Reprogramming the genome of phage for vaccination and immunotherapy applications

Abstract

Engineered filamentous bacteriophage particle are provided, and DNA extracted from such phage particles, including: (a) a filamentous bacteriophage capsid comprising a peptide antigen displayed on a surface of the capsid; and (b) a single stranded DNA molecule (ssDNA) encapsulated within the capsid, wherein the single stranded DNA molecule; wherein (i) the ssDNA comprises at least 12% unmethylated CG dinucleotides; (ii) the ssDNA comprises sequences encoding bacteriophage (A) fl-ori, (B) fl-term, and (C) packaging signal; and (iii) the ssDNA comprises less than 50% bacteriophage ssDNA.

Description

[0001] Reprogramming the Genome of Phage for Vaccination and Immunotherapy Applications

[0002] Sequence Listing Statement

[0003] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on October 28, 2025 having the file name “23-1016-WO.xml” and is 96,580 bytes in size.

[0004] Background

[0005] An effective vaccine formulation consists of three important factors: antigen for specificity, adjuvant to stimulate the innate immune system, and delivery’ vehicle for preferential taken up by antigen presentation cells (APCs) and efficient draining to lymph nodes (LNs) where immune response orchestrate. After the antigens are internalized by APCs, such as dendritic cells (DCs), they are processed and displayed on the MHC molecules, which then present them to the T receptors (TCRs). For efficient presentation, APCs should be activated by adjuvant with the elevated expression of co-stimulate molecules, such as CD80 and CD86, in order to ligase with the complementary' receptor on T cell surface. As this process takes place in the secondary’ lymphoid organs, such as LNs, the vaccine delivery systems that can promote accumulation in the draining LN would greatly enhance the vaccine efficiency.

[0006] Bacteriophages, or phages, are prokaryotic viruses that specifically infect bacteria, and are the most abundant life form on earth. Phage-based vaccines are the recombinant phages that display immunogenic peptide or proteins, belonging to the category of peptide-based vaccine.

[0007] The filamentous phages of Ff class (fl, fd, and Ml 3) are the predominant form of phage display vectors. The filamentous phages are essentially biological nanofibers that are ~7 nm in diameter and -1-2 pm in length. They are assembled from coat proteins and ssDNA genome, The five coat proteins consist of a few thousand copies of pVIII at tire side wall enclosing the ssDNA, and five copies of each of the minor coat proteins (pill, pVI, pVII, pIX) at the two tips. All five proteins can display antigens Little effort is made to improve the adjuvant effect of phage particles, to increase the epitope expression density on the major coat protein and to modulate the size of phage particles for better vaccination efficacy.

[0008] Filamentous phages have the feasibility and tolerance of displaying a large quantity of antigens on the major coat proteins, which can greatly increase the probability for APCs to present the epitopes to TCRs. Normally, multiple copies of antigens can be displayed as recombinant pVIIIs, forming “mosaic” phage particles, yet there has no report on studying how to modulate the expression density of the antigens for optimum vaccination efficiency. This is especially important for weak antigens, for example, neoantigens, that have low binding affinity towards the MHC molecules of APCs.

[0009] Summary

[0010] In one aspect, the disclosure provides engineered filamentous bacteriophage particles, comprising:

[0011] (a) a filamentous bacteriophage capsid comprising a peptide antigen displayed on a surface of the capsid; and

[0012] (b) a single stranded DNA molecule (ssDNA) encapsulated within the capsid, wherein the single stranded DNA molecule; wherein

[0013] (i) the ssDNA comprises at least 12% unmethylated CG dinucleotides; (ii) the ssDNA comprises sequences encoding bacteriophage (A) fl-ori, (B) fl -term, and (C) packaging signal; and

[0014] (iii) the ssDNA comprises less than 50% bacteriophage ssDNA.

[0015] In one embodiment, the ssDNA comprises between 12% unmethylated CG dinucleotides and about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% unmethylated CG dinucleotides. In another embodiment, the ssDNA is at least 700 nucleotides in length. In further embodiments, the ssDNA is between 700-21,000 nucleotides in length, or between 700-15,000 nucleotides in length, or between 700-10,000 nucleotides in length, or between 700-7,000 nucleotides in length, between 1000-21,000 nucleotides in length, or between 1000-15,000 nucleotides in length, or between 1000-10,000 nucleotides in length, or between 1000-7,000 nucleotides in length.

[0016] In one embodiment, the ssDNA comprises less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%. 10%. 9%, 8%, 7%, 6%, 5%, 4%, or 3%, bacteriophage ssDNA. In another embodiment, the ssDNA comprises between about 2.5% and about 75% bacteriophage ssDNA, between about 2.5% and about 50% bacteriophage ssDNA, 2.5% and about 25% bacteriophage ssDNA, 2.5% and about 15% bacteriophage ssDNA, or 2.5% and about 10% bacteriophage ssDNA. In further embodiments, (A) none of the unmethylated CG dinucleotides have a C residue immediately 5’ to the CG dinucleotide; and (B) none of the unmethylated CG dinucleotides have a G residue immediately 3’ to the CG dinucleotide.

[0017] In one embodiment, each unmethylated CG dinucleotide is independently separated from adjacent CG dinucleotides by a flanking nucleotide linker of between 1-50 nucleotides, or between about 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-18, 1-17, 1-16, 1-15, 1-12, or 1-10 nucleotides. In another embodiment, the ssDNA does not include any complementary sequences longer than 9, 10, 11, or 12 nucleotides in length. In a further embodiment, the ssDNA does not include any repeats of 9 nucleotides or longer.

[0018] In one embodiment, the ssDNA comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% CG dinucleotides. In another embodiment, the ssDNA does not encode any filamentous bacteriophage protein, or wherein the ssDNA does not encode any protein product. In a further embodiment, the ssDNA comprises a nucleotide sequence at least 50% identical to the nucleotide sequence of any one of SEQ ID NO:3-18 and 20.

[0019] In one embodiment, the filamentous bacteriophage capsid comprises an Ml 3 capsid. In another embodiment, the peptide antigen comprises a fusion protein of the peptide antigen fused to the N-terminus of the bacteriophage pVIII protein. In a further embodiment, the peptide antigen is 5-20 amino acids in length, or between 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 6- 20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 amino acids in length, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, or 8-12 amino acids in length. In a still further embodiment, the fusion protein further comprises 1-4 negatively charged amino acids (aspartic acid or glutamic acid) C -terminal and / or N-terminal to the antigen.

