KR100638503B1 - A polypetide comprising the amino acid of an N-terminal choline binding protein A truncate, vaccine derived therefrom and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention provides an isolated polypeptide comprising the amino acid sequence of the truncation of N-terminal choline binding protein A, having the amino acid sequence set forth in SEQ ID NO: 1, 3-7 or 9-11, fragments, mutants, variants thereof , Analogs or derivatives. The invention also provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation, wherein the amino acid sequence is set forth in SEQ ID NO: 24, and the polypeptide has its natural tertiary structure, and a method of making the same. The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation, wherein the polypeptide retains lectin activity and does not bind choline. The present invention provides an isolated immunogenic polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. The present invention provides an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. Finally, the present invention provides for use in pharmaceutical compositions, vaccines and diagnostic and therapeutic methods.

Pneumococcal, N-terminal Choline Binding Protein A Fragments, Choline, Lectin

Description

Polypeptide comprising the amino acid of an N-terminal choline binding protein A truncate, vaccine derived therefrom and pharmaceutical composition comprising the same}

The present invention generally relates to polypeptides of N-terminal choline binding protein A truncates. The invention also provides vaccines for providing protection against, or inducing protective antibodies against, bacterial infections, particularly pneumococcus , and antibodies to said polypeptides for use in diagnostic and passive immunotherapy. It is about antagonists. The polypeptides and / or nucleic acids encoding such polypeptides are also useful as competitive inhibitors of the bacterial adhesion of pneumococci. Finally, the present invention relates to therapies using such polypeptides.

Streptococcus pneumoniae is a Gram-positive bacterium that is a major cause of invasive infections such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen et al NEJM 322: 1280-1284, 1995). Pneumococci bind friendly to upper and lower airway cells. Like most bacteria, the attachment of pneumococcal to human cells is by the presentation of bacterial surface proteins that bind to eukaryotic carbohydrates in a lecithin-like manner. Cundell, D. & Tuomanen, E. (1994) Microb Pathog 17: 361-374. Pneumococci bind to inflamed epithelium, a process that can be considered as asymptomatic carriage. Switching to invasive disease has been suggested to involve the local generation of inflammatory factors that change the number and form of receptors available on such human cells while activating human cells. Cundell, D. et. al. (1995) Nature, 377: 435-438. One possibility suggested in this new setup is that pneumococci utilize these non-regulated receptors, namely platelet activating factor (PAF) receptors, and are believed to be associated with one of them. Cundell et al. (1995) Nature, 377: 435-438. Within a few minutes of the appearance of the PAF receptor, pneumococci will progress to more heightened adhesion and invasion. For example, inhibiting bacterial binding to cells activated by soluble receptor analogs blocks progression to disease in animal models. Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176: 704-712. Particularly effective in this regard are lacto-N-neotetraose with or without additional sialic acid, which prevents the attachment of pneumococcal to human cells in vitro and prevents colonization in the lungs in vivo. It is soluble carbohydrate to contain.

Choline Binding Proteins: Candidate Structural Attachment Genes:

Pneumococci produce a family of surface proteins capable of binding to bacterial surfaces by non-covalent binding to cell wall teichoic acid or lipoteichoic acid. The surface of Streptococcus pneumoniae is covered with a family of CBP (choline binding proteins) that covalently binds to phosphorylcholine. CbpA is a 75 kD surface-exposed choline binding protein exhibiting chimeric structure. There is a unique N-terminal domain proline rich region followed by a C-terminal domain consisting of 10 repeating regions involved in binding to choline.

CbpA is an attachment point (ligand) to a glycoconjugate containing receptor present on the surface of eukaryotic cells. Mutants deficient in cbpA exhibited decreased pathogenesis in infant rat models for nasopharyngeal colony formation. This binding is indicated for choline determinants covered with teichoic acid and is mediated by choline binding domains characteristic of each member of this protein family. These choline binding domains have been found in studies of autolytic enzymes such as Lopez and have been fully characterized (Ronda et al. (1987) Eur. J. Biochem, 164: 621-624. Other proteins containing these domains include autolysates of pneumococcal phage and pneumococcal surface protein A (PspA), a protective antigen. Ronda, C. et al. (1987) Eur. J. Biochem., 164: 621-624 and McDaniel, L.S., et al. (1992) Microb. Pathog, 13: 261-269. CbpA is shared with its other family member C terminus, but its binding activity to human cells does not colonize the nasal pharyngeal domain resulting from its unique N-terminal domain. Since this colony formation process and progression to disease depend on the attachment of pneumococcal to human cells as the first step, the competition of the N-terminal domain by competitive inhibition with cross-reactive antibodies or peptides that mimic these domains. Interfering with function can be crucial to blocking the disease.

Choline binding proteins for anti-pneumococcal vaccines are described in PCT International Patent Application PCT / US 97/07198, which PCT application is incorporated herein by reference. s. Recent vaccines against S. pneumoniae use purified carbohydrates from the 23 most common serotype capsules of the bacterium, but these vaccines have only about 50% protection [Shapiro et al. . NJEM 325: 1453, 1991], and are not immunogenic under 2 years of age. In addition, the therapeutic polypeptide may confer a therapeutic option in the case of infection with multiple resistant organisms. Therefore, the present invention satisfies the long-standing needs by providing a prophylactic vaccine.

Summary of the Invention

The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. Such polypeptides include the amino acid sequences set forth in SEQ ID NO: 1, 3-7 or 9-11, fragments, mutants, variants, analogs or derivatives thereof. The present invention also provides an isolated polypeptide exhibiting a tertiary structure comprising the amino acid sequence of an N-terminal choline binding protein A truncation having the amino acid set forth in SEQ ID NO: 24, and a method for producing such a polypeptide. The isolated polypeptide is suitable for use in immunizing animals and humans from bacterial infections, preferably pneumococcal infections.

In a further aspect, the invention relates to N-terminal choline binding protein A truncates with lectin activity but no choline binding activity. In addition, the present invention provides immunogenic N-terminal choline binding protein A truncates or fragments thereof.

The invention also relates to an isolated nucleic acid, such as a recombinant DNA molecule or cloned gene, or a degenerate variant, mutant, analog or fragment thereof that encodes or competitively inhibits the activity of the polypeptide. It is about. Preferably, an isolated nucleic acid comprising a degenerate, variant, mutant, analog or fragment thereof has the sequence set forth in SEQ ID NOs: 12, 14-17, 19-22 or 23. In a further aspect of the invention, the complete DNA sequence of the recombinant DNA molecule or cloned gene thus determined can be operably linked with expression control sequences that can be introduced into a suitable host. Thus, the present invention encompasses single cell hosts transformed with cloned genes or recombinant DNA molecules comprising the DNA sequences encoding the present invention, and more particularly DNA sequences or fragments thereof determined from sequences as set forth above.

Antibodies to such isolated polypeptides include naturally occurring antibodies and recombinantly produced antibodies. These include the ability to modulate bacterial attachment, including but not limited to polyclonal and monoclonal antibodies produced by known genetic engineering techniques, as well as bispecific (chimeric) antibodies, and competition agents. May include antibodies containing other functional groups for use in diagnostics.

It is a further object of the present invention to provide a method for the treatment of mammals which modulates the amount or activity of said bacteria or subunits thereof for treating or avoiding the adverse consequences of invasive, spontaneous or idiopathic pathological conditions. The present invention provides a pharmaceutical composition for use in therapy comprising or based on the isolated polypeptide, subunit or binding partner thereof.

Finally, the present invention provides the use of pharmaceutical compositions, vaccines, and methods of diagnosis and treatment thereof.

1 shows choline binding protein A (CbpA) and recombinant truncated R1 (amino acids 16 to 321 from the N-terminus of CbpA as shown in FIG. 2) and R2 (N- of CbpA as shown in FIG. 2). About 16 amino acids to 444 amino acids from the end) are shown schematically. Domain A is about 153 amino acids to 321 amino acids from the N-terminus of the CbpA amino acid sequence as shown in FIG. 2; Domain B is from about 270 amino acids to 326 amino acids from the N-terminus of the CbpA amino acid sequence as shown in FIG. 2; Domain C is amino acids 327 to 433 from the N-terminus of the CbpA amino acid sequence as shown in FIG. 2.

2A to B compare the homology of various serotypes of amino acid sequences with nucleic acids in the N-terminal region of CbpA.

3 shows expression and purification of recombinant R1 and R2.

4 shows the results of passive protection in mice. Immune serum for recombinant R2 is lethal S. Mice were protected from the Neumoniae challenge.

Figure 5 shows the titer of anti-R2 antibody for R6x attachment to LNnT-HSA coated plates.

FIG. 6 shows the titers of anti-CbpA and adsorbed anti-CbpA antibodies on activity that blocks the attachment of pneumococcal to LNnT-HSA coated plates.

7 shows the results of active protection in mice. Immune serum for recombinant R1 is lethal S. Mice were protected from Pneumoniae challenge (challenge 560cfu serotype 6B).

The present invention relates to an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. The polypeptide is suitable for use in immunizing an animal against pneumococcal infection. These polypeptides or peptide fragments thereof are used as prophylactic vaccines against pneumococci and other bacteria using cross-reactive proteins when they are formulated with a suitable adjuvant.

The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. In one embodiment, such polypeptides comprise the amino acid sequence set forth in SEQ ID NO: 1, 3 to 5, 7, or 9 to 11, fragments, mutants, variants, analogs or derivatives thereof. In another embodiment, the polypeptide has amino acid KXXE (SEQ ID NO: 6).

The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation as shown in FIG. In one embodiment, such polypeptides have an amino acid sequence that is a conserved region as shown in FIG. 2. For example, such conserved regions include amino acid sequences 158-210; 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; And 430 to 447, but is not limited thereto. Figure 2 shows the homology of the various serotypes of the amino acid sequence with the nucleic acid of the N-terminal region of CbpA contemplated by the present invention.

In addition, the present invention provides an isolated polypeptide showing a tertiary structure comprising the amino acid sequence of an N-terminal choline binding protein A truncation having the amino acid set forth in SEQ ID NO: 24. In one embodiment, the polypeptide is an analog, fragment, mutant or variant thereof. Considered variants are shown in FIG. 2. The invention also provides amino acid sequences of N-terminal choline binding protein A truncations having position 16 to about position 475 amino acids of serotype 4 as shown in FIG. 2 or the corresponding amino acids of serotype 4 as shown in FIG. Provided are isolated polypeptides that exhibit tertiary structure. In one embodiment, the tertiary structure corresponds to the structure present in natural protein.

The method for preparing a polypeptide is, for example, as follows: The total length of choline binding protein A is cleaved with hydroxylamine, which hydroxyl group is responsible for the position of serotype R6x and serotype 4 for choline binding protein A. N-terminal choline binding protein A truncates are prepared by cleaving at 475 amino acid asparagine (N) or cleaving the corresponding amino acid of serotype R6x or serotype 4 in the different sera shown in FIG. 2. Alternative methods of preparing truncated choline binding protein A or fragments thereof and maintaining the native tertiary structure (ie, the tertiary structure of full-length choline binding protein A) are contemplated and are known to those skilled in the art. Since the polypeptide maintains its tertiary structure, the isolated polypeptide is suitable for use as an immunogen to immunize animals and humans against bacterial infections, preferably pneumococci.

The amino acid sequence of choline binding protein A (CbpA) serotype type 4 is as follows:

“Polypeptide R2” refers to a polypeptide whose sequence comprises the amino acid sequence of positions 16 to 444 of the N-terminal truncation of choline binding protein A (CbpA) serotype 4 (FIG. 1) as follows.

The DNA sequence encoding polypeptide R2 of the N-terminal truncation of choline binding protein A (CbpA) serotype type 4 is as follows:

Amino acid sequence of CbpA of serotype 4:

DNA sequence encoding amino acid sequence of CbpA of serotype 4:

“Polypeptide R1” refers to a polypeptide whose sequence comprises the amino acid sequence of positions 16 to 321 of an N-terminal truncation of choline binding protein A (CbpA) serotype 4 as follows.

The DNA sequence encoding polypeptide R1 is as follows:

"Polypeptide C / R2" refers to a polypeptide comprising a repeat region C in R2, wherein the sequence has an amino acid sequence from positions 327 to 433 of the N-terminus of choline binding protein A (CbpA) serotype 4 as follows: do.

DNA sequence of polypeptide C / R2:

"Polypeptide A / R2" means a polypeptide comprising a repeat region A in R2, wherein the sequence has the amino acid sequence of positions 153 to position 269 of the N-terminus of the choline binding protein A (CbpA) serotype 4 as follows: do.

As shown in FIG. 1, region A of polypeptide R2 is identical to region A in R1.

The DNA sequence encoding polypeptide A / R2 is as follows:

The type or position of one or more amino acid residues may include variants, eg, deletions containing less than all residues specified for the protein, substitutions in which one or more specified residues are replaced by other residues, and one or more amino acid residues of the polypeptide It is changed or modified to include the addition added at the terminal or intermediate position (see FIG. 2). Such molecules include the following: insertion of "preferred" codons expressed by a host other than the selected mammal, provision of sites for cleavage by restriction endonuclease enzymes, and additional aids in the construction of readily expressed vectors. Providing initiation, terminal or intermediate DNA sequences. Specifically, examples of amino acid substitutions of serotype 4 include, but are not limited to: E at position 154 is substituted with K; P at position 155 is substituted with L; G at position 156 is substituted with E; E at position 157 is substituted with K; K at position 181 is substituted with E; D at position 182 is substituted with A; R at position 187 is substituted with Y, H or L; I at position 194 is substituted with N; E at position 200 is substituted with D; E at position 202 is substituted with D; E at position 209 is substituted with K; K at position 212 is substituted with E; V at position 218 is substituted with L; V at position 220 is replaced by K or E; K at position 221 is substituted with E; N at position 223 is replaced by D or K; P at position 225 is substituted with S, T or R; D at position 227 is substituted with N; E at position 228 is substituted with K; Q at position 229 is substituted with E, G or D; K at position 230 is substituted with T; K at position 232 is replaced by N; E at position 235 is substituted with K; A at position 236 is substituted with E; E at position 237 is substituted with K; S at position 240 is substituted with N; K at position 241 is substituted with E; Q at position 242 is substituted with K; K at position 249 is substituted with E; K at position 250 is substituted with N; E at position 257 is substituted with Q or K; A at position 263 is substituted with L; K at position 264 is substituted with E; R at position 265 is substituted with N; R at position 266 is substituted with I; A at position 267 is substituted with K or V; D at position 258 is substituted with T; A at position 269 is substituted with D; A at position 291 is substituted with T, V, P, G or X; G at position 294 is substituted with G, A or E; V at position 295 is substituted with D or A; P at position 295 is substituted with L or F; L at position 299 is substituted with P or Q; P at position 328 is substituted with S; E at position 329 is substituted with G; E at position 340 is substituted with A; K at position 343 is substituted with E or D; E at position 347 is substituted with K; D at position 349 is substituted with A; R at position 354 is substituted with H; E at position 366 is substituted with D; E at position 375 is substituted with K; K at position 378 is substituted with E; E at position 390 is substituted with G; P at position 391 is substituted with S; N at position 393 is substituted with D; V at position 397 is substituted with I; And K at position 408 is substituted with Q.

