of isotype-treated animals had between 10⁵- 10⁸ CFU/mL blood (FIGs. 12A-12C).

Passive immunization with 139G3, HOcsGl , HOG 1 1 and HOcsHl significantly reduced the mortality in animal experiments with S. pneumoniae NCTC- 1 1905 (PspA family 2, clade 4); along this line, the median blood CFU counts in anti-PspA treated mice was lower than in control mice 24 h after infection (FIGs. 13A-13C).

The results of all mouse passive immunization experiments of S. pneumoniae are summarized in FIG. 14.

The in vivo experimentation demonstrated that anti-PspA mAbs with broad binding spectrum and in vitro activity could display a wide range of in vivo efficacy when given alone or in combination with each other and thus have potential for the development of a passive immunization drug for the therapy of invasive pneumococcal disease.

Example 9: Therapeutically administered anti-PspA antibody HOcsHl confers CFU reduction and survival in S. pneumoniae mouse lung infection model.

A large proportion of community-acquired lung infections/pneumonias are caused by S. pneumoniae. To test whether anti-PspA mAbs confer protection in

experimental pneumonia, HOcsHl activity was tested in a model with S. pneumoniae PJ- 1324 (PspA family 2, clade 3) in which mice were infected intratracheally with therapeutic administration of mAbs.

6 h or 24 h after intratracheal infection with 0.75-2.7xl0⁶ CFU of S. pneumoniae PJ-1324 in 50 μΐ, PBS, CD-I mice were treated intraperitoneally with 200 μΐ, PBS containing 200 μg isotype IgG2a mAb C44 (open circles) or HOcsHl (closed circles). (A-B) 24 h after infection (18 h after treatment), CFU numbers in lungs and tail vein blood of the animals was determined. The detection limit of 100 CFU is indicated by dotted line. The combined results of three independent experiments are shown (*, /?<0.04, C44 vs. 140csHl treated mice, unpaired t-test). (C) Mice were treated 24 h after infection with C44 (open circles; n= 12 total) or 140csHl (closed circles; n= 13 total). The survival of the mice was followed for 15 days after infection. The combined results of two

independent experiments are shown in the graph. **,/?<0.004, C44 vs. 140csHl survival curve, Mantel-Cox test.

Treatment with 140csHl 6 h after infection resulted in >97% reduced median lung CFU numbers (Median: ~5.2xl0² CFU/lung) as compared to isotype treated mice (Median: ~1.8xl0⁴ CFU/lung) (FIGs. 15 and 15B). A median of 3.8x10^s CFU/mL blood were detected in isotype treated mice 24 h after the infection, whereas most mice receiving 140csHl did not have any detectable bacteria in their blood (FIGs. 15A and 15B) indicating that 140csHl inhibited the spread of P J- 1324 to extra-pulmonary sites. Further substantiating the therapeutic potential of anti-PspA mAbs, 140csHl significantly protected mice from mortality (FIG. 16) when administered 24 h after infection: 7/13 (-54%) of 140csHl treated mice survived, all isotype treated animals succumbed to infection.

The pneumonia model results with therapeutic application of 140csHl further substantiated the potential of anti-PspA mAbs as passive immunization drugs.

Example 10: Combination therapy of anti-PspA mAbs 140csHl and standard-of-care antibiotic ceftriaxone leads to survival benefit in a mouse passive immunization model using >20-fold the LDioo of S. pneumoniae PJ-1324.

We hypothesized that anti-PspA mAbs could have a therapeutic benefit when applied in conjunction with standard-of-care antibiotics such as ceftriaxone, which is used to treat severe pneumococcal infections. To that end, it was tested if a combination treatment of 140csHl and ceftriaxone shows a higher activity than any of the components alone in a mouse sepsis model of severe pneumococcal infection.

24 h after intraperitoneal (i.p.) infection with ~2.1-3.2xl0⁴ CFU of S. pneumoniae PJ-1324, which equals around 29-fold the LD₁₀₀, in 200 PBS, CD-I mice were treated i.p. with 200 iL PBS alone (vehicle), or 200 iL PBS containing 100 μg isotype IgG2a mAb C44 or 140csHl + 1 mg ceftriaxone (-50 mg/kg). Survival of the mice was followed for 15 days after infection (14 days after treatment). The combined results of three independent experiments are shown in FIGs. 17A and 17B. *, /?<0.05, survival curve comparison with Mantel-Cox test. As can be seen in FIGs. 17A and 17B, 100 μg of 140csHl + vehicle treatment did not confer any survival benefit in the mice compared to isotype/vehicle or vehicle alone treatment when given 24 h after infection with >25-fold the LDioo of S. pneumoniae PJ-1324. However, 100 μg 140csHl mediated 100% survival when given in combination with 50 mg/kg ceftriaxone (FIGs. 17A and 17B). This degree of protection was significantly higher than that of either 140csHl /vehicle, ceftriaxone/vehicle and

ceftriaxone/isotype treatment indicating that the anti-PspA mAb + antibiotic regimen worked better than any of the components alone.

