Immunogenic Compositions of Polysaccharide-Protein Pegylated Compounds
20230115072 · 2023-04-13
Assignee
Inventors
Cpc classification
A61K2039/6037
HUMAN NECESSITIES
International classification
Abstract
The disclosure describes compositions containing PEGylated compounds using linkers, bivalent polysaccharide covalent PEG compounds, and methods of bivalent polysaccharide-PEG compounds in the development of multivalent vaccines. PEGylated conjugation of capsular polysaccharides to carrier proteins is carried out using homo-bifunctional and/or hetero-bifunctional linkers of specific lengths. Incorporation of bifunctional PEG linkers induces higher titers of functional antibodies with high avidity, eliciting higher immunologic memory, and reduced carrier protein effect. This provides immunochemically cross-reactive capsular polysaccharides wherein one or more cross-reactive capsular polysaccharides are covalently PEG compounded sequentially or concurrently to carrier protein using bifunctional linkers bearing the same or different functional groups. Such a linker and the size of the capsular polysaccharides provides an effective multivalent vaccine with high antibody titers and a reduced carrier effect, with a reduction in the content of the capsular polysaccharide and protein per dose of vaccine which reduces reactogenicity.
Claims
1. An immunogenic complex comprising a first group of monovalent capsular polysaccharides and a second group of bivalent or multivalent capsular polysaccharides wherein: the first group of monovalent capsular polysaccharides comprise polysaccharides of S. pneumoniae serotypes 1, 2, 3,4, 5, 6A, 6B, 6C, 6D,7F, 8, 9V, 9N, 9A, 9B,10A, 11A, 12F, 14, 15B, 15A, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B; and the second group of bivalent or multivalent capsular polysaccharides comprises polysaccharides of S. pneumoniae serotypes 6A/6B/6C/6D, S. pneumoniae serotypes 9V/9N/9A/9B, S. pneumoniae serotypes 15B/15A/15C, or S. pneumoniae serotypes 19A/19F, wherein: the first group of monovalent capsular polysaccharides are each covalently coupled to a PEG linker and a first carrier protein, and the second group of bivalent or multivalent capsular polysaccharides are each covalently coupled to another PEG linker and another carrier protein.
2. The complex of claim 1, wherein the bivalent or multivalent capsular polysaccharides comprises two immunologically cross-reactive serotypes of S. pneumoniae.
3. The complex of claim 2, wherein the second group of bivalent or multivalent capsular polysaccharides comprises a structure polysaccharide-PEG-carrier protein-PEG-polysaccharide.
4. The complex of claim 2, wherein the bivalent or multivalent capsular polysaccharides are covalently coupled to the another carrier protein sequentially or concurrently.
5. The complex of claim 1, wherein the first group of monovalent capsular polysaccharides and/or the second group of bivalent or multivalent capsular polysaccharides comprise capsular polysaccharides of from about 10 kDa to about 50 kDa, from about 30 KDa to about 100 KDa, and/or from about 100 KDa to about 300 KDa.
6. The complex of claim 1, wherein the second group of bivalent or multivalent capsular polysaccharides comprise the structure 6A-PEG-CRM197-PEG-6B.
7. The complex of claim 1, wherein the carrier protein and/or the another carrier protein comprises tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragments (TTHc), N. meningitidis protein PorB, RSV virus proteins, B. pertussis proteins, Pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69 KDa protein, Human Papilloma viral protein antigens, Human Papilloma virus VLP forms, Hepatitis B virus core antigen, Hepatitis B virus VLP forms, derivatives of HBsAg, and/or combinations thereof.
8. The complex of claim 1, which comprises 4 micrograms or less of total polysaccharides per dose.
9. The complex of claim 1, wherein the carrier protein and the another carrier protein comprise from about 0.5% to about 0.7% by weight.
10. The complex of claim 1, which comprises about equal amount by weight of capsular polysaccharides to total carrier protein.
11. The complex of claim 1, which comprises a greater amount by weight of capsular polysaccharides to total carrier protein.
12. The complex of claim 1, further comprising of at least one adjuvant.
13. The complex of claim 12, wherein the adjuvant is selected from the group consisting of aluminum salt, calcium phosphate, a liposome of monophosphoryl lipid A (MPLA), saponin QS-21, a TLR7/8 agonist, and combinations thereof.
14. The complex of claim 13, wherein the aluminum salt is selected from the group consisting of aluminum phosphate, aluminum sulfate and/or aluminum hydroxide.
15. The complex of claim 1, comprising one or more serotypes of S. pneumoniae, H. influenza type a or b; S. pneumoniae, Group B Streptococcus, N. meningitis or combinations thereof.
16. The complex of claim 1, wherein the first group of monovalent capsular polysaccharides and/or the second group of bivalent or multivalent capsular polysaccharides are derived from Haemophilus influenzae serotypes a/b/c/d/e/f, non-typeable Haemophilus influenzae (NTHi) polysaccharides, Moraxella catarrhalis Lipooligosaccharides(LOS) or combinations thereof.
17. The complex of claim 1, wherein the first group of monovalent capsular polysaccharides and/or the second group of bivalent or multivalent capsular polysaccharides comprises capsular polysaccharides of N. meningitis serotypes A, B, C, Y, W-135 or X.
18. The complex of claim 1, wherein the first group of monovalent capsular polysaccharides and/or the second group of bivalent or multivalent capsular polysaccharides comprise capsular polysaccharides of Group B Streptococcus serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, IX, or N.
