Adhesion molecules for targeted infiltration of therapeutic cells

Abstract

The present disclosure pertains to a cell that expresses at least one binding protein, such as decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof. The cell may include immune cells, such as chimeric antigen receptor T-cells. The present disclosure also pertains to methods of treating or preventing a disease in a subject by administering a cell of the present disclosure to the subject. The present disclosure also pertains to methods of forming the cells of the present disclosure by expressing at least one binding protein in the cell.

Description

PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 TITLE ADHESION MOLECULES FOR TARGETED INFILTRATION OF THERAPEUTIC CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 678,403, filed on August 1, 2024. The entirety of the aforementioned application is incorporated herein by reference. SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF23853.P205.xml”. The date of the creation of the Sequence Listing is August 1, 2025. The size of the Sequence Listing is 10,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing. BACKGROUND

[0003] A need exists for more effective delivery of cell-based therapies to tumors cells. Numerous embodiments of the present disclosure aim to address the aforementioned need. SUMMARY

[0004] In some embodiments, the present disclosure pertains to a cell that expresses at least one binding protein that binds to one or more sulfated proteoglycans. In some embodiments, the binding protein includes, without limitation, decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof. In some embodiments, the cell includes immune cells, such as chimeric antigen receptor T-cells.

[0005] Additional embodiments of the present disclosure pertain to methods of treating or preventing a disease in a subject by administering a cell of the present disclosure to the subject. In some embodiments, the disease to be treated or prevented includes, without limitation, cancer, autoimmune diseases, autoimmune diseases of the heart, lupus nephritis, multiples sclerosis, rheumatoid arthritis, atherosclerotic plaque build-up, auto-immune pathologies with increased sulfated proteoglycan expression, or combinations thereof.

[0006] Further embodiments of the present disclosure pertain to methods of modifying a cell to form the cells of the present disclosure. In some embodiments, the cell modification method includes expressing at least one binding protein that binds to one or more sulfated proteoglycans in the cell.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A-1H demonstrate that biglycan is associated with angiogenesis and an increase in metastatic cancers. FIGS. 1A, 1C, 1E, and 1G provide scatter plots showing correlation between biglycan expression and angiogenesis markers in (FIG. 1A) Ovarian cancer, (FIG. 1C) Stomach adenocarcinoma, (FIG.1E) Kidney renal cell papillary carcinoma, and (FIG.1G) Skin cancer. FIGS. 1B, 1D, 1F, and 1H provide violin plots showing expression of biglycan in non-metastatic and metastatic cancer in (FIG. 1A) Ovarian cancer, (FIG. 1C) Stomach adenocarcinoma, (FIG. 1E) Kidney renal cell papillary carcinoma, and (FIG. 1G) Skin cancer. In FIGS. 1B, 1D, 1F and 1H, analysis was performed using two-tailed student’s t-test with Welsch’s correction. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0008] FIGS.2A-2T show Borrelia garinii decorin binding proteins A / B (DBPA / DBPB) expressed on PlatGP bind to biglycan. FIG.2A is a schematic showing possible orientation of DBPA / DBPB on plasma membranes. FIG.2B is a schematic showing design of plasmid constructs for DBPA / DBPB with C terminal flanking the extracellular domain (construct C) using a type II secretion signal. FIG. 2C is a schematic showing design of plasmid construct for DBPA / DBPB with N terminal flanking the extracellular domain (construct N) using a GPI anchoring signal. FIG.2D is a schematic showing the experimental design to confirm expression of functional DBPA / DBPB on plasma membrane of PlatGP cells.

[0009] FIGS.2E, 2G, 2I, and 2K show confocal microscopy images showing cytoplasmic expression of mCherry and extracellular staining of anti-myc-AF488 antibody on cells transfected with (FIG.2E) Construct C-DBPA-type II, (FIG. 2G) Construct N-DBPA-GPI, (FIG. 2I) Construct C-DBPB-type II, and (FIG.2K) Construct N-DBPB-GPI. Scale bar: 10 μm.

[0010] FIGS.2F, 2H, 2J, and 2L show density plots showing correlation between mCherry and anti- myc-AF488 antibody fluorescence intensity on cells transfected with (FIG.2F) Construct C-DBPA- type II, (FIG. 2H) Construct N-DBPA-GPI, (FIG. 2J) Construct C-DBPB-type II, and (FIG. 2L) Construct N-DBPB-GPI.

[0011] FIGS. 2M, 2O, 2Q, and 2S show confocal microscopy images showing cytoplasmic expression of mCherry and extracellular staining of anti-6xHIS-AF647 antibody on cells transfected with (FIG.2M) Construct C-DBPA-type II, (FIG.2O) Construct N-DBPA-GPI, (FIG.2Q) Construct C-DBPB-type II, and (FIG.2S) Construct N-DBPB-GPI. Scale bar: 10 μmPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0012] FIGS.2N, 2P, 2R, and 2T show density plots showing correlation between mCherry and anti- 6xHIS-AF647 antibody fluorescence intensity on cells transfected with (FIG. 2N) Construct C- DBPA-type II, (FIG.2P) Construct N-DBPA-GPI, (FIG.2R) Construct C-DBPB-type II, and (FIG. 2T) Construct N-DBPB-GPI.

[0013] FIGS. 3A-3O show that B. garinii DBPA / DBPB expression in human T cells enable their biglycan binding. FIG.3A provides a schematic showing a procedure for making retroviral particles carrying DBPA / DBPB constructs. FIGS. 3B, 3C, and 3D show a histogram showing mCherry fluorescent intensity in (FIG.3B) non-transduced T cells, cells transduced with (FIG.3C) Construct C-DBPA type II, and (FIG.3D) Construct C-DBPB type II. FIGS.3E, 3F, and 3G provide density plots showing a fraction of CD4 and CD8 T cells in (FIG.3E) non-transduced T cells, cells transduced with (FIG.3F) Construct C-DBPA type II, and (FIG.3G) Construct C-DBPB type II. FIGS.3H and 3L provide a schematic showing the experimental design to confirm expression of functional DBPA / DBPB on plasma membrane of T cells. FIGS.3I, 3J, and 3K provide density plots showing correlation between mCherry and anti-6xHIS-AF647 antibody fluorescence intensity on (FIG. 3I) non-transduced cells and cells transduced with (FIG.3J) Construct C-DBPA-type II, and (FIG.3K) Construct C-DBPB-type II in presence of biglycan. FIGS. 3M, 3N, and 3O show density plots showing correlation between mCherry and anti-6xHIS-AF647 antibody fluorescence intensity on (FIG.3M) non-transduced cells and cells transduced with (FIG.3N) Construct C-DBPA-type II and (FIG.3O) Construct C-DBPB-type II in absence of biglycan.

