A polypetide comprising the amino acid of an N-terminal choline binding protein A truncate, vaccine

专利摘要:
The present invention provides an isolated polypeptide, fragment, mutant, variant, homologue thereof comprising the amino acid sequence of a cleavage of N-terminal choline binding protein A having the amino acid sequence set forth in SEQ ID NO: 1, 3-7 or 9-11 Derivatives. The present invention also provides an isolated polypeptide comprising a amino acid sequence of an N-terminal choline binding protein A cleavage wherein the amino acid sequence is of 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 cleavage, 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 cleavage. The present invention provides an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage. Finally, the present invention provides for use in pharmaceutical compositions, vaccines and diagnostic and therapeutic methods.
公开号:KR20010052244A
申请号:KR1020007011206
申请日:1999-04-07
公开日:2001-06-25
发明作者:투오마넨엘레인아이；마수어에이치로버트；비제만테레사엠；존슨레슬릭시드너；쾨니히스콧
申请人:세인트 쥬드 칠드런즈 리써치 호스피탈；메디뮨 인코포레이티드；
IPC主号:

专利说明:

A polypetide comprising the amino acid of an N-terminal choline binding protein A truncate, vaccine derived therefrom and uses approximately}
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). Pneumococcus is friendly to the upper and lower airway cells. Like most bacteria, pneumococcus adhesion to human cells is achieved by the expression of bacterial surface proteins that bind to eukaryotic carbohydrates in a lecithin-like manner. Cundell, D. & Tuomanen, E. (1994) Microb Pathog 17: 361-374. Pneumococcus binds to inflamed epithelium, a process that can be thought of 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 in this new setting is that pneumococcus exhibits the benefits of and is believed to be associated with one of these non-regulated receptors, namely platelet activating factor (PAF) receptors. Cundell et al. (1995) Nature, 377: 435-438. Within minutes of the appearance of the PAF receptor, pneumococcus will progress to higher adhesion and invasion. For example, by soluble receptor homologues, inhibiting bacterial binding to activated cells blocks progression to disease in animal models. See Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176: 704-712. Particularly effective in this regard contains lacto-N-neotetrases with or without additional sialic acid which inhibits pneumococcal adhesion to human cells in vitro and inhibits metastasis in the lung in vivo. It is a soluble carbohydrate.
Choline Binding Proteins: Candidate Structural Adhesion Genes:
Pneumococcus produces a family of surface proteins capable of binding to bacterial surfaces by non-covalent binding to cell wall teicosan or lipoteichoic acid. The surface of Streptococcus pneumoniae is covered with a family of CBPs (choline binding proteins) that are non-covalently bound 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 adhesion (ligand) to a glycoconjugate containing receptor present on the surface of eukaryotic cells. Mutants deficient in cbpA exhibited decreased pathogenesis in immature rat models for nasopharyngeal metastasis. 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 such domains include the autolysin 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 families, but its binding activity to human cells does not metastasize the nasopharyngeal domains resulting from its unique N-terminal domain. Since this metastasis process and progression to disease depend on pneumococcal attachment 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 important in blocking 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. Bexin, currently used for S. pneumoniae, uses purified carbohydrates from the 23 most common serotype capsules of the bacterium, but these vaccines have only 50% protection [Shapiro]. et al. NJEM 325: 1455, 1991] under 2 years of age are not immunogenic. 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 cleavage. Such polypeptides include the amino acid sequences set forth in SEQ ID NO: 1, 3-7 or 9-11, fragments thereof, mutants, variants, homologs 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 cleavage having the amino acid set forth in SEQ ID NO: 24, and a method of making 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 present invention relates to an N-terminal choline binding protein A cleavage having lecithin activity but no choline binding activity. In addition, the present invention provides immunogenic N-terminal choline binding protein A cleavage or fragment thereof.
The invention also relates to an isolated nucleic acid, such as a recombinant DNA molecule or cloned gene, or a variant, mutant, homolog or fragment thereof that encodes the competitive polypeptide or competitively inhibits the activity of such polypeptide. will be. Preferably, an isolated nucleic acid comprising a variant, variant, mutant, homologue 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. Accordingly, the present invention encompasses single cell hosts transformed with cloned genes or recombinant DNA molecules, molecules comprising 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 polyclonal and monoclonal antibodies as well as bispecific (chimeric) antibodies produced by known genetic engineering techniques, and the ability to modulate bacterial adhesions including but not limited to acting as competitive agents. It may include antibodies that contain other functional groups for use in diagnostics in conjunction with.
It is a further object of the present invention to provide a method of treating a mammal to inhibit the amount or activity of said bacterium or subunits thereof to treat or avoid 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.
The present invention generally relates to polypeptides of N-terminal choline binding protein A cleavage. The invention also provides vaccines for providing protection against or inducing protection against bacterial infections, particularly pneumococcus, and antibodies for such polypeptides for use in diagnostic and passive immunotherapy and It is about antagonists. The polypeptides and / or nucleic acids encoding such polypeptides are also useful as competitive inhibitors of bacterial adhesion of pneumococcus. Finally, the present invention relates to therapies using such polypeptides.
1 shows choline binding protein A (CbpA) and recombinant cleavage 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.
5 shows titer measurements of anti-R2 antibodies on R6x adhesion to LNnT-HSA coated plates.
FIG. 6 shows titer measurements of anti-CbpA and adsorbed anti-CbpA antibodies on activity that blocks pneumococcal adhesion 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 cleavage. The polypeptide is suitable for use in immunizing an animal against pneumococcal infection. These polypeptides or peptide fragments thereof are used in vaccines against pneumococcus and vaccines against other bacteria using cross-reactive proteins when they are formulated with suitable adjuvants.
The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage. 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, homologs 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 cleavage 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 compares the homology of various serotypes of nucleic acid and amino acid sequences 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 cleavage 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 an amino acid sequence of an N-terminal choline binding protein A cleavage having an amino acid of about 16 to about 475 of serotype 4 as shown in FIG. 2 or a corresponding amino acid of serotype 4 as shown in FIG. It provides an isolated polypeptide showing a tertiary structure, including. In one embodiment, the tertiary structure corresponds to that present in the original 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. An N-terminal choline binding protein A cleavage form is prepared by cleaving at amino acid asparagine (N) at 475 or by cleaving the corresponding amino acid of serotype R6x or serotype 4 in the different serotypes shown in FIG. 2. Alternative methods of preparing cleaved choline binding protein A or fragments thereof or maintaining their intrinsic tertiary structure (ie, the full length of choline binding protein) A are foreseen and known to those skilled in the art. Since the polypeptide maintains its tertiary structure, the isolated polypeptide is suitable for use as an immunogen that is immune to 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” means 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 an N-terminal cleavage 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 serotype type 4:


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

The DNA sequence encoding polypeptide R1 is as follows:

“Polypeptide C / R2” is a repeat unit in R2 wherein the repeat region C comprises the amino acid sequence of positions 327 to 433 of the N-terminal truncation of choline binding protein A (CbpA) serotype 4 having the sequence It means a polypeptide comprising C.

DNA sequence of polypeptide C / R2:

“Polypeptide A / R2” is a repeat unit in R2 wherein the repeat region A comprises the amino acid sequence of positions 153 to 269 of the N-terminal truncation of choline binding protein A (CbpA) serotype 4 having the sequence A polypeptide comprising A is meant.

As shown in FIG. 1, region A of polypeptide R2 is identical to region A as in R1.
The DNA sequence encoding polypeptide A / R2 is as follows:

The kind or position of one or more amino acid residues may include a deletion containing less than all residues specified for the protein, a substitution in which one or more residues specified are replaced by another residue, and one or more amino acid residues at the terminal or central position of the polypeptide. Changes or variations are made to include variations such as added additions (see FIG. 2). Such molecules include the following: Further introduction of "preferred" codons expressed by a host other than the selected mammal, provision of a site for cleavage by restriction endonuclease enzymes, and further aiding in the construction of easily 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:


Amino acid sequence of CbpA of serotype R6x:


DNA sequence encoding amino acid sequence of CbpA of serotype R6x


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

DNA sequence encoding polypeptide R1:


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

DNA sequence of polypeptide C / R2 serotype R6x:

"Polypeptide A / R2 serotype R6x" refers to a polypeptide comprising repeat region A in R2 (see Figure 2), where repeat region A is the N- of choline binding protein A (CbpA) serotype R6X having the following sequence: Has an amino acid sequence from terminal 155 to position 265:

DNA sequence encoding polypeptide A / R2 serotype R6x

to be.
The present invention relates to an isolated polypeptide, wherein the isolated polypeptide consists of an amino acid sequence as set forth in SEQ ID NOS 22 or 23, including fragments, mutations, variants, analogs or derivatives thereof.