19. The complex of claim 1, wherein the first group of monovalent capsular polysaccharides is approximately equivalent by weight to and/or the second group of bivalent or multivalent capsular polysaccharides.
20. The complex of claim 1, which, upon administration to a subject, generates a lower immune response to carrier protein in comparison to monovalent conjugates comprised of the same capsular polysaccharides.
21. The complex of claim 1, which provides effective treatment or prevention of infection by Gram-positive and Gram-negative bacteria.
22. The complex of claim 1, comprising a therapeutically effective amount and a pharmacologically acceptable carrier.
23. The complex of claim 1, comprising capsular polysaccharides of Haemophilus influenza, N. meningitis, Group B Streptococcus, N. meningitis, H. influenza, and combination thereof.
24. The method for manufacture of the complex of claim 1, comprising: activating capsular polysaccharides of the first group of monovalent capsular polysaccharides and the second group of bivalent or multivalent capsular polysaccharides; coupling a PEG spacer to the activated capsular polysaccharides, wherein the PEG spacer is about 2.0 Å to about 40 Å; and coupling activated polysaccharide-PEG spacers to carrier proteins.
25. The method for manufacture of the complex of claim 1, comprising: activating carrier proteins to form activated carrier proteins; reducing a disulfide of each carrier protein to create a sulfhydryl group; coupling PEG spacers to the activated carrier proteins; and coupling capsular polysaccharides to the PEG spacers coupled with activated carrier proteins.
26. The method of claim 25, wherein the activated carrier proteins are selected from the group consisting of cross-reactive material (CRM197) obtained or derived from C. diptheriae, and recombinant CRM197 obtained or derived from P. fluorescens or E. coli.
Description
DESCRIPTION OF THE FIGURES
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DESCRIPTION OF THE INVENTION
[0061] Streptococcus pneumoniae is a Gram-positive bacterium which can cause diseases such as pneumonia, bacteraemia, meningitis, and acute Otitis media. Pneumococcus is encapsulated with a chemically linked polysaccharide which results in serotype specificity. At least 90 pneumococcal serotypes are known of which about 23 account for 90% of invasive diseases. The protection against invasive pneumococci disease is related to the antibody specific to the capsular polysaccharide, the protection is therefore serotype specific.
[0062] It was surprisingly discovered that multivalent S. pneumoniae vaccines comprising of a PEG linker between the carrier protein and the polysaccharide to form two groups of compounds, wherein group one comprises monovalent bacterial capsular polysaccharide PEG compounds and the other group comprises bivalent and/or multivalent carrier protein compounds to provide substantially improved results. Specifically, the bivalent or multivalent compounds and bivalent uni-molecular compounds are preferably synthesized by the reaction between carrier protein and bifunctional PEG linkers attached to cross reactive S. pneumoniae serotypes. Results achieved are enormously improved compared to vaccines containing multivalent S. pneumoniae vaccine containing monovalent bacterial capsular polysaccharide coupled with the same number of serotypes with a direct conjugation between the two instead of a linker.
[0063] The present disclosure is directed to multivalent PEGylated compounds, of immunogenic compositions, and vaccines comprising carrier protein compounded to bacterial capsular polysaccharides using PEG linker and uses thereof.
[0064] The linker is used to PEGylate both polysaccharide as well as protein by connecting to PEG via two hydrazine functional groups cable of covalently compounding with both carrier protein as well as polysaccharides. This creates a new class of covalently compounded PEG products that have the additional effect of PEG on their properties compared to conjugates made by established methods. PEG has an additional enhancing effect on the immunogenicity of polysaccharides compared to regular conjugates and a depressing effect on the Immune response of carrier proteins. As the compounds of the invention contain polysaccharide coupled to PEG which is coupled to carrier, there is no conjugation between polysaccharide and protein.
[0065] This disclosure provides a universal method of covalent PEGylated compounds with a high immune response which is unaltered in spite of increase in serotypes. This unexpected beneficial observation is critically important in developing immunogenic compounds such as vaccines.
[0066] Protection against pneumococcal disease is obtained by antibodies produced against the polysaccharide component. By PEGylation, the response observed is twice that of PREVNAR®. This means that the high antibodies observed after administration of the PEGylated form of the vaccine will fall slowly, much more slowly that the rapid reductions observed with PREVNAR®. This is an entirely unexpected and extremely beneficial outcome. This result eliminates any need of a third injection saving cost as well as pain to infants and others caused by multiple injections, and in addition, makes protection more widely available, especially for those unable to return for repeated injections.
[0067] In particular, compositions of the invention comprise two or more bacterial capsular polysaccharides covalently connected to same protein to form PEGylated compounds, of various serotype antigens wherein the bacterial capsular polysaccharides and oligosaccharides are derived from serotypes of Streptococcus pneumoniae. The carrier protein is covalently connected to bacterial capsular polysaccharides through mono functional as well as bi-functional PEG linkers, preferably of defined lengths and the bi-functional linkers are, homo-bi-functional (subgroup Serotypes like 6A-6B or 19A-19F, and/or all other serotypes).
[0068] One embodiment of the invention is directed to multivalent covalently connected compound vaccines comprised of bivalent-polysaccharide protein compounds with enhanced immunogenicity. Bivalent compounds with general structure PS1-PEG-carrier protein-PEG-PS2 have higher immunogenicity compared to similar monovalent conjugates wherein PS1 and PS2 are two different serotype polysaccharides from gram-negative and gram-positive bacterial pathogens. By developing a bi-valent covalently compounded vaccine, the efficacy of the vaccine increases and carrier immunogenicity is reduced. The chemistry disclosed herein substantially increases immunogenicity, at the same time reduces carrier protein load.
