Patent Grant
9248197
1. Field of Invention This invention is directed to reagents and methods of conjugating proteins to carbohydrates and, in particular, the processes and chemicals used in the manufacture of vaccines, and also the vaccines manufactured by the process.
2. Description of the Background
Vaccines that contain protein covalently linked to carbohydrate have proven remarkably successful in inducing an immune response to the carbohydrate moiety. Examples of such vaccines, known as “conjugates” are available for Haemophilus influenzae type b (e.g., ActHib, Hiberix), Neisseria meningiditis types A C W and Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar, Synflorix). In order for the protein to be linked to the carbohydrate, the latter generally needs to be activated so that it can be reacted with the protein, either directly or via a spacer (Dick, W. E. Jr and Beurret, M. Glyconjugates of bacterial carbohydrate antigens. A survey and consideration of design and preparation factors. In: Conjugate Vaccines (Eds Cruse, J. M. and Lewis, R. E.). Karger, Basel, 1989. One means of activation is through oxidation of the carbohydrate to produce aldehydes, which are then linked to lysines on protein through reductive amination. In other cases, the protein is first functionalized with hydrazides or aminooxy groups, which are subsequently reacted with aldehydes on the carbohydrate (Lees, A. Use of amino-oxy functional groups in the preparation of protein-polysaccharide (PS) conjugate vaccines United States Patent Publication No. 2005/0169941; which is incorporated by reference). Another method for activating polysaccharides is with the use of cyanogen bromide, to form a cyano-ester on the polysaccharide which is subsequently reacted with a spacer molecule such as adipic dihydrazide. The functionalized polysaccharide is then reacted with the protein. Improved methods for cyanating polysaccharides use 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (Lees, A., Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents. U.S. Pat. Nos. 5,651,971; 5,693,326; and 5,849,301). CDAP allows for protein to be linked directly to the polysaccharide. CDAP can also be used to functionalize the polysaccharide with a spacer, which is subsequently linked to the protein. Hydrazide or aminooxy functionalized proteins can also be linked to CDAP activated polysaccharides (U.S. Pat. No. 5,849,301; which is incorporated by reference).
The present invention, overcomes the problems and disadvantages associated with current strategies and designs in the manufacture of vaccines, and provides new tools and methods of protein conjugation especially for vaccine manufacture. One embodiment of the invention is directed to a process of conjugation of a carbohydrate comprising: mixing a compound such as 1-CBT, or 1-Cl, or CPPT, or 2-CPO, or a functional derivative or modification thereof with the chemical compound to create an activated chemical compound; and mixing the activated compound with a second compound to create a conjugate. Preferably the chemical compound is a natural or synthetic carbohydrate, polysaccharide, oligosaccharide, or combination thereof. Also preferably, the second compound is a peptide, a polypeptide or a protein, which may be an antigenic molecule. Conjugation may be direct or indirect with the additional of functional groups that facilitate conjugation. The process preferably further comprises removing components with a lower molecular weight than the conjugate for example, by dialysis filtration, chromatography, or a combination thereof. The resulting conjugate is preferably a vaccine or a diagnostic agent. The process may further comprise including a linking compound between the activated compound and the second compound. Preferably the steps of the process, as well as the inclusion of a linker, are performed together, but each may be performed independently.
Another embodiment of the invention is directed to a vaccine produced by the processes of the invention. Preferably the vaccine further comprises a pharmaceutically acceptable carrier which may include, but is not limited to water, saline, alcohol, saccharides, polysaccharides, oil, or combinations thereof.
Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention.
One embodiment of the invention is directed to a process of conjugation of a chemical compound comprising mixing CPPT, 1-CBT, 1-CI, or 2-CPO, or a functional derivative or conservative modification of any of the preceding compounds, with the chemical compound to create an activated chemical compound. The activated compound is mixed with a second compound to create a conjugate. These steps may be performed independently or together, and may include another compound as a linker between the two. Preferably the activated compound is mixed with a linker molecule which is subsequently reacted with a second compound to create a conjugate. Preferred linkers include but are not limited to hexanediamine, ethylenediamine, hydrazine, adipic dihydrazide, or 1,6-diaminooxyhexane.