"Polypeptide B / R2" refers to a polypeptide comprising the amino acid sequence from positions 270 to 326 of an N-terminal cut of choline binding protein A (CbpA) serotype 4 as shown in FIG. "Polypeptide B / R2 serotype-R6x" means a polypeptide comprising the amino acid sequence at positions 264-326 of the N-terminal cut 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.
The present invention also provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage, wherein the polypeptide has 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 cleavage, 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 amino acid sequences 158-172, 300-321, 331-339, 355-365, 367-374, 379-389, 409-427 and 430-447 It is not limited. Figure 2 shows the homology of the various serotypes of the nucleic acid and amino acid sequences of the N-terminal region of CbpA of interest in the present invention.
The present invention provides an isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage, 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 IN NO 1, 3 to 5, 7 or 9 to 11 comprising fragments, mutations, variants, analogs or derivatives thereof.
As used herein, the term "polypeptide with lectin activity" refers to a polypeptide, peptide or protein that is non-covalently bound to carbohydrates. "Adhesion" as defined herein means non-covalent binding of bacteria to human cells or secretion that is stable enough to withstand washing. As defined herein, "binding to LNnT" means binding to a substrate coated with Lacto-N-neotetraose better than albumin-regulation.
The present invention provides an isolated immunogenic polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage. Immunogenic polypeptides are contemplated by the present invention to have an amino acid sequence as set forth in SEQ IN NOS 1, 3 to 5, 7 or 9 to 11, including fragments, mutations, 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 cleavage as shown in FIG. 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. Analogue polypeptides have an N-terminal methionine or N-terminal polyhistidine optionally attached to the N or COOH terminus of a polypeptide comprising an amino acid sequence.
In another aspect, the present invention contemplates peptide fragments of polypeptides that result in proteolytic digestion products of the polypeptide. In other embodiments, derivatives of polypeptides have one or more chemical moieties attached thereto. 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 tetratrapylated. In other embodiments, the chemical moiety is N-terminal monopersilized.
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 as a treatment for severe combined immunodeficiency. The second advantage obtained 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 as disease treatment agents in other mammals without the risk of causing a severe immune response. Compounds of the invention can be delivered to microencapsulation devices to reduce or prevent host immune responses to the compound or to 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, especially leaving groups with N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitro Benzene-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 the reaction with aldehydes produced by 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 may replace certain L-amino acid residues so 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. The abbreviation used herein follows standard polypeptide nomenclature [J. Biol. Chem, 243: 3552-59 (1969).
It should be noted that all amino acid residue sequences are represented herein by the formula wherein the left and right directions are the common direction of the carboxy terminus from the amino terminus. Furthermore, a '-' at the beginning or end of an amino acid residue sequence indicates that the peptide binds to another sequence of one or more amino acid residues.
Synthetic polypeptides prepared using solid phase, liquid phase or peptide condensation techniques or known methods of mixing thereof may comprise natural or unnatural amino acids. The amino acids used for peptide synthesis were standard Boc (N ^α −) with standard deprotection, neutralization, coupling and washing protocols of the original solid phase method of Merrifield (1963, J. Am. Chem. Soc. 85: 2149-2154). Amino protected N ^α -t-butyloxycarbonyl) amino acid resin, or the base-labile N ^α -amino protected 9-described first in Carpino and Han (1972, J. Org. Chem. 37: 3403-3409). Fluorenylmethoxycarbonyl (Fmoc) amino acid. Thus, the polypeptides of the present invention may be formulated with a mixture of D-amino acids, D- and L-amino acids and various "designer" amino acids (e.g., β-methylamino acid, Cα-methylamino acid and Nα-methylamino acid, etc. ) May be included. Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine and norleucine for leucine or isoleucine. In addition, by placing certain amic acids in certain coupling steps, α-helices, β-turns, β-sheets, γ-turns and cyclic peptides can be produced.
In one aspect of the invention, the peptide may comprise a special amino acid at the C-terminus that simulates a free glycine or glycine amide group inserting a CO ₂ H or CONH ₂ side chain. Side chain D amino acid analogs or L amino acid analogs composed of a linker or linker to the beads may also be regarded as such special residues. In one example, the pseudo-free C-terminal residue is in D or L optical arrangement, and in other instances racemic mixtures of D and L-isomers may be used.
In another example, pyroglutamate may be included as the N-terminal residue of the peptide. By limiting only 50% of the peptide to be substituted on a bead given N-terminal pyroglutamate, the pyroglutamate cannot follow the sequence by Edman degradation, but sufficient non-pyroglutamate peptide on the bead for sequencing Will remain. Those skilled in the art will readily recognize that this technique can be used for sequencing peptides that insert residues that do not conform to Edman modifications at the N-terminus. Other methods of characterizing individual peptides that exhibit the desired activity are described in detail below. The specific activity of a peptide comprising a blocked N-terminal group, eg, pyroglutamate, indicates that if a specific N-terminal group is present within 50% of the peptide, then the fully blocked peptide (100%) is unblocked peptide. It can be shown easily by comparing with (0%).
In addition, the present invention seeks to use peptide mimetic bonds, such as peptide mimetics, ester bonds, to prepare peptides with more clearly specified structural properties and to produce peptides with novel properties. In another example, the peptide can generate a peptide by inserting a reduced peptide bond, ie, R ₁ -CH ₂ -NH-R ₂ , where R ₁ and R ₂ are amino acid residues or sequences. Reducing peptides can 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 breakdown of metabolism or resistance to protease activity. Moreover, it is well known that inhibitory peptides have increased structural activity in certain systems (Hurby, 1982, Life Sciences 31: 189-199; Hruby et al., 1990, Biochem J. 268: 249-262). The present invention provides a method for generating inhibitory peptides that insert random sequences at all possible positions.
Inhibited, cyclized peptides or rigidized peptides can be crosslinked to limit cyclization or stiffening of the peptide by inserting amino acids or amino acid analogs at two or more positions in the peptide's sequence to form crosslinks after treatment. It may be prepared by synthesis as long as it provides a chemical group. Cyclization would be preferred if an amino acid induced by rotation is inserted. Examples of amino acids capable of crosslinking peptides are cysteine, lactone, which forms disulfide bonds, aspartic acid, which forms lactase, and γ-carboxyl-glutamic acid (Gla) (Bachem), which chelates transition metals to form crosslinks. Chelates. Protected γ-carboxyl-glutamic acid can be prepared by modifying the synthesis described by Jiseng and Olsson (1980, Biophys. Biochem. Res. Commun. 94: 1128-1132). Peptides whose peptide sequences comprise at least two cross-linkable amino acids can be processed, for example, by oxidizing cysteine residues to form disulfides or by adding metal ions to form chelates, resulting in peptides. Crosslinking forms cyclic or rigid peptides that are inhibited.
The present invention provides a method by which crosslinking can be made structurally. For example, different protecting groups can be used when four cysteine residues are inserted into the 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. Optionally, 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-classical amino acids can be inserted into the peptide to provide a particular morphological motif: 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 (Kazmierski and Hruby, 1991, Tetrahedron Lett .)Reference); 2-aminotetrahydrolonnaphthalene-2-carboxylic acid (see Landis, 1989, Ph. D. "Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline- 3-carboxylate (see 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) (Dharanipragada).
The amino acid analogs and peptide mimetics described below can be inserted into peptides to induce or assist certain secondary structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid); β-rotating inducing dipeptide analogues (see Kemp et al., 1985, J. Org. Chem. 50: 5834-5838); β-sheet derived analogs (see Kemp et al., 1988, Tetrahedron Lett. 29: 5081-5082); β-rotation induced analogs (see Kemp et al., 1988, Tetrahedron Lett. 29: 5057-5060); Helix-induced analogs (see Kemp et al., 1988, Tetrahedron Lett. 29: 4935-4938); γ-rotation induced analogs (see Kemp et al., 1989, J. Org. Chem. 54: 109: 115) and analogs provided in Nagai and Sato, 1985, Tetrahededron Lett 26: 647 -650, DiMaio et al., 1989, J. Chem. Soc. Perkin trans. P. 1687); Gly-Ala-rotating analogs (see Kahn et al., 1989, Tetrahedron Lett. 30: 2317); Amide binding allotropes (see Jones et al., 1988, Tetrahedron Lett. 29: 3853-3856); Tretrazole (see Zabrocki et al., 1988, J. Am. Chem. Soc. 110: 5857-5880); DTC (see Samantha 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. Shape-limited mimetics of beta-rotation and beta-bulges 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 modifications or derivatives of the polypeptides or peptides 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 effected by chemical or enzymatic means. In another aspect, glycosylated or fatty acid acylated peptide derivatives can be prepared. The preparation of glycosylated or fatty acid acylated peptides is well known in the art. Fatty acid 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 example, an amino acid comprising a side chain of a fatty acid having the 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 can be made in the nucleic acid encoding the polypeptide to transform one codon into a codon encoding another amino acid. Such mutations are generally made with little change in nucleotides. Substitution of this class of mutations is conserved by a non-conservative manner 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 Can be altered in an appropriate 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 acid substitutions in the sequence may be selected from classes belonging to the amino acid from other classes. 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, histidine and the like. 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 the equipotential 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;
Gln Substituting Asn, Substituents That Can Retain Free NH ₂
to be.
Synthetic DNA sequences make it easy to create species that are homologous or "mutein." 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 make analogs of non-natural amino acids.
According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques in the art can be used. Such techniques are described in detail in the literature. Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM ed. (1994); "Cell Biology: A Laboratory Handbook" Vlumes I- III JE Celis, ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan, Je, ed. (1994); "Oligonucleotide Synthesis" MJ Gait ed. 1984; "Nucleic Acid Hybridization" BD Hames & SJ Higgins (1985); "Trasncription And Translation" BD Hames & SJ Higgins, eds. (1984); "Animal Cell Culture" RI Frenshney, 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. Although pyroglutamate cannot be contiguous by edmam degradation by limiting by substitution of only 50% peptide in a given bead with N-terminal pyroglutamate, sufficient bipyroglutamate peptide will remain on the beads for contiguity. Those skilled in the art will readily note that this technique can be used to sequence peptides that incorporate residual resistance to edmam degradation at the N-terminus. If a particular N-terminal group is present within 50% of the peptide, then each peptide describing the desired activity is described in detail under the specific activity of the peptide comprising the blocked N-terminal group (e.g., pyroglutamate). Another method for characterization is readily described by comparing the activity of fully (100%) blocked peptides with unblocked (0%) 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, for therapeutic use in the preparation of terminal products, 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 such component (s), they will be identified using the analytes provided herein.