[0014] FIGS.4A-4C show T-cells expressing functional DBPA / DBPB bind to plate bound biglycan. FIG. 4A provides a schematic showing experimental design to test whether T cell expressing DBPA / DBPB can bind to plate bound biglycan. FIG. 4B provides representative images of plate bound fluorescent DBPA / DBPB expressing T cells or non-transduced T cells after 2 washes with PBS. Scale bar: 300 μM. FIG.4C provides a violin plot showing number of T cells bound to plate per field of view. In FIG. 4C, analysis was performed using two-tailed student’s t-test with Welsch’s correction. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0015] FIGS. 5A-5F show DBPA / DBPB expressing human T cells roll on biglycan coated plates. FIG. 5A provides a schematic of experimental design to examine behavior of DBPA / DBPB expressing T cells flowing over plate bound biglycan. FIGS.5B-5D provide images showing path of (FIG. 5B) DBPA and (FIG. 5C) DBPB T cells or (FIG. 5D) non-transduced T cells over fourPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 consecutive time frames. Red blobs are fluorescently labelled T cells and the number associated with them. FIG. 5E provides a violin plot showing velocity distribution of T cells flowing over the plate bound biglycan surface. FIG. 5F provides a plot showing frequency distribution of T cell velocity over the plate bound biglycan surface. In FIG.5E, analysis was performed using two-tailed student’s t-test with Welsch’s correction. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant. their ID. Scale bar: 100 μm.

[0016] FIGS.6A-6E show that DBPA / DBPB expressing human T cells roll on HUVECs. FIG.6A provides a schematic of experimental design to examine behavior of DBPA / DBPB expressing T cells flowing over HUVECs. FIGS.6B-6D provide images showing path of (FIG.6B) DBPA and (FIG. 6C) DBPB T cells or (FIG.6D) non-transduced T cells over four consecutive time frames. Red blobs are fluorescently labelled T cells and the number associated with them is their ID. Scale bar: 100 μm. FIG.6E is a plot showing frequency distribution of T cell velocity over endothelial cells. DETAILED DESCRIPTION

[0017] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0018] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0019] Poor trafficking of therapeutic cells to a tumor is a major challenge in cell-based therapies for solid cancers. These cells are administered intravenously, requiring them to bind to tumor capillaryPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 vessels under high shear stress and velocity to successfully infiltrate the tumor. The intricate relationship between tumors and their microenvironment enables tumors to modulate their capillaries, secreting factors that hinder therapeutic cell binding.

[0020] Chimeric antigen receptor (CAR) T cells are genetically modified T cells designed to specifically recognize malignant cells and mount a cytotoxic immune response. While CAR-T cells show optimal response in liquid (hematological) tumors, their translation to solid tumors has largely been unsuccessful. In solid tumors, cancer cells are densely packed and outnumber CAR-T cells, resulting in high tumor antigen density and eventually CAR-T exhaustion. The number of T cells in tumors correlate strongly with overall responses, highlighting opportunities for enhancing CAR-T cell infiltration in solid tumors as a strategy to bolster efficacy.

[0021] CAR-T cells are typically administered intravenously. To reach the tumor through capillaries, CAR-T cells bind to and cross endothelial cells in a well-orchestrated pathway called diapedesis. Other immune cells (e.g., T-cells, neutrophils, and / or macrophages) also infiltrate tissues using diapedesis. In the process of diapedesis, when an immune cell comes in contact in endothelium, they start to roll on the surface by interacting with selectins (e.g., L-selectin and / or E-selectin) and selectin ligands. Upon rolling, cells sense chemokines (e.g., CCL21 and / or CCL5) via binding through G- protein coupled receptors (GPCRs) (e.g., CCR7 and / or CXCR3), which leads to integrin (e.g., LFA-1 and / or VLA-4) activation and leads to firm adhesion on endothelial cells. The cell further senses chemokine gradients and infiltrates the tissue where they can perform their effector functions. Tumors can modulate endothelial cells and downregulate molecules involved in diapedesis to inhibit CAR-T cell infiltration into tumors.

[0022] As cancer cells continue to proliferate, after reaching a certain size, they are nutrient deprived and secrete factors which remodel the tumor microenvironment (TME), ultimately leading to formation of new blood vessels. The process of new blood vessel growth is termed angiogenesis.

[0023] Angiogenesis is a major pathway involved in cancer metastasis, which is responsible for about 80% of cancer deaths. It is now well appreciated that angiogenic vessels are anergic and actively hamper immune cell infiltration in tumors. Research utilizing intravital microscopy revealed that leukocyte adhesion was significantly reduced in the tumor endothelium.

[0024] Blocking angiogenesis promoting vascular endothelial growth factor (VEGF) using anti- VEGF antibody increased lymphocyte infiltration and enhanced the effectiveness of adoptive cellPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 therapy in melanoma. Mechanistically, prolonged exposure to pro-angiogenic molecules like VEGF downregulate adhesion molecules (e.g., ICAM-1 and / or E-selectin) expressed on endothelial cells and favor immune exclusion. Moreover, tumor endothelial cells (TECs) have downregulated adhesion molecule (ICAM1) expression in lung cancers and hence actively exclude immune cells from infiltrating the tumor.

[0025] As such, a need exists for more effective delivery of cell-based therapies to tumors cells. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0026] Cells

[0027] In some embodiments, the present disclosure pertains to a cell. In some embodiments, the cell expresses at least one binding protein that binds to one or more sulfated proteoglycans.

[0028] Cell types

[0029] The cells of the present disclosure may be in various forms. For instance, in some embodiments, the cells include immune cells. In some embodiments, the immune cells include, without limitation, neutrophils, macrophages, leukocytes, B-cells, T-cells, chimeric antigen receptor T-cells, natural killer (NK) cells, regulatory T-cells, or combinations thereof. In some embodiments, the cells include chimeric antigen receptor T-cells. In some embodiments, the cells include NK cells.