Conjugation may be direct or indirect, meaning with or without the addition of functional groups that facilitate conjugation. Preferably, the chemical compound is a natural or synthetic carbohydrate, polysaccharide, oligosaccharide, or combination thereof. Preferably the second compound is a peptide, a polypeptide or a protein, and more preferably the second compound is an antigenic molecule for the preparation of a vaccine of as a diagnostic reagent.
This invention provides novel reagents for cyanating polysaccharides in aqueous or part aqueous solutions so that they may be covalently linked to proteins either directly or through a spacer. The examples illustrate the use of these reagents with a variety of polysaccharides and proteins showing that the methods are generally applicable.
Carbohydrate, polysaccharide and oligosaccharide are used interchangeably in this application. The method can employ either natural or synthetic carbohydrate.
Protein can refer to natural, recombinant or synthetic material. It can include peptides. Other molecules besides protein can be used as the second moiety to link either directly or indirectly to the activated carbohydrate.
Direct conjugation refers to linking of the protein to the activated carbohydrate without introducing additional functional groups. Indirect conjugation refers to the addition of functional groups which are used to facilitate conjugation. For example, carbohydrate can be functionalized with amines which are subsequently reacted with bromoacetyl groups. The bromoacetylated carbohydrate is then reacted with thiolated protein. (Hermanson, GT, Bioconjugate Techniques, Academic Press, 2nd ed, 2008). The term “functionalization” generally means to chemically attach a group to add functionality, for example, to facilitate conjugation. Examples include functionalization of proteins with hydrazides or aminooxy groups and functionalization of carbohydrate with amino groups.
Methods and Reagents
Conjugation of the protein to the carbohydrate increases its molecular weight, which can be monitored using analytical size exclusion chromatography (SEC HPLC). The earlier the material elutes the higher the molecular weight, and protein is generally monitored by its absorbance at 280 nm. Thus the shift of absorbance to an earlier time is an indication of an increase in molecular weight and hence of conjugation. A BioSep G4000 SEC column (Phenomenex) or similar column was used for SEC HPLC in a Waters 600 system equipped with Empower software. Amines and hydrazides were assayed using TNBS as generally described in: “Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid” Qi XY, Keyhani N O, Lee Y C. Anal Biochem. 1988 Nov 15;175(1):139-44 (incorporated by reference) and Vidal and Franci, J Immun. Meth 86:155, 1986.
Protein concentration was determined from its extinction coefficient and absorbance at 280 nm. Carbohydrate was assayed by the method of Monsigny M. et al. (Anal Biochem. 175(2):525-30, 1988; incorporated by reference).
1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) was prepared by Wilmington PharmaTech, Lot No. 1795-1536-10 and was greater than 97% pure as determined by reverse phase HPLC (see
The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.
CDAP and CPPT were each brought to 0.43 M in acetonitrile and 120 ul added to 2 ml of dextran (6 mg/ml in saline+0.02% sodium azide). After 30 sec, triethylamine was added to each (˜7u1) to maintain the pH at about pH 9.8. At 2 min, 1 ml aliquots of the activated dextran was removed ad added to either 1 ml of a 0.5 M solution of hexane diamine or to a solution of 1 ml of BSA (10 mg/ml). After 4 hours, the hexanediamine derivatized dextran solutions were dialyzed overnight into saline with frequent changes. To further remove low molecular weight reagents, the solutions were each brought to ˜6 ml, concentrated using a Corning spin filter device (Spin-X 10 kDa cutoff) and the process repeated. The filtrate from the second spin was assayed using trinitrobenzesulfonic acid (TNBS) and found to be essentially negative for amines. The desalted retentate was assayed for amines and dextran. The BSA-dextran conjugates were incubated overnight at 4° C. and then fractionated on a Superdex200 size exclusion column (GE Healthcare), equilibrated with PBS. The elution profile for the two conjugates is shown in
This data shows that CPPT activates dextran as well as CDAP and possibly better. It demonstrates that protein can be directly conjugated to CPPT activated polysaccharide.