The fact that 140csHl showed efficacy in combination with the standard-of- care antibiotic ceftriaxone when given 24 h after infection in a mouse passive

immunization model using >25-fold of the LDi₀₀ of the S. pneumoniae strain PJ-1324, further substantiated the potential of anti-PspA mAbs for the development of a protective passive immunization drug for the therapy of invasive pneumococcal disease. Specifically, anti-PspA antibodies could be given in conjunction with antibiotics thus have a therapeutic benefit for a patient suffering from invasive pneumococcal disease.

Example 11 : Activity of anti-PspA mAb 140csHl in a mouse passive immunization model with a multi-drug resistant pneumococcal strain.

The emergence of multi-drug resistant S. pneumoniae strains is of major concern. One example is the occurrence of pneumococci that are resistant to erythromycin (ERM), an antibiotic that is commonly used to treat pneumococcal pneumonia, e.g. in patients who have penicillin allergies. Mouse passive immunization experiments with 140csHl and the multi-drug resistant strain BAA-658, which is resistant to a variety of antibiotics including erythromycin (ERM), were conducted. Efficacy in such a model would further validate the potential of 140csHl PspA as drug candidate for the treatment of severe pneumococcal disease.

Approximately 30 min after intraperitoneal (i.p.) infection with 3.2-6.5x10⁷ exponential phase CFU of the ERM resistant pneumococcal strain BAA658, groups of 4-5 female CD-I mice were treated i.p. with 200 μΐ of PBS containing 300 μg of mouse isotype IgG2a or anti-PspA mAb 140csHl. Then, mice were treated intragastrically with either 200 μΐ, PBS, 1.5% ethanol (vehicle) or 200 μΐ. PBS, 1.5% ethanol, 1.5 mg/mL ERM (Dose: 15 mg/kg). The survival of the mice was monitored for 8 days. The results of three independent experiments were combined for the graph shown in FIG. 18. Of note, -3.75- 15 mg/kg is an often cited recommended dose a -70 kg human adult would receive within 24 h to treat pneumococcal pneumonia.

As can be seen in FIG. 18, a combination of 15 mg/kg ERM and 300 μg isotype mAb did not have significant activity compared to vehicle/isotype treatment which was expected as BAA-658 is high-level ERM-resistant. In contrast, a combination of therapeutically applied vehicle and 140csHl conferred significant protection relative to vehicle/isotype treatment. Unexpectedly, 140csHl efficacy was further enhanced when given in combination with ERM, which might be explained through minor activity of the antibiotic against BAA-658 and/or anti-inflammatory effects macrolides are known to have.

The fact that 140csHl showed efficacy in combination with the standard-of- care antibiotic ERM against a ERM-resistant pneumococcal strain further substantiated the potential of anti-PspA mAbs for the development of a protective passive immunization drug for the therapy of invasive pneumococcal disease.

Example 12: Increased activity of IgGl/IgG3 versions of mouse/human chimeric anti- PspA antibodies in complement deposition assays (CDAs) with human serum.

And,

Example 13: Increased activity of non-fucosylated mouse/human chimeric anti-PspA antibodies in opsonophagocytosis assays (OP As) with human phagocytes.

Several mouse/human chimeric versions of 140H1 and 140G1 were prepared to test if effector function of anti-PspA mAbs can be enhanced through antibody engineering.

It has been reported that human IgGl/IgG3 (hIgGl/IgG3) anti-cancer mAbs exhibit increased complement-dependent cytotoxicity towards cancer cells compared to human IgGl through enhancement of classical complement pathway activation. Based on this, we hypothesized that hIgGl/IgG3 type anti-PspA mAbs can confer enhanced activation of the classical complement pathway and deposition of C3b on the surface of pneumococci over hlgGl antibodies.

The Fc part of human IgG is usually fucosylated. It was shown that non- fucosylated anti-B cell lymphoma human IgGl had higher affinity to Fc gamma receptor Illb (FcgRIIIb) than fucosylated comparator antibodies thereby potentiating phagocytosis of cancer cells by human PMN. Therefore, we hypothesized that non-fucosylated (Fuc-) anti-PspA mAbs could confer increased uptake of pneumococci by human phagocytes compared to regular fucosylated (Fuc+) antibodies.