The water-soluble polymers are, for example, polyethylene glycol, copolymers 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, proylpropylene oxide / Ethylene oxide copolymers, polyoxyethylate 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 is about 2 to about 100 kDa (the term "about" indicates that some molecular weights are more or less than standard molecular weights when preparing polyethylene glycols). 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 effect 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 a combination 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 the polymer is not present) is determined by the degree of derivatization desired (eg mono, di, tri, etc.), the polymer selected between the side chain or the unbranched Will be measured by factors such as molecular weight and reaction conditions.
Polyethylene glycol molecules (or other chemical moieties) may be attached to the component (s) in view of the action of the protein or effect on the antigenic region. 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 tricell chloride). For example, polyethylene glycol can be covalently linked via amino acid residues via reactive groups (eg, free amino or carboxyl groups). Reactive groups that are activated polyethylene glycol molecules may be bonded. 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, sulfide groups can be used as reactive groups for attaching polyethylene glycol molecule (s). Preferred for therapeutic purposes is to attach at the amino group, such as to the N-terminal or lysine group.
The present invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of the N-terminal choline binding protein A truncated form. The present invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of N-terminal choline binding protein A truncated type, as shown in FIG. In one embodiment, nucleic acids are referred to hereinafter in SEQ ID NOS 12, 14-17 or 19-21, including fragments, mutations, variants, analogs or derivatives thereof. Nucleic acids are DNA, cDNA, genomic DNA, RNA. In addition, the isolated nucleic acid can be effectively 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 generate a copy of an attached fragment.
"DNA" is the polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) in single twisted form or double twisted strand form. These terms only refer to the first and second structures of the molecule and do not limit the specific tertiary form. Thus, the term includes the double-stranded DNA found, among others, linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes. When discussing the structure of certain double-twisted DNA molecules, the sequences are described herein according to standard delivery, which provides only sequences in the 5 'to 3' direction with non-transcribed DNA twists (ie, twists with sequences consistent with mRAN). It may be described in.
DNA sequences are “operably linked” to expression control sequences when they regulate expression control sequences and when they regulate transcription and translation of the DNA sequence. The term “operably linked” has a suitable starting signal (eg ATG) in front of the DNA sequence to be expressed and is encoded by the correct reading frame and DNA sequence to enable the expression of the DNA sequence under the control of the expression control sequence. To maintain the product of the desired product. If the gene desired to be inserted into the prefabricated DNA molecule contains a suitable starting signal, this starting 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 is the same as, a bacterial, yeast, gonzo or mammalian promoter. The vector can also be a plasmid, cosmid, yeast technical 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), simian virus 40 (SA40), cytomegalovirus (CMV), mouse breast cancer virus (MMTV), molony rat leukemia virus, DNA delivery systems, ie liposomes And expressing plasmid delivery systems. In addition, one class of vectors includes bovine papilloma virus, polyoma virus, rod virus, RNA tumor virus, and Semiki forest virus. Such vectors may be obtained conventionally or collected from sequences 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, primordial or mature nucleus 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.
Various host / expression vector combinations can be used to express the DNA sequences of the present invention. Useful expression vectors may, for example, consist of fragments 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 E. coli plasmid Cole El, pCR1, pBR322, pMB9 and derivatives thereof, PR4; Phage DNAS, eg, a number of derivatives of phage , Such as NM989 and other phage DNA such as M13 and islet single twisted phage DNA; Yeast plasmids (2μ plasmid) or derivatives thereof; Vectors useful in mature nucleus cells (eg, vectors useful in insect or mammalian cells); Vectors derived from a combination of plasmid and phage DNA (eg, plasmids modified to use phage DNA or other expression control sequences), and the like.
Various expression control sequences—sequences that regulate the expression of DNA sequences operably linked thereto—are used in such vectors to express the DNA sequences of the invention. Such useful expression control sequences are, for example, the main operation of SV40, CMV, vaccinia, polyoma or adenoma virus, lac systems, trap systems, TAC systems, TRC systems, LTR systems, phage λ Pores and promoters, regulatory regions of fd coated proteins, promoters for 3-phosphoglycerate kinases or other glycolic enzymes, promoters of acid phosphates (eg Pho 5), promoters of yeast α-crossing factors and primitive nucleus cells or Other sequences known to modulate the expression of a mature nucleus cell or virus thereof, and various combinations thereof.
Various single cell host cells are also useful for expressing the DNA sequences of the present invention. Such hosts include well-known mature and primitive nucleus 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; Kidney cells of African green monkeys (eg COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (eg Sf9) and plant cells in human cells and culture tissues.
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 will work equally 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, it must be considered, since the vector must act in the host. The number of vector copies, the ability to control copying numbers, and the expression of other proteins encoded by the vector (eg antibiotic indications) 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 its expressed genes, which are considered to be miscible or particularly effective second structures having a particular DNA sequence. Suitable single cell hosts are, for example, their miscibility with selected vectors, their secretion properties, their ability to fold proteins accurately, their fermentation needs, 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 producing a polypeptide comprising growing the host vector system described above under suitable conditions of limiting the production of 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 labels that are chemo, colorimetric, fluorescent or luminescent labels. Labeled antibodies can be polyclonal or monoclonal antibodies. In one embodiment, the labeled antibody is a purified labeled antibody. Methods of labeling antibodies 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 fancy antibodies and whole synthetic antibodies, and fractions thereof. Such antibodies include, but are not limited to, polyclonal, monoclonal, fancy, single chain, Fab fractions and Fab expression libraries.
Various procedures known in the art can be used to prepare polyclonal antibodies or derivatives or analogs thereof for polypeptides. Antibodies-A Laboratory Manual, Harlow and Lane, eds, Cold Spring Habor Laboratory Press: Cold Spring Harbor, Ne York, 1988]. To prepare antibodies, various host animals, including but not limited to rabbits, mice, rats, sheep, choline, and the like, can be immunized by injection with cleaved CbpA or derivatives thereof (eg, fractions or fusion proteins). Can be. In one embodiment, the polypeptide may be conjugated to an immunogen carrier such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response depending on the host type.
To prepare monoclonal antibodies, fractions, analogs, and derivatives thereof, the techniques provided for preparing 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-Hybridamoda 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 modify human B cells with EBV virus in vitro (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 conjugating genes from mouse antibody molecules specific for polypeptides with genes from human antibody molecules of appropriate biological activity [Morrison 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 disorders (described below), which are better than heterologous antibodies for human or humanized antibodies to elicit immune responses, especially allergic responses themselves. Because it is less suitable. Another aspect of the present invention is described in Fab expression literature, see Huse et al., 1989, Science 246; 1275-1281 allows for the rapid and easy identification of monoclonal Fab segments with the desired specificity for polypeptides or derivatives or analogs thereof using the techniques described.
Antibody fragments comprising the individual specificities of an antibody molecule can be produced by known techniques. For example, such segments include, but are not limited to: F (ab ') ₂ segments, which can be prepared for pepsin digestion of antibody molecules; Fab 'segments that can be produced by reducing the disulfide bridges of F (ab') ₂ segments, and Fab segments 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 immunoassay), "sandwich" immunoassay, immunoradiometric, agar diffusion sedimentation reactions. , Immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzymes or radioisotope labels), western blots, sedimentation, agglutination (e.g., agar aggregation, erythrocyte agglutination) , Complement binding assays, immunofluorescence assays, Protein A assays, and immunoelectrophoresis, etc.]. In one embodiment, antibody binding is achieved by detecting a label in the primary antibody. In another embodiment, the primary antibody is achieved 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 can be labeled using a radiopaque agent.
Almost 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, oramin, Texas red, AMCA blue and lucifer yellow. Particularly detectable substances are anti-rabbit antibodies made in choline and bound with fluorescein via isothiocyanate. Polypeptides are also labeled using radioactive elements or enzymes. The radioactive label may 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 bound 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. Peroxadases, β-glucuronidase, β-D-galactosidase, fenase, glucose oxidase and peroxidase and alkaline phosphatase are preferred. In US Pat. No. 3,654,090, US Pat. No. 3,850,752 and US Pat. No. 4,016,043, reference is made to examples of separate labeling materials and methods.
In another embodiment 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, comprise one or more labeled polypeptides or binding partners thereof, eg, according to the method selected (eg, "competition", "sandwich", "DASP", etc.). For example, antibody specificity and direction thereto. Such kits may also include peripheral 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) an 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,
Other reagents (b) and
Direction (c) for using the above kit.
The present invention provides antagonists or blockers including but not limited to peptide segments, nucleic acid molecules, ribozymes, polypeptides, small molecules, carbohydrate molecules, monosaccharides, oligosaccharides or antibodies. In addition, reagents that competitively block or inhibit pneumococcal bacterium are contemplated by the present invention. The present invention provides reagents comprising proteins that inhibit inorganic compounds, nucleic acid molecules, oligonucleotides, organic compounds, peptides, peptide-like compounds or polypeptides.
The present invention provides vaccines and pharmaceutically acceptable modulators or carriers comprising polypeptides having the amino acid sequences represented by any of SEQ ID NOS: 1, 3-7, 9-11, 22 and 23. The polypeptide may include the amino acid sequence of the proximal N-terminal choline binding protein A as shown in FIG. The present invention provides a vaccine and a pharmaceutically acceptable modulator or carrier comprising a polypeptide having an amino acid sequence comprising a conserved site as shown in FIG. 2. For example, conserved sites include amino acid sequences 158-172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427 and 430 to 447, but are not limited thereto. The present invention provides vaccines and pharmaceutically acceptable modulators or carriers comprising isolated nucleic acids encoding polypeptides.
Active immunity against gram positive bacteria, in particular pneumococcus, can be induced by immunization (inoculation) using immunogenic amounts of polypeptide or peptide derivatives or segments and adjuvants, wherein the polypeptide or antigen derivative or segment thereof is a vaccine. Antigen component of.
