Compositions and methods for tumor-penetrating solid tumor therapy

Abstract

A technology is described which provides a platform for improving the penetration into tumors in a subject of cancer treatment and cancer imaging agents for greater patient benefit. The invention in particular overcomes two challenges for use of tumor targeting methods for solid tumors: poor penetration and the immunosuppressive nature of the tumor microenvironment. The platform described here allows long-term, in vivo production of iRGD. In a preferred embodiment, CAR-T cells are engineered to secrete iRGD, a tumor-penetrating peptide to achieve tissue-penetrating delivery of drugs and imaging agents in a tumor-specific manner. The peptide acutely permeabilizes the tumor vasculature by binding to αv integrins and neuropilin, but in a tumor-specific manner. This method produces a modification in the tumor microenvironment that enhances drug access into the tumor, prevents distant metastasis of the tumor cells, and allows long-term iRGD therapy without the need for repeated injections of iRGD.

Description

Attorney Docket No. 15003-502PC0COMPOSITIONS AND METHODS FOR TUMOR-PENETRATING SOLID TUMOR THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application under the PCT which claims the benefit of United States provisional application serial no. 63 / 714,371, filed 31 October 2024. The entire contents of the aforementioned application(s) is / are hereby incorporated by reference as if fully set forth herein.BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the general field of medicine and more specifically to new therapeutic compounds and compositions for reduction of solid tumors. In particular, the invention provides a vector for in vivo translation of iRGD into a secreted peptide or protein that can reduce metastasis, enhance targeted delivery of therapeutics to tumors, increase susceptibility to therapeutics, and reduce the immunosuppressive environment in solid tumors and improve tissue permeablilization into the tumor.

[0004] 2. Background of the invention

[0005] Cancer is among the leading causes of death worldwide. Most existing cancer treatment programs include surgery, radiotherapy, and chemotherapy, targeted therapy and immunotherapy. Solid tumors continue to present a therapeutic challenge, however, due to their immunosuppressive and chemotherapy-resistant nature. Although small molecule chemotherapeutics, immune checkpoint blockade drugs, cellular immunotherapies, and biological drugs can target solid tumors. Many anti-cancer drugs are limited in efficacy due to inability of the drag to penetrate deep enough into the tumor parenchyma to reach blood vessels; they often fail to penetrate the tumor tissues or tumor cells sufficiently for optimal efficacy of therapeutic agent(s). Effective penetration of the dense and hypoxic tumor tissue and counteracting with the immunosuppressive solid tumor microenvironment (TME) thereforeAttorney Docket No. 15003-502PC0 continues to pose a significant hurdle for current cancer therapies which is not addressed by approaches currently available.

[0006] T cells play a crucial role in immunity against cancer and infections. CAR-T (chimeric antigen receptor T-cell) therapy is a type of cancer immunotherapy that involves genetically engineering a patient’s own T cells to attack cancer cells. CAR-T cells have shown promise for the treatment of hematologic malignancies but remain ineffective for solid tumors. CAR T-cell therapy for patients with solid tumors faces greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity in the tumor. In particular, CAR-T cell therapy has been ineffective against solid tumors given the lack of penetration into the tumor and the microenvironment causing exhaustion of the T cells. These challenges are pronounced in some cancers, such as pancreatic ductal adenocarcinoma (PDAC), given the desmoplastic and highly immunosuppressive TME of this particular tumor. Improving CAR-T therapy for solid tumors has been a major unmet need in cancer therapy.

[0007] iRGD peptides contain an RGD motif consisting of three amino acids- arginine, glycine, and aspartic acid- found in cell adhesion and signaling proteins that facilitate cell adhesion and penetration through integrin receptors, notably av integrins. These integrin receptors are abundantly expressed in tumor vasculature, malignant cells, stromal cells, and some immune cells and can be recognized by the RGD integrin recognition motif, which can facilitate absorption of drugs into cells. iRGD also contains an R / KXXR / K (SEQ ID NO:29) motif consisting of arginines (arg, R) and lysines (lys, K) (X: any amino acid), which binds to neuropilins (NRP).

[0008] It has been demonstrated that cancer therapeutic treatment combined with iRGD leads to better perfusion of the tumor, less desmoplasia, improved drug entry, infiltration and activation of T cells, and reduced metastasis. However, treatments with iRGD often requires continuous dosage along with these cancer therapies to remain efficacious. Currently, there are no FDA- approved approaches that greatly improve therapeutic response or prevent metastasis of PDAC.SUMMARY OF THE INVENTION

[0009] Thus, there is a need in the art for new compositions and methods, such as CAR-T cells and other cells, vectors, and peptides that have the ability to penetrate and image or treat solid tumors, particularly PDAC. This technology addresses these issues and the invention describedAttorney Docket No. 15003-502PC0 herein thus provides embodiments related to iRGD-secreting CAR-T cells. iRGD, a tumorpenetrating peptide, is explored here to enhance CAR-T cell therapy for solid tumors by improving T-cell infiltration and activation within the tumor microenvironment and enhance penetration into the tumor, leading to better anti-tumor activity.

[0010] In particular, the invention relates to an isolated iRGD peptide comprising an RGD sequence and an R / KXXR / K (SEQ ID NO:29) sequence. A preferred sequence is CRGDK / R / HGPD / EC (SEQ ID NO: 12) or a variant thereof comprising at least 95% identity therewith. The most preferred sequence is CRGDKGPDC (SEQ ID NO:8).

[0011] In certain other embodiments, the invention relates to a vector that comprises an expressible nucleic acid sequence that encodes the iRGD peptides above. Preferably, the vector comprises a promoter, which may be a mammalian vector, a viral vector or a bacterial vector, or an exosome, liposome, or other lipid based delivery agent that comprises the expressible sequence. In preferred embodiments, the vector an AAV, adenovirus, or lentivirus vector. A preferred vector is a plasmid vector comprising SEQ ID NO:9.

[0012] In certain other embodiments, the invention relates to a cell comprising the vector of claim 4 which expresses an iRGD peptide. This cell can be a mammalian cell, a yeast cell, a bacterial cell, or an insect cell, preferably a mammalian cell or a human cell. The cells used for the invention preferably comprise an antigen receptor that specifically binds to a cancer marker. Preferred markers are selected from the group consisting of MR1, B7H3, a claudin family members, mesothelin, a plastin, CEA, CD70, PSMA, HER2, MUC1, GD2, and EGDR.

[0013] In additional embodiments, the invention relates to a secretory iRGD peptide comprising SEQ ID NO: 12.

[0014] The invention also relates to, in certain embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptides disclosed herein, the vectors disclosed herein, or the cells disclosed herein.

[0015] In addition, the invention relates to a vaccine composition comprising a cell of claim 7 which secretes an iRGD peptide or an mRNA vaccine composition comprising mRNA encoding an iRGD peptide.

[0016] In other embodiments, the invention relates to a method of producing a cell as discussed herein, comprising obtaining a population of cells; optionally isolating specific cell population subsets from the cells; stimulating the cells or the cell population subsets using cytokines,Attorney Docket No. 15003-502PC0 antibodies, or both to promote activation, differentiation, and proliferation; and introducing to the cells one or more DNA or RNA nucleic acids encoding s-iRGD and a CAR construct using a gene delivery method, wherein the cells express and secrete an iRGD peptide. In this method, the population of cells preferably are selected from the group consisting of tumor-infiltrating lymphocytes, peripheral blood mononuclear cells, spleen cells, bone marrow cells, or cells from umbilical cord blood and the specific cell population subsets are selected from the group consisting of CD4+T cells, CD8+T cells, regulatory T cells, NK cells, and NKT cells. Isolation can be performed using magnetic beads or flow cytometry. The gene delivery method is selected from the group consisting of lentiviral, retroviral, adenoviral, or non-viral transfection.

[0017] The invention also relates to a programmed CAR-T cell produced by this method.

[0018] In other embodiments, the invention relates to a method of treating cancer comprising administering to a subject in need thereof the peptides, vectors, or cells discussed herein, preferably by intravenous injection. The cancer being treated can be a solid tumor. More specifically, the cancer being treated can be pancreatic ductal adenocarcinoma.

[0019] Another embodiment of the invention relates to a kit for cancer detection, comprising: (a) a population of cells that produce s-iRGD in vivo; (b) an imaging agent; and (c) instructions for use of the kit, wherein the imaging agent is compatible with computed tomography, magnetic resonance imaging, ultrasound, or positron emission tomography.

[0020] Further, the invention relates, in certain embodiments, a method for cancer imaging in a subject in need thereof, comprising: (a) obtaining a population of cells of claim 7 which produces s-iRGD;(b) injecting the cells into the subject in need, intravenously; (c) injecting an imaging agent into the subject in need and waiting for the imaging agent to penetrate into the cancer; and (d) imaging the subject in need using an imaging modality suitable for use with the injected imaging agent to detect the location of the imaging agent in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0022] FIG. 1 is a schematic drawing that shows how iRGD targets and extravasates into tumors in 3-steps mediated by av integrins, proteases, and NRP-1.Attorney Docket No. 15003-502PC0

[0023] FIG. 2 shows confocal images of BT474 orthotopic breast tumors from mice injected with IV Nab-paclitaxel (Nab-P) (green) either conjugated or co-injected with iRGD. Inset, Nab- P alone. Red, CD31 ; blue, DAPI.

[0024] FIG. 3 is a waterfall diagram showing data regarding the anti-tumor activity of iRGD + gemcitabine + Nab-P in patients with stage 4 pancreatic ductal adenocarcinoma (PDAC).

[0025] FIG. 4A is a set of confocal images PDAC from transgenic p48-Cre, LSL-KrasG12D, Ink4aflox(KRAS-Ink) mice that received IV anti-NRP-1 blocking antibody (Ab) or control IgG, followed by iRGD-coated phage (green).

[0026] FIG. 4B presents data for BT474 tumor mice treated with the indicated IV Nab-P formulations at 3 mg paclitaxel / kg or vehicle alone.

[0027] FIG. 5A is a western blot showing inhibition of transforming growth factor-p (TGF- ) activation by iRGD in PDAC cells using phosphorylated SMAD2 (pSMAD2) as a marker. FIG. 5B shows the data normalized to no inhibitor.

[0028] FIG. 6A is a western blot showing a lack of pSmad2 inhibition by iRGD in the presence of activated TGF- . FIG. 6B shows the data normalized to no inhibitor.

[0029] FIG. 7A is a photograph showing stromal fibers in PDAC tumors stained with fibroblast antigen ER-TR7 (red) and the fluorescent dye DAPI (blue). FIG. 7B is a bar graph showing data comparing the %ER-TR7+area in these PDAC cells.

[0030] FIG. 7C is a photograph showing angiogenic blood vessels in PDAC tumors stained with anti-CD31 antibody (red) and the fluorescent dye DAPI (blue). FIG. 7D is a bar graph showing a comparison of the %CD31+vessels in the PDAC.

[0031] FIG. 7E is a photograph showing angiogenic blood vessels in PDAC tumors stained with an anti-CD31 antibody (brown). FIG. 7F is a bar graph presenting the minor axis measured from FIG. 7E.

[0032] FIG. 7G is a set of photographs showing representative images of a blood vessel (red) perfused by tomato lectin (green) in an iRGD treated tumor. Blue staining is the fluorescent dye, DAPI. FIG. 7H is a bar graph showing the percentage of tomato lectin-positive vessels in FIG.7G.

[0033] FIG. 8A through FIG. 8E show data for KPC78 wild type or b5 integrin KO cells treated with TGF-p (FIG. 8 A. FIG. 8B, FIG. 8C) or LAP-TGF-p (FIG. 8D, FIG. 8E) ± LY2157299 (TGF-P inhibitor).Attorney Docket No. 15003-502PC0

[0034] FIG. 8F and FIG. 8G present data for KPC78 cells cultured with LAP-TGF-0 ± iRGD, or an iRGD variant with a disrupted RGD (iRGE).

[0035] FIG. 9A and FIG. 9B show a schematic drawing and results, respectively, from a reporter gene assay using av-deficient responder cells that selectively detect activated TGF- confirmed the results.

[0036] FIG. 10A and FIG. 10B show data for orthotopic PDAC mice bearing syngeneic KPC78 treated with IV PBS or iRGD 3x / week for 2 weeks. The tumors were stained and quantified for pSmad2 (blue) (FIG. 10A and FIG. 10B), and CD8+ T cells (arrow heads) in the periphery and core of the tumors (FIG. 10C and FIG. 10D).

[0037] FIG. 11A through FIG. 11G present data on spatial transcriptomics using human (FIG. 11A through FIG. 1 IF) and mouse (FIG. 11G) 5k pan tissue & pathways panel was run on huPDAC tumors treated with or without iRGD.

[0038] FIG. 11A presents gene ontology analysis showing T cell signatures in iRGD-treated tumors in contrast to control. FIG. 1 IB shows enhanced expression of T cell activation genes with iRGD. FIG. 11C through FIG. 1 IE show UMAPs of major T cell populations (FIG. 11C), CD4+ vs CD8+ T cells (FIG. 1 ID), and CD8 subtypes (FIG. HE) in the tumors. FIG. HF and FIG.l 1 show the spatial distribution of human (FIG. 1 IF) CD8 (CD8A), granzyme B (GZMB), and PD-1 (PDCD), and mouse (FIG. 11G) desmin (DES), FAP (FAP), PDGFR-P (PDGFRB), and CD31 (PEC AM- 1) genes in control and iRGD-treated tumors. FIG. 11H is a set of photographs showing examples of MISO-based clustering that reflects both histological and DGE features.

[0039] FIG. 12A shows the ratio of CD8+ T cells over regulatory T cells (Tregs) in PDAC tumors collected 3, 7, and 14 days after treatment with iRGD analyzed by flow cytometry. FIG. 12B and FIG. 12C provide representative images of immunofluorescence of PDAC tissue harvested after 14 days of PBS or iRGD treatment. FIG. 12D is a bar graph showing the CD8 / Treg ratio in the PDAC after the treatment.

[0040] FIG. 13A through FIG. 13E present data for PDAC mice treated with iRGD ± anti-PD-Ll mAb (FIG. 13A, FIG. 13B and FIG. 13C; n = 4-6)

[0041] FIG. 14A is a set of data showing the macroscopic appearance of the liver in PDAC mice with and without iRGD treatment. FIG. 14B is a bar graph showing the number of macroscopic liver metastases. FIG. 14C is a set of microphotographs showing H & E stained liver sectionsAttorney Docket No. 15003-502PC0 prepared from PDAC mice that were treated with or without iRGD. FIG. 14D is a bar graph showing the number of microscopic liver metastases. FIG. 14E is a set of microphotographs showing anti-CK19 antibody staining of liver sections to detect tumor cells in the liver of PDAC mice treated with or without iRGD. FIG. 14F is a bar graphs showing the number of CK19+macroscopic liver metastases.