Solutions of 7.5 mg/ml of Haemophilus influenza (Hib), Neisseria meningiditis A (MenA) and Neisseria meningiditis C (MenC) capsular polysaccharides was prepared. To 1.35 ml of each, 75 ul of 100 mg/ml CPPT in acetonitrile was added. At 30 sec, 5 ul of TEA was added to the Hib and MenA polysaccharides. 6 ul of TEA was added to the MenC solution. At 2.5 min, 0.6 ml of the activated PS was added to 0.6 ml of 0.5 M hexanediamine. After 3 hr, each was dialyzed extensively against saline.
Each solution was assayed for polysaccharide using the resorcinol sulfuric acid assay with the corresponding polysaccharide as the standard for MenA and MenC. Ribose was used as the standard for the Hib polysaccharide, with 1 mole ribose per repeat unit and a repeat unit MW of 243 g/mole (see Table 2).
This table further illustrates the use of CPPT to functionalize a variety of polysaccharides.
30 mg of 1-CI was added to 2 ml of 20 mg/ml solution of T2000 dextran (GE Healthcare). Five 100 ul aliquots of 0.2 M triethylamine were added to maintain the pH at 9-9.2. After about three minutes, 2 ml of 0.5 M hexanediamine was added and the pH adjusted to 9 with 0.5 M NaOH. After 2 hr of reaction, the solution was dialyzed exhaustively into saline. The solution was clarified by centrifugation and assayed for dextran and amines. The product was found to contain 27 amines/100 kDa of dextran, showing that 1-CI can be used to functionalize polysaccharides with amines.
A solution of 13.5 mg/ml of high molecular weight dextran was prepared. 67.5 ul of a 100 mg/ml solution of 1-CI in NMP was added to 1 ml and 30 sec later, 4 aliquots of 100 ul each of 0.2 M TEA added, followed by about 20 ul of 0.5 M NaOH. At about 3.5 min, 0.5 ml of a 20 mg/ml solution of BSA was added. The pH was raised to about 8.5 by the addition of 2 aliquots of 100 ul each 0.1 M sodium borate, pH 9.0. After two days at 4° C., the reaction mixture was analyzed by SEC HPLC. The figure shows that the BSA+1-CI activated dextran now elutes much earlier from the size exclusion column, indicating higher molecular weight due to conjugation to the polysaccharide.
Capsular polysaccharides from pneumococcal serotypes 1 PS (Ps1) and 6B PS (Ps6B) were prepared at 10 mg/ml in saline +0.02% sodium azide. 9.3 mg of 1-CI suspended in 93 ul NMP was added to 1 ml of Psi followed by 8 100 ul aliquots of 0.2 M TEA. At 2.5 min, 0.5 ml of 1 M hexanediamine was added, followed by 2 aliquots of 100 ul each of 0.5 M NaOH. The pH was approximately 9. 100 ul of a 100 mg/ml solution of 1-CI in NMP was added to 1 ml of the Ps6B solution. Two 100 ul aliquots of 0.2 M TEA were added, followed by 2 aliquots of 50 ul each of 0.5 M NaOH to maintain the pH at ˜9.5-10.8. At 2.5 min, 0.5 ml of 1 M hexanediamine was added along with 50 ul of 0.5 M NaOH. After about 3 hours, each was dialyzed extensively against saline. Each was then assayed for polysaccharide using the resorcinol sulfuric acid assay and for amines using TNBS (see Table 3).
Thus it is clear that 1-CI can be used to activate and functionalize polysaccharides.
239 ul of 1 M HEPES, pH 8 was added to a solution of about 9.5 mg in 2.2 ml of the Ps6B-NH2 prepared in Example 5. 170 ul of 0.1 M NHS bromoacetate in NMP was added. After 1 hr, the solution was desalted by repeated washes using an Amicon Ultra15 30 kDa cutoff spin filter, using 10 mM NaPO4+5mM EDTA pH 6.8 buffer. The retentate was then brought to a final volume of about 0.7 ml. Tetanus toxoid was thiolated as follows: 571 ul of tetanus toxoid (35 mg/ml) was brought to pH 8 by the addition of 64 ul 1 M HEPES. 27 ul of 0.1 M SPDP in NMP was slowly added. After about 2 hrs, the pH was reduced to 5.7 and 33 ul of 0.5 M DTT added to deprotect the thiol. After 30 min, the solution was desalted on a 1×15cm G25 column, equilibrated with 10 mM NaPO4+5mM EDTA pH 6.8 buffer. The void volume was pooled & concentrated to a final concentration of about 62 mg/ml. The thiol content was determined using DTNB. The tetanus had a ratio of about 6 mole thiols/mole TT. The bromoacetylated polysaccharide and thiolated TT were combined as follows: 0.66 ml Ps6B-BrAc+73 ul 1 M HEPES, pH 8+230 ul thiolated TT.
