US-12618074-B2 - CD40 specific DNA aptamers as vaccine adjuvants

Abstract

The present invention provides immunostimulatory nucleic acids that have an affinity to a specific target protein. The present invention also provides templates and methods for making and using the immunostimulatory nucleic acids. Further, methods for linking the immunostimulatory nucleic acids to antigens and using the resulting complexes to enhance an immune response are provided.

Inventors

• Adil Sabr AL-OGAILI
• Tieshan JIANG
• Christine N. VUONG
• Rohana Liyanage
• Jackson O. Lay, Jr.
• Suresh Kumar Thallapuranam
• Luc R. Berghman
• Young Min Kwon
• Billy Hargis

Assignees

• THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS
• THE TEXAS A&M UNIVERSITY SYSTEM

Dates

Publication Date

20260505

Application Date

20200413

Claims (18)

1. 1 . An immunostimulatory nucleic acid comprising a protein-binding sequence having at least 95% identity to any one of SEQ ID NOs: 1-8, wherein the immunostimulatory nucleic acid binds to CD40.
2. 2 . An immunostimulatory nucleic acid comprising a plurality of protein-binding units; wherein each of the protein-binding units comprise a first protein-binding sequence, a second protein-binding sequence, and a spacer sequence therebetween; wherein the second protein-binding sequence is a different sequence than the first protein-binding sequence; wherein (a) the first protein-binding sequence has at least 95% identity to any one of SEQ ID NOs: 1-8, (b) the second protein-binding sequence has at least 95% identity to any one of SEQ ID NOs: 1-8, or (c) both the first protein-binding sequence and the second protein-binding sequence have at least 95% identity to any one of SEQ ID NOs: 1-8; and wherein the immunostimulatory nucleic acid binds to CD40.
3. 3 . The immunostimulatory nucleic acid of claim 2 , wherein the first and the second protein binding sequence are selected from SEQ ID NO: 3 and SEQ ID NO: 4.
4. 4 . The immunostimulatory nucleic acid of claim 2 , wherein the immunostimulatory nucleic acid induces a CD40 signaling response when it is bound to CD40.
5. 5 . The immunostimulatory nucleic acid of claim 2 , further comprising a binding tag.
6. 6 . A method of binding CD40, the method comprising contacting CD40 with the immunostimulatory nucleic acid of claim 1 .
7. 7 . A template for producing a plurality of protein-binding units comprising in order from 5′ to 3′: i) a 3′ end of a primer-binding sequence, ii) a reverse complement of a second protein-binding sequence, iii) a reverse complement of a spacer sequence, iv) a reverse complement of a first protein-binding sequence, and v) a 5′ end of a primer-binding sequence, wherein the 5′ end of the primer-binding sequence and the 3′ end of the primer-binding sequence form a primer-binding site when the template is circularized.
8. 8 . The template of claim 7 , wherein the first protein-binding sequence comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 1-8, wherein the second protein-binding sequence comprises the sequence having at least 95% identity to any one of SEQ ID NOs: 1-8, or wherein both the first protein-binding sequence and the second protein-binding sequence comprise the sequence having at least 8095% identity to any one of SEQ ID NOs: 1-8, and wherein the plurality of protein-binding sequences bind to CD40.
9. 9 . The template of claim 7 , wherein both the first protein-binding sequence and the second protein-binding sequence have an affinity for an immune response signaling protein.
10. 10 . The template of claim 9 , wherein the immune response signaling protein is CD40.
11. 11 . A method of producing an immunostimulatory nucleic acid comprising (a) providing the template of claim 7 , (b) circularizing the template, and (c) amplifying the circularized template to produce the immunostimulatory nucleic acid.
12. 12 . The method of claim 11 , further comprising partially or completely hybridizing a complementary sequence to the spacer sequence of the immunostimulatory nucleic acid.
13. 13 . The method of claim 11 , wherein step (a) comprises (i) identifying two or more protein-binding sequences with an affinity for an immune response signaling protein and (ii) selecting the complement of the first protein-binding sequence and the complement of the second protein-binding sequence from the identified sequences.
14. 14 . A method of producing an immunostimulatory complex comprising: (a) amplifying the template of claim 7 to produce an immunostimulatory nucleic acid, (b) tagging the immunostimulatory nucleic acid with a binding tag, and (c) linking the immunostimulatory nucleic acid to an antigen.
15. 15 . An immunostimulatory complex comprising an adjuvant and an antigen, wherein the adjuvant is the immunostimulatory nucleic acid of claim 1 , and wherein the adjuvant and the antigen are linked.
16. 16 . The immunostimulatory complex of claim 15 , wherein the antigen is a bacterial antigen, viral antigen, or fungal antigen.
17. 17 . The immunostimulatory complex of claim 15 , wherein the antigen is a peptide.