[0030] Binding protein

[0031] Binding proteins generally refer to proteins that are operable to bind to one or more sulfated proteoglycans. In some embodiments, the sulfated proteoglycans include biglycans. In some embodiments, the binding protein includes, without limitation, decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof.

[0032] The cells of the present disclosure may express various binding proteins in various arrangements. For instance, in some embodiments, the binding protein is overexpressed by the cell. In some embodiments, the binding protein is heterologously expressed by the cell. In some embodiments, the binding protein is positioned on the outer cellular membrane of the cell.

[0033] In some embodiments, the binding protein is derived from a Borrelia bacterium species. In some embodiments, the Borrelia bacterium species includes, without limitation, B. garinii, B. burdorferi, B. afzelii, or combinations thereof. In some embodiments, the Borrelia bacterium species includes B. garinii.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0034] In some embodiments, the binding protein is fused to a signal sequence. In some embodiments, the signal sequence is a membrane targeting signal sequence. In some embodiments, the signal sequence is operable to direct the binding protein to the cell’s secretory pathway for association with the cell’s membrane. In some embodiments, the signal sequence includes, without limitation, a membrane targeting signal sequence from a mammalian origin, a type II membrane signal sequence, a signal sequence that targets the amino acid C terminus of the binding protein to the extracellular region of the plasma membrane, a signal sequence that associates the binding protein with the cell cytoskeleton, a signal sequence that associates the binding protein with the FERM domain of ERM binding proteins, a type II membrane protein signal sequence that associates the binding protein with the FERM domain of ERM binding proteins, a signal sequence encoded by the neprilysin protein, or combinations thereof.

[0035] In some embodiments, the signal sequence includes SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0036] In some embodiments, the binding protein includes DbpA and DbpB. In some embodiments, the binding protein includes DbpA.

[0037] In some embodiments, the DbPA is derived from a Borrelia bacterium species. In some embodiments, the Borrelia bacterium species includes, without limitation, B. garinii, B. burdorferi, B. afzelii, or combinations thereof. In some embodiments, the Borrelia bacterium species includes B. garinii.

[0038] In some embodiments, the DbpA includes SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments,PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 the DbpA includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the DbpA includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0039] In some embodiments, the binding protein includes DbpB. In some embodiments, the DbPB is derived from a Borrelia bacterium species. In some embodiments, the Borrelia bacterium species includes, without limitation, B. garinii, B. burdorferi, B. afzelii, or combinations thereof. In some embodiments, the Borrelia bacterium species includes B. garinii.

[0040] In some embodiments, the DbpB includes SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the DbpB includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.

[0041] In some embodiments, the binding protein is operable to associate with a sulfated proteoglycan (e.g., biglycan) present on tumor endothelial cells and thereby enhance the infiltration of the cell into the tumor. In some embodiments, the cell is suitable for use in treating or preventing a progression of a tumor in a subject, autoimmune diseases, autoimmune diseases of the heart, lupus nephritis, multiples sclerosis, rheumatoid arthritis, atherosclerotic plaque build-up, auto-immune pathologies with increased sulfated proteoglycan expression, or combinations thereof.

[0042] Methods of treating or preventing diseases in a subjectPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0043] Additional embodiments of the present disclosure pertain to methods of treating or preventing a disease in a subject. Such methods generally include administering a cell of the present disclosure to the subject.

[0044] Modes of administration

[0045] The cells of the present disclosure may be administered to subjects in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, oral administration, intratumor administration, local administration, or combinations thereof. In some embodiments, the administration occurs by intravenous administration.

[0046] Diseases

[0047] The methods of the present disclosure can be utilized to treat or prevent various diseases in subjects. For instance, in some embodiments, the disease includes, without limitation, cancer, autoimmune diseases, autoimmune diseases of the heart, lupus nephritis, multiples sclerosis, rheumatoid arthritis, atherosclerotic plaque build-up, auto-immune pathologies with increased sulfated proteoglycan expression, or combinations thereof.

[0048] In some embodiments, the methods of the present disclosure may be utilized to treat or prevent tumor progression in a subject in various manners. For instance, in some embodiments, the binding protein on the cells of the present disclosure associates with sulfated proteoglycan present on the tumor. Such association may then enhance the association of the cells with the tumor cells.

[0049] The methods of the present disclosure may be utilized to treat or prevent the progression of various tumors. For instance, in some embodiments, the tumor includes, without limitation, solid tumors, metastatic tumors, or combinations thereof.

[0050] In some embodiments, the tumor is associated with a cancer. As such, in some embodiments, the methods of the present disclosure may be utilized to treat or prevent a cancer. In some embodiments, the cancer includes, without limitation, metastatic cancer, non-metastatic cancer, ovarian cancer, medulloblastoma, glioblastoma, osteosarcoma, stomach cancer, stomach adenocarcinoma, kidney cancer, kidney renal cell papillary carcinoma, skin cancer, melanoma, breast cancer, pancreatic cancer, lung cancer or combinations thereof.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0051] Autoimmune diseases

[0052] In some embodiments, the methods of the present disclosure may be utilized to treat or prevent autoimmune diseases in a subject in various manners. For instance, in some embodiments, the binding protein on the cells of the present disclosure associates with a sulfated proteoglycan present on the endothelial cells of inflamed vessels in kidney, heart, brain and joints. Such association may then enhance the association of the anti-inflammatory cells (e.g., regulatory T-cells) with the endothelial cells.

[0053] The methods of the present disclosure may be utilized to treat or prevent the progression of various autoimmune diseases and buildup of atherosclerotic plaques. For instance, in some embodiments, the autoimmune diseases include, without limitation, lupus nephritis, rheumatoid arthritis, multiple sclerosis and autoimmune diseases of the heart.

[0054] Subjects

[0055] The methods of the present disclosure may be utilized to treat or prevent tumor progression in various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal, such as a dog or a cat. In some embodiments, the subject is suffering from tumor progression. In some embodiments, the subject is vulnerable to tumor progression.

[0056] Methods of modifying cells

[0057] Additional embodiments of the present disclosure pertain to methods of modifying a cell to form the cells of the present disclosure. In some embodiments, the cell modification method includes expressing at least one binding protein that binds to one or more sulfated proteoglycans in the cell.