[0020] In one embodiment, the capsid further comprises wild-type bacteriophage pVIII protein on the capsid surface. In another embodiment, a ratio of the fusion protein on the capsid surface to wild-type bacteriophage pVIII protein on the capsid surface is at least is 1:100, or at least 1:50, or at least 1:25, or at least 1:20, or at least 1:1 or at least 1: 10, or at least 1:2. In a further embodiment, the antigen is selected from the group consisting of SIINFEKL (SEQ ID NO: 39) (rS), ESIINFEKL (SEQ ID NO: 40) (rES), EESIINFEKL (SEQ ID NO: 41) (rEES), SVYDFFVWL (SEQ ID NO: 42), ESVYDFFVWLE (SEQ ID NO: 43), EGSRNQDWL (SEQ ID NO: 44), EGSRNQDWLE (SEQ ID NO: 45), KVPRNQDWL (SEQ ID NO: 46), EKVPRNQDWL (SEQ ID NO: 47), EKVPRNQDWLE (SEQ ID NO: 48), VKYTS (SEQ ID NO: 49), ASMTNMELM (SEQ ID NO: 50), ASMTNMELME (SEQ ID NO: 51), ASMTNMELM (SEQ ID NO: 52) and EEVKYTS (SEQ ID NO: 53). In one embodiment, the engineered filamentous bacteriophage particles further comprise a second fusion protein, wherein the second fusion protein comprises a fusion of bacteriophage pill protein fused to an immunomodulator, wherein the immunomodulator is displayed on a surface of the capsid. In one embodiment, the immunomodulatory comprises a checkpoint inhibitor. In another embodiment, the checkpoint inhibitor comprises anti-PDLl antibody or nanobody, and / or an anti-CTLA-4 antibody or nanobody. In a further embodiment the bacteriophage pill protein and the checkpoint inhibitor are separated by an amino acid linker, such as a cleavable linker.

[0021] The disclosure also provides pharmaceutical composition comprising:

[0022] (a) the engineered filamentous bacteriophage particle or the composition of any embodiment or combination of embodiments herein, and

[0023] (b) a pharmaceutically acceptable carrier.

[0024] The disclosure further provides methods for generating an immune response, comprising administering to a subject in need thereof an amount effective to generate an immune response against the antigen present in the engineered filamentous bacteriophage particle or the pharmaceutical composition of any embodiment herein.

[0025] The disclosure still further provides kits, comprising:

[0026] (a) a first plasmid encoding the ssDNA of embodiment herein; and

[0027] (b) a second plasmid comprising a gene encoding the fusion protein of any embodiment herein, operatively linked to a promoter. In one embodiment, the second plasmid also comprises genes encoding bacteriophage capsid proteins, which are operatively linked to a promoter. In another embodiment, the second plasmid also comprises a gene that encodes the second fusion protein of any embodiment herein, operatively linked to a promoter.

[0028] In another aspect, the disclosure provides compositions, comprising:

[0029] (a) a nucleic acid comprising or consisting of the nucleotide sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of any one of SEQ ID NO:21-36 and 38; and (b) a support to which the nucleic acid is attached.

[0030] In one embodiment, the support is selected from the group consisting of lipid nanoparticles, DNA origami, and DNA-based vaccines. In another embodiment, the composition further comprises an antigen. Description of the Figures

[0031] Figure 1. Tuning the antigen display by varying the number of negatively charged amino acids in the antigen peptide, a. Displaying SIINFEKL (SEQ ID NO: 39) at the N-terminus of a recombinant p8 by combining a phagemid and a helper plasmid rAg, in which the number of negatively charged amino acids were. b. Display of antigen p8 with varying number of negatively charged amino acids (rS: SIINFEKL (SEQ ID NO: 39), rES:

[0032] ESIINFEKL (SEQ ID NO: 40), rEES: EESIINFEKL (SEQ ID NO: 41)) is confirmed by MALDI. c,d. Quantification of the SIINFEKL (SEQ ID NO: 39) p8 display by HPLC. The antigen peak can be seen as the minor shoulder peak behind the major peak in the HPLC plot in Figure 1c. WT: wild type, recombinant p8:

[0033]

[0034] 0.0001, analyzed by one-way ANOVA (d) with Bonferroni post hoc test.

[0035] Figure 2. Increasing the antigen p8 display by increasing the expression rate of the antigen p8. a. Switching the expression site of the wild type p8 and the antigen p8 in the helper plasmid Ag. b. Confirmation of the antigen p8 display by MALDI. c, d. Quantification of the antigen pV8 display by HPLC. WT: wild type, recombinant p8: rp8,

[0036]

[0037] 0.0001, analyzed by two-tailed unpaired Student's t-test (d).

[0038] Figure 3. Decreasing the antigen pV8 display by introducing a regulatory mutation, a. A regulatory mutation, potentially a ribosome binding site mutation, is placed in front of the antigen p8 in the helper plasmid AgA1291T to decrease its expression rate. b. MALDI confirms the presence of the antigen p8. c,d. Quantification of the antigen pV8 with HPLC. WT: wild type, WT: wild type, recombinant p8: rp8, ****p< 0.0001, analyzed by two-tailed unpaired Student's / -test (d).

[0039] Figure 4. Tuning the adjuvanticity of the M13 phage ssDNA by programming its sequence, a. The ssDNA of the M13 phage is programmed (RP phages) to increase its stimulation of the TLR9 by incorporating more agonists, b. Three types of RP phages (CG0 / 09 / 27) of identical size with varying CG fractions in their ssDNAs are amplified, c. The activation of TLR9 increases with increasing CG fraction in the ssDNA. d. When mutating 40% of the CG in the CG27 phagemid to the CpG40 phagemid, the as assembled phages display higher TLR9 activation. WT: wild type, recombinant p8: rp8, ****?< 0.0001, analyzed by one-way ANOVA (c) with Bonferroni post hoc test and two-tailed unpaired Student's / -test (d). Scale bars represent 200 nm in (b).

[0040] Figure 5. Precise control over the length of the M13 phages by changing the size of the ssDNA. a. The length of the M13 phage is controlled by adjusting the ssDNA size in between the Fl ori and the packaging signal, b. M13 phages of ~ lOOnm, 200nm, 400nm and 800nm are amplified. WT: wild type, PS: packaging signal. Scale bars represent 200 nm in (b).

[0041] Figure 6. Shorter phages exhibit higher lymph node accumulation, a. Fluorescence images of the lymph nodes, after subcutaneous administration of M13 phages of lOOnm (left panel) and 800nm (right panel), b. Quantification of the fluorescence intensity of the inguinal, axillary', and brachial lymph nodes show phages accumulate preferentially in the inguinal lymph nodes, c. lOOnm phages display higher lymph node accumulation efficiency than 800nm phages. Inguinal: IN, axillary', brachial: BR. **p< 0.01, analyzed by two-way' AN OVA (b) with Bonferroni post hoc test.

[0042] Figure 7, Tailoring the adjuvanticity and antigen display of the RP phages for optimum vaccination efficiency, a. Dosing schedule of the in vivo vaccination studies using SIINFEKL (SEQ ID NO: 39) as the model antigen, b. SIINFEKL-specific (SEQ ID NO: 39) CD8+ T cell fraction increases with increasing adjuvanticity of the RP phages, c. Further increasing SIINFEKL (SEQ ID NO: 39) p8 display and / or adjuvanticity leads to no further improvement of the vaccination efficiency, d, e. The RP phage with the phagemid of CG27 and helper plasmid of rEES (CG27 / rEES) demonstrates better vaccination outcome than the free SIINFEKL / CpG (SEQ ID NO: 39) combinations. CpG: ODN1826. *p< 0.05, **p< 0.01, ***p< 0.001,

[0043]

[0044] 0.0001, analyzed by one-way ANOVA (b,c,d) and two-way ANOVA (e) with Bonferroni post hoc test.

[0045] Figure 8, Increasing the MC38 neoantigen Adpgk display on the RP phages, a.