To address these hypotheses, four chimeric versions of the anti-PspA mAbs 140H1 and 140G1 were prepared each containing the mouse variable region and: (1) a fucosylated human IgGl constant region (Fuc+ hlgGl); (2) a non-fucosylated human IgGl constant region (Fuc- hlgGl); (3) a fucosylated human IgGl/IgG3 constant region (Fuc+ hIgGl/IgG3); or (4) a non-fucosylated human IgGl/IgG3 constant region (Fuc- hIgGl/IgG3).

The pneumococcal strains used for CDAs with human serum were WU2

(highly encapsulated), BAA-658 (multi-drug resistant), and PJ-1324. Exponential phase cells were incubated for 15 min in 2.5% normal human serum or 40% absorbed human plasma in the presence or absence of 5 μ§/ηιΙ. of chimeric anti-PspA or control antibodies. Subsequently, the deposition of C3, which is indicative of complement deposition on the bacteria, was detected with a FITC-labeled anti-C3 antibody. The fluorescence intensity of the cells was measured with a BD FACSCalibur™ and analyzed with FlowJo software.

OPAs with human PMN and chimeric anti-PspA antibodies were conducted as follows. In 96-well polypropylene round bottom plates, 2.5x10⁶ fluorescein-labeled pneumococcal particles were incubated for 5-30 min with 5xl0⁵ purified human blood PMN in 200 μΐ, HBSS, 10% PBS containing 0-10 μg/mL isotype or chimeric anti-PspA antibody versions. Subsequently, the samples were washed once. Ethidium bromide at an endconcentration of 0.25 mg/mL was added to the samples to stain extracellular bacteria to differentiate between intra- and extracellular pneumococci by fluorescence microscopy using a triple dichroic filter for simultaneous visualization of green and red fluorescence. Samples were typically run in quadruplicate and 200 live PMN/sample analyzed for phagocytosis of pneumococci.

The activities of these antibodies were compared in CDAs with three 5".

pneumoniae strains and human serum or absorbed human plasma as complement source. Both Fuc+ and Fuc- hIgGl/IgG3 140H1 versions conferred increased C3 deposition on highly encapsulated S. pneumoniae WU2 as compared to Fuc+ IgGl and Fuc- IgGl comparator antibodies (FIGs. 19-20C). This shows that hIgGl/IgG3 type anti-PspA antibodies have higher activity than hlgGl antibodies in CDAs with human complement irrespective of their fucosylation status. Further validating this, the Fuc+ and Fuc- hIgGl/IgG3 type 140G1 and 140H1 had higher activity in CDAs with S. pneumoniae PJ- 1324 and BAA-658 (FIGs. 21 A-21H). Of note, the assays with the latter two strains were conducted with absorbed human plasma, because of high background activity of the human serum pool in CDAs with these bacterial strains. Examples for 140H1 are shown in FIGs. 19-21H.; similar results were obtained with the four chimeric 140G1 antibodies (data not shown). The CDA results are summarized in FIG. 22.

In OPAs without complement, Fuc- hlgGl and hIgGl/IgG3 140H1 and 140G1 versions conferred increased uptake of S. pneumoniae D39 cells by human blood PMN compared to the respective Fuc+ IgGl and IgGl/IgG3 molecules showing that the degree of anti-PspA antibody activity in complement-independent OPAs was heavily dependent on their fucosylation status (FIGs. 23 and 24).

The CDA and OPA results obtained with chimeric 140H1 and 140G1 demonstrated that the activity of anti-PspA antibodies can be significantly enhanced through antibody engineering and suggest that non-fucosylated hlgGl /IgG3 anti-PspA mAbs could exhibit higher therapeutic efficacy compared to regular fucosylated hlgGl- type antibodies.

Example 14: Creation of humanized anti-PspA antibody from clone 140H1

(1) Design of amino acid sequences of VH and VL of anti-PspA 140H1 humanized antibody

An amino acid sequence of VH of an anti-PspA 140H1 humanized antibody was designed in the following manner.

Firstly, the amino acid sequence of FR of VH of a human antibody for grafting amino acid sequences of CDRl to CDR3 of an anti-PspA mouse monoclonal antibody 140H1 VH represented by SEQ ID NOs: l, 2 and 3, respectively, was selected.

Using a GCG Package (manufactured by Genetics Computer Group) as a sequence analysis system, based on the amino acid sequence data base of conventional proteins by the BLASTP method [Nucleic Acids Res., 25, 3389 (1997)], a human antibody having a high homology with the anti-PspA mouse monoclonal antibody 140H1 was searched.

