Modulation of metalloproteinase or cathepsin to alter immune cell invasion in cancer

Abstract

Disclosed herein are materials and methods for the treatment of a disease or disorder, for example, cancer. The methods of the disclosure comprises, in some embodiments, treating cancer through the administration of genetically modified immune cells that overexpress a membrane-bound protease. In some aspects, the membrane-bound protease is a matrix metalloproteinase. In various aspects, the membrane-bound protease is a cathepsin.

Description

Docket No. 33794 / 70815MODULATION OF METALLOPROTEINASE OR CATHEPSIN TO ALTER IMMUNE CELL INVASION IN CANCER STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under CA050633 and CA239441 , awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION

[0002] The benefit of priority to U.S. Provisional Patent Application No. 63 / 772,320 filed March 14, 2025, is hereby claimed and the disclosure is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a XML file. The name of the XML file containing the Sequence Listing is “70815_SeqListing. xml”, which was created on March 10, 2026 and is 61 ,145 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.FIELD

[0004] The disclosure relates to pharmaceutical compositions and methods for treatment of diseases such as cancer comprising modified immune cells.BACKGROUND

[0005] Natural killer (NK) cells are important innate immunity effectors that play a critical role in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. T cells are adaptive immunity effectors; they possess antigen specificity, and must be primed by an antigen-presenting cell before exerting their cytotoxic effects. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells and T cells, with variable success.SUMMARY

[0006] A major hurdle in NK-cell focused therapies is NK cell recruitment and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined.

[0007] Disclosed herein are NK cells and T cells that overexpress a membrane-bound protease. In some embodiments, the membrane-bound protease is a heterologous proteaseDocket No. 33794 / 70815that is not endogenously expressed by the NK cells or the T cells. In other embodiments, the membrane-bound protease is homologous, but is expressed at levels above what would be expected for an endogenous NK cell or T cell. Forced overexpression of membrane-bound proteases promotes NK cell and T cell invasion through tumor matrix, ultimately increasing tumor cell lysis.

[0008] A pharmaceutical composition in accordance with the disclosure comprises genetically modified immune cells, wherein the modified immune cells overexpress one or more membrane-bound proteases. Further provided herein are pharmaceutical compositions for use in treating a disease comprising a therapeutically effective amount of genetically modified immune cells, wherein the genetically modified immune cells overexpress one or more membrane-bound proteases.

[0009] A method of enhancing the ability of immune cells to migrate into a tumor is provided, comprising genetically modifying the immune cells to overexpress a membranebound protease. A method of preparing one or more genetically modified immune cells is provided, comprising (i) transfecting a vector containing a gene for one or more membranebound proteases into one or more immune cells in medium; (ii) replicating the one or more immune cells transfected with the vector; and (iii) isolating the one or more immune cells transfected with the vector that overexpress the one or more membrane-bound proteases.

[0010] The pharmaceutical compositions and methods disclosed herein comprise genetically modified immune cells, wherein the modified immune cells overexpress one or more membrane-bound proteases. In some embodiments, at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease. In some aspects, at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound. In various aspects, the soluble protease is engineered to: i) remove the secretory domain; and ii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor. In some aspects, the secretory domain is a pro-MMP cleavage site.

[0011] In some embodiments, at least one of the one or more membrane-bound proteases is a matrix metalloproteinase. In various aspects, the membrane-bound matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25, or a combination thereof. In some aspects, the soluble protease is a matrix metalloproteinase selected from MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-13, MMP-18, MMP-19, MMP-20, MMP-21 , MMP-23A, MMP-23B, MMP-26, MMP-27, MMP-28, or a combination thereof.Docket No. 33794 / 70815

[0012] In various embodiments, at least one of the one or more overexpressed membrane-bound proteases is a cathepsin. In some aspects, the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof. In some embodiments, at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domaincontaining protein (ADAM). In various aspects, the disintegrin and metalloproteinase domain-containing protein (ADAM) is ADAM10, ADAM17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof. In various embodiments, at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof. In some embodiments, the at least one or more overexpressed membrane-bound proteases is a human protease. In various embodiments, the at least one or more overexpressed membrane-bound proteases is increased by 2-fold, 5-fold, 10-fold, or more compared to a suitable control.

[0013] In some embodiments, the genetically modified immune cells are natural killer (NK) cells, T cells, or a combination thereof. In various aspects, the T cells are CD4 T cells, CD8 T cells, gamma-delta T cells, or a combination thereof. In some aspects, the natural killer (NK) cells are derived from NK92, NK92-GFP, NKL, YT, KHYG-1, NK92-CD16V, or a combination thereof. In various aspects, the genetically modified natural killer (NK) cells are derived from a human donor, optionally wherein the human donor is a cancer patient. In some aspects, the modified NK cells are derived from induced pluripotent stem cells (iPSCs). In some aspects, the genetically modified natural killer (NK) cells are autologous or allogenic. In various aspects, prior to genetic modification, the natural killer (NK) cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof. In some aspects, the genetically modified natural killer (NK) cells are further modified to express a chimeric antigen receptor (CAR). In various aspects, the natural killer (NK) cells are further genetically modified to overexpress one or more chemokines or cytokines. In some aspects, the one or more chemokines or cytokines are CCL2, CCL5, CCL20, CXCL1 , CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof. In various aspects, the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

[0014] In various embodiments, the genetic modification of the immune cells is performed by transformation, transfection, or transduction. In some aspects, the pharmaceutical composition of the disclosure further comprises a pharmaceutically acceptable carrier. In certain aspects, the pharmaceutical composition of the disclosure is formulated for administration to a human subject. In various aspects, the composition comprises theDocket No. 33794 / 70815genetically modified immune cells in a therapeutically effective amount for infiltrating a solid cancer. In some aspects, infiltration is increased by at least 30% when compared to an unmodified immune cell. In various aspects, the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof. In some aspects, the cancer is pancreatic cancer. In various aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some aspects, the subject suffering from cancer is a human subject. In various aspects, the genetically modified immune cells treat the disease, such as cancer, by targeting extracellular matrix (ECM) components, by targeting the cells producing the ECM components, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 demonstrates the overexpression of MMP-1, MMP-2, and MMP-14 proteins in human donor NK and T cells. A significant increase in MMP protein levels is observed upon stable transduction with the MMP lentivirus compared to parental NK cells (Figure 1 A) and T cells (Figure 1 B), p = 0.0071 , one-way ANOVA test).

[0016] Figure 2 demonstrates that MMP overexpression enhances the invasive capacity of human donor NK cells through the matrix. The bar graphs represent the percentage of NK cell invasion, showing a significant increase in invasion upon MMP overexpression compared to parental cells (p < 0.001 , one-way ANOVA test).

[0017] Figure 3 discloses that protease overexpression enhances invasion of Jurkat cell lines (immortalized human T cells) through matrix. Figure 3A shows a schematic representation of the transwell assay. Figure 3B provides quantification of the percent of jurkat plus empty vector, jurkat plus FAP OE, jurkat plus MMP-1 OE, jurkat plus MMP-2 OE, jurkat plus MMP-3 OE, and jurkat plus MMP-14 OE cells that invaded to the lower portion of transwell chambers. Empty vector n=4, FAP n=3, MMP-1 n=2, MMP-2 n=4, MMP-3 n=3, MMP-14 n=3. *p<0.05, **p<0.01, as determined by unpaired two-tailed t-test.

[0018] Figure 4 provides representative images from a human PDAC tumor spheroid assay. PDAC cells were cultured as spheroids and embedded in a collagen matrix to mimic the tumor extracellular environment. Parental and MMP-1 / 2-overexpressing human donor NK cells were co-cultured in appropriate growth media for 24 hours. Spheroids were then stained with DAPI and Phalloidin to identify tumor structures, while NK cells were labeled with a fluorescent dye. Samples were mounted and imaged using Zeiss confocal microscopy. Fluorescent images reveal increased NK cell infiltration upon protease overexpression compared to control.Docket No. 33794 / 70815

[0019] Figure 5 provides representative images from a human PDAC tumor spheroid assay. PDAC cells were cultured as spheroids and embedded in a collagen matrix to mimic the tumor extracellular environment. Parental and MMP-14-overexpressing human donor NK cells were co-cultured in appropriate growth media for 24 hours. Spheroids were then stained with DAPI and Phalloidin to identify tumor structures, while NK cells were labeled with a fluorescent dye. Samples were mounted and imaged using Zeiss confocal microscopy. Fluorescent images revealed NKcell infiltration, and induction of apoptosis.

[0020] Figure 6 demonstrates the increased cytotoxic effects of anti-mesothelin mouse CAR T cells against PDAC mouse cell lines. Figure 6A provides a bar graph showing the cytotoxicity of mesothelin-targeting mCART cells against a panel of KPC-derived PDAC murine cell lines, for the purpose of evaluating the cell-killing capacity of the mCART cells.Figure 6B shows a plot representing the normalized percentage of mT3-2D cell death as a function of the anti-mesothelin CAR T cell-to-mT3-2D cell ratio.

[0021] Figure 7 shows that FAP overexpression enhances NK cell tumor infiltration in vivo. Figure 7A provides schematic representation of the murine experimental design. Figure 7B shows representative hematoxylin and eosin-stained images of PANC-1 tumors injected with 1x107NK92 or FAP OE NK92 cells, n = 3 mice. Figure 7C provides quantification of NK92 or FAP OE NK92 cells per mm2in PANC-1 tumors. Figure 7D shows representative hematoxylin and eosin-stained images of PANC-1 tumors injected with 5x107NK92 or FAP OE NK92 cells, n = 5 mice. Figure 7E provides quantification of NK92 or FAP OE NK92 cells per mm2in PANC-1 tumors, n = 5 mice. *p<0.05, ****p<0.0001 as determined by unpaired two-tailed t-test. All bar plots represent mean ± S.D.

[0022] Figure 8 shows that FAP overexpressing NK cells display enhanced anti-tumor effects in vivo. Figure 8A provides schematic representation of the murine therapeutic experimental design. Figure 8B shows growth curves of PANC-1 tumor measurements in mice treated with NK92 or FAP OE NK92 cells, n = 4. Figure 8C provides weights of tumors in mice treated with either NK92 or FAP OE NK92 cells extracted and measured on day 27. n = 4. Figure 8D shows representative hematoxylin and eosin-stained images of PANC-1 tumors injected with 4 consecutive injections of NK92 or FAP OE NK92 cells, n = 4 mice. Figure 8E provides quantification of the percent of NK92 and FAP OE NK92 cells compared to total cells within the tumor. *p<0.05, ***p<0.001 as determined by unpaired two-tailed t-test. All bar plots represent mean ± S.D.

[0023] Figure 9 describes the generation of MMP-14-overexpressing NK cells. Figures 9A-9B provide quantification of MMP14 protein expression in parental NK cells and NK cells engineered to overexpress MMP14 (MMP OE NK). Figure 9A provides the results of aDocket No. 33794 / 70815western blot analysis with protein lysates from parental and engineered NK cells, wherein total protein was extracted using RIPA buffer containing protease inhibitors. Equal amounts of protein (20-30pg) were resolved on SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with anti-MMP-14 primary antibody and HRP-conjugated secondary antibody. B-actin was used as a loading control. Bands were visualized using enhanced chemiluminescence. Figure 9B shows quantification of band intensity corresponding to MMP14, generated via densitometric analysis of the immunoblot and subsequent signal intensity quantification using Imaged, and expressed in arbitrary units (a.u.) corresponding to the integrated optical density of the MMP14 band. Figure 9C provides RNA isolation and quantitative real-time PCR (qRT-PCR), where total RNA was isolated using silica membrane spin columns and reverse-transcribed into cDNA. qRT-PCR was performed using SYBR Green chemistry with gene-specific primers for MMP-1 , MMP-2, and MMP-14. Expression levels were normalized to housekeeping gene (GAPDH) and calculated using the 2A-AACt method. Data are presented as relative gene expression compared to parental NK cells. All data are shown as mean ± SEM from at least three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t-test. A p-value <0.05 was considered significant.

[0024] Figure 10 shows a schematic of a transwell invasion assay with NK cells overexpressing MMP-1, MMP-2, MMP-14, or FAP.

[0025] Figure 11 provides the results of transwell migration and invasion assays. Figures 11 A and 11 E show the results of a transwell invasion assay using 24-well transwell inserts with porous membranes coated with Matrigel, which demonstrate that NK cells (Figure 11 A) and T cells (Figure 11E) overexpressing extracellular matrix (ECM)-remodeling enzymes showed increased invasion compared to parental cells. Figures 11 B and 11 F show that across multiple ECM barriers, including Matrigel, fibronectin, collagen IV, and collagen I, MMP-14-overexpressing NK cells (Figure 11 B) and T cells (Figure 11 F) consistently outperformed wild-type cells. Figures 11C and 11G demonstrate that when Matrigel polymerization time was extended, wild-type NK cell (Figure 11C) and T cell (Figure 11G) invasion dropped sharply, whereas MMP-14 overexpressing cells maintained high invasion rates. Figure 11 D and shows that MMP-14-overexpressing NK cells invaded collagen I more efficiently than WT cells, including at early time points following matrix stabilization. Data are presented as mean ± variability across replicates. Statistical significance was assessed using pairwise comparisons as indicated. P values are reported on the graphs.

[0026] Figure 12 demonstrates that a chimeric MMP-1 mirrors MMP-14 in driving T cell infiltration. A chimeric MMP-1 construct was designed, in which the catalytic domain of human MMP-1 was fused to the transmembrane and cytoplasmic domains of MMP-14. TheDocket No. 33794 / 70815chimeric MM P-1 construct was cloned into a lentiviral expression vector and used to transduce primary human T cells under standard activation conditions. Transduced cells were expanded and used directly in downstream invasion assays.

[0027] Figure 13 shows that MMP-14 overexpression enhances NK cell infiltration and collagen degradation in vivo. Figure 13A is an experimental schematic showing the timeframe for subcutaneous injection of MIA PaCa cells into immunodeficient mice to establish subcutaneous tumors, injection of a single dose of NK cells that were either wildtype or engineered to overexpress MMP-14, and tumor harvest and analysis. Each group included n = 8 mice. Figure 13B shows the result of tumor sections stained with Masson’s trichrome to assess collagen content (left panel), and tumor sections immunostained for CD56 to identify NK cells. Subcutaneous tumors were established in immunodeficient mice. Collagen area and NK cell infiltration were quantified using image analysis across multiple tumor regions per sample. Data are presented as percentage of total tumor area or total cells, respectively. Statistical comparisons were performed between WT and MM P-14-overexpressing NK cell groups.

[0028] Figure 14 provides results from treatment of an orthotopic PDAC model with NK cells. Figure 14A shows a schematic of the mouse treatment schedule, where orthotopic pancreatic tumors were established in NSG mice using ASPC1 cells. Mice were randomized to receive PBS, wild-type human donor NK cells, FAP-overexpressing cells, or MMP-14-overexpressing NK cells. NK cells were administered with systemic cytokine support using IL- 15 and IL-2 according to the indicated schedules. Tumors were harvested at the defined endpoint for histologic analysis. Figure 14B provides images of tumor sections immunostained for aSMA to identify cancer-associated fibroblasts, and immunostained for CD56 to detect infiltrating NK cells. Figures 14C-14D provide results of whole slide imaging and subsequent quantification as number of cells per mm2using standardized image analysis (Qupath software), where each data point represents an independent tumor region of interest for aSMA positive cells (Figure 14C) and CD56 positive cells (Figure 14D).Statistical comparisons were performed between treatment groups, with p values as indicated.

[0029] Figure 15 provides characterization and analysis of murine PushCART cells. Figure 15A shows flow cytometry analysis of activated murine T cells transduced with a mesothelin-targeting murine CAR (mCART). Transduction efficiency, lineage identity, and purity were assessed by flow cytometry using CD4, CD8, and NKp46 markers to confirm T cell identity and exclude NK cell contamination. Figure 15B confirms mesothelin expression in murine tumor cell lines as quantified via flow cytometry, using CD8 T cells as a control. Figure 15C provides confocal microscopy images of mesothelin-positive murine tumorDocket No. 33794 / 70815spheroids co-cultured with mCART cells, MMP-14-expressing T cells, or MMP-14-mCART PushCAR T cells. Tumor spheroids were fixed and stained with DAPI to visualize cellular architecture and cleaved caspase-3 to assess tumor cell apoptosis.