[0046] Amplification of RP phage displaying Adpgk neoantigen with CpG40 phagemid and rAE and AE helper plasmid, b. Confirmation of the Adpgk display on the RP phages with MALD1. c,d. Quantifying the Adpgk pVIII display ratio with HPLC. WT: wild type, rp8: recombinant p8. ***p< 0.001, analyzed by two-tailed unpaired Student's / -test (d).

[0047] Figure 9. RP phage-based MC38 colon cancer neoantigen vaccine, a. Dosing schedule of tire in vivo vaccination study, b. RP phages with higher Adpgk pVIII display exhibit higher CD8+ T cell activation, c. Activation of the Adpgk-specific CD8 f T cells is confirmed by the elevated expression of the PD1 molecules on the cell surface, d. Tumor volume of the vaccination study, e. Survival curve of the vaccination study, f. Individual mouse tumor volume in the vaccination study, ns, not significant, ***p< 0.001, ****p< 0.0001, analyzed by one-w'ay ANOVA (b) with Bonferroni post hoc test, two-tailed unpaired Student's / -test (c) and log -rank test (e). Figure 10. The activation of mouse TLR9 by the ssDNAs developed in the study, ns, not significant, **p< 0.01,

[0048]

[0049] 0.0001, analyzed by one-way ANOVA (b) with Bonferroni post hoc test.

[0050] Detailed Description

[0051] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0052] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0053] All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

[0054] As used herein, "about" means plus or minus 5% of the particular value.

[0055] In one aspect, the disclosure provides engineered filamentous bacteriophage particles, comprising:

[0056] (a) a filamentous bacteriophage capsid comprising a peptide antigen displayed on a surface of the capsid; and

[0057] (b) a single stranded DNA molecule (ssDNA) encapsulated within the capsid, wherein the single stranded DNA molecule; wherein

[0058] (i) the ssDNA comprises at least 12% unmethylated CG dinucleotides; (ii) the ssDNA comprises sequences encoding bacteriophage (A) fl-ori, (B) fl -term, and (C) packaging signal; and

[0059] (iii) the ssDNA comprises less than 75% bacteriophage ssDNA,

[0060] As described herein, the inventors have show' n that the engineered filamentous bacteriophage particles of the disclosure are useful provide significantly improved vaccination efficacy and immunotherapy outcomes compared to current vehicles.

[0061] A filamentous bacteriophage capsid is the set of proteins that encases a singlestranded DNA genome, as is known in the art. The capsid protein can be any proteins as long as they are able to form a stable ensemble with the ssDNA through electrostatic interaction, and with neighboring capsid proteins through protein-protein interactions, such as electrostatic, hydrophobic interactions. In one embodiment, the capsid may be from any filamentous bacteriophage as appropriate for an intended use, including but not limited to bacteriophage that can infect E. col. See, for example, Hay and Lithgow, EMBO Rep. 2019 Apr 5;20(6):e47427.

[0062] In one embodiment, the filamentous bacteriophage capsid comprises an M13, fl, or fd, capsid.

[0063] As used herein, the “ssDNA” molecule encapsulated within the capsid is the entire ssDNA within the phage capsid, including bacteriophage ssDNA and engineered ssDNA. The bacteriophage ssDNA includes at least FI -or, Fl -term, and the packaging signal, which have the following sequences:

[0064] fl-ori:

[0065] 5 ' tgggccatcgccctgatagacggtttttcgccctttgacgttggctttaatagtggactc ttgttccaaactggaacaa 3 ' (SEQ ID NO: 1)

[0066] fl- term:

[0067] 5' tgggccatcgccctgat gacggtttttcgccctttgacgttggagtccacgttctttaa tagtggactcttgttccaaacaacact 3 ' (SEQ ID NO: 2 )

[0068] packaging signal:

[0069] 5 ’ cgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgc tacacttgccagcgccctagcgcccgctcc 3 ' (SEQ ID NO; 54 )

[0070] Tire ssDNA comprises less than 75% bacteriophage ssDNA. Tire remainder of the ssDNA comprises non-bacteriophage ssDNA that comprises at least 12% unmethylated CG dinucleotides. The unmethylated CG dinucleotides serve to significantly increase the adjuvant effect of the particles for vaccination and immunotherapy applications.

[0071] In some embodiments, the ssDNA comprises less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%. 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%, bacteriophage ssDNA. In other embodiments, the ssDNA comprises between about 2.5% and about 75% bacteriophage ssDNA, between about 2.5% and about 50% bacteriophage ssDNA, 2.5% and about 25% bacteriophage ssDNA, 2.5% and about 15% bacteriophage ssDNA, or 2.5% and about 10% bacteriophage ssDNA. In various embodiments, the ssDNA comprises between 12% unmethylated CG dinucleotides and about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% unmethylated CG dinucleotides.

[0072] The ssDNA may be of any length as appropriate for a given capsid. In one embodiment, the ssDNA is at least 700 nucleotides in length. In various other embodiments, the ssDNA is between 700-21,000 nucleotides in length, or between 700-15,000 nucleotides in length, or between 700-10,000 nucleotides in length, or between 700-7,000 nucleotides in length, between 1000-21,000 nucleotides in length, or between 1000-15,000 nucleotides in length, or between 1000-10,000 nucleotides in length, or between 1000-7,000 nucleotides in length.

[0073] In some embodiments, (A) none of the unmethylated CG dinucleotides have a C residue immediately 5 ' to the CG dinucleotide; and (B) none of the unmethylated CG dinucleotides have a G residue immediately 3’ to the CG dinucleotide. In other embodiments, each unmethylated CG dinucleotide is independently separated from adjacent CG dinucleotides by a flanking nucleotide linker of between 1-50 nucleotides, or between about 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-18, 1-17, 1-16, 1-15, 1-12, or 1-10 nucleotides.

[0074] In a further embodiment, the ssDNA does not include any complementary’ sequences longer than 9, 10, 11, or 12 nucleo tides in leng th, to limit formation of any hairpin s tructures. In another embodiment, the ssDNA does not include any repeats of 9 nucleotides or longer.

[0075] In one embodiment, the ssDNA does not encode any filamentous bacteriophage protein. In other embodiments, the ssDNA does not encode any protein product.

[0076] As will be understood by those of skill in the art, the specific nucleotide sequence of the ssDNA is not critical, other than the limitations noted above. By way of example only, in some embodiments the ssDNA comprises a nucleotide sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%. 85%, 90%, 95%, or 100% identical to the nucleotide sequence of any one of SEQ ID NO:3-18 and 20. The sequences of SEQ ID NO:3-18 and 20 are shown in Table 1

[0077] Table 1

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] * lowercase leters indicated the original bacteriophage ssDNA, uppercase letters indicate the programmed ssDNA

[0089] The filamentous bacteriophage capsid also comprises a peptide antigen displayed on a surface of the capsid. A given capsid may comprise a large number of peptide antigens displayed on its surface; the peptide antigens may all be the same, or may differ. The peptide antigen may be any antigen as suitable for an intended purpose, including but not limited to tumor-associated antigens, neoantigens (i.e., new protein that forms on cancer cells when certain mutations occur in tumor DNA), and antigenic fragments of antigens from any pathogenic organism, including but not limited to bacteria, viruses, and fungi. The peptide antigen may be of any length as suitable for a given purpose. In various embodiments, the peptide antigen is 5-20 amino acids in length, or between 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 6- 20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 amino acids in length, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, or 8-12 ammo acids in length.