"Polypeptide R2 serotype-R6x" refers to a polypeptide whose sequence comprises the amino acid sequence of position 16 to position 444 of the N-terminal truncation of choline binding protein A (CbpA) serotype R6x.

The DNA sequence encoding polypeptide R2 serotype R6x is as follows:

delete

Amino acid sequence of CbpA of serotype R6x:

delete

The DNA sequence encoding the amino acid sequence of CbpA of serotype R6x is as follows:

“Polypeptide R1 serotype R6x” refers to a polypeptide whose sequence comprises the amino acid sequence of positions 16 to 321 of the N-terminal truncation of choline binding protein A (CbpA) serotype R6x.

The DNA sequence encoding polypeptide R1 is as follows:

delete

“Polypeptide C / R2 serotype R6x” comprises a repeat region C in R2, wherein the sequence has the amino acid sequence of positions 327 to 433 of the N-terminal truncation of choline binding protein A (CbpA) serotype R6x Means a polypeptide.

The DNA sequence of polypeptide C / R2 serotype R6x is as follows:

“Polypeptide A / R2 serotype R6x” comprises a polypeptide comprising a repeat region A in R2, having an amino acid sequence at position 155 to position 265 at the N-terminus of choline binding protein A (CbpA) serotype R6X having the sequence Means a polypeptide.

The DNA sequence encoding polypeptide A / R2 serotype R6x is as follows:

delete

The present invention relates to an isolated polypeptide, wherein the isolated polypeptide consists of an amino acid sequence as set forth in SEQ ID NO: 22 or 23, including fragments, mutants, variants, analogs or derivatives thereof.

“Polypeptide B / R2” refers to a polypeptide comprising the amino acid sequence of positions 270 to 326 of an N-terminal truncation of choline binding protein A (CbpA) serotype 4 as shown in FIG. 2. "Polypeptide B / R2 serotype-R6x" refers to a polypeptide comprising the amino acid sequence of positions 264 to 326 of the N-terminal truncation of choline binding protein A (CbpA) serotype R6x as shown in FIG. The present invention contemplates a polypeptide having an amino acid sequence of regions A, B, C, A + B, B + C, A + C as shown in FIG. 1.

The present invention also provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation, wherein the polypeptide has the amino acid KXXE (SEQ ID NO: 6).

The present invention relates to a polypeptide comprising an amino acid sequence of an N-terminal choline binding protein A truncation, the amino acid sequence of which is shown in FIG. In one embodiment, the polypeptide has an amino acid sequence that is a conserved region as shown in FIG. 2. For example, conserved regions include, but are not limited to, amino acid sequences 158-172, 300-321, 331-339, 355-365, 367-374, 379-389, 409-427 and 430-447 Do not. 2 shows homologs of various serotypes of nucleic acid and amino acid sequences of the N-terminal region of CbpA contemplated by the present invention.

The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation, wherein the polypeptide has lectin activity and does not bind choline. In one embodiment, the polypeptide has an amino acid sequence as set forth in SEQ ID NOs: 1, 3 to 5, 7 or 9 to 11, including fragments, mutants, variants, homologs or derivatives thereof.

As used herein, "polypeptide with lectin activity" refers to a polypeptide, peptide or protein that is non-covalently bound to carbohydrates. As defined herein, "attach" means non-covalent binding of bacteria to human cells or secretions that are stable enough to withstand washing. "Binding to LNnT" as defined herein means binding to a lacto-N-neotetraose-coated substrate better than an albumin-control.

The present invention provides an isolated immunogenic polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. Immunogenic polypeptides are contemplated herein as having an amino acid sequence as set forth in SEQ ID NOs: 1, 3 to 5, 7 or 9 to 11, including fragments, mutants, variants, analogs or derivatives thereof. The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation as shown in FIG. 2. In one embodiment, the polypeptide has an amino acid sequence that is a conserved region as shown in FIG. 2.

The present invention relates to analogues of polypeptides comprising amino acid sequences as set forth above. The analog polypeptide may have an N-terminal methionine or N-terminal polyhistidine optionally attached to the N or COOH terminus of the polypeptide comprising an amino acid sequence.

In another aspect, the present invention relates to a peptide fragment of a polypeptide obtained from the proteolytic product of the polypeptide. In other embodiments, derivatives of a polypeptide have one or more chemical moieties attached to the polypeptide. In other embodiments, the chemical moiety is a water soluble polymer. In another embodiment, the chemical moiety is polyethylene glycol. In other embodiments, the chemical moiety is mono, di-, tri- or tetrapegylated. In other embodiments, the chemical moiety is N-terminal monopegylated.

The attachment of polyethylene glycol (PEG) to compounds is particularly useful because PEG is very low in mammals (Carpenter et al., 1971). For example, PEG adducts of adenosine deaminase have been approved in the United States for use in humans for the treatment of severe combined immunodeficiency. The second advantage provided by PEG binding is to effectively reduce the immunogenicity and antigenicity of the heterologous compound. For example, PEG adducts of human proteins can be useful for treating diseases in other mammalian species without the risk of developing a severe immune response. Compounds of the present invention may be delivered to microencapsulation devices to reduce or prevent host immune responses against the compound or against cells capable of producing the compound. Compounds of the invention can also be transported microencapsulated (eg, liposomes) in the membrane.

Many active forms of PEG have been described that are suitable for direct reaction with proteins. Useful PEG preparations for reaction with protein amino groups include active esters or carbonate derivatives of carboxylic acids, in particular leaving groups with N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2- Nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful agents for modifying protein free sulfhydryl groups. Similarly, PEG preparations containing amino hydrazine or hydrazide groups are useful for reaction with aldehydes produced by the periodate oxidation of carbohydrate groups of proteins.

In one embodiment, the amino acid residues of the polypeptides described herein are preferably the "L" isoform. In other embodiments, residues of the "D" isoform can replace any L-amino acid residue as long as the desired functionality of lectin activity is retained by the polypeptide. NH ₂ represents a free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. Abbreviations used herein follow standard polypeptide nomenclature . See J. Biol. Chem , 243: 3552-59 (1969).

It should be noted that all amino acid residue sequences are represented herein by formulas in which the left and right directions are the common direction from the amino terminus to the carboxy terminus. It should also be noted that '-(dash)' at the beginning or end of an amino acid residue sequence indicates a peptide bond to another sequence of one or more amino acid residues.

Synthetic polypeptides prepared using well known methods of solid phase, liquid phase or peptide condensation techniques or any combination thereof may comprise natural or unnatural amino acids. The amino acids used for peptide synthesis are described in Merrifield [1963, J. Am. Chem. Soc. 85: 2149-2154 is a standard Boc (N ^α -amino protected N ^α -t-butyloxycarbonyl) amino acid resin used in the standard deprotection, neutralization, coupling and washing protocols of the original solid phase method, or Carpino and Han, 1972, J. Org. Chem. 37: 3403-3409, which may be the base-labile N ^α -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acid first described by Accordingly, the polypeptides of the present invention may be formulated with combinations of D-amino acids, D- and L-amino acids to convey particular properties and various "designer" amino acids (e.g., β-methylamino acids, Cα-methylamino acids and Nα-methylamino acids, etc.). ) May be included. Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine and norleucine for leucine or isoleucine. In addition, by assigning specific amino acids to specific coupling steps, α-helices, β-turns, β-sheets, γ-turns and cyclic peptides can be generated.

In one embodiment of the invention, the peptide may comprise at the C-terminus a particular amino acid that inserts a CO ₂ H or CONH ₂ side chain to simulate free glycine or glycine amide groups. Alternatively, D or L amino acid analogs having side chains composed of linkers or linkers to beads may also be considered as such particular residues. In one embodiment, the pseudo-free C-terminal residue is in D or L optical arrangement, and in other embodiments racemic mixtures of D and L-isomers may be used.

In another embodiment, pyroglutamate may be included as the N-terminal residue of the peptide. By limiting only 50% of the peptide to substitution on a given bead with N-terminal pyroglutamate, pyroglutamate cannot follow the sequence by Edman degradation, but sufficient non-pyroglutamate on the beads for sequencing The peptide will remain. Those skilled in the art will readily recognize that such techniques can be used to sequence any peptide that inserts residues resistant to Edman degradation at the N-terminus. Other methods of specifying individual peptides exhibiting the desired activity are described in detail below. The specific activity of a peptide comprising a blocked N-terminal group, eg, pyroglutamate, does not block a fully blocked peptide (100%) when a particular N-terminal group is present within 50% of the peptide. It can be shown easily by comparing with the peptide (0%).

The present invention also seeks to use peptido mimetic bonds, such as peptido mimetics, ester bonds, to prepare peptides with more clearly specified structural properties and to produce peptides with novel properties. In other embodiments, the peptides can be formed by inserting a reduced peptide bond, ie, R ₁ -CH ₂ -NH-R ₂ , wherein R ₁ and R ₂ are amino acid residues or sequences. Reducing peptide bonds may be introduced into the dipeptide subunit. Such molecules are resistant to hydrolysis of peptide bonds, for example protease activity. This peptide confers a special function and activity to the ligand, such as prolonging its half-life in vivo, due to its destruction of metabolism or resistance to protease activity. Moreover, it is well known that in certain systems constrained peptides exhibit vaporized functional activity (Hurby, 1982, Life Sciences 31: 189-199; Hruby et al., 1990, Biochem J. 268: 249-262. The present invention provides a method for generating constrained peptides that insert random sequences at all other positions.

Constrained, cyclic, or rigidized peptides are cross-linked such that amino acids or amino acid analogs are inserted at two or more positions in the peptide's sequence to form crosslinks after processing to form a crosslinked bond. It can be prepared synthetically as long as it provides a bondable chemical group. Cyclization would be preferred if an amino acid induced by rotation is inserted. Examples of amino acids capable of crosslinking peptides include cysteine forming disulfide bonds, aspartic acid forming lactones or lactase, and γ-carboxyl-glutamic acid (Gla) (Bachem) that chelates transition metals to form crosslinks. And chelate factors such as Protected γ-carboxyl-glutamic acid is described by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun. 94: 1128-1132 can be prepared by modifying the synthesis described. Peptides whose peptide sequences comprise two or more crosslinkable amino acids can be treated, for example, by the dissolution of cysteine residues to form disulfides or by the addition of metal ions to form chelates, resulting in crosslinking of the peptides. To form constrained, cyclic, or rigid peptides.

The present invention provides a method by which crosslinking can be made structurally. For example, different protection groups can be used when four cysteine residues are inserted into a peptide sequence. Hiskey, 1981, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross and Meienhofer, eds., Academic Press: New York, pp. 137-167; Ponsanti et al., 1990, Tetrahedron 46: 8255-8266). The first pair of cysteines can be deprotected and oxidized and the second set can be deprotected and oxidized. In this way certain disulfide crosslinks can be formed. Alternatively, a pair of cysteines and a pair of collating amino acid analogs can be inserted so that the crosslinks can have different chemical properties.

The following non-conventional amino acids can be inserted into the peptide to introduce specific structural motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991, J) Am. Chem. Soc. 113: 2275-2283; (2S, 3S) -Methyl-phenylalanine, (2S, 3R) -methyl-phenylalanine, (2R, 3S) -methyl-phenylalanine and (2R, 3R) -methyl-phenylalanine, see Kazmierski and Hruby, 1991, Tetrahedron Lett .]; 2-aminotetrahydronaphthalene-2-carboxylic acid [Landis, 1989, Ph.D. Thesis, University of Arizona; Hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Miyake et al., 1989, J. Takeda Res. Labs. 43: 53-76; β-carboline (D and L) [Kazmierski, 1988, Ph.D. Thesis, University of Arizona; HIC (histidine isoquinoline carboxylic acid) [See Zechel et al., 1991, Int. J. Pep. Protein Res. 43] and HIC (histidine cyclic urea) from Dharanipragada.

The following amino acid analogs and peptidomimetics can be inserted into peptides to induce or assist with specific secondary structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid); β-turn inducing dipeptide analogs [Kemp et al., 1985, J. Org. Chem. 50: 5834-5838; β-sheet derived analogs [Kemp et al., 1988, Tetrahedron Lett. 29: 5081-5082; β-turn inducing analogs [Kemp et al., 1988, Tetrahedron Lett. 29: 5057-5060; α-helix inducing analogs [Kemp et al., 1988, Tetrahedron Lett. 29: 4935-4938; γ-turn inducing analogues, see Kemp et al., 1989, J. Org. Chem. 54: 109: 115 and analogues provided in Nagai and Sato, 1985, Tetrahededron Lett 26: 647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p.1687; In addition, Gly-Ala-rotating analogs (Kahn et al., 1989, Tetrahedron Lett. 30: 2317; Amide-linked allotropes (Jones et al., 1988, Tetrahedron Lett. 29: 3853-3856; Tretrazole [Zabrocki et al., 1988, J. Am. Chem. Soc. 110: 5875-5880; DTC [Samanen et al., 1990, Int. J. Protein Pep. Res. 35: 501: 509; And Olson et al., 1990, J. Am. Chem. Sci. 112: 323-333 and Garvey et al., 1990, J. Org. Chem. 56: 436. Form-limited mimetics of beta turns and beta bulbs and peptides containing them are disclosed in US Pat. No. 5,440,013, filed by Kahn et al. On August 8, 1995.