[0030] Figure 16 provides an evaluation of primary human T cells engineered to overexpress fibroblast activation protein (FAP) and parental wild-type (WT) T cells for their invasive capacity, using 24-well transwell inserts with porous membranes coated with Matrigel. Cells were seeded in the upper chamber and allowed to migrate towards media containing CXCL9 in the lower chamber as a chemoattractant. After incubation, nonmigrated cells were removed from the upper surface of the membrane, and cells that traversed the ECM barrier were quantified. Invasion was calculated as the percentage of total input cells migrating through the membrane. Bars represent mean ± SEM of 2 biological repeats.DETAILED DESCRIPTION

[0031] Pharmaceutical compositions containing genetically modified immune cells that overexpress one or more membrane-bound proteases have been surprisingly and advantageously found to enhance the invasive capacity of the immune cells. Such surprisingly improved invasion capacity is advantageous for treatment of cancers, and particularly those involving solid tumors. The ability of the modified immune cells of the disclosure to be useful in the treatment of solid tumors is contrary to expectations in the art, as many proteases are associated with pro-tumorigenic activities. Indeed, cancer cells up-regulate proteases such as matrix metalloproteinases (MMPs) in order to metastasize (Reunanen, N., & Kahari, V. M. "Matrix Metalloproteinases in Cancer Cell Invasion" (PMC6598). Without wishing to be bound by any particular theory, it is contemplated herein that membrane-bound proteases promote proteolytic invasion of immune cells into the tumor stroma as well as the tumor itself, and that overexpression of membrane-bound proteases in immune cells is preferable to overexpression of soluble proteases due to (1) the potential metastatic consequences of flooding the tumor microenvironment with soluble proteases that degrade tumor matrix and scaffolding, and promote the migration of tumor cells into the tumor vasculature, and (2) the potential immunosuppressive consequences of soluble proteases breaking down the tumor stroma, releasing immunosuppressive cells and factors. In other words, an immune cell expressing a soluble protease will successfully invade the tumor microenvironment, but the consequences of flooding the tumor with immune cells shedding proteases may outweigh the benefits of immune cells successfully infiltrating the tumor microenvironment. Accordingly, the genetically modified immune cells of the disclosure are modified to express one or more membrane-bound protease, which isDocket No. 33794 / 70815believed to impart the benefits of enhanced immune infiltration without having to account for the negative effects of flooding the tumor with soluble proteases.

[0032] The ability of engineered immune cells to infiltrate tumor matrix and enter the tumor microenvironment is unexpected in view of the field’s understanding of the role of proteases in cancer development and progression. Tumors can upregulate protease expression to remodel the matrix in the tumor microenvironment and promote epithelial-to-mesenchymal transition of the tumor cells, eventually progressing from a primary tumor to a metastatic tumor. Based on current understanding of the role of proteases in cancer, overexpressing proteases in the tumor microenvironment would be expected to be a terrible strategy for patients, and yet overexpression of specifically membrane-bound proteases in immune cells unexpectedly promoted infiltration of the immune cells through model matrix (Figures 2-3), and into a tumor (Figures 7-8) and inhibits tumor progression. Surprisingly, overexpression of a membrane-bound protease yielded significantly greater infiltration of immune cells through a matrix than overexpression of soluble proteases, supporting a strategy of using membrane-bound proteases to improve immune cell infiltration and cytotoxic killing of tumors in subjects in need thereof.

[0033] Provided herein are pharmaceutical compositions comprising genetically modified immune cells, and pharmaceutical compositions for use in treating a disease comprising a therapeutically effective amount of genetically modified immune cells, wherein the genetically modified immune cells are altered to overexpress one or more membrane-bound protease. Further provided are methods of enhancing the ability of immune cells to migrate into a tumor comprising genetically modifying the immune cells to overexpress a membranebound protease, and a method of preparing one or more genetically modified immune cells comprising: transfecting a vector containing a gene for one or more membrane-bound proteases into one or more immune cells in a medium; replicating the one or more immune cells transfected with the vector, and; optionally isolating the one or more immune cells transfected with the vector that overexpress the one or more membrane-bound proteases.

[0034] Further disclosed herein are genetically modified immune cells overexpressing one or more membrane-bound proteases, wherein the genetically modified immune cell is capable of infiltrating extracellular matrix (ECM). In some embodiments, the genetically modified immune cell is capable of infiltrating ECM, with an increased invasive capacity when compared to a wild-type immune cell. In various embodiments, the genetically modified immune cell overexpresses one or more matrix metalloproteinases (MMPs). In some embodiments, the one or more genetically modified immune cell is capable of infiltrating ECM in the stroma of a tumor.Docket No. 33794 / 70815

[0035] Natural killer (NK) cells are innate lymphoid cells that influence many physiologic and pathologic conditions through their effector and regulatory functions1. NK cells are canonically known to recognize and kill aberrant cells, such as virus-infected or malignant cells, using a complex detection system comprised of multiple inhibitory and activating receptors1. Beyond their roles as effector cells, NK cells regulate the functions of other cell types, including dendritic cells, T cells, B cells, and endothelial cells, through the release of immunomodulating cytokines2-6. Due to their central role in the immune system and disease etiologies, efforts to manipulate NK cell activity have been sought to improve patient outcomes across many medical fields, but none of these efforts have identified an effective way to improve NK cell infiltration into solid tumors.

[0036] In cancer, patients with high tumoral NK cell content and activation have improved survival78and response to immunotherapy9-11. Thus, NK cells are emerging as major targets to promote cancer immunotherapy12. Current NK-focused immunotherapy approaches include autologous or allogeneic NK cell transfer13, chimeric antigen receptor-engineered (CAR) NK cells14, NK cell immune checkpoint inhibitors15, bi- or tri-specific killer engagers (BiKEs and TriKES)16, and cytokine super-agonists17. An impediment to these therapies is inadequate NK cell homing to and / or infiltration into solid tumors.

[0037] To date, strategies to increase NK cell infiltration have focused almost entirely on modulating NK chemokine receptors and chemoattractants18'19. However, lymphocyte migration depends on more than just chemotaxis. For NK cells to successfully infiltrate any tissue, including solid tumors, they must traverse complex microenvironments (e.g., extravasation from blood vessels and navigation through dense extracellular matrices)20. Beyond the chemokine / chemoattractant system, little is known about the mechanisms NK cells employe to physically migrate through these tissues. It has been surprisingly found that modification of immune cells, including but not limited to NK cells, to overexpress a membrane-bound protease significantly improved immune cell infiltration into solid tumors (see U.S. Patent Application No. 18 / 263,129, incorporated by reference herein).

[0038] As used herein, “nucleic acid” can include polynucleotides such as deoxyribonucleic acid and, where appropriate, ribonucleic acid (RNA).

[0039] Unless stated otherwise, the term “vector” is used herein to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectorsDocket No. 33794 / 70815having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In some embodiments, the genes to which the vectors are operably linked encode a gene product. In various aspects, the gene product is a peptide, a protein, or a combination thereof. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure contemplates such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0040] “Transduction” includes any process by which exogenous DNA enters a host cell. Transduction may occur under natural or artificial conditions using various methods well known in the art for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell, for example. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transduced” cells include stably transduced cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

[0041] “Transfection” includes methods for introducing bio-active materials, such as nucleic acids, proteins, enzymes, or small molecules, into a cell. The nucleic acids may be DNA, delivered as a plasmid or an oligonucleotide, RNA, or a combination thereof.

[0042] Unless stated otherwise, the term “expression” refers to the process by which genes are transcribed into RNA, and optionally, translated into peptides, polypeptides, or proteins. In some embodiments, “expression” refers to the transcription of a specific gene or specific gens or a specific nucleotide construct; the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA construct. If the gene is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the RNA.Docket No. 33794 / 70815

[0043] Overexpression, as is understood in the art, is an increased expression of a gene, or gene product thereof (e.g., the encoded protein) in a genetically modified immune cell as compared to the expression of said gene or gene product in a suitable control.Overexpression in the products and methods of the disclosure can include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1010%, 1020%, 1030%, 1040%, 1050%, 1060%, 1070%, 1080%, 1090%, 1100%, 1110%, 1120%, 1130%, 1140%, 1150%, 1160%, 1170%, 1180%, 1190%, 1200%, 1210%, 1220%, 1230%, 1240%, 1250%, 1260%, 1270%, 1280%, 1290%, 1300%, 1310%, 1320%, 1330%, 1340%, 1350%, 1360%, 1370%, 1380%, 1390%, 1400%, 1410%, 1420%, 1430%, 1440%, 1450%, 1460%, 1470%, 1480%, 1490%, or / to 1500% or more increased expression relative to a suitable control. For example, “overexpression” can be about a 100% increase in expression, or about a 50% to about a 200% increase in expression, or about a 100% to about a 250% increase in expression, or about a 250% to about a 500% increase in expression, or about a 300% to about a 700% increase in expression, or about a 500% to about a 1000% increase in expression, or about a 50% to about a 250% increase in expression, or about a 100% to about a 500% increase in expression, or about a 100% to about a 1000% increase in expression relative to a suitable control. Overexpression can be an increased expression of a gene or gene product thereof of about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more relative to a suitable control. For example, “overexpression” refers to about a 1 .5-fold to about a 100-fold, about a 2-fold to about a 100-fold, about a 3-fold to about a 100-fold, about a 4-fold to about a 100-fold, about a 5-fold to about a 100-fold, about a 10-fold to about a 100-fold, about 20-fold to about a 100-fold, about 30-fold to about a 100-fold, about a 40-fold to about a 100-fold, about a 50-fold to about a 100-fold, about a 50-fold to about a 100-fold, about a 60-fold to about a 100-fold, about a 70-fold to about a 100-fold, about a 80-fold to about a 100-fold, about a 90-fold to about a 100-fold increase, about a 2-fold to about a 50-fold, about a 3-fold to about a 50-fold, about a 4-fold to about a 50-fold, about a 5-fold to about a 50-fold, about a 6-fold to about a 50-fold, about a 7-fold to about a 50-fold, about an 8-fold to about a 50-fold, about a 9-fold to about a 50-fold, about a 10-fold to about a 50-fold, about a 20-fold to about a 50-fold, about a 30-fold to about a 50-fold,Docket No. 33794 / 70815about a 40-fold to about a 50-fold, about a 2-fold to about a 25-fold, about a 3-fold to about a 25-fold, about a 4-fold to about a 25-fold, about a 5-fold to about a 25-fold, about a 6-fold to about a 25-fold, about a 7-fold to about a 25-fold, about a 8-fold to about a 25-fold, about a 9-fold to about a 25-fold, about a 10-fold to about a 25-fold, about a 20-fold to about a 25-fold, about a 2-fold to about a 10-fold, about a 3-fold to about a 10-fold, about a 4-fold to about a 10-fold, about a 5-fold to about a 10-fold, about a 6-fold to about a 10-fold, about a 7-fold to about a 10-fold, about an 8-fold to about a 10-fold, about a 9-fold to about a 10-fold, about a 2-fold to about a 5-fold, about a 3-fold to about a 5-fold, or about a 4-fold to about a 5-fold in expression of a gene or gene product thereof relative to a suitable control. For example, “overexpression” can be about a 5-fold or more increase in expression of a gene or gene product thereof relative to a suitable control. A person of skill in the art can determine what is a “suitable control” given the overexpress described. For example, in the overexpression described herein, the suitable control can include a wild-type cell or an unmodified cell, wherein the wild-type cell or unmodified cell is the same type of cell as the genetically modified immune cell.

[0044] Overexpression can include the situation when a nucleotide sequence or protein is native to the host cell, whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter. Overexpression can also include when a nucleotide sequence or protein that is not native to the host cell is expressed in the host cell.

[0045] In some embodiments, the host cell is genetically modified to express a heterologous (e.g., non-native, exogenous, foreign, or recombinant) target regulatory protein. As used herein, “heterologous” can refer to a protein that is not native (or homologous, or endogenous) to the host organism into which it is introduced. As used herein, “overexpression” can also refer to a heterologous gene and corresponding protein. As used herein, host cells will have higher gene and protein levels of the heterologous protein than a control host cell, as the heterologous protein will be absent in the control host cell. “Overexpression” may refer to any levels of non-native protein expressed in the host cell.

[0046] In the present disclosure, an endogenous protein, e.g., native to the host cell, in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been made by recombinant DNA techniques to alter the native polypeptide may be referred to as an “overexpressed” protein in the host cell, if the modified protein levels are compared to levels of the corresponding endogenous protein.Docket No. 33794 / 70815

[0047] An “effective amount” or a “therapeutically effective amount” can refer to the amount of a therapeutic that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).

[0048] “At least one” can mean one or more, for example one, one to two, one to three, one to four, or more.

[0049] “Composition” as used herein can include a product comprising the specified components in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified components in the specified amounts.

[0050] The term “chemokine” can refer to a cytokine involved in chemotaxis.Immune Cells

[0051] “Immune cells” can refer to any cell that is part of the immune system and helps the body fight infections and other diseases. Immune cells develop from stem cells in the bone marrow and become different types of white blood cells. Immune cells can include, but are not limited to, neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells).

[0052] Disclosed herein are immune cells that are genetically modified to overexpress one or more membrane-bound proteases. In some embodiments, the immune cells are mammalian cells. In some embodiments, the mammalian cells are primary cells. In some embodiments, the immune cells are differentiated from stem cells. Exemplary stem cells include, without limitation, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (i.e., blood stem cells), mesenchymal stem cells (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the immune cells are cells derived or differentiated from stem cells of the present disclosure (e.g., from an ESC or iPSC). For example, immune cells can include, without limitation, cells of the lymphoid lineage. The lymphoid lineage provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Examples of immune cells of the lymphoid lineage include, without limitation, T cells, NK cells, and immune cells derived from embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells. T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immuneDocket No. 33794 / 70815system. In some embodiments, T cells of the present disclosure can be any type of T cells, including, without limitation, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal associated invariant T cells, and yd T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells may be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as chimeric TCRs or CARs. Further contemplated are immune cells derived from the myeloid lineage, which include, without limitation, dendritic cells, myeloid cells, macrophages, and monocytes.

[0053] Natural Killer or “NK” cells are a type of immune cell — large, granular lymphocytes with the central role of killing the virus-infected and malignantly transformed cells, without prior sensitization. A natural killer cell is the type of white blood cell that comprises part of the innate immune system. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells. Natural Killer cells are lymphocytes in the same family as B cells and T cells. In some embodiments, the NK cells are derived from NK92, NK29-GFP, NKL, YT, KHYG-1 , NK92-CD16V, or a combination thereof. In various embodiments, the NK cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof.

[0054] T cells are lymphoid cells that expresses a T cell receptor molecule. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof. T cells can be CD4+, CD8+, or CD4+and CD8+. T cells can be helper cells, for example helper cells of type Th1 , Th2, Th3, Th9, Th17, or TFH. T cells can be cytotoxic T cells. Regulatory T cells can be FOXP3+or FOXP3-. T cells can be alpha / Beta T cells or gamma / delta T cells. In some cases, the T cell is a CD4+CD25hiCD12710regulatory T cell. In some cases, the T cell is a regulatory T cell selected from the group consisting of Tr1 , Th3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or sub-population thereof. In some cases, the T cell is a FOXP3+T cell. In some cases, the T cell is a CD4+CD25l0CD127hieffector T cell. In some cases, the T cell is a CD4+CD25loCD127hiCD45RAhiCD45RO- naive T cell.Membrane-Bound and Soluble Proteases

[0055] Proteases can be differentiated based in part on whether they are bound to the surface of a cell, or whether they are secreted into the extracellular space. The activities ofDocket No. 33794 / 70815proteases, and specifically the substrates they can interact with, depend on whether they are diffused throughout the extracellular space or limited to the substrates proximal to the cell surface upon which they are expressed, with membrane-bound proteases having tighter substrate specificity due to the inherent restrictions on the number and type of substrates they will encounter while bound to the membrane. Membrane-bound proteases contain domains that tether them directly to the cell surface, with their catalytic domains exposed to the extracellular environment. Examples of tethering domains include, but are not limited to, transmembrane domains and glycosylphosphatidylinositol (GPI) anchors. In contrast, soluble proteases typically comprise a secretory domain that signals to cellular machinery. The secretory may comprise a pro-domain, wherein cleavage of the pro-domain from the catalytic site of the protease serves to sort the protease into the proper secretory pathway. The secretory domain may also comprise a cleavage site between the protease and a transmembrane domain or GPI anchor, wherein cleavage releases the protease from the cell membrane. In various embodiments, such as when the soluble protease is a matrix metalloproteinase, the secretory domain is a pro-MMP cleavage site. While proteases are generally categorized as “membrane bound” or “soluble”, membrane-bound proteases can be released from the membrane through a process known as ectodomain shedding, via cleavage of the protease on either side of the membrane.