[0090] In one embodiment, the peptide antigen comprises a fusion protein of the peptide antigen fused to the N-terminus of the bacteriophage pVIII protein. In other embodiments, the fusion protein further comprises 1-4 negatively charged amino acids (aspartic acid or glutamic acid) C-terminal and / or N-terminal to the antigen, lire inventors have shown that this embodiment permits tuning of antigen expression.

[0091] In one embodiment, amino acids 2-4 (counting from N terminus) of the wild type pVIII is replaced by the antigen peptide. In addition, the amino acid at position 12 is changed from asparagine(N) to aspartic acid(D), as exemplified below:

[0092] Wild type pVIII: AEGDDPAKAAFNSLQASATEYIGYAWAMWVIVGATIGIKLFKKFTSKAS Modified: A-Antigen- DPAKAAFDSLQASATEYIGYAWAMWVIVGATIGIKLFKKFTSKAS (SEQ ID NO: 55 ) In other embodiments, the peptide antigen is selected from the group consisting of SIINFEKL (SEQ ID NO: 39) (rS), ESIINFEKL (SEQ ID NO: 40) (rES), EESIINFEKL (SEQ ID NO: 41) (rEES), SVYDFFVWL (SEQ ID NO: 42), ESVYDFFVWLE (SEQ ID NO: 43), EGSRNQDWL (SEQ ID NO: 44), EGSRNQDWLE (SEQ ID NO: 45), KVPRNQDWL (SEQ ID NO: 46), EKVPRNQDWL (SEQ ID NO: 47), EKVPRNQDWLE (SEQ ID NO: 48), VKYTS (SEQ ID NO: 49), ASMTNMELM (SEQ ID NO: 50), ASMTNMELME (SEQ ID NO: 51), ASMTNMELM (SEQ ID NO: 52) and EEVKYTS (SEQ ID NO: 53). These antigens have all been tested in the examples that follow.

[0093]

[0094] In some embodiments, all of the bacteriophage pVIII protein on the capsid surface comprises fusions with peptide antigens as described herein, In another embodiment, the capsid further comprises wild-type bacteriophage pVIII protein (i.e., not present as a fusion protein) on the capsid surface. In this embodiment, the ratio of fusion protein to wild-type bacteriophage pVIII protein may be any that is suitable for an intended purpose. In some embodiments, a ratio of tlie fusion protein on the capsid surface to wild-type bacteriophage p VIII protein on the capsid surface is at least 1: 100. or at least 1:50, or at least 1:25, or at least 1:20, or at least 1: 15 or at least 1: 10, or at least 1:2.

[0095] In another embodiment, the engineered filamentous bacteriophage particle further comprises a second fusion protein, wherein the second fusion protein comprises a fusion of bacteriophage pill protein fused to an immunomodulator, wherein the immunomodulator is displayed on a surface of the capsid. As used herein, an “immunomodulatory” is a polypeptide that activates, enhances or promotes a response by an immune cell, or that inhibits, reduces or suppresses a response by an immune cell. Exemplary, non-limiting embodiments if immunomodulators include a cytokine, a chemokine, an activating ligand / receptor, an inhibitory’ ligand / receptor, or a combination thereof. In other embodiments, the immunomodulator comprises a checkpoint inhibitor. Any suitable checkpoint inhibitor may be used, including but not limited to an anti-PDLl antibody or nanobody, and / or an anti-CTLA-4 antibody or nanobody. The pill protein and the immunomodulatory' may be directly fused, or may be separated in the fusion protein by an amino acid linker. Any linker may be used as appropriate for an intended purpose, including but not limited to a cleavable linker.

[0096] In one embodiment, the second fusion protein comprises the following domain structure:

[0097] Immunomodulator-enzyme cleavable liker-flexible linker-pill. Ihe enzyme cleavable linker is included to cleave the immunomodulator by the enzyme rich in the target tissue. It can be a MMP2 cleavable linker such as GPLGIAGQ (SEQ ID NO: 56). Tire flexible linker is optional, and when present can be any suitable linker, including but not limited to a GS-rich linker (including but not limited to (GGGS)X, wherein x may7be 1-20. The sequence of wild type pill is shown below.

[0098] AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGWVCTGDETQCYGTW VPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPA NPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYW NGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGG SEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGD VSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEF SIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES (pill) (SEQ ID NO: 57)

[0099] In another aspect, the disclosure provides compositions, comprising: (a) a nucleic acid comprising or consisting of the nucleotide sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of any one of SEQ ID NO:21-36 and 38; and (b) a support to which the nucleic acid is attached.

[0100] The nucleotide sequence of SEQ ID NO:21-36 and 38 is provided in Table 2. These sequences are the designed sequences extracted from the nucleic acids of SEQ ID NO:3-18 and 20. The extracted ssDNA is rich with Toll-like receptor 9 agonists, and thus can be used as adjuvants. The support may be any suitable support, including but not limited to lipid nanoparticles, DNA origami, and DNA-based vaccines. The nucleic acids may be covalently attached, non-covalently attached, or present on or within the support.

[0101] For example, when the support comprises DNA origami, nucleic acid is incorporated into the structure through self-assembly driven by complementary base pairing, and the resulting composition can be self-adjuvanting, without incorporating additional adjuvants.

[0102] These compositions can be used as immunomodulators for immunotherapy applications,.

[0103] In some embodiments, the composition may further comprise an antigen as described in any embodiment or combination of embodiments above.

[0104] Table 2

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] The disclosure further provides pharmaceutical compositions, comprising:

[0113] (a) the engineered filamentous bacteriophage particle or composition of any embodiment or combination of embodiments disclosed herein; and

[0114] (b) a pharmaceutically acceptable carrier.

[0115] As used herein, the term ‘‘pharmaceutically acceptable carrier” refers to a pharmaceutically -acceptable material, composition or vehicle for administration of therapeutic described herein. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the active agent and are physiologically acceptable to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (i) sugars, such as lactose, glucose and sucrose; (ii) starches, such as com starch and potato starch; (iii) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin; (vii) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (viii) excipients, such as cocoa butter and suppository waxes; (ix) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (x) glycols, such as propylene glycol; (xi) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such as ethyl oleate and ethyl laurate; (xiii) agar; (xiv ) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid; (xvi) pyrogen-free water; (xvii) isotonic saline; (xviii) Ringers solution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi) polyesters, polycarbonates and / or polyanhydrides; (xxii) bulking agents, such as polypeptides and amino acids (xxiii) serum component, such as serum albumin, HDL and LDL; (xxiv) C2-C12 alcohols, such as ethanol; and (xxv) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

[0116] Pharmaceutically acceptable carriers can vary in a formulation described herein, depending on the administration route. The formulations described herein can be delivered via any administration mode known to a skilled practitioner. For example, the formulations described herein can be delivered in a systemic manner, via administration routes such as, but not limited to, oral, and parenteral, including intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous. In some embodiments, the formulations described herein are in a form that is suitable for injection.

[0117] The disclosure also provides methods for generating an immune response, comprising administering to a subject in need thereof an amount effective to generate an immune response against the antigen present in the engineered filamentous bacteriophage particle or the pharmaceutical composition of any embodiment or combination of embodiments herein. The methods can be used, for example, to vaccinate subjects in need thereof, or to treat cancer (such as when the peptide epitopes are tumor antigens or neoantigens).