When the homology score was compared with the homology of an actual amino acid sequence, SWISSPROT data base accession no. BAF64539, Production of

High- Affinity Human Monoclonal Antibody Fab Fragments to the 19-Kilodalton C- Terminal Merozoite Surface Protein 1 of Plasmodium falciparum (hereinafter referred to as

"BAF64539") exhibited a homology of 72.4%, and it was a human antibody which had the highest homology, therefore the amino acid sequence of FR of this antibody was selected.

An amino acid sequence of CDR of VH of the anti-PspA mouse monoclonal antibody 140H1 represented by SEQ ID NOs:l to 3 was grafted into an appropriate position of the thus determined amino acid sequence of FR of the human antibody. In this manner, an amino acid sequence HVO of VH of the anti-PspA humanized antibody represented by SEQ ID NO: 10 was designed.

Next, an amino acid sequence of VL of an anti-PspA humanized antibody was designed in the following manner.

An amino acid sequence of FR of VL of a human antibody for grafting amino acid sequences of CDRl to CDR3 of an anti-PspA mouse monoclonal antibody 140H1 VL represented by SEQ ID NOs:4 to 6, respectively, was selected.

Kabat et al. , have classified VL of conventionally known various human antibodies into four subgroups (HSG I to IV) based on the homology of their amino acid sequences and reported on the consensus sequences for each of the subgroups [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

Accordingly, the homology was examined between the amino acid sequences of FR of consensus sequences of subgroups I to IV of VL of the human antibody and the amino acid sequence of FR of VL of the anti-PspA mouse antibody 140H1.

As a result of the homology analysis, the homology of HSGI, HSGII, HSGIII, and HSGIV was 72.5%, 75.0%, 68.8%, and 77.5%, respectively. Therefore, the amino acid sequence of FR of 140H1 VL had the highest homology with subgroup IV.

Based on these results, the amino acid sequence of CDR of VL of the anti- PspA mouse monoclonal antibody 140H1 was grafted into an appropriate position of an amino acid sequence of FR of consensus sequences of subgroup IV of VL of the human antibody.

However, since Leu at position 1 10 in the amino acid sequence of VL of the anti-PspA mouse monoclonal antibody 140H1 represented by SEQ ID NO: 8 is not the amino acid residue having the highest use frequency in the region which corresponds to the amino acid sequence of the human antibody FR cited by Kabat, but is an amino acid residue which is used at a relatively high frequency, the above-mentioned amino acid residues which are recognized in the amino acid sequence of the anti-PspA mouse monoclonal antibody 140H1 were used.

In this manner, an amino acid sequence LV0 of VL of an anti-PspA humanized antibody represented by SEQ ID NO: 12 was designed.

The amino acid sequence HV0 of VH and amino acid sequence LV0 of VL of the anti-PspA humanized antibody designed in the above are sequences in which the CDR amino acid sequence of the anti-PspA mouse monoclonal antibody 140H1 alone was grafted into the selected human antibody FR amino acid sequence.

However, in general, when a humanized antibody is prepared, its binding activity is frequently lowered in the case of merely a simple grafting of CDR amino acid sequence of a mouse antibody to a human antibody FR. For these reasons, in order to avoid lowering of the binding activity, modifications of the amino acid residues considered to have an influence on the binding activity, among the FR amino acid residues which are different between human antibodies and mouse antibodies, are carried out together with the grafting of the CDR amino acid sequence.

Thus, the amino acid residues of FR considered to have an influence on the binding activity were identified in this Example in the following manner.

Firstly, a three-dimensional structure of an antibody V region (HV0LV0) comprising the amino acid sequence HV0 of VH and amino acid sequence LV0 of VL of anti-PspA humanized antibody designed in the above was constructed using a computer modeling technique.

In addition, preparation of the three dimensional structure coordinates and display of the three-dimensional structure were carried out using software Discovery Studio (manufactured by Accelrys) in accordance with instructions attached thereto. In addition, a computer model of the three-dimensional structure of the V region of the anti- PspA mouse monoclonal antibody 140H1 was also constructed in the same manner.

Further, by similarly constructing a three-dimensional structure model comprising an amino acid sequence in which the amino acid residues in the FR amino acid sequences of VH and VL of HVOLVO, which are different from those of the anti-PspA mouse antibody 140H1, were selected and modified into the amino acid residues of the anti-140Hl mouse monoclonal antibody 140H1, three-dimensional structures of the V regions of anti-PspA mouse monoclonal antibody 140H1, HVOLVO and modified product were compared, whereby the amino acid residues predicted to have an influence on the binding activity of the antibody were identified.