[0056] While endogenous processes can convert a membrane-bound protease into a soluble protease via ectodomain shedding, soluble proteases can be synthetically converted into recombinant membrane-bound versions of the otherwise soluble protease. The synthetic conversion typically involves engineered to create recombinant membrane-bound version of the otherwise soluble protease. Methods of engineering membrane-bound proteases are known in the field, for example Oda et al.56, which is incorporated herein in its entirety. In some embodiments, a modified soluble MMP coding sequence will be integrated into an E. coli plasmid vector. A bimodal modification is made to the protein-coding sequence: the first modification will be to integrate a GPI anchor or a transmembrane domain into the cell membrane binding domain of the MMP to ensure efficacious adherence in the tricomplex between cell membrane, GPI anchor or transmembrane domain, and MMP; the second modification will be to remove the soluble protease cleavage site that could be recognized and cleaved by, for example, a sheddase. Optionally, a third modification may be made to the protein-coding sequence; the introduction of a histidine-6 tag or a tdTomato tag to optimize vector size while preserving detection capability. Successful expression of a membrane-bound protease can be evaluated in culture media via spectrophotometric analysis of eluent after affinity-based chromatographic filtration through a cobalt resin column. Any dissociated MMP will be bound to resin via the histidine-6 tag.Docket No. 33794 / 70815

[0057] As a soluble protease can be engineered as a membrane-bound protease for the purposes of the disclosure, any protease (once modified to be membrane-bound) can be used in the modified immune cells and methods of the disclosure. In some embodiments, the one or more membrane-bound proteases is a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a metalloprotease. In some embodiments, the one or more membrane-bound proteases is a matrix metalloproteinase. In various embodiments, the membrane-bound matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25, or a combination thereof. In some embodiments, the membrane-bound matrix metalloproteinase is a soluble matrix metalloproteinase engineered to be membrane-bound, and is selected from MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP-26, MMP-27, MMP-28, or a combination thereof. In various embodiments, the membrane-bound matrix metalloproteinase is a protein encoded by a nucleotide sequence as set out in SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the membrane-bound matrix metalloproteinase comprises a protein encoded by a nucleotide sequence as set out in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the nucleotide sequence is further modified to encode a transmembrane domain, a GPI anchor, or a combination thereof. In various embodiments, the one or more membrane-bound proteases is a cathepsin. In some aspects, the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof. In various embodiments, the one or more membrane-bound proteases is a disintegrin and metalloproteinase domaincontaining protein (ADAM). In some aspects, the ADAM is ADAM10, ADAM17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof. In some embodiments, the one or more membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), fibroblast activation protein (FAP), or a combination thereof. In various embodiments, wherein the membrane-bound protease comprises FAP, FAP comprises a protein encoded by a nucleotide sequence as set out in SEQ ID NO: 5. In some embodiments, other proteases that can be used in conjunction with the disclosure are tumor-associated proteases. In some aspects, the tumor associated proteases include Cathepsin B, Cathepsin L, Cathepsin S, Cathepsin D, Cathepsin E, Cathepsin A, Cathepsin G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator, MMP-1, MMP-2, MMP-3, MMP-4, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-20, MMP-21, MMP-23, MMP-24, MMP-25, MMP-26, MMP-28, ADAM, ADAMTS, CD10 (CALLA), or prostate specific antigen. In various embodiments, the one or more membrane-bound proteases expressed or overexpressed by the immune cells of the disclosure are human proteases.Docket No. 33794 / 70815Matrix Metalloproteinases

[0058] Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the metzincin superfamily of metalloproteinases, which also includes the ‘a zinc and disintegrin metalloproteinase’ (ADAM) family. MMPs cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal.

[0059] MMPs can be sorted into six groups, based on substrate specificity and sequence similarity. These groups comprise collagenases (MMP-1, -8, -13, and -18), gelatinases (MMP-2 and -9), stromelysins (MMP-3, -10, and -11), matrilysins (MMP-7 and -26), enamelysins (MMP-20), a further diverse group (MMP-19, -21, -23A, -23B, -27, and -28)22, as well as membrane type (MT) MMPs (MMP-14, -15, -16, -17, -24, and -25)23. The MT-MMPs are also referred to by their MT-MMP numerical nomenclature, wherein MMP-14 is MT1-MMP, MMP-15 is MT2-MMP, MMP-16 is MT3-MMP, MMP-17 is MT4-MMP, MMP-24 is MT5-MMP, and MMP-25 is MT6-MMP. As referred to herein, “membrane-associated domains” are the domains which attach MT-MMPs to the cell surface, and include, but are not limited to, a transmembrane domain and a glycophosphatidylinositol (GPI) anchor.Endogenous MMP-14, -15, -16, and -24 comprise a transmembrane domain as a membrane-associated domain, and endogenous MMP-17 and-25 comprise a GPI anchor. However, this does not preclude the engineering of, for example, MMP-14 to further comprise a GPI anchor. Anchoring to the cell surface restricts the area of potential proteolytic activity of MT-MMPs to sites on or directly adjacent to the plasma membrane.

[0060] While a finely tuned array of MMPs is instrumental for orchestrating tissue development and homeostasis, the misregulation of MMPs is widespread in many pathological settings and especially in cancer. MMPs are the principal mediators of the alterations observed in the microenvironment during cancer progression (Kessenbrock et al., Cell, 141 : 52-67, 2010). It is generally expected that MMP overexpression contributes to tumorigenesis and tumor progression through multiple mechanisms. MMP proteolysis serves a path-clearing role in facilitating the movement of cells or groups of cells through the extracellular matrix (ECM)(Page-McCaw et al., Nat Rev Mol Cell biol, 8(3): 221-33, 2007); in this process, cleavage of some ECM components unmasks cryptic sites, generating fragments with new biological activities modulating migration, growth, or angiogenesis.MMPs also cleave cell-ECM adhesion proteins and cell-cell junction proteins, releasing individual epithelial cells from epithelial sheets, initiating outside-in signaling pathways thatDocket No. 33794 / 70815lead to widespread changes in gene transcription patterns, or generating soluble ectodomain fragments with novel activities.

[0061] Substantial evidence exists supporting the tumorigenic role of MMPs in cancer development and progression. For example, MMP-1 (a soluble MMP) cleaves and activates the protease activated receptor-1 (PAR-1), leading to increased migration and invasion of breast cancer cells (Boire et al., Cell, 120(3): 303-13, 2005). As another example, the targeting of E-cadherin by MMP-3 or MMP-7 (both soluble MMPs) generates a bioactive fragment that promotes invasion, and contributes to a cascade of molecular alterations leading to EMT in mammary epithelial cells (Lochter et al., J Cell Biol, 139(7): 1861-72, 1997). As yet another example, MMP-7 overexpression has been shown to correlate with an aggressive phenotype in many malignant tumors, including colorectal cancers (Klupp et al., BMC Cancer, 16: 494, 2016), gastric cancers (Koskensalo et al., Tumor Biol, 31 : 149-155, 2010), esophageal cancers (Miao et al., Clinical Biochemistry, 92: 9-18, 2021), and pancreatic cancers (Jones et al., Clin Cancer Res, 10(8): 2832-45, 2004).

[0062] MMP-14, also known as membrane-type MMP (MT-MMP), has been shown to be overexpressed in colorectal cancer and is associated with poor prognosis. It facilitates tumor invasion by activating pro-MMP-2, which subsequently degrades ECM components, promoting cancer cell migration and metastasis31. Additionally, MMP-14 expression correlates with a501 -integrin levels, further emphasizing its role in tumor cell adhesion and invasion32. In multiple myeloma, MMPs are implicated in the pathogenesis of bone disease associated with the malignancy. The broad-spectrum MMP inhibitor batimastat has been shown to prevent the degradation of mineralized trabeculae and reduce osteoclast recruitment, indicating that MMPs contribute to osteolytic lesions in this cancer type33. This highlights the multifaceted roles of MMPs not only in tumor progression but also in the associated bone pathology. MMP-11 has been linked to the progression of hepatocellular carcinoma, where it mediates metastasis. Studies have demonstrated that knockdown of MMP-11 in hepatocarcinoma cell lines inhibits metastatic proliferation, suggesting its critical role in cancer dissemination34. Similarly, MMP-3 has been identified as a stromal marker in ovarian cancer, where its overexpression is associated with increased invasion and poor prognosis35. MMP-3's involvement in the degradation of ECM components facilitates tumor cell migration, further supporting its role in cancer progression36. Moreover, MMP-9 has been extensively studied in various cancers, including breast and colorectal cancers. Elevated levels of MMP-9 have been associated with lymph node metastasis in breast cancer patients, indicating its potential as a prognostic biomarker37. In colorectal cancer, MMP-9 expression is significantly higher in tumor tissues compared to adjacent normal tissues, correlating with tumor aggressiveness and metastasis38. In gastric cancer, MMP-1 andDocket No. 33794 / 70815MMP-2 have been shown to play significant roles in peritoneal dissemination. MMP-1 facilitates cancer cell motility by degrading ECM components, while MMP-2 is involved in the degradation of type IV collagen, further promoting tumor invasion39. The expression of these MMPs is crucial for understanding the metastatic potential of gastric cancer. In summary, the literature provides substantial evidence of the involvement of various MMPs, including MMP-1 , MMP-2, MMP-3, MMP-9, MMP-11 , and MMP-14, in cancer development and progression across multiple cancer types. Their roles in ECM remodeling, tumor cell migration, and metastasis underscore their potential as therapeutic targets and prognostic biomarkers in oncology.

[0063] Given the pro-tumorigenic role MMPs play in a multitude of cancers, there is an expectation of potential complications with a cancer treatment strategy involving MMP overexpression within the tumor microenvironment, such as transitioning a primary tumor to a metastatic tumor. Epithelial-to-mesenchymal transition (EMT), for example, is a process integral to the formation of many tissues and organs during development, termed developmental EMT (Radisky et al., J Cell Sci, 118(19): 4325-6, 2005). However, in the context of tumorigenesis EMT is pathological EMT, defined as the acquisition of mesenchymal phenotype by malignant epithelial cells to allow for increased migration and invasion ultimately required for metastasis. MMPs have been implicated as promoters and mediators of developmental and pathogenic EMT processes in cancer, as MMPs degrade and modify the extracellular matrix (ECM) and cell-ECM and cell-cell contacts, facilitating detachment of epithelial cells from the surrounding tissue. MMPs are specifically associated with EMT in cancer progression through three distinct mechanisms: (1) elevated levels of MMPs in the tumor microenvironment can directly induce EMT in epithelial cells, (2) cancer cells that undergo EMT can produce more MMPs, facilitating cell invasion and metastasis, and (3) EMT can generate activated stromal-like cells that drive cancer progression via further MMP production. Given the number of tumor-progression and metastatic mechanisms MMPs are implicated in, MMP subtype expression is a diagnostic and prognostic biomarker in many types and stages of cancer (Roy et al., J Clin Oncol, 27: 5287-97, 2009). The current understanding of MMPs in cancer is as a poor prognostic indicator, with significant increases in MMP expression correlating with worse outcomes in colon (Murashige et al., Jpn J Clin Oncol, 26:303-309, 1996), ovarian (Young et al., Gynecol Oncol, 62: 89-99, 1996), breast (Talvensaari-Mattila et al., Cancer (Phila), 83: 1153-62, 1998), prostate (Stearns et al., Oncol Res, 8: 69-75, 1996), lung (Kawano et al., Hum Pathol, 28: 613-22, 1997), gastric (Nomura et al., Cancer Res, 55: 3263-66, 1995), and pancreatic cancer (Jones et al., Clin Cancer Res, 10(8): 2832-45, 2004). In pancreatic cancer specifically, MMP-7, MMP-8, MMP-9, MMP-10, and MMP-11 overexpression has beenDocket No. 33794 / 70815reported, with both MMP-7 and MMP-11 demonstrating prognostic significance (Jones et al., Clin Cancer Res, 10(8): 2832-45, 2004). Thus, any therapeutic intervention based on MMP expression in cancer would be a strategy of MMP inhibition. Increasing MMP expression, in the context of MMP overexpression being correlated with poor prognosis, would be viewed by those with skill in the field as being contrary to the state of the art.

[0064] Despite the tumor-promoting implications of MMP expression in cancer, MMPs do not act as universal tumor promoters under all circumstances, and the effects observed can also vary depending upon the model and upon the genetic background of a subject suffering from cancer (Martin et al., Cancer Res, 68(15):6251 -9, 2008). For example, mammary-directed overexpression of MMP-1 in mice has a tumor-promoting effect (Masson et al., J Cell Biol, 140(6): 1535-41, 1998), while MMP-1 had a growth suppressing effect in a MMTV-PyMT multistage mammary tumorigenesis model (Szabova et al., Oncogene, 27(23): 3274-81 , 2008). For a large variety of MMPs, MMP overexpression in cancer is generally expected in the art to be associated with increased tumor growth, angiogenesis and vascularization, EMT and ECM breakdown, and subsequent metastasis. Nevertheless, MMP inhibition has been evaluated as a therapeutic point of intervention in cancer. (Coussens et al., Science, 295(5564): 2387-92, 2002).

[0065] MMPs are not the only proteases upregulated in cancer to promote tumor progression and metastasis. Other tumor associated proteases, i.e., proteases expressed by tumors, include, for example, a cathepsin, a cysteine protease, an aspartyl protease, a serine protease, or a metalloprotease. Specific examples of tumor associated proteases include Cathepsin B, Cathepsin L, Cathepsin S, Cathepsin D, Cathepsin E, Cathepsin A, Cathepsin G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator, Metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-4, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-20, MMP-21, MMP-23, MMP-24, MMP-25, MMP-26, MMP-28, ADAM, ADAMTS, CD10 (CALLA), or prostate specific antigen. As with MMPs, the conventional treatment of cancers would target inhibiting these protease activity. In view of the known pro-tumor activities of various proteases, the skilled person would not have explored the overexpression of proteases in a pharmaceutical composition for treatment of cancer. Rather, overexpression may be considered to be deleterious to treatment of the cancer. It has surprisingly been found, however, that immune cells engineered to overexpress one or more membrane-bound protease improved cell infiltration and, thereby, improves the pharmaceutical compositions cytotoxicity against the tumor and efficacy in treating cancers. The findings of the disclosure demonstrating that upregulating proteases on immune cells to improve tumor infiltration was contrary to the expectations in the art.Docket No. 33794 / 70815Immune Cells Expressing One or More Immunomodulatory Factor

[0066] In addition to genetically modifying an immune cell to express one or more membrane-bound protease, disclosed herein is the further modification of an immune cell to express one or more immunomodulatory factors. In some embodiments, a genetically modified immune cell expressing one or more membrane-bound protease further expresses a chimeric antigen receptor (CAR). In various embodiments, a genetically modified immune cell expressing one or more membrane-bound protease further overexpresses one or more chemokines or cytokines.

[0067] Chimeric Antigen Receptors (CARs). Certain aspects of the present disclosure relate to genetically modified immune cells further engineered to express a chimeric antigen receptor (CAR). In general, CARs are chimeric proteins that include an antigen-binding domain and polypeptide molecules that are heterologous to the antigen-binding domain, such as peptides heterologous to an antibody that an antigen-binding domain may be derived from. Polypeptide molecules that are heterologous to the antigen-binding domain can include, but are not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or combinations thereof.

[0068] In some embodiments, the genetically modified immune cells of the disclosure is an isolated immune cell comprising one or more chimeric receptors. In some embodiments, the genetically modified immune cells of the disclosure comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more chimeric receptors.

[0069] In some embodiments, CARs are engineered receptors that graft or confer a specificity of interest onto an immune effector cell. In certain embodiments, CARs can be used to graft the specificity of an antibody onto an immune cell, such as a T cell. In some embodiments, CARs of the present disclosure comprise an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, fused to one or more intracellular signaling domains.

[0070] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR, also generally referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of the immune cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of the immune cell. In some embodiments, activation of an immune cell results in killing of target cells. In some embodiments, activation of an immune cell results in cytokine or chemokineDocket No. 33794 / 70815expression and / or secretion by the immune cell. In some embodiments, stimulation of an immune cell results in cytokine or chemokine expression and / or secretion by the immune cell. In some embodiments, stimulation of an immune cell induces differentiation of the immune cell. In some embodiments, stimulation of an immune cell induces proliferation of the immune cell. In some embodiments, activation and / or stimulation of the immune cell can be combinations of the above responses.

[0071] A CAR of the present disclosure may be a first, second, or third generation CAR. "First generation" CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. "First generation" CARs generally have the intracellular signaling domain from the CD3-zeta (CD3£) chain, which is the primary transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3 chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1 BB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. "Second generation" CARs provide both costimulation (e.g., CD28 or 4- 1 BB) and activation (CD3 ). Preclinical studies have indicated that "Second Generation" CARs can improve the anti-tumor activity of immunoresponsive cell, such as a T cell. "Third generation" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4-1 BB) and an intracellular activation signaling domain (CD3 ).

[0072] In some embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure binds to one or more antigens with a dissociation constant (Kd) of about 2 x 10-7M or less, about 1 x 10-7M or less, about 9 x 10-8M or less, about 1 x 10-8M or less, about 9 x 10-9M or less, about 5 x 10-9M or less, about 4 x 10-9M or less, about 3 x 10-9M or less, about 2 x 10-9M or less, or about 1 x 10-9M or less. In some embodiments, theKdranges from about is about 2 x 10-7M to about 1 x 10-9M.