[0058] Cells

[0059] The methods of the present disclosure may be utilized to modify various cells. For instance, in some embodiments, the cells include immune cells. In some embodiments, the immune cells include, without limitation, neutrophils, macrophages, leukocytes, B-cells, T-cells, chimeric antigen receptor T-cells, natural killer (NK) cells, regulatory T-cells, or combinations thereof. In some embodiments, the cells include chimeric antigen receptor T-cells.

[0060] Binding proteins

[0061] The methods of the present disclosure may be used to express binding proteins in various manners. For instance, in some embodiments, the expression includes the introduction of a nucleotidePCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 sequence encoding the binding protein into a cell. In some embodiments, the nucleotide sequence is positioned on an expression vector. In some embodiments, the expression vector is a plasmid. In some embodiments, the nucleotide sequence is associated with a particle. In some embodiments, the particle includes, without limitation, a viral particle, retroviral particle, a liposome, or combinations thereof.

[0062] In some embodiments, the nucleotide sequence encodes a binding protein and a signal sequence that becomes fused to the binding protein after expression. In some embodiments, the signal sequence includes, without limitation, a membrane targeting signal sequence from a mammalian origin, a type II membrane signal sequence, a signal sequence that targets the amino acid C terminus of the binding protein to the extracellular region of the plasma membrane, a signal sequence that associates the binding protein with the cell cytoskeleton, a signal sequence that associates the binding protein with the FERM domain of ERM binding proteins, a type II membrane protein signal sequence that associates the binding protein with the FERM domain of ERM binding proteins, a signal sequence encoded by the neprilysin protein, or combinations thereof.

[0063] In some embodiments, the signal sequence encodes SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the signal sequence encodes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0064] In some embodiments, the expressed binding protein includes, without limitation, decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof.

[0065] In some embodiments, the nucleotide sequence encodes DbpA. In some embodiments, the DbpA nucleotide sequence includes SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In somePCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 embodiments, the DbpA nucleotide sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.

[0066] In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 65% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 70% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 75% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 80% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 85% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 90% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 95% codons encoding the same amino acid as in SEQ ID NO: 3. In some embodiments, the DbpA nucleotide sequence includes a sequence with at least 99% codons encoding the same amino acid as in SEQ ID NO: 3.

[0067] In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence from mammalian origin. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a type II membrane signal sequence. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence that targets the amino acid C terminus of the encoded protein to the extracellular region of the plasma membrane. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence that associates the encoded protein with the cell cytoskeleton. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence that associates the encoded protein with FERM domain of ERM bindingPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 proteins. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a type II membrane protein signal sequence that associates the encoded protein with FERM domain of ERM binding proteins. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoded by the neprilysin protein.

[0068] In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding sequence SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 65% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 70% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 75% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 80% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 85% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 90% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 95% identity to SEQ ID NO: 5. In some embodiments, the DbpA nucleotide sequence SEQ ID NO: 3 is fused to a signal sequence encoding a sequence with at least 99% identity to SEQ ID NO: 5.

[0069] In some embodiments, the DbpA nucleotide sequence includes a fusion of a DbpA nucleotide sequence and a nucleotide sequence for a CD69 signal sequence. In some embodiments, the DbpA- CD69 nucleotide sequence includes SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, thePCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 DbpA-CD69 nucleotide sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 6.

[0070] In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 65% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA- CD69 nucleotide sequence includes a sequence with at least 70% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 75% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 80% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 85% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 90% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA- CD69 nucleotide sequence includes a sequence with at least 95% codons encoding the same amino acid as in SEQ ID NO: 6. In some embodiments, the DbpA-CD69 nucleotide sequence includes a sequence with at least 99% codons encoding the same amino acid as in SEQ ID NO: 6.

[0071] In some embodiments, the nucleotide sequence encodes DbpB. In some embodiments, the DbpB nucleotide sequence includes SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleotide sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 4.

[0072] In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 65% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpBPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 nucleotide sequence includes a sequence with at least 70% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 75% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 80% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 85% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 90% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 95% codons encoding the same amino acid as in SEQ ID NO: 4. In some embodiments, the DbpB nucleotide sequence includes a sequence with at least 99% codons encoding the same amino acid as in SEQ ID NO: 4.

[0073] In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence from mammalian origin. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a type II membrane signal sequence. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence that targets the amino acid C terminus of the encoded protein to the extracellular region of the plasma membrane. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence that associates the encoded protein with the cell cytoskeleton. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence that associates the encoded protein with FERM domain of ERM binding proteins. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a type II membrane protein signal sequence that associates the encoded protein with FERM domain of ERM binding proteins. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoded by the Neprilysin protein.

[0074] In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding sequence SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 65% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 70% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 75% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 ID NO: 4 is fused to a signal sequence encoding a sequence with at least 80% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 85% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 90% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 95% identity to SEQ ID NO: 5. In some embodiments, the DbpB nucleotide sequence SEQ ID NO: 4 is fused to a signal sequence encoding a sequence with at least 99% identity to SEQ ID NO: 5.

[0075] In some embodiments, the DbpB nucleotide sequence includes a fusion of a DbpB nucleotide sequence and a nucleotide sequence for a CD69 signal sequence. In some embodiments, the DbpB- CD69 nucleotide sequence includes SEQ ID NO: 7.

[0076] In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 7.

[0077] In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 65% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB- CD69 nucleotide sequence includes a sequence with at least 70% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 75% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 80% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 85% codons encoding the same amino acid as in SEQ IDPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 90% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB- CD69 nucleotide sequence includes a sequence with at least 95% codons encoding the same amino acid as in SEQ ID NO: 7. In some embodiments, the DbpB-CD69 nucleotide sequence includes a sequence with at least 99% codons encoding the same amino acid as in SEQ ID NO: 7.

[0078] Expression of binding proteins

[0079] The methods of the present disclosure may be utilized to express binding proteins in cells in various manners. For instance, in some embodiments, the methods of the present disclosure may be utilized to heterologously express binding proteins in cells. In some embodiments, the methods of the present disclosure may be utilized to overexpress binding proteins in cells.