The invention also provides for the modification or derivatization of a polypeptide or peptide of the invention. Modifications of peptides are well known to those skilled in the art and include phosphorylation, carboxymethylation and acylation. The modification can be made by chemical or enzymatic means. In other embodiments, glycosylated or fatty acylated peptide derivatives can be prepared. The preparation of glycosylated or fatty acylated peptides is well known in the art. Fatty acyl peptide derivatives may also be prepared. For example, without limitation, free amino groups (N-terminus or lysyl) may be acylated, for example myristoylated. In another embodiment, an amino acid comprising an aliphatic side chain of structure (CH ₂ ) _n CH ₃ may be inserted into the peptide. These peptide fatty acid conjugates and other peptide fatty acid conjugates suitable for use in the present invention are described in British Patent No. 8809162.4, International Patent Application PCT / AU89 / 00166 and Reference 5, supra.

Mutations in the nucleic acid encoding the polypeptide can be made to transform a particular codon into a codon encoding a different amino acid. Such mutations are generally made with almost no possible change in nucleotides. Substitution of this class of mutations is accomplished by a non-conservative manner of the amino acids in the resulting protein (ie, changing the codon from an amino acid belonging to an amino acid group with a particular size and characteristic to an amino acid belonging to another group), or It can be altered in a conservative manner (ie, by changing the codon from an amino acid belonging to an amino acid group having a particular size and characteristic) to an amino acid belonging to the same group. Such conservative changes generally cause the resulting protein to change less in structure and function. Non-conservative changes further alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences that contain conservative changes that do not significantly alter the binding properties or activity of the resulting protein. Substituents for amino acids in the sequence may be selected from the kinds belonging to other kinds of members. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine and the like. Amino acids having aromatic rings include phenylalanine, tryptophan and trypsin. Polar natural amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is believed that such modifications will not affect the apparent molecular weights measured by polyacrylamide gel electrophoresis or isoelectric point method.

Particularly preferred substituents are:

Lys substituting Arg, i.e., a substituent capable of retaining a positive charge;

Glu, which substitutes for Asp, ie, a substituent capable of maintaining a negative charge;

Ser substituting Thr, a substituent wherein free -OH may be maintained; And

Substituting Asn Gln, a substitution with a glass NH ₂ it can be maintained.

delete

Synthetic DNA sequences allow convenient construction of genes expressing analogs or “muteins”. General methods for site-specific insertion of non-natural amino acids into proteins are described in Noren, et al. Science, 244: 182-188 (April 1989). This method can be used to construct analogs of non-natural amino acids.

According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the art can be used. Such techniques are described in detail in the literature. See Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M. ed. (1994); "Cell Biology: A Laboratory Handbook" Volumes I-III J.E. Celis, ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan, J.E., ed. (1994); "Oligonucleotide Synthesis" M.J. Gait ed. 1984; "Nucleic Acid Hybridization" B.D. Hames & S.J. Higgins eds. (1985); "Trasncription And Translation" B.D. Hames & S.J. Higgins, eds. (1984); "Animal Cell Culture" R.I. Freshney, ed. (1986); "Immobilized Cells And Enzymes" IRL Press, (1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984)].

In further embodiments, pyroglutamate may be included as the N-terminal residue of the peptide. By limiting only 50% of the peptide to substitution on a given bead with N-terminal pyroglutamate, pyroglutamate cannot follow the sequence by Edman degradation, but sufficient non-pyroglutamate peptide remains on the bead for sequencing something to do. Those skilled in the art will readily recognize that this technique can be used to sequence any peptide that incorporates residues resistant to Edman degradation at the N-terminus. Other methods of specifying individual peptides exhibiting the desired activity are described in detail below. If a particular N-terminal group is present within 50% of the peptide, the specific activity of the peptide comprising a blocked N-terminal group (eg pyroglutamate) is completely (100%) with the unblocked (0%) peptide. It is easily illustrated by comparing the activity of blocked peptides.

Chemical residues for derivatization

Suitable chemical moieties for derivatization may be selected from water soluble polymers. The polymer selected is water soluble so that the components to which it is attached do not precipitate in an aqueous environment (eg, a physiological environment). Preferably, in the therapeutic use of the terminal product preparation, the polymer may be pharmaceutically acceptable. Those skilled in the art will be able to use the polymer / component conjugates therapeutically and will be able to select the desired polymer in view of the desired dosage, cycle time, resistance to proteolysis and other considerations. For the component (s) in question, these can be identified using the assays provided herein.

The water-soluble polymer is, for example, polyethylene glycol, copolymer of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1, 3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, prolylpropylene oxide / Ethylene oxide copolymers, polyoxyethylated polyols and polyvinyl alcohols. Polyethylene glycol propionaldehyde may have advantages in manufacturing because of its stability in water.

The polymer may be of specific molecular weight and may be branched or unbranched. For polyethylene glycols, the preferred molecular weight for ease of handling and preparation is about 2 to about 100 kDa (the term "about" indicates that some molecular weights are more or less than standard molecular weights in the production of polyethylene glycol). Other sizes may affect the desired therapeutic profile (e.g., desired duration of sustained release, effect, optionally, biological activity, ease of handling, degree or lack of antigenicity, and the effects of other known polyethylene glycols on therapeutic proteins). It can be used depending on.

The number of attached polymer molecules can vary and those skilled in the art will be able to assure the effect upon action. It may be a mono-derivative or may be several combinations of di-, tri-, tetra- or derivatives having the same or different chemical moieties (eg polymers such as polyethylene glycol of different weights). Because of their concentration in the reaction mixture, the ratio of polymer molecules to components or component molecules can vary. Generally. The optimum ratio (in excess of unreacted component (s) and the efficiency of the reaction in which no polymer is present) determines the desired degree of derivatization (eg mono-, di-, tri-, etc.), whether the polymer is branched or unbranched. It will be determined by factors such as molecular weight and reaction conditions of the polymer selected in the liver.

Polyethylene glycol molecules (or other chemical moieties) should be attached to the component (s) in view of the action of the protein or effect on the antigenic domain. Many methods of attachment useful to those skilled in the art are described, for example, in EP 0 401 384 (coupling PEG to G-CSF), Malik et al. See 1992, Exp. Hematol, 20: 1028-1035 (reporting PEGylation of GM-CSF using trisil chloride). For example, polyethylene glycol can be covalently linked through amino acid residues via reactive groups such as free amino or carboxyl groups. Activated polyethylene glycol molecules can be bound to reactive groups. Amino acid residues with free amino groups include lysine residues and terminal amino acid residues, and amino acid residues with free carboxyl groups include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. In addition, sulfhydryl groups can be used as reactive groups for attaching the polyethylene glycol molecule (s). Preferred for therapeutic purposes is attachment at the amino group, such as at the N-terminus or lysine group.

The present invention provides an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation. The present invention provides an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A truncation as shown in FIG. 2. In one embodiment, the nucleic acid is set forth in SEQ ID NOs: 12, 14-17 or 19-21, including fragments, mutants, variants, analogs or derivatives thereof. Nucleic acids are DNA, cDNA, genomic DNA, RNA. In addition, the isolated nucleic acid may be operably linked to a promoter of RNA transcription. It is believed that nucleic acids are used to competitively inhibit lectin activity.

A "vector" is a replicon (eg, plasmid, phage or cosmid), which is another DNA fragment that can be attached to replicate an attached fragment.

"DNA" refers to the polymer form of single- or double-stranded deoxyribonucleotides (adenine, guanine, thymine or cytosine). These terms only refer to the primary and secondary structures of the molecule and are not limited to other specific tertiary structures. Thus, the term includes double stranded DNA, including linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes, among others. When discussing the structure of a particular double stranded DNA molecule, the sequence is herein described according to a normal transformation that provides only the sequence in the 5 'to 3' direction along an untranscribed DNA chain (ie, a chain having a sequence homologous to mRAN). It may be described.

The DNA sequence is "operably linked" to the expression control sequence when the expression control sequence controls the transcription and translation of the DNA sequence. The term "operably linked" has a suitable initiation signal (eg ATG) in front of the DNA sequence to be expressed and is encoded by the correct reading frame and the DNA sequence to enable expression of the DNA sequence under the control of the expression control sequence. To maintain the production of the desired product. If the gene of interest to be inserted into a recombinant DNA molecule does not contain a suitable initiation signal, such an initiation signal may be inserted before the gene.

The present invention further provides a vector comprising the nucleic acid molecule described above. The promoter may be or be a bacterial, yeast, insect or mammalian promoter. The vector can also be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.

Many other vector backbones known in the art, useful for expression proteins, can be used. Such vectors include adenovirus (AV), adeno-associated virus (AAV), monkey virus 40 (SA40), cytomegalovirus (CMV), mouse breast cancer virus (MMTV), and molony rat leukemia. Viruses, DNA delivery systems, ie liposomes and expression plasmid delivery systems, but are not limited thereto. In addition, one class of vectors includes bovine papilloma virus, polyoma virus, baculo virus, retrovirus or Semliki Forest virus. Such vectors can be assembled from sequences that are commercially available or described by methods well known in the art.

The invention also provides a host vector system for producing a polypeptide comprising a vector of a suitable host cell. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells such as bacterial cells (including gram positive cells), yeast cells, fungal cells, insect cells and animal cells. Many mammalian cells include, but are not limited to, mouse fibroblast cells NIH 3T3, CHO cells, HeLa cells, Ltk cells, Cos cells, and the like.

A wide variety of host / expression vector combinations can be used to express the DNA sequences of the invention. Useful expression vectors can, for example, consist of segments of chromosomes, non-chromosomes and synthetic DNA sequences. Suitable vectors are derivatives of SV40 and known bacterial plasmids such as E. coli. Plasmids such as the E. coli plasmid col El, pCR1, pBR322, pMB9 and derivatives thereof, PR4; Phage DNAS, eg, a number of derivatives of phage?, Such as NM989 and other phage DNAs such as M13 and filamentary single stranded phage DNA; Yeast plasmids (eg 2μ plasmid) or derivatives thereof; Vectors useful in eukaryotic cells (eg, vectors useful in insect or mammalian cells); Vectors derived from the combination of plasmid and phage DNA (eg, plasmids modified to use phage DNA or other expression control sequences), and the like.

A wide range of any expression control sequence—a sequence that controls the expression of a DNA sequence operably linked thereto—can be used in such vectors to express the DNA sequence of the invention. Such useful expression control sequences are, for example, SV40, CMV, vaccinia, polyoma or adenovirus, lac system, trp system, TAC system, TRC system, LTR system, phage λ main operator and promoter, fd coat Regulatory regions of proteins, promoters for 3-phosphoglycerate kinases or other glycolytic enzymes, promoters of acid phosphatase (such as Pho 5), promoters of yeast α-crossing factors and expression of prokaryotic or eukaryotic cells or viruses thereof Other sequences known to control, and various combinations thereof.

A wide range of single cell host cells are also useful for expressing the DNA sequences of the present invention. Such hosts include well known eukaryotic and prokaryotic cells such as E. coli, Pseudomonas, Bacillus, Streptomyces; Fungi such as yeast; Animal cells such as CHO, R1.1, B-W and L-M cells; Renal cells of African Green Monkey (eg, COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (eg Sf9) and plant cells in human cells and tissue culture.

It is understood that all vectors, expression control sequences and hosts will work uniformly well in expressing the DNA sequences of the present invention. Not all hosts work uniformly well using the same expression system. However, one of ordinary skill in the art would be able to select suitable vectors, expression control sequences and hosts without experimentation inadequate to achieve the desired expression without departing from the scope of the present invention. For example, in selecting a vector, the host must be considered because the vector must act in the host. Vector copy number, ability to regulate copy number, and expression of other proteins encoded by the vector (eg antibiotic markers) will also be considered.

In selecting an expression control sequence, various factors will generally be considered. This includes, for example, the relative strength of the system, its controllability, and in particular its suitability with particular DNA sequences or genes to be expressed as potential secondary structures. Suitable single cell hosts are, for example, their suitability with a selected vector, their secretory properties, their ability to accurately fold proteins, their fermentation requirements and their toxicity to the host of the product encoded by the DNA sequence to be expressed, And ease of purification of the expression product.

The invention also provides a method of making a polypeptide comprising the step of growing the host vector system described above under conditions suitable for producing the polypeptide and recovering the polypeptide thus produced.

The invention also provides antibodies that can specifically recognize or bind an isolated polypeptide. The antibody may be monoclonal or polyclonal. In addition, antibodies can be labeled using detection markers that are radioactive, chromogenic, fluorescent or luminescent markers. The labeled antibody can be a polyclonal or monoclonal antibody. In one embodiment, the labeled antibody is a purified labeled antibody. Antibody labeling methods are well known in the art.

The term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies. In particular, the term “antibody” includes polyclonal or monoclonal antibodies and fractions thereof. The term “antibody” also includes chimeric antibodies and whole synthetic antibodies, and fractions thereof. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and Fab expression libraries.

Various methods known in the art can be used to prepare polyclonal antibodies or derivatives or analogs thereof to polypeptides. See Antibodies-A Laboratory Manual, Harlow and Lane, eds, Cold Spring Habor Laboratory Press: Cold Spring Harbor, Ne York, 1988]. To prepare antibodies, immunization is achieved by injecting various host animals, including but not limited to rabbit, mouse, rat, sheep, goat, etc., using truncated CbpA or derivatives thereof (eg, fractions or fusion proteins). You can. In one embodiment, the polypeptide may be conjugated to an immunogenic carrier such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants can be used to increase the immunogenic response depending on the host type.

To prepare monoclonal antibodies, fractions, analogs or derivatives thereof, the techniques provided for the production of antibody molecules by continuous cell lines in culture can be used. Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Habor Laboratory Press: Cold Spring Harbor, Ne York, 1988]. It was originally developed by Kohler and Milstein (1975, Nature 256: 495-497), hybridoma technology, trioma technology, human B-cell hybridoma technology [Kozbor et al. , 1983, Immunology Today 4:72] and EBV-hybridoma technology for preparing human monoclonal antibodies (Col et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.). , pp. 77-96, but is not limited to such. In a further aspect of the invention, monoclonal antibodies can be prepared in sterile animals using the latest technology (PCT / US90 / 02545). According to the present invention, human antibodies can be used and human hybridomas can be used (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 2026-2030] or transforming human B cells with in vitro EBV virus (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96]. Indeed, according to the present invention, techniques developed for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules specific for polypeptides with genes from human antibody molecules of appropriate biological activity. et al., 1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454 can be used; Such antibodies are within the scope of the present invention. Such human or humanized chimeric antibodies are preferred for use in treating human diseases or conditions (described below), which are heterologous for human or humanized antibodies to elicit an immune response, especially an allergic response itself. This is because they are less suitable than antibodies. Additional aspects of the invention include Fab expression libraries [Huse et al., 1989, Science 246; 1275-1281 enables the rapid and easy identification of monoclonal Fab fragments with the desired specificity for polypeptides or derivatives or analogs thereof using the techniques described.