[0073] Binding of the extracellular antigen-binding domain of a CAR of the present disclosure can be determined by, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or a Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in an RIA assay. The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or byDocket No. 33794 / 70815autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific for the extracellular antigen-binding domain and wherein the secondary antibody is labeled (e.g., radioactively or with a fluorescent marker).

[0074] In some embodiments, a CAR of the present disclosure comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component may be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any suitable natural killer cell receptor, including without limitation, a killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1 , KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS DPI; a natural cytotoxicity receptor (NCR), such as NKp30, NKp44, NKp46; a signaling lymphocyte activation molecule (SLAM) family of immune cell receptor, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; an Fc receptor (FcR), such as CD16, and CD64; and an Ly49 receptor, such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adaptor molecule or intracellular signaling domain, such as DAP12. Exemplary configurations and sequences of CARs comprising NKR components are described in International Patent Publication WO 2014 / 145252.

[0075] Cytokines and Chemokines. Maturation, activation, survival, and recruitment of lymphoid cells, including NK cells and T cells, are influenced by cytokines and chemokines. For example, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and type I interferons positively regulate NK cell function, and activated NK cells endogenously produce large quantities of IFN-y. In the tumor microenvironment, TGFp, IL-10, and IL-6 can suppress NK activity directly, and antagonize the effect of stimulatory cytokines, thereby dampening the antitumor response of NK cells and promoting subsequent tumor evasion and progression (Konjevic et al., Cytokine, 117L 30-40, 2019). Furthermore, NK cells infiltrate the tumor site via chemokine receptors, including CXCR3 that binds tumor-derived chemokine ligands CXCL9, CXCL10, and CXCL11. In some embodiments, genetically modified immune cells are further modified to overexpress one or more chemokines or cytokines. In such embodiments, the one or more cytokines or chemokines are CCL2, CCL5, CCL20, CXCL1 , CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof.Docket No. 33794 / 70815

[0076] Tumor infiltrating immune cells often exhibit a reduced function and phenotype, and accumulating evidence indicates that tumor cells, various immunosuppressive cells, and tumor resident cells produce microenvironmental factors such as cytokines and other immunosuppressive mediators that negatively affect immune effector cell function.Immunosuppressive cytokines, including TGFp, IL-10, and IL-6, can directly inhibit immune effector cells, in addition to indirectly by affecting antigen presenting cells (APCs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) to produce additional immunosuppressive factors. Without wishing to be bound by any particular theory, infiltration of modified immune cells into the tumor microenvironment is believed to be improved by coexpressing one or more membrane-bound proteases and one or more cytokines or chemokines. In some embodiments, the one or more cytokines or chemokines is constitutively expressed. In various embodiments, the one or more cytokines or chemokines are overexpressed upon engagement with a cancer cell.

[0077] The genetically modified immune cells of the present disclosure may be used, in some instances, to treat cancer, such as pancreatic cancer. For example, the genetically modified immune cells may be used to treat lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof. In some embodiments, the cancer is pancreatic cancer. In various embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).Compositions and Methods

[0078] Pharmaceutical Composition. The disclosure further provides a pharmaceutical composition comprising genetically modified immune cells, wherein the modified immune cells overexpress one or more membrane-bound proteases. In some embodiments, the genetically modified immune cells overexpress one or more membrane-bound proteases, two or more membrane-bound proteases, three or more membrane-bound proteases, four or more membrane-bound proteases, five or more membrane-bound proteases, six or more membrane-bound proteases, seven or more membrane bound proteases, or more. In various embodiments, the modified immune cells overexpress a heterologous protease, a homologous protease, or a combination thereof. In some embodiments, the modified immune cells overexpress a membrane-bound protease, a soluble protease engineered to be membrane-bound, or a combination thereof. A cell for producing a genetically modified immune cell can be isolated from a human donor, such as a subject known or suspected to have cancer, or a human donor not suffering from cancer. In various embodiments, the human donor is suffering from cancer, wherein the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer,Docket No. 33794 / 70815kidney cancer, esophageal cancer, liver cancer, or a combination thereof. In some embodiments, the pharmaceutical composition of the disclosure is formulated for administration to a human subject. In various embodiments the pharmaceutical composition of the disclosure further comprises a pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.

[0079] A “dose” as used herein refers to a composition comprising one or more of the genetically modified immune cells of the disclosure. In some embodiments, the methods of the disclosure comprise administering a first dose of a pharmaceutical composition comprising the genetically modified immune cells. In various embodiments, the methods of the disclosure further comprise administering a second dose, a third dose, a fourth dose, a fifth dose, and / or a sixth dose of a pharmaceutical composition comprising the genetically modified immune cells. In some embodiments, the methods may further comprise additional doses of a pharmaceutical composition comprising genetically modified immune cells. In some embodiments, a dose comprises from about 1x106genetically modified immune cells to about 1.5x107genetically modified immune cells. In various embodiments, a dose comprises about 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 7x106, 8x106, 9x106, 1 x107, or 1.5x107genetically modified immune cells.

[0080] Pharmaceutical Composition for Use in Treating a Disease or Disorder. In this regard, the disclosure contemplates the genetically modified immune cells overexpressing one or more membrane-bound proteases for use as a medicament for treating a disease or disorder in a subject in need thereof. The disclosure also provides use of genetically modified immune cells overexpressing one or more membrane-bound proteases for the treatment of a disease or disorder in a subject in need thereof. In various embodiments, the uses disclosed herein include delivering, or administering, to a subject (e.g., a human subject) genetically modified immune cells as provided herein as a cell therapy to produce in vivo at least one membrane-bound protease (e.g., a matrix metalloproteinase). Further provided herein are uses that include delivering, or administering, to a subject (e.g., a human subject) genetically modified immune cells as provided herein to produce in vivo at least two membrane-bound proteases, e.g., a matrix metalloproteinase and a cathepsin. Further provided herein are uses that include delivering, or administering, to a subject (e.g., a human subject) any of the pharmaceutically acceptable carriers described herein, wherein the pharmaceutical composition comprises any of the genetically modified immune cells described herein comprising any of the membrane-bound proteases described herein and a pharmaceutically acceptable carrier.

[0081] In some embodiments, the genetically modified immune cells, alone or formulated with a pharmaceutically acceptable carrier, are administered via intravenous, intraperitoneal,Docket No. 33794 / 70815intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (e.g., packed in a delivery particle), or arterial (e.g., internal carotid artery) routes. Thus, the modified immune cells, with or without a pharmaceutically acceptable carrier, may be administered systemically or locally (e.g., to a TME or via intratumoral administration).

[0082] A cell for producing a genetically modified immune cell can be isolated from a subject, such as a subject known or suspected to have cancer. An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic genetically modified cells can be HLA-matched to the subject being administered a treatment.

[0083] Modified immune cells, with or without a pharmaceutically acceptable carrier, can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, modified immune cells can be administered in combination with one or more immunomodulatory drugs described herein. FDA-approved immunomodulatory drugs can be administered in their approved fashion. In another example, the genetically modified immune cells can be administered in combination with a checkpoint inhibitor therapy. Exemplary checkpoint blockade inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIG IT antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-triggering receptor expressed on myeloid cells 1 (anti-TREMI) antibodies, and anti-triggering receptor expressed on myeloid cells 2 (anti-TREM2) antibodies. Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD-1 ; MK-3475 / Keytruda® - Merck), nivolumamb (anti-PD-1 ; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / Cu reTech), AMP224 (anti-PD-1 ; NCI), avelumab (anti-PD-L1 ; Bavencio® - Pfizer), durvalumab (anti-PD-L1 ; MEDI4736 / lmfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-L1 ; Tecentriq® - Roche / Genentech), BMS-936559 (anti-PD-L1 - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0084] Some uses comprise selecting a subject (or patient population) having a tumor (or cancer) and treating that subject with genetically modified immune cells that modulate tumor-mediated immunosuppressive mechanisms.

[0085] The genetically modified immune cells of the present disclosure may be used, in some instances, to treat cancer, such as pancreatic cancer. For example, the geneticallyDocket No. 33794 / 70815modified immune cells may be used to treat lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof. In some embodiments, the cancer is pancreatic cancer. In various embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the disease to be treated with the pharmaceutical compositions, methods, and uses of the disclosure is a disease present in a human subject.

[0086] Animal models are well known to serve as basic translational models in preclinical studies, serving to elucidate key biochemical and physiological processes in many diseases, including cancer onset and progression. Experimental tumors raised in animals, particularly in rodents, constitute a major preclinical tool for evaluating anticancer therapeutics prior to clinical trials in humans. Indeed, the use of mice as animal models of human disorders has long been considered essential for elucidating the underling mechanisms of disease, as well as for translational research from bench to bedside. Physiological and anatomical similarities between mice and humans are matched by substantial genetic homology; the protein-coding regions of human and mouse genomes share approximately 85% identity (Uhl et al., Curr Pathobiol Rep. 3:219-223, 2015).

[0087] Immunocompetent mouse models have been used in decades in cancer research for their ability to generate antitumor-specific immune responses, with these tumors having the advantage of growing in a physiologically pertinent tumor microenvironment (TME). While immunocompetent models have been limited in their translational efficacy, genetically engineered mouse models and immunodeficient mouse models have already been developed and utilized in cancer research. Genetically engineered mouse models are engineered to express an oncogene or incapacitate a tumor suppressor gene, which can favor tumor development in a tissue-specific fashion. Genetically engineered mouse models provide the advantage of slower tumor growth, allowing for prolonged immunotherapy treatment and complete microenvironments. Immunocompromised mouse models can be engrafted with human tumors, either in the form of cell-line derived xenografts (CDXs) or patient-derived xenografts (PDXs). Immunocompromised mouse models can also be humanized via engraftment with human immune cells, such as peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), human fetal bone, liver, and thymic tissue (BLT), and splenic mononuclear cells (SPMCs) (Cogels et al. Front Oncol. 11:784947, 2021).

[0088] For example, the NOD.Cg-PrkdcscidI I2rgtmlWjl (NSG) mouse model is well suited for generating data that can be translated into humans, especially in the context of oncological research and precision medicine. NSG mice lackT, B, functional NK cells asDocket No. 33794 / 70815well as both alleles of the IL2 receptor common gamma chain, thus lacking cytokine signaling through multiple receptors. The NSG mouse model has already seen success in translating preclinical animal studies to human clinical trials. For example, administration of anti-PD1 antibodies to NSCLC-bearing NSG mice humanized with either HSCs or PBMCs caused significant tumor growth inhibition, in line with the efficacy observed in NSCLC patients (Meraz et al., Cancer Immunol Res. 7:1267-79, 2019; Traggiai et al., Science 304:104-07, 2004). As another example, pembrolizumab treatment led to significant tumor growth inhibition and increased activity of tumor-infiltrating lymphocytes (TILs) in adrenocortical carcinoma (ACC) engrafted into humanized mice, with these results supported by a phase II trial (NCT02673333) that showed a partial response to pembrolizumab in 23.1% of enrolled ACC patients, suggesting that humanized mice could efficiently model human response to treatment (Lang et al., J Clin Endrocrinol Metab.105:26-42, 2020). Altogether, animal models are widely recognized as a tool for evaluating therapeutic efficacy at the preclinical stage with potential for translation of the preclinical data into human clinical trials.

[0089] Preclinical data can also be modeled with three-dimensional organoids, cultures derived from stem cells that closely mimic the architecture and functionality of native organs (Yang et al., MedComm. 4(3), 2023). Organoids are considered to provide a more accurate representation of human tissues than certain animal models or two-dimensional cell cultures, enabling more reliable and efficient drug screening and functional validation (Taverna et al., Lung Cancer 190:107533, 2024). Patient-derived organoids, which resemble the original tissues, typically retain their genetic complexity, and offer a rapid and cost-effective way to study diseases, in contrast to patient-derived primary cell cultures, which closely resemble the original tumor but are hindered by a finite lifespan and limited replicative capacity.Furthermore, the drug sensitivity of tumor organoids has been demonstrated to be well-matched with specific cancer’s molecular subtypes. For example, van de Wetering et al utilized colorectal cancer (CRC) organoids to investigate a group of compounds, and found that organoids carrying TP53 mutations were resistant to nutlin-3a (inhibitor of MDM2) and those with RAS mutations were insensitive to EGFR inhibition (Van de Wetering et al., Cell 161(4):933-45, 2015), which reflected observations made in the clinic (De Roocket al., Lancet Oncol. 11 : 753-62, 2010). As additional examples, organoids have demonstrated predictive capacity for colorectal and gastroesophageal cancer responses to anticancer agents (Vlachogiannis et al., Science 359(6378):920-26, 2018), as well as rectal cancer responses to chemoradiation therapy (Ganesh et al., Nat Med. 25(10):1607-14, 2019). Thus, in vitro organoid models are recognized as recapitulating patient responses in disease models, including cancer (Hu et al., J Hematol Oncol. 11:116, 2018).Docket No. 33794 / 70815

[0090] Method of Enhancing the Ability of Immune Cells to Migrate Into a Tumor. Increased immune cell infiltration into tumors is associated with improved patient survival and predicts response to immune therapies. The density and spatial distributions of immune infiltrates into tumors, prior to treatment, are associated with patient survival and responses to immune therapy in various cancers, including pancreatic cancer (Carstens et al., Nat Commun, 8: 15095, 2017). Barriers to immune cell extravasation and infiltration include multiple mechanisms such as vascular limitations, absence of critical immune cell homing chemokines, extracellular matrix barriers, and cell-associated barriers. The disclosure provides a method of enhancing the ability of immune cells to migrate into a tumor, wherein the method comprises genetically modifying the immune cells to overexpress a membranebound protease. In tumors, activated cancer-associated fibroblasts (CAFs), inflammation, high interstitial pressure, and increased expression of collagen-processing lysyl oxidases can increase collagen deposition, cross-linking, and disorganized matrix structure, which can obstruct immune cell infiltration into the tumor (Heneke et al., Front Mol Biosci, 6:160, 2019).

[0091] The genetically modified immune cells of the disclosure have the potential to circumvent the physical and chemical barriers that prevent endogenous immune cell infiltration into the tumor microenvironment. In some embodiments, the genetically modified immune cell overexpresses one or more membrane-bound proteases, enhancing the ability of the cell to migrate through the physical barriers erected by the tumor. In various embodiments, the genetically modified immune cell further comprises an immunomodulatory factor, wherein an immunomodulatory factor includes, but is not limited to, a CAR, a cytokine, a chemokine, or a combination thereof. In some aspects, a CAR imparts a specificity of interest onto the modified immune cell, wherein the CAR is capable of binding a tumor-associated antigen, tumor specific antigen, or neoantigen expressed by a tumor. First generation, second generation, and third generation CARs are contemplated. In some aspects, a modified immune cell expresses one or more cytokines or chemokines, wherein the one or more cytokines or chemokines can improve activation of the cell itself, other modified immune cells, and / or endogenous immune cells within the vicinity of the tumor microenvironment, and can combat the presence of immuosuppressive factors within the tumor microenvironment (e.g., TGF0, IL-6, IL-10).

[0092] In various embodiments, the ability of modified immune cells can be evaluated with a transwell assay, which are well described in the field, for example, which is incorporated herein by reference.

[0093] The genetically modified immune cells of the disclosure are modified to overexpress one or more membrane-bound protease. In various embodiments, the genetically modified immune cells exhibit an enhanced ability to invade extracellular matrixDocket No. 33794 / 70815(ECM). In some embodiments, the ECM is tumor-associated ECM present in a tumor’s microenvironment. In various embodiments, the genetically modified immune cells target and breakdown the ECM components, such as collagen, elastin, fibronectin, laminin, proteoglycans, or glycosaminoglycans. The ability of genetically modified immune cells in accordance with the disclosure to invade ECM can be evaluated via transwell migration and invasion assays, wherein cells are seeded in transwell inserts with porous membranes. The assays can optionally include having the transwell insert overlaid with an ECM barrier, such as Matrigel, fibronectin, collagen IV, or collagen I. The density of the ECM barrier for testing can be modified by adjusting the time allowed for polymerization of the ECM component (typically between 30-90 minutes).

[0094] In some embodiments, the genetically modified immune cells target the cells that produce the ECM, such as fibroblasts. In various embodiments, the cells that produce the ECM are cancer-associated fibroblasts (CAFs). In such embodiments, the ability of genetically modified immune cells to target ECM-producing cells can be assessed via immunostaining tumor cross-sections. In certain embodiments, wherein the ECM-producing cells of interest are CAFs, tumor sections can be immunostained for aSMA, and optionally tumor sections can be costained for an immune cell marker (e.g., CD8 for T cells, or CD56 for NK cells) to detect genetically modified immune cell infiltration.