18. 18 . A method for enhancing an immune response in a subject comprising administering an effective amount of the immunostimulatory complex of claim 15 to the subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This patent application is a national stage filing under 35 U.S.C. § 371 of International Patent Application No. PCT/US2020/027970, filed Apr. 13, 2020, which claims the benefit of priority of U.S. Provisional Patent Application No. 62/832,725, filed Apr. 11, 2019, and U.S. Provisional Patent Application No. 62/952,802, filed Dec. 23, 2019, all of which are incorporated herein by reference in their entirety. SEQUENCE LISTING A Sequence Listing accompanies this application and is submitted as an ASCII text file of the sequence listing named “169946-00549_ST25.txt” which is 28.5 kb in size and was created on Apr. 13, 2020. The sequence listing is electronically submitted via EFS-Web with the application and is incorporated herein by reference in its entirety. BACKGROUND Cluster of differentiation 40 (CD40) is a costimulatory receptor that is expressed on antigen presenting cells (APCs) for regulation of the acquired immune response. The ligand for the CD40 receptor (CD40L/CD154) is transiently expressed by activated CD4+ T helper (TH) lymphocytes15. Four signals are required to initiate an adaptive immune response. The first signal occurs during an innate immune response when pattern recognition receptors (PRRs) detect cognate pathogen-associated molecular patterns (PAMPs) associated with microbial pathogens, and initiate chemokine signaling and engulfment of foreign peptides by immature dendritic cells (DCs) in peripheral tissues6, 7. This first signal initiates DC maturation, in which DCs process and present the foreign peptide on major histocompatibility complex class II (MHC class II) molecules at their surface. Maturing DCs change their cytokine production profile and migrate to T zones at draining lymph nodes, where they exhibit less phagocytic capacity and more antigen-presenting activity. The second signal results from the engagement of a naïve T cell receptor with a processed foreign peptide presented by a DC. This engagement activates the T cell and causes it to transiently express CD40L along with other costimulatory molecules and cytokines. Crosslinking of these costimulatory molecules serves as the third signal, leading to downstream cell signaling in both the DC and T cell. Cytokines produced by the DC, such as interleukin (IL)-1 and IL-2, serve as the fourth signal, stimulating T cell differentiation. At the same time, activated T cells produce cytokines, such as IL-2 and IL-4, which influence the activity of local cells. The type of cytokines generated by the T cells depends on the type of foreign peptide that was initially presented by the DC. Importantly, the second, antigen-specific signal alone is not sufficient to stimulate full differentiation of naïve T cell into effector TH cells, regulatory T cells, or memory T cells. T cells need all four signals to complete differentiation into the full array of effector cells8-11. In addition to this role in T cell activation, the CD40-CD40L interaction is also essential for T-cell-dependent B-cell activation. Moreover, CD40 participates in several other immune processes, including T-lymphocyte-dependent antibody class switching, antibody affinity maturation, development of memory B cells, and formation of the germinal centers12-16. Thus, this costimulatory protein plays a central role in initiating an adaptive immune response. Recent studies have shown that CD40 agonists can be used to induce an enhanced immune response. These agonists, which include both anti-CD40 monoclonal antibodies (mAbs) and soluble CD40L, mimic the outcome of the naturally occurring CD40-CD40L interaction. In previous work, our group demonstrated the ability of a novel anti-chicken CD40 mAb to activate a chicken HD11 macrophage cell line. When this CD40 agonist was conjugated with an antigenic peptide and administered by several routes, it induced a robust immune response and several desired effects, including immunoglobulin class switching17-19. These results suggest that this mAb would be useful as a vaccine adjuvant. However, the high cost and the difficulty of producing mAb make it impractical to commercialize this antibody for use in industries with tight profit margins, such as the poultry industry. Further, mAbs are highly immunogenic and have the potential to interfere with vaccine action. Thus, there is a need in the art for alternative CD40-stimulating biomolecules with low-immunogenicity for use as vaccine adjuvants. SUMMARY The present invention provides immunostimulatory nucleic acids that comprise a protein-binding sequence having at least 80% identity to any one of SEQ ID NOs: 1-8. In another aspect, the present invention provides immunostimulatory nucleic acids comprising a plurality of protein-binding units, wherein each of the protein-binding units comprise a first protein-binding sequence, a second protein-binding sequence, and a spacer sequence therebetween, and wherein the second protein-binding sequence is a diff