[0080] The methods of the present disclosure may be utilized to express various binding proteins in cells. Suitable binding proteins were described supra and are incorporated herein by reference.

[0081] Advantages and Applications

[0082] Previous technologies aimed at increasing therapeutic cell infiltration in solid tumors were mostly based on overexpression of chemokine receptors. However, a prerequisite to migration towards the tumor is adhesion of cells to the endothelium. To the best of Applicant’s knowledge, prior efforts have not targeted the increased expression of sulfated proteoglycans (e.g., biglycans) on tumor endothelial cells for promoting the binding of therapeutic cells. Additionally, there are no reports of heterologous expression of Borrelia DbpA / B in human cells. As such, the cells and methods of the present disclosure present numerous advantages over existing cell-based therapy methods.

[0083] Additional embodiments

[0084] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0085] Example 1. Heterologous expression of functional Borrelia garinii DbpA and DbpB in human cells and their efficient binding to biglycan on tumor cells

[0086] Tumor endothelial cells (TECs) are phenotypically altered and hinder the binding of therapeutic cells by downregulating key adhesion molecules (e.g., ICAM-1, VCAM-1, selectins, etc).PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 Studies have shown that biglycan (BGN), a secreted proteoglycan, is overexpressed on tumor endothelial cells.

[0087] Bacteria of genus Borrelia bind to biglycan via their adhesion molecules decorin binding protein A and B (DbpA / B). Biglycan and decorin are secreted proteoglycans consisting of a 42 kDa leucine rich repeat domains. While biglycan is attached to two glycosaminoglycans (GAG) chains at the S42 and S47 sites of the propeptide, decorin is attached to only S34. Although biglycan and decorin are structurally similar, their spatial expression and presence is distinct.

[0088] Biglycan is present on endothelial cells while decorin is not, rather decorin is expressed in connective tissues. Biglycan is overexpressed in multiple tumors and TECs. Moreover, biglycan is strongly correlated to angiogenesis related proteins and increased metastasis in multiple tumor types.

[0089] Biglycan binds to TLR2 / TLR4 and induces expression of VEGF on endothelial cells. Because biglycan is overexpressed on TECs, engineering CAR-T cells to bind biglycan should increase their infiltration in tumors.

[0090] Bacterium of genus borrelia (B. garinii, B. burdorferi, B. afzelii) are causative agents of Lyme disease via a tick vector. The bacteria can spread from the site of tick bite that is skin to multiple organs like heart, brain and joints. Borrelia express multiple adhesion molecules on their surface (OspA, OspC, DbpA / B, BBA64, RevA) which have been reported to aid their binding to endothelial cells and spread systemically to multiple organs. Decorin binding proteins (DbpA / B) are two distinct proteins expressed on borrelia which bind to GAG chains of decorin and biglycan. Borrelia DbpA / B promote bacterial adherence to endothelial cells under flowing as well as static conditions. Furthermore, silencing biglycan expression on endothelial cells by siRNA abrogated enhanced DbpA / B mediated binding of bacteria to endothelial cells.

[0091] Since borrelia DbpA / B bind to biglycan, Applicant envisioned that heterologous expression of functional DbpA / B in T cells should promote their binding to biglycan expressed on endothelial cells. In this Example, Applicant has engineered functional Borrelia. garinii DbpA and DbpB to be expressed on T cells and show their enhanced binding on endothelial cells under flowing conditions in vitro.

[0092] Example 1.1. Biglycan is overexpressed in metastatic cancers

[0093] Biglycan is a secreted proteoglycan, which is present in the glycocalyx of endothelial cells. Multiple studies have reported increased expression of biglycan on endothelial cells in metastaticPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 cancer compared to endothelial cells in normal tissues, utilizing RT-PCR and immunohistochemical staining. Mechanistically, biglycan binds to TLR2 / TLR4 and promotes VEGF expression in endothelial cells.

[0094] Applicant investigated multiple human cancers in the TCGA dataset to determine whether biglycan expression is associated with increased expression of angiogenesis-related genes. Applicant tested biglycan expression against the normalized expression of an angiogenesis-related gene set consisting of VEGFB, VEGFC, KDR, FLT1, FGF2, PDGFC, ANGPT1, and ANGPTL220. Applicant found that biglycan expression strongly correlated with the expression of the angiogenesis gene set in ovarian cancer (Pearson R=0.68, p<0.0001), stomach adenocarcinoma (Pearson R=0.71, p<0.0001), kidney renal cell papillary carcinoma (Pearson R=0.16, p=0.004), and skin cancer (Pearson R=0.54, p<0.0001) (FIGS. 1A, 1C, 1E and 1G). Furthermore, mean biglycan expression was significantly higher in patients with metastasis in these cancers (ovarian cancer: p=0.004, stomach adenocarcinoma: p=0.02, kidney renal cell papillary carcinoma: p=0.008, and skin cancer: p=0.006) (FIGS. 1B, 1D, 1F, and 1H). Such analyses on human cancers suggest that biglycan is overexpressed in metastatic cancers and is expressed on endothelial cells in these cancers.

[0095] Example 1.2. Borrelia garinii decorin binding protein A / B (DBPA / DBPB) expressed on mammalian cells binds to biglycan

[0096] Bacterium Borrelia garinii is among one of the causative agents of lyme disease. Its decorin binding proteins (DBPA / DBPB) contribute to its systemic spread via facilitating binding to biglycan expressed on endothelial cells. As endothelial cells in metastatic cancers express biglycan, Applicant aimed to investigate whether a functionally active heterologous membrane bound DBPA / DBPB could be expressed on mammalian cells and whether the biglycan binding of B. garinii could be imitated. As a membrane protein can be expressed either with N or C terminal flanking the extracellular domain (FIG. 2A), Applicant made two plasmid constructs to express membrane bound DBPA and DBPB. To make DBPA / DBPB with C terminal flanking the extracellular domain, Applicant fused the sequence encoding the first 73 amino acids of CD69, a type II membrane protein, before the codon optimized gene sequence of DBPA / DBPB (FIG.2B) (Construct C). To make DBPA / DBPB with N terminus flanking the extracellular domain, Applicant fused sequence encoding CD55 secretion signal before DBPA / DBPB gene sequence and GPI anchor signaling domain after DBPA / DBPB gene sequence (FIG. 2C) (Construct N). Applicant fused a myc-tag to the extracellular domain ofPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 DBPA / DBPB and P2A-mCherry post DBPA / DBPB sequence in both constructs to aid in confirming expression and selection (FIGS.2B-2C).

