IP Library › Granted Patent US 12,280,097
Granted Patent B2
US 12,280,097 · App. 17/492,190 · Granted Apr 22, 2025

Optimized cell-free synthesis of invasion plasmid antigen B and related compositions and methods of use

Inventors: Neeraj Kapoor (Foster City, CA); Jeffery C. Fairman (Mountain View, CA)
Assignee: Vaxcyte, Inc.
A61K39/0283A61P31/04C07K14/25
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Quick Facts
Patent No.
US 12,280,097
App. No.
17/492,190
Granted
Apr 22, 2025
Kind
B2
Abstract

The present disclosure provides a cell-free method for synthesizing an Invasion Plasmid Antigen B (IpaB) antigen associated with a Shigella bacterium comprising exogenous addition of the purified chaperone protein IpgC to the cell-free synthesis mixture. The disclosure further provides IpaB antigen mutants comprising non-natural amino acids incorporated during cell-free synthesis, enabling covalent conjugation to a Shigella O-antigen polysaccharide. Further provided are Ipa B antigens and conjugates thereof, as well as immunogenic compositions prepared with the synthesized IpaB antigens and conjugates thereof and methods of use.

Claims (36)

1. An Invasion Plasmid Antigen B (IpaB) polypeptide antigen comprising 3 or 4 non-natural amino acids (nnAA) incorporated into the IpaB polypeptide antigen amino acid sequence, wherein the nnAA is incorporated at position K395; at least one of positions K299 and K368, and a position selected from K289, K436, and K470 of SEQ ID NO: 1.

2. The IpaB antigen of claim 1 , wherein the IpaB polypeptide antigen comprises an nnAA incorporated at:

(a) each of positions K289, K368, and K395 of SEQ ID NO: 1;

(b) each of positions K299, K395, and K436 of SEQ ID NO: 1;

(c) each of positions K299, K368, and K395 of SEQ ID NO: 1;

(d) each of positions K289, K368, K395, and K436 of SEQ ID NO: 1;

(e) each of positions K299, K395, K436, and K470 of SEQ ID NO: 1; or

(f) each of positions K299, K368, K395, and K436 of SEQ ID NO: 1.

3. The IpaB antigen of claim 2 , wherein the IpaB polypeptide antigen comprises

(a) the amino acid sequence of SEQ ID NO: 2;

(b) the amino acid sequence of SEQ ID NO: 3;

(c) the amino acid sequence of SEQ ID NO: 4;

(d) the amino acid sequence of SEQ ID NO: 5;

(e) the amino acid sequence of SEQ ID NO: 6; or

(f) the amino acid sequence of SEQ ID NO: 7.

4. The IpaB antigen of claim 1 , wherein the nnAA is selected from 2-amino-3-(4-azidophenyl) propanoic acid (pAF), 2-amino-3-(4-(azidomethyl) phenyl) propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl) pyridin-2-yl) propanoic acid, 2-amino-3-(4-(azidomethyl) pyridin-2-yl) propanoic acid, 2-amino-3-(6-(azidomethyl) pyridin-3-yl) propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl) phenyl) propanoic acid, or any combination thereof.

5. The IpaB antigen of claim 4 , wherein the nnAA is pAMF.

6. The IpaB antigen of claim 1 , conjugated to one or more of an O-antigen Shigella polysaccharide (OPS).

7. The IpaB polypeptide antigen of claim 6 , wherein the one or more OPS is selected from serotypes 1a, 1b, 2a, 2b, 3b, 4a, 4b, 5a, 5b, 6, 7a, 7b, or combinations of the foregoing.

8. An immunogenic composition comprising the IpaB antigen of claim 1 .

9. The immunogenic composition of claim 8 , further comprising an adjuvant.

10. The immunogenic composition of claim 8 , formulated as a sterile injectable solution or a lyophilized form.

11. A method for expressing an Invasion Plasmid Antigen B (IpaB) polypeptide antigen from a Shigella bacterium comprising expressing the IpaB polypeptide antigen using cell-free protein synthesis in the presence of an exogenous IpgC chaperone protein.

12. The method of claim 11 , wherein the Shigella bacterium comprises a Shigella species selected from S. dysenteriae, S. flexneri, S. boydii , and S. sonnei.

13. The method of claim 11 , wherein the IpaB polypeptide antigen comprises an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.

14. The method of claim 11 , wherein at least one non-natural amino acid (nnAA) is incorporated into the IpaB polypeptide antigen amino acid sequence.

15. The method of claim 14 , wherein the nnAA is incorporated at one or more positions selected from K241, K262, K269, K283, K289, K299, C309, K312, 5329, 5333, D347, E360, K368, E372, K376, D380, K384, E387, D392, K394, K395, K397, K424, K429, K436, K440, K448, K451, K470, and K482 of SEQ ID NO: 1.