[0042] FIG. 15 is a drawing of a plasmid vector with the DNA sequence of an iRGD peptide attached to an endoplasmic reticulum (ER) signal peptide.

[0043] FIG. 16A is a diagram showing the process whereby the ER signal peptide directs the product to the ER, Golgi and cell exterior. FIG. 16B is a schematic drawing depicting the trafficking and production / secretion of secretory proteins, which is directed by an ER signal peptide that becomes detached before secretion of the protein from the Golgi.

[0044] FIG. 17A is a table presenting DNA and peptide sequences as indicated: ER signal of human cerebral dopamine neurotrophic factor (DNA, SEQ ID NO:1; peptide, SEQ ID NO:2); HA tag (DNA. SEQ ID NO:3; peptide. SEQ ID NO:4); Myc tag (DNA. SEQ ID NO:5; peptide, SEQ ID NO:6); and iRGD (DNA, SEQ ID NO:7; peptide, SEQ ID NO:8).

[0045] FIG. 17B lists the DNA (SEQ ID NO:9) and peptide (SEQ ID NO: 10) sequences of the full length protein, and the secretory form of the peptide (SEQ ID NO: 11).

[0046] FIG. 18 is a set of photomicrographs showing TCON gastric cancer cells producing HA- Myc-iRGD detected by anti-HA and anti-Myc antibodies. Left panel, HA staining; center panel, Myc staining, right panel, merge, with DAPI staining.

[0047] FIG. 19A is a bar graph showing sandwich ELISA that detects HA-Myc-iRGD in conditioned media (CM) of gastric cancer cells from FIG. 18, or control cells. FIG. 19B is a schematic diagram of the sandwich ELISA.

[0048] FIG. 20 is a bar graph providing relative fluorescence intensity results that represent the amount of HA-Myc-iRGD in CM collected from various cells transduced with the plasmid vector. The sandwich ELISA in FIG. 19B was used.

[0049] FIG. 21 is a bar graph providing relative fluorescence intensity results for HA-Myc- iRGD in the plasma of nude mice bearing siRGD- secreting or control PANC-1 human PDAC tumors measured by ELISA in FIG. 19B.

[0050] FIG. 22A is a schematic diagram showing an ELISA design used in FIG. 22B. FIG. 22B is a bar graph showing the ELISA results for the CM from cells that produce a various secretoryAttorney Docket No. 15003-502PC0 iRGD peptides, which were assessed for the ability to compete with the binding of biotinylated iRGD to immobilized avP5 integrin.

[0051] FIG. 23A shows the avP5 expression on Tregs and CD8+T cells in orthotopic PDAC (Tu) and spleen (Spl) of syngeneic KPC78 tumor mice (Tu Ms) and the spleen of normal mice (N Ms) analyzed by flow cytometry. FIG. 23B and FIG. 23C show the homing of FAM-iRGD to Tregs in PDAC and spleen in KPC78 mice quantified by flow cytometry.

[0052] FIG. 24A and FIG. 24B, respectively present data for Treg / CD4 and CD8 / Treg ratios in orthotopic KPC78 and spleen after 1 week of PBS or iRGD therapy analyzed by flow cytometry. FIG. 24C shows data for orthotopic KPC78 mice were treated with iRGD and / or anti-PD-Ll Ab 3x a week for 2 weeks.

[0053] FIG. 25 shows H&E and IHC for CD3 (brown) of on-treatment biopsies from a patient treated with iRGD + Gem + Nab-P + durvalumab in the iLSTA trial.

[0054] FIG. 26 shows an HLA-A2+huPDAC mouse with an orthotopic PANC-1 tumor and spontaneous liver metastases (arrows).

[0055] FIG. 27 is a diagram of immune cell components in the primary tumors analyzed by RNA-seq and CYBERSORTx.

[0056] FIG. 28A and FIG 28B are a set of photographs and a bar graph showing that iRGD therapy (12 pmol / kg, 3x / week x 8 weeks) increased the CD8 / Treg ratio in the tumor.

[0057] FIG. 29 presents immunohistochemistry data showing that iRGD opened blood vessels and reduced hypoxia by detection of a hypoxic marker, carbonic anhydrase (FIG. 29). See also FIG. 7E and FIG. 7F and the description relating to these figures.

[0058] FIG. 30A and FIG. 30B are photomicrographs of multiplex immunofluorescence of tumors showing more vascular coverage with a-smooth muscle actin (a-SMA) (FIG. 30 A) and increased CD8 / Treg ratio (FIG. 30B) in the tumor.

[0059] FIG. 31A and FIG. 31C, respectively, show a macroscopic view and H&E staining of liver metastasis. FIG. 31 B and FIG. 31 D are bar graphs showing analysis of these data.

[0060] FIG. 32 presents flow cytometry data showing cell surface MR1 expression across various solid tumor cell lines including PDAC cells, but not normal cells (n=3).

[0061] FIG. 33 A through FIG. 33C and FIG. 34 show data for MR1 CAR-T cells kill MRP tumor cells (FIG. 33a through FIG. 33C), but have minimal effect on cells that lack MR1 (FIG. 34).Attorney Docket No. 15003-502PC0

[0062] FIG. 35 presents data for IV MR1 CAR-T cells, which inhibit the growth of bilateral sq SNU16 human gastric cancer tumors in NSG mice (n=5 mice per group).

[0063] FIG. 36A and FIG. 36B show anti-tumor effects of IC MR1 CAR-T against orthotopic U87 glioblastoma (GBM) (FIG. 36A) and IV MR1 CAR-T against sq U87 GBM (FIG. 36B). n=5 mice per group.

[0064] FIG. 37 and FIG. 39A present data for human B7H3-specific nanobody CAR introduced into human (FIG. 37) and mouse (FIG. 39A) T cells analyzed by flow cytometry 5 days posttransduction (bottom).

[0065] FIG. 38AB and FIG. 39B present data for in vitro cytotoxicity of the human (FIG. 38AB) and mouse (FIG. 39B) B7H3 CAR-T cells against GFP-positive solid tumor cancer cells monitored in real time using the Incucyte live-cell imaging platform.

[0066] FIG. 40 A through FIG. 40C and FIG. 41 A through FIG. 41C present flow cytometry data for MR1 (FIG. 40A through FIG. 40C) or B7H3 (FIG. 41A through FIG. 41C) CAR expression on human primary T cells with or without s-iRGD co-transduction. UTD-T, untransduced T cells.

[0067] FIG. 42 and FIG. 43 show ELISA results which detect s-iRGD released into the CM of the T cells bearing MR1 (FIG. 42) or B7H3 (FIG. 43) CAR.

[0068] FIG. 44 and FIG. 45 show and in vitro cytotoxicity assay showing that s-iRGD expression does not impair the ability of MR1 (FIG. 44) or B7H3 (FIG. 45) CAR-T to kill cocultured tumor cells.

[0069] FIG. 46 is a set of micrographs showing that parental MR1 CAR-T cells minimally entered the tumors in this study. No difference in tumor size was noted in this short time frame, but pilot H&E revealed increased patchy necrosis in the s-iRGD CAR-T arm.

[0070] FIG. 47A through FIG. 47D present micrographs of s-iRGD B7H3 CAR-T cells that infiltrated sq PANC-1 tumors in NSG mice. FIG. 47E is a bar graph presenting data for the cells shown in FIG. 47A through FIG. 47D.

[0071] FIG. 48, shows 0779E sq PDAC tumors from NSG mice treated with IV MR1 CAR-T cells ± s-iRGD production for 5 days. Representative images of CAR-T cells (human CD3, blue) in the periphery and core of the tumors and H&E are shown.

[0072] FIG. 49A through FIG. 49D are bar graphs showing the differential response of M21 variants to active TGF- and LAP-TGF-0. M21 (FIG. 49A and FIG. 49B) and M2 IL (FIG. 49CAttorney Docket No. 15003-502PC0 and FIG. 49D). Expression of pSmad2, Smad2, and GAPDH was analyzed by IB and quantitated as shown, n.s., not significant; *, p<0.05; **, pcO.Ol; ***, pcO.OOl.

[0073] FIG. 50 shows the differential response of M21L4 cells cultured in the presence of active TGF-P or LAP-TGF-P at the indicated concentrations for 3 hours. Expression of pSmad2, Smad2, and GAPDH was analyzed by IB and quantitated as shown, n.s., not significant; *, p<0.05; **, pcO.Ol; ***, pcO.OOl.

[0074] FIG 51A and FIG. 5 IB present a set of photomicrographs for tissue subjected to spatial transcriptomics performed using a 5k human pan tissue & pathways panel on an orthotopic PANC-1 from huPDAC mice treated with or without iRGD as indicated. A limited set of immune cell signatures is shown with and without H&E overlay.DETAILED DESCRIPTION OF THE INVENTION

[0075] 1. Overview

[0076] The present invention relates to therapeutic products and methods that involve using plasmid vectors or any suitable vector as discussed herein that comprises a nucleic acid molecule that encodes internalizing RGD (iRGD), which can be transduced into mammalian tumors or turn or- adjacent mammalian cells to limit aspects of the immunosuppressive environment, lessen tumor growth, suppress metastasis, modify the tumor, and deliver other drugs, imaging agents and cells into the tumor.

[0077] iRGD peptides carry the RGD motif consisting of three amino acids: arginine (arg, R) glycine (gly, G), and aspartic acid (asp, D), which binds to av integrins, and an R / KXXR / K (SEQ ID NO:29) motif consisting of arginines (arg, R) and lysines (lys. K) (X: any amino acid), which binds to neurolipins (NRP). iRGD selectively penetrates solid tumors and inhibits signaling pathways of TGF-P cytokines. This mechanism mitigates metastasis, tumor fibrosis, angiogenesis and hypoxia, and enables more immune activity within the tumor. This peptide also can significantly improve penetration of drugs into the tumor when administered concurrently with them or by directly attaching them to iRGD. A vector which enables translation of the peptide can be transduced into many cancer types, including kidney cancer cells, pancreatic ductal adenocarcinoma, gastric cancer cells, prostate cancer cells, and immune cells, for example. Any cell of the body can be used according to the treatment to be given. ThisAttorney Docket No. 15003-502PC0 technology therefore can be used to greatly improve the efficacy of existing therapeutics to suppress tumor growth and metastasis.

[0078] iRGD peptides can improve drug absorption through tumor- specific interactions with integrin and neuropilin receptors. However, current treatments with iRGD peptides require constant administration to remain efficacious. Many anti-cancer drugs cannot penetrate deep enough in solid tumors to reach blood vessels, reducing their effectivity. av-Integrins, notably avP3 and avP5, are abundantly expressed in tumor vasculature and malignant cells. RGD mediates tumor infiltration through binding to av integrins found on cell surfaces. In alternative embodiments, traditional RGD peptides (i.e. those that do not bind to neuropilin) may be implemented in place of iRGD. It is believed that traditional RGD peptides may have similar, but weaker properties as iRGD based on their binding to integrins.

[0079] The methods described here also can be used to produce a vaccine that can induce in vivo production of soluble iRGD which is useful for cancer treatment. One embodiment relates to a viral delivery vector that is engineered to express iRGD upon administration. This technology can serve as a preventative medicine that improves anti-tumor immunity and inhibits metastasis over time. In addition, the invention may have potential to increase the efficacy and longevity of many current therapeutic treatments and to improve visualization of tumors when combined with imaging modalities.

[0080] 2. Definitions

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.Attorney Docket No. 15003-502PC0

[0082] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element, or step modified by the article.

[0083] As used herein, the term “about” means plus or minus 20 percent of the recited value so that, for example, “about 0.125” means 0.125 ± 0.025, and “about 1.0” means 1.0 ± 0.2. Notwithstanding that the numerical ranges and parameters setting for the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing.

[0084] As used herein, the term “iRGD” refers to internalizing RGD, peptides that carry an RGD integrin recognition motif (arginine, glycine, aspartic acid) and an R / KXXR / K (SEQ ID NO:29) motif (consisting of arginines (arg, R) and lysines (lys, K) (X: any amino acid), which binds to neuropilins (NRP). The term “s-iRGD” refers to secretory internalizing RGD.

[0085] As used herein, the term “subject” is used interchangeably with “patient,” “individual,” and “host” to refer to any animal, and can include simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for the invention preferably is a human that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be ameliorated, treated or prevented by administration of one or more iRGD peptides. In particular, the conditions to be treated in a subject can include a solid tumor, hematologic malignancies, and the like. The preferred condition for treatment is a solid tumor. Conditions amenable to treatment by the invention can define an appropriate subject in need or patient, and can be discerned easily by the person of skill in the art based on the disclosures herein. AAttorney Docket No. 15003-502PC0“subject in need” refers to a subject that has or is suspected of having a cancerous condition, or a solid tumor.

[0086] As used herein, the term “administering” and all its cognates refers to contacting a compound, agent, or pharmaceutical composition with a tissue of the body of a subject. This can be accomplished by any means known in the art, and can be performed using any of the various methods or delivery systems for administering agents, pharmaceutical compositions or delivering gene vectors known to those skilled in the art. Modes of administering include, but are not limited to oral administration or intravenous, subcutaneous, intramuscular, intraocular, intraosseous, intracerebral, intraspinal, intravesicular, or intraperitoneal injections, rectal or intestinal administration by way of suppositories, enema or endoscopy, administration into the airway by way of inhalation or instillation, local administration directly into or onto a target tissue to which it is targeted. Administration can refer to introducing the nucleic acid construct to the subject as DNA or mRNA, introducing a vector containing the nucleic acid to the subject, or introducing cells that have been transduced ex vivo with a construct, such as by electroporation or using a vector as described herein, to the subject. As used herein, the term “long-term” refers to administration of 1-3 or more days, preferably about 7 or more days.

[0087] As used herein, the term “treatment” and its cognates refers to action taken to obtain a desired pharmacologic and / or physiologic effect. "Treatment," includes: (a) preventing (i.e., reducing the chance of) the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression or remission of the condition or disease or symptom thereof.

[0088] As used herein, the term “solid tumor” refers to any abnormal mass of tissue that grows uncontrollably from cells and may or may not contain liquid areas or cysts. A solid tumor can be benign or malignant, and can occur in any part of the body, such as breast, lung, heart, colon, small intestine, stomach, esophagus, appendix, prostate, kidney, ureter, urethra, bladder, skin, bone, cartilage, fat, muscle, blood vessels, pancreas, liver, bile duct, gallbladder, thyroid, salivary glands, nervous system, lymph nodes, reproductive systems, and the like. This class of tumors includes carcinomas, sarcomas, lymphomas, and the like.Attorney Docket No. 15003-502PC0

[0089] As used herein, the term “variant” with respect to an iRGD peptide refers to an iRGD peptide with at least about 90% identity to the reference peptide. In the case of a nonomer, this includes one amino acid deletion, addition, or substitution, generally.

