Bacterial antigen presenting cells, fusion proteins, and methods of use

Abstract

Bacterial antigen presenting cells are provided. Fusion proteins comprising a peptide-MHC complex, linker, and an autotransporter can be expressed on the surface of gram-negative bacteria, which can selectively stimulate T-cells reactive to the peptide-MHC complex. The bacterial antigen presenting cells can be used, e.g., to stimulate T-cells in vitro or in vivo.

Description

DESCRIPTIONBACTERIAL ANTIGEN PRESENTING CELLS, FUSION PROTEINS, ANDMETHODS OF USE

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 696,946, filed September 20, 2024, and United States Provisional Patent Application No. 63 / 804,270, filed May 12, 2025, the entirety of each of which are incorporated herein by reference.

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on September 3, 2025, is named UTFBP1382WO.xml and is 28,467 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns fusion proteins for expression in bacteria that can activate T-cells.2. Description of Related Art

[0004] Although progress has been made for activating immune cells including T cells to treat a disease in a mammalian subject (e.g. , cancer), challenges remain. In Honda et al. (2014), a MHC I / peptide complex was expressed in Magnetospirillum magneticum', however, these modified bacteria did not result in any T-cell activation. These results illustrate the challenges of using gram-negative bacteria to present MHC-peptide complexes that can modulate T-cell responses, and support the idea that methods do not presently exist for using gram-negative bacteria to cause T-cell activation to a MHC-peptide complex expressed by the gram-negative bacteria. There is a need for new methods for activating T-cells from a mammalian subject.4911-1898-8134, v. 1 1SUMMARY

[0005] The present disclosure overcomes limitations in the prior art by providing, in some aspects, fusion proteins and bacterial antigen presenting cells. The present disclosure is based, in part on the generation of fusion proteins comprising: (i) a MHC, linker, and an antigenic peptide (e.g., wherein the MHC, linker and antigenic peptide can preferably function similar to or form a peptide-MHC complex), and (ii) an outer membrane display protein, preferably an autotransporter. The fusion protein can be expressed on the surface of gramnegative bacteria, to result in bacterial antigen presenting cells, and these bacterial antigen presenting cells can selectively stimulate T-cells reactive to the peptide-linker-MHC portion of the fusion protein. Methods of using the bacterial antigen presenting cells are also provided. For example, the bacterial antigen presenting cells can be used to stimulate T-cells in vitro that are reactive to the peptide-linker-MHC portion of the fusion protein. Thus, T-cells (e.g. , autologous T-cells from a mammalian subject such as a human) can be obtained and contacted with the bacterial antigen presenting cell to increase T-cell reactivity to the peptide that is relevant for treating a disease (e.g., cancer) or infection (e.g., viral, fungal, or bacterial). The activated T-cells may then be administered to a mammalian subject to treat the disease or infection. In some aspects, the bacterial antigen presenting cells can be directly administered to a mammalian subject in vivo to selectively stimulate T-cells in the subject. The bacterial antigen presenting cell may comprise a gram-negative bacteria that is live, attenuated, or inactivated. In some aspects, the bacterial antigen presenting cells can be used to screen a peptide library for peptides that are therapeutically relevant for immunological recognition of a diseased cell (e.g., cancerous cell) or a immunological foreign invader (e.g. , virus, fungi, bacteria, protozoan). The gram-negative bacteria may further express (e.g., recombinantly or episomally) a co-stimulation factor (e.g., CD80) and / or one or more cytokines that may enhance the T-cell stimulating properties. In contrast to Honda el al. (2014), the present disclosure provides compositions and methods for expressing MHC-peptide complexes in gram-negative bacteria that can activate mammalian T-cells (e.g., human T-cells) that selectively recognize the MHC-peptide complex.

[0006] As shown in the below examples and figures, fusion proteins are provided that comprise (i) a MHC (e.g., preferably MHC-I), linker, and peptide, and (ii) an outer membrane display protein, preferably an autotransporter. The fusion protein may be expressed on the outer cell membrane of a gram-negative bacteria. Based on previous studies, the fusion protein4911-1898-8134, v. 1 2would not have been expected to display on the cell surface of gram-negative bacteria for at least two critical reasons. First, single chain MHC proteins are generally thought to require expression in eukaryotic systems for proper folding. Expressing single chain MHC proteins in E. coli results in protein aggregation and MHCs are found as insoluble inclusion bodies (Cohen et al., 2003). Second, MHC proteins include 3-4 disulfide bonds and are comprised of amino acids located 30-60 residues apart. These disulfide bonds are required for proper protein folding and would generally be considered to appear in the periplasm of a gram-negative bacteria (Jose et al. , 1996; Leyton et al. , 201 1). These disulfide bonds are generally considered to block transit of proteins through outer membrane display systems like autotransporters and thus MHC proteins encoding disulfide bonds would be expected to be stuck in the periplasm and never reach the gram-negative cell surface. Thus, requirements of eukaryotic processing and presence of disulfide bonds would both generally be expected to prevent the display of single chain MHC molecules on the surface of gram-negative bacteria. Nonetheless, it was surprisingly observed that the fusion protein comprising an autotransporter was able to express on the outer membrane of gram-negative bacteria including E. coli. Further and in contrast to previous work, the MHC-peptide complex was observed to be able to selectively activate mammalian T-cells. The gram-negative bacteria may comprise one or more genetic features, mutations, chaperone proteins etc. from E. coli W3110. In some aspects, the E. coli W3110 bacteria are used to express the fusion protein. The inventors observed that expression of the fusion protein in gram-negative bacteria, with or without co-stimulation of CD28 was sufficient to selectively stimulate T-cells extracted from OT1 mice that are reactive to the OVA peptide (e.g., see FIGS. 3A-B, FIGS. 4A-B). Based on expression of CD69, a dose-dependent and selective activation of T-cells was observed when contacted with the antigen presenting bacterial cells using T cells extracted from OT-1 mice. These results support the idea that gram-negative bacteria, such as E. coli, expressing the fusion protein can be used to selectively activate mammalian T-cells (e.g., from rat, mouse, non-human primate, or humans) in vitro and / or in vivo. Without wishing to be bound by any theory, it is anticipated that bacteria may further act as an adjuvant for stimulating the immune system or T-cells, and this effect may further enhance the T-cell activating effects of the expressed fusion protein. It is anticipated that a variety of autotransporters and additional gram-negative surface display proteins may be used with the approaches and fusion proteins provided herein. The MHC region of the fusion protein may preferably be MHC-I or MHCII. A variety of linkers can be used including glycine-serine linkers or poly-Glycine-Serine linkers such as, e.g., (GGGGS)nwherein n=3-4 (SEQ ID NO:6 and SEQ ID NO:7). The fusion protein may optionally comprise an epitope4911-1898-8134, v. 1 3tag (e.g., epitope tag E) that may be used for detection or analysis (e.g., flow cytometry) if desired. Nucleic acids encoding the fusion proteins were optimized for expression in bacterial cells. Bacterial antigen presenting cells were fixed and inactivated (i.e. , killed) using formalin, and even after inactivation the bacterial antigen presenting cells were able to stimulate T-cells (e.g., FIG. ID). These results support the idea that live, attenuated, or inactivated bacteria in the bacterial antigen presenting cells can be used to promote T-cell activation.