[0069] Another embodiment of the invention is directed to vaccines with lower molecular weight polysaccharides and longer arm bifunctional linkers preferably with enhanced immunogenicity. Another embodiment of the invention is directed to providing higher immunogenicity and avidity of bivalent compounds as well as lower carrier protein immunogenicity. Another embodiment of the invention is directed to reducing covalently compounded vaccine dose with higher immunogenicity.
[0070] As disclosed herein, four parameters have been introduced to minimize the disadvantages of conventional vaccines: [0071] Polysaccharide size is preferably 10-50 KDa. [0072] Cross-reactive polysaccharides concurrent covalent connection to carrier protein. [0073] Two or more cross reactive serotypes are covalently compounded concurrently with carrier proteins. [0074] A long hetero- or homo-bifunctional PEG spacer arm is preferably from 2-40 Å (but may be from 2-40 Å, 4-40 Å, 10-40 Å, 20-40 Å, 9-20 Å, 5-20 Å, 5-30 Å).
[0075] These four parameters taken together are profoundly effective to increase the polysaccharide/protein ratio in the covalent compound to reduce carrier protein load, and to provide several folds of increase in immunogenicity and avidity.
[0076] The present invention is directed to polysaccharide-protein PEG compounds with enhanced immunogenicity displaying significantly high antibody titers. The carrier protein is obtained from, for example, tetanus toxoid, diphtheria toxoid, CRM197, tetanus toxoid fragments (TTHc), N. meningitidis protein PorB, RSV virus proteins, B. pertussis proteins like pertussis toxoid (PT), adenylate cyclase toxin (ACT), 69 KDa protein and Human Papilloma viral protein antigens or its VLP form, Hepatitis B core antigen or its VLP form or derivatives of HBsAg, and other conventional carriers. Polysaccharide fragment is obtained from group of group of gram positive bacteria and gram-negative bacteria, preferably from immunochemically cross-reactive polysaccharides of S. pneumoniae. The present invention is also directed to a process of preparing the polysaccharide-protein covalent PEG compound in which carrier protein reacts with cleaved and depolymerized polysaccharide fragments of optimum chain length.
[0077] Immunogenic compositions of the present invention provide improved protection against S. pneumoniae serotypes not found in PREVNAR-13®, and SYNFLORIX-10®.
[0078] Bivalent compounds with cross-reactive polysaccharides of S. pneumoniae serotypes (6A/6B, 9V/9N, 15A/15B and 19A/19F and similar cross-reactive serotypes) with short chain molecular size (10-50 KDa) were used to prepare 16-26-valent pneumococcal CPS PEG covalent compound vaccine in the present study. Pneumococcus type 6A and 6B polysaccharide was used as the model cross-reactive CPSs. CRM197 was used as the carrier protein for its clinical acceptance.
[0079] Multivalent mono-compounds have also been prepared using shorter PS chain length (0-50 KDa), long spacer arm (9-40 Å) with homo or hetero-bifunctional PEG or non-PEG linker with carrier protein CRM197.
[0080] CPS was activated either by oxidation or by cyanylation chemistry and oxidized by sodium periodate and introduced with either -reactive aldehyde or isothiocyanate (-OCN) groups in CPS.
[0081] Two strategies (short and long linker, short and long CPSs) were used to introduce, respectively. Physicochemical and immunological characteristics of the bivalent covalent compound vaccines were then investigated independently or combining with multivalent compound formulation.
[0082] The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.
EXAMPLES
Example 1 Polysaccharide Size Reduction, Activation and PEGylation Compounding Process for Multiple S. Pneumoniae Serotypes -1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F and 35B.
6A and 6B Polysaccharide
[0083] 100 mg each of capsular polysaccharides of S. pneumoniae 6A and 6B is dissolved in 10 ml of aqueous solution containing 10 mM of Acetic acid or 0.1 M HCl at pH 2.5-3.0 and hydrolysis is carried out by maintaining the solution at a temperature of 60-85° C. for a period of 60-120 mins. The so-obtained oligosaccharides after neutralization, diafiltered using 3-10 KDa TFF Centricon filters. Upon .sup.1H NMR analysis (
[0084] CPS (50 mg) moiety (native polysaccharides of size between ≥200-500 KDa or size -reduced polysaccharides of size between 10-50 KDa) were activated cyanylation reagents commonly used in activation process (Table 1). Polysaccharide molecular size distributions were determined using SEC-HPLC (Shodex SB-405 and SB-406 SEC columns) with analysis using (10-1000 KDa) Pollulan mixture as reference standard (Pollulan standards from Shodex, USA).
[0085] Short spacer arm was introduced to PS by reaction with 5-8-fold molar excess of ADH (Sigma) at pH 5.6-6.0 for 3-5 hr. Long spacer arm (bifunctional linker or long 4-arm linker) was introduced into PS by reaction with 5-10-fold molar excess of at pH 5.6-6.0 for 3-5 h.
TABLE-US-00001 Polysaccharide size distribution (KDa) used for conjugation PS Polysaccharide KDa 6A 10-30 KDa 6B 20-50 KDa 15B 20-40 KDa 18C 20-50 KDa 22F 10-30 KDa
[0086] Activated PS is further derivatized with short arm linker (adipic acid di-hydrazide, ADH, 174.2 g/mole), one more spacer arm linkers with varying size from 2-4 Å to 8-20 Å (600 g/mol-3.5 g/mole).