As a result, as the amino acid residues among amino acid residues of FR of HVOLVO, which are considered to change a three-dimensional structure of the antigen binding region and therefore have an influence on the binding activity of the antibody, Val at position 2, Ser at position 9, Val at position 20, Arg at position 38, Gin at position 39, Glu at position 46, Met at position 48, Phe at position 68, Val at position 93, Tyr at position 95 and Ala at position 97 in the HVO sequence, and He at position 2, Leu at position 15, Ala at position 19, He at position 21, Pro at position 49 and Leu at position 84 in the LVO sequence were respectively selected.

By modifying at least one or more amino acid sequences of these selected amino acid residues into the amino acid residues which are present at the same positions of the amino acid sequence of the anti-PspA mouse monoclonal antibody 140H1, VH and VL of a humanized antibody having various modifications were designed.

Specifically, regarding the antibody VH, at least one modification was introduced among the amino acid modifications for substituting Val at position 2 with He, Ser at position 9 with Pro, Val at position 20 with He, Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with lie, Val at position 93 with Thr, Tyr at position 95 with Phe and Ala at position 97 with Gly in the amino acid sequence represented by SEQ ID NO: 10.

Further, regarding the antibody VL, at least one modification was introduced among the amino acid modifications for substituting He at position 2 with Thr, Leu at position 15 with Val, Ala at position 19 with Val, lie at position 21 with Met, Pro at position 49 with Ser and Leu at position 84 with Val in the amino acid sequence represented by SEQ ID NO: 12.

By designing amino acid sequences of variable regions of HV9LV0, HV1 1LV0, HV1 1LV4, and HV11LV6 with modifications of at least one of amino acid residues present in FR of HVOLVO, amino acid sequences of H chain variable regions HV9 and HVl 1 are represented by SEQ ID N0s: 14 and 16, and amino acid sequences of L chain variable regions LV4 and LV6 are represented by SEQ ID NOs: 18 and 20, respectively. (2) Preparation and evaluation of anti-PspA humanized antibody

DNA encoding the amino acid sequence of the variable region of the anti-PspA humanized antibody was constructed in mammalian cells using a codon which is used at a high frequency, when amino acid modification(s) are carried out using a codon which is used as DNA encoding the amino acid sequence of VH or VL of the anti-PspA mouse monoclonal antibody 140H 1.

The DNA sequences encoding the amino acid sequence of HVO and LVO of the anti-PspA humanized antibody are respectively represented by SEQ ID NOs:9 and 1 1, whereas the DNA sequences encoding the amino acid sequences of variable regions HV9, HVl 1, LV4 and LV6 on which amino acid modification(s) were made are respectively represented by SEQ ID NOs: 13, 15, 17 and 19.

(3) Construction of cDNA coding for VH of anti-PspA humanized antibody

A cDNA encoding the amino acid sequence HVO, HV9 or HVl 1 of the VH of the anti-PspA humanized antibody represented by SEQ ID NO: 10, 14 or 16 designed in the above (1) of this Example was prepared by total synthesis.

(4) Construction of cDNA encoding VL of anti-PspA humanized antibody

A cDNA encoding the amino acid sequence LVO of the VL of the anti-PspA humanized antibody represented by SEQ ID NO: 12, 18 or 20 designed in item (1) of this Example was prepared by total synthesis.

(5) Construction of anti-PspA humanized antibody expression vector

Various ant-PspA humanized antibody expression vectors were constructed by inserting a cDNA encoding any one of the HVO, HV9 and HVl 1 and a cDNA encoding the LVO, LV4 and LV6 obtained in the above (2) and (3) of this Example, into appropriate positions of the humanized antibody expression vector pKANTEX93 described in

WO97/10354.

(6) Expression of anti-PspA humanized antibody in animal cell

Stable expression of the anti-PspA humanized antibody using an animal cell and purification of the antibody from culture supernatant were carried out in the same manner as the methods described in the following manner. Using the anti-PspA humanized antibody expression vectors obtained in the above (5), expression of anti-PspA humanized antibody in an animal cell was carried out by a general method [Antibody Engineering, A Practical Guide, W.H. Freeman and Company (1992)] to obtain anti-PspA humanized antibody producing transformants.

(7) Preparation of purified antibodies

Each of the transformants obtained in the above (6) was cultured by a general culturing method, and the cell suspension was recovered and centrifuged for 15 minutes under a condition of 3000 rpm at 4°C to recover the culture supernatant, and then the culture supernatant was sterilized by filtration using MillexGV filter (manufactured by Millipore Corp) having a pore size of 0.22 μηι.