Antibody fragments containing the idiotype of an antibody molecule can be produced by known techniques. For example, such fragments include, but are not limited to: F (ab ') ₂ fragments that can be prepared by pepsin digestion of antibody molecules; Fab 'fragments that can be produced by reducing the disulfide bridges of F (ab') ₂ fragments, and Fab fragments that can be produced by treating antibody molecules with papain and a reducing agent.

In the preparation of antibodies, screening for preferred antibodies can be carried out using techniques known in the art, such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion sedimentation reactions, Immunodiffusion assay, immunoassay in situ (e.g. using colloidal gold, enzyme or radioisotope labeling), western blot, sedimentation reaction, agglutination assay (e.g. gel aggregation assay, hemagglutination assay), complement Fixed assays, immunofluorescence assays, Protein A assays and immunoelectrophoresis, etc.]. In one embodiment, antibody binding is detected by detecting a label in the primary antibody. In another embodiment, the primary antibody is detected by binding the secondary antibody or detecting a reagent for the primary antibody. In another embodiment, the secondary antibody is labeled. Many means are known for detecting binding in immunoassays and are within the scope of the present invention.

Antibodies can be labeled in vitro using, for example, labels (eg, enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidal gold, latex particles and chemiluminescent agents). Alternatively, the antibody may be used in vivo for detection, for example, radioisotopes (preferably technetium or iodine); Magnetic resonance transfer agents such as gadolinium and magnesium; Or by using a radiopaque agent.

The most commonly used labels for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and the like. Many fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramin, Texas red, AMCA blue and lucifer yellow. Particular detection agents are anti-rabbit antibodies prepared in goats and bound to fluorescein via isothiocyanates. Polypeptides are also labeled using radioactive elements or enzymes. Radioactive labels can be detected by any counting process currently used. Preferred isotopes can be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re.

Enzyme labels are likewise useful and can be detected by colorimetry, spectrophotometry, fluorescence spectrophotometry, amperage or gas measurement methods currently used. Enzymes are conjugated to selected particles by reaction with bridge molecules such as carbodiimide, diisocyanate, glutaraldehyde and the like. Many enzymes that can be used in this process are known and can be used. Peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase and peroxidase and alkaline phosphatase are preferred. In US Pat. Nos. 3,654,090, 3,850,752 and 4,016,043, reference is made to examples of separate labeling materials and methods.

In another aspect of the present invention, conventional test kits suitable for use by a medical practitioner can be prepared to determine the presence or absence of a given binding activity or a given binding activity capacity to a suspected target cell. In accordance with the test techniques discussed above, one group of such kits may, of course, depend upon one or more labeled polypeptides or their binding partners, eg, according to the method selected (eg, “competitive”, “sandwich”, “DASP”, etc.). For example, it will contain antibody specificity and use for it. Such kits may also contain ambient reagents (eg, buffers, stabilizers, etc.).

Thus, test kits can be prepared to demonstrate the presence or capacity of cells for certain bacterial binding activities, including:

(a) a predetermined amount of one or more labeled immunochemically reactive components obtained by direct or indirect binding to the presence of a polypeptide or specific binding partner thereto,

(b) other reagents and

(c) Instructions for using the kit above.

The present invention provides antagonists or blockers including but not limited to peptide fragments, mimetics, nucleic acid molecules, ribozymes, polypeptides, small molecules, carbohydrate molecules, monosaccharides, oligosaccharides or antibodies. In addition, reagents that competitively block or inhibit pneumococcal bacteria are contemplated by the present invention. The present invention provides reagents comprising proteins that inhibit inorganic compounds, nucleic acid molecules, oligonucleotides, organic compounds, peptides, peptidomimetic compounds or polypeptides.

The present invention provides a vaccine comprising a polypeptide having the amino acid sequence set forth in any of SEQ ID NOs: 1, 3 to 7, 9 to 11, 22 and 23 and a pharmaceutically acceptable adjuvant or carrier. The polypeptide may comprise the amino acid sequence of the N-terminal choline binding protein A truncation shown in FIG. 2. The present invention provides a vaccine comprising a polypeptide having an amino acid sequence comprising the conserved region shown in FIG. 2 and a pharmaceutically acceptable adjuvant or carrier. For example, the conserved region may comprise amino acid sequences 158-172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409-427 and 430-447. The present invention provides a vaccine comprising an isolated nucleic acid encoding a polypeptide and a pharmaceutically acceptable adjuvant or carrier.

Active immunity against Gram-positive bacteria, in particular pneumococcus, can be induced by immunization (vaccination) using immunogenic amounts of polypeptides or peptide derivatives or fragments and adjuvants, wherein the polypeptides or antigen derivatives thereof or The fragment is the antigenic component of the vaccine.

Polypeptides, derivatives thereof or fragments thereof of the invention can be prepared in admixture with an adjuvant to prepare a vaccine. Preferably, the derivative or fragment thereof thereof used as the antigenic component of the vaccine is an adhesion. More preferably, the polypeptide or peptide derivative or fragment thereof used as the antigenic component of the vaccine is an antigen common to all or a plurality of strains of Gram-positive bacteria or antigens common to a class of closely related bacteria. Most preferably, the antigenic component of the vaccine is an attachment site that is a common antigen.

The vector containing the nucleic acid-based vaccine of the present invention may be prepared by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion). ), By the use of gene guns or by DNA vector transporters (Wu et al., 1992, J. Biol. Chem. 267: 963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990] can be introduced into the desired host.

The vaccine can be administered by any parenteral route, including but not limited to intramuscular, intraperitoneal, intravenous, and the like. Preferably, since the desired outcome of vaccination is to describe the immune response to the antigen, it is preferred by direct administration to pathogenic organisms or indirectly by selection or targeting of viral vectors to lymphoid tissue (eg lymph nodules or spleen). Do. Because immune cells continue to replicate, they are ideal targets for retroviral vector-based nucleic acid vaccines, since retroviruses require replicating cells.

Passive immunization is achieved by administering an antiserum, polyclonal antibody, or neutralizing monoclonal antibody to a polypeptide of the invention to a patient suspected of being infected with Gram-positive bacteria, preferably Streptococcus, and more preferably pneumococci. It may be provided to. Passive immunity is not protected for a long time, but may be a useful means for treating bacterial infections in vaccinated individuals. Passive immunity is particularly important for the treatment of antibiotic resistant strains of Gram-positive bacteria, since no other therapy is available. Preferably, the antibody administered for passive immunotherapy is an autoantibody. For example, if the individual is a human, preferably the antibody is of human origin or is "humanized" to minimize the possibility of an immune response to the antibody. Administration of the active or passive vaccines, or attachments of the invention, can be used to protect animal subjects from infection of Gram-positive bacteria, preferably Streptococci, more preferably pneumococci.

The present invention provides a pharmaceutical composition comprising an amount of the described polypeptide and a pharmaceutically acceptable carrier or diluent.

For example, such pharmaceutical compositions for preventing pneumococcal adhesion to mucosal surfaces may include antibodies against lectin domains and / or antibodies against soluble excess lectin domain proteins. Blocking adhesion by any mechanism blocks the early stage of infection, reducing colony formation. This, in turn, reduces human-borne transmission and prevents the development of symptomatic disease.

The present invention provides a method of inducing an immune response in an individual infected with or exposed to pneumococcal, comprising administering to the individual an amount of a pharmaceutical composition.

The present invention provides a method for preventing infection by pneumococcal in a subject comprising administering to the subject a certain amount of a pharmaceutical composition in an amount effective to prevent pneumococcal bacterial attachment. do.

The present invention provides a method for preventing infection by pneumococcal in a subject comprising administering to the subject a predetermined amount of a pharmaceutical composition comprising an antibody and a pharmaceutically acceptable carrier or diluent. To provide.

The present invention provides a method of treating a subject infected with or exposed to pneumococcal bacteria, comprising treating the subject by administering to the subject a therapeutically effective amount of a vaccine of the present invention.

The present invention is directed to or infected with pneumococcal bacteria, comprising inducing an immune response by administering to a subject an amount of a pharmaceutical composition comprising a polypeptide consisting of the amino acid sequences set forth in SEQ ID NO: 1, 3 to 5, 7 or 9 to 11. Provided are methods for inhibiting colony formation in host cells of an exposed individual. Therapeutic peptides that block colony formation are delivered by the respiratory mucosa. The pharmaceutical composition comprises a polypeptide consisting of the amino acid sequence shown in FIG.

As used herein, a "pharmaceutical composition" refers to a suitable diluent, preservative, solubilizer, emulsion, adjuvant and / or carrier useful for providing a therapeutic effect or benefit, for example, by preventing colony formation of pneumococci. Together means a therapeutically effective amount of a polypeptide product of the invention. As used herein, “therapeutically effective amount” refers to an amount that provides a therapeutic effect in a given condition and method of administration. Such compositions are liquid or lyophilized or dried formulations, which have varying buffer contents, pH and ionic concentrations (e.g. tris-HCl, acetate, phosphate), additives such as albumin or gelatin to prevent adsorption to the surface. , Detergents (eg Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (eg glycerol, polyethylene glycerol), antioxidants (eg ascorbic acid, sodium metabisulfite) Preservatives (e.g. thimerosal, benzyl alcohol, parabens), bulking substances or osmotic agents (e.g. lactose, mannitol), covalent bonds of polymers with proteins such as polyethylene glycol, complexation of metal ions , Or incorporation of a substance into or on a particulate preparation of a polymeric compound, such as polylactic acid, polyglycolic acid, hydrogel, or the like, or liposomes, microemulsions, micelles, single or multilayer vesicles, red blood It includes the incorporation of a ghost or spheroplast (spheroplast) up. Such compositions affect the physical state, solubility, stability, rate of in vivo release, and rate of in vivo removal of the therapeutic agent. The choice of composition will depend on the physical and chemical properties of the protein having therapeutic activity. For example, products derived from membrane bound active forms may require formulations containing detergents. Controlled or sustained release compositions include formulations in lipophilic reservoirs (eg, fatty acids, waxes, oils). In addition, active forms that are particulate compositions coated with a polymer, such as poloxamer or poloxamine, and that are coupled to an antibody directed against a tissue specific receptor, ligand or antigen, or coupled to a ligand of a tissue specific receptor, are also disclosed herein. Is understood by. In another embodiment of the compositions of the invention, microparticles are introduced that form protective coatings, protease inhibitors or penetration enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.

In addition, as used herein, “pharmaceutically acceptable carrier” is well known to those skilled in the art and includes 0.01 to 0.1 M, preferably 0.05 M of phosphate buffer or 0.8% saline, It is not limited. In addition, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or nonvolatile oils. Intravenous vehicles include fluid nutritional supplements, electrolyte supplements such as Ringer's dextrose, and the like. Preservatives and other additives may also be present and may be, for example, antibacterial agents, antioxidants, cholating agents, inert gases, and the like.

The term “adjuvant” refers to a compound or mixture that enhances an immune response to an antigen. Adjuvants can serve as tissue reservoirs that slowly release antigens and as lymphatic activators that nonspecifically enhance immune responses. Hood et al., Immunology, Second Ed., 1984, Benjamin / Cummings: Menlo Park, California, p. 384]. Often the major challenge of antigen alone in the absence of an adjuvant fails to elicit a humoral or cellular immune response. The adjuvant may be a complete Freund's adjuvant, an incomplete Freund's adjuvant, a saponin, a mineral gel (e.g. aluminum oxide), a surface active substance (e.g. lysolecithin, fluoronate polyol, polyanion, peptide, oil or hydrocarbon Emulsions), keyhole limpit hemocyanin, dinitrophenol and potentially useful human adjuvant (eg BCG (Basyl Calmette-Guerin)] and Corynebacterium parboom. Preferably, the adjuvant is pharmaceutically acceptable.

Controlled or sustained release compositions include lipophilic reservoirs (eg fatty acids, waxes, oils). In addition, a compound that is a particulate composition coated with a polymer such as poloxamer or poloxamine and that is coupled to a tissue-specific receptor, ligand or antibody directed against an antigen, or that is coupled to a ligand of a tissue-specific receptor It is understood by the present invention. Other embodiments of the compositions of the present invention incorporate particulate protective coatings, protease inhibitors or penetration enhancers for the administration of various routes including parenteral, pulmonary, nasal and oral.
When administered, compounds are often quickly removed from the mucosal surface or circulation and thus can elicit relatively short lifespan of pharmaceutical activity. As a result, often relatively large doses of bioactive compounds may be necessary to sustain therapeutic efficiency. Compounds modified by covalent attachment of water soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran polyvinyl alcohol, polyvinylpyrrolidone or polyproline are not modified correspondingly. It is known to exhibit substantially longer half-life in blood after intravenous injection than uncompounded compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987. Such modifications can also increase the solubility of the compound in aqueous solution, remove aggregates, improve the physicochemical stability of the compound and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity is achieved by administering the aforementioned polymer-chemical by-products at a less frequent or lower dosage than the unmodified chemicals.

Dosage. An effective amount can be from about 1 μg / kg to about 1000 mg / kg, but is not limited thereto. The dosage can be 10 mg / kg. Pharmaceutically acceptable composition forms include pharmaceutically acceptable carriers.

As mentioned above, the present invention includes pharmaceutical compositions comprising vectors, vaccines, polypeptides, nucleic acids and antibodies, anti-antibodies and agents for combating the pathogenic activity of pneumococci, such as attachment to host cells. It provides a therapeutic composition.

The preparation of therapeutic compositions containing the active ingredient is well understood in the art. Typically, such compositions are prepared as aerosols of polypeptides delivered to the nasopharynx, as injections, as liquid solutions or suspensions, but may also be prepared as solid forms suitable for preparation as solutions or suspensions which are liquid prior to injection. The formulations may also be emulsified. The therapeutically active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like or combinations thereof. In addition, if desired, the composition may contain small amounts of wetting or emulsifying agents, pH buffers, which enhance the effect of the active ingredient.