[0087] Homo -bifunctional PEG linkers with diamine functional groups attached, e.g. NH.sub.2-PEG0.6K-NH.sub.2, NH.sub.2-PEG3.5K-COOH (Table 2).
TABLE-US-00002 Short and long chain linker used for polysaccharide or carrier protein derivatization used (several other linkers either in pegylated form or non-pegylated form have also been used Linker No. Linker Structure Chemical Structures/KDa or A used 1 NH.sub.2-PEG- NH.sub.2/NHS H.sub.2N-(CH.sub.2CH.sub.2O).sub.N-CH.sub.2CH.sub.2-NH.sub.2 1K and 3.5K 2 NHS/NH.sub.2- PEG--COOH
[0088] Two aliquots of 2 ml each of the derivatized CPS (10 mg/ml) were mixed with 1 ml aliquot of the two CRM197 protein samples (10 mg/ml) at 4° C. for 8-12 hrs. The compounds with long and short spacer arm were purified by a 100-300 KDa Centricon filters (EMD Millipore) (Table 3).
TABLE-US-00003 Physicochemical Characterization of mono-valent Compounds PS Activated PS KDa by SEC-HPLC Compounds KDa by SEC-HPLC PS: Protein ratio Free PS% 6A 10-30 KDa, 200-300 KDa >200-300, >2500 0.5-2, 1-2 <2 6B 20-50 KDa,200-400 KDa >300-500, >2500 0.5-2, 1-2 <1 15B 20-40 KDa >300-500 0.5-2, 1:1 <1 18C 20-50 KDa >300-500 0.5-2, 1:1 <2 22F 10-30 KDa >200-300 0.5-2, 1:1 <1 Note: Internal std. for KDa determination of PS for SEC-HPLC:Pullulan std. mixture (2 KDa-2500 KDa).
Example 2 Activation of Size Reduced Polysaccharides of Different Molecular Weights Oligosaccharides of Different Molecular Weights Synthesized as Described in Example 1 Were Activated Cyanylation Reagents Commonly Used in Activation Process.
[0089] CDAP Cyano-4-dimethylaminopyridinium tetrafluoroborate (Sigma Aldrich, USA)) cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) or cyanogen bromide (CNBr) and coupling carrier protein (see
[0090] Polysaccharide solution (10 mg/ml) was incubated with 10 mg/ml CDAP (100 mg/ml in acetonitrile) in 2 M NaCl or 200-300 mM bicarbonate buffer at RT for 4-6 minutes. pH was maintained at 10-10.5 using either 1 N NaOH or 1 N HCl. Then, pH was adjusted to 8.1-8.2, pegylated linkers (Hz—PEG—HZ) were allowed to react with CDAP treated PS. For 8-12 hrs at RT. The reaction mixtures were depth filtered followed by 100-300 KDa cutoff centricon fiters 5-8 times using 150 mM NaCl.
Derivatization of Activated Size Reduced Polysaccharides
[0091] Activated oligosaccharides were further derivatized with short chain homo-bifunctional PEG hydrazide linker. Typical reagent was adipic Acid di-hydrazide, ADH, Molecular weight 174.2 g/mole). Homo or hetero-bifunctional PEG linkers bearing di -amine, di-hydrazide, or amine or hydrazide-carboxylic acid/aldehyde functional groups, e.g. NH2-PEG(1 K-3.5 K)-NH2, HZ-PEG(1-3.5 K)-HZ, NH2-PEG3.5 K-COOH were used. (Table 2). Several other homo-or hetero-bifunctional spacer arms can also be used for derivatization (Table 2). Short spacer arm was introduced to oligosaccharide by reaction with 5-8 fold molar excess of adipic acid di-hydrazide (Sigma) at pH 5.8-6.0 for 3-5 hr. longF chain PEG linker (bifunctional linker or long tetra functional linker (Table 2), No. 12 four arm PEG linker) was introduced into Polysaccharide by reaction with 5-10-fold molar excess of the linker to the oligosaccharide at pH 5.8-6.0 for 3-5 hrs. at RT.
Derivatization of Carrier Protein with Short or Long-Linkers
[0092] Carrier protein CRM197 was further derivatized with short chain homo-bifunctional PEG hydrazide linker. Typical reagent was adipic Acid di-hydrazide, ADH, molecular weight 174.2 g/mole). Homo or hetero-bifunctional PEG linkers bearing di-amine, di-hydrazide, or amine or hydrazide-carboxylic acid/aldehyde functional groups, e.g., NH2-PEG(1 K-3.5 K)-NH2, HZ-PEG(1-3.5 K)-HZ, NH2-PEG3.5 K-COOH were used. (Table 2). Several other homo-or hetero-bifunctional spacer arms can also be used for derivatization as listed in Table 2). Short spacer arm was introduced to carrier protein CRM197 by reaction with 5-8 fold molar excess of adipic Acid di-hydrazide (Sigma) at pH 5.8-6.2 in 300-600 mM MES buffer for 3-5 hr at RT. Long chain PEG linker (bifunctional linker or long tetra functional linker (Table 2, No. 12 four arm PEG linker) was introduced into carrier protein by reaction with 5-10-fold molar excess of the linker to the oligosaccharide at pH 5.8-6.2 in 300-600 mM MES buffer for 3-5 hr at RT (room temperature).
Example 3 Cross-Reactive Polysaccharide Serotypes Activation and Attachment of Short or Long-PEG Spacer Arm Linkers (Serotypes of Interest are 6A/6B, 9V/9N, 15A/15B and 19A/19F or Any Other Cross-Reactive Serotypes).