From the obtained culture supernatants, anti-PspA humanized antibodies HV9LV0, HVl 1 LV0, HV l 1LV4 and HVl 1LV6 were purified using a MabSelect SuRe Resin (manufactured by GE healthcare) column and in accordance with the instructions attached thereto.

Purification degree and expressed molecular size of the thus obtained purified samples of HV9LV0, HVl 1LV0, HVl 1 LV4 and HVl 1 LV6 were confirmed by SDS- PAGE using a gradient gel (manufactured by Atto Corp., catalogue number: E-T520L) and in accordance with the instructions attached thereto.

Regarding the electrophoresis pattern of the thus purified anti-PspA humanized antibodies, a single band was found at around a molecular weight of 150 kilodalton (hereinafter referred to as kDa) to 200 kDa under non-reducing condition, and two bands of about 50 kDa and about 25 kDa under reducing condition.

Such an electrophoresis pattern coincided with a result in which SDS-PAGE of an IgG class antibody was carried out under the same condition.

Accordingly, it was confirmed that the anti-PspA humanized antibodies HV9LV0, HVl 1 LV0, HVl 1 LV4 and HVl 1LV6 were expressed as antibody molecules having correct structures. Example 15 : Binding of humanized 140H1 antibody variants to live pneumococci.

Four humanized versions of the anti-PspA antibody 140H1 were prepared, which were named KM8120 (HV9LV0), KM8121 (HVl 1 LV0), KM8122 (HVl 1 LV4), and KM8123 (HVl 1 LV6). The antibodies were compared with the parental mouse/human chimeric version of 140H1 (KM5080) for binding to live S. pneumoniae whole cells by flow cytometry in order to evaluate if they retained their ability to bind to their antigen on live pneumococcal whole cells. Live, exponential phase S. pneumoniae cells were washed, and then incubated for 1 h with purified isotype human IgGl or the indicated chimeric or humanized anti- PspA antibodies at 4°C with shaking. Then, cells were washed and bound primary antibody detected with PE-labeled anti-human IgG. Subsequently, the FL-2 mean fluorescence intensities (MFI) of > 20,000 bacterial particles per sample were measured after excitation with a 488 nm laser with a FACSCalibur™. Samples were run in duplicate and average MFI values for each data point are shown in the graphs.

As can be seen in FIGs. 25A-25D, the four humanized antibodies show similar binding pattern to S. pneumoniae D39 and BAA-658 cells as the parental antibody.

However, KM8120 and KM8121 show reduced binding compared to parental antibody M5080 and KM8122 and KM8123 to S. pneumoniae TIGR4 and PJ-1324 cells.

The flow cytometry results obtained with the parental chimeric and humanized 140H1 versions suggest that KM8122 and KM8123 show similar binding profile as the parental antibody, whereas KM8120 and KM8121 seemed to have lost affinity to certain PspA proteins on live pneumococcal whole cells (TIGR4 and PJ-1324).

Example 16: Activity of humanized 140H1 antibody variants in opsonophagocytosis assays with human phagocytes.

The activity of two humanized versions of the anti-PspA antibody 140H1 , KM8122 (HV 1 1 LV4) and KM8123 (HV 1 1 LV6), were compared with that of the parental mouse/human chimeric version of 140H1 (KM5080) in opsonophagocytosis assays (OPAs) with S. pneumoniae D39 cells and purified human peripheral blood

polymorphonuclear neutrophils (PMN), which are considered as a major professional phagocytic cell population in humans.

In 96-well polypropylene round bottom plates, 2.5x10⁶ fluorescein-labeled pneumococcal particles were incubated for 15 min with 5xl0⁵ purified human blood PMN in 200 μΐ, HBSS, 10% PBS containing 100 μg/mL human IgGi antibody +/- 10 μg/mL of the indicated negative control or chimeric or humanized anti-PspA antibody versions. Subsequently, the samples were washed once. Ethidium bromide at an endconcentration of 0.25 mg/mL was added to the samples to stain extracellular bacteria to differentiate between intra- and extracellular pneumococci by fluorescence microscopy using a triple dichroic filter for simultaneous visualization of green and red fluorescence. Samples were run in hexuplicate and at least 100 live PMN/sample analyzed for phagocytosis of pneumococci.