[0090] As used herein, the term “CAR-T cells” refers to T cells that have been genetically engineered to express a CAR protein (specific antigen receptor) that let them recognize and destroy cancer cells. A CAR protein typically includes an antigen-binding domain, a transmembrane region, a co-stimulatory domain and a CD3(^ signaling domain. When a CAR binds its target antigen, the intracellular donmains trigger T cell activation, cytokine release, and proliferation.

[0091] 3. Embodiments of the Invention

[0092] A. Introduction

[0093] Arginine glycine aspartic acid (RGD) is an oligopeptide that binds to several integrins, on several types of cells, including cancer cells, cancer stromal cells such as pericytes and cancer fibroblasts, some immune cells, and particularly on endothelial cells(ECs). RGD was originally identified as the minimal recognition sequence in fibronectin, and functions as an adhesion sequence. It also has been found to play a role as an inhibitor of tumor growth, metastasis, and angiogenesis due to its agonist function in the cells in which it exists, such as tumor endothelial cells. iRGD is a modified version of a cyclic RGD peptide with particular affinity to neuropilins, receptors that are highly expressed on cells such as tumor endothelial cells, cancer cells, stromal cells, and some immune cells, and which mediates cell and tissue penetration, mediated by macropinosome-like vesicles. iRGD penetrates tumors and allows other molecules to penetrate tumors well due to the RGD motif on top of the NRP-binding R / KXXR / K (SEQ ID NO:29) motif. Peptides with only the R / KXXR / K (SEQ ID NO:29) motif penetrate any cell or tissue that expresses NRP, and peptides with only the RGD sequence also can be used.

[0094] iRGD itself has an immunomodulatory effect that is selective to cancer, and the addition of anti-cancer agents further enhances the effect. The result is sensitization of the cancer to immunotherapy, such as immune checkpoint inhibitors. The technology of this invention depletes Tregs selectively in the cancer tissue, enhancing the efficacy of immunotherapy against only the cancer. This avoids non-specific depletion of Tregs, which can lead to a series of inflammatory side effects. The technology also includes co-administration of iRGD to Standard-Attorney Docket No. 15003-502PC0 of-care chemotherapy to potentiate immune checkpoint inhibitors. Therefore, it does not require the generation of new drug entities for each application, enabling immediate application of the technology to various anti-cancer therapies in combination with immunotherapy. Application of the technology can sensitize a wide variety of cancers other than PDAC, and can be administered as intraperitoneal chemotherapy in addition to systemic chemotherapy. In some aspects, this technology allows spontaneous production of iRGD, secreted in vivo, which eliminates the need of repeated injections of exogenous iRGD to maintain the therapy.

[0095] The compounds, compositions and methods discussed herein provide tools and methods which can modify the tumor microenvironment with local production of secretory “internalizing” RGD, referred to herein as “iRGD” or “iRGD peptide.” As a monotherapy, iRGD can reduce angiogenesis, fibrosis, tumor volume in some cases, induce vascular normalization, reduce Tregs in the tumor, expand CD8+ T cells, and inhibit metastasis. When co-administered with drugs, iRGD can enhance penetration of these agents into the tumor and thus significantly enhance their efficacy. The products of the present invention can be transduced into cancer cells, normal cells, or immune cells to allow in vivo manufacture of a steady supply of iRGD at the tumor site, without the need for multiple injections, as a short- or long-term cancer therapy. Any cell in the body can be programmed to produce iRGD using this technology. For example, in a preferred embodiment, CAR-T cells can be programmed to secrete iRGD to improve their tumor penetration and anti-tumor activity.

[0096] The iRGD peptide efficiently penetrates solid tumors in a turn or- specific manner. The penetration is mediated by macropinosome-like vesicles that engulf both the peptide and agents that are attached to the peptide. The vesicles also engulf bystander agents allowing free drags and imaging agents that are simply co-injected with the peptide to also penetrate cells and tissues in the tumor. Thus, this iRGD technology is not generally toxic and provides a simple and versatile platform to achieve tumor-specific drug delivery, such as, for example, gemcitabine and nab-paclitaxel, used in cancers such as pancreatic ductal adenocarcinoma.

[0097] Thus, the iRGD method can achieve drug delivery through a simple co-injection without the need to chemically conjugate it to the cargo. The extravasated cargo spreads deeper into the tumor mediated by various cells that express iRGD receptors, such as cancer-associated fibroblasts, cancer epithelial cells, and some of the immune cells like regulatory T cells (Tregs). Logical next steps for improving iRGD co-administration methods would be to optimize theAttorney Docket No. 15003-502PC0 dosing schedule to achieve steady plasma concentration of iRGD, prolong the half-life of iRGD by attaching elements such as a polyethylene glycol tail or an extra-cysteine that allows iRGD to piggy-back on albumin, or use a material or device that sustainably delivers iRGD. None of these approaches have been able to provide more than an incremental advance in treatment, however.

[0098] Alternatively, the invention here provides a technology to program cells to secrete iRGD in vitro, ex vivo, in vivo, or to form a source of iRGD in, around, or away from the tumor over a period of time to assist in tumor treatment. In this manner, the invention can provide an iRGD factory anywhere inside the body.

[0099] Because iRGD inhibits signaling pathways of TGF-P cytokines, the peptide can exert multitude of effects on the TME that counter tumor progression and increase the benefits of anticancer therapies. For example, TGF-P induces a chaotic network of abnormal blood vessels through angiogenesis, which can lead to poor tissue perfusion, hypoxia, and high interstitial fluid pressure. TGF-P also activates carcinoma-associated fibroblasts (CAFs), which produce various cytokines, growth factors, and extracellular matrix components, and contribute to the formation of a desmoplastic TME. A TME rich in TGF-P often has an immunosuppressive nature because TGF-P suppresses various immune cells such as CD4+ and CD8+ T cells that contribute to antitumor immunity, and facilitates the development and functions of immunosuppressive Tregs. These features make PDAC and other TGF-P-rich tumors resistant to therapies because poor perfusion and desmoplasia prevent drugs from entering the tumor, and the immunosuppressive TME is not sensitive to immune checkpoint blockades (ICBs).

[0100] These effects are exaggerated in desmoplastic tumors, such as PDAC, because these cancers generally express avP5 integrin and NRP-1 at high levels. These receptors are activators of TGF-P, which plays a fundamental role in mediating the immunosuppressive nature of the TME. iRGD blocks the TGF-P activation mechanism in PDAC mice causing vascular normalization, stromal fiber remodeling, Treg depletion, CD8 T cell expansion and activation, and significant metastasis suppression over time. These TME changes, in addition to the acute penetration effects, may greatly benefit CAR-T cells (or other programmed cells) by providing them a conduit to enter the tumor and preventing dysfunction.

[0101] To provide sustained iRGD effects over the course of cancer therapy, engineered CAR-T cells were made to self-produce soluble iRGD (s-iRGD). This approach provides sufficient amounts of s-iRGD locally in the tumor as the CAR-T cells expand in response to tumorAttorney Docket No. 15003-502PC0 antigens. This method also reduces the burden on patients by eliminating the need to repetitively inject synthetic iRGD, which has a half-life of only minutes. Human CAR-T cells can be engineered to produce s-iRGD in vitro and in vivo. These s-iRGD-producing CAR-T cells effectively penetrate PDAC in mice where they produce and secrete the iRGD tumor-penetrating peptide, thereby acutely enhancing vascular / tissue permeability in the tumor and chronically impair the tumor microenvironment (TME) to enable T cells and other therapeutics better access into tumors.

[0102] The effects of the treatment modalities discussed herein include, but are not limited to, tumor penetration, modification of the TME, reduction of metastasis of the tumor cells, production of cancer vaccines, and an increase in the tumor’s susceptibility to other anti-cancer agents. Without wishing to be bound by theory, these effects are believed to be due to the ability of iRGD to inhibit the activation of TGF-p. TGF-P is secreted in an inactive form known as the large latent complex (LLC) consisting of the latency-associated protein (LAP), TGF-p, and latent TGF-P-binding protein. Active TGF-P is released from the LLC by cytoskeletal force, which is induced when LLC binds to proteins such as av integrins and neuropilin- 1 (NRP-1). The data show that iRGD effectively blocks this activation process as it is a ligand of these proteins.

[0103] B. Compounds

[0104] The iRGD tumor-penetrating peptide (CRGDK / RGPD / EC; SEQ ID NO: 12) carries a tumor-specific RGD motif and an NRP-l-binding RXXR / K (SEQ ID NO:28) motif (see schematic in FIG. 1A). After intravenous (IV) injection, iRGD targets av integrins on tumor blood vessel ECs. It is then cleaved to expose a C-terminal RXXR / K (SEQ ID NO:28), which binds to NRP- 1. This binding triggers an active transport pathway mediated by vesicles that engulf the peptide and bystander molecules. FIG. 1 A is a schematic showing that iRGD targets and extravasates into tumors in 3-steps mediated by av integrins, proteases, and NRP-1. iRGD triggers the formation of vesicles that deliver cargos attached to, or co-injected with iRGD, widely into the tumor tissue. The inset shows gold nanoparticles in an NRP-1 -induced vesicle imaged by transmission electron micrography.

[0105] The PDAC TME consists of a fibrotic stroma and a chaotic network of non-perfusing blood vessels. The result is a stiff and hypoxic tumor with high interstitial fluid pressure (IFP),Attorney Docket No. 15003-502PC0 which is difficult for systemic drugs to reach and infiltrate. The iRGD peptide (CRGDK / RGPD / EC; SEQ ID NO: 12) delivers drugs deep into tumors beyond these barriers.

[0106] The iRGD peptide acutely induces tumor- specific vascular and tissue permeability, allowing drugs chemically attached to the peptide, and even those freely co-injected with the peptide, to enter tumors. See FIG. 2, which shows confocal images of of BT474 orthotopic breast tumors from mice injected with IV Nab-paclitaxel (Nab-P) (green) either conjugated or co-injected with iRGD. Inset, Nab-P alone. Red, CD31; blue, DAPI. Scale bars, 100 pm.. iRGD in combination with chemotherapy for patients with pancreatic ductal adenocarcinoma (PDAC) also showed a remarkable preliminary efficacy in a phase lb trial. The anti-tumor activity of iRGD + Gemcitabine + Nab-P in patients with stage 4 PDAC is shown in a waterfall plot of the best objective response compared to baseline by RECIST 1.1 criteria is shown in FIG. 3.

[0107] iRGD-secreting CAR-T cells can be used as a generalized platform for targeting a wide variety of solid tumors. This platform can help to resolve a long-standing issue in CAR-T therapy, which currently is ineffective against solid tumors.

[0108] Various approaches have been taken to improve the efficacy of CAR-T for solid tumors, such as expression of multiple CARs on T cells, adjunct therapy with cytokine inhibitors and ICIs, and direct injection of CAR-T cells into or in the vicinity of tumors. However, none of them have led to a breakthrough that makes CAR-T truly effective against solid tumors. The methods described here use iRGD to develop a breakthrough platform that makes CAR-T widely applicable to solid tumor therapy. In particular, the development of iRGD-secreting CAR-T cells that have specificity to different tumor antigens (including, but not limited to, MR1, Claudinl8.2, and B7-H3) can be used for their ability to penetrate and treat solid tumors such as pancreatic cancer. The same technology can be used to engineer other immune cells such as CAR-NK cells and tumor infiltrating lymphocytes.

[0109] In certain embodiments, the invention provides an iRGD peptide therapeutic for treatment and reduction of the size of solid tumors. The iRGD peptides of this invention efficiently penetrate solid tumors in a tumor- specific manner. Examples of peptides contemplated for use in this invention include those listed herein or any length of peptide or protein that contains an RGD sequence and / or an R / KXXR / K (SEQ ID NO:29) sequence or a sequence resembling the sequences based on the amino acid nature such as RXXH. Traditional RGD peptides also can be used, but may have weaker effects without the addition of SEQ IDAttorney Docket No. 15003-502PC0NO:29. Peptides containing only SEQ TD NO:29 also can be used, for example the Lyp-1 peptide.

[0110] In certain embodiments, the invention provides a plasmid vector, or any suitable vector, for in vivo or ex vivo translation of iRGD to produce a secreted peptide in the tumor to be treated, in the TME, or elsewhere in the body with penetration into the tumor. A preferred protein is SEQ ID NO: 14 (see Table 1, below), where X is an iRGD peptide, preferably one selected from the group consisting of SEQ ID NOs:8, 12, or 15-26. Any cell, for example skeletal muscle cells or liver cells and the like can be used as the site of iRGD production.

[0111] The peptide and the vector that allows production of the peptide reduce metastasis of a solid tumor and increase the susceptibility of the tumor to therapeutics with targeted delivery to solid tumors. These products also can reduce the immunosuppressive environment in and around solid tumors by limiting fibrosis, angiogenesis, hypoxia and the presence of regulatory T cells. The peptide acts mechanistically by inhibiting the activation of TGF- in cells by NRP-l / av integrins and large latent complex (LLC).

[0112] The compounds discussed here can be caused to be expressed and secreted by various cancer cells (e.g„ kidney cancer cells, pancreatic ductal adenocarcinoma, gastric cancer cells, and prostate cancer) as shown in in vitro studies and also can be transfected into and expressed by chimeric antigen receptor (CAR) T cells or other immune cells as part of a dual treatment of the solid tumor. CAR-T cells that target major histocompatibility complex class I- related molecule 1 (MR1) or B7H3 can be engineered to produce functional soluble iRGD (s-iRGD) in vitro and in vivo, and that effectively penetrate into PDAC in mice. For example, CAR-T cells that secrete iRGD achieve sustained in vivo iRGD delivery to acutely and chronically modify the TME to facilitate their own tumor entry and activation, and can provide added benefits such as metastasis inhibition. This approach can reliably provide increasing amounts of iRGD as the CAR-T cells expand in response to tumor antigens, and eliminate the need to repetitively inject iRGD for the duration of therapy.

[0113] iRGD targets v integrins and neuropilin to induce the tumor- specific entry of drugs that are attached to or even simply co-injected with iRGD by enhancing vascular permeability in the tumor. iRGD enriches at tumor blood vessels by targeting av integrins where it is cleaved into a neuropilin (NRP)-binding fragment. It then triggers NRP-dependent formation of intracellular vesicles that engulf and carry more iRGD along with drugs either attached to or freely existingAttorney Docket No. 15003-502PC0 by iRGD into tumor cell layers that further propel the penetration cascade. iRGD widely penetrates desmoplastic tumors because various cells in the tumor such as endothelial cells (ECs), cancer-associated fibroblasts (CAFs), cancer epithelial cells, and some of the immune cells (like Tregs) express avP5 integrin and NRP-1, the iRGD receptors. The penetration is propelled deeper into the tumor tissue by the cells that express the iRGD receptors.