[0093] Activation of the oligosaccharide derived from the capsular polysaccharide of S. Pneumoniae Type 6A and 6B covalently compounding with CRM197 and introduction of the primary amino groups to the oligosaccharides concurrently.
[0094] Native or size reduced polysaccharide of serotype 6A and 6B (≥200-400 KDa) were covalently compounded using the same procedure as described in Examples 1 and 2.
[0095] The oligosaccharides mixture thus obtained as reported in Example 1 are dissolved in WFI, to an end concentration of 10 mg/ml. At the end of the reaction, the Oligosaccharide are purified by diafiltration using 3-10 KDa Centricon filters.
[0096] The Oligosaccharides into which the amino groups have been introduced are diluted to a concentration of 10 mg/ml in an aqueous solution of DMSO (at 20-30% v/v) to DMSO containing ADH short PEG linker or long spacer arm PEG linkers in molar excess relatively to the amino groups introduced into the oligosaccharide (usually 5-10:1). The reaction was carried out by keeping the solutions at RT for a time of 4-12 hours. At the end of the period, oligosaccharide was again purified using 3-10 KDa Centricon filters.
Example 4 Synthesis of Pneumococcal Polysaccharide Monovalent PEG Compounds
[0097] Two separate aliquots of same or differently size reduced and derivatized size reduced Polysaccharides (with short PEG spacer arm ADH and Long. PEG spacer arm HZ-PEG-HZ) as synthesized in example 3 (10 mg/ml) were mixed with 1 ml aliquot of the CRM197 protein sample (10 mg/ml) at 4° C. for 8-12 hrs. The compounds containing both long and short chain linkers were purified using 100-300 KDa Centricon filters (EMD Millipore). Monovalent compounds were assayed for total polysaccharide content by either anthrone or uronic acid assay, total protein content by BCA or Lowry assay (Table 4).
[0098] All other cross-reactive Polysaccharide covalent compounds are made using the same procedure as above.
TABLE-US-00004 Physicochemical Characterization of Bi-valent Compounds of general structure 6A-CRM197-6B PS Activated Oligosaccharide KDa Covalent. compound KDa Oligosaccharide: Protein ratio (Weight ratio) Free Oligosaccharide % by weight 6A ≥100-300 KDa >200-300 KDa, >2500 KDa 0.5-2, 1-2 <2 6B ≥200-400 KDa >300-500 KDa,>2500 KDa 0.5-2, 1-2 <1 6C ≥200-400 KDa >300-500 KDa,>2500 KDa 0.5-2, 1-2 <1 15B ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <1 15A ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <1 18C ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <2 22F ≥100-300 KDa >200-300 KDa, >1000 KDa 0.5-2, 1:1 <1 1 ≥100-300 KDa >200-300 KDa, >2500 KDa 0.5-2, 1-2 <2 3 ≥200-400 KDa >300-500 KDa,>2500 KDa 0.5-2, 1-2 <1 4 ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <1 7F ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <2 9V ≥100-300 KDa >200-300 KDa, >1000 KDa 0.5-2, 1:1 <1 9N ≥100-300 KDa >200-300 KDa, >1000 KDa 0.5-2, 1:1 <1 14 >100-300 KDa >200-300 KDa, >2500 KDa 0.5-2, 1-2 <2 18C ≥200-400 KDa >800 KDa,>2500 KDa 0.5-2, 1-2 <1 19A ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <1 19F ≥100-300 KDa >300-500 KDa, >1500 KDa 0.5-2, 1:1 <2 23F ≥100-300 KDa >200-300 KDa, >1000 KDa 0.5-2, 1:1 <1 33F ≥100-300 KDa >200-300 KDa, >2500 KDa 0.5-2, 1-2 <2 Note: Internal std. for SEC-HPLC (KDa): Pollulan std mixture (2 KDa-1200 KDa)
Example 4 Investigational Formulation of 16V-or Higher Valent Pneumococcal Covalent Compound Vaccine
[0099] Pneumo polysaccharide-CRM197 covalent compounds for serotypes containing 1, 3, 5, 7F, 14, 15B, 18C, 22F, 23F, 33F, 35B and cross-reactive polysaccharide compounds 6A, 6B, 9V, 9N, 15A, 15B, 19A, and 19F were combined to yield final antigen concentration of 4.0 .Math.g PS/mL. Sodium chloride (150 mM) solution, 10-20 mM Histidine, succinic acid and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant. 16-V compound was aseptically filled in 2 mL sterile vials. PNEUMOVAX® (Merck, USA) or PREVNAR-13® (Pfizer, USA) was used as two control commercial vaccine formulation.
Example 5 Immunogenicity Studies of Covalent Compounds
[0100] A New Zealand white rabbit model (NZW) was selected in this work to compare the immunogenicity of the Pneumo PS-CRM197 covalent compounds. Rabbits from all groups (16-V {valent}, PREVNAR-13®, and PNEUMOVAX®) were examined for clinical signs before and after immunization periods. For all groups, pre-immunization, booster dose (7 and 14-days) and terminal bleed (28 days) were collected and aliquoted and store at minus 80° C. until use. Multiplexed Immunogenicity assay for the determination of Total IgG were performed according to the standard protocol using reference standard serum 007 (CBER, FDA, USA). Reference serum and rabbit serum were diluted and pre-adsorbed for cross-reacting antibodies by treatment with pneumococcal CWPS and either 22 F PS or 25 PS. Human monoclonal anti-polysaccharide antibodies (Pamlico Biopharma, USA) were used for total IgG estimation. Bio-Plex 200 (Bio-Rad). Multiplex reader was used as per manufacturer’s instructions (see
Example 5 S. Pneumoniae Cross-Reactive Capsular Polysaccharide Serotypes Activation and Attachment of Short and Long-Spacer Linkers.