[0007] An aspect of the present disclosure relates to a gram-negative bacteria, wherein the gram-negative bacteria expresses a fusion protein comprising: (i) a single chain trimer comprising: (a) an antigenic peptide, (b) a first linker, (c) a P-microglobulin, (d) a second linker, and (e) a mammalian MHC heavy chain, and (ii) an outer membrane display protein, preferably an autotransporter; wherein the antigenic peptide is preferably 8-1 1 amino acids in length. The fusion protein is preferably expressed on the surface of the outer membrane of the gramnegative bacteria. The gram-negative bacteria may activate mammalian T cells that selectively bind the antigenic peptide or complex between the antigenic peptide and the mammalian MHC heavy chain. The MHC may be a human MHC, such as a human class I MHC heavy chain (e.g., HLA-A, HLA-B, or HLA-C). In some aspects, the human class I MHC is HLA-A, preferably HLA-A2. The mammalian MHC , may be a mouse MHC, a rat MHC, a rabbit MHC, or a non-human primate MHC. The MHC may comprise P-2 microglobulin. The antigenic peptide may further defined as a human MHC-restricted peptide. The antigenic peptide may be randomized or may be from a MHC restricted peptide library, such as for example preferably from a library of antigenic peptides from a cancer, a virus, or an infectious bacteria. The gram-negative bacteria may be an E. coli (e.g., an E. coli strain W3110). The gram-negative bacteria may further express a co- stimulation factor. The co-stimulation factor may be CD80, CD86, IL2, B7-H1, B7RP-1, CD27, CD28, CD70, TNFR1, TNFR2, HVEM, CD30, 4-1BB, 0X40, GITR, CD27, CD40, or TIM-1. In some preferred aspects, the costimulation factor is CD80. The gram-negative bacteria may be a live bacteria, an attenuated bacteria, or an inactivated bacteria. The gram-negative bacteria may be inactivated, e.g., via exposure to ionizing radiation, chemical inactivation (e.g., exposure to formaldehyde or formalin), or exposure to ultrasound. The gram-negative bacteria may be inactivated via chemical inactivation by formaldehyde or formalin. In some aspects, the gram-negative bacteria may be a vaccine bacteria or a probiotic bacteria. The antigenic peptide may be an antigen from a cancerous cell, an infectious bacterium, a virus, or a protozoan. The cancerous cell may be a melanoma, colorectal cancer, prostate cancer, breast cancer, or lung cancer. The4911-1898-8134, v. 1 4antigenic peptide may be Trpl, Trp2, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), Her2 / neu, or MUC 1. The autotransporter may be an adhesin involved in diffuse adherence (AIDA). The autotransporter may be ShdA, IgAP, NalP, Bru A, VacA, YfaL, intimin, or MisL autotransporter. The autotransporter may be EhaA autotransporter (e.g. , GenBank WP_424703195). The first linker and / or the second linker may be a serine-glycine linker. The first linker and / or the second linker may comprise or consist of (GGGGS)n, wherein n=3-4 (SEQ ID NO:6 and SEQ ID NO:7). The fusion protein may comprise an epitope tag. The epitope tag may be epitope E. The gram-negative bacteria may express or secrete a cytokine (e.g. , IL-12, IL-18, IL-2, IL-7, IL-15, IL-4, an interferon, or TGF-0). The fusion protein, co-stimulation factor, and / or cytokine may be recombinantly expressed. The fusion protein, co-stimulation factor, and / or cytokine may be episomally expressed. The fusion protein may comprise one or more sequences provided herein or below, or a polypeptide having at least 90% sequence identity, more preferably 95% sequence identity thereto. In some aspects, the gram-negative bacteria is not Magnetospirillum magneticum, and preferably not a magnetotactic bacterium. The gram- negative may be E. coli e.g. , E. coli strain W3110) .

[0008] Aspects of the present disclosure can be understood by the following numbered sentences:1. A gram-negative bacteria, wherein the gram-negative bacteria expresses a fusion protein comprising:(i) a single chain trimer comprising:(a) an antigenic peptide,(b) a first linker,(c) a P-microglobulin,(d) a second linker, and(e) a mammalian MHC heavy chain, and(ii) an outer membrane display protein, preferably an autotransporter; wherein the antigenic peptide is preferably 8- 11 amino acids in length.2. The gram-negative bacteria of sentence 1 , wherein the fusion protein is expressed on the surface of the outer membrane of the gram-negative bacteria.3. The gram-negative bacteria of sentence 2, wherein the gram-negative bacteria can activate mammalian T cells that selectively bind the antigenic peptide.4911-1898-8134, v. 1 54. The gram- negative bacteria of any of sentences 1-3, wherein the MHC is a human MHC.5. The gram-negative bacteria of any of sentence 4, wherein the human MHC is a human class I MHC heavy chain.6. The gram-negative bacteria of sentence 5, wherein the human class I MHC is HLA-A, HLA-B, or HLA-C.7. The gram-negative bacteria of sentence 6, wherein the human class I MHC is HLA-A, preferably HLA-A2.8. The gram-negative bacteria of any of sentences 1-3, wherein the mammalian MHC is a mouse MHC, a rat MHC, a rabbit MHC, or a non-human primate MHC.9. The gram-negative bacteria of any one of sentences 1-8, wherein the MHC comprises P-2 microglobulin.10. The gram- negative bacteria of any one of sentences 1-9, wherein the antigenic peptide is further defined as a human MHC-restricted peptide.11. The gram- negative bacteria of any one of sentences 1-10, wherein the antigenic peptide has been randomized or is from a MHC restricted peptide library, preferably a library of antigenic peptides from a cancer, a virus, or an infectious bacteria.12. The gram-negative bacteria of any one of sentences 1-11, wherein the gram-negative bacteria is an E. coli.13. The gram-negative bacteria of sentence 12, wherein the gram-negative bacteria is an E. coli strain W3110.14. The gram-negative bacteria of any one of sentences 1-13, wherein the gram-negative bacteria further expresses a co- stimulation factor.15. The gram-negative bacteria of sentence 14, wherein the co-stimulation factor is CD80, CD86, IL2, B7-H1, B7RP-1, CD27, CD28, CD70, TNFR1, TNFR2, HVEM, CD30, 4-1BB, 0X40, GITR, CD27, CD40, or TIM-1.16. The gram-negative bacteria of sentence 15, wherein the co-stimulation factor is CD80.4911-1898-8134, v. 1 617. The gram-negative bacteria of any one of sentences 1-16, wherein the gram-negative bacteria is a live bacteria.18. The gram-negative bacteria of any one of sentences 1-16, wherein the gram-negative bacteria is an attenuated bacteria.19. The gram-negative bacteria of any one of sentences 1-16, wherein the gram-negative bacteria is an inactivated bacteria.20. The gram-negative bacteria of sentence 19, wherein the gram-negative bacteria has been inactivated via exposure to ionizing radiation, chemical inactivation, or exposure to ultrasound.21. The gram-negative bacteria of sentence 20, wherein the gram-negative bacteria has been inactivated via chemical inactivation by formaldehyde or formalin.22. The gram-negative bacteria of any one of sentences 1-21, wherein the gram-negative bacteria is a vaccine bacteria or a probiotic bacteria.23. The gram- negative bacteria of any one of sentences 1-22, wherein the antigenic peptide is an antigen from a cancerous cell, an infectious bacterium, a virus, or a protozoan.24. The gram-negative bacteria of sentence 23, wherein the cancerous cell is a melanoma, colorectal cancer, prostate cancer, breast cancer, or lung cancer.25. The gram-negative bacteria of sentence 24, wherein the antigenic peptide is Trpl , Trp2, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), Her2 / neu, or MUC1.26. The gram-negative bacteria of any one of sentences 1-25, wherein the autotransporter is an adhesin involved in diffuse adherence (AIDA).27. The gram-negative bacteria of sentence 26, wherein the autotransporter is ShdA, IgAP, NalP, BruA, VacA, YfaL, intimin, or MisL autotransporter.28. The gram-negative bacteria of sentences 26, wherein the autotransporter is EhaA autotransporter.29. The gram-negative bacteria of any one of sentences 1 -28, wherein the first linker and / or the second linker is a serine-glycine linker.4911-1898-8134, v. 1 730. The gram-negative bacteria of sentence 29, wherein the first linker and / or the second linker comprises or consists of (GGGGS)n, wherein n=3-4.31. The gram-negative bacteria of any one of sentences 1-30, wherein the fusion protein comprises an epitope tag.32. The gram-negative bacteria of sentence 31 , wherein the epitope tag is epitope E.33. The gram-negative bacteria of any one of sentences 1-32, wherein the gram-negative bacteria expresses or secretes a cytokine.34. The gram-negative bacteria of sentence 33, wherein the cytokine is IL-12, IL-18, IL-2, IL-7, IL- 15, IL-4, an interferon, or TGF-p.35. The gram-negative bacteria of any one of sentences 1-34, wherein the fusion protein, co-stimulation factor, and / or cytokine is recombinantly expressed.36. The gram-negative bacteria of any one of sentences 1-34, wherein the fusion protein, co-stimulation factor, and / or cytokine is episomally expressed.37. The gram-negative bacteria of sentence 1 , wherein the fusion protein comprises one or more sequences provided herein, or a polypeptide having at least 90% sequence identity, more preferably 95% sequence identity thereto.38. The gram-negative bacteria of any one of sentences 1-37, wherein the gram-negative bacteria is not Magnetospirillum magneticum, and preferably not a magnetotactic bacterium.39. The gram-negative bacteria of any one of sentences 1-37, wherein the gram-negative bacteria is E. coli.40. The gram-negative bacteria of sentence 39, wherein the E. coli is E. coli strain W3110.41. A composition comprising the gram-negative bacteria of any one of sentences 1-38 and a carrier.42. The composition of sentence 41, wherein the composition is a pharmaceutical composition and wherein the carrier is an excipient.4911-1898-8134, v. 1 843. The composition of sentence 42, wherein the composition is formulated for intravenous, oral, mucosal, parenteral, or intratumoral administration.44. A nucleic acid encoding the fusion protein of any one of sentences 1-38.45. The nucleic acid of sentence 44, wherein the nucleic acid is comprised in a vector.46. The nucleic acid of any one of sentences 44-45, wherein the nucleic acid encoding the fusion protein is operably connected to a promoter.47. The nucleic acid of sentence 46, wherein the promoter is a prokaryotic promoter or is active in bacteria.48. The nucleic acid of sentence 47, wherein the promoter is an inducible promoter or a constitutive promoter.49. The nucleic acid of sentence 47, wherein the promoter is a lac, taq, tet, arabinose, or a constitutive promoter.50. The nucleic acid of any one of sentences 44-49, wherein the nucleic acid is optimized for expression in a gram-negative bacteria.51. The nucleic acid of sentence 50, wherein the gram- negative bacteria is E. coli.52. An in vitro method for activating mammalian T cells comprising:(i) obtaining mammalian T-cells, and(ii) contacting the T-cells with the gram-negative bacteria of any one of sentences 1- 38.53. The method of sentence 52, wherein the T-cells are contacted with a co-stimulator factor or a cytokine.54. The method of sentence 53, wherein the co-stimulator factor or cytokine is exogenous.55. The method of any one of sentences 52-54, wherein the mammalian T cells are human T cells.4911-1898-8134, v. 1 956. A method of providing activated T cells to a mammalian subject, comprising generating activated mammalian T cells via the method of any one of sentences 52-55, and administering the T cells to the mammalian subject.57. The method of sentence 56, wherein the mammalian subject is a human.58. The method of sentence 57, wherein the T cells are administered intravenously, orally, mucosally, parenterally, or intratumorally to the subject.59. A method for activating T cells in a mammalian subject comprising administering the gram-negative bacteria of any one of sentences 1-38 or the composition of any one of sentences 41-43 to the mammalian subject.60. The method of sentence 59, wherein the mammalian subject is a human.61. The method of sentence 60, wherein the subject has a cancer, the antigenic peptide is a cancer antigen, and wherein the method comprises treating cancer in the subject.