[0118] As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s). When the method comprises treating cancer, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the size or volume of tumors and / or metastases in the subject; (b) limiting any increase in the size or volume of tumors and / or metastases in the subject; (c) increasing survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing development of symptoms associated with cancer; and (f) inhibiting worsening of symptoms associated with cancer. The subject may be any subject that can benefit from the methods of the disclosure. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. In one embodiment, the subject is a human subject. The disclosure also provides kits, comprising

[0119] (a) a first plasmid encoding the ssDNA of any embodiment or combination of embodiments herein, operatively linked to a promoter; and

[0120] (b) a second plasmid comprising a gene encoding the fusion protein of any embodiment or combination of embodiments herein, operatively linked to a promoter.

[0121] As described in the examples that follow, the kits may be used to generate the engineered filamentous bacteriophage particles of the disclosure. In one embodiment, the second plasmid also comprises genes encoding bacteriophage capsid proteins, which are operatively linked to a promoter. In this embodiment, the bacteriophage capsid proteins that are encoded on the second plasmid comprise pVI, pVII, and pIX, In some embodiments, the bacteriophage capsid proteins also comprise wild type pVIII (i.e., as opposed to the pVIII present in the fusion protein). In some embodiments, the bacteriophage capsid proteins that are encoded on the second plasmid also comprise pill. In other embodiments, the second plasmid also comprises a gene that encodes the second fusion protein of any embodiment disclosed herein, operatively linked to a promoter. All or some of the proteins encoded on the second protein may be under the control of a single promoter, or all may be under the control of separate promoters.

[0122] Examples

[0123] Overview

[0124] We developed a programming code and programmed the genome of MI3 phage with well-defined rules to enhance the TLR9 activation aiming to enhance the adjuvant effect of recombinant phage-based vaccine (RP phage). In addition, we also developed a systematic way of tuning the antigen density on the phage capsid protein. Furthermore, we studied how the size of MI3 phage impacts the vaccination efficiency. Our in vitro TLR9 cell assay and in vivo animal vaccination study have demonstrated the enhanced activation of TLR9 and improved cancer vaccination efficacy by using our RP phages.

[0125] Dual-vector system to amplify hybrid M13 phage displaying cancer antigen Materials and Methods

[0126] Capsid plasmid

[0127] In contrast to conventional M13 phage amplification approach, we use a two-vector system to amplify the hybrid M13 phage that displays the antigen of interest. The capsid plasmid has a pl 5a origin of replication and kanamycin resistance. It is responsible for coding 5 capsid proteins. The RP phagemid has a colEl origin and ampicillin resistance. It has fl-ori, fl -term and packaging signal (PS), which is able to produce highly pure flanked ssDNA segment with well-defined length for assembling M13 phage.

[0128] RP phagemid design

[0129] The DNA sequences of interest were constructed with custom-developed python code. A set of rules is defined to design the DMA sequence. First, the flanking DMA sequence in between the CG consists of ATCG but cannot begin with C and end with G. Second, if there are repeating sequence that are 9 bases or longer, the A / T in the first repeating sequence is mutated to T / A until all the repeating sequences are removed. Third, the hairpin structures are removed. Hairpin structures are defined as complementary DMA sequences that are longer than 12 bases in the DNA sequences. They are removed by mutating A to T or T to A in the hairpin structures.

[0130] Then gblocks of the designed DNA sequences were synthesized by Integrated DNA Technologies. An inho-phagemid was used as backbone to construct the RP phagemid. Specifically, an inho-phagemid was truncated after the fl-ori and before the PS with mutagenesis, then the gblock of the designed DNA was inserted with Gibson assembly.

[0131] Hybrid M13 phage amplification

[0132] A typical phage amplification process is described below:

[0133] Chemical competent XL 1 blue cells are co-transformed with the hybrid capsid protein and the RP phagemid and plated under the selection of kanamycin (50pg / mL) and ampicillin(100pg / mL). Then single colony is picked and amplified in 800mL LB broth under the selection of kanamycin(50pg / mL) and ampicillin (lOOpg / mL) with for two consecutive overnights at 37 °C with continuous 215 rpm shaking. Then the bacterial growths are spun at 8000 rpm (Beckman Coulter JLA 8, 1000) for 1 hr to pellet out bacterial cells. The phage supernatants are collected, and PEG / NaCl (final 10w / v% PEG / 0.5 M NaCl) are added to facilitate the phage precipitation at 4 °C overnight. Post precipitation, phage supernatants are spun at 8000 rpm (Beckman Coulter, JLA 8.1000) for 1 hr to pellet out the phage particles. The phage pellets are resuspended in 1 w TBS / MgCL / DNase I solution (final 5 pg mL1DNase I, 10 mM Tris-HCl, and 10 mM MgCl₂; 25 °C) and incubate for a minimum of 40min on a benchtop shaker to digest DMA contaminants. After digestion, phage precipitation is facilitated through the addition of PEG / NaCl (final 10w / v% PEG / 0.5 M NaCl) and incubate on ice for at least 2hrs. Post precipitation, the phage particles are collected by centrifuging at 14krpm for lOmin and the phage pellets are resuspended in PBSlx and further purified under CsCl gradient ultracentrifugation (1,2-1.6 g mL1gradient, SW32, 30000 rpm, 4 °C for 4 h). After ultracentrifuge, the extracted phage band (positioned at ~1,3g / mL density) is further purified of salts via dialysis (50kDa cut-off membrane) against PBSlx for at least 24hrs with frequent buffer exchange. Finally, the phage might be concentrated again by adding PEG / NaCl (final 10w / v% PEG / 0.5 M NaCl), centrifuged and redispersed in PBSlx for in vitro or in vivo applications.

[0134] Results

[0135] Antigen expression modulation

[0136] The model antigen of interest is OVA257-264 (SIINFEKL(SEQ ID NO: 39)), which has been well studied to stimulate T cell through binding to H-2Kbof MHC class I. To express the SIINFEKL (SEQ ID NO: 39) peptide, we engineered the helper plasmid construct, in which, we inserted a recombinant pVIII, controlled by Lac operon and Tac promoter and the expression of the recombinant pVIII is induced by the presence of IPTG (Figure la). The resulting Ml 3 phage would be a mosaic phage, with pVIII consisting of wild type pVIIIs (wpVIII) and SIINFEKL (SEQ ID NO: 39) pVIIIs. This helper plasmid construct is named rAg.

[0137] Thermodynamically controlling the recombinant pVIII display

[0138] The pVIII capsid protein consists of three main sections: a positively charged C-terminal that is rich with positively charged amino acids, a hydrophobic section that is buried in the capsid protein and a negatively charged N-tenninal. There exist multiple interactions among the pVIII capsid proteins, including and not limited to, hydrophobic interactions and electrostatic interactions. For N-terminal phage display, we are focusing on engineering amino acids 1-15, which are highly polar and negatively charged to interact with the surrounding solvent molecules such as water. Hence, the inserted peptide should be highly polar in order for better thermodynamically interact with the neighboring pVIIIs. Thus, the first approach we developed for improving the antigen pression on pVIII, is to vary the number of negatively charged amino acid at the N-terminal altogether with the antigen of interest.