The reaction was allowed to proceed overnight at 4° C. and then analyzed by SEC HPLC (
Capsular polysaccharide from pnueumococcal serotype 6A (Ps6A) was solubilized in water at 10 mg/ml. Each of 5 1 ml tubes was heated to 80° C. for 2 min and 1 ml 0.1 M sodium borate, pH 9 added to each. After 2.5 hr, the tubes were cooled on ice and then dialyzed. 25 ul of TEA was added to 10 ml of the hydrolyzed Ps6A at 5 mg/ml and 50 mg of 1-CI added while vortexing. At 2.5 min, 30 ml of CRM at 17.2 mg/ml was added and the pH maintained at about 9. After an overnight reaction, the solution was concentrated to about 1 ml using an Amicon Ultra 15 30 kDa cutoff spin device. 0.5 ml was loaded onto a Superdex 200 column (1×30cm), equilibrated with 10 mM sodium borate, 150 mM NaCl, pH 9 and the column run at 0.5 ml/min. The chromatogram is shown in
12 mg of 1-CBT were suspended in 240 ul of acetonitrile+120 ul of NMP. 120 ul of 0.2 M were added to 0.5 ml of 20mg/ml T2000 dextran (10 mg) followed by the addition of 120 ul of the 1-CBT suspension. At about 3 min, 0.5 ml of 0.5 M adipic dihydrazide was added. After 2 hrs of reaction, the solution was dialyzed extensively against saline over 2 days. The solution was then centrifuged and assayed for dextran and hydrazides.
Approximately 9.5 mg of dextran was recovered with a ratio of about 11 hydrazides per 100 kDa of dextran. To confirm that the hydrazides were linked to the dextran, an aliquot of TNBS labeled hydrazide-dextran was examined by SEC HPLC with monitoring at 500 nm, where the TNBS-hydrazide adduct adsorbs. The chromatogram indicated that the trinitrobenzene was associated with the high molecular weight dextran, showing that the hydrazide was indeed linked to the dextran. This shows that 1-CBT can be used to functionalize polysaccharides.
120 ul of 0.2 M TEA was added to 500 ul of a 20 mg/ml solution of T2000 dextran and 160 ul of a 50 mg/ml suspension of 1-CBT in acetonitrile added. At about 2 min, 200 ul of a 47 mg/ml solution of BSA in saline was added and the solution became more viscous. After an overnight reaction, the conjugate was analyzed by SEC HPLC. The majority of the BSA eluted at the void volume of the column, indicating that it was high molecular weight. This demonstrates that 1-CBT can be used to directly conjugate proteins to polysaccharides.
The carbohydrate is activated using 2-CPO (X=Cl, Y=Cl) and is then directly reacted with a protein or functionalized, e.g., with amino groups. Reagent 2a (See Scheme 1 Kim et al.) is made up at 100 mg/ml in acetonitrile and added to a 10 mg/ml solution of pneumococcal type 14 polysaccharide (Ps14) at ratio of 1 mg reagent to 1 mg carbohydrate. The pH is raised to pH 9.5 using triethylamine and maintained. After 2.5 min, half the solution is added to an equal volume of 0.5 M hexanediamine and the pH adjusted to about 8. The other half of the solution is combined with an equal weight of tetanus toxoid in 10 mg/ml solution. After an overnight incubation at 4° C., the hexanediamine polysaccharide solution is extensively dialyzed against saline and then assayed for amines and polysaccharide. A ratio of 10 amines per 100 kDa polysaccharide is found. The tetanus polysaccharide solution is fractionated on a S400HR column (GE Healthcare) to remove unconjugated protein. The protein and polysaccharide concentrations are determined. A ratio of about 0.75 mg protein per mg polysaccharide is found.