Polypeptides, derivatives thereof or fragments thereof of the invention can be prepared in admixture with an adjuvant to produce a vaccine. Preferably, the derivative thereof or segment thereof used as the antigenic component of the vaccine is adhenzine. More preferably, the polypeptide or peptide derivative thereof or fragment thereof used as an antigen component of a vaccine is an antigen common to all or a plurality of strains of a gram positive bacterium or an antigen common to a class of closely related bacteria. Most preferably, the antigenic component of the vaccine is adhegen, which 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, such as transfection, electroporation, microinjection, transformation, cell fusion, DEAE dextrin, calcium phosphate precipitation, lipofection ( Lysosomal fusion), a preferred host or DNA vector carrier by the use of a genetic gum (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.
The vaccine can be administered by any oral route, including but not limited to intramuscular, intraperitoneal, intravenous, and the like. Preferably, since the desired outcome of vaccination describes an immune response to the antigen, either direct administration to pathogenic organisms or indirect selection or targeting of viral vectors to lymphoid tissue (eg lymph nodes or spleen). Is preferred. Because immune cells repeat repeatedly, they are the actual targets for retroviral vector-based nucleic acid vaccines, since retroviruses require repeat cells.
Passive immunity is presumed to be infectious by Gram-positive bacteria, preferably Streptococci, and more preferably pneumococci, by administering to the patient an immune serum, polyclonal or neutralizing monoclonal antibody against a polypeptide of the invention. Can be compared to an animal subject. Passive immunity is not protected for long periods of time, but may be a valuable means for the treatment of bacterial infections in unvaccinated subjects. Passive immunity is particularly important for the treatment of antibody resistant strains of Gram-positive bacteria, since no other therapy is used. Preferably, the antibody administered for passive immunotherapy is an autoantibody. For example, if the subject is a male and female, preferably the antibody is of human origin or is "humanized" to minimize the possibility of an immune response against the antibody. Administration of the active or passive vaccines, or adhegens of the present 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 can include antibodies against lectin domains and / or antibodies against soluble excess lectin domain proteins. Blocking adhesion by any mechanism blocks the initial stage of infection, reducing colony production. This, in turn, reduces human-borne transmission and prevents the development of symptomatic disease.
The present invention provides a method for inducing an immune response in an individual infected with or exposed to pneumococcal, including administering an amount of a pharmaceutical composition to the individual to induce an immune response.
The present invention provides a method for preventing infection by pneumococcal of an individual by administering to the individual a predetermined amount of a pharmaceutical composition in an amount effective to prevent pneumococcal bacterial attachment.
The present invention provides a method for preventing infection of an individual by pneumococcal, including preventing an infection by pneumococcal by administering to the individual a pharmaceutical composition comprising an antibody and a pharmaceutically acceptable carrier or diluent. do.
The present invention provides a method of treating a patient infected with or exposed to pneumococcal bacteria, comprising treating the patient by administering to the patient a therapeutically effective amount of a vaccine of the present invention.
Pneumococcal bacteria comprising inducing an immune response by administering to a patient a predetermined amount of a pharmaceutical composition comprising a polypeptide consisting of the amino acid sequence represented by amino acid sequence identification numbers 1, 3 to 5, 7 or 9 to 11 Provided are methods for inhibiting colonization of host cells in a patient infected with or exposed to. Therapeutic peptides that block colonization are delivered by the respiratory mucosa. The pharmaceutical composition comprises a polypeptide consisting of the amino acid sequence shown in FIG.
As used herein, the term “pharmaceutical composition” refers to the treatment of the present invention with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers useful for providing a therapeutic effect or benefit of preventing pneumococcal colonization. Means a pharmaceutically effective amount of polypeptide product. As used herein, the term “therapeutically effective amount” refers to an amount that provides a therapeutic effect on a given disease and dosing regimen. Such compositions may be liquid or lyophilized or dried formulations with 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 (e.g. Tween 20, tween 80, Pluronic F68, bile salts), solubilizers (e.g. glycerol, polyethylene glycerol), antioxidants (e.g. ascorbic acid, sodium metabisulfite), Preservatives (e.g. thimerosal, benzyl alcohol, parabens), bulking substances or tonic modifiers (e.g. lactose, mannitol), covalent bonding of polymers such as polyethylene glycol to proteins, complexation with metal ions, or Incorporation into or above particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, or liposomes, microemulsions, micelles, monolayer or multilayer vesicles, critrosite It includes the introduction of a cast or spiro plast-up. Such compositions affect the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the therapeutic agent. The choice of composition depends on the physical and chemical properties of the protein having therapeutic activity. For example, products derived from membrane bound active forms require formulations containing detergents. Controlled or delayed release compositions include formulations as lipophilic low oils (eg fatty acids, waxes, oils). In addition, the composition of the present invention is a particulate composition coated with a polymer (eg poloxamer or poloxamine) and is an active form that is bound to an antibody to a tissue specific receptor, ligand or antigen or to an antibody to a tissue specific receptor. . 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.
The term "pharmaceutically acceptable carrier" as used herein is also 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 to this. 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 ethylene oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or nonvolatile oils. Intravenous carriers 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" may be a compound or mixture that enhances an immune response to an antigen. Adjuvants can be provided as tissue hypoxia that slowly release antigens and as lymphatic system 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 adjuvants fails to induce a humoral or cellular immune response. Adjuvant may include complete Freund's adjuvant, incomplete Freund's adjuvant, saponins, mineral gels (e.g. aluminum oxide), surface active substances (e.g. lysorecithin, pluronic glycols, polyanions, peptides, oils or hydrocarbon emulsions) , Keyhole limpit hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (Basil Calmete-Guerin) and Corynebacterium parboom. Preferably, the adjuvant is pharmaceutically acceptable.
Controlled or delayed release compositions include lipophilic depots (eg fatty acids, waxes, oils). Also shown are particulate compositions coated with polymers such as poloxamers or poloxamines and compounds coupled to antibodies directed against tissue-specific receptors, ligands or antigens or to ligands of tissue-specific receptors. It is understood by the 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, the compound often brightens rapidly from the mucosal surface or circulation and thus can elicit relatively short lifespan of pharmaceutical activity. As a result, often relatively high 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 polypyrrole are not modified It has been shown to represent the next intravenous injection of substantially half the life in the blood than the compound [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 polymer-chemical adducts described above at a less frequent or lower dosage than the unmodified chemicals.
Dosage. Sufficient dosages can be from about 1 μg / kg to about 1000 mg / kg, but are not limited thereto. The dosage can be 10 mg / kg. Formulations of a therapeutically acceptable composition include a pharmaceutically acceptable carrier.
As mentioned above, the present invention provides a therapeutic comprising a pharmaceutical composition comprising mediators, vaccines, polypeptides, nucleic acids and antibodies, anti-antibodies and agents for combating the pathogenic activity of pneumococci, such as adhering to host cells. To provide a 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 solid forms suitable for solutions or suspensions that are liquid prior to injection can also be prepared. 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 or mixtures thereof. In addition, if desired, the compositions 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 organic acids such as acetic acid, oxalic acid, tartaric acid and mandelic acid. Is formed. Salts formed from free carboxylic acids can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or or iron hydroxides and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine.
When at least about 75% by weight of the protein, DNA, mediator (depending on the category of species A and B) in the composition is "A", "A" where "A" is a single protein, DNA molecule, Compositions comprising mediators, etc.), are substantially free of "B", where "B" is one or more contaminating proteins, DNA molecules, mediators, and the like. 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.
A "therapeutically effective amount" is herein used in an amount sufficient to reduce the activity, action and response of the strain by at least about 15%, preferably at least 50%, more preferably at least 90%, most preferably clinically significant. It is used to mean a sufficient amount to prevent the shortage. In addition, a therapeutically effective amount is sufficient to enhance clinically significant conditions in the strain. In the present invention, the deficiency in the response of the strain is evidenced by the continuation or dispersion of the bacterial infection. Improvements in clinically significant conditions of the strain include reduction of bacterial load, clearing of bacteria from colonized strain cells, reduction of fever or inflammation associated with infection, or reduction of certain symptoms associated with bacterial infection.
According to the invention, the components or components of the therapeutic compositions of the invention can be introduced parenterally, for example, orally, nasal, lung, rectal or transdermal. Parenteral, for example, 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 because pneumococci generally colonize the nasopharynx 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 can be delivered to vesicles, in particular liposomes. Langer, Science 249: 1527-1533 (1990); Treat atal., 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, implanted 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 may 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. 22, pp. 115-138 (1984). Preferably, the controlled release device is introduced near an inappropriate immune activity or tumor site of the subject. Other controlled release systems are discussed in Langer, Science 249: 1527-1533 (1990).
Subjects to which the administration of the active ingredient mentioned above is an effective therapeutic regime for bacterial infections are preferably humans, 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 all animals, particularly domestic animals such as cats or dogs, including but not limited to cattle, horses, choline, sheep, and pigs. It is particularly suitable for administration to mammals, including such breeding animals, wild animals (wild or zoo animals), mice, rats, rabbits, choline, 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. A therapeutically effective amount can be determined by a medical practitioner based on patient characteristics (age, weight, sex, symptoms, complications, other diseases, etc.), as is well known in the art. In addition, as another route study is conducted, more specific information will be clarified about the dosages suitable for the treatment of various symptoms of various patients, and those skilled in the art will consider the treatment history, age and general health of the receptor. To find the appropriate dose. Generally, for intravenous injection or infusion, the dosage may be small from 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 needed to administer is dependent on the judgment of the practitioner and is specific to the individual. However, suitable dosages can be about 0.1 to 20, preferably about 0.5 to about 10, more preferably 1 to several mg per kg of individual body weight per day and depends on the route of administration. The regimens suitable for initial administration and for two-time immunization also vary, but are typed as repeated administrations by injection or other administration at intervals of one hour or more after the initial administration. Also contemplated are continuous intravenous infusions sufficient to maintain 10 nanomolar to 10 micromolar concentrations in the blood.