[0139] We varied the number of glutamic acid (E), developing three different antigen constructs: SIINFEKL (SEQ ID NO: 39) (rS), ESIINFEKL (SEQ ID NO: 40) (rES) and EESIINFEKL (SEQ ID NO: 41) (rEES). The MALDI data in Figure lb shows the coexistence of the wpVIII and SIINFEKL (SEQ ID NO: 39) pVIII. HPLC data in Figure 1c shows that the SIINFEKL (SEQ ID NO: 39) pVIII expression level changes as a function of the number of negatively charged amino acids. The highest being rES construct with only one glutamic acid inserted (ESIINFEKL(SEQ ID NO: 40)) at N-terminal, and the SIINFEKL (SEQ ID NO: 39) pVIII display ratio is about -18% (Figure Id),

[0140] Kinetically control the expression rate of the pVIIIs

[0141] During the assembly of M13 phages in E. coli, the pVIIIs are being produced and then transported to the inner membrane of the E. coli, where they are assembled into the phage particles. If more antigen pVIII are being generated, more antigen pVIIIs will be assembled into the phage particle under the condition that the antigen pVIII are thermodynamically favorable for phage assembly. With this, we propose to increase the antigen pVIII display by either increasing the antigen pVIII synthesis rate or decreasing the wpVIII synthesis rate.

[0142] In the previous section, we selected the most thermodynamic favorable antigen sequence of ESIINFEKL (SEQ ID NO: 40) with a maximum antigen pVIII display ratio of 18%. The expression of the antigen pVIII is controlled by a Tac promoter, w'hile the expression of the w pVIII is controlled by the innate promoter of the helper plasmid. Hence, we speculate that the protein expression rate of the innate promoter is higher than the Tac promoter. We then propose to increase the antigen pVIII display by switching the expression site of the antigen pVIII and the wpVIII. This will increase the expression rate of the antigen pVIII and decrease the expression rate of the wpVIII. To do this, we engineered a different helper plasmid, named Ag as in Figure 2a. The coexistence of antigen pVIII and w pVIII is confirmed by MALDI in Figure 2b. HPLC data in Figure 2c shows that the antigen display is increased when switching the expression site of the antigen pVIII and the wpVIII, reaching a display ratio of 70% (Figure 2c), which is considered high for pVIII N-terminus phage display of 9 amino acids.

[0143] However, with more antigen pVIIIs being assembled into the phage particles, the yield of the phage drops substantially due to the unfavorable interactions among the pVIIIs. Hie low yield will limit tire practical applications of M13 phages, for example, as vaccines. Hence, we need to further control the expression rate of pVIII to reach a balance between the antigen display ratio and the phage yield. We propose to do this by introducing a regulatory mutation, potentially a ribosome binding site (RBS) mutation in front of the starting codon of the antigen pVIII to put a brake on the antigen p VIII expression. An RBS mutation named A1291T is thus introduced to decrease the expression rate of the antigen pVIIl, and the helper plasmid is named AgA1291T (Figure 3a). With the introduction of the mutation, the antigen pVIIl display ratio is reduced to about 35% with an improved phage yield (Figure 3b, c and d).

[0144] In summary, we are able to tune the expression of the antigen pVIIl from a thermodynamic and kinetic perspective. Specifically, we select the thermodynamically favorable antigen sequence by varying the number of negatively charged amino acids. Based on that, we change the expression rate of the antigen pVIIl and the wpVIII to kinetically control the incorporation rate of the antigen pVIIl Finally, we achieve a balance between the antigen display ratio and the phage yield for the purpose of applying phage for vaccination and immunotherapy applications. In addition to the SIINFEKL (SEQ ID NO: 39) peptide, the systematic approach developed here is also applied to adjust the expression density of other antigen peptides of interest, as shown in the table above. This method can support the development of various cancer vaccines, such as those targeting melanoma-associated antigens like Trp2 and gplOO, and fungal vaccines, such as those using Sap2 for Candida albicans

[0145] Improved adjuvanticity by reprogramming the ssDNA of the phages

[0146] In order for a vaccination formulation to work, an adjuvant which can stimulate the innate immune pathways should be included. M13 has a ssDNA enclosed in the phage capsid and ssDNA with unmethylated CG (CpG) islands is a well-known agonist of TLR9. With the activation of TLR9, inflammatory cytokines will be secreted for the maturation of APCs and activation of I' cells. Hence, we propose to increase the adjuvanticity of Ml 3 by programming its ssDNA for better activation of TLR9. We developed a python programming code with our design rules to either increase the CG fraction or include the canonical CpG hexamers (AACGTT and GACGTT) with better TLR9 activation efficiency (Figure 4a). In addition, the length of the phage is proportional to the number of nucleotides in between the Fl-ori and Fl-term, we can precisely control the length of the phage. The M13 phages with reprogrammed ssDNA are named as RP phages.

[0147] As shown in Figure 4b, we have developed a series of RP phages with equivalent size (ssDNA has the same number of nucleotides) but differs in the CG content of their ssDNA. Based on the cell assay using HEKBlue mTLR9, with increasing CG fraction, the activation of TLR9 increases. In addition, for the RP phage of CG27, when mutating 40% of the CG to the canonical CpG hexamers, the TLR9 activation is further increased. In summary, we are able to tune the adjuvanticity of the RP phages with our custom-built python script.

[0148] Size effect on lymph node draining efficiency

[0149] The size of nanoparticle can impact its draining efficiency to LN where the antigen presentation and T cell priming orchestrate. It has been reported that the higher the draining efficiency of the vaccine formulations into the draining LNs, the better the vaccination efficiency. In addition, smaller nanoparticles with size less than 200nm tend to drain freely into the LN via fenestrated LN vessels. Furthermore, as the potent APCs, DCs tend to uptake small-sized nanoparticles better. However, all those studies are based on spherical nanoparticles, and M13 phage has an ultra-high aspect ratio. Hence, we seek to explore the size effect of Ml 3 phage on LN draining efficiency and immune cell distributions for optimum vaccination efficiency.

[0150] We developed a series of RP phages with length ranging from lOOnm to 800nm, the length of the RP phages is precisely controlled by the number of nucleotides of the ssDNA as illustrated in Figure 5a and b.

[0151] In order to study the draining efficiency of the RP phages, the RP phages were chemically stained with FITC for fluorescence imaging. Post 24hrs of administration of the RP phages with length of lOOnm and 800nm via tail -base, the inguinal LNs (IN), axillary’ LNs (AX), and brachial LNs were excised and then imaged (Figure 6a).

[0152] As shown in Figure, the phages (both lOOnm and 800nm) tend to accumulate in IN LNs more than in AX and BR LNs. In addition, smaller phages (lOOnm) have better accumulation efficiency than bigger phages (800nm).

[0153] Adjuvanticity and antigen display optimization for maximum vaccination efficacy Based on the antigen display studies and the adjuvanticity modulation studies in the previous sections, we plan to apply the RP phages with precisely controlled adjuvanticity and antigen pVIII display for in vivo vaccination applications. We use SIINFEKL (SEQ ID NO: 39) as a model antigen for the studies.