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. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. 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.
This application is a continuation of and claims priority to U.S. application Ser. No. 13/514,356 filed Jun. 7, 2012, which is a national stage submission of International Application No. PCT/US2010/061133 filed Dec. 17, 2010, which claims priority to U.S. Provisional Application No. 61/287,593 filed Dec. 17, 2009, and the entirety of each is hereby incorporated by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4356170 | Jennings et al. | Oct 1982 | A |
| 4540573 | Neurath et al. | Sep 1985 | A |
| 4663160 | Tsay et al. | May 1987 | A |
| 4678553 | Mandle et al. | Jul 1987 | A |
| 4695624 | Marburg et al. | Sep 1987 | A |
| 5039607 | Skold et al. | Aug 1991 | A |
| 5306492 | Porro | Apr 1994 | A |
| 5360897 | Anderson et al. | Nov 1994 | A |
| 5425946 | Tai et al. | Jun 1995 | A |
| 5585100 | Mond et al. | Dec 1996 | A |
| 5651971 | Lees | Jul 1997 | A |
| 5693326 | Lees | Dec 1997 | A |
| 5747663 | Colpan et al. | May 1998 | A |
| 5849301 | Lees | Dec 1998 | A |
| 5955079 | Mond et al. | Sep 1999 | A |
| 6087328 | Lees | Jul 2000 | A |
| 6146902 | McMaster | Nov 2000 | A |
| 6248334 | Lees et al. | Jun 2001 | B1 |
| 6284250 | Lees et al. | Sep 2001 | B1 |
| 6299881 | Lees et al. | Oct 2001 | B1 |
| 6309646 | Lees | Oct 2001 | B1 |
| 6428703 | Zinn et al. | Aug 2002 | B1 |
| 6432679 | Mond et al. | Aug 2002 | B1 |
| 6585973 | Lees | Jul 2003 | B1 |
| 6596172 | Kopf | Jul 2003 | B1 |
| 6756041 | Lees et al. | Jun 2004 | B2 |
| 6765091 | Bencomo et al. | Jul 2004 | B1 |
| 7094883 | Cassels et al. | Aug 2006 | B1 |
| 7101562 | Lees et al. | Sep 2006 | B1 |
| 7166708 | Lees et al. | Jan 2007 | B2 |
| 7250494 | Stinson et al. | Jul 2007 | B2 |
| 7452533 | Walsh et al. | Nov 2008 | B2 |
| 7470441 | Van Der Giessen et al. | Dec 2008 | B2 |
| 7566540 | Cassels et al. | Jul 2009 | B2 |
| 7777017 | Stinson et al. | Aug 2010 | B2 |
| 20020054879 | Lees et al. | May 2002 | A1 |
| 20020119529 | Mond et al. | Aug 2002 | A1 |
| 20030215436 | Walsh et al. | Nov 2003 | A1 |
| 20030224000 | Kokai-Kun et al. | Dec 2003 | A1 |
| 20030235578 | Stinson et al. | Dec 2003 | A1 |
| 20040052779 | Stinson et al. | Mar 2004 | A1 |
| 20050074460 | Lees et al. | Apr 2005 | A1 |
| 20050075486 | Cassels et al. | Apr 2005 | A1 |
| 20050169941 | Lees | Aug 2005 | A1 |
| 20050222434 | Bessodes et al. | Oct 2005 | A1 |
| 20060165822 | Lees | Jul 2006 | A1 |
| 20070065465 | Lees | Mar 2007 | A1 |
| 20070292404 | Lees | Dec 2007 | A1 |
| 20080019976 | Lees | Jan 2008 | A1 |
| 20090081180 | Lees | Mar 2009 | A1 |
| 20110263834 | Lees et al. | Oct 2011 | A1 |
| 20120214187 | Lees | Aug 2012 | A1 |
| 20130302877 | Lees | Nov 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 1163000 | Feb 2008 | EP |
| 2004501869 | Jan 2004 | JP |
| 2008510210 | Apr 2008 | JP |
| WO 9521177 | Aug 1995 | WO |
| WO 9521179 | Aug 1995 | WO |
| WO9617941 | Jun 1996 | WO |
| WO 9617941 | Jun 1996 | WO |
| WO 9617941 | Jun 1996 | WO |
| WO 9629094 | Sep 1996 | WO |
| WO 9640662 | Dec 1996 | WO |
| WO 0170685 | Sep 2001 | WO |
| WO 0178787 | Oct 2001 | WO |
| WO03054511 | Jul 2003 | WO |