[0009] Another aspect of the present disclosure relates to a composition comprising the gram-negative bacteria described above or herein and a carrier. In some aspects, the composition is a pharmaceutical composition and the carrier is an excipient. The composition may be formulated for intravenous, oral, mucosal, parenteral, or intratumoral administration.

[0010] Yet another aspect of the present disclosure relates to a nucleic acid encoding the fusion protein described above or herein. The nucleic acid may be comprised in a vector. The nucleic acid encoding the fusion protein may be operably connected to a promoter. The promoter may be a prokaryotic promoter or may be active in bacteria. The promoter may be an inducible promoter or a constitutive promoter. The promoter may be, e.g. , a lac, taq, tet, arabinose, or a constitutive promoter. The nucleic acid may be optimized for expression in a gram-negative bacteria. The gram-negative bacteria may be E. coli.

[0011] Another aspect of the present disclosure relates to an in vitro method for activating mammalian T cells comprising: (i) obtaining mammalian T-cells, and (ii) contacting the T-cells with the gram-negative bacteria described above or herein. The T-cells are contacted with a co-stimulator factor or a cytokine. The co-stimulator factor or cytokine may be exogenous. The mammalian T cells may be human T cells.4911-1898-8134, v. 1 10

[0012] Yet another aspect of the present disclosure relates to a method of providing activated T cells to a mammalian subject, comprising generating activated mammalian T cells via the method described above or herein, and administering the T cells to the mammalian subject. The mammalian subject may be a human. The T cells may be administered intravenously, orally, mucosally, parenterally, or intratumorally to the subject.

[0013] Another aspect of the present disclosure relates to a method for activating T cells in a mammalian subject comprising administering the gram-negative bacteria described above or herein or the composition described above or herein to the mammalian subject. The mammalian subject may be a human. In some aspects, the subject may have a cancer, wherein antigenic peptide is a cancer antigen, and wherein the method comprises treating cancer in the subject.

[0014] As used herein “T-cell activation” and “T-cell stimulation” are interchangeably used to refer to the process in which mature T cells, which express antigen-specific T-cell receptors on their surfaces, recognize an antigenic peptide bound by a MHC and respond by one or more of entering the cell cycle, secreting cytokines or lytic enzymes, and / or initiating one or more cell-based functions of the immune system.

[0015] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0016] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0017] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0018] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of114911-1898-8134, v. 1the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0019] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0021] The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e. carrier, or vehicle.

[0022] The term “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth or development in an immunocompromised non-human animal model, or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell” or “cancerous cell” and encompasses cancer cells of a solid tumor and a liquid tumor. “Cancer” may be used interchangeably herein with “tumor”.

[0023] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or4911-1898-8134, v. 1 12prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0024] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0025] The terms “in operable combination”, “in operable order”, and “operably linked” refer to a linkage wherein the components so described are in a relationship permitting them to function in their intended manner, for example, a linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene or the synthesis of desired protein molecule, or a linkage of amino acid sequences in such a manner so that a fusion protein is produced.

[0026] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0027] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.4911-1898-8134, v. 1 13BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0029] FIGS. 1A-D: Confirming Folded MHC I Display in W3110; Efficient cell expression and folded MHC I display in W3110. (FIG. 1A) Expression confirmation of the E- tagged MHC I protein in W3110. Western blot of W3110 transformed with the indicated pHEA MHC I construct after induction, probed by anti-E-tag antibody. (FIG. IB) Flow cytometry with PE-conjugated mAbs specific for ovalbumin-derived peptide SllNFEKL (SEQ ID NO:8) bound to H-2Kb of MHC class I. Surface staining histograms are shown for induced W3110 MHC I transformants (arrow indicates) and wild-type W3110. (FIG. 1C) Flow cytometry with FITC-conjugated pAbs specific for E-tag. Surface staining is shown for induced W3110 MHC I transformants (arrow indicates) and wild-type W3110. (FIG. ID) Flow cytometry with APC- conjugated mAbs specific for ovalbumin-derived peptide SllNFEKL (SEQ ID NO: 8) bound to H-2Kb of MHC class I. Surface staining is shown for induced and formaldehyde fixed W3110 MHC I transformants (arrow indicates) and formaldehyde fixed wild-type W3110.

[0030] FIGS. 2A-C: Display of Disordered MHC I in BL21; Efficient cell expression and disordered display of MHC I in BL21. (FIG. 2A) Western blot of BL21 transformed with the indicated pHEA MHC I construct after induction, probed by anti-E-tag antibody. Lane 2 shows expression of the E-tagged MHC I protein (FIG. 2B) Flow cytometry with PE-conjugated mAbs specific for ovalbumin-derived peptide SllNFEKL (SEQ ID NO:8) bound to H-2Kb of MHC class I. Surface staining is shown for induced BL21 MHC I transformants (arrow indicates) and wild-type BL21. (FIG. 2C) Flow cytometry with FITC- conjugated pAbs specific for E-tag. Surface staining is shown for induced BL21 MHC I transformants (arrow indicates) and wild-type BL21.

[0031] FIGS. 3A-B: Early Activation Marker T Cell Activation; T cell activation by W3110 MHC I transformants. (FIG. 3A) Flow cytometry exemplary plots showing live OT-1 CD8+ lymphocyte subset surface stained with an anti-CD69 antibody following a 6-hour T cell activation assay. Row 1 is a set of standardized positive and negative controls. Row 2 compares the amount of surface stained CD69 marker after incubation with exogenously added4911-1898-8134, v. 1 14Anti-CD28 and induced W3110 MHC I and wild-type W3110, respectively. Representative data are shown for one of three replicates. (FIG. 3B) Graphs represent the quantification of the percentage of cells of the indicated subset that express surface CD69.

[0032] FIGS. 4A-B: Early Activation Marker T Cell Activation Assay without CD28; T cell activation by W3110 MHC I transformants. (FIG. 4A) Flow cytometry exemplary plots showing live OT-1 CD8+ lymphocyte subset surface stained with an anti- CD69 antibody following a 6-hour T cell activation assay. Row 1 is a set of standardized positive and negative controls. Row 2 compares the amount of surface stained CD69 marker after incubation with induced W3110 MHC I and wild-type W3110. Representative data are shown for one of three replicates. (FIG. 4B) Graphs represent the quantification of the percentage of cells of the indicated subset that express surface CD69.

[0033] FIGS. 5A-B: OT-1 Specific T Cell Activation; Evaluating the specificity of CD8 T cell activation. (FIG. 5A) Graph represents the quantification of the percentage of cells of live OT-1 CD8 cell subset that express surface CD69 after T cell activation assay with induced W3110 MHC I and wild-type W3110. (FIG. SB) Graph represents the quantification of the percentage of cells of live polyclonal CD8 cell subset that express surface CD69 after T cell activation assay with induced W3110 MHC I and wild-type W3110.