[0114] Desmoplastic tumors, such as pancreatic ductal adenocarcinoma (PDAC), are excellent targets for iRGD since these tumors often express high levels of iRGD receptors, av integrins and neuropilin- 1. These receptors are known as activators of TGF-p, which facilitates the formation of a desmoplastic, hypoperfused, and immunosuppressive TME. iRGD competes with the TGF-P activation mechanism to widely reduce TGF-P signaling in PDAC causing dynamic TME changes over time, such as vascular normalization, less desmoplasia, Treg depletion, CD8 infiltration and activation, and strikingly reduced metastasis.

[0115] Long-term iRGD therapy, even as monotherapy, normalizes blood vessels, reduces hypoxia, and increases tumor infiltration of CD8+T cells in the PDAC tissue. iRGD monotherapy also leads to a striking inhibition of metastasis in PDAC mice. Mechanistic studies revealed that iRGD competitively blocks the activation of transforming growth factor (TGF)-P mediated by the avP5 integrin in the PDAC tissue, suggesting it as one of the causes of the dynamic TME modification. In addition, iRGD preferentially depletes regulatory T cells (Tregs) in the PDAC tissue because PDAC-infiltrating Tregs express the otvP5 integrin (and NRP-1 as a Treg marker) allowing iRGD to effectively target them. This mechanism further facilitates the expansion of CD8+T cells in the tumor tissue.

[0116] An ongoing analysis in humanized PDAC (huPDAC) mice using spatial transcriptomics shows that activated cytotoxic CD8+T cells are increased in the tumor upon iRGD therapy. As a result. iRGD sensitizes PDAC to an anti-programmed cell death ligand 1 (PD-L1) antibody (Ab) in PDAC mice. Strategically inducing the TME changes with iRGD can improve CAR-T cell therapy because normalized vasculature serves as a conduit for T cells to enter the tumor, and reversal of the immunosuppressive TME can help maintain T cell activity and hinder exhaustion.

[0117] In summary, in vivo production of iRGD peptides offers an innovative way to induce TME modifications for improved drug penetration, reduced immunosuppression, and suppressed metastasis. Examples of iRGD peptides are listed in Table 1. Other iRGD peptides include those that contain an RGD sequence as well as a R / KXXR / K (SEQ ID NO:29) sequence within theAttorney Docket No. 15003-502PC0 same peptide regardless of the length of the peptide chain. Other peptides that contain only an RGD or an R / KXXR / K (SEQ ID NO:29) sequence can also provide similar effects based on our mechanistic studies.

[0118] C. Cells

[0119] In certain embodiments, the invention provides cells that have been transduced and can express an iRGD peptide as described herein. Any cell suitable for use for targeting and treatment of a solid tumor can be used. For example, any type of cancer cell, normal cell, or immune cell is contemplated for use with this invention. Preferably, CAR-T cells (chimeric antigen receptor T cells), and the like, are used to secrete the iRGD peptide(s), but any biocompatible cell can be used.

[0120] To obtain CAR-T cells for use in the invention, T cells generally are taken from the blood of a subject requiring treatment. The cells then are genetically modified to express a chimeric antigen receptor (CAR), a protein that binds to a specific antigen on the surface of the cancer cells to be treated. This allows the T cells to target a particular cell type, such as a particular tumor. Thus, the CAR should be chosen based on the markers that the specific tumor to be treated. For example, markers on pancreatic cancer cells targetable with CAR-T cells include, but are but limited to, MR1, Claudin 18.2, and B7H3. However, any suitable marker can be selected by the practitioner based on testing of the specific tumor in the patient. Similarly, any cell surface marker that allows CAR-T cells to target a particular type of cancer can be used. These cells are additionally modified so that they secrete an iRGD peptide according to the invention here and infused or injected back into the patient. They target to the tumor(s) being treated so that the iRGD peptide is released in the area of the tumor. The constructs generally are designed to express a CAR or CARs on T cells and to express secretory iRGD can be combined into a single construct or a vector. Thus, the s-iRGD expression construct can be embedded into a CAR-expression vector, or another vector.

[0121] iRGD has been used to deliver T cells into tumors mainly by chemically coating it onto T cells using lipid tails. Chemically coating CAR-T cells with synthetic iRGD has been used in the past to assist the cells to enter the tumor environment and the tumor itself, but is not optimal for the purpose of cancer treatment because under those conditions the number of iRGD molecules on the cell surface continuously decreases as the cells undergo cytokinesis, diminishing theAttorney Docket No. 15003-502PC0 iRGD effects. Giving a bolus of iRGD with but separate from the cells to be targeted (the coinjection approach) has been tested, but it did not effectively facilitate tumor entry, suggesting that inducing acute penetration alone is not sufficient. Sustained TGF-P antagonism by iRGD, which modifies the vasculature to provide T cells a conduit for tumor entry and a less immunosuppressive TME, is likely an additional requirement.

[0122] The s-iRGD CAR-T cells discussed here as part of this invention provide an innovative way to induce acute penetration and maintain TGF-P antagonism during CAR-T therapy. This therapy can have certain advantages: (1) it allows continuous delivery of iRGD throughout the duration of the CAR-T therapy, which can last years in some cases, (2) it eliminates the need of repetitive iRGD injections over a long duration, (3) it allows direct iRGD therapy toward the tumor tissue, and (4) it can serve as a switch that accelerates s-iRGD production in response to tumor cells and then decelerates upon remission. It can also serve as a biosensor that reports the presence of cancer cells as increasing amounts of s-iRGD detected in the biological samples such as the blood would indicate CAR-T expansion in response to an existing tumor. TME changes induced by s-iRGD CAR-T cells may also facilitate entry of adjunct therapies into the tumor and prevent metastasis, providing additional benefits to the patients. Since repeated injections of synthetic iRGD in PDAC mice or PDAC human patients have not caused injury to normal tissue or enhanced the side effects of co-injected cytotoxic agents or ICBIs, extended in vivo treatment with s-iRGD should be safe. Synthetic iRGD has shown no dose limiting toxicities.

[0123] CAR-T cells according to this invention were designed to produce secretory iRGD (s- iRGD) to achieve sustained plasma levels of iRGD reliably while CAR-T therapy is in effect. This feature is important for the maintenance of TGF-P antagonism to induce continuing beneficial TME changes and potentially suppress metastasis. By using s-iRGD CAR-T cells, the treatment can be more effective because CAR-T cells can be engineered to secrete avP5 integrin- binding s-iRGD in vitro and in vivo, and the resulting CAR-T cells effectively penetrate PDAC tumors. Therefore, developing safe and effective s-iRGD CAR-T cells (or other programmed cells) can lead to a novel CAR-T platform that can be applied to the treatment of a wide range of solid tumors.

[0124] In summary, the types of cells that can be programmed to secrete an iRGD for use in the invention can be any cell of the body. Examples include but are not limited to stem cells, bone cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, epithelial cells, sex cells, andAttorney Docket No. 15003-502PC0 cancer cells. Immune cells such as CAR-T cells, TILs (tumor-infiltrating lymphocytes), macrophages, and natural killer (NK) cells are most preferred.

[0125] D. Vector Constructs, Intracellular Delivery and Transduced Cells as Therapeutic Products

[0126] In a preferred embodiment, the iRGD peptide compounds are administered to a patient in the form of a vector for transduction of cell therapy products, however the constructs can be delivered to the patients and transduce cells in situ for treatment purposes. Any suitable vector can be used according to the preference of the practitioner. Other methods also can be used, such as lipid nanoparticles and the like, or any suitable delivery system. The technology of the invention programs cells to produce the iRGD tumor-penetrating peptide (e.g., including the amino acid sequence: CRGDK / RGPD / EC (SEQ ID NO: 12) or any one of the sequences SEQ ID NO:8 or 15-26, for example). Other iRGD peptides include those that contain an RGD sequence as well as a R / KXXR / K (SEQ ID NO:29) sequence within the same peptide regardless of the length of the peptide chain. Other peptides that contain only an RGD or an R / KXXR / K (SEQ ID NO:29) sequence can also provide similar effects based on mechanistic studies. These peptides, vectors and cells produce a steady supply of iRGD in vivo to modify the TME to enhance the efficacy of various cancer therapeutics such as chemotherapy, biological drugs, targeted therapy, and immunotherapy, and to inhibit metastatic spread of cancer cells as discussed herein. Thus, the invention relates to a method of introducing a vector to cells in vivo, in vitro, or ex vivo to deliver iRGD peptide by expression to a subject.

[0127] Any suitable vector can be used to transduce cells to express one or more iRGD peptides. See FIG. 15 for an example. Any kind of vector that can introduce a gene may be used.

[0128] The expressed peptide preferably is G6-HA-G6-Myc-G6-iRGD peptide (GGGGGGYPYDVPDYAGGGGGGEQKLISEEDLGGGGGGX; SEQ ID NO: 14) where X is an iRGD peptide such as SEQ ID NO:8. SEQ ID NO: 12 or SEQ ID NO: 15-26. A preferred sequence is GGGGGGYPYDVPDYAGGGGGGEQKLISEEDLGGGGGGCRGDKGPDC; SEQ ID NOT E The most preferred sequence is SQ ID NO: 12.

[0129] The vectors are constructed using methods known in the art by a person of ordinary skill. Briefly, the vectors can be constructed as follows: A mammalian expression vector such as an ampicillin-resistant pRP plasmid vector is used. The vector contains a DNA sequence encodingAttorney Docket No. 15003-502PC0 an ER signal peptide of choice followed by a DNA sequence encoding an iRGD peptide sequence driven under an EFl A promotor or any other suitable promoter. The iRGD peptide sequence can incorporate DNA sequences that provide a tag that allows the detection of the peptide in vivo or toxicity that affects tumor cells. The vector can simultaneously carry DNA information that encodes other peptides or proteins, such as cell death domains, any type of CAR when the vector is intended for the production of CAR-bearing immune cells, other tumorpenetrating peptides such as LyP-1 that targets receptors that are not recognized by iRGD, regulators of gene expression such as a Tet-on Tet-off system, and the like.

[0130] Cells, such as CAR-T cells, may be transduced ex vivo, in situ or in vivo, according to other known methods in the art as well. Suitable cells include bacterial or mammalian cells, however human cells are preferred for treatment of humans. Briefly, transduction is achieved as follows: transfection using electroporation, virus vectors, transfection reagents, lipid constructs including but not limited to lipid nanoparticles (LNPs), cell-penetrating peptides used for transfection, and any other agent that can be used to deliver genetic materials into cells. Other agents include the following:(i) Exosomes (such as those described in Lakhal et al., “Intranasal Exosomes for Treatment of Neuroinflammation? Prospects and Limitations,” Mol. Ther. 19: 1754-1756 (2011); Zhang et al., “Newly Developed Strategies for Multifunctional Mitochondria-Targeted Agents In Cancer Therapy,” Drug Discovery Today 16: 140-146 (2011), each of which is hereby incorporated by reference in its entirety).(ii) Lipid-based delivery systems (such as those described in Bildstein et al. / ‘Transmembrane Diffusion of Gemcitabine by a Nanoparticulate Squalenoyl Prodrug: An Original Drug Delivery Pathway,” I. Controlled Release 147: 163- 170 (2010); Foged, “siRNA Delivery with Lipid-Based Systems: Promises and Pitfalls,” Curr. Top. Med. Chem. 12:97-107 (2012); Holpuch et al.. “Nanoparticles for Local Drug Delivery to the Oral Mucosa: Proof of Principle Studies,” Pharm. Res. 27: 1224-1236 (2010); Kapoor et al., “Physicochemical Characterization Techniques for Lipid Based Delivery Systems for siRNA,” Int. J. of Pharm. 427, 35-57 (2012), each of which is hereby incorporated by reference in its entirety), including microtubules, such as those described in (Kolachala et al., “The Use of Lipid Microtubes as a Novel Slow-Release Delivery System forAttorney Docket No. 15003-502PC0Laryngeal Injection,” The Laryngoscope 121 : 1237- 1243 (201 1), which is hereby incorporated by reference in its entirety).(iii) Liposome or liposome-based delivery systems.(iv) Micelles, including disulfide cross-linked micelles, such as those described in (Li et al., “Delivery of Intracellular- Acting Biologies in Pro-Apoptotic Therapies,” Curr. Pharm. Des. 17:293-319 (2011). which is hereby incorporated by reference in its entirety). Carriers with disulfide bonds can be formulated so that one or more disulfide bonds link to the to the antiviral knockout agent, such as oligonucleotide based inhibitors. A variety of micelles have been described, such as phospholipid-polyaspartamide micelles for pulmonary delivery.(v) Microparticles, such as those described in (Ateh et al., “The Intracellular Uptake of CD95 Modified Paclitaxel-Loaded Poly(Lactic-Co-Glycolic Acid) Microparticles,” Biomater. 32:8538-8547 (2011), which is hereby incorporated by reference in its entirety).(vi) Molecular carriers, such as those described in (Hettiarachchi et al., “Toxicology and Drug Delivery by Cucurbit[n]uril Type Molecular Containers,” PloS One 5:el0514 (2010), which is hereby incorporated by reference in its entirety).(vii) Nanoparticles referred to as ‘nanocarriers’, such as those described in (Gu et al., “Tailoring Nanocarriers for Intracellular Protein Delivery,” Chem. Soc. Rev. 40:3638-3655 (2011), which is hereby incorporated by reference in its entirety), some of which have been formulated for delivery of agents to HIV infected cells, such as those described in (Gunaseelan et al., “Surface Modifications of Nanocarriers for Effective Intracellular Delivery of Anti-HIV Drugs,” Adv. Drug Delivery Rev. 62:518-531 (2010), which is hereby incorporated by reference in its entirety).(viii) Nanoscopic multi- variant carriers.(ix) Nanogels (such as those described in Zhan et al., “Acid-Activatable Prodrug Nanogels for Efficient Intracellular Doxorubicin Release,” Biomacromolecules 12:3612-3620 (2011) and Zhang et al., “Folate-Mediated poly(3-hydroxybutyrate- co-3-hydroxyoctanoate) Nanoparticles for Targeting Drug Delivery,” Eur. J.Attorney Docket No. 15003-502PC0Pharm. Biopharm. 76: 10-16 (2010), each of which is hereby incorporated by reference in its entirety).(x) Hybrid nanocarrier systems, which consist of components of two or more particulate delivery systems (such as those described in Pittella et al., “Enhanced Endosomal Escape of siRNA-incorporating Hybrid Nanoparticles from Calcium Phosphate and PEG-Block Charge- Conversional Polymer for Efficient Gene Knockdown With Negligible Cytotoxicity,” Biomater. 32:3106-3114 (2011), which is hereby incorporated by reference in its entirety). Copolymeric micelle nanocarriers (such as those described in Chen et al„ “pH and Reduction DualSensitive Copolymeric Micelles for Intracellular Doxorubicin Delivery,” Biomacromolecules 12:3601- 3611 (2011), which is hereby incorporated by reference in its entirety); liposomal nanocarriers, (such as those described in (Kang et al., “Design of a Pep-1 Peptide-Modified Liposomal Nanocarrier System for Intracellular Drug Delivery: Conformational Characterization and Cellular Uptake Evaluation,” I. of Drug Targeting 19:497-505 (2011), which is hereby incorporated by reference in its entirety).(xi) Peptide-based drug delivery systems, which include a variety of cell penetrating peptides and including (but not limited to) TAT-based delivery systems (such as those described in Johnson et al.. “Therapeutic Applications of Cell-Penetrating Peptides,” Methods Mol. Biol. 683 :535-551 (2011), which is hereby incorporated by reference in its entirety). Such peptides can be chemically linked to an antiviral knockout agent.(xii) Polymers or copolymer-based delivery systems, such as those described in (Edinger et al., “Bioresponsive Polymers for the Delivery of Therapeutic Nucleic Acids,” Wiley Interdiscip. Rev. Nanomed. and Nanobiotechnol. 3 :33-46 (2011), which is hereby incorporated by reference in its entirety).The term vector is intended to include reference to plasmids, viral vectors, bacterial vectors as well as other agents such as those enumerated above that are suitable for intracellular delivery and which comprise an expressible nucleic acid sequence that encodes an iRGD or other sequence described herein.Attorney Docket No. 15003-502PC0

[0131] Results presented here show that long-term treatment with iRGD antagonizes the TME features in PDAC mice, causing less fibrosis, less angiogenesis, more open and functional blood vessels, and less hypoxia. In addition, the data showed an increased ratio of cytotoxic CD8+ T cells over Tregs in the tumors. As a result, iRGD enhances the efficacy of immunotherapy. Under conditions in which neither iRGD nor an ICB (anti-PD-Ll mAb) alone inhibited tumor growth, iRGD in combination with ICB led to a significant decrease in tumor volume. Similar results were noted when iRGD was combined with the anti-PD-Ll mAb and Gem.