The active ingredient may be formulated into a therapeutic composition in the form of a neutralized pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts (formed from free amino groups of polypeptides or antibody molecules), which include, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. It is formed with an organic acid. Salts formed from free carboxylic acid groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. .

When at least about 75% by weight of the protein, DNA, vector (depending on the category of species A and B) in the composition is "A", "A" where "A" is a single protein, DNA molecule, vector Compositions, etc., are substantially free of "B" (where "B" is one or more contaminating proteins, DNA molecules, vectors, etc.). Preferably, "A" comprises at least about 90% by weight of the A + B species in the composition and most preferably at least about 99% by weight.

As used herein, a “therapeutically effective amount” is an amount sufficient to reduce the activity, action, and response of the host by at least about 15%, preferably at least 50%, more preferably at least 90%, most preferably clinically. As such, it means an amount sufficient to prevent significant deficiency. Alternatively, a therapeutically effective amount is sufficient to enhance a clinically significant condition in the host. In the present invention, deficiency in the response of the host is evidenced by the continuation or dispersion of the bacterial infection. Improvement of clinically significant status in a host includes reduction of bacterial load, clearing of bacteria from colonized strain cells, reduction of fever or inflammation associated with infection, or reduction of any symptoms associated with bacterial infection.

According to the invention, the components or components of the therapeutic compositions of the invention may be introduced parenterally, mucosa, for example oral, nasal, pulmonary, rectal or transdermal. Parenteral, such as intravenous injection, and, but not limited to, intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration are preferred. Oral or pulmonary delivery may be desirable to activate mucosal immunity, and mucosal immunity may be a particularly effective prophylactic since pneumococci generally form colonies in the nasal pharynx and pulmonary mucosa. The term “unit dose”, when used for the therapeutic compositions of the present invention, means physically discrete units suitable as single doses for humans, each unit having the desired treatment in association with the required diluent, ie, carrier or vehicle. It contains a predetermined amount of active substance calculated to produce the effect.

In another embodiment, the active compounds may be delivered as vesicles, in particular liposomes. Langer, Science 249: 1527-1533 (1990); Treat at al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (des.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., Pp. 317-327].

In another aspect, the therapeutic composition can be delivered to a controlled release system. For example, the polypeptide can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes or other modes of administration. In one embodiment, a pump may be used. See Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989). In another embodiment, polymeric materials can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability. Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105 (1989). In another embodiment, a controlled release system may require only fragments of the system dose located near the therapeutic target, ie the brain. Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Preferably, the controlled release device is introduced near an inappropriate immune activation or tumor site of an individual. Other controlled release systems are discussed in Langer, Langer, Science 249: 1527-1533 (1990).

The individual whose administration of the active ingredient mentioned above is an effective therapeutic diet for bacterial infection is preferably a human, but can be any animal. Thus, as will be readily appreciated by those skilled in the art, the methods and pharmaceutical compositions of the present invention may be applied to any animal, particularly livestock such as cats or dogs, including but not limited to cattle, horses, goats, sheep, and pigs. It is particularly suitable for administration to mammals, including such domestic animals, wildlife (wild or zoo animals), mice, rats, rabbits, goats, sheep, pigs, dogs, cats and the like.

In the methods and compositions of the invention, a therapeutically effective amount of the active ingredient is provided. The therapeutically effective amount can be determined based on patient characteristics (age, weight, sex, condition, complications, other diseases, etc.) by a healthcare practitioner as is well known in the art. In addition, as another route study is conducted, more specific information will be clarified about the dosage levels suitable for the treatment of various conditions of various patients, and those skilled in the art will consider the beneficiary's treatment history, age and general health. A suitable dose can be found. In general, the dose for intravenous injection or infusion may be less than that for intraperitoneal, intramuscular or other routes of administration. Dosing schedules may vary depending on the circulatory half-life and the formulation used. The composition is administered in a manner suitable for a therapeutically effective amount of a dosage form. The exact amount of active ingredient required to administer depends on the judgment of the practitioner and is personally specific. However, a suitable dose may be about 0.1 to 20, preferably about 0.5 to about 10, more preferably 1 to several mg of active ingredient per kg of body weight per day and depends on the route of administration. Methods suitable for initial administration and booster injection also vary, but are typified by repeated administration by injection or other administration at intervals of one hour or more after the initial administration. Alternatively, continuous intravenous infusion sufficient to maintain 10 nanomolar to 10 micromolar concentrations in the blood is contemplated.

Administration with other compounds: For the treatment of bacterial infections, the active compounds of the present invention may be administered without limitation (1) antibiotics, (2) soluble carbohydrate bacterial attachment inhibitors, (3) other small molecular weight bacterial attachment inhibitors, (4) Administered with one or more pharmaceutical compositions used to treat bacterial infections, including bacterial metabolism, transport or transformation inhibitors, (5) bacterial lysis stimulants or (6) antibacterial agents, antibodies or vaccines against other bacterial antigens can do. Other potential active ingredients include anti-inflammatory agents such as steroids and nonsteroidal anti-inflammatory drugs. It may be administered simultaneously (e.g., administration of a mixture of the active ingredient and antibiotic) or separately.

Thus, in certain embodiments, the therapeutic composition may further comprise an effective amount of the active ingredient and one or more of the following active ingredients: antibiotics, steroids, and the like. Exemplary formulations are as follows:

Intravenous Formulation I

Ingredient mg / ml

Celltaxim 250.0

Polypeptide 10.0

Dextrose USP 45.0

Sodium sulfite USP 3.2

Edetate Disodium USP 0.1

1.0ml water for injection

Intravenous Formulation II

Ingredient mg / ml

Ampicillin 250.0

Polypeptide 10.0

Sodium sulfite USP 3.2

Disodium Edetate USP 0.1

1.0ml water for injection

Intravenous Formulation III

Ingredient mg / ml

Gentamicin (charged as sulfate) 40.0

Polypeptide 10.0

Sodium sulfite USP 3.2

Disodium Edetate USP 0.1

1.0ml water for injection

Intravenous Formulation IV

Ingredient mg / ml

Polypeptide 10.0

Dextrose USP 45.0

Sodium sulfite USP 3.2

Edetate Disodium USP 0.1

1.0ml water for injection

Intravenous Formulation V

Ingredient mg / ml

Polypeptide Antagonist 5.0

Sodium sulfite USP 3.2

Disodium Edetate USP 0.1

1.0ml water for injection

Thus, in certain cases where an antibody against, or a ligand thereof, or an antibody against, the ligand is required to reduce or inhibit infection resulting from bacterial mediated binding of the host cell to the bacterium, the polypeptide is introduced into the bacterium and the host cell. Block the interaction of

Pulmonary delivery of the present polypeptide having lectin activity, which acts as an attachment inhibitor (or derivative thereof) of the present invention, is also contemplated. Adhesion inhibitors (or derivatives) can be delivered to the lungs of a mammal to inhibit the binding of bacteria, ie streptococci and preferably pneumococci, to host cells. Reports on the manufacture of proteins for other lung delivery are found in the prior art. Adjei et al. Pharmaceutical Research, 7: 565-569 (1990); Adjei et al., International Journal of Pharmaceutics, 63: 135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology, 13 (suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine, Vol. III, pp. 206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (α-1-proteinase); Oswein et al., “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (1990) (recombinant human growth hormone); Debs et al., J. Immunol. 140: 3482-3488 (1988) from interferon-γ and tumor necrosis factor alpha; Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor)]. Methods for pulmonary delivery of the drug and compositions therefor are described in US Pat. No. 5,451,569 (1995.9.19) to Wong et al.

All such devices require the use of a formulation suitable for the dispersion of the attachment inhibitor (or derivative). Typically, each formulation may contain suitable propellant materials in addition to conventional diluents, adjuvants and / or carriers that are specific to the type of device used and are particularly useful for treatment. In addition, the use of liposomes, microcapsules or microspheres or other types of carriers, including complexes, is also contemplated. Chemically modified attachment inhibitors may be prepared in different formulations depending on the type of chemical modification or the type of device used.

Formulations suitable for use with nebulizers, jets or ultrasounds typically include an attachment inhibitor (or derivative) dissolved in water at a concentration of about 0.1-25 mg of biologically active attachment inhibitor per ml of solution. The formulations may also include buffers and simple sugars (eg, for stabilizing inhibitors and controlling osmotic pressure). Nebulizer formulations may also contain surfactants to reduce or prevent surface induced aggregation of adhesion inhibitors caused by spraying of the solution upon aerosol formation.

Formulations for use with a metered dose inhalation device generally comprise a powder of fine powder containing an attachment inhibitor (or derivative) suspended in a propellant in combination with a surfactant. Propellants can be any materials conventionally used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoro Hydrocarbons including roethanol and 1,1,1,2-tetrafluoroethane, or mixtures thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant.

Liquid aerosol formulations contain adjuvant inhibitors and dispersants in physiologically acceptable diluents. Anhydrous powder aerosol formulations of the present invention consist of an attachment inhibitor and a dispersant in the form of a finely divided solid. In liquid or dry powder aerosol formulations, the formulation is aerosolized. That is, liquid or solid particles must also be crushed so that the aerosolized dose actually reaches the nasal or lung mucosa. The term “aerosol particles” is used herein to describe liquid or solid particles suitable for nasal or pulmonary administration, ie reaching the mucosa. Other considerations such as the configuration of the delivery device, the additional components in the formulation and the particle properties are important. Such aspects of pulmonary administration of the drug are known in the art and the construction of formulation treatments, aerosolization means and delivery devices is required by most of the general experimentation in the art. In certain embodiments, the mass mean dynamic diameter will be 5 micrometers or less to ensure that drug particles reach the alveoli. Wearley, L. L., Crit. Rev. in Ther. Drug Carrier Systems 8: 333 (1991).

Aerosol delivery systems such as pressurized measurand inhalation and dry powder inhalation are described in Newman, S.P., Hewman S.P., Aerosols and the Lung, Clarke, S.W. and Davia, D. editors, pp 197-22 and can be used in connection with the present invention.

In a further embodiment, the aerosol formulations of the invention, as described in detail below, may include therapeutic or pharmacologically active factors, in addition to, but not limited to, attachment inhibitors such as antibiotics, steroids, non-steroidal anti-inflammatory agents, and the like. have.

Liquid Aerosol Formulations. The present invention provides an aerosol formulation and the dosage is intended for use in the treatment of a subject suffering from a bacterial, eg streptococcal, in particular pneumococcal infection. In general, such formulations contain an attachment inhibitor in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include, but are not limited to, distilled water, saline, buffered saline, dextrose solution, and the like. In certain embodiments, the diluent or pharmaceutical formulation of the present invention that may be used in the present invention is phosphate buffered saline or buffered salt solutions or water, generally in the range of pH 7.0 to 8.0.

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Liquid aerosol formulations of the present invention may comprise, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers, surfactants and excipients. The formulation may comprise a carrier. Carriers are macromolecules that are soluble in the circulatory system and are physiologically acceptable, where physiologically acceptable means that the carrier can be injected into the patient as part of therapeutic eating. Preferably, the carrier is relatively stable in the circulation having an acceptable plasma half-life for removal. Such macromolecules include, but are not limited to, soybean lecithin, oleic acid and sorbitan trioleate, with sorbitan trioleate being preferred.

Formulations of embodiments of the present invention may also include other agents useful for pH maintenance, solution stabilization, or osmotic pressure control. Examples of such agents include, but are not limited to, salts such as sodium chloride or potassium chloride and carbohydrates such as glucose, galactose or mannose, and the like.

The present invention also contemplates liquid aerosol formulations comprising attachment inhibitors and other therapeutically effective drugs such as antibiotics, steroids, nonsteroidal anti-inflammatory agents, and the like.

Aerosol Anhydrous Powder Formulation. It is contemplated that the aerosol formulations can be prepared as anhydrous powder formulations, including the finely divided powder form of the attachment inhibitor and the dispersant.

Formulations for dispersing from a powder inhalation device include finely divided anhydrous powders containing attachment inhibitors (or derivatives), and also a bulking agent such as lactose, sorbitol, sucrose or mannitol to facilitate dispersion of the powder from the device. Amount, such as from 50 to 90% by weight of the formulation. Attachment inhibitors (or derivatives) should be most advantageously prepared in particulate form with an average particle size of less than 10 mm (or μ), most preferably from 0.5 to 5 mm, for the most effective delivery to terminal lungs. In another embodiment, anhydrous powder formulations may include finely divided anhydrous powders, dispersants and also bulking agents containing an attachment inhibitor. Bulking agents useful as mixtures with the formulations of the present invention include agents such as lactose, sorbitol, sucrose or mannitol in amounts that promote dispersal of the powder from the device.

The present invention also contemplates anhydrous powder formulations comprising attachment inhibitors and therapeutically effective drugs such as antibiotics, steroids, nonsteroidal anti-inflammatory agents, and the like.

In general, Remington's Pharmaceutical Sciences, 18th Ed. Oral solid dosage forms described in 1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89 are contemplated for use herein. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Liposomal or protenoid encapsulation can also be used to formulate the compositions of the present invention (eg, protenoid microspheres as reported in US Pat. No. 4,925,673). Liposomal encapsulation can be used and liposomes can be derived using a variety of polymers (eg US Pat. No. 5,013,556). A description of possible solid formulations for treatment can be found in Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979. Generally, formulations include inert ingredients and component (s) (or chemically modified forms thereof) that protect the gastric environment and release the biologically active substance into the intestine.

Also contemplated are oral formulations of the derived component (s). Component (s) may be chemically modified to be effective for oral delivery of derivatives. In general, the chemical modification contemplated is the attachment of one or more residues to the component molecule itself, which residues (a) inhibit proteolysis and (b) allow uptake into the bloodstream from the stomach or intestine. It is also desirable to increase the overall stability of the component (s) and to increase the circulation time in the body. Examples of such residues include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts "In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4: 185-189. Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-trioxocane. As mentioned above polyethylene glycol moieties are preferred for pharmaceutical use.

The release site of the component (or derivative) may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. One skilled in the art can use a formulation that does not dissolve in the stomach but releases the substance in the duodenum or other intestines. Preferably, the release avoids the deleterious effects of the gastric environment by protection of the protein (or derivative) or by releasing a biologically active substance outside the gastric environment, for example in the intestine.

It is essential to ensure complete gastrointestinal resistance of coatings that are impermeable above pH 5.0. Examples of more common inert ingredients used as enteric skin include cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit (Eudragit) ) L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and Shellac. Such coatings can be used as mixed films.