16. The method of claim 14 , wherein the nnAA is selected from 2-amino-3-(4-azidophenyl) propanoic acid (pAF), 2-amino-3-(4-(azidomethyl) phenyl) propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl) pyridin-2-yl) propanoic acid, 2-amino-3-(4-(azidomethyl) pyridin-2-yl) propanoic acid, 2-amino-3-(6-(azidomethyl) pyridin-3-yl) propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl) phenyl) propanoic acid, or any combination thereof.

17. The method of claim 16 , wherein the nnAA is pAMF.

18. The method of claim 11 , wherein the IpgC chaperone protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 8.

19. The method of claim 11 , further comprising purifying the IpaB polypeptide antigen.

20. The method of claim 19 , wherein the IpaB polypeptide antigen is purified in the presence of a detergent effective to degrade the IpgC chaperone protein without substantially affecting the IpaB polypeptide antigen.

21. A purified IpaB antigen prepared by the method of claim 11 , wherein at least one non-natural amino acid (nnAA) is incorporated into the IpaB polypeptide antigen amino acid sequence.

22. A method of immunizing a subject against Shigella dysentery, comprising administering to the subject an effective amount of the IpaB polypeptide antigen of claim 1 .

23. A method of reducing the risk of Shigella dysentery infection developing in a subject, the method comprising administering to the subject an effective amount of the IpaB polypeptide antigen of claim 1 .