[0132] Any cell of the body, especially including immune cells can be used with this invention. Most preferred is CAR-T cells, but other immune cells including CAR-NK cells and TILs (tumor-infiltrating lymphocytes) also can be used. Other cells contemplated for use with this invention include, but are not limited to: stem cells, bone cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, epithelial cells, sex cells, cancer cells, and the like.

[0133] Any suitable promoter as determined by the practitioner can be used with the invention. However, preferred promoters include but are not limited to EFla, CMV, CAG, and other constitutive promoters, as well as drug-inducible systems (such as Tet-On / Tet-Off, steroid- inducible, lac operon). Signal peptides also optionally can be used, including but not limited to human cerebral dopamine neurotrophic factor, albumin, IL-2, tPA, or any functional secretion signal. Fusion tags also can be used, including but not limited to HA, Myc, FLAG, His, and fluorescent proteins (GFP, mCherry).

[0134] E. Pharmaceutical Compositions

[0135] In certain embodiments of the invention, the compounds including iRGD peptides as disclosed herein are formulated as a pharmaceutical composition. These compositions are made up of one or more peptides as discussed here, one or more vectors that can be expressed in different cell types, cells that have been modified to secrete one or more iRGD peptide, a vaccine composition, or other compounds and compositions, together with a pharmaceutical carrier that allows them to be administered to a subject as a treatment or prophylaxis. As is known in the art, most treatment modalities are formulated in order to produce a product that can effectively deliver the compounds to the body or the tissues in need of treatment.

[0136] Therefore, in preferred embodiments, the compounds described herein are formulated and are administered as a pharmaceutical composition that includes a pharmaceutically acceptableAttorney Docket No. 15003-502PC0 carrier and one or more pharmaceutical agent, including one or more of the inventive compounds described herein, and also including one or more of the inventive compounds described herein with an additional agent, such as a traditional anticancer agent in combination with a source of iRGD peptide.

[0137] A pharmaceutically acceptable carrier refers to any convenient compound or group of compounds that is not toxic and that does not destroy or significantly diminish the pharmacological activity of the therapeutic agent with which it is formulated. Such pharmaceutically acceptable carriers or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers known in the art. A suitable carrier depends on the route of administration contemplated for the pharmaceutical composition and the nature of the pharmaceutical or treatment modality. For example, solid pharmaceuticals such as peptides can be formulated advantageously as a tablet or for injection, whereas peptides and vectors that express peptides can be formulated advantageously in a liquid vehicle suitable for injection.Cells designed to secrete peptides can be administered in a liquid vehicle suitable for injection as well.

[0138] Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated. Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated. For example, routes of administration can include, but are not limited to: local or parenteral, including: oral, intravenous, intraarterial, intrathecal, subcutaneous, intradermal, intraperitoneal, rectal, vaginal, topical, nasal, local injection, buccal, transdermal, sublingual, inhalation, transmucosal, wound covering, direct injection into a tumor or the area surrounding a tumor, and the like. The administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art.

[0139] Therefore, the forms which the pharmaceutical composition can take will include, but are not limited to: tablets, capsules, caplets, lozenges, dragees, pills, granules, oral solutions, powders for dilution, powders for inhalation, vapors, gases, sterile solutions or other liquids for injection or infusion, transdermal patches, buccal patches, inserts and implants, rectal suppositories, vaginal suppositories, creams, lotions, oils, ointments, topical coverings (e.g.,Attorney Docket No. 15003-502PC0 wound coverings and bandages), suspensions, emulsions, lipid vesicles, cells (such as T cells or other immune cells) and the like.

[0140] Treatment regimens include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including administration for the remainder of the subject’s life. The regimen can include multiple (bolus) doses per day, one dose per day, week or month, for example, or a long infusion administration lasting for an hour, multiple hours, a full day, or longer.

[0141] Dosage amounts per administration include any amount determined by the practitioner, and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the condition to be treated or prevented, the form of the product being administered, and the like. In general, it is contemplated that for the majority of subjects, a dose in the range of about 0.01 mg / kg to about 100 mg / kg is suitable, preferably about 0.1 mg / kg to about 50 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg, and most preferably about 0.2 mg / kg to about 5 mg / kg are useful. This dose can be administered weekly, daily, or multiple times per day. A dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered. When cells are injected, a preferred dose is about 1-500 milliion cells per kilogram body weight, about 5-250 million cells per kilogram body weight, or about 10-200 million cells per kilogram body weight.Alternatively, the compounds can be administered in sufficient amount(s) to achieve a plasma level as determined by the clinician, based on the needs of the patient.

[0142] Pharmaceutical compositions can be formulated to contain the inventive peptide, vectors, cells, one or more imaging agents, etc. and also may contain additional active agents in addition to the pharmaceutical carrier(s). The additional active agent for co-administration in a single pharmaceutical composition can be any agent needed by the patient, but preferably is an anticancer agent. In preferred embodiments, the anticancer agent for co-administration is a small molecule cancer chemotherapy drug or an immune checkpoint blockade drug, however any active agent can be used in the invention. In other embodiments, the inventive composition and the additional agent are administered to a patient in separate compositions administered separately but either concomitantly or sequentially, or separated by time. In other embodiments, the pharmaceutical composition can contain one or more imaging agent, which would cause theAttorney Docket No. 15003-502PC0 agent to penetrate the tumor to enhance tumor-specific visualization by delivering the imaging agent into the tumor.

[0143] An iRGD vaccine or other treatment for cancer can be given periodically or seasonally to maintain effective plasma concentration of iRGD over the duration of cancer therapy, which can become necessary even for 5 years or longer in some patients.

[0144] F. Methods of Programming Cells

[0145] Collection of Immune CellsImmune cells are obtained from one or more sources, including but not limited to TILs, peripheral blood mononuclear cells (PBMCs), spleen, bone marrow, or umbilical cord blood.

[0146] Isolation of Specific Cell Populations (optional)Specific immune cell subsets, such as CD4+or CD8+T cells, regulatory T cells, NK cells, or NKT cells, may be isolated using magnetic beads, flow cytometry, or other suitable separation techniques.

[0147] Activation and Expansion (optional)The collected or isolated immune cells are stimulated using cytokines and / or antibodies — for example, IL-2, IL-7, IL-15, IL-21, anti-CD3, anti-CD28, or other immune-modulating agents — to promote activation, differentiation, and proliferation.

[0148] Gene IntroductionA nucleic acid encoding s-iRGD (DNA or RNA) and a CAR construct, either as a combined or separate sequence, is introduced into the immune cells using a gene delivery method such as lentiviral. retroviral, adenoviral, or non- viral transfection (e.g., electroporation, lipid-based transfection).

[0149] Cell Expansion and Functional ValidationThe modified immune cells are expanded ex vivo and assessed for transgene expression, viability, cytotoxic activity, or tumor-targeting functionality prior to in vitro or in vivo use.

[0150] G. Vaccines and Therapeutic Products

[0151] The vaccine product according to the invention delivers expression vectors of s-iRGD to cause cells to produce s-iRGD in vivo. s-iRGD vaccine will administered through various routes that include, but are not limited to: local or parenteral, including: oral, intravenous, intraarterial,Attorney Docket No. 15003-502PC0 intrathecal, subcutaneous, intradermal, intraperitoneal, rectal, vaginal, topical, nasal, local injection, buccal, transdermal, sublingual, inhalation, transmucosal, wound covering, direct injection into a tumor or the area surrounding a tumor, and the like. The vaccine will program various cells to produce s-iRGD in vivo to serve as a local factory of s-iRGD. The cells can be any cell, including but not limited to: stem cells, bone cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, epithelial cells, sex cells, cancer cells, and the like.

[0152] The iRGD vaccine can be produced using any biocompatible vector, including but not limited to AAV-9 and is able to provide sustained in vivo production of soluble iRGD through a single shot of iRGD vaccine, which leads to beneficial TME changes with no off-target toxicity. The iRGD vaccine serves as a medicine that improves anti-tumor immunity and inhibits metastasis over time.

[0153] H. Methods of Treating Cancer

[0154] The invention preferably is used to treat disease, cancer in particular, but also can be used as a research model. Solid tumors, for example, are treated by administering cells transduced with genetic material that allows the cells to secrete iRGD, administering secretory iRGD- encoding genetic materials directly into the body using agents that facilitate transduction, or administering secretory iRGD isolated from cells that produce the peptide. Methods of administration include oral administration, intravenous, subcutaneous, intramuscular, intraocular, intraosseous, intraocular, intraosseous, intracerebral, intraspinal, intravesicular, or intraperitoneal injections, rectal or intestinal administration by way of suppositories, enema, or endoscopy, local administration directly into or onto a target tissue (such as the pancreas), administration into the airway by way of inhalation or instillation, or administration by any route or method that delivers transduced cells that secrete the peptide, genetic materials into cells, or therapeutically effective amount of the peptide or composition to the cells or tissue to which they are targeted. The person of skill is able to determine the most efficacious route of administration, but preferably the CAR-T cells are administered intravenously, intraperitoneally, or directly into the pancreas or the tumor(s) itself.

[0155] The therapy can be combined with other treatment modalities, for example, small molecule drugs, biological agents such as antibodies, nanodrugs, immunotherapy such as immune checkpoint inhibitors, vaccines, and cytokines, targeted agents such as KRAS inhibitorsAttorney Docket No. 15003-502PC0 and tyrosine kinase inhibitors, radiation therapy, surgery, and any other modality that can facilitate tumor treatment. Alternatively, the invention comprises an imaging agent, alone or in combination with other modalities, for improved visualization of tumors.

[0156] The inventive compositions can be administered alone or co-administered with any beneficial drug or composition, such as chemotherapy or immune checkpoint blockade drugs, for example, to obtain long-term production of iRGD peptide at the site of a solid tumor or other tissue for treatment. In addition, the techniques of the invention also can achieve long-term production of pro-inflammatory or pro-proliferative (regarding to immune activity), etc. peptides at the tumor site for other therapeutic purposes.

[0157] By transduction of T cells, NK cells, or other immune cells, the invention can be used for treatment of solid tumors, hematologic malignancies, and other diseases that benefit from immune cell surveillance and targeting, by extending the reach of immune cells to target problematic cells in a patient, facilitating the development and maintenance of beneficial immune cells in the diseased tissue, reducing immunosuppressive cells, or using them as iRGD producers with a built-in switch of iRGD production that turns on / off in response to antigen recognition. A preferred embodiment includes modified CAR-T cells that secrete an iRGD peptide. This product preferably is injected into a suitable subject as a method of treating cancer.

[0158] iRGD vaccine is intended to produce the iRGD tumor-penetrating peptide in vivo, which modifies the tumor microenvironment (TME) to enhance tumor- specific delivery of various cancer therapeutics and protect patients against metastasis. Although iRGD is a tumorpenetrating peptide originally developed to acutely cause vascular and tissue permeabilization in solid tumors, we have discovered that iRGD also has chronic effects that lead to better perfusion of the tumor, improved drug entry, infiltration and activation of T cells, and reduced metastasis. The iRGD vaccine can strategically induce these beneficial effects by producing sustained concentrations of iRGD without the need of repetitive injections of the iRGD peptide itself.

[0159] The iRGD vaccine can be given periodically or seasonally to maintain effective plasma concentration of iRGD over the duration of cancer therapy, which can be lengthy and even for 5 years or longer in some patients. Clinical data from the phase lb trial for iRGD as an enhancer of standard-of-care therapy in patients with pancreatic ductal adenocarcinoma (PDAC) confirmed that iRGD was safe to administer to patients for at least 2 years.Attorney Docket No. 15003-502PC0

[0160] Furthermore, this technology has the potential to increase the efficacy and longevity of many current therapeutic treatments, as well as tumor imaging techniques. A prototype of iRGD vaccine using an adeno-associated virus (AAV)-9 has been generated and currently is being tested. This technology can serve as a preventative medicine that improves anti-tumor immunity, could inhibit metastasis, could improve tumor imaging, and can be combined with multiple existing tumor therapies to increase their longevity and effectiveness.

[0161] I. Diseases and Conditions

[0162] This invention is contemplated for use in treatment of solid tumors, however the following conditions also may be treated by the iRGD peptide technology described herein: hematologic malignancies, diseases related to fibrosis, and diseases related to vascular abnormalities. Solid tumors which can be treated using this invention include tumors of the pancreas, breast, lung, heart, colon, small intestine, stomach, esophagus, appendix, prostate, kidney, ureter, urethra, bladder, skin, bone, cartilage, fat. muscle, blood vessels, liver, bile duct and gallbladder (e.g., cholangiocarcinima), thyroid, salivary glands, nervous system (e.g., glioblastoma), lymph nodes (including lymphomas an other hematologic malignancies that form masses), reproductive systems, and the like. It is contemplated that any solid tumor can be treated according to this invention. The tumor to be treated can be any size ranging from a micrometastasis to a large established tumor. When immune cells are used to produce s-iRGD, the invention can be used to detect, prevent, and treat recurrence (e.g., local recurrence, metastatic recurrence) because there will be increased s-iRGD production when the immune cells encounter a recurrent lesion.