[0101] Serotypes of 6A/6B, 9V/9N, 15A/15B and 19A/19F which are cross-reactive serotypes are used for the synthesis of bi-valent covalent compounds containing capsular poly saccharides and carrier protein. Bivalent covalent compounds by definition contain two capsular polysaccharide attached to CRM 197 simultaneously or concurrently.
[0102] Activation of the size reduced polysaccharide derived from the capsular polysaccharide of S. pneumoniae Type 6A and 6B, covalently compounded with CRM197 and introduction of the primary amino or hydrazide groups to the oligosaccharides carried out concurrently.
[0103] Native polysaccharides or size reduced oligosaccharide of serotype 6A and 6B (≥200-500 KDa) were covalent compounded using the same procedure as described in Example 1 -4.
[0104] The size reduced polysaccharides mixtures thus obtained were dissolved in water for injection, so that the final concentration was 10 mg/ml. The size reduced polysaccharides into which the amino or hydrazide groups were introduced were diluted to a concentration of 10 mg/ml in an aqueous solution of dimethyl sulfoxide (DMSO) so the percentage of DMSO was in the range of 20-30% (v/v). This was added to DMSO containing short chain linker such as ADH or long chain linkers as described in Table 2 in molar excess relatively to the amino/hydrazide groups introduced into the size reduced polysaccharides (usually 5:1 or 10:1), more specifically 8:1.
[0105] The reaction was carried out at room temperature for a duration of 4-12 hours. At the end of the reaction period, the reaction product was again purified using 3-10 KDa Centricon filters.
Example 6 Simultaneous or Concurrent Covalent Compounds of S. Pneumoniae Oligosaccharides of Type 6A and Type 6B with CRM197 Carrier Protein as Bivalent Compound Manufacturing.
[0106] The aqueous solution containing 15 mg/ml of CRM197, was added to DMSO containing the linker attached oligosaccharide (20-30% in water) derived from the capsular polysaccharide of S. pneumoniae Type 6A. The ratio of PEG linker attached oligosaccharide to CRM197 was selected from 1:1,2:1,1:2. The mixture so obtained was kept, under mild stirring, at room temperature for 8-12 hrs. At the end of said time, the solution containing the derivatized oligosaccharide derived from the capsular polysaccharide of S. pneumoniae 6B was added. The molar ratio of capsular polysaccharide of S. pneumoniae 6B to the CRM197, was selected from 1:1,2:1,1:2). The resulting mixture was kept for 8-12 hrs at room temperature (Table 5). The covalent compounding reaction can also be carried out by adding, at the same time (concurrently), to the CRM197-containing solution, the two -activated oligosaccharide respectively derived from the capsular polysaccharide of S. pneumoniae Type 6A and from the capsular polysaccharide of S. pneumoniae Type 6B. The oligosaccharide-protein covalent compounds so obtained were dialyzed using 100-300 KDa dialysis membrane (Spectrum lab, USA), conditioned in 0.01 M phosphate buffer containing 0.2 M NaCl (pH=6.6-7.0) and finally filtered through a 0.22 .Math.m filter.
[0107] All other cross-reactive polysaccharide compounds were made using the same procedure as used above. Reaction sequences are depicted in
TABLE-US-00005 Comparisons of PS Contents Bivalent Oligosaccharide Activated oligosaccharide KDa Covalent compounds KDa Total Polysaccharide Protein ratio by weight Free oligosaccharide % by weight 6A-6B ≥100-300 2.0:1.5 2-1.5 (1:0.75) <2 6A-6B ≥100-300, ≥300 >1200-2500 KDa 2-1.4 (1: 0.7) <3 19A-19F ≥100-300 >500-800 KDa 2-1.6 (1:0.8) <2 15A-15B ≥100-300, ≥300 >500-1000 KDa 2-1.3 (1: 0.65) <3 9V-9N ≥100-300, ≥300 >500-1000 KDa 2-1.3 (1: 0.65) <3
Example 7 Investigational Formulation of 18-Valent or Higher Valent Pneumococcal Covalent Compound Vaccine.
[0108] Pneumococcal polysaccharide-CRM197 covalent. Compounds for serotypes containing 1, 3, 5, 7F, 14, 18C, 22F, 23F, 33F, 35B (10 serotypes polysaccharides) and cross-reactive polysaccharide compounds of (6A, 6B), (9V, 9N), (15A, 15B) and (19A, 19F) (8 serotypes) were combined to yield final polysaccharide concentration of 2.2-4.4 .Math.g PS/mL (1.1-2.2 .Math.g/human dose, 0.5 mL). Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant.
[0109] 18-valent or higher valent (>20 V-24 V) covalent compound was aseptically filled in 2 mL sterile vials. PNEUMOVAX® (Merck, USA) and/or PREVNAR-13® (Pfizer, USA) were used as controls.