24. A method of inducing a protective immune response against a Shigella bacterium in a subject comprising administering the IpaB polypeptide antigen of claim 1 to the subject.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 11, 2022
From: KAPOOR, NEERAJ; FAIRMAN, JEFFERY C.
To: SUTROVAX, INC.
Reel/Frame 058623/0159 →
CHANGE OF NAME Recorded Jan 11, 2022
From: SUTROVAX, INC.
To: VAXCYTE, INC.
Reel/Frame 058701/0933 →
Continuity (3)
Continuation PCTUS2020025384 · Mar 27, 2020
Provisional Application 62828364 · Apr 2, 2019
Related Publication 20220125907A1 · Apr 28, 2022
References Cited (92)
US 5679782A · Rosenberg et al. · 1997 [cited by applicant]
US 5972899A · Zychlinsky et al. · 1999 [cited by applicant]
US 9040253B2 · Roy et al. · 2015 [cited by applicant]
US 9492523B2 · Picking · 2016 [cited by examiner]
US 9650621B2 · Thanos et al. · 2017 [cited by applicant]
US 10716839B2 · Tennants et al. · 2020 [cited by applicant]
US 20160257946A1 · Zimmerman et al. · 2016 [cited by applicant]
US 20180333484A1 · Fairman et al. · 2018 [cited by applicant]
CN 1912106A · 2007 [cited by applicant]
CN 101711165A · 2010 [cited by applicant]
TW 200848426A · 2008 [cited by applicant]
WO WO2010049806A1 · 2010 [cited by applicant]
WO WO2013020090A2 · 2013 [cited by applicant]
WO WO2013020090A3 · 2013 [cited by applicant]
WO WO2015054587A1 · 2015 [cited by applicant]
WO WO2016020413A1 · 2016 [cited by applicant]
WO WO2018126229A3 · 2018 [cited by applicant]
WO WO2018126229A2 · 2018 [cited by examiner]
WO WO2020010000A1 · 2020 [cited by applicant]
WO WO2020010016A1 · 2020 [cited by applicant]
Berti et al. 2018 (Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification; Chem Soc. Rev. 47: 9015) (Year: 2018). [cited by examiner]
Barta, M.L. et al. (2016). “Recombinant Expression and Purification of the [cited by applicant]
Desalegn, G. et al. (2023). “Novel [cited by applicant]
Arabshahi, S. et al. (2018). “In silico design of a novel chimeric shigella IpaB fused to C terminal of clostridium perfringens enterotoxin as a vaccine candidate,” [cited by applicant]
Ashkenazi, S. et al. (1999). “Safety and immunogenicity of Shigella sonnei and Shigella flexneri 2a O-specific polysaccharide conjugates in children,” J. Infect. Dis. 179:1565-1568. [cited by applicant]
Baliban, S.M. et al. (2019). “Maternal antibodies elicited by immunization with an O-polysaccharide glycoconjugate vaccine protect infant mice against lethal [cited by applicant]
Ballban, S.M. et al. (2017). “Development of a glycoconjugate vaccine to prevent invasive [cited by applicant]
Baliban, S.M. et al. (2017). “Development of a glycoconjugate vaccine to prevent invasive [cited by applicant]
Bertl, F. et al. (2018). “Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification,” Chem. Soc. Rev. 47:9015-9025. [cited by applicant]
Birket, S.E. et al. (2007). “Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri,” Biochemistry 46:8128-8137. [cited by applicant]
Bode, C. et al. (2011). “CpG DNA as a vaccine adjuvant,” Expert Rev. Vaccines 10:499-511. [cited by applicant]
Chong, S. (2014). “Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications,” Curr. Protoc Mol Biol. 108:16.30.1-11. [cited by applicant]
Cohen, D. et al. (2021). “Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-c… [cited by applicant]
Cohen, D. et al. (1996). “Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers,” Infection and immunity 64:4074-4077. [cited by applicant]
Cohen, D. et al. (2019). “Serum IgG antibodies to Shigella lipopolysaccharide antigens—a correlate of protection against shigellosis,” Hum. Vaccin. Immunother. 15:1401-1408. [cited by applicant]
Dagan, R. et al. (2010). “Glycoconjugate vaccines and immune interference: A review,” Vaccine 28:5513-5523. [cited by applicant]
Hegerle, N. et al. (2018). “Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers… [cited by applicant]
Heine, S.J. et al. (2014). “Intradermal delivery of Shigella IpaB and IpaD type III secretion proteins: kinetics of cell recruitment and antigen uptake, mucosal and systemic immunity, and protection across serotypes,” J… [cited by applicant]
Heine, S.J. et al. (2013). “Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD,” Vaccine 31:2919-2929. [cited by applicant]
Heine, S.J. et al. (2015). “Shigella IpaB and IpaD displayed on L. lactis bacterium-like particles induce protective immunity in adult and infant mice,” Immunol Cell Biol. 93:641-652. [cited by applicant]
Henikoff, S. et al. (1992). “Amino acid substitution matrices from protein blocks,” PNAS 89:10915-10919. [cited by applicant]
Hosangadi, D. et al. (2019). “Considerations for using ETEC and Shigella disease burden estimates to guide vaccine development strategy,” Vaccine 37:7372-7380. [cited by applicant]
International Search Report mailed on Mar. 28, 2018, for PCT Application No. PCT/US2020/025384, filed on Mar. 27, 2020, 5 pages. [cited by applicant]
Islam, D. et al. (1995). “Immunoglobulin subclass distribution and dynamics of Shigella-specific antibody responses in serum and stool samples in shigellosis,” Infect Immun. 63:2054-2061. [cited by applicant]
Kapoor, N. et al. (2018). “Malaria Derived Glycosylphosphatidylinositol Anchor Enhances Anti-Pfs25 Functional Antibodies That Block Malaria Transmission,” Biochemistry 57:516-519. [cited by applicant]
Kim, E. et al. (2019). “Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo,” Chemical Science 10:7835-7851. [cited by applicant]
Kirchman, D. et al. (1985). “Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems,” Applied and Environmental Microbiology 49:599-607. [cited by applicant]
Kotloff, K.L. et al. (1995). “Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid [cited by applicant]
Kotloff, K.L. et al. (2004). “Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208,” J. Infect. Dis. 190:1745-1754. [cited by applicant]
Lin, J. et al. (2016). “Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production,” Clin. Vaccine Immunol. 23:681-688. [cited by applicant]