[0095] Method of Preparing One or More Genetically Modified Immune Cells.Immune cells, such as T cells, NK cells, and macrophages, can be modified using gene transfer techniques to directly and stably express proteins on their surface. A method of producing an immune cell overexpressing one or more protease, more specifically one or more membrane-bound protease, is also provided herein.

[0096] For instance, the disclosure provides a method of making natural killer (NK) cells expressing one or more membrane-bound proteases, wherein the method comprises: transfecting a vector containing a gene for one or more membrane-bound proteases into one or more immune cells in medium; replicating the one or more immune cells transfected with the vector, and; isolating the one or more immune cells transfected with the vector that overexpress the one or more membrane-bound proteases.

[0097] Kits and containers thereof suitable for storage and transportation of any one of the compositions are also disclosed herein. Such a kit can comprise an immune cell expressing one or more membrane-bound proteases according to the methods disclosed herein; a sterile container; and a syringe. In some embodiments, a kit further comprises instructions for use. In various embodiments, a kit further comprises a cryopreservation medium. In some embodiments, the sterile container is able to withstand freezing at liquid nitrogenDocket No. 33794 / 70815temperatures. In some embodiments, the kit further comprises a larger container, referred to herein as a “transportation container”, that is capable of transporting frozen cells at dry ice or liquid nitrogen temperatures.

[0098] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define a scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLESExample 1

[0099] Adopted from a previously published method (Neron, S., Thibault, et al., 2007), peripheral blood mononuclear cells (PBMCs) were isolated from leukoreduction system (LRS) chambers obtained from normal donors. The LRS chambers were flushed with PBS containing 1U / mL heparin (Sigma-Aldrich#H3149) and 2% fetal bovine serum (FBS, Gibco#10082147), and the collected product was diluted to 40 mL. White blood cell (WBC) count was determined using a Hemavet analyzer. RosetteSep (StemCell Technologies#! 5025, #15061 , 0.5 pL per 1x106WBCs) was added to enrich for NK cells, and T cells, respectively, followed by Ficoll-Paque Plus (Cytiva#17144002) density gradient centrifugation (400xg, 30 min, no brake). The PBMC layer was collected, washed with PBS + 0.5% bovine serum albumin (BSA, Sigma-Aldrich#A9418), and subjected to RBC lysis using a commercially available RBC lysis buffer. Cells were resuspended and cultured in 10% Human AB serum (HAB) media consisting of RPMI 1640 (Gibco#11875093), 10% human AB serum (Sigma#H4522), 2 mM Penicillin-Streptomycin / L-Glutamine(Gibco#10378-016), 1% HEPES buffer (Gibco#15630-080), 1% non-essential amino acids (NEAA, Gibco#11140-050), and 1 mM sodium pyruvate (Gibco#11360070), filtered through a 0.22 pm sterile filter. NK and T cells were cultured in this media supplemented with IL-2 (500U / mL, PeproTech#200-02) and IL-15 (1 ng / mL, PeproTech#200-15).

[0100] NK cell purity was assessed using flow cytometry for CD3 (Biolegend, cat#300411 ) and CD56 (BD Bioscience, cat#555516). Donor cells were maintained in culture with IL-2 and IL-15.

[0101] cDNAs for FAP, MMP-1 , -2 and -14 were cloned into the CSII-EF lentiviral vector. HEK 293T cells were transfected with the previously mentioned lentiviral vectors asDocket No. 33794 / 70815previously described (Afifi et al., 2023). Viral supernatants were collected and further filtered and centrifuged at 2600 rpm for 20 mins after growing for 48 hours in Opti-MEM media (Life Technologies). Human donor T and NK cells were transduced with the lentiviral FAP, MMP-1 , -2, and MMP-14 lentiviral vectors and centrifuged for 1.5 hours at 3000 rpm in the proper full growth culture media. Mix-populations were obtained and validated for expression of FAP, MMP-1 , -2, and MMP-14 using both Flow cytometry analysis and western blot.

[0102] NK cells (Figure 1A) and T ceils (Figure 1B) expressing the indicated constructs were fixed in 4% paraformaldehyde for 10 min at room temperature, washed 3 times in PBS supplemented with 2% FBS, then permeabiiized by adding ice-cold 100% methanol for 30 min on ice. Cells were washed and stained with anti-MMP-1 anti-MMP-2, and anti-MMP- 14 (invitrogen, MA5-15872, 43600, and MA-532076, respectively) diluted in the same PBS buffer for 1 h at room temperature. Cells were then stained with secondary fluorescent tagged antibodies (Invitrogen, 62249) and analyzed using an LSR Fortessa SORP I cytometer (BD Biosciences). Figure 1 provides bar graphs representing the percentage of MMP-positive cells post-transduction, wherein the ceils are human NK cells (Figure 1 A) or human T cells (Figure 1B).

[0103] To evaluate the ability of modified NK and T cells to invade matrix, matrigel was diluted 1 :4 in RPMI media. 50pL of this mixture was plated on the underside of a 5pm pore transwell insert (Corning, cat#CLS421). This was allowed to solidify for 20 minutes at room temperature. 2x105cells in 200pL media were plated in the top well of the plate. 10Ong / mL CXCL9 (R&D systems, cat# 350-NS-010 / CF) was added to 400pL media plated in the lower well of the plate. The cells were allowed to invade for 24 hours and the number of cells in the bottom well was counted using a hemocytometer. Data were compiled and assessed for statistical significance using GraphPad Prism 10. As shown in Figure 2, overexpression of every protease evaluated herein improved immune cell infiltration through Matrigel. In NK cells, overexpression of membrane-bound MMP-14 yielded NK cells with the greatest invasive capacity when compared to parental NK cells (Figure 2A). However, all overexpressed proteases improved NK cell invasive capacity when compared to parental NK cells. In T cells, overexpression of MMP-2 yielded T cells with the greatest invasive capacity, although all overexpressed proteases improved T cell invasive capacity when compared to parental T cells (Figure 2B).Example 2

[0104] Whether MMP overexpression enhances T cell invasion was investigated in the human T cell line Jurkat. FAP, MMP-1 , -2, -3, and -14 were overexpressed in this cell lineDocket No. 33794 / 70815utilizing the method described below. Retrovirus was generated and GFP positive cells were sorted. To analyze the impact of FAP on the invasiveness of these cells, Jurkat cells (WT and FAP OE) were plated in the top well of a transwell chamber. CXCL12, a known T cell chemoattractant55, was placed in the lower well to stimulate T cell invasion. T cells were allowed to invade through the membrane coated with a Matrigel barrier for 24 hours (Figure 3A). Overexpression of FAP as well as all the selected MMPs resulted in enhanced invasion through Matrigel (Figure 3B).

[0105] Generation of overexpressing cells. In jurkat cells, overexpression of FAP, MMP-1 , -2, -3, and -14 were induced by retroviral transduction. Phoenix amphotropic cells were transfected with either the pBMN plasmid containing the FAP gene or a retroviral vector containing MMP-1, MMP-2, MMP-3 and MMP-14 (received from vectorbuilder) using Lipofectamine and Plus reagent (Life Technologies) as previously described (Kikuchi-Maki et al., 2003). Supernatants were collected from these cells after growing for 48 hours in Opti-MEM media (Life Technologies). The supernatant was mixed with Lipofectamine and Plus reagent and added to 2x106jurkat cells in a 6-well plate. These cells were centrifuged for 45 min at 2000xg. This process was repeated two consecutive times. For FAP overexpression, cells were flow sorted for GFP positivity three days after the final transduction using the BD FACS Aria Hu cell sorter in the Georgetown Lombardi Comprehensive Cancer Center Flow Cytometry and Cell Sorting Shared Resource (FCSR). For MMP-1, -2, -3, and -14, cells were selected using puromycin resistance and were confirmed using GFP expression.

[0106] Transwell assay. Matrigel was diluted 1 :4 in RPMI media. 50pL of this mixture was plated on the underside of a 5pm pore transwell insert (Corning, cat#CLS421). This was allowed to solidify for 20 minutes at room temperature. 2x105cells in 200pL media were plated in the top well of the plate. 100ng / mL CXCL12 (R&D systems, cat# 350-NS-010 / CF) was added to 400pL media plated in the lower well of the plate. The cells were allowed to invade for 24 hours and the number of cells in the bottom well was counted using a hemocytometer. Data were compiled and assessed for statistical significance using GraphPad Prism 9.Example 3

[0107] 3D clusters were generated, embedded and stained as previously described (Lin et al., 2020; Westcott et al., 2020). To generate the clusters, 1 ,000 PANC-1 or BXPC-3 cells were plated in each well of a 96-well Nunclon Sphera low adhesion plates (Thermo Scientific, cat#174925) and incubated overnight at 37°C. 24 hours later, 8 clusters were embedded into ECM containing 2,000 donor NK cells. Donor NK cells included parental NKDocket No. 33794 / 70815cells, MM P-1 -overexpressing NK cells, and MMP-2-overexpressing NK cells (Figure 4). In a separate experiment, donor NK cells included parental NK cells and MMP-14-overexpressing NK cells (Figure 5).

[0108] These clusters were then plated on top of a base layer of ECM in one well of a Nunc Lab-Tek II 8-well chamber slide (ThermoScientific, cat#154534PK). The ECM mixture consisted of 20% growth factor reduced Matrigel (Corning, 10-12 mg / ml stock concentration, #354230) and 80% rat tail collagen type I at 3mg / mL (Gibco, A1048301). Cells were allowed to invade for 24 hours and the following day, they were fixed with 4% paraformaldehyde for 1 hour. They were then permeabilized with 0.5% Triton-X and blocked using BSA. Donor NK cells were stained with DiO prior to plating per manufacturer’s instructions. To detect cell lysis, clusters were also stained for anti-cleaved caspase-3 (Cell Signaling, cat#9661)(Figure 5). DAPI, phalloidin, and secondary antibodies labeled with Alexa Fluor 488 nm or 647 nm (Invitrogen) were used. To analyze cleaved caspase-3, mean fluorescent intensity was 50 measured using Image-J software. To account for varying background levels between experiments with donor NK cells, background was subtracted from each image and a common threshold was set for fluorescent intensity across conditions.Example 4

[0109] The cytotoxicity of mesothelin-targeting mCART cells against a panel of KPC-derived PDAC murine cell lines was evaluated, for the purpose of evaluating the cell-killing capacity of the mCART cells. Target cells for the assays disclosed herein included mT3-2D cells, mT4-2D cells, mT5-2D cells, and KP1 (murine pancreatic cancer cell line expressing mesothelin) cells. The mCART cells tested herein include engineered CD4+ T cells and CD8+ T cells. mCART cells were initially quantified, and incubated overnight in appropriate complete growth media supplemented with IL-2. Separately, the PDAC cell lines to be evaluated were plated at an appropriate density (-50% confluence per well) in a multiwell plate. After overnight incubation, the T cells were quantified, concentrations adjusted, and populations combined to bring the CD4+:CD8+ T cell ratio to 1:1. The mCART cells were then added to the multiwell plate containing the PDAC cell lines, with a ratio of mCART cells: PDAC cells of: 10:1 , 5:1 , 1 :1 , and 0.5:1. The mCART cells and PDAC cells were incubated together in the multiwell plate for 24 hours. After completion of the timecourse, flow cytometry was used to determine the killing efficacy of mCART cells against the KPC-derived pancreatic cell lines. The results of this assay are shown in Figure 6.

[0110] To isolate mononuclear T cells, a cell strainer is placed in a petri dish with 5 mL of FPBS, and spleen or lymph node tissues are ground using a syringe plunger until no visibleDocket No. 33794 / 70815chunks remain. The strainer is then washed with FPBS, and the suspension is homogenized using a 21 G needle and 5 mL syringe before transferring it into a 15 mL tube. After centrifugation at 1900 rpm for 5 minutes, the supernatant is removed, and the pellet is resuspended in 1 mL of FPBS. A 6 mL cell suspension is carefully layered over 3 mL of Ficoll and centrifuged at 1900 rpm for 12 minutes without braking. The top 4 mL is discarded, and mononuclear cells from the mid-layer are collected and washed with 10 mL of FPBS. After a final centrifugation at 1900 rpm for 5 minutes, the supernatant is removed, and the cell pellet is resuspended in 1 mL of FPBS before counting the cells. According to the manufacturer’s recommendations (Gibco catalog#11452D) Dynabeads Mouse T-Activator CD3 / CD28 were used to activate and expand mouse T cells without the need for antigen-presenting cells. For activation, purified T cells were cultured with pre-washed Dynabeads at a 1 :1 bead-to-cell ratio in a humidified CO2incubator at 37°C. The activated cells were then utilized for downstream applications such as transduction, gene expression studies, or T cell receptor signaling analysis. Expansion was carried out by culturing T cells with Dynabeads and IL-2, with regular monitoring and splitting when the cell density exceeded 2.5 x 106cells / mL. Restimulation was performed by removing used beads and reintroducing fresh Dynabeads and IL-2 when cell proliferation declined. The protocol ensured proper bead handling, culture conditions, and monitoring for optimal T cell activation and expansion. T cells were then transduced with anti-mesothelin CAR constructs using a previously published method and maintained in complete growth media supplemented with recombinant IL-2. Anti-mesothelin CART cells were then cocultured with mouse pancreatic cell lines (highly expressing mesothelin) for 24 hours before staining with cell death markers using Annexin-V assay and analyzed using LSR Fortessa SORP I cytometer (BD Biosciences).

[0111] This shows the ability to isolate and generate CAR T cells, and further assessment of these cells with overexpression of one or more proteases in accordance with the disclosure will be performed to assess the extent of tumor infiltration in vivo.Example 5

[0112] Fibroblast activating protein (FAP) is a proteolytic enzyme expressed by various NK subsets, and is involved in NK cell migration and invasion. To analyze whether FAP overexpression enhances tumor infiltration by NK cells in vivo, NSG mice bearing subcutaneous PANC-1 tumors were injected with either NK92 or FAP OE NK92 cells. NK92 cells are a NK cell line, and FAP OE NK92 cells are a NK cell line overexpressing FAP. Tumors were allowed to grow to at least 100mm3, and then were injected with either 1x107Docket No. 33794 / 70815NK92 or FAP OE NK92 cells into the tail vein (Figure 7A). The mice were euthanized after 24 hours and tumors were collected, fixed, and stained with an anti-CD56 antibody to detect NK cells. Tumors from mice injected with FAP OE NK92 cells contained more than three times as many NK cells when compared to tumors from mice injected with NK92 cells (Figure 7B-7C). In Figure 7, there is a notable difference in NK cells within the tumor as a function of therapy with either NK92 or FAP OE NK92 cells. This data suggests that FAP does in fact promote NK cell tumor infiltration in vivo.

[0113] To ensure that these results were not cell dose-specific, and to establish optimal dosing conditions for NK cell infiltration of tumors, 5x107NK92 or FAP OE NK92 cells were injected into NSG mice bearing subcutaneous PANC-1 tumors. The mice were euthanized 24 hours following treatment, and tumors were collected. Again, there was a statistically significant increase in NK infiltration in the tumors treated with FAP OE NK92 cells when compared to tumors treated with NK92 cells (Figure 7D-7E). There was a five-fold overall increase in NK cell infiltration in tumors treated with 5x107cells compared to the 1x107cells.

[0114] To assess potential off-target effects, NK cell trafficking to non-tumor locations was analyzed. Spleen, liver, and lung samples were collected from mice treated with 1x107NK92 or FAP OE NK92 cells, and subsequently stained. There was no significant difference in NK content in any of the aforementioned organs (data not shown). These results suggest that under the described experimental conditions, NK92 cells preferentially invade into tumors rather than other organs; these findings further support the translational potential of such an approach.

[0115] As FAP overexpression led to enhanced NK cell infiltration, it was next determined whether this increased NK content in the tumor translated to enhanced anti-tumor efficacy. Both male and female mice were injected with PANC-1 tumors subcutaneously. Mice were then treated once weekly for four weeks with either 1x107NK92 cells plus IL-2, or 1x107FAP OE NK92 cells plus IL-2. Tumor volumes were measured throughout the experiment. One week following the fourth injection, mice were euthanized, and tumors were collected and weighed (Figure 8A). A significant reduction in tumor growth was observed in mice treated with FAP OE NK92 cells compared to those treated with NK92 cells (Figure 8B). Further, tumors treated with FAP OE NK92 cells weighed significantly less than those treated with NK92 cells at the experimental end point (Figure 80). This experiment was replicated both with PANC-1 , and with additional PDAC cell line MIA PaCa-2, which yielded similar results.

[0116] As there was no differential cytotoxic effect observed in vitro (data not shown), it was hypothesized that the increased antitumor effects seen in vivo is due to the increase in NK cell content in the tumor. To determine whether NK cells were successfully invading theDocket No. 33794 / 70815tumor, tumors were collected, fixed, and stained with anti-CD56 antibody. An increase in NK cell content was observed in tumors from mice treated with FAP OE NK92 cells when compared to those treated with NK92 cells (Figure 8D-8E). This data indicate that FAP overexpression can enhance NK cell infiltration and efficacy in solid tumors.