Coatings or coating mixtures may be used on tablets, but this is not intended as a protection against the stomach. This may include dragees, or coatings that make swallowing easier to swallow. Capsules may consist of hard shells (such as gelatin) for delivery of anhydrous therapeutics (eg powders), and soft gelatin shells may be used for the liquid form. The shell material of casein may be rich starch or other edible paper. In the case of pills, lozenges, molded tablets, or refined abrasive powders, wet mass techniques can be used.

Peptide therapeutic agents can be included in the formulation as fine multiparticles in the form of granules or pellets of about 1 mm particle size. The formulation of the substance for capsule administration may also be a powder, a lightly compressed plug or a tablet. Therapeutic agents may be prepared by compression.

Both colorants and flavoring agents may be included. For example, proteins (or derivatives) can be formulated (eg liposomes or microspheres encapsulation) and then further contained in refrigerated beverages containing edible products, such as colorants and flavoring agents.

The inert material may dilute or increase the volume of the therapeutic agent. These diluents may include carbohydrates, in particular mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercial diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicel.

Disintegrants may be added during formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include, but are not limited to, starches containing commercially available disintegrant Explotabs based on starch. Sodium starch glycolate, Amberlite, Sodium Carboxylmethylcellulose, Ultramilopectin, Sodium Alginate, Gelatin, Orange Peel, Acid Carboxymethyl Cellulose, Natural Sponges and Bentonite can all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as disintegrants and binders and they can include powdered gums such as agar, karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders can be used to combine therapeutic agents to form hard tablets and can include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Both polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic.

Antifriction agents may be included in the therapeutic formulation to prevent sticking during the formulation process. Lubricants can be used as layers between the therapeutic agent and the mold walls, which include but are not limited to stearic acid, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes including magnesium and calcium salts. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000 can be used.

Glidants can be added during formulation to enhance the flow properties of the drug and to aid rearrangement during compression. Glidants can include starch, talc, pyrogenic silica, and hydrated silicoaluminates.

Surfactants may be added as wetting agents to help the therapeutic agent degrade into the aqueous environment. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents may be used, including benzalkonium chloride or benzetomium chloride. Examples of potential nonionic detergents that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hardened castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60 , 65 and 80, sucrose fatty acid esters, methyl cellulose and carboxymethyl cellulose. These surfactants may be present alone or as a mixture in different proportions in the formulation of the protein or derivative.

Additives that can potentially enhance the uptake of polypeptides (or derivatives) are, for example, fatty acids oleic acid, linoleic acid and linolenic acid.

Lung delivery. The present invention also contemplates pulmonary delivery of polypeptides (or derivatives thereof). The polypeptide (or derivative thereof) is delivered to the lungs of a mammal during inhalation, covering the mucosal surface of the alveoli. This is included in the following references: Adjei et al., 1990, Pharmaceutical Research, 7: 565-569; Adjei et al., 1990, International Journal of Pharmaceutics, 63: 135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13 (suppl. 5): 143-146 (Endothelin-I); Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp 206-212 (aI-antitrypsin): Smith et al., 1989, J. Clin. Invest. 84: 1145-1146 (a-1-proteinases); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140: 3482-3488 (interferon-g and tumor necrosis factor alpha) and Platz et al., US Patent No. 5,284,656 (granulocyte colony stimulating factor)]. Methods and compositions for pulmonary delivery of drugs for systemic effects are described in US Pat. No. 5,451,569 to Wong et al., September 19, 1995.

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The use of various mechanical devices designed for pulmonary delivery of therapeutic agents is contemplated for carrying out the present invention, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Formulations suitable for use with a nebulizer (jet or ultrasonic) typically include a polypeptide (or derivative) dissolved in water at a concentration of about 0.1-25 mg of biologically active protein per ml of solution. The formulation may also include buffers and simple sugars (eg, for protein stabilization and osmotic pressure control). Nebulizer formulations may also contain a surfactant to prevent or reduce aggregation of surface induced proteins caused by spraying of the solution in preparing the aerosol.

Formulations for use as metered dose inhalers will generally comprise finely divided powders containing the polypeptide (or derivative) suspended in the propellant with the aid of a surfactant. Propellants can be used for this purpose with conventional materials, for example chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or trichlorofuluromethane, dichlorodifluoromethane, dichlorotetrafluoro Hydrocarbons including roethanol and 1,1,1,2-tetrafluoroethane or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may be useful as a surfactant.

Formulations for dispersing from powder inhalers will include finely divided anhydrous powders containing polypeptides (or derivatives), and may also contain bulk agents such as lactose, sorbitol, sucrose or mannitol to facilitate dispersion of the powder from the device ( For example 50 to 90% by weight of the formulation). Proteins (or derivatives) should be prepared in particulate form most advantageously below 10 mm (or microns), most preferably 0.5 to 5 mm, for the most efficient delivery to terminal lungs.

Intranasal delivery. Intranasal or nasal pharynx delivery of polypeptides (or derivatives) is also contemplated. Intranasal delivery allows the polypeptide to be passed directly onto the upper airway mucosa after administration of the therapeutic agent in the nose, without the need to deposit pulmonary products. Formulations for intranasal administration include dextran or cyclodextran.

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The following examples are presented to more fully illustrate preferred embodiments of the present invention. However, they cannot be regarded as limiting the broad scope of the invention.
EXAMPLE

Experiment detail

Example 1: Peptide Truncates of Choline Binding Protein A (CbpA)

A polypeptide comprising a truncated N-terminal fragment of CbpA (serum type 4) is generated. Full length CbpA is amplified using PCR primers SJ533 and SJ537, and primers are designed based on the derived N-terminal amino acid sequence of the CbpA polypeptide. 5 'forward primer SJ533 = 5' GGC GGA TCC ATG GA (A, G) AA (C, T) GA (A, G) GG 3 '. Both these degenerate primers BamHI and NcoI restriction sites and ATG start codons designed from the amino acid sequence XENEG are inserted. 3 'reverse primer SJ537 = 5' GCC GTC GAC TTA GTT TAC CCA TTC ACC ATT GGC 3 '. This primer inserts a natural stop codon from the SalI restriction site and CbpA for cloning purposes and is based on two types of 4 and R6x sequences.

PCR products are generated from genomic DNA using primers SJ533 and SJ537 amplified 30 cycles using a High Fidelity enzyme (Boehringer Mannheim) at annealing temperature 50 ° C. as a template. The resulting PCR product was purified using QIAquick PCR Purification Kit (Qiagen, Inc.), then digested with BamHI and SalI restriction enzymes, and digested with pQE30 expression vector (Qiagen, Inc.) with BamHI, XbaI and SmaI restriction enzymes. Clone).

Polypeptide R2:

A naturally occurring PvuII site at the end of the second repeat region, ie, the C region (nucleic acid 1228 of type 4 sequence) as shown in FIG. 1, is used to generate a truncated cbpA gene containing only the 5 'region of the gene. . To generate truncated clones, full-length clones PMI580 (type 4) or PMI581 (R6x) are digested using PvuII and XbaI, and the resulting fragments are bound to the expression vector, PQE30, and transformed into a suitable host. The protein is expressed and purified. In this case the stop codon used by the expression vector is downstream of the insert and the expressed protein is larger than the insert expected size due to the additional nucleic acid at the 5 'end of the cloning site. The amino acid sequence of polypeptide R2 is shown in SEQ ID NO: 1.

Polypeptide R1:

 Similar strategies are used to express only the first repeating region in the N-terminal region of CbpA, the A region of polypeptide R1. Here, a naturally occurring XmnI site between two amino repeats (nucleic acid 856 of type 4 sequence) is used. cbpA full length clone PMI580 is digested using XmnI and AatII. Vector pQE30 is digested using AatII and SmaI. Once again combine two constant size fragments. E. coli is transformed and clones are screened for inserts. One positive clone is selected and the recombinant protein is purified from the strains described above.

All polypeptides. Expression and purification using the Qia expression system (Qiagen) using E. coli and pQE30 vectors. The amino terminus of His tagged proteins is detected using Western analysis using host and anti-histine antibodies and protein specific antibodies.

Purification of R1 and R2

this. To prepare and purify recombinant proteins from E. coli, single colonies are selected from smeared bacteria containing recombinant plasmids and grown overnight in 6.0 ml of LB buffer with 50 ug / ml kanamycin and 100 ug / ml ampicillin at 37 ° C. This 6.0 ml culture is added to 1 L LB with this concentration of antibiotic. The culture is shaken at 37 ° C. until A ₆₀₀ = ˜0.400. 1 M IPTG is added to 1 L culture at a final concentration of 1 mM. The culture is then shaken at 37 ° C. for 3-4 hours. The 1 L culture is spun at 4000 rpm for 15 minutes in a model J-6B centrifuge. Discard the supernatant and store the pellet at -20 ° C.

1 L pellet is resuspended in 25 ml of 50 mM NaH ₂ PO ₄ , 10 mM Tris, 6M GuCl, 300 mM NaCl, pH 8.0 (buffer A). Rotate the mixture for 30 minutes at room temperature and set the discharge setting to 7 at 50% cuty cycle for 30 seconds twice using an ultrasonicator (VibraCell Sonicator (Sonics and Materials, Inc., Dnabury, CT)) using a micro tip. Ultrasonic grinding. The mixture is spun for 5 minutes at 10K on a JA20 rotator and the supernatant is removed and discarded. The supernatant is loaded into a 10 ml Talon (Clonetech, Palo Alto, CA) resin column attached to GradiFrac System (Pharmacia Biotech, Upsala Sweden). The column is equilibrated with 100 ml Buffer A and washed with 200 ml of additional buffer. The volume was based on a pH gradient using 100% 50 mM NaH ₂ PO ₄ , 8M urea, 20 mM MES, pH 6.0 (buffer B), running over a total volume of 100 ml as the final desired buffer. Protein eluted in ˜30% buffer B. Eluted peaks were collected and collected.

For refolding, dialysis is performed with 2 L volume of PBS at room temperature for about 3 hours using a dialysis tube of molecular weight 14,000. The sample is then dialyzed overnight in 2 L of PBS at 4 ° C. Further buffer exchange is performed by adding and re-spinning the stock solution spun in PBS using a Centrirep-30 spin column while the protein is concentrated. Protein concentration is determined by BCA protein analysis and purity is visualized using a Coomassie stained 4-20% SDS-PAGE gel (FIG. 3).

Example 2: Lectin Activity of Polypeptides R1 and R2

LNnt is a carbohydrate analog of the receptor for pneumococci present on eukaryotic cells. It is known that CbpA incomplete pneumococcal mutants cannot attach to eukaryotic cells or fixed sugars known as CbpA's attachment ligands. CbpA is a regulatory protein that can be divided into two regions: the N-terminal action domain and the C-terminal choline adhesion domain (FIG. 1). Polypeptides R1 and R2 are analyzed for biological activity to determine whether the activity of complete CbpA is present at a specific N terminus (eg R2) or fragment thereof (eg R1). It is determined whether only the N terminal domain (R2) has biological activity for lectin binding without the choline binding domain (CBD). This is tested using CbpA full length and polypeptide R2 (a truncated loss of the CBD region under the Pvu II site in the proline rich region).

Assays are performed in coat tissue culture wells using glycoconjugates known to be recognized by CbpA: LNnT-albumin, 3 'sialyl lactose-albumin and negative control albumin. The plate was then blocked with albumin and washed, followed by addition of full length CbpA polypeptide R2 or polypeptide R1 over 15 minutes (0.8 μg / ml) followed by addition of R6 pneumococcal labeled with fluorescein over 30 minutes without washing. After washing, the attached bacteria are counted visually.

The binding of R6 to carbohydrates without the addition of any peptides is a positive control and this corresponds in 100% (Table 1). In three separate experiments, CbpA full length or polypeptide R2 competitively inhibits pneumococcal binding to LNnT coated surfaces. Full length CbpA is inhibited by 71, 64% and 63% of the control group: Polypeptide R2 is inhibited by 65%, 53% and 74% of the control group. Equivalent activity with CbpA and R2 indicates that the choline binding domain is not required for LNnT lectin activity of CbpA and that R2 is a candidate LNnT lectin.

In contrast to binding to LNnT, attachment of pneumococcal to 3 'sialyl lactose is inhibited by R2 (79 and 101%) compared to full length CbpA (74 and 66%). This means that sialic acid cognitive activity is lost when CBD is lost. In contrast, R1 is active in sialic acid recognition and this property is shared with CbpA but completely hidden in R2. This indicates that folding the polypeptide in the functional domain is affected by the length of the composition and the polypeptide. Some sequence modifications are found in other strains (see FIG. 2). When the homology of the sequences between R1 and R2 is high, it is possible that both R1 and R2 are lectins that are required for lectin activity or that they have slightly different properties (± sialic acid).

Inhibition of R6 Pneumococci and Purified Glycoconjugates by Soluble CbpA Form LNnT 3 'sialyl lactose Cbp form Pneumococcal count (SD) per monolayer Control group (%) Number of pneumococci per monolayer % Control Per Well Peptide-free 3282 2421 (489) 2210 (350) 100% 2611 2115 (125) 100% Full length CbpA 2075 1740 (167) 1415 (50) 63, 71, 64 1933 1405 (240) 74 66 Polypeptide R2 2461 1288 (672) 1440 (530) 74, 53, 65 2639 1670 (420) 101 79 Polypeptide R1 3002 2245 (182) 2500 (310) 91, 92, 112 1052 1445 (526) 40 68

N is 3, measuring LNnt in each of three wells

N is 2 and measures the SiL of each 3 wells

Lectin activity associated with cell binding activity

Human cells contain surface molecules containing carbohydrates (glycoproteins and glycolipids) and bacteria attach to glycoconjugates by these carbohydrates, despite being very different protein and lipid backbones. Therefore, bacteria containing polypeptides having bacterial activity in vitro can attach to the surface of human cells. This direct connection between the activity of lectins and cell binding activity in vitro is known for pneumococci. LNnT, for example, competitively inhibits the attachment of pneumococcal to TNF activated by human lung cells in A549 and blocks the progression of pneumonia in vivo. To establish that the lectin activity of CbpA truncation reflects cell binding activity, it is tested for CbpA and truncation to inhibit pneumococcal binding to lung cells (Table 2). Full length CbpA and polypeptide R2 competitively inhibit the attachment of pneumococcal to lung cells with 58% and 63% of controls, respectively. Polypeptide R1 is not valid, indicating that L2 binding activity of R2 is required and describes the binding of pneumococcal to lung cells.