Livio, S. et al. (2014). “Shigella Isolates from the Global Enteric Multicenter Study Inform Vaccine Development,” Clinical Infectious Diseases 59:933-941. [cited by applicant]
Lokareddy, R.K. et al. (2010). “Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone lpgC and translocator protein IpaB,” J. Biol. Chem. [cited by applicant]
Martinez-Becerra, F.J. et al. (2013). “Characterization of a novel fusion protein from IpaB and IpaD of [cited by applicant]
Martinez-Becerra, F.J. et al. (2012). “Broadly protective Shigella vaccine based on type III secretion apparatus proteins,” Infection and immunity 80:1222-1231. [cited by applicant]
Maggio, E.T. (2014). “Absorption enhancing excipients in systemic nasal drug delivery,” [cited by applicant]
Merkus, F.W. et al. (1999). “Cyclodextrins in nasal drug delivery,” [cited by applicant]
Micoli, F. et al. (2018). “Protein Carriers for Glycoconjugate Vaccines: History, Selection Criteria, Characterization and New Trends,” Molecules 23:1451. [cited by applicant]
Murray, C.J. et al. (2013). “Cell-free translation of peptides and proteins: from high throughput screening to clinical production,” Current Opin. Chem. Biol. 17:420-426. [cited by applicant]
Nahm, M.H. et al. (2018). “Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput Shigella Serum Bactericidal Assay,” mSphere 3:e00146-18. [cited by applicant]
Ndungo, E. et al. (2018). “A novel Shigella proteome microarray discriminates targets of human antibody reactivity following oral vaccination and experimental challenge,” Msphere 3:e00260-18. [cited by applicant]
Ndungo, E. et al. (2020). “Functional antibodies as immunological endpoints to evaluate protective immunity against Shigella,” Hum. Vaccin. Immunother. 16:197-205. [cited by applicant]
Noriega, F.R. et al. (1999). “Strategy for Cross-Protection among [cited by applicant]
Oberhelman, R.A. et al. (1991). “Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides,” Infection and immunity 59:2341-… [cited by applicant]
Passwell, J.H. et al. (2010). “Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1-4-year-old Israeli children,” Vaccine 28:2231-2235. [cited by applicant]
Passwell, J.H. et al. (1995). “Shigella lipopolysaccharide antibodies in pediatric populations,” Pediatr. Infect. Dis J. 14:859-865. [cited by applicant]
Passwell, J.H. et al. (2003). “Safety and immunogenicity of Shigella sonnei-CRMs and Shigella flexneri type 2a-rEPA [cited by applicant]
Picking, W.L. et al. (1996). “Cloning, expression, and affinity purification of recombinant Shigella flexneri invasion plasmid antigens IpaB and IpaC,” [cited by applicant]
Ravenscroft, N. et al. (2019). “Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate,” Glycobiology 29:669-680. [cited by applicant]
Riddle, M.S. et al. (2016). “Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella flexneri 2a administered to healthy adults: a single-blind, randomized phase I study,” Clinical land Vaccine Im… [cited by applicant]
Sayem, M.A. et al. (2011). “Differential host immune responses to epidemic and endemic strains of Shigella dysenteriae type I,” J. Health Popul. Nutr. 29:429-437. [cited by applicant]
Shen, D-K. et al. (2016). “MxiA, MxiC and IpaD Regulate Substrate Selection and Secretion Mode in the T3SS of Shigella flexneri,” PLoS One 11:e0155141. [cited by applicant]
Shigella Vaccine Data Presentation (2020). 29 total pages. [cited by applicant]
Shimanovich, A.A. et al. (2017). “Functional and Antigen-Specific Serum Antibody Levels as Correlates of Protection against Shigellosis in a Controlled Human Challenge Study.” [cited by applicant]
Shimizu, Y. et al. (2006). “Cell-free translation systems for protein engineering,” FEBS Journal 273:4133-4140. [cited by applicant]
Simon, R. et al. (2011). “ [cited by applicant]
Thompson, C.N. et al. (2016). “The transfer and decay of maternal antibody against Shigella sonnei in a longitudinal cohort of Vietnamese infants,” Vaccine 34:783-790. [cited by applicant]
Turbyfill, K.R. et al. (2018). “Assembly, biochemical characterization, immunogenicity, adjuvanticity, and efficacy of [cited by applicant]
UniProtKB ID: Q03945 (1994). Shigella dysenteriae, 3 total pages. [cited by applicant]
UniProtKB ID: P18011 (1990). Shigella flexneri, 4 total pages. [cited by applicant]
UniProtKB ID: Q8KXT4 (2002). Shigella boydii, 2 total pages. [cited by applicant]
UniProtKB ID: Q3YTQ2 (2005). Shigella sonnei (strain Ss046), 3 total pages. [cited by applicant]
Van Sorge, N.M. et al. (2014). “The classical lancefield antigen of group a [cited by applicant]
Van de Verg, L.L. et al. (1992). “Age-specific prevalence of serum antibodies to the invasion plasmid and lipopolysaccharide antigens of [cited by applicant]
Vaxcyte Presentation (2021). Development of an anti-Shigella vaccine using IpaB-OPS conjugates, Shigella Vaccine Program, 19 total pages. [cited by applicant]
Wahid, R. et al. (2013). “Shigella antigen-specific B memory cells are associated with decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a,” Clinical Immunology 148:35-43. [cited by applicant]
WHO (2017). “WHO publishes list of bacteria for which new antibiotics are urgently needed,” located at https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-neede… [cited by applicant]
Written Opinion of the International Searching Authority mailed on Mar. 28, 2018, for PCT Application No. PCT/US2020/025384, filed on Mar. 27, 2020, 7 pages. [cited by applicant]
Yang, S-C. et al. (2015). “The roles of the virulence factor IpaB in [cited by applicant]
Yin, G. et al. (2017). “RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates,” Sci. Rep. 7:3026. [cited by applicant]
Zawada, J.F. et al. (2011). “Microscale to manufacturing scale-up of cell-free cytokine production—a new approach for shortening protein production development timelines,” Biotechnol. Bioeng. 108:1570-1578. [cited by applicant]
Zhao, Y. et al. (2019). “Synthesis and immunological studies of a Group A [cited by applicant]
Zimmerman, E.S. et al. (2014). “Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system,” Bioconjugate Chemistry 25:351-361. [cited by applicant]