Example 9 Immunogenicity Studies of the Covalent Compounds
[0110] A New Zealand white rabbit model (NZW) was selected in this work to compare the immunogenicity of the Pneumococcal PS-CRM197 covalent compounds. Rabbits from all groups (18 or higher-valent compounds, PREVNAR-13®, Pfizer and PNEUMOVAX®-23 (Merck USA) were examined for serological titers before and after immunization periods. For all groups, pre-immunization, booster dose (7 and 14-days) and terminal bleed (28 days) were collected and aliquoted and store at minus 80° C. until use. Immunogenicity assay for the determination of Total IgG were performed according to the standard protocol using reference standard serum 007 (CBER, FDA, USA). Reference serum and Rabbit serum were diluted and pre-adsorbed for cross-reacting antibodies by treatment with Pneumococcal CWPS and non-vaccine serotype 25 PS. Human/rabbit/mouse monoclonal anti-polysaccharide antibodies were used for total IgG estimation. Bio-Plex 200 (Bio-Rad) reader were used as per the manufacturer’s instructions.
[0111] Immunogenicity of the compounds, i.e. capsular polysaccharide specific antibodies (total IgG) were measured using bead-based ELISA assay method were given in Table 6. Total IgG values were compared head to head with PREVNAR-13® in rabbit immunogenicity data. 14- day data shows significant increase in titer in IVT-18V-1 vaccine compared to PREVNAR-13® vaccine. Similarly, IVT-18V-1 data has significant booster on IgG values as compared to PREVNAR-13® (Table 6).
TABLE-US-00006 Capsular Polysaccharides specific antibodies (Total IgG in .Math.g/ml) using Multiplex bead-based ELISA assay for 18 V-monovalent covalent compound vaccines PREVNAR ®-13® 2.2 .Math.g/dose (IgG) 14 day/zero day (IgG) 28 day /Zero day IVT-18V-1 2.2 .Math.g/dose (IgG) 14 day /Zero day (IgG) 28 day /Zero day 1 45 350 1 375 1500 3 47 200 3 48 480 6A 188 560 6A 775 3775 6B 165 780 6B 662 3662 18C 50 280 18C 306 3560 19A 45 235 19A 233 2500 19F 29 290 19F 72 720 4 49 230 4 150 750 5 186 700 5 550 3550 7F 180 680 7F 332 3860 9V 52 520 9V 212 2400 9N - - 9N 200 2200 14 85 400 14 272 2890 15A - - 15A 672 3900 15B - - 15B 750 4000 18C 175 800 18C 550 5500 22F - - 22F 1000 8000 23F 53 450 23F 212 2420 Note: IVT-18 V == 18-V compound vaccine (monovalent covalent PEG compounds mixed together); 9N, 15A, 15B, 22F and 23F serotype are not present in PREVNAR-13®, so IgG values not measured; 18-V formulation as monovalent covalent compounds were prepared using 2.2 .Math.g for each serotype except 4.4 .Math.g of 6B covalent PEG compounds. Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant; capsular polysaccharides antibodies (total IgG) using bead-based ELISA: 18-V covalent PEG compounds vaccine formulation-2 (IVT-18V-2): 10-V formulation as monovalent covalent compounds and remaining 8-V added as bivalent-covalent PEG compounds which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F. (vaccine dose used as 2.2 .Math.g for each serotype except 4.4 .Math.g of 6B) 10-V formulation as monovalent compounds and remaining 8-V added as bivalent-compounds which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F. 6A-6B bivalent unimolecular compounds are used as 2.2 .Math.g/dose, remaining bivalent compounds are used as 2.2 .Math.g/dose. Sodium chloride (150 mM) solution, 10-20 mM histidine, 20 mM HEPES or MOPS buffer and 0.001% Tween-20 was also used during the formulation process as diluent, and aluminum phosphate (Adju-Phos, Brenntag, USA) was used as investigational adjuvant.
[0112] Immunogenicity of the covalent PEG compounds, capsular polysaccharide specific antibodies (total IgG) were measured using bead-based ELISA assay method were given in Table 7. Total IgG values were compared head to head with PREVNAR-13® in rabbit immunogenicity data. 14-day data shows significant increase in titer in IVT-18V-2 vaccine compared to PREVNAR-13® vaccine. Interestingly, IVT-18V-2 total IgG data for bivalent compounds serotypes (for example. 6A/6B, 9V/9N, 15A/15B, and 19A/19F) has significant booster on IgG values as compared to IVT-18V-1 formulation with monovalent covalent PEG compounds. Therefore, it can be concluded that Bivalent covalent PEG compounds have better immunogenicity in comparison to monovalent covalent PEG compounds (Table 7). Therefore, IVT-18V-2 covalent PEG compounds vaccine formulation has superior immunogenicity not only against PREVNAR-13® but also against IVT-18V-1 formulation. Polysaccharide covalent PEG compounds with either 1-3.5 K linker (HZ-PEG-HZ) elicits much higher immunogenicity in compared to short linker (ADH) or no linker conjugates as in PREVNAR-13®.
TABLE-US-00007 Capsular Polysaccharides antibodies (total IgG) using Multiplex bead-based ELISA PREVNAR -13® 2.2 .Math.g/dose Ratio 14 day/0 day Ratio 28 day/0 day IVT-18V-2 2.2 .Math.g/dose Ratio 14 day/0 day Ratio 28 day/0 day 1 45 350 1 375 1500 3 47 200 3 50 530 6A 188 560 6A/6B 875/762 4375/4662 6B 165 780 18C 50 280 18C 316 3600 19A 45 235 19A/19F 300/198 3500/2700 19F 29 290 4 49 230 4 180 1000 5 186 700 5 550 3600 7F 180 680 7F 360 4100 9V 52 520 9V/9N 350/300 3400/3200 9N - - 14 85 400 14 310 32000 15A - - 15A/15B 872/850 5900/5600 15B - - 18C 175 800 18C 600 6800 22F - - 22F 1020 8150 23F 53 450 23F 300 3200 Note: 1VI-18V-2 = 10-monovalent compounds and 4 bivalent compounds mixed together; 18-V compound vaccine formulation (IVT-18V-3): 10-V formulation as monovalent compounds used as 2.2 .Math.g/dose and remaining 8-V added as bivalent-compounds which includes 6A/6B, 9V/9N, 15A/15B and 19A/19F used as 1.1 .Math.g/dose, except 6B 2.2 .Math.g/dose.