[0163] Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult cancers to treat given its resistance to various therapies and highly metastatic nature. One of its features that makes PDAC difficult to treat is the stiffness of the tumor, which prevents drugs from reaching the tumor cells. Blood vessels in PDAC are often collapsed, irregular and non-functional, and tumor cells are protected by fibrotic cells that fill in the gaps. Fifty percent of PDAC patients present with metastasis at the time of diagnosis, limiting their 5-year survival to a mere 3.2%. Even after an R0 resection, 75% of the patients experience metastasis in the future making the disease incurable. Currently, there are no clinically proven approaches that prevent or even reduce the chance of metastasis, greatly limiting the prognosis of PDAC patients.Attorney Docket No. 15003-502PC0

[0164] iRGD binds to receptors expressed on tumor blood vessels and various cells in the tumor tissue allowing it to condense at tumor sites and effectively spread into the tumor tissue. It opens a pathway through blood vessels and cell layers selectively in tumors, and allows other agents to enter the tumor together.

[0165] Long-term iRGD treatment induces various TME changes in PDAC represented by disrupted stromal fibers, less angiogenesis, more functional blood vessels, and more CD8+ T cells, features that make tumors physically more accessible to drugs, less hypoxic, and more immunoreactive. iRGD is an antagonist of av[35 integrin-dependent activation of TGF-0, leading to reduced TGF-0 signaling in the PDAC tissue. The iRGD peptide also results in dramatic inhibition of liver metastasis. While extensive macroscopic and microscopic liver masses are found in control mice, iRGD-treated mice had minimal metastatic foci, if any, suggesting that iRGD prevents the formation of metastasis, not the growth.

[0166] Thus, the tumor-directed efficacy of nearly any type of anti-cancer drug can be enhanced simply by co-administering iRGD. iRGD-based chemotherapy has shown remarkable preliminary efficacy in phase lb clinical trials in PDAC patients, and is showing promising results in multiple phase 2 trials. No alarming side effects of iRGD has been noted so far even in PDAC patients who received it for more than 3 years. This invention therefore can be developed to create a “smart” technology that continues to secrete s-iRGD in the patient’s body while tumor cells are present, but halts once remission is achieved. This one general method therefore allows the practitioner to deliver siRGD therapy only when necessary (when cancer cells are present), potentially allowing patients to receive life-long siRGD therapy with minimal drawbacks.

[0167] Other applications for the peptides, vectors, and cells of the invention also include tumor- targeted drug delivery, diagnostic imaging, cell therapy, immunotherapy combinations, inhibition of metastasis.

[0168] G. Summary

[0169] In summary, the data presented here show that cells can be programmed to produce and secrete functional iRGD peptides using the constructs disclosed herein (and others with similar designs that allow production of secretory peptides). The technology can be used for various applications such as treatment or vaccines for solid tumors or any tumor. While certain cells were used here as the producers of soluble iRGD as described herein, any cell in the body can beAttorney Docket No. 15003-502PC0 programmed to produce iRGD and used with the invention. The constructs can also be delivered in vivo in various forms (including mRNA vaccines) to program cells in situ.

[0170] This technology enables the practitioner to program cells to produce and secrete an iRGD peptide into, around, or away from a tumor, which improves solid tumor penetration, induces beneficial tumor microenvironment changes, and activates and prevents the exhaustion of immune cells (such as CAR-T cells). In this way, new treatment modalities are now available for cancer treatment, cancer immunotherapy, cancer imaging, and cancer vaccines. This invention is a platform for widely using CAR-T cells and any other cellular therapy as a solid tumor therapy which improves the effectiveness of adoptive cancer immunotherapy.

[0171] 5. Examples

[0172] This invention is not limited to the particular processes, compounds, compositions, or methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described.

[0173] Example 1: iRGD Tumor-Penetrating Peptide.

[0174] Molecules both attached to the peptide and freely existing in the vicinity are transported through the ECs leading to extravasation. See FIG. 4A, which provides confocal images of PDAC from transgenic p48-Cre, LSL-KrasGl2D, Ink4cfox(KRAS-Ink) mice that received IV anti- NRP-1 blocking antibody (Ab) or control IgG, followed by iRGD-coated phage (green). Note the NRP-1 -dependent spreading of iRGD phage. Red, CD31; blue, DAPI. Arrows, blood vessels; “d”, tumor ducts. Scale bars, 50 pm. The effect is tumor specific as the vesicles do not form unless iRGD is cleaved at the tumor vasculature.

[0175] This mechanism allows drug delivery of a drag into tumors by simply co-injecting iRGD. See FIG. 2, which provides confocal images of BT474 orthotopic breast tumors from mice injected with IV Nab-P (green) either conjugated or co-injected with iRGD. Inset. Nab-P alone. Red, CD31; blue, DAPI. Scale bars, 100 pm. The iRGD co-injection leads to an improvedAttorney Docket No. 15003-502PC0 therapeutic window (see FIG. 4B). For these data, BT474 tumor mice were treated with the indicated IV Nab-P formulations at 3 mg paclitaxel / kg or with vehicle alone. Statistics: oneway analysis of variance (ANOVA); n.s., not significant; ***, p < 0.001. The effects have been confirmed using various drugs, e.g., small molecules, antibodies (Abs), and nanodrugs, in numerous tumor mouse models.

[0176] Intravenous (IV) iRGD enriches at tumor blood vessels by targeting ccv integrins with its RGD, proteolytically exposes an RXXK / R neuropilin- 1 (NRP-l)-binding motif, and triggers NRP-1 -dependent formation of vesicles that carry more iRGD into subsequent cell layers. See FIG. 5A and FIG. 5B. The result is a greatly improved therapeutic window. See FIG. 6B. PDAC is an excellent target for iRGD as the thick stroma facilitates the penetration. In the extravascular tumor tissue, carcinoma-associated fibroblasts (CAFs) transport iRGD and drugs in an av05-mediated manner, and induce av[35 expression on PDAC cells to develop a TME that further propagates the penetration cascade.

[0177] Example 2: iRGD Example Sequences..

[0178] See Table 1, below for sequences of peptides. iRGD is defined with respect to this invention as a peptide that carries both an RGD motif and an R / KXXR / K (SEQ ID NO:29) motif without limiting it to the CRGDK / RGPD / EC-related sequence. This includes other R / KXXR / K- containing (SEQ ID NO:29) peptides with tumor specificity such as iNGR (CRNGRGPD / EC), LyP-1 (CGNKRTRGC), and more as they may have similar properties. Any tumor- specific tissue-penetrating peptides that carry an R / KXXR / K (SEQ ID NO:29) peptide motif are suitable for use with the invention.Table 1. Example iRGD Peptides.Attorney Docket No. 15003-502PC0X = any one of SEQ ID NO: 8, 12, or 15-26

[0179] Example 3: iRGD Inhibits TGF-0 Activation.

[0180] Mouse pancreatic ductal adenocarcinoma (PDAC) cells were incubated in the presence of latent (inactive) TGF-0 with or without iRGD or a variant of iRGD that has a disrupted integrin- binding RGD motif (iRGE). The results in FIG. 5A and FIG. 5B show that iRGD significantly inhibited the phosphorylation of Smad 2 (pSmad 2), a downstream molecule of the TGF- pathway, while iRGE (arginine, glycine, glutamic acid) had less effect.

[0181] For the data shown in FIG. 6A and FIG. 6B, mouse PDAC cells were incubated in the presence of activated TGF- with or without iRGD. iRGD did not inhibit pSmad 2 here, indicating that it inhibits the activation of TGF-P rather than the function of activated TGF-p.

[0182] Example 4: iRGD Effects in PDAC on the Tumor Microenvironment.

[0183] iRGD modifies the TME in PDAC mice. C57B6129SF1 / J hybrid mice (FIG. 7A and FIG. 7B, FIG. 7C and FIG. 7D, FIG. 7G) or humanized mice (FIG. 7E and FIG. 7F and FIG. 7H) bearing orthotopic PDAC tumors were treated intravenously with PBS or iRGD 3 times a week for 2 weeks (FIG. 7A and FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7G) or 8 weeks (FIG. 7E and FIG. 7H). Tumors were collected at the end of the treatment.

[0184] FIG. 7 A shows stromal fibers in PDAC tumors stained with fibroblast antigen ER-TR7 (red) and the fluorescent dye DAPI (blue). FIG. 7B provides a comparison of the %ER-TR7+area in these PDAC cells, hi FIG. 7B, angiogenic blood vessels are shown stained with an anti- CD31 antibody (red). Blue, DAPI.

[0185] FIG. 7C shows angiogenic blood vessels stained with anti-CD31 antibody (red) and the fluorescent dye DAPI (blue). Note that the blood vessels in the iRGD group are circular whileAttorney Docket No. 15003-502PC0 those in the PBS group are linear. FIG. 7D provides representative images of a blood vessel (red) perfused by tomato lectin (green) in an iRGD treated tumor. Blue, DAPI. The figure shows a comparison of the %CD31+vessels in the PDAC. For this figure, the mice received intravenous tomato lectin before the tumors were collected. Blood vessels in PBS-treated tumors were not functional. FIG. 2E shows angiogenic blood vessels from 2C, stained for a hypoxic marker carbonic anhydrase 9 (CA9). CA9+ areas were computationally quantified. *. p<0.05. Note that the blood vessels in the iRGD group in FIG. 7E are circular while those in the PBS group are linear.

[0186] For FIG. 7G, the mice received intravenous tomato lectin before the tumors were collected. FIG. 7G shows representative images of a blood vessel (red) perfused by tomato lectin (green) in an iRGD treated tumor. Blood vessels in PBS-treated tumors were not functional. Blue staining is the fluorescent dye, DAPI.

[0187] FIG. 7H is a bar graph showing data for tumors from FIG. 7E that were stained for a hypoxic marker carbonic anhydrase 9 (CA9). CA9+areas were computationally quantified. *, p<0.05.

[0188] Example 5: iRGD Inhibits TGF-B Activation and Modifies the TME in PDAC.

[0189] TGF-P is secreted in an inactive form bound to the latency-associated protein (LAP), which is tom open to release TGF-P when it binds to av integrins via its RGD motif. For the data presented in FIG. 8A through FIG. 8F, K.PC78 cells were treated with TGF-P (FIG. 8A and FIG. 8D or LAP-TGF-p (FIG. 8C, FIG. 8E, and FIG. 8F) ± LY2157299 (TGF-P inhibitor), iRGD, or an iRGD variant with a disrupted RGD (iRGE). In FIG. 8C through FIG. 8E, P5-KO cells were also used. (p)Smad2 was quantitated by IB (FIG. 8A through FIG. 8D and FIG. 8F and FIG. 8G) and SOX4 was quantitated by qPCR (FIG. 8E). Representative IB images are shown in some panels. Wild type (WT) K.PC78 PDAC cells derived from KPC mice respond to endogenous and exogenous TGF-P evidenced by increased phosphorylation of Smad2C (pSmad2), which is abolished by a TGF-P inhibitor LY2157299 based on immunoblotting. See FIG. 8A.

[0190] WT KPC78 effectively activates exogenous LAP-TGF-P (left 2 bars, FIG. 8C). P5 integrin-knock out (KO) cells, which respond to active TGF-P (FIG. 8D), failed to activate LAP- TGF-P based on pSmad2 (right 2 bars, FIG. 8C) and SOX4 expression (FIG. 8E). Thus, avP5Attorney Docket No. 15003-502PC0 integrin activates TGF-P, which in turn enhances avP5 expression, forming a vicious cycle that maintains an avP5- and TGF-p-rich TME in PDAC.

[0191] iRGD inhibits the avP5-mediated activation of TGF-P in vitro and in vivo. iRGD reduced pSmad2 induced by LAP-TGF-P, but not that by active TGF-P (not shown), in KPC78 cells in an RGD-dependent manner (FIG. 8F).

[0192] A reporter gene assay using av-deficient responder cells that selectively detect activated TGF-P confirmed the results. KPC78 cells were cultured with LAP-TGF-P ± iRGD. The supernatant was transferred to M2 IL cells that carried a TGE-p / Smad responsive luciferase gene. The M2 IL cells respond to activated TGF-P, but not to LAP-TGF-P, given the lack of cell surface av integrins. See EIG. 9A and EIG. 9B).

[0193] In EIG. 10, orthotopic PDAC mice bearing syngeneic KPC78 were treated with IV PBS or iRGD 3x / week for 2 weeks. See also PIG. 7. In PIG. 7G and PIG. 7H, the mice received IV tomato lectin at the end. The tumors were stained and quantified for pSmad2 (blue) (PIG. 10A and FIG. 10B), ER-TR7+ stromal fibers (red) (FIG. 7A and FIG. 7B), CD31+ blood vessels (red) (FIG. 7C and FIG. 7D), width of CD31+ vessels (FIG. 7F), CD31+ vessels (red) perfused with tomato lectin (green) (FIG. 7G and FIG. 7H), and CD8+ T cells (brown, arrow heads) in the periphery and core of the tumors (FIG. 10C and FIG. 10D). Blue, DAPI (FIG. 7A, FIG. 7C, FIG. 7G). Bars, 50 pm. ANOVA (FIG. 10A and FIG. 7F); Student’s t-test was used; n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0194] The results show that iRGD monotherapy in syngeneic orthotopic KPC78 PDAC mice drastically reduced pSmad2 in the tumor (see FIG. 10A and FIG. 10B). The tumors also underwent a dynamic TME change showing reduced stromal fibers (see FIG. 7A), less angiogenesis (see FIG. 7C and FIG. 7D), wider lumen of remaining blood vessels (see FIG. 7H), and better perfusion (see FIG. 7H). Enhanced CD8 infiltration into the tumor core was noted in the iRGD arm (see FIG. 10B). These studies show that iRGD can both drive T cell penetration into solid tumors and help to overcome the immunosuppressive TME, at least partially, through inhibition of effects from active TGF-p, strongly suggesting its potential to dramatically improve CAR-T therapy for solid tumors.

[0195] Therefore, these test show that, in addition to its acute tumor penetration properties, iRGD has chronic effects on the TME, making cancers such as PDAC less metastatic and more susceptible to various therapies. Without wishing to be bound by theory, the effect appears to beAttorney Docket No. 15003-502PC0 induced by the ability of iRGD to inhibit the activation of transforming growth factor-0 (TGF-0). TGF-0 is secreted in an inactive form bound to the latency-associated protein (LAP) and released / activated by cytoskeletal force when LAP binds to av integrins via its RGD motif. Wild type (WT) KPC78 mouse PDAC cells respond to endogenous and exogenous TGF-0 evidenced by increased phosphorylation of Smad2C (pSmad2), which is abolished by a TGF-0 inhibitor LY2157299 based on immunoblotting. The KPC78 cells highly express av05. allowing them to effectively activate exogenous LAP-TGF-0. 05 integrin-KO cells, which respond to active TGF- 0, failed to activate LAP-TGF-0 based on pSmad2, and SOX4 expression. This mechanism to activate TGF-0 helps PDAC to maintain an av05- and TGF-0-rich TME because TGF-0 triggers the expression of av05.