[0117] To again assess NK cell trafficking to non-tumor locations, spleen, liver, and lung samples were collected from tumor-bearing mice and stained with anti-CD56 antibody.There was no significant difference in NK content in any of the examined organs (data not shown). Thus, under the aforementioned experimental conditions NK92 cells preferentially invade into tumors rather than organs, supporting the translational potential of this approach.Example 6

[0118] Primary human NK cells were isolated from leukoreduction system (LRS) chambers using negative selection (magnetic bead-based isolation). NK cells were activated with IL-2 (200 ILJ / mL) for 20 hours prior to genetic modification. Cells were transduced with a lentiviral vector encoding human MMP-14 or an empty vector control (parental NK).Transduction was performed in the presence of polybrene (8 pg / mL), followed by spinoculation. Transduced cells were expanded for 5-7 days in IL-2-supplemented media. Total protein was extracted from parental wild-type NK cells and MMP-14-overexpressing NK cells using RIPA buffer containing protease inhibitors. Equal amounts of protein (20-30pg) were resolved on SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with anti-MMP-14 primary antibody and HRP-conjugated secondary antibody, p-actin was used as a loading control. Bands were visualized using enhanced chemiluminescence (see Figure 9A). Band intensity corresponding to MMP14 was quantified by densitometric analysis of the immunoblot, with Figure 9B representing the relative MMP14 signal detected in parental NK cells compared with NK cells engineered to overexpress MMP14. The densitometric analysis of the Western blot demonstrated substantially increased MMP14 protein levels in NK cells engineered to overexpress MMP14 compared with parental NK cells, confirming successful genetic modification and elevated expression of MMP14 in the engineered NK cell population (Figure 9B).

[0119] Total RNA was isolated from the parental NK controls and the MMP-14-overexpressing NK cells using silica membrane split columns, and reverse-transcribed into cDNA. qRT-PCR was performed using SYBR Green chemistry with gene-specific primers for MMP-1 , MMP-2, and MMP-14, and expression levels were normalized to housekeeping gene (GAPDH) and calculated using the 2A-AACt method (Figure 9C). Overall, engineeringDocket No. 33794 / 70815of primary human NK cells to overexpress MMP14 resulted in robust and stable MMP-14 protein expression compared with parental NK cells, as confirmed by immunoblotting.

[0120] In the following experiments, primary T cells were assessed in parallel to the NK cells using identical lentiviral transduction methods to generate MMP overexpressing cells, and identical ECM components to compare invasion capacity relative to NK cells. For donor-derived experiments, wild-type or MMP-overexpressing cells were evaluated under matched conditions. In all of the following experiments, the invasive capacity of NK cells and T cells was evaluated using 24-well transwell inserts with porous membranes using the experimental set-up shown in Figure 10. Cells were seeded in the upper chamber in serum-free media. The lower chamber contained media supplemented with CXCL9 as a chemoattractant. After incubation, non-migrated cells were removed from the upper surface. Cells that traversed the membrane were quantified. Invasion was calculated as a percentage of total input cells migrating across the barrier. In Figure 11 , each dot represents an independent biological replicate.

[0121] NK and T cells were engineered to overexpress MMP-1 or MMP-2 using the same lentiviral transduction methods used to engineer NK and T cells to overexpress MMP-14. Engineered NK cells and T cells, and parental wild-type control cells for each (labeled WT in the figures), were assessed using 24-well transwell inserts with porous membranes. The porous membranes were coated with ECM components prior to the invasion assay, where the ECM layer was allowed to polymerize for approximately 30 minutes at 37°C before cells were seeded the upper chamber. Both NK cells and T cells overexpressing ECM-remodeling enzymes showed increased invasion compared to parental cells in a transwell invasion assay where the porous membrane was coated with a Matrigel overlay to model ECM components (Figures 11 A and 11 E). MMP-14 overexpression produced the most pronounced effects, significantly increasing the percentage of invading NK cells (Figure 11 A). NK and T cells were engineered to overexpress MMP-1 , MMP-2, or MMP-14. NK cells engineered to overexpress MMP-1 and MMP-2 were generated using the same transduction approach described above for producing NK cells engineered to overexpress MMP-14. Engineered NK cells and T cells, and parental wild-type control cells for each (labeled WT in the figures), were assessed using 24-well transwell inserts with porous membranes. Both NK cells and T cells overexpressing ECM-remodeling enzymes showed increased invasion compared to parental cells (Figures 11 A and 11 E). In particular, MMP-14 overexpression significantly increased NK cell invasion compared to parental cells (p = 0.0076), producing the most pronounced effect among the proteases tested (Figure 11 A).

[0122] Next, a separate experiment was prepared where the invasive capacity of engineered NK and T cells, and WT controls for each, were assessed using 24-wellDocket No. 33794 / 70815transwell inserts either left uncoated (labeled “No ECM” in the figures) or overlaid with defined ECM barriers including Matrigel, fibronectin, collagen IV, or collagen I, where the defined ECM barriers were allowed to polymerize for approximately 30 minutes at 37°C before cells were seeded in the upper chamber. Across multiple ECM barriers, MMP-overexpressing NK cells and T cells consistently outperformed wild-type cells (Figures 11 B and 11F).

[0123] Next, Matrigel overlays were polymerized for different periods of time to modulate barrier density. The polymerization duration was extended to 60 and 90 minutes to generate a denser and less porous matrix barrier. These denser conditions create a more restrictive environment that may more accurately approximate the dense fibrotic stroma observed in tumors, such as pancreatic ductal adenocarcinoma (PDAC). The barriers used to evaluate NK cells were polymerized for either 60 minutes or 90 minutes. The barriers used to evaluate T cells were polymerized for either 30 minutes, 60 minutes, or 90 minutes. When Matrigel polymerization time was extended, wild-type NK cell and T cell invasion dropped sharply, whereas MMP-14-overexpressing cells maintained high invasion rates (Figures 11 C and 11G). This divergence increased with longer polymerization times, consistent with enhanced matrix degradation capacity.

[0124] Finally, the invasive capacity of MMP-14-overexpressing NK cells was assessed in a porous membrane comprising no overlay, and a porous membrane overlaid with collagen I polymerized for 90 minutes. MMP-14-overexpressing NK cells invaded collagen I more efficiently than wild-type NK cells, including at early time points following matrix stabilization. This effect persisted after prolonged collagen setting, demonstrating resistance to barrier stiffening (Figure 11 D).Example 7

[0125] To mimic the membrane-bound activity of MMP-14 while preserving MMP-1 catalytic function, a chimeric MMP-1 construct was designed in which the catalytic domain of human MMP-1 was fused to the transmembrane and cytoplasmic domains of MMP-14. This design was intended to prevent enzymatic shedding, which is characteristic of native MMP1 , and to enforce cell-surface localization of proteolytic activity. The chimeric construct was cloned into a lentiviral expression vector and used to transduce primary human T cells under standard activation conditions. Transduced cells were expanded and used directly in downstream invasion assays.

[0126] Primary T cells expressing chimeric MMP-1 exhibited a significant increase in invasion compared to parental and MMP-1 -overexpressing cells. While overexpression ofDocket No. 33794 / 70815native MMP-1 led to only a modest enhancement of infiltration, it was surprisingly discovered that membrane-tethered chimeric MMP-1 substantially increased the proportion of invading T cells, reaching levels comparable to those observed with MMP-14 overexpression (Figure 12). These findings demonstrate that significant improvement in infiltration can be achieved through the specific selection of overexpression of membrane-bound proteases and that success can be achieved by preventing shedding and enforcing membrane localization of non-membrane bound proteases, thereby converting it from a weakly pro-invasive protease into a potent driver of infiltration. It has, thus, been surprisingly and beneficially found, that spatial confinement of proteolytic activity at the immune cell surface, rather than protease identity alone, governs effective ECM traversal.Example 8

[0127] After the demonstrated efficacy of MMP-14 overexpression in vitro, it was determined whether MMP overexpression increases NK cell infiltration in vivo, in a model of pancreatic cancer. Subcutaneous tumors were established by injecting 2x106MIA PaCa-2 cells into immunodeficient mice. Once tumors were established, mice received a single intratumoral injection of 1x106human donor NK cells, that were either wild-type or engineered to overexpress MMP-14 (Figure 13A). Control animals received PBS. Tumors were harvested five days after NK cell injection for histological analysis. Each group consisted of 8 mice.

[0128] T umor sections were stained with Masson’s trichrome to assess collagen content and immunostained for CD56 to identify infiltrating NK cells. Collagen area and NK cell infiltration were quantified using image analysis across multiple tumor regions per sample. The data in Figure 13B is presented as a percentage of total tumor area (Figure 13B, left panel) or total cells (Figure 13B, right panel). Statistical comparisons were performed between wild-type and MMP-14-overexpressing NK cell groups.

[0129] Figure 13B, right panel, shows that MMP-14 overexpression enhances NKcell infiltration in vivo. Tumors receiving MMP-14-overexpressing NK cells exhibited significantly higher NK cell infiltration compared to tumors injected with wild-type NK cells, as evidence by increased CD56-positive staining (Figure 13B, right panel). Quantitative analysis confirmed a marked increase in the percentage of CD56-positive cells in tumors treated with MMP-14-overexpressing NK cells (p < 0.01).

[0130] Figure 13B, left panel, shows that MMP-14-overexpressing NK cells promote collagen degradation in vivo. Masson’s trichrome staining revealed substantial collagen depletion in tumors infiltrated by MMP-14-overexpressing NK cells relative to wild-typeDocket No. 33794 / 70815controls. Quantification showed a significant reduction in collagen content in the MMP-14 group (p = 0.01), indicating active ECM remodeling in vivo.Example 9

[0131] NK cell treatment was evaluated in an orthotopic pancreatic ductal adenocarcinoma (PDAC) model. Orthotopic pancreatic tumors were established in NSG mice using ASPC1 cells. Once tumors were established, mice were randomized to receive PBS, wild-type human donor NK cells, FAP-overexpressing NK cells, or MMP-14-overexpressing NK cells. NK cells were administered with systemic cytokine support using IL-15 and IL-2 according to the schedule set out in Figure 14A. Tumors were harvested at the defined endpoint for histological analysis.

[0132] After collection, tumor sections were immunostained for a-smooth muscle actin (aSMA) to identify cancer-associated fibroblasts (CAFs), and immunostained for CD56 to detect infiltrating NK cells. Whole slide imaging was performed, and positive cells were quantified as the number of cells per mm2using standardized image analysis (Qupath software). Representative images of tumor sections from mice treated with PBS, wild-type human donor NK cells, FAP-overexpressing NK cells, and MMP-14-overexpressing NK cells are presented in Figure 14B. Each data point represents an independent tumor region of interest. Statistical comparisons were performed between treatment groups, with p values indicated.

[0133] Tumors treated with PBS or wild-type NK cells retained a dense aSMA-positive fibroblast network, consistent with a CAF-rich stroma. In contrast, tumors treated with MMP-14-overexpressing NK cells showed a marked reduction in aSMA-positive cells (Figure 14C). Quantification confirmed a significant decrease in CAF density in the MMP-14 NK group compared to PBS and wild-type NK controls.

[0134] CD56 staining demonstrated increased NK cell accumulation in tumors treated with the protease-engineered NK cells, with the highest infiltration observed in the MMP-14-overexpressing NK group (Figure 14D). Quantitative analysis showed significantly higher CD56-positive cell density in MMP-14 NK-treated tumors compared to PBS, and wild-type NK cells.

[0135] The reduction in aSMA-positive CAFs and increase in NK cell infiltration indicates that MMP-14-overexpressing NK cells actively target and disrupt the fibroblast-rich tumor stroma. These data support a model in which membrane-bound MMP-14 enables NK cells toDocket No. 33794 / 70815overcome CAF- mediated physical barriers and directly or indirectly suppress CAF abundance, thereby remodeling the tumor microenvironment to favor immune infiltration.Example 10

[0136] Immune cells were engineered to co-express a membrane-bound protease and a chimeric antigen receptor (CAR) to determine whether the combination could enhance both tumor invasion and anti-tumor activity. Murine T cells were isolated and activated ex vivo, and then transduced with a mesothelin-targeting murine CAR (mCART). For PushCAR constructs, MMP-14 was expressed either alone in T cells, or together with the mCAR. Transduction efficiency, lineage identity, and purity were assessed via flow cytometry using CD4, CD8, and NKp46 markers to confirm T cell identity and exclude NK cell contamination (Figure 15A).

[0137] Mesothelin expression on murine tumor cell lines was quantified by flow cytometry prior to functional assays to confirm suitability for mCAR recognition (Figure 15B).

[0138] Mesothelin-positive murine tumor spheroids were generated and co-cultured with mCART cells, MMP-14-overexpressing T cells, or MMP-14-mCART PushCAR T cells.Spheroids were fixed and stained with DAPI to visualize cellular architecture and cleaved caspase-3 to assess tumor cell apoptosis. Imagining was performed via confocal microscopy.

[0139] Flow cytometry confirmed high transduction efficiency with preservation of CD4+ and CD8+ T cell populations and negligible NKp46 expression, indicating minimal NK cell contamination (Figure 15A). Target tumor cells expressed high levels of mesothelin (Figure 15B), validating CAR specificity.

[0140] In three-dimensional spheroid models, conventional mCART cells remained largely restricted to the spheroid periphery (Figure 15C). In contrast, MMP-14-expressing T cells demonstrated deeper infiltration, while MMP-14-mCART PushCAR T cells showed the most extensive and uniform penetration throughout the tumor mass.

[0141] Cleaved caspase-3 staining revealed limited apoptosis in spheroids treated with mCART cells alone. MMP-14-expressing T cells induced increased tumor cell apoptosis, which was maximal in spheroids treated with MMP-14-mCART PushCAR T cells. These data indicated that coupling antigen-specific targeting with localized ECM remodeling markedly enhanced CAR T cell infiltration and cytotoxic efficacy in three-dimensional tumor models.Example 11Docket No. 33794 / 70815

[0142] Primary human T cells were engineered to overexpress fibroblast activation protein (FAP) using the same lentiviral transduction strategy described for donor-derived NK cells. FAP overexpression was achieved using a third-generation lentiviral system (packaging plasmids pMDIg, pRSV-rev, and pCMV-VSVG). Lentivirus was produced in HEK293 cells and concentrated using Amicon Ultra centrifugal filter units. Activated primary human T cells were transduced with lentiviral particles under conditions smilar to those used for NK cells, and successfully transduced cells were enriched based on GFP expression by flow cytometric sorting. Parental wild-type T cells cultured under identical conditions served as controls. To assess invasion, engineered and parental cells were evaluated using 24-well transwell inserts with porous membranes coated with a Matrigel overlay to model ECM components. Cells were seeded in the upper chamber and allowed to migrate toward media containing a chemoattractant, CXCL9, in the lower chamber to establish a chemotactic gradient. After incubation, non-migrated cells were removed from the upper membrane surface and cells that migrated through the membrane were quantified. Invasion was calculated as the percentage of total input cells traversing the ECM barrier.

[0143] T cells engineered to overexpress FAP demonstrated increased invasion through ECM-coated transwell membranes relative to parental wild-type T cells. Across independent biological replicates, FAP-overexpressing cells showed a higher proportion of cells migrating through the ECM barrier compared with WT controls, indicating an enhanced lymphocyte invasive capacity (Figure 16). These findings demonstrate that expression of FAP enhances the ability of T cells to penetrate ECM barriers. Dense ECM structures and fibrotic stroma often restrict immune cell access to tumor cells in solid tumors, and engineering immune cells to express ECM-remodeling enzymes (such as FAP) may improve immune cell infiltration into stromal-rich tissues, enhancing the effectiveness of adoptive immune cell therapies in cancers characterized by dense fibrotic tumor microenvironments.