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 adhesion inhibitors, (3) other small molecular weight bacterial adhesion inhibitors, (4) bacterial metabolism And one or more pharmaceutical compositions used to treat bacterial infections, including transport or modification inhibitors, (5) bacterial lysis stimulants, or (6) antibacterial agents, antibodies, or vaccines against other bacterial antigens. . Other potent 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. 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 this ligand is required to reduce or inhibit infection resulting from bacterial mediated binding of strain cells, the polypeptide may be introduced to interact with bacteria and strain cells. To block.
Pulmonary delivery of the present polypeptide having lectin activity that acts as an adhesion inhibitor (or derivative thereof) is also contemplated herein. Adhesion inhibitors (or derivatives) can be delivered in mammalian fertilizers 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 Interna 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., US. 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 formulations suitable for the dispersion of adhesion inhibitors (or derivatives). 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 adhesion 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 adhesion inhibitors (or derivatives) dissolved in water at a concentration of about 0.1-25 mg of biologically active adhesion inhibitor per ml of solution. The formulation may also include buffering agents and simple sugars (eg for adhesion inhibitor stabilization and osmotic pressure control). 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 fine powder containing an adhesion inhibitor (or derivative) suspended in a propellant together with a surfactant. Propellants include materials commonly used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and Hydrocarbons including 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 adhesion inhibitors and dispersants in physiologically acceptable diluents. The dry powder aerosol formulations of the present invention consist of adhesion inhibitors and dispersants in the form of finely divided solids. For liquid or dry powder aerosol formulations, the formulation is aerosolized. That is, the liquid or solid particles are also crushed to ensure 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 and particle properties in the formulation are important. These aspects of pulmonary administration of the drug are known and the structure of the formulation treatment, aerosolization means and delivery device is sufficient for most general experiments known to those skilled in the art. In specific embodiments, the mass mean dynamic diameter to ensure that the drug particles reach the alveoli will be less than 5 micrometers [Wearley, L.L., Crit. Rev. in Ther. Drug Carrier Systems 8: 333 (1991).
Aerosol delivery systems such as pressurized metered dose inhalation and dry powder inhalation are described in Newman, 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 another embodiment, the aerosol formulations of the invention, as described in detail below, may be used in addition to adhesion inhibitors to therapeutic or pharmaceutically active factors such as, but not limited to, antibiotics, steroids, non-steroidal anti-infective agents, and the like. It may include.
Liquid aerosol formulations.
The present invention provides an aerosol formulation and the dosage is intended for use in the treatment of patients suffering from bacterial, eg streptococal, in particular pneumococcal infections. In general, such dosage forms include adhesion inhibitors in pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, but are not limited to, distilled water, saline, buffered saline, dextrose solutions, and the like. In certain embodiments, the carrier 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 7.0-8.0.
Liquid aerosol formulations of the present invention may include, as additional materials, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, surfactants, and excipients. The formulation may comprise a carrier. Carriers are macromolecules that are soluble and pharmaceutically acceptable in the circulatory system, where pharmaceutically acceptable means that the carrier can inject the carrier into the patient as part of the treatment. Preferably the carrier is relatively stable in the circulation system with an acceptable plasma half-life for clearance. Such macromolecules include, but are not limited to, soya 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 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 adhesion inhibitors and other therapeutically effective drugs such as antibiotics, steroids, nonsteroidal anti-inflammatory agents, and the like.
Aerosol Anhydrous Powder Formulation. It is also contemplated that such aerosol formulations may be prepared as anhydrous powder formulations comprising finely divided powder forms of adhesion inhibitors and dispersants.
Formulations for dispersing from a powder inhalation device include finely divided anhydrous powders containing adhesion inhibitors (or derivatives), and an amount that facilitates dispersion of the powder from the device with a bulking agent such as lactose, sorbitol, sucrose or mannitol For example, 50 to 90% by weight of the formulation. Adhesion inhibitors (or derivatives) should most preferably be made in particulate form with an average particle size of less than 10 mm (or μ), most preferably 0.5 to 5 mm, for the most effective delivery to the peripheral lungs. In another embodiment, anhydrous powder formulations may include finely divided anhydrous powders, dispersants and also bulking agents containing adhesion inhibitors. 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 adhesion 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 (Mark Publishing Co. Easten 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 centroids 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 dosage forms for treatment is provided in Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979. In general, the formulations comprise the ingredient (s) and ingredient (s) (or chemically modified forms thereof) and inactive ingredients which protect the surrounding environment of the stomach and release biologically active substances into the intestine.
In addition, oral dosage forms of the derived component (s) are specifically contemplated. The component (s) may be chemically modified to be effective for oral delivery of the derivative. In general, the expected chemical modification 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 an object 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; Newmmark, 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 formulations that do not dissolve in the stomach but release the substance in the duodenum or other intestines. Preferably, the release avoids the deleterious effects of the peri gastric periscope by the protection of the protein (or derivative) or by releasing a biologically active substance in the intestine, for example, other than the peri-ambient environment.
It is essential to ensure complete gastrointestinal resistance of impermeable coatings at 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 to protect the stomach. This may include dragees, or coatings that make swallowing easier to swallow. Capsules may consist of hard shells (such as gelatin) for the delivery of anhydrous therapeutics (eg powders), and soft gelatin shells may be used for the liquid form. The shell material of casee may be rich starch or other edible paper. In the case of fillers, 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. Formulations of the capsule administration material may also be powders, lightly pressed flocs or tablets. Therapeutic agents may be prepared by compression.
Both pigments 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 pigments and flavoring agents.
The inert material may dilute or increase the volume of the therapeutic agent. These diluents can include carbohydrates, in particular mannitol, 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 carboxymethylcellulose, ultramilfectin, 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 the layer between the therapeutic agent and the die wall, 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.
A lubricant may be added during formulation to enhance the flow properties of the drug and aid in rearrangement during pressurization. The lubricant may 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, octanyl 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 cured castor oils 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 absorption of the polypeptide (or derivative) 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 mammalian lung during inhalation to coat the mucosal surface of the bubble. This is included in the following literature: 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 Plaz 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.
It is contemplated to use various machinery designed for pulmonary delivery of therapeutic agents, 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, for example, conventional materials such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or trichlorofuluromethane, dichlorodifuluromethane, dichlorotetrafluoro Hydrocarbons including ethanol and 1,1,1,2-tetrafluoroethane or mixtures thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid is useful as a surfactant.
Formulations for dispensing from powder inhalers will include finely divided dry powders containing polypeptides (or derivatives), and also to facilitate dispersion of the powder from the device with a bulking agent such as lactose, sorbitol, sucrose or mannitol (Eg from 50 to 90% by weight of the formulation). Proteins (or derivatives) should be prepared in the form of particulates most advantageously less than 10 mm (or microns), most preferably 0.5 to 5 mm in average particle size, for the most efficient delivery to the distant lungs.
Intranasal delivery
Intranasal or pharyngeal delivery of polypeptides (or derivatives) may also be 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.
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.
Experiment detail
Example 1 Peptide Trunnions of Choline Binding Protein A (CbpA)
Polypeptides are generated comprising the truncated N-terminal fragment of CbpA (serum type 4). 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 '. Such altered primers are designed from the amino acid sequence XENEG, which introduces both BamHI and NcoI restriction sites and ATG start codons. 3 'reverse primer ST537 = 5' GCC GTC TTA GTT TAC CCA TTC ACC ATT GGC 3 '. This primer introduces a natural stop codon from the SaLL restriction position and CbpA for cloning purposes and is based on two forms of 4 and R6x sequences.
PCR products are generated using primers SJ533 and SJ537 amplified 30 cycles using High Fidelity enzyme (Boehringer Mannheim) at annealing temperature 50 ° C. as a template from genomic DNA. The resulting PCR product was purified using the QIAquich PCR Purification Kit (Qiagen, Inc.), then digested using BamHI and SaLL restriction enzymes, and digested with pQE30 expression vector (Qiagen, Inc.) with BamHI, XbaI and SmaI restriction enzymes. .)
Polypeptide R2:
The native PvuII position at the end of the second repeating region, ie the C region (nucleic acid 1228 of type 4 sequence) as shown in FIG. 1, is used to generate a cleavage modification of the cbpA conservator. To generate cleavage clones, full-length clones PMI580 (type 4) or PMI581 (R6x) are digested using PvuII and XbaI and the resulting fragments ligated into the expression vector, PQE30, and transformed into suitable hosts. The protein is expressed and purified. The stop codon used by the expression vector in this case is the insert downstream and the expressed protein is larger than the expected size of the insert 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, the native XmnI position 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 two normalized fragments are ligated, transformed into E. coli. And clones are screened for inserts. One positive clone is selected and the recombinant protein is purified from the stains described above.
All polypeptides are expressed and purified using 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
To prepare and purify the recombinant protein from this coli, a single colony is selected from smeared bacteria containing the recombinant plasmid 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 in 1 L LB with this concentration of antibody. 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 output 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, Upsula 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) as the final desired buffer was run over 100 ml total volume. Protein was produced in ˜30% buffer B. The resulting peaks are accumulated and stored.
For the recontact layer, dialysis is carried out with a volume of 2 L 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 Coomassie stained 4-20% SDS-PAGE gel (FIG. 3).
Example 2: Lectin Activity of Polypeptides R1 and R2