[0196] Example 6: iRGD Effects on huPDAC TME.

[0197] FIG. 11 shows data for spatial transcriptomics using human (FIG. 11A through FIG. 1 IF) and mouse (FIG. 11G) 5k pan tissue & pathways panel run on huPDAC tumors treated with or without iRGD.

[0198] See FIG 11A for GSEA showing significant T cell signatures in iRGD-treated tumors in contrast to control. FIG. 11B shows elevated T cell activation genes in the iRGD arm. FIG. 11C through FIG. HE present UMAPs of major T cell populations (FIG. 11C) and CD8 subtypes (FIG.1 ID) in the tumors. The left UMAPs indicate the cell types, which are reflected to the right UMAPs and circled with matching colors. The right UMAPs indicate DGE between control (blue) and iRGD (orange) arms. The bar diagram in FIG. 1 ID shows the proportion of activated CD8+T cells. Student’s t-test; **, p<0.01.

[0199] FIG. HE and FIG. 1 IF show the spatial distribution of human (FIG. HE) CD8A, GZMB, and PDCD, and mouse (FIG. 1 IF) DES (desmin), FAP (fibroblast activation protein, FAP), PDGFRB, and PECAM-1 (CD31) genes in control and iRGD tumors. The yellow cell in F coexpresses FAP and PDGFRB. The red arrow in FIG. 1 IF points to PDGFRB+and PECAM-1+cells next to each other. FIG. 11G shows examples of MISO-based clustering that reflects both histological and DGE features are shown.

[0200] Differential gene expression (DGE) and gene set enrichment analysis (GSEA) on the human panel shows that iRGD alters T cell functions (FIG. 11A and FIG. 1 IB) and increases some of the T cells (e.g., activated and cytotoxic CD8) while it reduces Tregs (FIG. 11C andAttorney Docket No. 15003-502PC0FIG. 1 ID). Spatial analysis shows that CD8+T cells in iRGD tumors express G7.MB (granzyme B) rather than PDCD (PD-1), depicting an activated and less exhausted phenotype (FIG. HE). Analysis of the mouse panel shows that iRGD upregulates PDGFRB (platelet-derived growth factor receptor-P), a marker of perivascular progenitor cells and pericytes in line with increased pericyte coverage of blood vessels (see FIG. 11F). In-depth spatial analysis using multi-modal spatial oniics is shown in FIG. 11G. These data show that iRGD can both drive T cell entry into solid tumors and overcome the immunosuppressive TME, at least partially, through inhibition of TGF- effects, suggesting its potential to vastly improve CAR-T therapy for solid tumors. So far, despite the high in vitro activity of CAR-T for solid tumors, clinical results have been disappointing with a reported 9% RR as compared to »50% for CAR-T products for B cell malignancies and multiple myelom. Poor penetration / activation of CAR-T in fibrotic and immunosuppressive TME of solid tumors for which TGF- is a dominant component have widely been reported as barriers to efficacy.

[0201] Example 7: iRGD Enhances the Ratio of CD8+ T cells over Trees.

[0202] C57B6129SF1 / J hybrid mice bearing orthotopic PDAC tumors were intravenously treated with PBS or iRGD 3 times a week for 2 weeks. Tumors were collected 3, 7 and 14 days after the treatment was started.

[0203] The ratio of CD8+ T cells over Tregs (CD8 / Treg ratio) in the tumor was analyzed by flow cytometry. See FIG. 12A (n = 3 (day 3, PBS), 4 (day3, iRGD; day 7) or 5 (day 14) per group. FIG. 12B is a representative image of immunofluorescence performed on the PDAC tissue harvested after 14 days of treatment with PBS (control). FIG. 12C is a representative image after 14 days of treatment with iRGD. Green, CD8+ T cells; red, Foxp3+ Tregs; blue, DAPI. Scale bars, 50 pm. FIG. 12D shows the CD8 / Treg ratio, which was quantitated by counting the cells in 5 random high-power fields per section for 3 mice per group. *, p<0.05.

[0204] Example 8: iRGD Enhances the Anti-Tumor Effects of an ICB (anti-PD-Ll mAb).

[0205] PDAC mice were treated with iRGD ± anti-PD-Ll mAb (FIG. 13A through FIG. 13C; n = 4-6) or iRGD + Gem ± anti-PD-Ll mAb (FIG.13D; n = 4) 3 times a week for 2 weeks.Primary tumors were weighed at the end of the study, n.s., not significant; *, p<0.05, Thus, using a condition in which neither iRGD nor an ICB (anti-PD-Ll mAb) alone inhibited tumorAttorney Docket No. 15003-502PC0 growth (see FTG. 13A), iRGD in combination with the ICB led to a significant decrease in tumor volume (FIG. 13B). Similar results were noted when iRGD was combined with the anti-PD-Ll mAb and Gem (see FIG. 13D and FIG. 13E).

[0206] These results strongly suggest that iRGD modifies the TME by normalizing the vasculature to create a conduit for T cells to enter the tumor to improve anti-tumor immunity. Normalized vasculature is known to have improved integrity and is resistant to tumor cell dissemination, which is in line with the observation that long-term iRGD therapy leads to strikingly reduced liver metastasis. TGF-0 inhibitors can indeed cause these changes to create an overall protective TME for cancer patients.

[0207] Example 9: iRGD Monotherapy Inhibits Metastasis in PDAC Mice.

[0208] Humanized PDAC (huPDAC) mice bearing an orthotopic PANC-1 tumor were treated with or without intravenous injections of 1.2 mg / kg of iRGD 3 times a week for 8 weeks. The effect of systemic iRGD monotherapy on liver metastasis in PDAC-1 huPDAC mice was characterized based on the macroscopic appearance of the liver (see FIG. 14A and FIG. 14B) and microscopic analyses of the liver sections stained with H&E (see FIG. 14C (DAPI, blue) and FIG. 14D) or an anti-CK.19 antibody (red) that detects tumor cells (see FIG. 14E and FIG. 14F; dotted circles: metastases). Scale bars for FIG. 14 are 5 mm (FIG. 14A), 500 pm (FIG. 14C), and 100 pm (FIG. 14E). The number of liver metastases under each analytical condition was counted either macroscopically or under a microscope. Error bars, mean ± standard error: *p < 0.05.

[0209] Without wishing to be bound by theory, it is believed that iRGD monotherapy inhibits metastasis by a mechanism of_TGF-p antagonism. iRGD-treated PDAC mice had no macroscopic liver nodules, while all the control mice had multiple of them (see FIG. 14A through FIG. 14D, which shows macroscopic and H&E-stained microscopic analysis of treated and untreated livers). The analysis revealed no obvious micrometastasis in iRGD-treated mice, while many were noted in each of the control mice. Similar results were obtained with liver sections stained for CK19 (see FIG. 14E and FIG. 14F. As iRGD monotherapy did not affect the weight of the primary tumors, delayed tumor growth was not the cause of the metastasis inhibition.Attorney Docket No. 15003-502PC0

[0210] Example 10: Design of Secretory iRGD Plasmid Vector.

[0211] The data above show that iRGD therapy induces changes in the TME that would both provide beneficial effects in cancer therapy and prevent metastasis. Thus, this invention has established a technology to produce iRGD in vivo in a subject. Accordingly, a plasmid vector containing the DNA sequence of an iRGD peptide attached to an endoplasmic reticulum (ER) signal peptide fused with an HA tag. a Myc tag. and iRGD controlled under an EFl A promoter (see FIG. 15). This mammalian expression vector for secretory iRGD production was generated using an ampicillin-resistant pRP plasmid vector as the backbone. The ER signal peptide directs the product to the ER and becomes detached upon secretion of iRGD (see FIG. 16A, which shows a scheme depicting the trafficking of the production and secretion of secretory peptides). To allow easy detection of the resulting soluble iRGD (siRGD) peptide, we also included HA and Myc tags in the initial construct (see FIG. 16B). DNA and amino acid sequences of the ER signal, HA tag, Myc tag, and iRGD used in the vector are shown in FIG. 17A, including, in order, the DNA and protein sequences of the ER signal (SEQ ID NO:1 and SEQ ID NO:2), the HA tag (SEQ ID NO:3 and SEQ ID NO:4), and the Myc tag (SEQ ID NO:5 and SEQ ID NO:6), and iRGD (SEQ ID NO:7 and SEQ ID NO:8). FIG. 17B provides the DNA sequence of the full- length protein (SEQ ID NO:9), the amino acid sequence of the full-length protein (7 kDa, SEQ ID NO: 10) and the amino acid sequence secretory form of the protein (4kDa, SEQ ID NO: 11).

[0212] Example 11: Production of Secretory HA-Myc-iRGD by Transduced TCON 3077 Cells.

[0213] Transducing TCON 3077 mouse gastric cancer cells with the vector above resulted in the production of HA-Myc-iRGD in the cells. This was confirmed by immunofluorescence using anti-HA and anti-Myc antibodies. See FIG. 19, which shows mouse TCON gastric cancer cells producing secretory iRGD tagged with HA (green) and Myc (red). Blue, DAPI.

[0214] HA-Myc-iRGD released into the culture media from cultured gastric cancer cells was confirmed by a sandwich ELISA using anti-HA and anti-Myc antibodies (see FIG. 20A and FIG. 20B). HA-Myc-iRGD was not detected in the culture media of transduced TCON 3077 cells treated with GolgiPlug™, confirming that the HA-Myc-iRGD detected in FIG. 20A was a result of active secretion of the peptide and not other causes such as cell death that can result in accidental release of the peptide.Attorney Docket No. 15003-502PC0

[0215] Various cells were successfully programmed to secrete HA-Myc-iRGD into culture media using the construct as indicated in FIG. 21. When PANC-1 human PDAC cells transduced with the construct were subcutaneously implanted into mice, large amounts of HA- Myc-iRGD became detectable by the sandwich ELISA assay of FIG. 7C in the plasma within 3 weeks (see FIG. 21).

[0216] Finally, see FIG. 22, which shows the amount of secreted HA-Myc-iRGD in the plasma of nude mice bearing siRGD-secreting or control PANC-1 human PDAC tumors, measured by ELISA. The plasma samples were diluted 40x. ***, p<0.001.

[0217] Example 12: Additional iRGD-Producing Constructs.

[0218] The HA and Myc tags were removed from the vector, leaving either an iRGD peptide preceded by a G6 peptide sequence (GGGGGG; SEQ ID NO:27) or plain iRGD. KPC-derived mouse PDAC cells derived from Kras-LSLG12D; p53-LSL172H; Pdx-l-cre KPC mice were stably transduced with secretory HA-Myc-iRGD, G6-iRGD or iRGD plasmid vectors using lentiviruses carrying the constructs. Using an ELISA assay (depicted in the schematic of FIG. 23A), conditioned media (CM) prepared from both the cells that were transduced with the G6- iRGD- and plain iRGD-producing constructs competed with the binding of biotinylated iRGD to immobilized avP5 integrin confirming the secretion of functional iRGD. CM from the cells was assessed for the ability to compete with the binding of biotinylated iRGD to immobilized av05 integrin. CM from wild type (WT) cells and synthetic iRGD (final concentration 500 umol) were used as controls.

[0219] The results are shown in FIG. 23B. CM from cells transduced with the initial HA-Myc- iRGD construct also competed with the binding. The results indicated that the iRGD peptides released from the cells retained a proper cyclic structure because iRGD otherwise fails to retain its integrin-binding capacity.

[0220] See also Table 1, above for sequence information.

[0221] Example 13: iRGD Targets PDAC Tregs and Potentiates Anti-PD-Ll Antibodies.

[0222] iRGD depletes tumor-infiltrating Tregs owing to its anti-TGF-P properties, and because the Tregs express avp5. FIG. 24A shows avp5 expression on Tregs and CD8+T cells in orthotopic PDAC (Tu) and spleen (Spl) of syngeneic KPC78 tumor mice (Tu Ms) and the spleenAttorney Docket No. 15003-502PC0 of normal mice (N Ms) analyzed by flow cytometry. The cells were magnetically isolated from dissociated tissues. In FIG. 24B and FIG. 24C, homing of FAM-iRGD to Tregs in PDAC and spleen in KPC78 mice quantified by flow cytometry. FIG. 25 A shows data for Treg / CD4 and CD8 / Treg ratios in orthotopic KPC78 and spleen after 1 week of PBS or iRGD therapy analyzed by flow cytometry.

[0223] Under a condition that neither iRGD nor anti-PD-Ll Ab was effective, iRGD + anti-PD- L1 Ab inhibited the growth of syngeneic KPC78 tumors in mice. See FIG. 25B, which presents data on orthotopic KPC78 mice treated with iRGD and / or anti-PD-Ll Ab 3 times a week for 2 weeks. See also FIG. 25C.

[0224] See also FIG. 26, which shows H&E and IHC staining for CD3 (brown) of on-treatment biopsies from a patient treated with iRGD + Gem + Nab-P + durvalumab in the iLSTA trial.Blue dotted circle, remaining cancer duct. Student’s t-test: n.s., not significant; *,p<0.05; **, j><0.01. This staining of on-treatment biopsies shows that 4 / 4 PR and 1 / 3 SD cases underwent 2- lOx increase in CD3+T cells.

[0225] Example 14: Effects of iRGD on huPDAC Tumors.

[0226] Human leukocyte antigen (HLA)-A2+PANC-1 huPDAC mice, whose immunity, up to 90%, was replaced by HLA-matched human immune cells that recognize tumor antigens in an HLA-restricted manner. See FIG. 27. This figure shows an HLA-A2+huPDAC mouse with an orthotopic PANC-1 tumor and spontaneous liver metastases (arrows). Representative H&E and multiplex immunofluorescence (IF) images of the tumors are shown. Blue, DAPI; aqua, CD 19; green, CD4; red, CD8; purple, CD 163; yellow, granzyme B. Bars. 100 pm. The composition of their immune cells resembles that of human peripheral blood mononuclear cells (PBMC: e.g., B cells 25%; T cells 60%; CD4 / CD3, 65%; CD8 / CD3, 30%). They develop fibrotic tumors in the pancreas and metastatic liver tumors, which are infiltrated by human immune cells recapitulating the signatures of the human disease.

[0227] PANC-1 huPDAC mice were treated with or without IV iRGD 3x / week for 8 weeks. The opening of CD31+vessels based on the length of minor axes (FIG. 30A) and hypoxia based on carbonic anhydrase 9 (CA9) positivity (FIG. 30B) in the tumors were analyzed by IHC. a-SMA (green) coverage of CD31+vessels (red) (FIG. 31 A) and immune cells (FIG. 3 IB; red. Foxp3; aqua, CD8; green, CD3) in the tumors were analyzed by IF. Blue, DAPI. Macroscopic view andAttorney Docket No. 15003-502PC0H&E staining of liver metastasis (FIG. 32). Yellow circles, tumors. Student’s t-test; n.s., not significant; *, p<0.05.