As can be seen in FIG. 26, the two humanized versions conferred a similar degree of uptake of S. pneumoniae D39 cells by human phagocytes. The OPA results indicate that the humanized anti-PspA antibodies KM8122 (HV1 1LV4) and KM8123 (HV1 1LV6) retained their functionality in terms of mediating Fc receptor mediated uptake of antibody-opsonized pneumococci. Example 17: Activity of chimeric and humanized 140H1 antibody variants in mouse passive immunization model of S. pneumoniae sepsis.

The activity of two humanized versions of the anti-PspA antibody 140H1, KM8122 (HV11LV4) and KM8123 (HV1 1LV6) and that of the parental mouse/human chimeric version of 140H1 (KM5080) was tested in a mouse passive immunization of pneumococcal sepsis in order to determine whether the antibodies are able to confer protection in vivo.

24 h before intraperitoneal (i.p.) infection with ~7.1xl0³ CFU of S. pneumoniae strain PJ-1324, groups of 3-5 female CD-I mice were pretreated i.p. with PBS containing 100 μg of the indicated antibodies in PBS. Survival of the mice was followed for 9 days after infection.

KM5080, KM8122 and KM8123 showed clear trend towards protecting mice from mortality at 100 μg dose. Parental antibody KM5080 and two humanized antibodies KM8122 and KM8123 showed clear protective trend in the mouse passive immunization experiment at 100 ig dose.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. All references cited herein are incorporated in their entirety. This application is based on U.S. provisional patent application No. 61/682,378 filed on August 13, 2012, the entire contents of which are incorporated hereinto by reference.

Industrial Applicability

According to the present invention, the embodiments relating to bacteriology and antibody drug development are provided. More specifically, the various embodiments relating to monoclonal antibody against Streptococcus pneumoniae surface protein A (PspA) which has both preventive (protective) and therapeutic efficacy against

Streptococcus pneumonia-induced infectious diseases including pneumonia are provided. Sequence Listing Free Text

SEQ ID NO: l : VH CDR1 amino acid sequence of 140H1

SEQ ID NO:2: VH CDR2 amino acid sequence of 140H1 SEQ ID NO:3: Description of the artificial sequence: VH CDR3 amino acid

SEQ ID NO:4: VL CDR1 amino acid sequence of 140H1

SEQ ID NO:5: VL CDR2 amino acid sequence of 140H1

SEQ ID NO:6: VL CDR3 amino acid sequence of 140H1

SEQ ID NO:9: HVO nucleotide sequence

SEQ ID NO: 10: Synthetic construct

SEQ ID NO: 1 1 : LVO nucleotide sequence

SEQ ID NO: 12: Synthetic Construct

SEQ ID NO: 13: HV9 nucleotide sequence

SEQ ID NO: 14: Synthetic Construct

SEQ ID NO: 15: HV 11 nucleotide sequence

SEQ ID NO: 16: Synthetic Construct

SEQ ID NO: 17: LV4 nucleotide sequence

SEQ ID NO: 18: Synthetic Construct

SEQ ID NO : 19 : LV6 nucleotide sequence

SEQ ID NO: 20: Synthetic Construct

SEQ ID NO:21 : pelB-PspA(D39)-His nucleotide sequence

SEQ ID NO:22: pelB-PspA(TIGR4)-His nucleotide sequence

SEQ ID NO:23: MBP-PspA-BAA658-FL nucleotide sequence

SEQ ID NO:24: MBP-PspA-BAA658-Sl nucleotide sequence

SEQ ID NO:25: MBP-PspA-BAA658-S2 nucleotide sequence

SEQ ID NO:26: MBP-PspA-BAA658-S3 nucleotide sequence

SEQ ID NO:27: MBP-PspA-BAA658-S4 nucleotide sequence

SEQ ID NO:38: Recombinant human IgGl/IgG3 constant domain nucleotide sequence SEQ ID NO:39: pelB-PspA(D39)-His amino acid sequence

SEQ ID NO:40: pelB-PspA(TIGR4)-His amino acid sequence

SEQ ID NO:41 : MBP-PspA-BAA658-FL amino acid sequence

SEQ ID NO:42: MBP-PspA-BAA658-Sl amino acid sequence

SEQ ID NO:43: MBP-PspA-BAA658-S2 amino acid sequence

SEQ ID NO:44: MBP-PspA-BAA658-S3 amino acid sequence

SEQ ID NO:45: MBP-PspA-BAA658-S4 amino acid sequence

SEQ ID NO:55: Recombinant human IgGl/IgG3 constant domain amino acid sequence SEQ ID NO:56: BAA658-PR1 amino acid sequence