[0097] Applicant transiently transfected PlatGP cells with these constructs and imaged the cells using confocal microscopy (FIG. 2D). Applicant could detect cytoplasmic mCherry expression for all constructs (FIGS.2E, 2G, 2I and 2K). To confirm whether DBPA / DBPB was expressed on the cell membrane, Applicant imaged the cells after staining with anti-myc-AF488 antibody. Applicant could detect fluorescence on the cell membrane for all constructs (FIGS. 2E, 2G, 2I and 2K). Applicant further confirmed the expression of mCherry and myc-tag on all the constructs using flow cytometry (FIGS. 2F, 2H, 2J and 2L) and found that the expression of DBPA / B and mCherry was strongly correlated. In sum, the aforementioned results confirmed that DBPA / DBPB with extracellular N or C terminal domain could be expressed on the cell membrane of human cells.

[0098] After confirming the expression of DBPA / DBPB on the cell membrane, Applicant sought to assess the functionality of DBPA / B expressed on PlatGP cells and its ability to bind to biglycan. Applicant devised a sandwich assay where Applicant initially incubated transiently transfected PlatGP cells with recombinant biglycan containing a 6xHIS-tag on the C-terminus. Following cell washing, Applicant stained them with anti-6xHIS AF647 antibody and subsequently imaged the cells using confocal microscopy (FIG.2D). While the DBPA / DBPB constructs with the C-terminal flanking the extracellular domain (Construct C) exhibited an AF647 fluorescence signal on the cell membrane (FIGS.2M and 2Q), the constructs with the N-terminal flanking the extracellular domain (Construct N) did not (FIGS.2O and 2S).

[0099] Utilizing flow cytometry, Applicant observed a strong correlation between AF647 and mCherry fluorescence for construct C (FIGS. 2N and 2R), whereas construct N did not display positive AF647 staining even at high mCherry fluorescence intensities (FIGS. 2P and 2T). This finding aligns with a prior report emphasizing the significance of the free C-terminal of DBPB for biglycan binding. Additionally, considering that DBPA and DBPB are anchored to the bacterial membrane at the N-terminus, Applicant’s results suggest that a flanking C-terminus on the extracellular domain might be crucial for biglycan binding in these proteins. In summary, Applicant’s data indicates that, when expressed with an extracellular C domain on human cells, DBPA / DBPB can facilitate biglycan binding.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0100] Example 1.3. B. garinii DBPA / DBPB expression in human T cells enable their biglycan binding

[0101] After confirming that DBPA / DBPB with a type II signal sequence could be expressed on human cells and bind to biglycan, Applicant’s next aim was to investigate whether functional DBPA / DBPB could be expressed in T cells. Applicant generated retroviral particles for Construct C (FIG.3A) and transduced human T cells derived from peripheral blood mononuclear cells (PBMCs). The efficiency of transduction for DBPA and DBPB constructs reached 94% and 89%, respectively, as indicated by mCherry fluorescence intensity plots (FIGS.3B-3D), without impacting the phenotype of the T cells (FIGS.3E-3G).

[0102] To verify if DBPA / DBPB expressed on T cells could bind to biglycan, Applicant replicated the biglycan-anti-6xHIS-AF647 sandwich immunofluorescence assay (FIG. 3H). Like PlatGP cells, AF647 fluorescence intensity was directly correlated to mCherry fluorescence intensity (FIGS.3I-K). Notably, DBPA exhibited higher avidity for biglycan compared to DBPB.

[0103] To eliminate the possibility of nonspecific binding of the anti-6xHIS antibody to T cells, Applicant stained DBPA / DBPB-expressing T cells with anti-6xHIS antibody (FIG. 3L). No appreciable AF647 fluorescence was observed, and mCherry and AF647 fluorescence intensity were not correlated (FIGS.3M-3O).

[0104] Having established that T cells expressing DBPA / DBPB could bind to biglycan, Applicant’s subsequent goal was to determine if DBPA / DBPB expression on T cells could enhance their adherence to plate-bound biglycan. Applicant coated 10 μg / ml biglycan on 24-well plates, blocked the plates with bovine serum albumin (BSA), and added an equal number of DBPA, DBPB, and non-transduced T cells fluorescently labeled with BioTracker green dye to biglycan-coated wells. After incubating the cells for 1 hour, Applicant washed each well thrice with PBS, imaged the wells, and counted the number of adherent T cells (FIG. 4A). The DBPA-expressing construct displayed the highest number of cells bound to the plate per field of view, followed by the DBPB-expressing constructs, and then the non-transduced T cells (FIGS.4B-4C). Applicant’s results demonstrate that T cells expressing functional DBPA / DBPB can robustly bind to free and plate-bound biglycan.

[0105] Example 1.4. DBPA / DBPB expressing human T cells roll on biglycan coated plates

[0106] Applicant hypothesized that T cells expressing DBPA / DBPB would facilitate their rolling and adhesion on endothelial cells. As such, Applicant proceeded to test whether DBPA / DBPB-PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 expressing T cells could roll on biglycan-coated plates under flowing conditions (FIG.5A). Applicant coated 10 μg / ml biglycan on ibidi μ slides (microfluidic flow channel, channel height: 0.4 mm). Applicant fluorescently labeled DBPA / DBPB or non-transduced T cells using BioTracker red and allowed them to flow through the microfluidic channel at a shear stress of 1 dyne / cm2. Applicant imaged the cells using an inverted fluorescence microscope (FIG. 5A) and analyzed the cells under flowing conditions. DBPA and DBPB expressing T cells exhibited a significantly lower velocity and higher frequency of rolling compared to non-transduced T cells (FIG.5B). The mean rolling velocity for cells expressing DBPA and DBPB was 14 μm / s and 27 μm / s, respectively, while that of non- transduced T cells was 54 μm / s (FIG.5C).

[0107] The frequency of cells with rolling velocity between 0-20 μm / s was 15% and 7% for DBPA and DBPB expressing constructs, while it was 0% for non-transduced T cells (FIG. 5D). Hence, the expression of functional DBPA / DBPB on T cells enabled their rolling on biglycan-coated surfaces under flowing conditions.