[0034] FIG. 6: OVAp Mutants on T cell Activation. OVAp mutants have different effects on T cell activation. OVAp mutants and irrelevant peptide N7p were displayed on single chain MHCI and tested in the T cell activation assay. CD69 levels were measured as a marker for T cell activation. Inset shows sequences of OVAp mutants; top to bottom: SEQ ID NOS:8, 13-17.4911-1898-8134, v. 1 15DESCRIPTION OF ILLUSTRATIVE EMBODIMENTSI. Fusion Proteins and Bacterial Antigen Presenting Cells

[0035] It is anticipated that a variety of single-chain MHC-fusion proteins can be included in a fusion protein comprising an outer membrane display protein, preferably an autotransporter. In some aspects, a MHCI-peptide complex can include a fusion protein as described in Mottez et al. (1995) or Fremont et al. (2007). The display of signal chain MHCI using an autotransporter can include any of the sequences provided herein.

[0036] In some aspects, the fusion protein includes a signal peptide as described in Rusch and Kendall, (2007). Signal peptides are short amino-terminal parts of exported precursor proteins that direct the respective proteins to the protein export systems in the cytoplasmic membrane. Sec signal peptides generally do not show sequence similarities; however a conserved tripartite overall structure can be recognized, which generally include a positively charged amino-terminal n-region, a central hydrophobic core (h-region), and a polar carboxyl-terminal domain (c -region) that contains the signal peptidase recognition site (consensus motif A-X-A). The signal sequence incorporated in the engineered fusion protein described herein can include but are not limited to OmpA; PhoA; PelB; DsbA; or TorT. In some preferred aspects the signal sequence is provided in SEQ ID NO: 1.

[0037] MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 1) is provided. In some embodiments, the fusion protein is a single-chain trimer (SCT), comprising a peptide antigen, P2-microglobulin (P2m), and an MHC class I heavy chain covalently linked in a single polypeptide chain. The SCT format permits the use of distinct flexible or rigid linkers between individual domains, including but not limited to Gly-Ser linkers (e.g., (G4S)X(e. . , where x is 2, 3, 4, or 5 (SEQ ID NOS:26, 6, 7, and 27))), a-helical linkers (e.g., EAAAK repeats (SEQ ID NOS:28-31)), or engineered protease-cleavable linkers. In some embodiments, distinct linker sequences are used between the P2m domain and MHCI heavy chain, and between the MHCI heavy chain and the autotransporter, providing differential flexibility or spatial arrangement.

[0038] The P2-microglobulin domain may be derived from human or non-human sources and may comprise native or engineered variants designed to enhance expression, stability, or immunogenicity. In certain embodiments, human P2m e.g., UniProt P61769) is utilized. Alternative P2m sequences include murine P2m (UniProt P01887) and other speciesspecific variants as appropriate for xenogeneic or comparative studies. Engineered P2m4911-1898-8134, v. 1 16mutants may include alterations at positions that improve peptide-MHC stability or reduce aggregation (see, e.g., Shields et al., JBC 1998). In some preferred aspects, the P2m sequence is provided in SEQ ID NO: 2.IQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSK DWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDM (SEQ ID NO: 2).

[0039] The MHC class I heavy chain domain may be selected from any class I HLA allele or ortholog, including but not limited to human HLA- A, HLA-B, and HLA-C molecules, as well as class I MHC molecules from non-human species, such as murine H-2 alleles. Exemplary human alleles suitable for use include HLA-A02 / 07, HLA-A 11:01, HLA-B07:02, and HLA-C07 :01. Representative murine alleles include H-2Kb, H-2Db, H-2Kd, and H-2Dd. Additional alleles may be selected based on population coverage, immunodominant epitope presentation, or specific disease associations relevant to therapeutic or preclinical models. Specifically, the fusion protein described herein may comprise MHC class I heavy chain domain having a sequence of SEQ ID NO. 3.GPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEG PEYWERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRG YQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVE WLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNG EELIQDMELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPP PSTVSNM (SEQ ID NO. 3).

[0040] The antigenic peptide component of the construct is selected based on the intended therapeutic or diagnostic application. While ovalbumin-derived peptide SIINFEKL (SEQ ID NO:8) is used in model systems, other clinically relevant peptide epitopes may be incorporated. Such peptides may be tumor-associated antigens e.g., NY-ESO-1, gplOO, WT1), viral antigens (e.g., CMV pp65, HIV Gag), bacterial epitopes (e.g., Listeria-derived peptides), or autoantigens relevant to autoimmune diseases (e.g., insulin B chain). Representative peptides include: SIINFEKL (OVA257-264; SEQ ID NO:8); NLVPMVATV (CMV pp65; SEQ ID NO:9); SLLMWITQC (NY-ESO-1 ; SEQ ID NO: 10); KTWGQYWQV (WT1 ; SEQ ID NO:11); YLEPGPVTA (HIV Gag; SEQ ID NO: 12). SAINFEKL (N4; SEQ ID NO:13); SIYNFEKL (Y3; SEQ ID NO:14); SIIQFEKL (Q4; SEQ ID NO: 15); SIITFEKL (T4; SEQ ID174911-1898-8134, v. 1NO: 16); RGYVYQGL (VSV N7; SEQ ID NO: 17); FAPGNYPAL (SVNP; SEQ ID NO: 18); SVYDFFVWL (TRP2; SEQ ID NO: 19).

[0041] In some embodiments, the fusion protein may further comprise an epitope tag to facilitate detection, purification, or quantification of the expressed protein. While an E-tag (GAPVPYPDPLEPR; SEQ ID NO:20) has been used in certain configurations, the disclosure is not limited to this sequence. Alternative epitope tags that may be incorporated include but are not limited to: FLAG-tag (DYKDDDDK; SEQ ID NO:21), HA-tag (YPYDVPDYA; SEQ ID NO:22), Myc-tag (EQKLISEEDL; SEQ ID NO:23), V5-tag (GKPIPNPLLGLDST; SEQ ID NO:24), and His-tag (e.g., HHHHHH; SEQ ID NO:25), among others. These tags may be positioned at the N-terminus, C-terminus, or internally, depending on the structural and functional constraints of the fusion protein. In some preferred aspects the fusion protein described herein may comprise an epitope tag having a sequence of SEQ ID NO. 4.VDGAPVPYPDPLEPIDNSAAISMANPRPPTPR (SEQ ID NO. 4).Single Chain Trimers

[0042] Single chain trimer (SCT) molecules, as described herein, may be unbranched polypeptide chains comprising, in amino-to-carboxy terminal order: (i) an antigenic peptide sequence; (ii) a P-2 microglobulin ( 2m) sequence; (iii) a major histocompatibility complex (MHC) class I heavy chain sequence comprising one or more of the extracellular (al, a2, a3), transmembrane, and cytoplasmic domains; and (iv) an outer membrane display or targeting domain, such as a bacterial autotransporter domain. The SCT construct is capable of forming a stable, single polypeptide chain that can mimic the structure and function of a native peptide- loaded MHC class I molecule.

[0043] In certain embodiments, the SCT further comprises a first linker sequence positioned between the antigenic peptide and the 2m domain, and / or a second linker sequence positioned between the P2m domain and the class I heavy chain domain. Each linker may independently comprise a flexible motif, such as a (GGGGS)Xrepeat, where x is an integer from 2 to 5 (e.g., 2, 3, 4, or 5 (SEQ ID NOS:26, 6, 7, and 27), or any range derivable therein), or may alternatively include rigid or semi-rigid motifs such as (EAAAK)x (SEQ ID NOS:28- 31), or protease-sensitive sequences enabling post-translational cleavage. Combinations of flexible and rigid linkers may be used in a single SCT molecule to optimize folding, antigen presentation, or expression efficiency.4911-1898-8134, v. 1 18

[0044] Various SCT modifications may be employed, including but not limited to truncations, in which the transmembrane and / or cytoplasmic tail domains are omitted to facilitate soluble expression or secretion; engineered disulfide bonds, to enhance peptide / MHC complex stability; bispecific or tandem SCTs, comprising multiple peptide-P2m-MHC units in a single polypeptide for multiplexed antigen presentation; SCTs fused to immunomodulatory or trafficking domains, such as CD80, PD-L1, or cell-penetrating peptides; protease-sensitive SCTs, comprising cleavage or switchable domains to allow conditional MHC engagement; multimeric SCT formats, such as dimeric or tetrameric SCTs fused to multimerization domains (e.g., streptavidin, Fc domains, leucine zippers) for avidity enhancement.Outer Membrane Display Proteins and Autotransporters

[0045] In certain embodiments, the fusion constructs described herein may be expressed and displayed on the surface of gram-negative bacterial cells via the Type V secretion system. The Type V secretion system encompasses multiple subclasses, including classical autotransporters (Type Va), two-partner secretion systems (Type Vb), and trimeric autotransporters (Type Vc). Among these, classical autotransporters (ATs) are most commonly utilized for the translocation and surface display of heterologous polypeptides (Finton, et al., Frontier in Immunology 2013).