[0154] We first test the hypothesis that the vaccination efficiency can be improved by increasing the adjuvanticity of the RP phage while keeping the antigen display the same. The RP phages are amplified by combining the phagemid (CG0 / 09 / 27) in Figure 4c and the helper plasmid in Figure la (rEES). With this combination, the TLR9 activation is modulated by the varying CG fraction in the phagemid, while the antigen is expressed by the recombinant pVIII. We have shown that the activation of TLR9 increases with increasing CG fraction in the ssDNA in Figure 4c. We then confirmed with HPLC that the antigen display ratio is independent of the ssDNA and is -15%. Following the vaccination schedule in Figure 7a. The FACS study with MHC tetramer staining shows that the CD8+ T cell activation increases with the adjuvanticity of the ssDNA in the RP phages (Figure 7b). When keeping the ssDNA the same (CG27) and increasing the antigen expression by changing the helper plasmid from the rAg (Figure la) to the AgA1291T (Figure 3a), the antigen expression increases from about -15% to -35%, however, the tetramer staining shows no increase in the CD8+ T cell activation (Figure 7c). Similarly, when further increasing the adjuvanticity by changing tire phagemid from CG27 to CpG40 (Figure 4d), while keeping the helper plasmid the same (Agl291T), the CD8+ T cell activation increases little. Overall, the results indicate that there is an optimum ratio between the adjuvant and the antigen and vaccination efficiency doesn’t always increase by increasing antigen and adjuvant. Next, we demonstrate that our RP phages exhibit about three times of SIINFEKL-specific (SEQ ID NO: 39) CD8+ T cell (Figure 7d) and show better tumor prevention compared to free antigen / adjuvant combinations (Figure 7e).

[0155] RP phage-based MC38 neoantigen vaccine

[0156] In the above studies, we use SIINFEKL (SEQ ID NO: 39) as a model antigen to study how to increase antigen pVIII display on the phage capsid. We then further use SIINFEKL (SEQ ID NO: 39) as a model antigen to study the effect of increasing adjuvanticity and antigen display on vaccination efficiency. Next, we aim to develop RP phage-based MC38 colon cancer vaccine by displaying MC38 colon cancer neoantigen Adpgk on the pVIII. Neoantigens are type of tumor associated antigens due to the gene mutations in the tumor tissue and they are only present in tire tumor tissue, which makes them an ideal target for cancer vaccine and therapies. Adpgk is one of the neoantigens present in the MC38 colon cancer. It occurs due to tire mutation of one single nucleotide acid, which changes the peptide from ASMTNRELM (SEQ ID NO: 58) to ASMTNMELM (SEQ ID NO: 50). Because neoantigens typically are weak antigens, the RP phages are amplified by combining the phagemid CpG40 and the helper plasmid Ag to ensure that they have the highest TLR9 activation and the highest antigen pVIII display on the phage capsid as illustrated in Figure 8a. The successful amplification of the Adpgk RP phages is confirmed by MALDI in Figure 8b. HPLC results in Figure 8b show that the Adgpk pVIII display ratio (ratio of Adpgk pVIII to the sum of Adpgk pVIII to wild type pVIII) increases from -i 3% to -60% (Figure 8c) when changing the helper plasmid from rAE to AE.

[0157] Following the dosing schedule in Figure 9a, the in vivo vaccination studies show that the CD8+ T cell activation increases with the antigen pVIII display as in Figure 9b. In addition, the elevated PD1 expression level in the Adpgk+ CD8+ T cells, indicating the activation of the antigen specific T cells (Figure 9c). Furthermore, tlie tumor prophylatic study demonstrates that the Adpgk RP phages amplified with the phagemid of CpG40 and the helper plasmid AE have successfully prevented the occurrence of the MC38 colon cancer.

[0158] Finally, we summarized the TLR9 activation of all the ssDNAs we developed (Figure 10) and all of them has a similar size of - 1400-1600 nucleotides, lire results demonstrate that we can tune up to 4-fold enhancement in the TLR9 activation (RP1415CG0mut 33 vs CpG40).

[0159] In summary, we have developed an RP phage constiuct in which the adjuvanticity of the phage is controlled by the programmable ssDNA sequence, and the antigen expression is tuned by the helper plasmid architecture. We successfully applied this construct for developing a personalized vaccine against MC38 colon cancer.

[0160] Advantages and improvements over existing methods, devices or materials.

[0161] The phage display technology has enabled tire application of phage particles for vaccination and immune therapy applications. In our work, we focus on reprogramming the genome of phage particles to enhance its adjuvant efficiency. Furthermore, we develop a systematic approach to tuning the expression level of the antigen on the phage capsid for use in vaccination and therapeutic applications, significantly improving the vaccination efficacy and immunotherapy outcomes.

[0162] Commercial applications

[0163] With the reprogrammed ssDNA of the RP phages, and more antigen peptide displayed on the phage capsid, the multifunctional RP phages can be used as vaccine against cancer, bacterial or fungi infection to activate cellular or humoral immune response. In addition, the multifunctional RP phages is an ideal platform for personalized cancer vaccine. Utilizing the technology of RNA sequencing etc., the tumor antigen of a specific patient can be identified and displayed on the phage capsid. Then the RP phages fused with the antigen can be used as personalized cancer vaccine or as immunotherapy agents. In addition, the RP phages can also be used for cancer treatment when applied alone or combining with other treatment modalities, such as chemotherapy, photothermal therapy, photodynamic therapy, and immunotherapy for example, immune checkpoint blockade (ICB) based immunotherapy. Furthermore, the ssDNAs with controllable adjuvanticity could be leveraged to design other therapeutic platforms, such as self-adj wanting DNA origami, for immune stimulation.

[0164] References

[0165] 1. Wang, Y. et al. Nontoxic engineered virus nanofibers as an efficient agent for the prevention and detection of fungal infection. Nano Res. 11, 2248-2255 (2018).

[0166] 2. Yu, C. & Irudayaraj, J. Multiplex biosensor using gold nanorods. Anal. Chem. 79, 572- 579 (2007).

[0167] 3. Dalpke, A., Frank, J., Peter, M. & Heeg, K. Activation of toll-like receptor 9 by DNA from different bacterial species. Infect. Immun. 74, 940-946 (2006).

[0168] 4. Wipf, P. & George, K. M. Regulation of Autoreactive B Cell Responses to Endogenous TLR Ligands. Bone 23, 1-7 (2008).

[0169] 5. Krieg, A. M. et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc. Natl. Acad. Sci. U. S. A. 95, 12631-12636 (1998). 6. Stunz, L. L. et al. Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur. J. Immunol. 32, 1212-1222 (2002).

[0170] 7. Bachmann, M. F. & Jennings, G. T. Vaccine delivery': A matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787-796 (2010).

[0171] 8. Slutter, B. & Jiskoot, W. Sizing tire optimal dimensions of a vaccine delivery system: A particulate matter. Expert Opin. Drug Deliv. 13, 167-170 (2016).

Claims

We claim:

1. An engineered filamentous bacteriophage particle, comprising:(a) a filamentous bacteriophage capsid comprising a peptide antigen displayed on a surface of the capsid; and(b) a single stranded DNA molecule (ssDNA) encapsulated within the capsid, wherein the single stranded DNA molecule; wherein(i) the ssDNA comprises at least 12% unmethylated CG dinucleotides; (ii) the ssDNA comprises sequences encoding bacteriophage (A) fl-ori, (B) fl -term, and (C) packaging signal; and(iii) the ssDNA comprises less than 50% bacteriophage ssDNA.