[0228] The results showed that iRGD (12 pmol / kg) given 3x / week over 8 weeks induced wider blood vessels (FIG. 30A), less hypoxia (FIG. 30B), more vascular coverage with a-smooth muscle actin (a-SMA) (FIG. 31A), and increased CD8 / Treg ratio (FIG. 31B) in the tumor. iRGD also strongly inhibited spontaneous liver metastasis (FIG. 32) without affecting the growth of primary tumors.

[0229] Example 15: MR1 CAR-T Cells have Promising Anti-Tumor Effects against Solid Tumors.

[0230] As the next step toward establishing s-iRGD-producing CAR-T cells. CAR-T products were prepared that target two different pan-cancer antigens, MR1 and B7H3. MR1 is a non- classical MHC class 1 protein with ubiquitous cell surface expression across cancer types, but nearly none on normal cells. It resides in the ER, but translocates to the cell surface when it binds to microbial metabolites to present them to mucosal associated invariant T cells in the gut. In cancer cells, MR1 binds to cancer-derived metabolities that induce the surface translocation, making it an innovative CAR target.

[0231] Flow cytometry results in FIG. 33, showed cell surface MR1 expression across various solid tumor cell lines including PDAC cells, but not normal cells (n=3). In our hands, MR1 was expressed on the surface of various solid tumor cells including PDAC cells, but not on normal cells, by flow cytometry (see FIG. 33). Controls were isogenic AML cell lines (derived from OCI-AML3 cells) with MR1 expression (WT) or MR1 KO achieved with CRISPR-Cas9.

[0232] The results also show that MR1 CAR-T cells kill MR1+tumor cells (FIG. 34), but have minimal effect on cells that lack MR1 (FIG. 35). ***, pcO.OOl. A second generation CAR targeting MR1 comprising a CD28 intracellular domain, showed its activity against solid tumor cancer cells in vitro (see FIG. 34). The killing was dependent on cell surface MR1 as the MR1- KO AML cells were resistant (see FIG. 35).

[0233] As shown in FIG. 36, IV MR1 CAR-T cells inhibit the growth of bilateral sq SNU16 human gastric cancer tumors in NSG mice (n=5 mice per group). IV therapy with the MR1 CAR-T cells slowed the growth of (though did not eliminate) human gastric cancer sq tumors in NSG mice (see FIG. 36).Attorney Docket No. 15003-502PC0

[0234] The anti-tumor effects of IC MR1 CAR-T against orthotopic U87 glioblastoma (GBM) (FIG. 37A) and IV MR1 CAR-T against sq U87 GBM (FIG. 37B) are shown. n=5 mice per group. The results indicate that, in an intracranial (IC) orthotopic model of U87 glioblastoma (GBM), IC injection of MR1 CAR-T cells fully eradicated the tumors (see FIG. 37 A). However, IV dosing of MR1 CAR-T failed to control the growth of sq U87 tumors (see FIG. 37B). These results suggest that the improvements in CAR-T cell trafficking and / or maintenance of CAR-T activity could lead to measureable improvements of efficacy of systemic CAR-T therapy.

[0235] Example 16: B7H3 Human and Mouse CAR-T.

[0236] For FIG. 38 and FIG. 40A, human B7H3-specific nanobody CAR introduced into human (FIG. 38) and mouse (FIG. 40A) T cells analyzed by flow cytometry 5 days post-transduction (bottom). For FIG. 39 and FIG. 40B, in vitro cytotoxicity of the human (FIG. 39) and mouse (FIG. 40B) B7H3 CAR-T cells against GFP-positive solid tumor cancer cells monitored in real time using the Incucyte™ live-cell imaging platform. The CAR-T cells were co-cultured with the cancer cells at the indicated effector-to-target (E:T) ratio. Mean ± SEM. n=3.

[0237] As a secondary CAR target, B7H3, a widely used traditional pan-cancer antigen was chosen. A human B7H3-specific nanobody CAR was successfully introduced into primary human T cells (see FIG. 38). The B7H3 human CAR-T cells effectively killed solid human tumor cells including PDAC cells (see FIG. 39). To allow studies in immunocompetent mice, we also introduced the human B7H3 nanobody CAR into mouse T cells harvested from C57BL6 mice (see FIG. 40A). The B7H3 mouse CAR-T cells also killed solid tumor cells in vitro (see FIG. 40B).

[0238] Example 17: Co-expression of s-iRGD with MR1 or B7H3 in Primary Human T Cells.

[0239] Flow cytometry for MR1 (FIG. 41) or B7H3 (FIG. 42) CAR expression on human primary T cells with or without s-iRGD co-transduction was performed. UTD-T, untransduced T cells. ELISA was used to detect s-iRGD released into the CM of the T cells bearing MR1 (FIG. 43) or B7H3 (FIG. 44) CAR. An in vitro cytotoxicity assay was performed, and showed that s-iRGD expression does not impair the ability of MR1 (FIG. 45) or B7H3 (FIG. 46) CAR-T to kill co-cultured tumor cells. *, p<0.05; ****, p<0.0001.Attorney Docket No. 15003-502PC0

[0240] MR1 and B7H3 human CAR-T cells that secrete s-iRGD thus have been prepared. We co-expressed s-iRGD and validated by flow cytometry that it did not affect the expression of MR1 (FIG. 41) or B7H3 (FIG. 42) CAR on the T cells. s-iRGD secretion from the MR1 (FIG. 43).

[0241] ) and B7H3 (FIG. 44) CAR-T cells into the CM was confirmed by ELISA. The coexpression of s-iRGD also did not adversely impact the cytotoxicity of MR1 (FIG. 45) or B7H3 (FIG. 46) CAR-T cells based on in vitro cell killing assays using human PANC-1 cells.

[0242] To test whether s-iRGD production enhances CAR-T entry into PDAC, we injected s- iRGD MR1 CAR-T cells into the tail vein of NSG mice bearing orthotopic 0779E human PDAC tumors. Infiltration of the CAR-T cells into the extravascular tumor tissue was noted five days after the injection. Parental MR1 CAR-T cells minimally entered the tumors. No difference in tumor size was noted in this short time frame, but H&E revealed increased patchy necrosis in the s-iRGD CAR-T arm. See FIG. 47. s-iRGD B7H3 CAR-T cells infiltrated sq PANC-1 tumors in NSG mice. See FIG. 48.

[0243] Example 18: Tumor Entry of s-iRGD Human CAR-T Cells.

[0244] 0779E sq PDAC tumors from NSG mice were treated with IV MR1 CAR-T cells ± s- iRGD production for 5 days. Representative images of CAR-T cells (human CD3, blue) in the periphery and core of the tumors and H&E are shown in FIG. 47. Insets show a magnified view of dotted areas in matching colors; arrowheads, disintegrated cancer duct.

[0245] PANC-1 sq tumors from NSG mice were treated with IV B7H3 CAR-T cells ± s-iRGD production for 7 days. Representative images of human CD3 staining (brown, arrowheads) and CD3 quantification based on the IHC are shown in FIG. 49. UTD, untransduced T cells. Scale bars, 50 pm.

[0246] To test whether s-iRGD production enhances CAR-T entry into tumors, we injected IV s- iRGD MR1 CAR-T into the tail vein of NSG mice bearing sq 0779E human PDAC. In 5 days, the CAR-T cells widely entered the periphery as well as the core of the tumors (see FIG. 47). The cells were often found to infiltrate cancer ducts, which were disintegrated in some areas. Parental MR1 CAR-T was only found in the periphery. No difference in tumor size was noted in this time frame, but H&E showed increased ductal distortion in the s-iRGD CAR-T arm in lineAttorney Docket No. 15003-502PC0 with the THC. IV s-iRGD B7H3 CAR-T cells infiltrated sq PANC-1 tumors in NSG mice (see FIG. 49).

[0247] The results strongly suggest that s-iRGD CAR-T cells have amplified the capacity to penetrate solid tumors. The cells were often found to infiltrate cancer ducts, which were disintegrated in some areas. Parental MR1 CAR-T was only found in the periphery. No difference in tumor size was noted in this time frame, but H&E showed increased ductal distortion in the s-iRGD CAR-T arm in line with the IHC.

[0248] Example 19: Differential Response of M21 Variants to Active TGF-P and LAP-TGF-P.

[0249] M21 (FIG. 50), M21L (FIG. 50), and M21L4 (FIG. 51) cells were cultured in the presence of active TGF-P or LAP-TGF-P at the indicated concentrations for 3 hours. Expression of pSmad2, Smad2, and GAPDH was analyzed by IB and quantified as shown, n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

[0250] To further distinguish whether siRGD inhibits the activation or the function of TGF-p, further tests can be performed using M21 cells, which transmit TGF-P signals evidenced by increased p-Smad2 in response to active TGF-P (see FIG. 50). They also respond to LAP-TGF-P as they can activate TGF-p. In contrast, M21L cells that lack cell surface av integrins respond to active TGF-P but not to LAP-TGF-P, indicating that they can no longer activate TGF-P (see FIG. 50). The M21L cells regain the ability to activate TGF-P when they are forced to re-express av integrins (M21L4; FIG. 51).

[0251] Example 20: Spatial Transcriptomics Data.

[0252] Spatial transcriptomics was performed using a 5k human pan tissue & pathways panel on an orthotopic PANC-1 from huPDAC mice treated with or without iRGD. FIG. 53 shows a limited set of immune cell signatures with and without H&E overlay. These preliminary data show changes in the level and distribution of immune cell signatures and TGF-P target molecules. This type of study allows direct comparison of the effects of s-iRGD and synthetic iRGD on PDAC TME to study the difference / similarity of the two peptides.Attorney Docket No. 15003-502PC0

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Claims

1. Attorney Docket No. 15003-502PC0Claims1. An isolated iRGD peptide comprising an RGD sequence and an R / KXXR / K (SEQ ID NO:29) sequence.

2. An isolated iRGD peptide of claim 1 comprising CRGDK / R / HGPD / EC (SEQ ID NO: 12) or a variant thereof comprising at least 95% identity therewith.

3. An isolated iRGD peptide of claim 2 comprising CRGDKGPDC (SEQ ID NO:8).

4. A vector that comprises an expressible nucleic acid sequence that encodes the iRGD peptide of any of claims 1-3.

5. A vector of claim 4 which comprises a promoter operatively linked to the nucleic acid sequence.

6. The vector of claim 5 which is a mammalian vector, a viral vector or a bacterial vector, or an exosome, liposome, or other lipid based delivery agent that comprises the expressible nucleic acid sequence.

7. The vector of claim 4 which is an AAV, adenovirus, or lentivirus vector.

8. The vector of claim 4 which is a plasmid vector comprising SEQ ID NO:9.

7. A cell comprising the vector of claim 4 which expresses an iRGD peptide.

8. The cell of claim 7 which is selected from a mammalian cell, a yeast cell, a bacterial cell and an insect cell.

9. The cell of claim 8 which is a mammalian cell.Attorney Docket No. 15003-502PC010. The cell of claim 8 which further comprises an antigen receptor that specifically binds to a cancer marker.

11. The cell of claim 10 wherein the cancer marker is selected from the group consisting of MR1, B7H3, a claudin family members, mesothelin, a plastin, CEA, CD70, PSMA, HER2, MUC1. GD2, and EGDR.

12. A secretory iRGD peptide comprising SEQ ID NO: 12.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the peptide of claim 1.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the vector of claim 4.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the cell of claim 7.

16. A vaccine composition comprising a cell of claim 7 which secretes the peptide of claim 1.

17. An mRNA vaccine composition comprising mRNA encoding the iRGD peptide of claim 1.

18. A method of producing a cell of claim 7, comprising:(a) obtaining a population of cells;(b) optionally isolating specific cell population subsets from the cells;(c) stimulating the cells or the cell population subsets using cytokines, antibodies, or both to promote activation, differentiation, and proliferation; and(d) introducing to the cells one or more DNA or RNA nucleic acids encoding s-iRGD and a CAR construct using a gene delivery method, wherein the cells express and secrete an iRGD peptide.Attorney Docket No. 15003-502PC019. The method of claim 18 wherein the population of cells are selected from the group consisting of tumor-infiltrating lymphocytes, peripheral blood mononuclear cells, spleen cells, bone marrow cells, or cells from umbilical cord blood.

20. The method of claim 18 wherein the specific cell population subsets are selected from the group consisting of CD4+T cells, CD8+T cells, regulatory T cells, NK cells, and NKT cells.

21. The method of claim 20 wherein the isolating is performed using magnetic beads or flow cytometry.

22. The method of claim 18 wherein the gene delivery method is selected from the group consisting of lentiviral, retroviral, adenoviral, or non-viral transfection.

23. A programmed CAR-T cell produced by the method of claim 18.

24. A method of treating cancer comprising administering to a subject in need thereof the peptide of claim 1.

25. A method of treating cancer comprising administering to a subject in need thereof the vector of claim 4.

26. A method of treating cancer comprising administering to a subject in need thereof the cell of claim 7.

27. A method of claim 24 wherein the administering is by intravenous injection.

28. The method of claim 24 wherein the cancer is a solid tumor.

29. The method of claim 28 wherein the cancer is pancreatic ductal adenocarcinoma.

30. A kit for cancer detection, comprising:Attorney Docket No. 15003-502PC0(a) a population of cells that produce s-iRGD in vivo;(b) an imaging agent; and(c) instructions for use of the kit, wherein the imaging agent is compatible with computed tomography, magnetic resonance imaging, ultrasound, or positron emission tomography.

31. A method for cancer imaging in a subject in need thereof, comprising:(a) obtaining a population of cells of claim 7 which produces s-iRGD;(b) injecting the cells into the subject in need, intravenously;(c) injecting an imaging agent into the subject in need and waiting for the imaging agent to penetrate into the cancer; and(d) imaging the subject in need using an imaging modality suitable for use with the injected imaging agent to detect the location of the imaging agent in the subject.

32. A method comprising transfecting a cell with an expressible nucleic acid sequence that encodes an RGD peptide, wherein the transfecting occurs ex vivo, in vitro or in vivo.

33. The method of claim 32, wherein when the transfecting occurs ex vivo or in vitro, the method further comprises delivering the resulting transfected cell into a subject.

34. The method of claims 32 or 33, wherein the RGD peptide is iRGD.

35. The method of any of claims 32, wherein the expressible nucleic acid is expressed in a subject.

36. The vector of claim 4, wherein the promoter is selected from EFla, CMV, CAG, or other constitutive promoter.

37. The vector of claim 4, wherein the expressible nucleic acid sequence further comprises a signal peptide.

38. The vector of claim 37, wherein the signal peptide is selected from fusion tags: HA, Myc, FLAG, His, or fluorescent protein.

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