LNnt is a carbohydrate homologue of the receptor for pneumococci present on eukaryotic cells. It is known that CbpA incomplete pneumococcal mutants cannot attach to eukaryotic cells or immunized sugars known as CbpA's attachment ligands. CbpA is a regulatory protein that can be divided into two regions: the N-terminal functional region and the C-terminal choline attachment region (FIG. 1). Polypeptides R1 and R2 are analyzed for biological activity to determine whether complete CbpA activity is present at a specific N terminus (eg R2) or fragment thereof (eg R1). It is determined whether only the N terminal region (R2) has biological activity for lectin binding without the choline binding site (CBD). This is experimented with CbpA total length and polypeptide R2 (fragment with a loss of CBD site under the Pvu II site in the proline enrichment region).
Assays are performed in coat tissue culture wells using glucoconjugates 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 CbpA polypeptide R2 or polypeptide R1 over 15 minutes (0.8 μg / min) followed by the addition of fluorescein labeled with R6 pneumococci over 30 minutes without washing and washing. Count the attached bacteria with your eyes.
Attaching R6 without adding any peptide was a positive control and it was calibrated at 100% (Table 1). In three separate experiments, the CbpA total length or polypeptide R2 competitively inhibited the attachment of pneumococcal to the LNnT coating surface. CbpA total length is inhibited by 71, 64% and 63% controls: polypeptide R2 is inhibited by 65%, 53% and 74% controls. Equivalent activity with CbpA and R2 does not require the LNnT lectin activity of CbpA at the choline binding site and indicates that LNnT lectin is superior to R2.
In contrast to binding to LNnT, attachment of pneumococcal to 3 ′ sialyl lactose inhibits with R2 (79 and 101%) compared to total 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 laminating the polypeptide into the functional region is affected by the length of the composition and the polypeptide. Some sequence modifications are found in other strains (see FIG. 2). When the homogeneity of the sequence 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 adhesion to purified glycoconjugates between R6 pneumococci by soluble CbpA formsLNnT 3 'sialyl lactose Cbp form Pneumococcal count (SD) per monolayer Control group (%) Number of pneumococci per monolayer % Control Per Well Peptide-free 32822421 (489) 2210 (350) 100% 26112115 (125) 100% CbpA of total length 20751740 (167) 1415 (50) 63, 71, 64 19331405 (240) 7466 Polypeptide R2 24611288 (672) 1440 (530) 74, 53, 65 26391670 (420) 10179 Polypeptide R1 30022245 (182) 2500 (310) 91, 92, 112 10521445 (526) 4068
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 include surface molecules that include hydrocarbons (glycoproteins and glycolipids) and bacteria are attached to glycojugates by these hydrocarbons, despite being very different protein and lipid backbones. Therefore, bacteria, including polypeptides having bacterial activity in vivo, can be attached to human cell surface. This direct association between the activity of nectins and cell binding properties in vivo is known for pneumococci. For example, LNnT competitively inhibits the attachment of pneumococcal to TNF activated by A549 human lung cells and inhibits the progression of pneumonia in vitro. To establish that the lectin activity of the CbpA cleavage site reflects cell binding activity, it is tested whether CbpA and the cleavage site inhibit the attachment of pneumococcal to the lung cells (Table 2). Total length CbpA and polypeptide R2 inhibit the attachment of pneumococci to ischemic cells competitively for 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.
Binding of R6 Pneumococcal to TNF-activated Human Lung CellsA549 lung Cbp form Number of pneumococci per monolayer (average) Control% Peptide-free 697, 704, 674702, 722 (700) 100% Total length CbpA 376, 431 (403) 58% Polypeptide R2 517, 693314, 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 comprises 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 regions to viability R1, including only region A is compared with R2 and total length CbpA. In the adhesion assay, polypeptide R1 did not convey any attachment to LNnT (unmodified 91, 92 and 112%). However, polypeptide R1 has shown to inhibit some attachment to silallyl lactose (control 68% and 40%). This indicates that polypeptide R2 is required for LNnT lectin activity and R2 is a good example for the LNnT lectin region. In contrast, R1 seems to be active for sialic acid recognition.
Antibodies to the N-terminal Region of CbpA Block Cell Binding
When binding the N-terminal region of CbpA to the cell, a conflict with N-terminal region activity will inhibit or prevent the bacteria from attaching to the cell or purified glycoconjugate. One such contact mechanism is an antibody.
Inhibition of Binding of L6 Pneumococci to LNnT Coated Surfaces by Anti-CbpA R2 AntibodiesPneumococcal count (SD) per monolayer Control% (Average) Prelmmune antibodies 198 (64). 88 (4) 100% Antibodies Against R2 Fragments 56 (11); 9 (2) 28%; 10%
LNnT coated wells are added for attachment analysis after preincubation for 6x30 min at 5μl + 2 x 10 ⁷ R6 5μl x RT undiluted rabbit antibody. Perform two different experiments.
Antisera highlighted in the recombinant N-terminal region of CbpA (R2) is tested for the ability of pneumococcal to inhibit adhesion to LNnT. Rabbit polyclonal anti CbpA antiserum (5 μl) and 5 μl of labeled bacteria 2 × 10 ⁷ are incubated for 30 minutes at room temperature. The mixture is added dropwise onto immobilized LNnT for 30 minutes and then washed three times with PBS to remove unbound bacteria. The bacteria bound to the plate are counted under the microscope 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 titration curves of preimmune versus anti-CpbA R2 antibodies for inhibition of attachment to pneumococcal R6x to the illustrated receptor LNnT. Pneumococcal adhesion above 70% is inhibited 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 strain used in the adhesion inhibition assay is extracted from serotype 2. The ability of the antibody to block the attachment of heterologous serotype bacteria means that there is cross-protective activity across the serotypes. This activity is highly desirable for effective vaccine immunogens.
Antibody Activity Against the Original Structure of CbpA N-terminus
As described in a paper by Rosenow et al., A choline affinity column can be used to purify CbpA from a natural host, Pneumococcus. Alternatively, polyhistidine tags can be engineered at the ends of the genes so that the transcribed protein is extended by several histidine residues. These residues facilitate purification of the full length polypeptide by nickel affinity matrix purification, in contrast to allowing shorter truncates to maintain their natural structure. CbpA, specifically purified from pneumococcal coli (E. coli) or other host bacteria by this biochemical method, retains its natural structure. CbpA, a naturally occurring double friend, produces antibodies when used as immunogens, which are potentially different from those induced by immunization with truncates that may overlap in other ways. Likewise, CbpA used as a therapeutic agent may have a different structure than the cleavage branch, which may enhance the ability to block adhesion. Given these considerations, it may be advantageous to biochemically cleave the C terminus (CBD) by producing CbpA as a full-length protein that overlaps its native tertiary structure. 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 portion 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 defective-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 antibody, the ability of the absorbed antiserum to block the attachment of pneumococcal to the model receptor LNnT was measured. R6x pneumococci were incubated with antiserum diluted 1: 600 and then placed in wells coated with LNnT albumin.
Block adhesion of absorbed anti-CbpA antiserum Antiserum (1: 600) Pneumococcal pneumoniae 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%)
These results indicate that antibodies to the native Cbp / Adml N terminal region strongly block adhesion. This activity is greater than the cleavage branch activity of FIG. 5 which is inactive at 1: 600 dilution. This activity of the absorbed anti-CbpA antiserum is further demonstrated by the titration study of FIG. 5. Baseline attachment of pneumococcal type 4 to LNnT-coated wells is indicated by a triangle. Pre-incubation of pneumococcal pneumoniae and unabsorbed (square) or absorbed (rhombic) antiserum at various dilutions meant a reduction in adhesion. The fact that the two antisera showed a similar decrease in adhesion demonstrates that most of the blocking activity of the antibody against CbpA is at the N-terminus (ie removing the antibody to the choline binding region by absorption) Does not reduce sex).
Example 3: Passive Protection with Anti-R2 Antiserum
Immune Antibody Production in Rabbits
Rabbit immune antibodies against polypeptides R2 (CbpA truncated branches) and CbpA were produced in Covants (Denver, PA). After collecting pro-immune antibodies, 250 ug of R2 in Complete Freund's Adjuvant, containing both amino acid terminal repeats (manufactured 483: 58 above), were immunized to New Zealand white rabbits. The rabbit was infused with 125 ug increments of R2 in Incomplete Freund's Adjuvant on day 21 and bled on day 31. Similarly, the second rabbit was immunized with purified CbpA.
Manual protection on mouse
100 μl of pre-immune serum in 1: 2 diluted rabbit anti-R2 or sterile PBS (pre-immune and 31-day immune serum) were 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 pre-immune rabbit serum died within 7 days.
This data shows that CbpA specific antibodies protect systemic pneumococcal infection. The data also show that choline-binding sites are not essential for protection, since antibodies specific for polypeptide R2, a truncated branched protein without choline binding repeat retention, were also sufficient for protection. In addition, sera against CbpA of serotype 4 showed protective effect against serotype 6B inoculation.
Example 4 Active Protection with Anti-R1 Antisera
CbpA cleaved branched protein R1 (15 ug in 50 ug PBS, plus 50 ul of the Complete Prepond Adjuvant) was immunized by intraperitoneal administration of C3H / HeJ mice (10 / 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 ug protein and Incomplete Prep 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 cleavage branch titer of serum from 10 CbpA immunized mice was 4,096,000. No antibody was detected in serum from gastric-immunized mice. At 10 weeks mice were inoculated with 560 CFU Streptococcus pneumoniae serotype 6B (SPSJ2P strain, provided by P. Pflin at St. Jude Children's Hospital in Memphis, Tennessee). Survival of the mice was monitored for 14 weeks. 80% of mice immunized with CbpA cleaved branched protein R1 survived. All gastric-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. This data also shows that choline-binding sites are not essential for protection because the immunogen used is the truncated branched 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 because recombinant pneumococcal protein was generated based on serotype 4 DNA sequence and a protective effect was observed after inoculation with serotype 6B isolates.
Example 5 Prevention of Colony Formation in Nasopharyngeal Rats
In vitro, the N-terminal region of CbpA competitively inhibited pneumococcal adhesion. To demonstrate the therapeutic utility of peptides having this activity, colonies in the nasopharynx were measured after inoculation of pneumococcal pneumoniae after administering truncated branched peptides to pups.
10 ul of PBS containing 0.8 ug of 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 adhesion and colony formation, the number of pneumococcal strains found after nasal wash at 72 hours was quantified for each 4 animals. Rats receiving SIII alone showed 2200, 6500, 6900 and 8700 (average 6075) colonies per 10 ul. Animals treated with truncated branches R2 had the lowest bacterial count (3600, 3500, 2500, 2100) with an average of 2925 per 10 ul. Animals treated with truncated branches R1 also showed reduced colony formation (5000, 4800, 3500, 1600) with an average of 3725 (61% control).
This experiment shows that administration of the peptides of the present invention to animals of therapeutic study has a protective effect against subsequent pneumococcal inoculation.
Argument
As demonstrated by the experiments, polypeptide R2 is a preferred composition as a vaccine formulation because it 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 adhesion of pneumococcal conjugates and is therefore a preferred composition for preventing and treating colony formation or invasive diseases. In addition, CbpA truncated branches function as lectins without CBD. Two carbohydrates are recognized: LNnT recognition by peptides containing both N-terminal repeats (A and C) (FIG. 1) and sialic acid by peptides containing only a single N-terminal repeat (A). . Polypeptides R1 and R2, which are cleavage branches containing N-terminal repeats, also exhibit lectin activity in cell culture assays.
Important properties of polypeptide R2 activity are: 1) complete correlation of polypeptide R2 and full-length CbpA bioavailability for recognition of purified glycoconjugate receptors in lung cells of animal models. Lose. 2) Cross-protective properties between type 4 inducers and bacteria in in vitro tests 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 that it relates to the disclosure.
[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
130 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