[0108] Example 1.5. DBPA / DBPB expressing human T cells roll on human endothelial cells under flow

[0109] As DBPA / DBPB expressing T cells could roll on biglycan coated plates, Applicant went on to investigate if they could bind to endothelial cells under flow. Human umbilical cord endothelial cells (HUVEC) express biglycan on their surface, and Applicant aimed to test whether DBPA / DBPB expressing T cells could roll on HUVECs. Applicant cultured HUVECs on ibidi μ slides (microfluidic flow channel) and when the HUVECs were confluent, Applicant imaged DBPA / DBPB expressing T cells flow over HUVECs at 1 dyne / cm2(FIG. 6A). DBPA expressing cells rolled more often compared to DBPB expressing and control T cells (FIGS.6B-6D). While 21% of DBPA expressing cells rolled (velocity < 244 μm / s) on endothelial cells, only 7% of DBPB and control cells rolled on the endothelial cells (FIG. 6D). Compared to biglycan coated plates, DBPA and DBPB expressing cells rolled less often and at a higher velocity on endothelial cells. Furthermore, DBPA expressing T cells rolled more effectively (12% cells between 0-40 μm / s) compared to DBPB (5% cells between 0-40 μm / s) and control T cells (5% cells between 0-40 μm / s) (FIG. 6D). In conclusion, DBPA expressing T cells roll efficiently on HUVECs compared to DBPB and control T cells.

[0110] Example 1.6. DiscussionPCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0111] Several factors, including CAR-T cell exhaustion, antigen escape and lack of adequate number of CAR-T cells in the tumor, can lead to CAR-T therapy failure in solid tumors. Solid tumors often evolve to have an immunosuppressive state characterized by either exclusion or absence of anti- tumor immune cells in the tumor. Since number of T cells in the tumors correlate strongly with overall survival in multiple cancers, engineering T cells to enhance their tumor infiltration efficacy could lead to favorable responses in CAR-T cell therapies. Intratumoral injections of CAR-T cells, which although is infeasible in many solid tumors, provides an avenue to evaluate the impact of CAR-T cells when there is no resistance to infiltration. Intratumoral CAR-T cell injections against c-met protein expressed in breast cancers lead to extensive tumor necrosis and complete antigen depletion in phase 0 trial of breast cancers in human.

[0112] Upon intravenous administration of CAR-T cells in mice, only 1-2% of these cells reach tumors at day 2 and day 7, while the majority of cells end up in lungs and liver. Hence, engineering CAR-T cell to specifically reach tumor would not only increase efficacy of CAR-T therapy, but also open a window for lower doses and hence lower therapy related toxicity.

[0113] The tumor microenvironment (TME) can exclude immune cells by downregulating adhesion molecules on endothelial cells through which T cells infiltrate the tumor. Angiogeneic vessels are known to downregulate ICAM-1, VCAM-1 upon stimulation with angiogenic factors like VEGF, FGF and ANGPT. Because angiogenic vessels are a pathway for systemic cancer cell dissemination and metastasis, Applicant hypothesize that increasing CAR-T infiltration near angiogenic vessels, which are often immune excluded, would lead to reduced metastasis and better overall survival. Because angiogenic vessels reduced expression of conventional cell adhesion molecules but have increased levels of other molecules like the proteoglycan biglycan, Applicant aimed to test whether T cells could be engineered to bind to biglycan.

[0114] To engineer T cells to bind biglycan, Applicant drew inspiration from bacterium of the genus borrelia, which have been shown to bind to endothelial cells via binding to biglycan by decorin binding proteins DBPA / B. In support of Applicant’s hypothesis, in a study, 28% of invasive breast cancer specimens stain positive for Borrelia. burgdorferi. Applicant showed that human cells expressing Borrelia. garinii DBPA / B fused to type II membrane protein (CD69) signal sequence bound to biglycan while those expressing DBPA / B fused to GPI anchor (CD55) did not bind to biglycan.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074

[0115] Given that borrelia DBPA / B is anchored to periplasm with C terminal flanking the extracellular domain and a free C terminus is required for binding to biglycan, Applicant’s results align well with these observations and reiterate the importance of correct orientation of DBPA / B to bind biglycan. Among DBPA and DBPB, Applicant observed that DBPA was more efficient in binding to biglycan under static and flowing conditions.

[0116] Higher binding efficiency for DBPA could be attributed to its higher expression on T cells as evidenced by increased mCherry expression or better efficacy of DBPA in binding to biglycan. Nevertheless, DBPA and DBPB expressing T cells rolled on biglycan coated plates, with DBPA rolling more frequently and slowly compared to DBPB. On endothelial cells, where the density of biglycan on the cell surface might not reach the levels on the plate, there was a decrease in the frequency of rolling cells and an increase in their speed. This effect was observed for both DBPA and DBPB expressing cells.

[0117] In conclusion, Applicant has engineered B. garinii DBPA / DBPB for heterologous expression in T cells and shown that these T cells roll on endothelial cells. Although Applicant has demonstrated that T cells expressing DBPA / DBPB can undergo rolling on both biglycan-coated plates and endothelial cells, there remains room for enhancing these constructs. Unlike CD62L, which can facilitate T cell rolling on high endothelial venules (HEVs) under shear stresses of up to 30 dyne / cm2, the DBPA / DBPB-mediated rolling on endothelial cells could not be sustained at high shear stresses. This discrepancy can be attributed to the absence of intracellular domains in DBPA / B constructs capable of binding to the actin cytoskeleton, a crucial component for CD62L mediated efficient binding to HEVs.

[0118] The process of diapedesis encompasses multiple critical interactions, including rolling, activation, and firm adhesion, all of which have been targeted in efforts to mitigate leukocyte extravasation in autoimmune disorders. In vitro studies have demonstrated that blocking selectins, pivotal for rolling, can reduce leukocyte adhesion by ~80%. Additionally, inhibition of CD62L has been shown to diminish T cell homing to lymph nodes. Human squamous cell carcinomas have downregulated of E-selectin expression on endothelial cells, hence hampering T cell infiltration. Furthermore, intravital microscopy has revealed that T cells exhibit a lower frequency of rolling within tumors compared to adjacent tissue. These findings strongly suggest that promoting T cell rolling enhance their binding to tumor endothelium, thus enhancing their T cell infiltration into tumors.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 Conclusively, Applicant has successfully engineered the heterologous expression of B. garinii DBPA / B on human T cells, enabling them to roll over endothelial cells by binding to biglycan. This achievement holds promise for enhancing CAR-T infiltration in metastatic tumors.