[0046] A classical autotransporter molecule generally comprises three functionally distinct regions: (i) an N-terminal signal peptide that directs secretion through the Sec translocon of the inner membrane; (ii) a central passenger domain, which may include the fusion construct or antigen of interest and which is ultimately displayed on the extracellular surface of the host cell; and (iii) a C-terminal translocator domain, typically forming a P-barrel structure, which inserts into the outer membrane and facilitates the translocation of the passenger domain to the bacterial surface. The modularity of the autotransporter architecture permits replacement of the native passenger domain with a heterologous polypeptide sequence, such as a single chain trimer (SCT) construct, without impairing translocation functionality.

[0047] The autotransporter may be selected from any one of the autotransporters disclosed herein or homologs thereof having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%) identity with an autotransporters sequence disclosed herein. In some preferred aspects the fusion protein described herein may comprise an autotransporter having the sequence of SEQ ID NO. 5.4911-1898-8134, v. 1 19TPTPGPDLNVDNDLRPEAGSYIANLAAANTMFTTRLHERLGNTYYTDMVTGEQKQT TMWMRHEGGHNKWRDGSGQLKTQSNRYVLQLGGDVAQWSQNGSDRWHVGVMA GYGNSDSKTISSRTGYRAKASVNGYSTGLYATWYADDESRNGAYLDSWAQYSWFD NTVKGDDLQSESYKSKGFTASLEAGYKHKLAEFNGSQGTRNEWYVQPQAQVTWM GVKADKHRESNGTLVHSNGDGNVQTRLGVKTWLKSHHKMDDGKSREFQPFVEVN WLHNSKDFSTSMDGVSVTQDGARNIAEIKTGVEGQLNANLNVWGNVGVQVADRG YNDTSAMVGIKWQF* (SEQ ID NO. 5).

[0048] In certain embodiments, the fusion constructs described herein may comprise a recombinant fusion protein comprising the Escherichia coli lipoprotein Lpp covalently linked to a truncated form of outer membrane protein A (OmpA), preferably residues 46-159. The Lpp component may anchor the fusion to the outer membrane via its N-terminal lipid moiety, while the OmpA domain may serve as a transmembrane scaffold capable of presenting heterologous polypeptides on the surface of gram-negative bacterial cells. This design can leverage the ability of Lpp to covalently link the outer membrane to the peptidoglycan layer, thereby preserving membrane integrity (Tucker et al., Cell 2018), and can enable the extracellular display of otherwise periplasmic proteins such as |3-lactamase (Georgiou et al., Protein Eng. 1996). Lpp-ompA typically utilizes peptides from E. coli (e.g., GenBank V00302. 1 and EFK49281.1). The Lpp-ompA sequence may be substituted for an autotransporter in fusion proteins described herein.

[0049] The Lpp-OmpA fusion protein can be expressed using bacterial secretion systems capable of localizing membrane proteins, including the Sec-dependent translocation pathway. Once inserted into the outer membrane, the OmpA P-barrel structure may facilitate stable insertion and display of fused domains, while Lpp’s covalent interaction with the peptidoglycan may contribute to outer membrane rigidity and spatial organization. The mechanical integrity of the outer membrane may be critically dependent on Lpp’s tethering role (Tucker et al., Cell 2018), and its incorporation into chimeric constructs can help ensure host viability during heterologous protein expression.4911-1898-8134, v. 1 20Table 1. Autotransporters

[0050] Suitable gram-negative bacterial hosts for SCT display include, but are not limited to, Escherichia coli, Salmonella enterica, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In particular, E. coli strains such as MCI 061 and C41 are widely utilized due to their well-characterized genetics and availability of established autotransporter systems (e.g., AIDA-I, Ag43, IgA protease). Additionally, probiotic or commensal strains such as Escherichia coli Nissle 1917 and attenuated Salmonella strains e.g., VNP20009) may be employed for therapeutic purposes, including mucosal vaccination and cancer immunotherapy.

[0051] The use of bacterial display systems can enable high-density surface presentation of SCT molecules and may facilitate T cell engagement in vitro or in vivo, screening of peptide / MHC combinations, and / or development of live vector vaccines. Such systems may be adapted to co-display additional immunostimulatory molecules, such as co-4911-1898-8134, v. 1 21stimulatory ligands, cytokines, or Toll-like receptor agonists, to further enhance immunogenicity or modulate immune responses.Nucleic Acids

[0052] Also provided are nucleic acid molecules that encode a protein described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules that encode only the ectodomain of the protein are also contemplated. The nucleic acid molecule can be incorporated into a vector, such as an expression vector.

[0053] The nucleic acid may be a self-replicating RNA molecule. The nucleic acid may include a modified RNA molecule. The nucleic acid may include a circular RNA molecule. Also provided are compositions comprising a nucleic acid described herein.

[0054] A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0055] Nucleic acids can include one or more expression control or regulatory elements operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like.

[0056] In a bacterial system, expression control / regulatory elements may include bacterial promoters, which can be constitutive or inducible depending on the desired level and timing of expression. Bacterial promoters that may be utilized include, but are not limited to: Constitutive promoters (e.g., Anderson series) or inducible promoters (e.g., lac promoter, T7 promoter, P_BAD promoter, TET promoter, P con promoter or N25 promoter).

[0057] In a mammalian system, expression control / regulatory elements may include mammalian promoters, which can be constitutive or inducible depending on the desired level, tissue specificity, and timing of expression. Mammalian promoters that may be utilized include, but are not limited to: Constitutive promoters (e.g., cytomegalovirus (CMV) immediate early4911-1898-8134, v. 1 22promoter, elongation factor-1 alpha (EF-la) promoter, SV40 early promoter, or ubiquitin C (UbC) promoter) or inducible promoters (e.g. , tetracycline (Tet)-responsive promoter, mifepristone-inducible promoter, ecdysone-inducible promoter, or heat shock protein (HSP) promoter).

[0058] Expression control / regulatory elements can be obtained from the genome of any suitable organism, including bacterial species such as Escherichia coli, Salmonella enterica, Shigella flexneri, Vibrio cholerae, and others, as well as from phages or synthetic sources. The choice of promoter, whether constitutive or inducible, can be tailored based on the desired application, ensuring that the expression of the polypeptide is optimized for the system in use.

[0059] In various embodiments, additional regulatory elements, such as mRNA stability sequences (e.g. , ribosomal binding sites or untranslated regions), may also be included to enhance expression or to allow for fine-tuning of translation efficiency in the host cell.Diseases

[0060] Bacterial antigen presenting cells provided herein can be used to selectively activate T-cells in or from a mammalian subject (e.g., a mouse, rat, non-human primate, or human). It is anticipated that bacterial antigen presenting cells provided herein can be used to induce a selective T cell immune response and can be used in vaccine compositions to generate a T cell response to treat a disease such as, e.g., a bacterial infection, viral infection, or cancer. For example, it is anticipated that bacterial antigen presenting cells provided herein can be administered to a subject to treat a cancer, e.g., using intravenous methods of administration as described on Toso et al. (2001) or Luke et al. (2023). It is anticipated that the bacteria may be attenuated or inactivated (e.g., using chemical inactivation with formaldehyde or formalin, irradiation, or ultrasound) prior to administration to a mammalian subject in vivo or use in an in vitro experiment to activate T cells. In some aspects, the bacterial antigen presenting cells can be used to activate or eliminate select T cell populations and / or treat an autoimmune disease. For example, T cells can be activated with both MHCI and co-stimulatory factors (CD80, IL2 etc), but if T-cells only interact with MHCI then it may drive the interacting T cell into anergy (e.g., quiescence or cell death); thus, select bacteria antigen expressing cells that express MHCI or a MHCI-peptide complex may promote anergy, which may be observed in mice. As shown in the below examples, in vitro (bacteria + purified T cells) show that bacteria expressing the MHCI-peptide fusion protein comprising an autotransporter alone cause4911-1898-8134, v. 1 23activation. Without wishing to be bound by any theory, these results suggest that the E. coli bacteria used (e.g., strain W3110) may inherently process a co-stimulatory factor, and removing the costimulatory factor from the bacteria may enable T cell elimination, if desired.