[0144] Table 1 : SequencesSEQ ID Description SequenceNO1 MMP1 GCGGATCCTCTAGAGTTAACATCGAGGGATCAAGCTTATCGATAATCAACC TCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CC I 1 1 IACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCAI 1 1 1 CTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCA CTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCDocket No. 33794 / 70815CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCC TTTGGGCCGCCTCCCCGCATCGATACCGTCGAGACCTAGAAAAACATGGA G CAATCACAAGTAG CAATACAGC AG CTACCAATG CTG ATTGTGCCTGG CTA GAAGCACAAGAGGAGGAGGAGGTGGG I I I I CCAGTCACACCTCAGGTAC CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC I I I I IAA AAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCA GAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGT GCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGA AGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATG ACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTT CATCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCAGTGTG GAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTG CC AG CC ATCTGTTGTTTG CCCCTCCCCCGTG CCTTCCTTG AC CCTG G AAG GTG CC ACTCCC ACTGTCCTTTCCTAATAAAATG AG G AAATTG C ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTC TAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG TG GTG GTTACG CGC AG CGTG ACCG CTACACTTGCC AGCG CCCTAG CGCCC G CTCCTTTCGCTTTCTTCCCTTCCTTTCTCG CC ACGTTCG CCG GCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGG I I I I I CGCCCTTTGACGTTGGAGTCCACGTTC TTTAATAGTG G ACTCTTGTTCC AAACTG G AACAACACTCAACCCTATCTCG G TCTATTC I I I I GAI I IAIAAGGGAI I I I GCCGATTTCGGCCTATTGGTTAAAA AATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAG G CTCCCC AG C AG GC AG AAGTATG CAAAGC ATGC ATCTCAATTAGTC AG CA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT CCGCCCATTCTCCGCCCCATGGCTGACTAAI I I I I I I IATTTATGCAGAGGC CGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC I l l i TTGGAGGCCTAGGC I l l i GCAAAAAGCTCCCGGGAGCTTGTATATCCATTT TCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGC ATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGT GCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGC CGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACC AGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA CG AG CTGTACG CCG AGTG GTCGG AG GTCGTGTCCACG AACTTCCGG G AC GCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGDocket No. 33794 / 70815AGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTC TATGAAAGGTTGGGCTTCGGAATCG I I I I CCGGGACGCCGGCTGGATGAT CCTCC AG CG CGGG G ATCTC ATGCTG G AGTTCTTCG CCCACCCCAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA AATAAAGCAI I I I I I I CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA ATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGA GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG GTAATACG GTTATCCACAG AATC AG GG G ATAACG CAG G AAAG AACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTG G AAG CTCCCTCGTG CGCTCTCCTGTTCCG ACCCTG CCG CTTAC CG G ATACCTGTCCG CCTTTCTCCCTTCGG G AAG CGTG G CGCTTTCTC ATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTG AGTCC A ACCCG GTA AG AC ACG ACTTATCG CC ACTG G C AG C AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG GTATCTG CG CTCTG CTG AAG CC AGTTACCTTCG G A A A A AG AGTTG GTAG C TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGC AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT C I I I I CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA I I I I GG I CAI GAGAI I Al CAAAAAGGAI C I I CACC I AGAI CC I I I IAAATTA AAAATGAAG I I I IAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC G CAG AAGTG GTCCTG CAACTTTATCCG CCTCC ATCCAGTCTATTAATTGTTG CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG TTG CC ATTG CTAC AG G C ATCGTG GTGTC ACG CTCGTCGTTTG GTATG G CTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGC I I I I CTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC I I I IACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG CCG CAAAAAAG GG AATAAG GG CG ACACG G AAATGTTG AATACTCATACTCTTCC I I I I I CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGDocket No. 33794 / 70815ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGAT CCCCTATGGTG CACTCTC AGTAC AATCTG CTCTG ATG CCG CATAGTTAAGCC AGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGC AAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAA TCTGCTTAGGGTTAGGCG I I I IGCGCTGCTTCGCGATGTACGGGCCAGATA TACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTGATGCGG I I I IGGCAGTACATCA ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTG I I I IGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCG CCCC ATTG ACG CAAATG G GCG GTAG GCGTGT ACGGTGGGAGGTCTATATAAGCAGCGCG I I I IGCCTGTACTGGGTCTCTCT GGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCA CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCA GTGTG G AAAATCTCTAGCAGTG GCG CCCG AAC AG G G ACTTG AAAGCG AA AGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG CGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATT TTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTA TTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCC AGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGG AGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCT GTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAA GAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAG GATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAG C AAAAC AAAAGTAAG ACC ACCG CAC AG CAAG GG CCGCTG ATCTTCAG AC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATA AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGA AGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGAC GCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGA ACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACA GTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCA TTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG AACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAA GAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTG GAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGA TAGTAGGAGGCTTGGTAGGTTTAAGAATAG I I I I IGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCDocket No. 33794 / 70815AACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTACAAATG GCAGTATTCATCCACAAI I I IAAAAGAAAAGGGGGGATTGGGGGGTACAG TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAG AATTACAAAAACAAATTACAAAAATTCAAAAI I I I CGGGTTTATTACAGGG ACAGCAGAAATTCACTTTGAATTAATTCAAGCTTCGTGAGGCTCCGGTGCC CGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC I I I I I CCCGAGGGTGGG GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC I I I I I CGCAAC GGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC CTG G CCTCTTTACG G GTTATG G CCCTTG CGTG CCTTG A ATTACTTCC ACCTG GCTCCAGTACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTG CGCTTTAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAAI I I I I GATGACCTGCTGCGACGC I I I I I I I CTGGCAAGATAGTCTTGTAAATGCGGGCCAGGATCTGCACACTG GTATTTCGG I I I I I GGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCG CGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGC ACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTCCAGGG GGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAGGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCT TTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG I I I IATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTT GATGTAATTCTCCTTGGAATTTGCCC I I I I I GAGTTTGGATCTTGGTTCATT CTCAAGCCTCAGACAGTGGTTCAAAG I I I I I I I CTTCCATTTCAGGTGTCG TGAACACGCTACCGGTCATGCACAGCTTTCCTCCACTGCTGCTGCTGCTGT TCTGGGGTGTGGTGTCTCACAGCTTCCCAGCGACTCTAGAAACACAAGAG CAAGATGTGGACTTAGTCCAGAAATACCTGGAAAAATACTACAACCTGAAG AATGATGGGAGGCAAGTTGAAAAGCGGAGAAATAGTGGCCCAGTGGTTG AAAAATTGAAGCAAATGCAGGAATTCTTTGGGCTGAAAGTGACTGGGAA ACCAGATGCTGAAACCCTGAAGGTGATGAAGCAGCCCAGATGTGGAGTG CCTGATGTGGCTCAGTTTGTCCTCACTGAGGGGAACCCTCGCTGGGAGCA AACACATCTGACCTACAGGATTGAAAATTACACGCCAGATTTGCCAAGAGC AGATGTGGACCATGCCATTGAGAAAGCCTTCCAACTCTGGAGTAATGTCAC ACCTCTGACATTCACCAAGGTCTCTGAGGGTCAAGCAGACATCATGATATC I I I I G I CAGGGGAGAI CAI CGGGACAAC I C I CC I I I I GATGGACCTGGAG GAAATCTTGCTCATGC I I I I CAACCAGGCCCAGGTATTGGAGGGGATGCTCAl I I I GATGAAGATGAAAGGTGGACCAACAATTTCAGAGAGTACAACTTA CATCGTGTTGCAGCTCATGAACTCGGCCATTCTCTTGGACTCTCCCATTCTA CTGATATCGGGGCTTTGATGTACCCTAGCTACACCTTCAGTGGTGATGTTCA GCTAGCTCAGGATGACATTGATGGCATCCAAGCCATATATGGACGTTCCCA AAATCCTGTCCAGCCCATCGGCCCACAAACCCCAAAAGCGTGTGACAGTAAGCTAACCTTTGATGCTATAACTACGATTCGGGGAGAAGTGATGTTCTTTADocket No. 33794 / 70815AAGACAGATTCTACATGCGCACAAATCCCTTCTACCCGGAAGTTGAGCTCA ATTTCATTTCTG I l l i CTGGCCACAACTGCCAAATGGGCTTGAAGCTGCTTA CGAATTTGCCGACAGAGATGAAGTCCGG I I I I I CAAAGGGAATAAGTACT GGGCTGTTCAGGGACAGAATGTGCTACACGGATACCCCAAGGACATCTAC AGCTCCTTTGGCTTCCCTAGAACTGTGAAGCATATCGATGCTGCTCTTTCTG AGGAAAACACTGGAAAAACCTACTTCTTTGTTGCTAACAAATACTGGAGG TATGATGAATATAAACGATCTATGGATCCAGGTTATCCCAAAATGATAGCAC ATGACTTTCCTGGAATTGGCCACAAAGTTGATGCAG I l l i CATGAAAGATG GAI I I I I CTATTTCTTTCATGGAACAAGACAATACAAATTTGATCCTAAAAC GAAGAGAAI I I I GACTCTCCAGAAAGCTAATAGCTGGTTCAACTGCAGGA AAAATTGA MMP2 GCGGATCCTCTAGAGTTAACATCGAGGGATCAAGCTTATCGATAATCAACC TCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CC I I I IACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCAI I I I CTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCA CTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGC TCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCC TTTGGGCCGCCTCCCCGCATCGATACCGTCGAGACCTAGAAAAACATGGA G CAATCACAAGTAG CAATACAGC AG CTACCAATG CTG ATTGTGCCTGG CTA GAAGCACAAGAGGAGGAGGAGGTGGG I I I I CCAGTCACACCTCAGGTAC CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC I I I I IAA AAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCA GAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGT GCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGA AGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATG ACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTT CATCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCAGTGTG GAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC CCTGG AAG GTG CC ACTCCC ACTGTCCTTTCCTAATAAAATG AG G AAATTG C ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTC TAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG TG GTG GTTACG CGC AG CGTG ACCG CTACACTTGCC AGCG CCCTAG CGCCC G CTCCTTTCG CTTTCTTCCCTTCCTTTCTCG CC ACGTTCG CCG GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACDocket No. 33794 / 70815GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGG I I I I I CGCCCTTTGACGTTGGAGTCCACGTTC TTTAATAGTG G ACTCTTGTTCC AAACTG G AACAACACTCAACCCTATCTCG G TCTATTC I I I I GAI I IAIAAGGGAI I I I GCCGATTTCGGCCTATTGGTTAAAA AATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAG G CTCCCC AG C AGG CAG AAGTATG CAAAGC ATGC ATCTCAATTAGTC AG CA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT CCGCCCATTCTCCGCCCCATGGCTGACTAAI I I I I I I IATTTATGCAGAGGC CGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC I l l i TTGGAGGCCTAGGC I l l i GCAAAAAGCTCCCGGGAGCTTGTATATCCATTT TCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGC ATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGT GCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGC CGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACC AGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA CGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGAC GCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGG GAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCG AGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTC TATGAAAGGTTGGGCTTCGGAATCG I I I I CCGGGACGCCGGCTGGATGAT CCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA AATAAAGCAI I I I I I I CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA ATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGA GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG GTAATACG GTTATCCACAG AATC AG GG G ATAACG CAG G AAAG AACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTG G AAG CTCCCTCGTG CGCTCTCCTGTTCCG ACCCTG CCG CTTAC CG G ATACCTGTCCG CCTTTCTCCCTTCGG G AAG CGTG G CGCTTTCTC ATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTG AGTCC A ACCCG GTA AG AC ACG ACTTATCG CC ACTG G C AG C AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG GTATCTG CG CTCTG CTG AAG CC AGTTACCTTCG G A A A A AG AGTTG GTAG C TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATDocket No. 33794 / 70815C l I I I C IACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA I I I I GG I CAI GAGAI I Al CAAAAAGGAI C I I CACC I AGAI CC I I I IAAATTA AAAATGAAG I I I IAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC G CAG AAGTG GTCCTG CAACTTTATCCG CCTCC ATCC AGTCTATTAATTGTTG CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG TTG CC ATTG CTAC AG G C ATCGTG GTGTC ACG CTCGTCGTTTG GTATG G CTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGC I I I I CTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC I I I IACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG CCG CAAAAAAG GG AATAAG GG CG ACACG G AAATGTTG AATACTCATACTC TTCC I I I I I CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGAT CCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC AGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGC AAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAA TCTGCTTAGGGTTAGGCG I I I I GCGCTGCTTCGCGATGTACGGGCCAGATA TACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTGATGCGG I I I I GGCAGTACATCA ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTG I I I I GGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCG CCCC ATTG ACG CAAATG G GCG GTAG GCGTGT ACGGTGGGAGGTCTATATAAGCAGCGCG I I I I GCCTGTACTGGGTCTCTCT GGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCA CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCA GTGTG G AAAATCTCTAGCAGTG GCG CCCG AAC AG G G ACTTG AAAGCG AA AGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG CGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATT TTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCDocket No. 33794 / 70815AGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGG AGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCT GTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAA GAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAG GATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAG C AAAAC AAAAGTAAG ACC ACCG CAC AG CAAG GG CCGCTG ATCTTCAG AC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATA AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGA AGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGAC GCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGA ACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACA GTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCA TTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG AACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAA GAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTG GAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGA TAGTAGGAGGCTTGGTAGGTTTAAGAATAG I I I I I GCTGTACTTTCTATAGT GAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCC AACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTACAAATG GCAGTATTCATCCACAAI I I IAAAAGAAAAGGGGGGATTGGGGGGTACAG TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAG AATTACAAAAACAAATTACAAAAATTCAAAAI I I I CGGGTTTATTACAGGG ACAGCAGAAATTCACTTTGAATTAATTCAAGCTTCGTGAGGCTCCGGTGCC CGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC I I I I I CCCGAGGGTGGG GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC I I I I I CGCAAC GGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC CTG G CCTCTTTACG G GTTATG G CCCTTG CGTG CCTTG A ATTACTTCC ACCTG GCTCCAGTACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTG CGCTTTAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAAI I I I I GATGACCTGCTGCGACGC I I I I I I I CTGGCAAGATAGTCTTGTAAATGCGGGCCAGGATCTGCACACTG GTATTTCGG I I I I I GGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCG CGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGC ACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTCCAGGG GGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAGGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG I I I IATGCGATGGAGDocket No. 33794 / 70815TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTT GATGTAATTCTCCTTGGAATTTGCCC I I I I I GAGTTTGGATCTTGGTTCATT CTCAAGCCTCAGACAGTGGTTCAAAG I I I I I I I CTTCCATTTCAGGTGTCG TGAACACGCTACCGGTCATGGAGGCGCTAATGGCCCGGGGCGCGCTCAC GGGTCCCCTGAGGGCGCTCTGTCTCCTGGGCTGCCTGCTGAGCCACGCCG CCGCCGCGCCGTCGCCCATCATCAAGTTCCCCGGCGATGTCGCCCCCAAAA CGGACAAAGAGTTGGCAGTGCAATACCTGAACACCTTCTATGGCTGCCCC AAGGAGAGCTGCAACCTGTTTGTGCTGAAGGACACACTAAAGAAGATGC AGAAGTTCTTTGGACTGCCCCAGACAGGTGATCTTGACCAGAATACCATCG AGACCATGCGGAAGCCACGCTGCGGCAACCCAGATGTGGCCAACTACAAC TTCTTCCCTCG CAAG CCC AAGTGG G ACAAG AACC AG ATCACATAC AG G AT CATTGGCTACACACCTGATCTGGACCCAGAGACAGTGGATGATGCCTTTGC TCGTGCCTTCCAAGTCTGGAGCGATGTGACCCCACTGCGG I I I I CTCGAAT CCATGATGGAGAGGCAGACATCATGATCAACTTTGGCCGCTGGGAGCATG GCGATGGATACCCCTTTGACGGTAAGGACGGACTCCTGGCTCATGCCTTCG CCCCAGGCACTGGTGTTGGGGGAGACTCCCAI I I I GATGACGATGAGCTA TGGACCTTGGGAGAAGGCCAAGTGGTCCGTGTGAAGTATGGGAACGCCG ATG GG G AGTACTG CAAGTTCCCCTTCTTGTTCAATG GC AAG G AGTAC AAC AGCTGCACTGATACCGGCCGCAGCGATGGCTTCCTCTGGTGCTCCACCACC TACAACTTTGAGAAGGATGGCAAGTACGGCTTCTGTCCCCATGAAGCCCT GTTCACCATGGGCGGCAACGCTGAAGGACAGCCCTGCAAGTTTCCATTCC GCTTCCAGGGCACATCCTATGACAGCTGCACCACTGAGGGCCGCACGGAT GGCTACCGCTGGTGCGGCACCACTGAGGACTACGACCGCGACAAGAAGT ATGGCTTCTGCCCTGAGACCGCCATGTCCACTGTTGGTGGGAACTCAGAA GGTGCCCCCTGTGTCTTCCCCTTCACTTTCCTGGGCAACAAATATGAGAGC TG CACC AG CG CCGG CCGC AGTG ACG G AAAG ATGTGGTGTG CG ACC AC AG CCAACTACGATGATGACCGCAAGTGGGGCTTCTGCCCTGACCAAGGGTAC AGCCTGTTCCTCGTGGCAGCCCACGAGTTTGGCCACGCCATGGGGCTGGA GCACTCCCAAGACCCTGGGGCCCTGATGGCACCCATTTACACCTACACCAA G AACTTCCGTCTGTCCC AG G ATG AC ATC AAG GG CATTC AG G AGCTCTATG G GGCCTCTCCTGACATTGACCTTGGCACCGGCCCCACCCCCACGCTGGGCC CTGTCACTCCTGAGATCTGCAAACAGGACATTGTATTTGATGGCATCGCTC AGATCCGTGGTGAGATCTTCTTCTTCAAGGACCGGTTCATTTGGCGGACTG TGACGCCACGTGACAAGCCCATGGGGCCCCTGCTGGTGGCCACATTCTGG CCTGAGCTCCCGGAAAAGATTGATGCGGTATACGAGGCCCCACAGGAGG AG AAG G CTGTGTTCTTTG C AG G G A ATG A ATACTG G ATCTACTC AG CC AG C ACCCTGGAGCGAGGGTACCCCAAGCCACTGACCAGCCTGGGACTGCCCC CTG ATGTCC AG CG AGTG G ATG CCG CCTTTAACTG G AG CAAAAACAAG AAG ACATACATCTTTGCTGGAGACAAATTCTGGAGATACAATGAGGTGAAGAA GAAAATGGATCCTGGCTTCCCCAAGCTCATCGCAGATGCCTGGAATGCCAT CCCCG ATAACCTG G ATG CCGTCGTG G ACCTG CAGG G CGG CGGTC AC AG CT ACTTCTTCAAGGGTGCCTATTACCTGAAGCTGGAGAACCAAAGTCTGAAG