权利要求:
Claims (53)
[1" claim-type="Currently amended] An isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage.
[2" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence comprises a fragment, mutant, variant, homologue or derivative of the polypeptide as set forth in SEQ ID NO: 1.
[3" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence is shown in SEQ ID NO: 3, comprising a fragment, mutant, variant, homologue, or derivative of the polypeptide.
[4" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence is set forth in SEQ ID NO: 6.
[5" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence comprises a fragment, mutant, variant, homologue or derivative of the polypeptide as set forth in SEQ ID NO: 7.
[6" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence comprises a fragment, mutant, variant, homologue, or derivative of the polypeptide as set forth in SEQ ID NO: 9.
[7" claim-type="Currently amended] An isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage having the amino acid set forth in SEQ ID NO: 24, and showing a tertiary structure.
[8" claim-type="Currently amended] 8. The isolated polypeptide of claim 7 wherein the tertiary structure corresponds to the tertiary structure present in the native protein.
[9" claim-type="Currently amended] The isolated polypeptide of claim 7, wherein the polypeptide is produced by cleaving full-length choline binding protein A with hydroxylamine, which cleaves choline binding protein A at amino acid 475 to form an N-terminal choline binding protein A cleavage. Polypeptide.
[10" claim-type="Currently amended] An isolated homologue of the polypeptide of claim 1.
[11" claim-type="Currently amended] The isolated polypeptide of claim 10, wherein the homologue comprises an amino acid sequence having an N-terminal methionine or an N-terminal polyhistidine.
[12" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein said fragment is a proteolytic product of the polypeptide.
[13" claim-type="Currently amended] An isolated polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage, wherein the polypeptide has lectin activity and does not bind choline.
[14" claim-type="Currently amended] An isolated immunogenic polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage.
[15" claim-type="Currently amended] The immunogenic polypeptide of claim 14, wherein the amino acid sequence comprises a fragment, mutant, variant, homolog or derivative of the polypeptide.
[16" claim-type="Currently amended] The immunogenic polypeptide of claim 14, wherein the amino acid sequence comprises a fragment, mutant, variant, homolog, or derivative of the polypeptide.
[17" claim-type="Currently amended] The immunogenic polypeptide of claim 14, wherein the amino acid sequence comprises a fragment, mutant, variant, homolog, or derivative of the polypeptide.
[18" claim-type="Currently amended] The immunogenic polypeptide of claim 14, wherein the amino acid sequence comprises a fragment, mutant, variant, homolog, or derivative of the polypeptide.
[19" claim-type="Currently amended] An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of an N-terminal choline binding protein A cleavage.
[20" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid comprises a fragment, mutant, variant, homologue or derivative of the nucleic acid.
[21" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid comprises a fragment, mutant, variant, homologue or derivative of the nucleic acid.
[22" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid comprises a fragment, mutant, variant, homologue or derivative of the nucleic acid.
[23" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid comprises a fragment, mutant, variant, homologue or derivative of the nucleic acid.
[24" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid is DNA.
[25" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid is cDNA.
[26" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid is genomic DNA.
[27" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid is RNA.
[28" claim-type="Currently amended] The isolated nucleic acid of claim 19, wherein the nucleic acid is operably linked to a promoter of RNA transcription.
[29" claim-type="Currently amended] A vector comprising the nucleic acid molecule of claim 19.
[30" claim-type="Currently amended] The vector of claim 29, wherein the promoter comprises a promoter of bacteria, yeast, insect or mammal.
[31" claim-type="Currently amended] The vector of claim 30, wherein the vector is a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.
[32" claim-type="Currently amended] A host vector system for producing a polypeptide comprising the vector of claim 30 in a suitable host cell.
[33" claim-type="Currently amended] The host vector system of claim 32, wherein the suitable host cell is a prokaryotic or eukaryotic cell.
[34" claim-type="Currently amended] A cell line comprising the nucleic acid of claim 19.
[35" claim-type="Currently amended] (a) introducing the vector of claim 19 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.
[36" claim-type="Currently amended] An antibody capable of specifically binding to the polypeptide of claim 1.
[37" claim-type="Currently amended] The antibody of claim 36, wherein the antibody is a monoclonal antibody.
[38" claim-type="Currently amended] The antibody of claim 36, wherein the antibody is a polyclonal antibody.
[39" claim-type="Currently amended] The antibody of claim 36, wherein the antibody is a chimeric (bispecific) antibody.
[40" claim-type="Currently amended] A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier or diluent.
[41" claim-type="Currently amended] A method of inducing an immune response in a patient exposed to or infected with pneumococcal bacterium, including inducing an immune response by administering to the patient the pharmaceutical composition of claim 40.
[42" claim-type="Currently amended] A method for preventing infection by pneumococcal in a patient, comprising administering to the patient the pharmaceutical composition of claim 40 effective to prevent pneumococcal infection.
[43" claim-type="Currently amended] The method of claim 42, wherein the pharmaceutical composition is delivered to the airway or nasopharynx.
[44" claim-type="Currently amended] A method of preventing infection by pneumococci in a patient, comprising administering to the patient a pharmaceutical composition comprising the antibody of claim 36 and a pharmaceutically acceptable carrier or diluent.
[45" claim-type="Currently amended] A vaccine comprising the polypeptide of claim 1 and a pharmaceutically acceptable adjuvant or carrier.
[46" claim-type="Currently amended] 46. The vaccine of claim 45, wherein the polypeptide has an amino acid sequence as set forth in any of SEQ ID NOs: 1, 3 to 7, 9 to 11, 22, and 23.
[47" claim-type="Currently amended] 46. The vaccine of claim 45, wherein the polypeptide comprises the amino acid sequence of the N-terminal choline binding protein A cleavage set forth in FIG.
[48" claim-type="Currently amended] A vaccine comprising a polypeptide having an amino acid sequence comprising the conserved region set forth in FIG. 2 and a pharmaceutically acceptable adjuvant or carrier.
[49" claim-type="Currently amended] 49. The composition of claim 48, wherein the conserved region is comprised of the amino acid sequences 158-172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; And a vaccine selected from 430 to 447.
[50" claim-type="Currently amended] A vaccine comprising an isolated nucleic acid encoding a polypeptide of claim 1 and a pharmaceutically acceptable adjuvant or carrier.
[51" claim-type="Currently amended] A vaccine comprising the isolated nucleic acid of claim 19 and a pharmaceutically acceptable adjuvant or carrier.
[52" claim-type="Currently amended] A vaccine comprising the vector of claim 29 and a pharmaceutically acceptable adjuvant or carrier.
[53" claim-type="Currently amended] 52. A method of treating a patient infected with or exposed to pneumococcal, comprising treating the patient by administering to the patient a therapeutically effective amount of the vaccine of any of claims 45-51 or 52.