[0061] The bacterial antigen presenting cells provided herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. The pharmaceutical composition may preferably comprise a therapeutically effective amount of the bacterial antigen presenting cells. Also included is a method of treating a disease or condition in a subject in need thereof comprising administering to the subject a bacterial antigen presenting cell provided herein.

[0062] In cancer immunotherapy applications, live or attenuated gram-negative strains, including those engineered for tumor targeting, can be used to colonize hypoxic tumor microenvironments and stimulate innate immune activation. For example, attenuated S. typhimurium strains such as VNP20009 and Al-R have been explored as delivery vehicles for therapeutic proteins, including tumor antigens and immune-modulating agents (Zhang, et al., Oncotarget 2017). Such bacteria can be used to express a fusion protein comprising a single chain trimer and an autotransporter as described herein.

[0063] In some embodiments, the bacterial host may be live, attenuated, or inactivated ( .g., heat-killed or chemically treated) depending on the desired safety profile and immunological outcome. Optionally, the bacterial strain may be engineered with a biological containment feature, such as a genetically encoded “kill switch” or programmed lysis mechanism, to enable temporal or environmentally triggered elimination of the bacteria after administration. A variety of kill-switch strategies have been developed, including inducible toxin-antitoxin systems, temperature-sensitive replication, and synthetic gene circuits responsive to host-specific cues

[0064] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g. ,4911-1898-8134, v. 1 24allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0065] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0066] The bacterial antigen presenting cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0067] In certain embodiments, bacterial antigen-presenting cell may be employed in the treatment of solid tumors through intratumoral or systemic administration. Bacterial species such as E. coli strains e.g., W3110) may be used due to their tumor-targeting capabilities and ability to selectively replicate within hypoxic and necrotic tumor cores. These bacteria can serve as delivery vehicles for SCT molecules that stimulate tumor- specific cytotoxic T lymphocyte (CTL) responses. In order to minimize the risk of systemic infection, the bacteria may be genetically attenuated and / or administered in a carefully killed e.g., heat- or chemically-inactivated) form, or may include an inducible kill-switch to enable bacterial clearance following therapeutic delivery4911-1898-8134, v. 1 25

[0068] In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a leukemia. In one embodiment, the cancer is a solid tumor.

[0069] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0070] Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine4911-1898-8134, v. 1 26carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0071] Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0072] In another aspect, bacterial antigen-presenting cell may be utilized to present one or more pathogen-derived epitopes in order to prime antigen-specific T cell responses for the treatment or prevention of viral infections. Bacterial vectors may be engineered to express SCTs loaded with viral peptides derived from HIV Gag, HBV core, or HPV E6 / E7 antigens. Attenuated or inactivated strains of Salmonella or E. coll may be employed for mucosal immunization strategies ( .g., delivery to the gastrointestinal tract of a mammalian subject), leveraging the bacteria's natural ability to interact with the gut- associated lymphoid tissue (GALT) to stimulate systemic and mucosal immunity.

[0073] In some embodiments, bacterial antigen-presenting cell may be designed to induce tolerance rather than immunity. When expressed on the surface of probiotic or commensal gram-negative bacteria, such SCTs may present self-peptides in a manner that promotes regulatory T cell induction or deletion of autoreactive T cells. For example, antigens associated with pancreatic P-cells (e.g., insulin, GAD65) may be presented to suppress autoimmunity in Type 1 Diabetes. Probiotic strains such as E. coli Nissle 1917 or Lactobacillus spp. engineered to express outer membrane protein constructs may be employed for oral or mucosal delivery.

[0074] The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, orally, mucosally, subcutaneously, intradermally, intratumorally, intranasally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the4911-1898-8134, v. 1 27subject, a local disease site in the subject, such as a lymph node, an organ, a tumor, and the like.Gram-Negative Bacteria

[0075] It is anticipated that a variety of gram-negative bacteria may be used as bacterial antigen presenting cells. In some preferred aspects, the bacteria is E. coli. The gram-negative bacteria may be attenuated to harbor one or more deletions in the guanine nucleotide synthesis pathway, e.g., similar to or corresponding with Shigella flexneri 2a as described in Korloff et al. (2004).

[0076] The gram-negative bacterial host may be live, attenuated, or inactivated (e.g., heat-killed, UV-treated, or chemically fixed), and may optionally be modified to include one or more deletions in essential metabolic pathways, such as the guanine nucleotide synthesis pathway. In addition to E. coli and Shigella, other suitable gram-negative bacterial species for use include, but are not limited to: Salmonella enterica, Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Neisseria meningitidis, Haemophilus influenzae or Francisella tularensis.

[0077] In various embodiments, such bacterial vectors may also incorporate genetically encoded containment systems or "kill-switches," including inducible toxin-antitoxin modules, metabolite-dependent survival circuits, or synthetic gene circuits responsive to host environmental cues (e.g., temperature, pH, or oxygen tension). These systems allow temporal control over bacterial viability and minimize the risk of unintended systemic infection (Stirling, et al., Molecular cell 2017).

[0078] In various embodiments the bacteria may be modified to increase or decrease the immunogenicity of its endogenous cellular components.IL Combination therapies

[0079] In order to increase the effectiveness of the bacterial antigen presenting cells of the embodiments, it may be desirable to combine these compositions with other agents effective in the treatment of the disease of interest.

[0080] As a non-limiting example, the treatment of cancer may be implemented with a modified cell composition of the present embodiments along with other anti-cancer agents. An4911-1898-8134, v. 1 28“anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the anti-cancer peptide or nanoparticle complex and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the bacterial antigen presenting cells composition and the other includes the second agent(s).A. Chemotherapy

[0081] Cancer therapies also include a variety of combination therapies. In some aspects a bacterial antigen presenting cell composition of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent. For example, in some aspects the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erbl, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD- 002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A- 05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.

[0082] Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine;4911-1898-8134, v. 1 29acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophy cin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);4911-1898-8134, v. 1 30cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, famesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac {e.g., from about 400 to about 800 mg / day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein.B. Radiotherapy

[0083] Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0084] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.C. Gene Therapy

[0085] In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic4911-1898-8134, v. 1 31composition. Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these method are well known in the art (see, e.g. Sambrook et al. , 1989; Ausubel et al. , 1998; Ausubel, 1996).D. Surgery

[0086] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0087] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs’ surgery). It is further contemplated that the present embodiments may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. In some aspects, following tumor resection a bacterial antigen presenting cell composition of the embodiments is administered to lymphoid tissue that drained the previous site for the tumor.E. Co-stimulation Factors and Cytokines

[0088] In some aspects, one or more co-stimulation factors or cytokines can be administered to a mammalian subject in combination with a bacterial antigen presenting cell provided herein. The co-stimulation factor(s) or cytokine(s) may stimulate or promote proliferation of T cells in the subjectIII. Pharmaceutical and Probiotic Compositions

[0089] The pharmaceutical formulations provided herein may further include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include4911-1898-8134, v. 1 32normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7, 0-8.0. Suitable pharmaceutical carriers include, but are not limited to sterile water, salt solutions (such as Ringer’s solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, tale, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be mixed with auxiliary agents, e.g., lubricants, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, e.g., ileal brake hormone regulatory substances to improve metabolism and ameliorate metabolic syndromes.

[0090] Cells provided herein may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, duents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the cells.

[0091] Pharmaceutical compositions may also include one or more active ingredients such as, anti-inflammatory agents, anesthetics, and the like. Formulations for oral or intratumoral administration may include buffers, liposomes, diluents and other suitable additives. The compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., statins, linaclotide, ileal brake hormone releasing substances, anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, antioxidants, opacifiers, thickening agents and stabilizers. Depending on the particular active ingredients, the formulations may be administered in the same pill or tablet or as a distinct pill or tablet as part of a co-administration protocol. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.4911-1898-8134, v. 1 33

[0092] Cells provided herein may be used in combination with one or more additional active agent and encapsulated in a particulate dosage form. In this manner, certain cells provided here, alone or in combination with other active agents, are released at that site over time to provide a sustained therapeutic benefit.