Coupling of R6 Pneumococci with TNF-activated Human Lung Cells A549 lung Cbp form Number of pneumococci per monolayer (average) Control% Peptide-free 697, 704, 674 702, 722 (700) 100% Full length CbpA 376, 431 (403) 58% Polypeptide R2 517, 693 314, 342, 350 (443) 63% Polypeptide R1 696, 642, 552 (630) 90%

N is 2 and tested in each 2 or 3 wells

R2-dependent LNnT lectin activity

The N-terminal region of CbpA contains two repeat units of each ˜110 amino acids (see FIG. 1, regions A and C in polypeptide R2). To study the relative contribution of the two domains to the bioactive R1, those containing only domain A are compared to R2 and full length CbpA. In the adhesion assay, polypeptide R1 did not inhibit adhesion to LNnT at all (91, 92 and 112% of wild type). However, polypeptide R1 has been shown to partially inhibit adhesion to sialyl lactose (68% and 40% of the control). This indicates that polypeptide R2 is required for LNnT lectin activity and that R2 is a candidate LNnT lectin domain. In contrast, R1 seems to be active in sialic acid recognition.

Antibody against N-terminal domain of CbpA blocking cell binding

When binding the N-terminal domain of CbpA to a cell, inhibition with N-terminal domain activity will prevent or prevent bacteria from attaching to the cell or purified glycoconjugate. One such inhibitory mechanism is an antibody.

Inhibition of binding of R6 pneumococci to LNnT coated surfaces by anti-CbpA R2 antibodies Pneumococcal count (SD) per monolayer Control% (Average) Pre-immune antibodies 198 (64). 88 (4) 100% Antibody Against Frustum R2 56 (11); 9 (2) 28%; 10%

LNnT coated wells are added for adhesion analysis after 5 × l of undiluted rabbit antibody + 2 × 10 ⁷ R6 × 5μl, pre-incubation at RT for 6 × 30 min. Perform two different experiments.

Antisera highlighted in the recombinant N-terminal domain of CbpA (R2) is tested for the ability of pneumococcal to inhibit adhesion to LNnT. Rabbit polyclonal anti CbpA antiserum (5 μl) and labeled bacteria 2 × 10 ⁷ 5 μl are incubated for 30 minutes at room temperature. The mixture is added dropwise onto fixed LNnT for 30 minutes and then washed three times with PBS to remove unbound bacteria. Bacteria bound to the plate are counted microscopically and the results are shown as mean and standard deviation from six wells. From the results shown in Table 3, it can be seen that antisera raised against the R2 polypeptide inhibits pneumococcal attachment to LNnT. FIG. 5 shows titer curves of preimmune versus anti-CpbA R2 antibodies for inhibition of adhesion to pneumococcal R6x to the illustrated receptor LNnT. Pneumococcal adhesion above 70% is blocked by anti-R2 in dilutions of 1: 100 and 1: 200. Dilution at 1: 400 also removes the activity that has a specific effect.

The CbpA used to prepare the antisera shown in Table 3 and FIG. 5 is increased for CbpA from serotype 4. The R6x pneumococcal strain used in the adhesion inhibition assay is derived from serotype 2. The ability of the antibody to block adhesion of heterologous serotype bacteria means that there is cross-protective activity across serotypes. This activity is highly desirable for effective vaccine immunogens.

Antibody Activity Against the Original Stereostructure of the CbpA N-terminus

As described in a paper by Rosenow et al., Choline affinity columns can be used to purify CbpA from natural hosts, ie pneumococci. Alternatively, the polyhistidine tag can be engineered at the end of the gene so that the transcribed protein is extended by several histidine residues. These residues facilitate the purification of the full length polypeptide by nickel affinity matrix purification, in contrast to allowing shorter truncations to maintain their natural tertiary structure. By these biochemical methods, pneumococcal as well as e. CbpA purified from E. coli or other host bacteria maintains its natural tertiary structure. CbpA, a naturally folding structure, produces antibodies when used as immunogens, which are potentially different from those derived by immunization with truncates that may otherwise be folded. Likewise, CbpA used as a therapeutic agent may have a different tertiary structure as a truncation, which may enhance its ability to block adhesion. Given these considerations, it may be advantageous to produce CbpA as a full-length protein that allows folding into its natural tertiary structure to biochemically cleave the C terminus (CBD). For example, treatment with hydroxylamine will cleave the CbpA at amino acid position 475 of choline binding protein A, serotypes R6x and serotype 4, separating the N- and C-terminals. This N terminal fragment is suitable as a therapeutic or immunogen.

Alternatively, native CbpA can be used as an immunogen and antiserum for active structures. Bioactive anti-N-terminal antibodies in this mixture can be enriched by removing the antibody to BD by adsorption. Such antibodies can be prepared by incubating 200 ul of serum for 1 hour at R1 with 1 × ^{10 8} CbpA defect-bacteria. Other choline binding proteins on this variant are removed from the antiserum by absorbing anti-CBD antibodies and then centrifugation and bacterial removal.

To demonstrate the bioactivity of the absorbed anti-CbpA antibodies, the ability of the absorbed antiserum to block the attachment of pneumococci to the model receptor LNnT was measured. R6x pneumococci were incubated with antiserum diluted 1: 600 and then added to wells coated with LNnT albumin.

Block adhesion of absorbed anti-CbpA antiserum Antiserum (1: 600) Number of pneumococci per well ± SD (% control) No antibodies 563 ± 11 (100%) Pre-immune antibodies 479 ± 11 (85%) Anti-CbpA antiserum 294 ± 72 (52%) Absorbed Anti-CbpA Antiserum for CBD Antibody Removal 175 ± 38 (31%)

This result means that the antibody to the N-terminal domain of Cbp / A in the natural three-dimensional structure strongly blocks the adhesion. This activity is greater than the truncated activity of FIG. 5 which is inactive at 1: 600 dilution. This activity of the absorbed anti CbpA antiserum is further demonstrated by the titer study in FIG. 5. The lowest attachment of pneumococcal type 4 to LNnT-coated wells is indicated by a triangle. Pre-incubation of pneumococcal and unabsorbed (square) or absorbed (rhombic) antisera at various dilutions meant a reduction in adhesion. The fact that the two antisera show a similar decrease in adhesion demonstrates that most of the blocking activity of the antibody against CbpA is at the N-terminus (ie, removal of the antibody to the choline binding domain by absorption) Does not reduce activity).

Example 3: Passive Protection with Anti-R2 Antiserum

Rabbit's immune antibody generation

Rabbit's immune antibodies against polypeptides R2 (CbpA truncated) and CbpA were produced in Covance (Denver, PA). After pre-immunization antibodies were collected, 250 ug of R2 in Complete Freund's Adjuvant, containing both amino acid terminal repeats (manufactured 483: 58 above), were immunized with New Zealand white rabbits. This rabbit was injected with 125 ug increments of R2 in Incomplete Freund's Adjuvant on day 21 and bled on day 31. Likewise, the second rabbit was immunized with purified CbpA.

Manual protection on mouse

100 ul of pre-immune serum in rabbit anti-R2 or sterile PBS (pre-immune and 31-day immune serum) diluted 1: 2 was passively immunized into C3H / HeJ mice (5 / group) intraperitoneally. One hour after serum administration, mice were inoculated with 1600 CFU Streptococcus pneumoniae serotype 6B (SP317 strain). Mouse survival was monitored for 14 days. 80% of mice immunized with rabbit immune sera against polypeptide R2 survived (FIG. 4). All mice immunized with preimmune rabbit serum died within 7 days.

This data shows that CbpA specific antibodies protect systemic pneumococcal infection. The data also show that choline-binding regions are not essential for protection because antibodies specific for polypeptide R2, truncated proteins without choline binding repeat retention, were also sufficient for protection. In addition, sera against CbpA of serotype 4 showed a protective effect against challenge with serotype 6B.

Example 4 Active Protection with Anti-R1 Antisera

CbpA truncated protein R1 (15 μg in 50 μl PBS and 50 μl complete Freund's adjuvant) was immunized by intraperitoneal administration of C3H / HeJ mice (10 mice / group). Ten groups of sham-immunized mice received PBS and adjuvant. Four weeks later, a second immunization was performed through the abdominal cavity with 15 μg protein and incomplete Freund's adjuvant (IFA) (gastroimmunization with PBS and IFA). At 3, 6 and 9 weeks, blood collection (half-track blood collection) was performed to analyze the immune response. At week 9, the ELISA endpoint anti-CbpA truncated titer of serum from 10 CbpA immunized mice was 4,096,000. No antibody was detected in serum from false-immunized mice. At 10 weeks the mice were challenged with 560 CFU Streptococcus Pneumoniae serotype 6B (SPSJ2P strain, provided by P. Pflin of St. Jude Children's Hospital in Memphis, Tennessee). Survival of the mice was monitored for 14 days. 80% of mice immunized with CbpA truncated protein R1 survived. All false-immunized mice died within 8 days (FIG. 7).

This data demonstrates that immunization with CbpA recombinant fragments leads to the production of specific antibodies that can protect against systemic pneumococcal infection and death. The data also show that choline-binding regions are not essential for protection because the immunogen used is truncated protein R1. The results also suggest that a single amino acid terminal repeat may be sufficient to induce a protective response. Cross-protective effects were also demonstrated, since recombinant pneumococcal proteins were generated based on serotype 4 DNA sequences and protective effects were observed after inoculation with serotype 6B isolates.

Example 5 Prevention of Colony Formation in Nasal Pharynx in Cub Rats

In vitro, the N-terminal domain of CbpA competitively inhibited pneumococcal adhesion. To demonstrate the therapeutic utility of peptides having this activity, colonies in nasal pharynx were measured after challenge with pneumococci after administering the truncated peptides to pups.

10 ul of PBS containing 0.8 μg polypeptide R2 or R1 or no protein was administered intranasally. After 15 minutes, type 3 pneumococcal (SIII strain) ( ¹⁰ ul containing 1 × 10 ⁵ cfer) was introduced intranasally. To determine the ability of polypeptides to competitively inhibit pneumococcal attachment and colony formation, the number of pneumococci found after nasal wash at 72 hours was quantified for each 4 animals per group. Rats receiving SIII alone showed 2200, 6500, 6900 and 8700 (average 6075) colonies per 10 ul. Animals treated with truncated R2 had the largest bacterial count reduction (3600, 3500, 2500, 2100) with an average of 2925 per 10 ul (48% of control). Animals treated with truncated R1 also showed reduced colony formation (5000, 4800, 3500, 1600) with an average of 3725 (61% of the control).

This experiment shows that administration of the peptides of the invention to the animals in the therapeutic study design have a protective effect against subsequent pneumococcal challenge.

Argument

As evidenced by the experiments, polypeptide R2 is a preferred composition as a vaccine formulation since 1) induces a protective antibody when administered as a vaccine; 2) When delivered as a peptide to the respiratory and / or nasopharyngeal receptors, the composition prevents the adhesion of pneumococci and thus is a preferred composition for the prevention and treatment of colony formation or invasive diseases. In addition, CbpA truncates act as lectins without CBD. Two carbohydrates are recognized: sialic acid by a peptide containing only a single N-terminal repeat (A) and LNnT (FIG. 1) by a peptide containing both N-terminal repeats (A and C). The truncated polypeptides R1 and R2, which contain N-terminal repeats, also exhibit lectin activity in cell culture assays.

Important properties of polypeptide R2 activity include: 1) complete correlation of polypeptide R2 and full-length CbpA bioactivity for recognition of purified glycoconjugate receptors in lung cells of animal models; . 2) Cross-protective properties between type 4 inducers and bacteria in in vitro assays with other serotypes (eg 6B and 2) are important for useful vaccine, prophylactic and therapeutic models.

While the invention has been illustrated and described herein by reference to various specific materials, methods, and examples, it is to be understood that the invention is not limited to these specific materials and combinations thereof and methods selected for this purpose. Various changes in these details will be appreciated by those skilled in the art. Likewise, any reference cited herein should be considered as described herein to the extent relevant to the disclosure of the present invention.

[Sequence list]

<110> Jude Children's Research Hospital; Medimmune, Inc.