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AU3479799A|1999-10-25|

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JP4739521B2|2011-08-03|

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TR200003498T2|2001-10-22|

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NO20005081L|2000-12-06|

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WO1999051188A2|1999-10-14|

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BR9909910A|2002-01-15|

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NO20005081D0|2000-10-09|

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WO1999051188A3|2001-04-19|

引用文献:

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法律状态:
1998-04-07|Priority to US9/056,019

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1998-04-07|Priority to US8087898P

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1998-04-07|Priority to US09/056,019

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1998-04-07|Priority to US60/080,878

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1998-04-07|Priority to US09/056,019

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1999-04-07|Application filed by 세인트 쥬드 칠드런즈 리써치 호스피탈, 메디뮨 인코포레이티드

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2001-06-25|Publication of KR20010052244A

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2006-10-26|Application granted

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2006-10-26|Publication of KR100638503B1

优先权:

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申请号 | 申请日 | 专利标题

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US8087898P| true| 1998-04-07|1998-04-07|

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US09/056,019|1998-04-07|

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US60/080,878|1998-04-07|

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US09/056,019|US6858706B2|1998-04-07|1998-04-07|Polypeptide comprising the amino acid of an N-terminal choline binding protein a truncate, vaccine derived therefrom and uses thereof|

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US9/056,019|1998-04-07|