[0119] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 CLAIMS 1. A cell, wherein the cell heterologously expresses at least one binding protein that binds to one or more sulfated proteoglycans.

2. The cell of claim 1, wherein the cell comprises an immune cell.

3. The cell of claim 2, wherein the immune cell is selected from the group consisting of neutrophils, macrophages, leukocytes, B-cells, T-cells, chimeric antigen receptor T-cells, natural killer (NK) cells, regulatory T-cells, or combinations thereof.

4. The cell of claim 1, wherein the cell comprises chimeric antigen receptor T-cells.

5. The cell of claim 1, wherein the binding protein is overexpressed by the cell.

6. The cell of claim 1, wherein the binding protein is positioned on the outer cellular membrane of the cell.

7. The cell of claim 1, wherein the binding protein is derived from a Borrelia bacterium species selected from the group consisting of B. garinii, B. burdorferi, B. afzelii, or combinations thereof.

8. The cell of claim 1, wherein the binding protein is fused to a membrane targeting signal sequence.

9. The cell of claim 8, wherein the signal sequence comprises SEQ ID NO: 5 or a sequence with at least 65% sequence identity to SEQ ID NO:

5.

10. The cell of claim 1, wherein the binding protein is selected from the group consisting of decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof.

11. The cell of claim 1, wherein the binding protein comprises DbpA and DbpB.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 12. The cell of claim 1, wherein the binding protein comprises DbpA.

13. The cell of claim 12, wherein the DbpA comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO:

1.

14. The cell of claim 12, wherein the DbpA is fused to a membrane targeting signal sequence.

15. The cell of claim 14, wherein the signal sequence comprises SEQ ID NO: 5 or a sequence with at least 65% sequence identity to SEQ ID NO:

5.

16. The cell of claim 1, wherein the binding protein comprises DbpB.

17. The cell of claim 16, wherein the DbpB comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO:

2.

18. The cell of claim 16, wherein the DbpB is fused to a membrane targeting signal sequence.

19. The cell of claim 18, wherein the signal sequence comprises SEQ ID NO: 5 or a sequence with at least 65% sequence identity to SEQ ID NO:

5.

20. The cell of claim 1, wherein the cell is suitable for use in treating or preventing a progression of a tumor in a subject, autoimmune diseases, autoimmune diseases of the heart, lupus nephritis, multiples sclerosis, rheumatoid arthritis, atherosclerotic plaque build-up, auto-immune pathologies with increased sulfated proteoglycan expression, or combinations thereof.

21. A method of treating or preventing a disease in a subject, said method comprising administering a cell to the subject, wherein the cell heterologously expresses at least one binding protein that binds to one or more sulfated proteoglycans.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 22. The method of claim 21, wherein the administration comprises intravenous administration.

23. The method of claim 21, wherein the subject is a human being.

24. The method of claim 21, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, autoimmune diseases of the heart, lupus nephritis, multiples sclerosis, rheumatoid arthritis, atherosclerotic plaque build-up, auto-immune pathologies with increased sulfated proteoglycan expression, or combinations thereof.

25. The method of claim 21, wherein the disease is cancer.

26. The method of claim 25, wherein the cancer is selected from the group consisting of metastatic cancer, non-metastatic cancer, ovarian cancer, medulloblastoma, glioblastoma, osteosarcoma, stomach cancer, stomach adenocarcinoma, kidney cancer, kidney renal cell papillary carcinoma, skin cancer, melanoma, breast cancer, or combinations thereof.

27. The method of claim 21, wherein the cell comprises an immune cell selected from the group consisting of neutrophils, macrophages, leukocytes, B-cells, T-cells, chimeric antigen receptor T- cells, natural killer (NK) cells, regulatory T-cells, or combinations thereof.

28. The method of claim 21, wherein the cell comprises chimeric antigen receptor T-cells.

29. The method of claim 21, wherein the binding protein is derived from a Borrelia bacterium species selected from the group consisting of B. garinii, B. burdorferi, B. afzelii, or combinations thereof.

30. The method of claim 21, wherein the binding protein is fused to a membrane targeting signal sequence.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 31. The method of claim 21, wherein the binding protein is selected from the group consisting of decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof.

32. The method of claim 21, wherein the binding protein comprises DbpA and DbpB.

33. The method of claim 21, wherein the binding protein comprises DbpA.

34. The method of claim 33, wherein the DbpA comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO:

1.

35. The method of claim 21, wherein the binding protein comprises DbpB.

36. The method of claim 35, wherein the DbpB comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO:

2.

37. A method of modifying a cell, said method comprising heterologously expressing at least one binding protein that binds to one or more sulfated proteoglycans in the cell.

38. The method of claim 37, wherein the cell comprises an immune cell selected from the group consisting of neutrophils, macrophages, leukocytes, B-cells, T-cells, chimeric antigen receptor T- cells, natural killer (NK) cells, regulatory T-cells, or combinations thereof.

39. The method of claim 37, wherein the cell comprises chimeric antigen receptor T-cells.

40. The method of claim 37, wherein the binding protein is selected from the group consisting of decorin binding protein A (DbpA), decorin binding protein B (DbpB), or combinations thereof.

41. The method of claim 37, wherein the expressing comprises introducing a nucleotide sequence encoding the binding protein into the cell.PCT Application Attorney Docket No. AF23853.P205WO UH ID No.2024-074 42. The method of claim 41, wherein the nucleotide sequence encodes DbpA.

43. The method of claim 42, wherein the DbpA nucleotide sequence comprises SEQ ID NO: 3 or a sequence with at least 65% sequence identity to SEQ ID NO:

3.

44. The method of claim 41, wherein the nucleotide sequence encodes DbpB.

45. The method of claim 44, wherein the DbpB nucleotide sequence comprises SEQ ID NO: 4 or a sequence with at least 65% sequence identity to SEQ ID NO: 4.

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