[0093] In preferred embodiments, the pharmaceutical composition of the invention is administered orally, mucosally, parenterally, sublingually, rectally or intratumorally. Dosing can be dependent on a number of factors, including severity and responsiveness of the disease state to be treated, and with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0094] Suitable dosage amounts for gram-negative bacteria or probiotic organisms may, for example, vary from about 105to 1012organisms, typically about 106based on the numbers of organisms found in the ileum of said patient. Similarly, delivery of gram-negative bacteria provided herein will be specific to particular cells, conditions, and locations, such as ileum. In general, dosage is from tablets, capsules, granules and microgranules, powders, liquids and alike, and which may be given once or more daily, weekly, monthly or yearly, or even less frequently. In the treatment or prevention of certain conditions, an appropriate dosage level will generally be as above per day which can be administered in single or multiple doses. Live, killed or attenuated microorganisms or therapeutic compounds according to the invention (e.g. live, killed or attenuated organisms) may be formulated into pharmaceutical compositions for administration according to known methodologies, including for example using immediate- release, as well as pulsatile-release, and delayed-release technologies. Pharmaceutical compositions may, for example, comprise one or more constructs, in combination with a pharmaceutically acceptable carrier, excipient or diluent. Such carriers will be non-toxic to recipients at the dosages employed. A suitable dosage may be from about as above, per species at least 105to 1012oral and various ranges within these amounts being still more typical for administration. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, saline and phosphate-buffered saline at physiological pH may be used.4911-1898-8134, v. 1 34Stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.

[0095] "Pharmaceutically acceptable salt" refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

[0096] However, pharmaceutical compositions provided herein may be in any form which allows for the composition to be administered to a patient by the oral route and less commonly by sublingual or rectal routes. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable at the site targeted upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where tablet may be a single dosage unit, and a container of one or more compounds of the invention in oral form may hold a plurality of dosage units.

[0097] For oral administration, an excipient and / or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethyl cellulose and ethyl cellulose. Coloring and / or flavoring agents may be present. A coating shell may be employed, applying common membranes used for microencapsulation and suitable for the microencapsulation of live, killed or attenuated probiotic organisms include biodegradable synthetic "polymers" such as poly-lactide, polyglycolic acid, and polyanhydride. Established "polymers" for live encapsulation and enzyme encapsulation include alginate-polylysine- alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), Multilayered HEMA-MMA- MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile / sodium methally-Isulfonate (AN-69), polyethylene glycol / poly pentamethylcyclopentasiloxane / polydimethylsiloxane (PEG / PD5 / PDMS), poly N,N-dimethyl acrylamide (PD-MAAm), Siliceous encapsulates and cellulose sulphate / Sodium alginate / polymethylene-co-guanidine (CS / A / PMCG). Other materials that are useful include, without limitation, cellulose acetate phthalate, calcium alginate and k-carrageenan-Locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co- glycolides), carrageenan, starch poly- anhydrides, starch polymethacrylates, polyamino acids, enteric coating polymers.4911-1898-8134, v. 1 35

[0098] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: diluents such as water, preferably fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glyc-erin, propylene glycol or other solvents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.IV. Examples

[0099] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Generation and Testing of Bacterial Antigen Presenting Cells

[0100] To determine successful expression of the pHEA MHC I plasmid in E. coli W3110, we investigated for the presence of the MHC I protein by immunoblotting with anti-E-tag antibody. A protein band corresponding to the theoretical size of the MHC I was detected in W3110 after induction and not in the Wild-type sample (Fig. 1 A).

[0101] Cell surface display of MHC I by W3110 was detected by flow cytometry both with mAh 25 -DI .16, which binds specifically to the ovalbumin-derived peptide SIINFEKL (SEQ ID NO: 8) bound to H-2Kb of MHC class I and with pAb anti-E-tag antibody. The induced W3110 MHC I transformants showed equal positive surface staining with both the H2-kb / OVA-specific antibody and anti-E-tag antibody compared to the wild- type strain, suggesting the efficient and successful display of the E-tagged MHC I / peptide complex on the surface of the engineered W3110 cells (Fig. IB and C). We then confirmed display with post4911-1898-8134, v. 1 36induced formaldehyde fixed W3110 MHC I cells and observed a comparable positive shift with the H2-kb / OVA specific antibody to the unfixed results (Fig. ID).

[0102] To test strain specificity of our display, we transformed the pHEA MHC I plasmid into the well characterized lab strain BL21. The expression of MHCI protein in B121 was confirmed by immunoblotting with anti-E-tag antibody (Fig. 2A). Flow cytometry of induced BL21 MHC I transformants with the H2-kb / OVA-specific antibody and anti-E-tag antibody showed differing positive surface staining results. When stained with the H2- kb / OVA-specific antibody BL21 MHC I showed majority overlapping fluorescence intensity (Fig. 2B). However, when stained with the E-tag antibody BL21 MHC I showed an obvious positive shift (Fig. 2C). The appearance of the E-tag antibody, with the absence of the H2- kb / OVA-specific antibody in the same quantity, indicates the possible display of disordered MHC I. These results support the idea that that the BL21 strain cannot successfully display proper folded MHC I on its surface with our pHEA MHC I plasmid. The MHC I display system used here does require certain strain specific characteristics that enable the folded display.

[0103] The ability of the W3110 MHC I display system to directly activate T cells was tested. To accomplish this goal, we used a T cell activation assay coupled with flow cytometry to measure the levels of CD69, a known early activation marker, on the surface of murine OT-1 CD8+ T cells. OT-1 CD8+ T cells are T cells enriched from an OT-1 transgenic mouse that primarily recognize ovalbumin-derived peptide SIINFEKL (SEQ ID NO:8) when presented by the MHC I complex. These cells should specifically recognize our folded complex displayed by W3110. First, to test the levels of CD69 expression with T cell activation, a positive control containing native splenocytes and OVA peptide was incubated with OT-1 CD8+ T cell for 6 hours, followed by subsequent anti-CD69 staining and flow cytometry. Our positive control showed robust activation of T cells with about 60% of the population expressing the early activation marker (FIGS. 3A-B). When we co-incubated our induced W3110 MHC I at different seeding cell counts with the same amount of OT-1 CD8+ T cells treated with exogenous anti-CD28, a dose dependent increase of the early activation marker can be seen (Fig. 3b). The addition of CD28 in these tests was decided based on previous evidence showing the treatment of T cells with monoclonal anti-CD28 antibodies provide a costimulatory signal that engages the T cell receptor for antigen induced activation. Markedly, T cell incubation with wild-type W3110 nor incubation with just anti-CD28 show a positive shift4911-1898-8134, v. 1 37towards activation. Together, these data indicate that induced W3110 MHC I display can cause early lymphocyte activation with anti-CD28 co-stimulation.

[0104] Next, the ability of this display system to activate T cells was further tested by removing the exogenous anti-CD28 co-stimulation. The inventors ran the same activation assay with induced W3110 MHC I in similar conditions and with the same controls without the addition of anti-CD28 and saw an increase in the early activation marker, CD69 (FIGS. 4A-B). This suggests that co-stimulation is not necessary to initiate an activation response from T cells when co incubated with our display system.

[0105] Importantly, the specificity of the interaction between the displayed MHC I OVA peptide complex and the targeted OT-1 CD8+ T cells to initiate activation must be evaluated. For this we used enriched polyclonal CD8+ T cells from a common B6 mouse, which do not specifically recognize the OVA peptide for activation and ran the activation assay. The polyclonal CD8+ T cells did not show an increase in activation marker, like what was observed with the OT-1 CD8+ T cells (FIGS. 5A-B). Furthermore, this activation can be titrated by varying the affinity of the MHCI bound peptide and T cell receptor interaction (FIG. 6). This clearly demonstrates the specificity for the displayed peptide within the complex and not just activation of any T cell receptor.

[0106] Results were further validated by replicating data from previous literature showing OT-I T cell activation with OVAp mutants. Peptide avidity refers to the strength and stability of the interaction between the T cell receptor (TCR) and the pMHC complex. When a T cell encounters a high-avidity peptide, the TCR forms a more stable and prolonged interaction with the pMHC, resulting in stronger TCR signaling. It has been shown that although the altered peptide ligands bound with similar capacity as the original OT-1 ligand SIINFEKL (N4; SEQ ID NO:8) they differed in potency for stimulating T cells. Four ovalbumin peptide mutants were tested, each with a single amino acid mutation at various locations, that have shown differing T cell activation capabilities in literature, and an irrelevant antigenic peptide, N7p RGYVYQGL (SEQ ID NO: 17), from the vesicular stomatitis virus (VSV) in the previously described bacterial display regime and activation assay. Results showed no activation of OT-I CD8+T cells with mAPC displaying the N7p and differing T cell activation dependent on the OVAp mutant that closely matches the literature (FIG. 6). These results confirmed the activation of OT-I CD8+T cells by the mAPC is specific to the peptide presented.4911-1898-8134, v. 1 38Example 2 - Materials and Methods

[0107] Cloning: Single chain MHCI encoding ova peptide was cloned in plasmid pHEA (Addgene (Plasmid #168297) to generate a fusion protein to the membrane portion of autotransporter pHEA.