SEQ ID NO:57: BAA658-PR1.2 amino acid sequence

SEQ ID NO:58: BAA658-PR2 amino acid sequence

SEQ ID NO:59: BAA658-PR3 amino acid sequence

SEQ ID NO:60: BAA658-PR4 amino acid sequence

Claims

1. A monoclonal antibody or antigen-binding fragment thereof which recognizes one epitope comprising a peptide selected from TPAPAPKPEQPA,

KPAPAPQP and DDQQAEEDYARRSEEEYNRLPQQQPPKAE of a Proline-rich domain, and binds to pneumococcal surface protein (hereinafter described as Psp).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein Psp is at least one Psp selected from PspA and PspC.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to Streptococcus pneumoniae which expresses PspA.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to PspA polypeptide derived from Streptococcus pneumoniae strain selected from D39 and BAA-658 at the affinity less than 10 nM.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to Streptococcus pneumoniae strains consisting of ATCC-6301, ATCC-49619, NCTC-1 1888, BAA-658, ATCC-700675, NCTC-11886, ATCC-700905, PJ-1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-1 1906, ATCC-700673, NCTC-

1 1897, BAA-612 and ATCC-700671.

6. The antibody, or antigen-binding fragment thereof according to claim 1 , which binds to Streptococcus pneumoniae strains consisting of ATCC-6301, NCTC-1 1910, ATCC-49136, ATCC49619, NCTC-1 1888, EF3030, BAA-658, ATCC-700675, ATCC- 6305, WU2, NCTC-7978, D39, NCTC-11886, BAA-475, BAA-340, ATCC-700905, PJ- 1324, TIGR4, NCTC-1 1902, NCTC-1 1905, NCTC-1 1906, ATCC-700673, NCTC-1 1897, BAA-612, ATCC-49150, ATCC-700671 and DS2341-94.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises complementarity determining regions (hereinafter, described as CDR) 1 to 3 of heavy chain variable region (hereinafter described as VH) comprising amino acid sequences of SEQ ID NOs: l to 3, respectively, and CDRs 1 to 3 of light chain variable region (hereinafter described as VL) comprising amino acid sequences of SEQ ID NOs:4 to 6, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises VH comprising amino acid sequence consist of SEQ ID NO: 7 and VL comprising amino acid sequence consist of SEQ ID NO:8.

9. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is any recombinant antibodies selected from a chimeric antibody, humanized antibody and human antibody.

10. A humanized antibody or antigen-binding fragment thereof according to claim 9, is selected from (a) and (b) as followed,

(a) wherein VH of the humanized antibody comprises an amino acid sequence in which at least one substitution selected from substitutions of Val at position 2 with He, Ser at position 9 with Pro, Val at position 20 with He, Arg at position 38 with Gin, Gin at position 39 with Lys, Glu at position 46 with Gin, Met at position 48 with He, Phe at position 68 with He, Val at position 93 with Thr, Tyr at position 95 with Phe, and Ala at position 97 with Gly is introduced in the amino acid sequence represented by SEQ ID NO: 10; and

1 1. A method for detecting Psp polypeptide or Psp-expressed on Streptococcus pneumoniae comprising a use of the antibody or antigen-binding fragment thereof according to claim 1.

12. A method for treating a Streptococcus pneumoniae-^' dxxccd infectious disease of a subject, comprising administration of the antibody or antigen-binding fragment thereof according to claim 1 to the subject.

13. The method for treating the infectious diseases according to claim 12, wherein the infectious disease is selected from pneumonia, sepsis, septic shock, otitis media, pericarditis, peritonitis, bronchitis, bacteremia and meningitis.

14. A method for treating the infectious diseases according to claim 12, wherein the infectious disease is caused by at least one of Streptococcus pneumoniae strains being resistant to complement activation.

15. A method for treating the infectious diseases according to claim 12, wherein the infectious disease is caused by at least one of Streptococcus pneumoniae strains being resistant to multi-drugs.

16. A method for treating the infectious diseases according to claim 12, wherein the administration of the antibody is combined with at least one antibiotic.

17. A method for treating a Streptococcus pneumoniae-induced infectious disease of a subject described in claim 16, wherein the antibiotic is selected from cephem antibiotic and macrolide antibiotic.

18. A nucleotide encoding amino acid sequence of the antibody and antigen- binding fragment thereof according to claim 1.

19. A recombinant vector which comprises the DNA according to claim 18.

20. A transformant obtainable by introducing the recombinant vector according to claim 19 into a host cell.

21. A process for producing the antibody or the antibody fragment thereof according to claim 1, comprising culturing the transformant described in claim 20 in a medium to form and accumulate the antibody or the antibody fragment thereof described in claim 1 in the culture, and collecting the antibody or the antibody fragment thereof from the culture.