[0113] Immunogenicity of the compounds, i.e. capsular polysaccharide specific antibodies (total IgG) were measured using multiplex bead-based ELISA assay method were given in Table 8. Total IgG values were compared head to head with PREVNAR-13® in rabbit immunogenicity data. 14-day data shows significant increase in titer in IVT-18V-3 vaccine compared to PREVNAR-13® vaccine. Interestingly, IVT-18V-3 formulations with lower dose (2.2 vs 1.1 ug dose), total IgG data for bivalent compounds serotypes (for example. 6A/6B, 9V/9N, 15A/15B, and 19A/19F) has comparable IgG values as compared to IVT-18V-2 formulations for bivalent compound serotypes. Therefore, it can be concluded that bivalent compounds have better immunogenicity in comparison to monovalent compounds with lower dose. Therefore, IVT-18V-2 compound vaccine formulation has superior immunogenicity not only against PREVNAR-13® but also against IVT-18V-1 formulation. Polysaccharide compounds with either 1-3.5 K linker (HZ-PEG-HZ) elicits much higher immunogenicity in compared to short linker (ADH) or no linker conjugates as in PREVNAR-13@ (Table 8).
TABLE-US-00008 Total IgG data for bivalent compounds serotypes PREVNAR ®-13® 2.2 .Math.g/dose Ratio 14 day/0 day Ratio 28 day/0 day IVT-18V-2 2.2 .Math.g/dose Ratio 14 day/0 Ratio 28 day/0 day 1 45 350 1 375 1500 3 47 200 3 50 530 6A 188 560 6A/6B 825/860 4275/4900 6B 165 780 18C 50 280 18C 316 3600 19 A 45 235 19A/19F 275/250 3400/3000 19F 29 290 4 49 230 4 180 1000 5 186 700 5 550 3600 7F 180 680 7F 360 4100 9V 52 520 9V/9N 320/380 3300/3800 9N - - 14 85 400 14 310 32000 15 A - - 15A/15B 790/900 5800/6200 15B - - 18C 175 800 18C 600 6800 22F - - 22F 1020 8150 23F 53 450 23F 300 3200 Note: 1VI-18V-3 = 10-monovalent compounds and 4 bivalent compounds mixed together.
Example 10
[0114] Table 9 shows immune response with PEG and without PEG covalent compounds.
TABLE-US-00009 13-v comparative IgG Analysis of PEGylated Componds vs. Conventional Compounds (IgG .Math.g/ml) Serotype PEGylated Conventional PREVNAR® Pneomovax 13 V 13 V 13 V 23 V 2.2 .Math.g/dose 2.2 .Math.g/dose 2.2 .Math.g/dose 25 .Math.g/dose 1 6.07 3.4 3.43 0.21 3 0.95 0.36 0.34 0.2 4 36.64 8.436 7.30 0.4 5 20.22 7.552 2.40 0.35 6A 41.44 32.38 30.35 0.5 6B 42.86 18.06 22.50 <0.4 7F 112.22 27.592 33.79 0.4 9V 36.17 3.756 4.20 0.35 14 16.34 12.3 6.06 <0.37 18C 27.72 4.5 4.26 <0.5 19A 24.72 5.773 5.61 <0.14 19F 32.86 10.384 11.05 <0.35 23F 67.9 25.31 26.86 <0.5
PEGylated compounds involve PEGylated Bis-hydrazide (1-2 kDa) - HZ-PEG2K-HZ. Conventional means conjugation with CRM197.
[0115] Increasing the number of serotype has a detrimental effect on the potencies of the same conjugates when there is an increase in serotypes. This is seen for PREVNAR® 13 compared with PREVNAR® 7.
[0116] Surprisingly the use of PEG demonstrates prevention of such an effect and the immune responses remains elevated and the same in spite of an increased number of serotypes.
Example 11 Anti-CRM197 Antibody Response of IVT-25Valent PEGylated Compounds of 28D Sera Measured and Compared with PREVNAR®-13 28D Sera.
[0117] Anti-CRM197 antibodies were 50% of PREVNAR®-13 antibodies. Although IVT-25 has 25-serotypes compared to the 13 serotypes of PREVNAR®-13, meaning IVT-25 has almost twice the protein content, IVT-25 had 50% less antibodies than PREVNAR®-13. A rise in the numbers or serotypes does not affect the polysaccharide response which is believed due to the PEGylation (see Table 10).
TABLE-US-00010 Total Rabbit IgG Data of IVT-25V and PREVNAR®-13 Individual Rabbit Analysis | GMC Serotype 1 3 4 5 6B 7F 9V 14 18C 19A 19F 23F Prevenar 2.54 2.49 6.08 8.38 12.98 17.86 4.49 4.03 7.51 9.64 15.79 12.88 13-28D PCV25V 9.44 3.92 11.58 19.25 25.46 39.93 13.5 31.08 13.32 21.07 29.8 26.61 IVT-G3-28D
[0118] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims. Furthermore, the term “comprising of” includes the terms “consisting of” and “consisting essentially of.”