| WO03057849 | Jul 2003 | WO |
| WO 03057849 | Jul 2003 | WO |
| WO 03097699 | Nov 2003 | WO |
| WO 2004104811 | Dec 2004 | WO |
| WO 2005056608 | Jun 2005 | WO |
| WO 2006032475 | Mar 2006 | WO |
| WO2007109129 | Sep 2007 | WO |
| WO 2008021076 | Feb 2008 | WO |
| WO 201105397 | Jan 2011 | WO |
| Entry |
|---|
| Canada Office Action for PCT/US10/61133, dated Jan. 9, 2015. |
| Suárez, et al, “Improved conjugation and purification strategies for the preparation of protein-polysaccharide conjugates,” J. Chromatogr A, 1213, pp. 169-175, 2008. |
| Bystrický, et al, “Candida albicans mannan-protein conjugate as vaccine candidate,” Immunol Lett., 85, pp. 251-255, 2003. |
| Wu, et al, “1-Cyanomidazole as a mild and efficient electrophilic cyanating agents,” J Org Lett, pp. 795-797, 2000. |
| Mexico Office Action for PCT/US10/61133, dated Nov. 18, 2014. |
| R. Wong & H, Tse, Lateral Flow Immunoassay, Humana Press (2009). |
| Bioconjugate Protocols: Strategies and Methods (Methods in Moldecular Biology) Christof M. Niemeyer (Editor) Humana Press (2009). |
| Canadian Office Action for Application No. 2758323, dated Sep. 30, 2013. |
| PCT Patenbility Report for PCT/US13/45413, dated Dec. 2, 2013. |
| Webb, R.R., et al., “Synthesis of 1-(aminooxy)-4-A(3-nitro-2-pyridyl) dithiou butane and 1-(aminooxy)-4-A(3-nitro-2-pyridyl) dithiou but-2-ene, novel heterobifunctional cross-linking reagents,” Bioconjugate Chemistry, ACS, Washington, DC, US, vol. 1, No. 2, p. 96-99 (1990). |
| Brask, J. et al., “Carbopetptides: chemoselective ligation of peptide aldehydes to an aminooxyfunctionalized D-galactose template,” Journal of Peptide Science, vol. 6, p. 290-299 (2000). |
| Zeng, W. et al., “Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological activity,” Vaccine, Butterworth Scientific, Guildford, GB, vol. 19, No. 28-29, p. 3843-3852 (Jul. 16, 2011). |
| Kubler-Kielb, J., et al., “A new method for conjugation of carbohydrates to proteins uing an aminoxy-thiol heterobifunctional linker,” J. Org. Chem., vol. 70, p. 6987-6990 (Jul. 2005). |
| PCT Search Report for PCT/US2010/055107, dated Jul. 28, 2011. |
| PCT Patentability Report for PCT/US2010/055107, dated Jul. 28, 2011. |
| PCT Patentability Report for PCT/US2010/055107, dated May 8, 2012. |
| PCT Patentability Report for PCT/US2010/043387, dated Apr. 27, 2011. |
| Chinese Office Action for Chinese application No. 200580010225.4, dated May 18, 2012. |
| D. C. Watson, J. B. Robbins, and S. C. Szu, “Protection of mice against Salmonella typhimurium with an O-specific polysaccharide-protein conjugate vaccine,” Infection and Immunity 60:4679-4686, 1992. |
| E. Konadu, J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu, “Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid, with emphasis on the role of O-acetyls,” Infect Immun. Jul. 1996; 64(7):2709-15. |
| Schneerson R, Barrera O, Sutton A, Robbins JB. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med. Aug. 1, 1980;152(2):361-376. |
| PCT Search Report for PCT/US2010/043387, dated Apr. 27, 2011. |
| PCT Search Report for PCT/US10/61313, dated Feb. 15, 2011. |
| PCT Patentability Report for PCT/US10/61313, dated Feb. 15, 2011. |
| Chu, et al., “Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates,” Inf. & Imm., 40:245-256 (1983). |