<120> POLYPEPTIDE COMPRISING THE AMINO ACID OF AN N-TERMINAL CHOLINE BINDING

      PROTEIN A TRUNCATE, VACCINE DERIVED THEREFROM AND USES THEREOF

5-2000-045336-1; 5-2000-043861-8

<140> 60 / 080,878

<141> 1998-04-07

<140> 09 / 056,019

<141> 1998-04-07

<160> 39

<170> KOPATIN 1.5

Attach sequence list electronic file

Claims (53)

An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 4, 5, 7, 9, 10, 11, 22, 23, or 24. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 3, 4, 5, 22, or 24 and having 1 to 57 amino acid substitutions selected from the group consisting of the following amino acid substitutions: (a) lysine substitution at a position corresponding to amino acid residue 154 of SEQ ID NO: 40, (b) a leucine substitution at a position corresponding to amino acid residue 155 of SEQ ID NO: 40, (c) glutamic acid substitution at the position corresponding to amino acid residue 156 of SEQ ID NO: 40, (d) lysine substitution at the position corresponding to amino acid residue 157 of SEQ ID NO: 40, (e) glutamic acid substitution at the position corresponding to amino acid residue 181 of SEQ ID NO: 40, (f) alanine substitution at the position corresponding to amino acid residue 182 of SEQ ID NO: 40, (g) tryptophan, histidine or leucine substitution at a position corresponding to amino acid residue 187 of SEQ ID NO: 40, (h) asparagine substitution at a position corresponding to amino acid residue 194 of SEQ ID NO: 40, (i) aspartic acid substitution at a position corresponding to amino acid residue 200 of SEQ ID NO: 40, (j) aspartic acid substitution at a position corresponding to amino acid residue 202 of SEQ ID NO: 40, (k) lysine substitution at the position corresponding to amino acid residue 209 of SEQ ID NO: 40, (l) glutamic acid substitution at the position corresponding to amino acid residue 212 of SEQ ID NO: 40, (m) a leucine substitution at a position corresponding to amino acid residue 218 of SEQ ID NO: 40, (n) lysine or glutamic acid substitution at the position corresponding to amino acid residue 220 of SEQ ID NO: 40, (o) glutamic acid substitution at the position corresponding to amino acid residue 221 of SEQ ID NO: 40, (p) aspartic acid or lysine substitution at the position corresponding to amino acid residue 223 of SEQ ID NO: 40, (q) a serine, threonine or arginine substitution at the position corresponding to amino acid residue 225 of SEQ ID NO: 40, (r) asparagine substitution at a position corresponding to amino acid residue 227 of SEQ ID NO: 40, (s) lysine substitution at a position corresponding to amino acid residue 228 of SEQ ID NO: 40, (t) glutamic acid, glycine or aspartic acid substitution at a position corresponding to amino acid residue 229 of SEQ ID NO: 40, (u) threonine substitution at a position corresponding to amino acid residue 230 of SEQ ID NO: 40, (v) asparagine substitution at a position corresponding to amino acid residue 232 of SEQ ID NO: 40, (w) lysine substitution at a position corresponding to amino acid residue 235 of SEQ ID NO: 40, (x) glutamic acid substitution at the position corresponding to amino acid residue 236 of SEQ ID NO: 40, (y) lysine substitution at the position corresponding to amino acid residue 237 of SEQ ID NO: 40, (z) asparagine substitution at a position corresponding to amino acid residue 240 of SEQ ID NO: 40, (aa) glutamic acid substitution at the position corresponding to amino acid residue 241 of SEQ ID NO: 40, (bb) lysine substitution at a position corresponding to amino acid residue 242 of SEQ ID NO: 40, (cc) glutamic acid substitution at the position corresponding to amino acid residue 249 of SEQ ID NO: 40, (dd) asparagine substitution at a position corresponding to amino acid residue 250 of SEQ ID NO: 40, (ee) a glutamine or lysine substitution at a position corresponding to amino acid residue 257 of SEQ ID NO: 40, (ff) a leucine substitution at a position corresponding to amino acid residue 263 of SEQ ID NO: 40, (gg) glutamic acid substitution at a position corresponding to amino acid residue 264 of SEQ ID NO: 40, (hh) asparagine substitution at a position corresponding to amino acid residue 265 of SEQ ID NO: 40, (ii) an isoleucine substitution at a position corresponding to amino acid residue 266 of SEQ ID NO: 40, (jj) lysine or valine substitution at a position corresponding to amino acid residue 267 of SEQ ID NO: 40, (kk) threonine substitution at a position corresponding to amino acid residue 258 of SEQ ID NO: 40, (ll) aspartic acid substitution at a position corresponding to amino acid residue 269 of SEQ ID NO: 40, (mm) threonine, valine, proline or glycine substitution at the position corresponding to amino acid residue 291 of SEQ ID NO: 40, (nn) alanine or glutamic acid substitution at the position corresponding to amino acid residue 294 of SEQ ID NO: 40, (oo) aspartic acid or alanine substitution at the position corresponding to amino acid residue 295 of SEQ ID NO: 40, (pp) leucine or phenylalanine substitution at the position corresponding to amino acid residue 295 of SEQ ID NO: 40, (qq) a serine substitution at a position corresponding to amino acid residue 328 of SEQ ID NO: 40, (rr) glycine substitution at the position corresponding to amino acid residue 329 of SEQ ID NO: 40, (ss) alanine substitution at the position corresponding to amino acid residue 340 of SEQ ID NO: 40, (tt) glutamic acid or aspartic acid substitution at a position corresponding to amino acid residue 343 of SEQ ID NO: 40, (uu) lysine substitution at a position corresponding to amino acid residue 347 of SEQ ID NO: 40, (vv) an alanine substitution at the position corresponding to amino acid residue 349 of SEQ ID NO: 40, (ww) histidine substitution at a position corresponding to amino acid residue 354 of SEQ ID NO: 40, (xx) aspartic acid substitution at a position corresponding to amino acid residue 366 of SEQ ID NO: 40, (yy) lysine substitution at the position corresponding to amino acid residue 375 of SEQ ID NO: 40, (zz) glutamic acid substitution at a position corresponding to amino acid residue 378 of SEQ ID NO: 40, (aaa) glycine substitution at a position corresponding to amino acid residue 390 of SEQ ID NO: 40, (bbb) a serine substitution at a position corresponding to amino acid residue 391 of SEQ ID NO: 40, (ccc) aspartic acid substitution at a position corresponding to amino acid residue 393 of SEQ ID NO: 40, (ddd) isoleucine substitution at a position corresponding to amino acid residue 397 of SEQ ID NO: 40, and (eee) glutamine substitution at the position corresponding to amino acid residue 408 of SEQ ID NO: 40. delete delete An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, 9, 10, 11 or 23 and having 1 to 57 amino acid substitutions selected from the group consisting of the following amino acid substitutions: (a) lysine substitution at a position corresponding to amino acid residue 154 of SEQ ID NO: 40, (b) a leucine substitution at a position corresponding to amino acid residue 155 of SEQ ID NO: 40, (c) glutamic acid substitution at the position corresponding to amino acid residue 156 of SEQ ID NO: 40, (d) lysine substitution at the position corresponding to amino acid residue 157 of SEQ ID NO: 40, (e) glutamic acid substitution at the position corresponding to amino acid residue 181 of SEQ ID NO: 40, (f) alanine substitution at the position corresponding to amino acid residue 182 of SEQ ID NO: 40, (g) tryptophan, histidine or leucine substitution at a position corresponding to amino acid residue 187 of SEQ ID NO: 40, (h) asparagine substitution at a position corresponding to amino acid residue 194 of SEQ ID NO: 40, (i) aspartic acid substitution at a position corresponding to amino acid residue 200 of SEQ ID NO: 40, (j) aspartic acid substitution at a position corresponding to amino acid residue 202 of SEQ ID NO: 40, (k) lysine substitution at the position corresponding to amino acid residue 209 of SEQ ID NO: 40, (l) glutamic acid substitution at the position corresponding to amino acid residue 212 of SEQ ID NO: 40, (m) a leucine substitution at a position corresponding to amino acid residue 218 of SEQ ID NO: 40, (n) lysine or glutamic acid substitution at the position corresponding to amino acid residue 220 of SEQ ID NO: 40, (o) glutamic acid substitution at the position corresponding to amino acid residue 221 of SEQ ID NO: 40, (p) aspartic acid or lysine substitution at the position corresponding to amino acid residue 223 of SEQ ID NO: 40, (q) a serine, threonine or arginine substitution at the position corresponding to amino acid residue 225 of SEQ ID NO: 40, (r) asparagine substitution at a position corresponding to amino acid residue 227 of SEQ ID NO: 40, (s) lysine substitution at a position corresponding to amino acid residue 228 of SEQ ID NO: 40, (t) glutamic acid, glycine or aspartic acid substitution at a position corresponding to amino acid residue 229 of SEQ ID NO: 40, (u) threonine substitution at a position corresponding to amino acid residue 230 of SEQ ID NO: 40, (v) asparagine substitution at a position corresponding to amino acid residue 232 of SEQ ID NO: 40, (w) lysine substitution at a position corresponding to amino acid residue 235 of SEQ ID NO: 40, (x) glutamic acid substitution at the position corresponding to amino acid residue 236 of SEQ ID NO: 40, (y) lysine substitution at the position corresponding to amino acid residue 237 of SEQ ID NO: 40, (z) asparagine substitution at a position corresponding to amino acid residue 240 of SEQ ID NO: 40, (aa) glutamic acid substitution at the position corresponding to amino acid residue 241 of SEQ ID NO: 40, (bb) lysine substitution at a position corresponding to amino acid residue 242 of SEQ ID NO: 40, (cc) glutamic acid substitution at the position corresponding to amino acid residue 249 of SEQ ID NO: 40, (dd) asparagine substitution at a position corresponding to amino acid residue 250 of SEQ ID NO: 40, (ee) a glutamine or lysine substitution at a position corresponding to amino acid residue 257 of SEQ ID NO: 40, (ff) a leucine substitution at a position corresponding to amino acid residue 263 of SEQ ID NO: 40, (gg) glutamic acid substitution at a position corresponding to amino acid residue 264 of SEQ ID NO: 40, (hh) asparagine substitution at a position corresponding to amino acid residue 265 of SEQ ID NO: 40, (ii) an isoleucine substitution at a position corresponding to amino acid residue 266 of SEQ ID NO: 40, (jj) lysine or valine substitution at a position corresponding to amino acid residue 267 of SEQ ID NO: 40, (kk) threonine substitution at a position corresponding to amino acid residue 258 of SEQ ID NO: 40, (ll) aspartic acid substitution at a position corresponding to amino acid residue 269 of SEQ ID NO: 40, (mm) threonine, valine, proline or glycine substitution at the position corresponding to amino acid residue 291 of SEQ ID NO: 40, (nn) alanine or glutamic acid substitution at the position corresponding to amino acid residue 294 of SEQ ID NO: 40, (oo) aspartic acid or alanine substitution at the position corresponding to amino acid residue 295 of SEQ ID NO: 40, (pp) leucine or phenylalanine substitution at the position corresponding to amino acid residue 295 of SEQ ID NO: 40, (qq) a serine substitution at a position corresponding to amino acid residue 328 of SEQ ID NO: 40, (rr) glycine substitution at the position corresponding to amino acid residue 329 of SEQ ID NO: 40, (ss) alanine substitution at the position corresponding to amino acid residue 340 of SEQ ID NO: 40, (tt) glutamic acid or aspartic acid substitution at a position corresponding to amino acid residue 343 of SEQ ID NO: 40, (uu) lysine substitution at a position corresponding to amino acid residue 347 of SEQ ID NO: 40, (vv) an alanine substitution at the position corresponding to amino acid residue 349 of SEQ ID NO: 40, (ww) histidine substitution at a position corresponding to amino acid residue 354 of SEQ ID NO: 40, (xx) aspartic acid substitution at a position corresponding to amino acid residue 366 of SEQ ID NO: 40, (yy) lysine substitution at the position corresponding to amino acid residue 375 of SEQ ID NO: 40, (zz) glutamic acid substitution at a position corresponding to amino acid residue 378 of SEQ ID NO: 40, (aaa) glycine substitution at a position corresponding to amino acid residue 390 of SEQ ID NO: 40, (bbb) a serine substitution at a position corresponding to amino acid residue 391 of SEQ ID NO: 40, (ccc) aspartic acid substitution at a position corresponding to amino acid residue 393 of SEQ ID NO: 40, (ddd) isoleucine substitution at a position corresponding to amino acid residue 397 of SEQ ID NO: 40, and (eee) glutamine substitution at the position corresponding to amino acid residue 408 of SEQ ID NO: 40. The isolated polypeptide of claim 1, 2 or 5 comprising a chemical moiety selected from polyethylene glycol or a water soluble polymer attached to the polypeptide. delete delete delete delete The isolated polypeptide of claim 1, 2 or 5 comprising N-terminal methionine or N-terminal polyhistidine attached to the polypeptide. An isolated polypeptide consisting of a fragment consisting of 138 to 428 consecutive amino acids of SEQ ID NO: 24, which does not bind choline. delete The isolated polypeptide of claim 1, 2, 5 or 12 which interacts with an antibody capable of interacting with the full length CbpA polypeptide. delete delete delete delete An isolated nucleic acid molecule consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 4, 5, 7, 9, 10, 11, 22, 23 or 24 and encoding a polypeptide that does not bind choline. The isolated nucleic acid molecule of claim 19, wherein the nucleic acid molecule consists of SEQ ID NO: 12, 14, 15, 16, 17, 19, 20, or 21. An isolated nucleic acid molecule consisting of nucleotides consisting of 138-428 consecutive amino acids of SEQ ID NO: 24 and encoding a polypeptide that does not bind choline. An isolated nucleic acid molecule encoding a polypeptide that does not bind choline and consisting of a nucleotide sequence consisting of 318-1219 consecutive nucleotides of SEQ ID NO: 12. delete 23. The isolated nucleic acid molecule of any one of claims 19-22, which is DNA. 23. The isolated nucleic acid molecule of any of claims 19-22, which is cDNA. delete The isolated nucleic acid molecule of claim 19, wherein the nucleic acid molecule is operably linked to a promoter. The isolated nucleic acid molecule of claim 27, wherein the promoter is a promoter of RNA transcription. 23. A vector comprising the nucleic acid molecule of any one of claims 19-22. delete The vector of claim 29 which is a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA. A host vector system for producing a polypeptide comprising the vector of claim 29 in a suitable non-human host cell. 33. The host vector system of claim 32, wherein suitable non-human host cells comprise prokaryotic or eukaryotic cells. A non-human cell line comprising the vector of claim 29. (a) introducing a vector according to claim 29 into a suitable host cell; (b) culturing the resulting host cell to produce a polypeptide; (c) recovering the polypeptide produced in step (b); And (d) purifying the polypeptide recovered in step (c), to obtain the polypeptide in purified form. A polypeptide capable of specific binding to a polypeptide according to any one of claims 1, 2, 5 or 12 and comprising the amino acid sequence of an N-terminal choline binding protein A truncate. Pharmaceuticals for preventing or treating infection with pneumococcal bacterium, wherein the isolated antibody does not bind to the choline binding domain and the antibody that binds to the choline binding domain is substantially absent. Composition. The pharmaceutical composition of claim 36, wherein the antibody is a monoclonal antibody. The pharmaceutical composition of claim 36, wherein the antibody is a polyclonal antibody. The pharmaceutical composition of claim 36, wherein the antibody is a chimeric (bispecific) antibody. delete A pharmaceutical composition for preventing or treating infection by pneumococcal, comprising the polypeptide according to any one of claims 1, 2, 5 or 12 and a pharmaceutically acceptable carrier or diluent. delete The pharmaceutical composition of claim 36, which is delivered to the airways or nasopharynx. delete A vaccine comprising the polypeptide according to any one of claims 1, 2, 5 or 12 and a pharmaceutically acceptable adjuvant or carrier. delete A vaccine comprising a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, and 39 and a pharmaceutically acceptable adjuvant or carrier. delete delete 23. A vaccine comprising the isolated nucleic acid molecule according to any one of claims 19, 20, 21 or 22 and a pharmaceutically acceptable adjuvant or carrier. delete A vaccine comprising the vector according to claim 29 and a pharmaceutically acceptable adjuvant or carrier. A pharmaceutical composition for treating or preventing a subject infected with or exposed to pneumococcal, comprising the vaccine according to claim 45.

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