[0108] Western Blotting: To detect expressed MHC I protein, E. coli cells were inoculated in 1 mL of LB medium and cultured for 1 h at 37°C with shaking. A 500 uL amount of culture was taken and diluted in 500 uL of fresh LB medium and induced with IPTG for a final concentration of ImM for 1 h at 37°C and normalized to an ODeoo of 0.2. Cells were washed and resuspended in SDS sample buffer and boiled. Western blotting was performed after SDS-PAGE separation of proteins. Rabbit anti-E-tag HRP-conjugated pAb (GenScript) was used as the primary antibody, diluted 1:2000, followed by an ultra-sensitive enhanced chemiluminescent substrate. The Bio-Rad ChemiDoc imager was used for visualization.

[0109] E. coli Cell Flow Cytometry: To detect display of MHC I protein on the surface of cells, E. coli cells were inoculated in 1 mL of LB medium and cultured for 1 h at 37°C with shaking. A 500 uL amount of culture was taken and diluted in 500 uL of fresh LB medium and induced with IPTG for a final concentration of ImM for 1 h at 37 °C and normalized to an ODeoo of 0.2, approximately 1x10scells per sample. Cells were washed and resuspended in FACS wash buffer (PBS buffer + 50 mM glucose). For surface staining, the appropriate fluorescently conjugated antibody was added to the cells and incubated for 1 h at 4°C in the dark. Stained cells were washed and resuspended in FACS wash buffer. Flow cytometry data were acquired using a Sony S A3800 spectral cell analyzer. A minimum of 10,000 events were collected for analysis. Data was analyzed using FlowJo software.

[0110] T cell Activation Assay: The spleen of an OT-1 TCR transgenic mouse was harvested, and OT-1 CD 8 T cells were purified from single cell suspension of splenocytes using MojoSort Mouse CD8 T cell isolation kit (Bioledgend) according to the manufacturer’s instructions. Purified CD8+ T cells were counted then resuspended in cell culture medium (Supplemented RPMI media) at 20,000 cells per well. Simultaneously, W3110 E. coli cells were induced with IPTG, as previously described above, normalized to the specified seeding cell number and washed and resuspended in cell culture medium supplemented with IPTG. W3110 cells and CD8+ T cells were co-incubated with anti-CD28 (unless specified) for 6 hours4911-1898-8134, v. 1 39at 37°C and 5% CO2. Positive control was CD8+ T cells co-incubated with splenocytes and 1 uM of OVA peptide.

[0111] For the analysis of T cells by flow cytometry, the cells were stained with Anti- CD45.2, Anti-CD8, Anti-CD69, and propidium iodide and prepared for flow cytometry as previously described above. Flow cytometry data were acquired using a FACSAria Fusion SORP cell sorter. Data was analyzed using FlowJo software.

[0112] For the analysis of T cell activation by OVAp mutants, cloning of the pHEA MHC display construct with different peptide sequences was completed. Expression of the construct was verified with the E-tag antibody with a western blot and display was confirmed with FACS. T cell activation was performed and analyzed via the described activation assay.* * *

[0113] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.4911-1898-8134, v. 1 40REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Cohen CJ, et al. J Immunol Baltim Md 1950. 2003 Apr 15; 170(8):4349-4361. PMID: 12682272Jose J, et al. Gene. 1996 Oct 31 ; 178(1-2): 107-110. PMID: 8921899Leyton DL, et al. J Biol Chem. 2011 Dec 9;286(49):42283-42291. PMCID: PMC3234927Honda, Torn, et al. Protein Engineering, Design & Selection 28.2 (2015): 53-58.Mottez, Estelle, et al. The Journal of experimental medicine 181.2 (1995): 493-502.Mitaksov, Vesselin, et al. Chemistry’ & biology 14.8 (2007): 909-922.Toso, John F., et al. Journal of clinical oncology 20.1 (2002): 142-152.Kotloff, Karen L., et al. The Journal of infectious diseases 190.10 (2004): 1745-1754.Luke, Jason J., et al. Clinical Cancer Research 29.13 (2023): 2435-2444.Dautin N, Bernstein HD. Annu Rev Microbiol. 2007;61 :89-112. doi: 10.1146 / annurev.micro.61.080706.093233. PMID: 17506669.Rusch SL, Kendall DA. 2007;46:9665-73.Shields, M. J., et al. Journal of Biological Chemistry, 1998, 273(35), 22015-22022.Finton, K. et al. Frontiers in Immunology, 2023, 14, 1170462.Zhang, Y., et al. Oncotarget, 2017, 8(33), 54616-54628.Hansen, T. H., et al. Trends in Immunology, 2010, 31(10), 363-370. https: / / doi.org / 10.1016Zj.it.2010.07.003.Stirling, F., et al. Molecular Cell, 2017,68(4), 686-697. e3.Georgiou, G., et al. Protein Engineering, 1996, 9(2), 239-247.Tucker, A. T„ et al. Cell, 2018, 172(3), 618-628.el3. https: / / doi.Org / 10.1016 / j.cell.2017.12.009.4911-1898-8134, v. 1 41

Claims

WHAT IS CLAIMED IS:

1. A gram-negative bacteria, wherein the gram-negative bacteria expresses a fusion protein comprising:(i) a single chain trimer comprising:(a) an antigenic peptide,(b) a first linker,(c) a 0 -microglobulin,(d) a second linker, and(e) a mammalian MHC heavy chain, and(ii) an outer membrane display protein, preferably an autotransporter; wherein the antigenic peptide is preferably 8-11 amino acids in length.

2. The gram-negative bacteria of claim 1 , wherein the fusion protein is expressed on the surface of the outer membrane of the gram-negative bacteria, preferably wherein the gramnegative bacteria can activate mammalian T cells that selectively bind the antigenic peptide.

3. The gram-negative bacteria of any of claims 1-2, wherein the MHC is a human MHC, such as a human class I MHC heavy chain.

4. The gram- negative bacteria of claim 3, wherein the human class I MHC is HLA-A, preferably HLA-A2.

5. The gram-negative bacteria of any one of claims 1-4, wherein the antigenic peptide is further defined as a human MHC -restricted peptide.

6. The gram-negative bacteria of any one of claims 1 -5, wherein the gram-negative bacteria is an E. coli.

7. The gram-negative bacteria of claim 6, wherein the gram-negative bacteria is an E. coli strain W3110.4911-1898-8134, v. 1 428. The gram-negative bacteria of any one of claims 1-7, wherein the gram-negative bacteria further expresses a co- stimulation factor, such as CD80, CD86, IL2, B7-H1, B7RP-1, CD27, CD28, CD70, TNFR1, TNFR2, HVEM, CD30, 4- IBB, 0X40, GITR, CD27, CD40, or TIM-1.

9. The gram-negative bacteria of any one of claims 1-8, wherein the gram-negative bacteria is a live bacteria, an attenuated bacteria, or an inactivated bacteria.

10. The gram-negative bacteria of any one of claims 1-9, wherein the gram-negative bacteria is a vaccine bacteria or a probiotic bacteria.1 1. The gram-negative bacteria of any one of claims 1-10, wherein the antigenic peptide is an antigen from a cancerous cell, an infectious bacterium, a virus, or a protozoan.

12. The gram-negative bacteria of any one of claims 1- 11, wherein the autotransporter is an adhesin involved in diffuse adherence (AIDA).

13. The gram-negative bacteria of claim 12, wherein the autotransporter is ShdA, IgAP, NalP, BruA, Vac A, YfaL, intimin, MisL autotransporterl, or an EhaA autotransporter.

14. The gram-negative bacteria of any one of claims 1-13, wherein the first linker and / or the second linker is a serine-glycine linker, preferably (GGGGS)n, wherein n=3-4.

15. The gram-negative bacteria of any one of claims 1-14, wherein the fusion protein, costimulation factor, and / or cytokine is recombinantly expressed or episomally expressed.

16. A composition comprising the gram-negative bacteria of any one of claims 1-15 and a carrier.

17. A nucleic acid encoding the fusion protein of any one of claims 1-15.

18. An in vitro method for activating mammalian T cells comprising:(i) obtaining mammalian T-cells, and(ii) contacting the T-cells with the gram-negative bacteria of any one of claims 1-15.4911-1898-8134, v. 1 4319. A method of providing activated T cells to a mammalian subject, comprising generating activated mammalian T cells via the method of any one of claim 18, and administering the T cells to the mammalian subject.

20. A method for activating T cells in a mammalian subject comprising administering the gram-negative bacteria of any one of claims 1-15 or the composition of any one of claim 16to the mammalian subject.444911-1898-8134, v. 1

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