2. The engineered filamentous bacteriophage particle of claim 1, wherein tire ssDNA comprises between 12% unmethylated CG dinucleotides and about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% unmethylated CG dinucleotides.

3. The engineered filamentous bacteriophage particle of claim 1 or 2, wherein the ssDNA is at least 700 nucleotides in length,4. The engineered filamentous bacteriophage particle of any one of claims 1-3, wherein the ssDNA is between 700-21,000 nucleotides in length, or between 700-15,000 nucleotides in length, or between 700-10,000 nucleotides in length, or between 700-7,000 nucleotides in length, between 1000-21,000 nucleotides in length, or between 1000-15,000 nucleotides in length, or between 1000-10,000 nucleotides in length, or between 1000-7,000 nucleotides in length.

5. Tlie engineered filamentous bacteriophage particle of any one of claims 1-15, wherein the ssDNA comprises less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%, bacteriophage ssDNA.

6. The engineered filamentous bacteriophage particle of any one of claims 1-15, wherein the ssDNA comprises between about 2.5% and about 75% bacteriophage ssDNA, between about 2.5% and about 50% bacteriophage ssDNA, 2.5% and about 25% bacteriophage ssDNA, 2.5% and about 15% bacteriophage ssDNA, or 2.5% and about 10% bacteriophage ssDNA.

7. The engineered filamentous bacteriophage particle of any one of claims 1-6, wherein (A) none of the unmethylated CG dinucleotides have a C residue immediately 5’ to the CG dinucleotide; and(B) none of the unmethylated CG dinucleotides have a G residue immediately 3’ to the CG dinucleotide,8. The engineered filamentous bacteriophage particle of any one of claims 1-7, wherein each unmethylated CG dinucleotide is independently separated from adjacent CG dinucleotides by a flanking nucleotide linker of between 1-50 nucleotides, or between about 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-18, 1-17, 1-16, 1-15, 1-12, or 1-10 nucleotides.

9. The engineered filamentous bacteriophage particle of any one of claims 1-8, wherein the ssDNA does not include any complementary sequences longer than 9, 10, 11, or 12 nucleotides in length.

10. The engineered filamentous bacteriophage particle of any one of claims 1-9, wherein the ssDNA does not include any repeats of 9 nucleotides or longer.

11. The engineered filamentous bacteriophage particle of any one of claims 1 -10, wherein the ssDNA comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% CG dinucleotides.

12. Tire engineered filamentous bacteriophage particle of any one of claims 1-11, wherein the ssDNA does not encode any filamentous bacteriophage protein, or wherein the ssDNA does not encode any protein product.

13. The engineered filamentous bacteriophage particle of any one of claims 1-12, wherein the ssDNA comprises a nucleotide sequence at least 50% identical to the nucleotide sequence of any one of SEQ ID NO: 3- 18 and 20.

14. Tire engineered filamentous bacteriophage particle of any one of claims 1-13, wherein the filamentous bacteriophage capsid comprises an M13 capsid.

15. The engineered filamentous bacteriophage particle of any one of claims 1-14. wherein the peptide antigen comprises a fusion protein of the peptide antigen fused to the N-terminus of the bacteriophage pVIII protein.

16. The engineered filamentous bacteriophage particle of claim 15, wherein the peptide antigen is 5-20 amino acids in length, or between 5-19, 5-18, 5-17, 5-16, 5-1, 5-14, 5-13, 5-12, 6- 20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12 amino acids in length, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, or 8-12 ammo acids m length.

17. The engineered filamentous bacteriophage particle of claim 15 or 16, wherein the fusion protein further comprises 1-4 negatively charged amino acids (aspartic acid or glutamic acid) C-terminal and / or N-terminal to the antigen.

18. The engineered filamentous bacteriophage particle of any one of claims 15-17, wherein the capsid further comprises wild-type bacteriophage pVlll protein on the capsid surface.

19. Tire engineered filamentous bacteriophage particle of claim 18, wherein a ratio of the fusion protein on the capsid surface to wild-type bacteriophage pVIII protein on the capsid surface is at least is 1:100, or at least 1:50, or at least 1:25, or at least 1:20, or at least 1:15 or at least 1:10, or at least I:2.

20. The engineered filamentous bacteriophage particle of any one of claims 1-19 wherein the antigen is selected from the group consisting of SIINFEKL (SEQ ID NO: 39) (rS), ESIINFEKL (SEQ ID NO: 40) (rES), EESIINFEKL (SEQ ID NO: 41) (rEES), SVYDFFVWL (SEQ ID NO: 42), ESVYDFFVWLE (SEQ ID NO: 43), EGSRNQDWL (SEQ ID NO: 44), EGSRNQDWLE (SEQ ID NO: 45), KVPRNQDWL (SEQ ID NO: 46), EKVPRNQDWL (SEQ ID NO: 47), EKVPRNQDWLE (SEQ ID NO: 48), VKYTS (SEQ ID NO: 49), ASMTNMELM (SEQ ID NO: 50), ASMTNMELME (SEQ ID NO: 51), ASMTNMELM (SEQ ID NO: 52) and EEVKYTS (SEQ ID NO: 53).

21. Tire engineered filamentous bacteriophage particle of any one of claims 1 -20, further comprising a second fusion protein, wherein the second fusion protein comprises a fusion ofbacteriophage pill protein fused to an immunomodulator, wherein the immunomodulator is displayed on a surface of the capsid.

22. The engineered filamentous bacteriophage particle of claim 21, wherein the immunomodulatory comprises a checkpoint inhibitor.

23. The engineered filamentous bacteriophage particle of claim 22, wherein the checkpoint inhibitor comprises anti-PDLl antibody or nanobody, and / or an anti-CTLA-4 antibody or nanobody.

24. The engineered filamentous bacteriophage particle of claim 22 or 23, wherein the bacteriophage pill protein and the checkpoint inhibitor are separated by an amino acid linker, such as a cleavable linker,25. A pharmaceutical composition comprising:(a) the engineered filamentous bacteriophage particle of any one of claims 1-24 or the composition of claim 30 or 31; and(b) a pharmaceutically acceptable carrier.

26. A method for generating an immune response, comprising administering to a subject in need thereof an amount effective to generate an immune response against the antigen present in the engineered filamentous bacteriophage particle of any one of claims 1-24 or the pharmaceutical composition of claim 25.

27. A kit, comprising:(a) a first plasmid encoding the ssDNA of any one of claims 1-24; and(b) a second plasmid comprising a gene encoding the fusion protein of any one of claims 15-24, operatively linked to a promoter.

28. The kit of claim 27, wherein the second plasmid also comprises genes encoding bacteriophage capsid proteins, which are operatively linked to a promoter.

29. The kit of claim 27 or 28, wherein the second plasmid also comprises a gene that encodes the second fusion protein of any one of claims 22-24, operatively linked to a promoter.

30. A composition, comprising:(c) a nucleic acid comprising or consisting of the nucleotide sequence at least 25%, 30%, 35%, 40%. 45%, 50%, 60%. 70%, 75%, 80%. 85%, 90%, 95%. or 100% identical to the nucleotide sequence of any one of SEQ ID NO:21-36 and 38; and (d) a support to which the nucleic acid is attached.

31. The composition of claim 30, wherein the support is selected from the group consisting of lipid nanoparticles, DNA origami, and DNA-based vaccines.

32. The composition of claim 30 or 31, further comprising an antigen.

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