| Jennings, et al., “Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as a route to a potential vaccine against group B Neisseria meningitides,” Inf. & Immun., 43:407-412 (1984). |
| Laferriere et al., “The Synthesis of Streptococcus pnemoniae polysaccharide-tetanus toxoid conjugates and the effect of chain length on immunogenicity,” Vaccine, 15:179 (1997). |
| Kohler, et al., “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, 256:495-7 (1975). |
| Morath, et al., “Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus,” J. Exp. Med. 193(3), p. 393-397 (2001). |
| PCT Search Report for PCT/US05/003040, dated Mar. 8, 2006. |
| Marcaurelle, L., et al., “Synthesis of oxime-linked mucin mimics containing tumor-related Tn and sialyl Tn antigens,” Organic Letters, vol. 3, No. 23, p. 3691-3694 (2001). |
| Webb, R.R., et al., “Synthesis of 1-(aminooxy)-4-A(3-nitro-2-pyridyl) dithiou butane and 1- (aminooxy)-4-A(3-nitro-2-pyridyl) dithiou but-2-ene, novel heterobifunctional cross-linking reagents,” Bioconjugate Chemistry, ACS, Washington, DC, US, vol. 1, No. 2, p. 96-99 (1990). |
| Brask, J. et al., “Carbopetptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template,” Journal of Peptide Science, vol. 6, p. 290-299 (2000). |
| Kubler-Kielb, J., et al., “A new method for conjugation of carbohydrates to proteins uing an aminooxy-thiol heterobiofunctional linker,” J. Org. Chem., vol. 70, p. 6987-6990 (Jul. 2005). |
| PCT Patentability Report for PCT/US05/003040, dated Jul. 29, 2006. |
| EPO Exam Report for PCT/US05/003040, dated Feb. 22, 2008. |
| EPO Exam Report for PCT/US05/003040, dated Jul. 11, 2007. |
| AU Exam Report for PCT/US05/003040, dated Mar. 4, 2011. |
| JP Exam Report for PCT/US05/003040, dated May 19, 2011. |
| Renaudet & Dumy, “Chemoselectively template-assembled glycopeptides presenting clustered cancer related t-antigen,” Tetrahedron Letters, vol. 45, No. 1, p. 65-68 (Jan. 2004). |
| JP Exam Report for PCT/US05/003040, dated Nov. 22, 2010. |
| Chinese Exam Report for PCT/US05/003040, dated Oct. 24, 2011. |
| Chinese Exam Report for PCT/US05/003040, dated Apr. 3, 2009. |
| CA Exam Report for PCT/US05/003040, dated Jun. 11, 2010. |
| CA Exam Report for PCT/US05/003040, dated Jul. 13, 2009. |
| AU Exam Report for PCT/US05/003040, dated Sep. 14, 2007. |
| India Exam Report for PCT/US05/003040, dated Sep. 14, 2007. |
| PCT Patentability Report for PCT/US2010/061133, dated Jun. 19, 2012. |
| Arkivoc, 2007(iii), pp. 5-12. |
| Tetraheddron, 2005, 61, pp. 5889-5894. |
| Korean Office Action for Application No. 10-2014-7007579, dated Oct. 15, 2014. |
| EP Search Report for PCT/US2010/061133, dated Sep. 7, 2015. |
| Zuber, et al., “Enhanced ligation of DNA with a synthetic effector molecule,” Journal of the American Chemical Society, vol. 115, No. 11, pp. 4939-4940, Jan. 1, 1993. |
| Kim, et al, “2-Cyanopyridazin-3(2H)-ones: effective and chemoselective electrophilic cyanating agents,” Tetradedron 61, pp. 5889-5894, May 10, 2005. |
| Number | Date | Country | |
|---|---|---|---|
| 20140316110 A1 | Oct 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61287593 | Dec 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13514356 | US | |
| Child | 14312748 | US |