AGCGTGAAGTTTGGAAGCATCAAATCCGACTGGCTAGGCTGCTGAchiMMPl TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAG G G ACTTTCC ATTG ACGTC A ATG G GTG G AGTATTTACG GTA A ACTG CDocket No. 33794 / 70815CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC GTC AATG ACGGTAAATG GCCCG CCTG GC ATTATG CCC AGTAC ATG ACCTTAT GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG GTGATGCGG I I I I GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTC ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG I I I I G GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATT GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGA GCTGGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGG AGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGC CTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACT AGAGATCCCTCAGACCC I I I IAGTCAGTGTGGAAAATCTCTAGCAGTGGCG CCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCG ACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCG GCGACTGGTGAGTACGCCAAAAAI I I I GACTAGCGGAGGCTAGAAGGAG AGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGC GATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATT AAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATC CTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTA CAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAG TAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGG AAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGC ACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGA CAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTA GGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAA AGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG GAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACA ATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGA G G CG C A AC AG C ATCTGTTG C A ACTC AC AGTCTG G G G CATC A AG C AG CTCC AGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTG GGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTG GAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGAC CTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTC CTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTAT TGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATT GGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTT AAGAATAG I I I I I GCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATAT TCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAG GCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATC CATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGG GGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAA CAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATT TTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGAGGCTAGCCA ACTTTGTATAGAAAAGTTGGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCA CATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC CGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTG TACTGGCTCCGCC I I I I I CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC I I I I I CGCAACGGGTTTGCCGCCAGAACACDocket No. 33794 / 70815AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTAT GGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTT GATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCG CTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGC TGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGC TTTCGATAAGTCTCTAGCCATTTAAAAI I I I IGATGACCTGCTGCGACGCTT I I I I I CTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGT ATTTCGG I I I I IGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGC GCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCG GACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCG CCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCAC CAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACC C AC AC AAAG G AAAAGG G CCTTTCCGTCCTC AG CCGTCGCTTCATGTG ACT CCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTT TGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG I I I IATGCGATGGAGTT TCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGA TGTAATTCTCCTTGGAATTTGCCC I I I I IGAGTTTGGATCTTGGTTCATTCTC AAGCCTCAGACAGTGGTTCAAAG I I I I I I ICTTCCATTTCAGGTGTCGTGA CAAGTTTGTACAAAAAAGCAGGCTGCCACCATGAGCCCGGCGCCGCGCCC GCCGCGCTGCCTGCTGCTGCCGCTGCTGACCCTGGGCACCGCGCTGGCGA GCCTGGGCAGCGCGCAGAGCAGCAGCTTTAGCCCGGAAGCGTGGCTGCA GCAGTATGGCTATCTGCCGCCGGGCGATCTGCGCACCCATACCCAGCGCA GCCCGCAGAGCCTGAGCGCGGCGATTGCGGCGATGCAGAAAI I I IATGGC CTGCAGGTGACCGGCAAAGCGGATGCGGATACCATGAAAGCGATGCGCC GCCCGCGCTGCGGCGTGCCGGATAAATTTGGCGCGGAAATTAAAGCGAA CGTGCGCCGCAAACGCGGCAACCCGCGCTGGGAACAGACCCATCTGACC TATCGCATTGAAAACTATACCCCGGATCTGCCGCGCGCGGATGTGGATCAT GCGATTGAAAAAGCGTTTCAGCTGTGGAGCAACGTGACCCCGCTGACCTT TACCAAAGTGAGCGAAGGCCAGGCGGATATTATGATTAGCTTTGTGCGCG GCGATCATCGCGATAACAGCCCGTTTGATGGCCCGGGCGGCAACCTGGCG CATGCGTTTCAGCCGGGCCCGGGCATTGGCGGCGATGCGCAI I I IGATGA AGATGAACGCTGGACCAACAACTTTCGCGAATATAACCTGCATCGCGTGGC GGCGCATGAACTGGGCCATAGCCTGGGCCTGAGCCATAGCACCGATATTG GCGCGCTGATGTATCCGAGCTATACCTTTAGCGGCGATGTGCAGCTGGCGC AGGATGATATTGATGGCATTCAGGCGATTTATGGCCGCAGCCAGAACCCG GTGCAGCCGATTGGCCCGCAGACCCCGAAAGCGTGCGATAGCAAACTGA CCTTTGATGCGATTACCACCATTCGCGGCGAAGTGATGTTTTTTAAAGATC GC I I I IAI Al GCGCACCAACCCG I I I IATCCGGAAGTGGAACTGAACTTTAT TAGCGTG I I I IGGCCGCAGCTGCCGAACGGCCTGGAAGCGGCGTATGAAT TTGCGGATCGCGATGAAGTGCGCTTTTTTAAAGGCAACAAATATTGGGCG GTGCAGGGCCAGAACGTGCTGCATGGCTATCCGAAAGATATTTATAGCAGC TTTGGCTTTCCGCGCACCGTGAAACATATTGATGCGGCGCTGAGCGAAGA AAACACCGGCAAAACCTAI I I I I I IGTGGCGAACAAATATTGGCGCTATGA TGAATATAAACGCAGCATGGATCCGGGCTATCCGAAAATGATTGCGCATGAI l l i CCGGGCATTGGCCATAAAGTGGATGCGGTGTTTATGAAAGATGGCTTI I I I IAI I I I I I ICATGGCACCCGCCAGTATAAATTTGATCCGAAAACCAAADocket No. 33794 / 70815CGCATTCTGACCCTGCAGAAAGCGAACAGCTGGTTTAACTGCCGCAAAAA CCCGAGCGGCGGCCGCCCGGATGAAGGCACCGAAGAAGAAACCGAAGT GATTATTATTGAAGTGGATGAAGAAGGCGGCGGCGCGGTGAGCGCGGCG GCGGTGGTGCTGCCGGTGCTGCTGCTGCTGCTGGTGCTGGCGGTGGGCC TGGCGGTG I I I I I I I I I CGCCGCCATGGCACCCCGCGCCGCCTGCTGTATT GCCAGCGCAGCCTGCTGGATAAAGTGGGCGGCGGCAGCGGCGGCGGCA GCATGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTG GAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGCGGCGAAG G CG AAGG CG ATG CG ACC AACGG CAAACTG ACCCTG AAATTTATTTG CACC ACCGGCAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGACCTA TGGCGTGCAGTGCTTTAGCCGCTATCCGGATCATATGAAACGCCATGAI I I I TTTAAAAGCGCGATGCCGGAAGGCTATGTGCAGGAACGCACCATTAGCTT TAAAGATGATGGCACCTATAAAACCCGCGCGGAAGTGAAATTTGAAGGCG ATACCCTGGTGAACCGCATTGAACTGAAAGGCATTGAI I I IAAAGAAGATG GCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAACGTGTA TATTACCG CG G ATA A AC AG A A A A ACG G C ATTA A AG CG A ACTTTA A A ATTCG CCATAACGTGGAAGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGA ACACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAACCATTATCTGA GCACCCAGAGCGTGCTGAGCAAAGATCCGAACGAAAAACGCGATCATATG GTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCATGGCATGGATGA ACTGTATAAATAAACCCAGCTTTCTTGTACAAAGTGGTGATAATCGAATTCC GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTA ACTATGTTGCTCC I I I IACGCTATGTGGATACGCTGCTTTAATGCCTTTGTAT CATGCTATTGCTTCCCGTATGGCTTTCAI I I I CTCCTCCTTGTATAAATCCTG GTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCG TGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCA CGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCT TTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCT TCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCC TGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGA GTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGGAATTCCCGCGGTTCG AATTCTACCGGGTAGGGGAGGCGC I l l i CCCAAGGCAGTCTGGAGCATGC GCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCC TCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGG TGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCC G CCCCG CAGCTCG CGTCGTGC AG G ACGTG AC AAATG G AAGTAG CACGTCT C ACTAGTCTCGTG CAG ATG G ACAGC ACCG CTG AGC AATG G AAGCG GGTA GGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGC TCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGC TCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCT GCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTC CG GG CCTTTCGACCTC ACGTG GCCACCATGG CCAAGTTGACCAGTG CCGT TCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACC GACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTDocket No. 33794 / 70815GGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAG CTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTC CGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTT CGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAG CAGGACTGAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCT TAGCCAC I I I I IAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCC AACGAAGACAAGATCTGC I I I I I GCTTGTACTGGGTCTCTCTGGTTAGACC AGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG CCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCAGTGTGGAAA ATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCA AAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAAT GGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCAI I I I I I I CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTC TGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCC CAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAAI I I I I I I IATTTATGCAG AGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC l I I I I I G G AGG CCTAG G G ACGTACCC AATTCGCCCTATAGTG AGTCGTAT TACGCGCGCTCACTGGCCGTCG I I I IACAACGTCGTGACTGGGAAAACCC TG GCGTTACCCAACTTAATCG CCTTG CAG CACATCCCCCTTTCG CC AG CTG GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA GCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGC G G GTGTG GTG GTTACG CG C AG CGTG ACCG CTAC ACTTG CC AG CG CCCTAG CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTT TCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGC TTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAG TGGGCCATCGCCCTGATAGACGG I I I I I CGCCCTTTGACGTTGGAGTCCAC GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC TCGGTCTATTC I I I I GAI I IAIAAGGGAI I I I GCCGATTTCGGCCTATTGGTT AAAAAATGAGCTGATTTAACAAAAATTTAACGCGAAI I I IAACAAAATATTA ACGCTTACAATTTAGGTGGCAC I l l i CGGGGAAATGTGCGCGGAACCCCT ATTTGTTTAI I I I I CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC ATTTCCGTGTCGCCCTTATTCCC I I l l i I GCGGCAI I I I GCC I I CC I G I I I I I G CTC ACCCAG AAACGCTG GTG AAAGTAAAAG ATG CTG AAG ATCAGTTG G GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAG I I I I CGCCCCGAAGAACG I I I I CCAAI GAI GAGCAC I I I IAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTC GGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAAC GACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAA ACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCDocket No. 33794 / 70815GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAA GTTTACTCATATATACTTTAGATTGATTTAAAACTTCAI I I I IAATTTAAAAGG ATCTAGGTGAAGATCC I I I I IGATAATCTCATGACCAAAATCCCTTAACGTG AG I I I I CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT TCTTGAGATCC I I I I I I I CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTT TTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTC TAGTGTAG CCGTAGTTAG GCCACCACTTC AAG AACTCTGTAG CACCG CCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC GCCACGCTTCCCGAAGAGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC GCGGCC I I I I IACGG I ICC IGGCC I I I IGC IGGCC I I I I GCTCACATGTTCT TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG AGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGC GTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAA GCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTG AGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAG CGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTT MMP14 GCGGATCCTCTAGAGTTAACATCGAGGGATCAAGCTTATCGATAATCAACC TCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CC I I I IACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCAI I I I CTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCA CTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGC TCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCC TTTGGGCCGCCTCCCCGCATCGATACCGTCGAGACCTAGAAAAACATGGA G CAATCACAAGTAG CAATACAGC AG CTACCAATG CTG ATTGTGCCTGG CTA GAAGCACAAGAGGAGGAGGAGGTGGG I I I I CCAGTCACACCTCAGGTAC CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC I I I I IAA AAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCA GAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGADocket No. 33794 / 70815AGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATG ACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTT CATCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCAGTGTG GAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC CCTGG AAG GTG CC ACTCCC ACTGTCCTTTCCTAATAAAATG AG G AAATTG C ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTC TAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG TG GTG GTTACG CGC AG CGTG ACCG CTACACTTGCC AGCG CCCTAG CGCCC G CTCCTTTCGCTTTCTTCCCTTCCTTTCTCG CC ACGTTCG CCG GCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGG I I I I I CGCCCTTTGACGTTGGAGTCCACGTTC TTTAATAGTG G ACTCTTGTTCC AAACTG G AACAACACTCAACCCTATCTCG G TCTATTC I I I I GAI I IAIAAGGGAI I I I GCCGATTTCGGCCTATTGGTTAAAA AATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAG G CTCCCC AG C AGG CAG AAGTATG CAAAGC ATGC ATCTCAATTAGTC AG CA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT CCGCCCATTCTCCGCCCCATGGCTGACTAAI I I I I I I IATTTATGCAGAGGC CGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC I l l i TTGGAGGCCTAGGC I l l i GCAAAAAGCTCCCGGGAGCTTGTATATCCATTT TCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGC ATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGT GCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGC CGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACC AGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA CGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGAC GCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGG GAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCG AGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTC TATGAAAGGTTGGGCTTCGGAATCG I I I I CCGGGACGCCGGCTGGATGAT CCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA AATAAAGCAI I I I I I I CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA ATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGADocket No. 33794 / 70815GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG GTAATACG GTTATCCACAG AATC AG GG G ATAACG CAG G AAAG AACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTG G AAG CTCCCTCGTG CGCTCTCCTGTTCCG ACCCTG CCG CTTAC CG G ATACCTGTCCG CCTTTCTCCCTTCGG G AAG CGTG G CGCTTTCTC ATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTG AGTCC A ACCCG GTA AG AC ACG ACTTATCG CC ACTG G C AG C AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG GTATCTG CG CTCTG CTG AAG CC AGTTACCTTCG G A A A A AG AGTTG GTAG C TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGC AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT C I I I I CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA I I I I GG I CAI GAGAI I Al CAAAAAGGAI C I I CACC I AGAI CC I I I IAAATTA AAAATGAAG I I I IAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC G CAG AAGTG GTCCTG CAACTTTATCCG CCTCC ATCC AGTCTATTAATTGTTG CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG TTG CC ATTG CTAC AG G C ATCGTG GTGTC ACG CTCGTCGTTTG GTATG G CTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGC I I I I CTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC I I I IACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG CCG CAAAAAAG GG AATAAG GG CG ACACG G AAATGTTG AATACTCATACTC TTCC I I I I I CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGAT CCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC AGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGC AAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAA TCTGCTTAGGGTTAGGCG I I I I GCGCTGCTTCGCGATGTACGGGCCAGATA TACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGDocket No. 33794 / 70815GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTGATGCGG I I I IGGCAGTACATCA ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTG I I I IGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCG CCCC ATTG ACG CAAATG G GCG GTAG GCGTGT ACGGTGGGAGGTCTATATAAGCAGCGCG I I I IGCCTGTACTGGGTCTCTCT GGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCA CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCA GTGTG G AAAATCTCTAGCAGTG GCG CCCG AAC AG G G ACTTG AAAGCG AA AGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG CGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATT TTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTA TTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCC AGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGG AGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCT GTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAA GAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAG GATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAG C AAAAC AAAAGTAAG ACC ACCG CAC AG CAAG GG CCGCTG ATCTTCAG AC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATA AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGA AGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGAC GCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGA ACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACA GTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCA TTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG AACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAA GAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTG GAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGA TAGTAGGAGGCTTGGTAGGTTTAAGAATAG I I I I IGCTGTACTTTCTATAGT GAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCC AACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTACAAATG GCAGTATTCATCCACAAI I I IAAAAGAAAAGGGGGGATTGGGGGGTACAG TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAG AATTACAAAAACAAATTACAAAAATTCAAAAI I I ICGGGTTTATTACAGGG ACAGCAGAAATTCACTTTGAATTAATTCAAGCTTCGTGAGGCTCCGGTGCC CGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC I I I I I CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC I I I I I CGCAACDocket No. 33794 / 70815GGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC CTG G CCTCTTTACG G GTTATG G CCCTTG CGTG CCTTG A ATTACTTCC ACCTG GCTCCAGTACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTG CGCTTTAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAAI I I I I GATGACCTGCTGCGACGC I I I I I I I CTGGCAAGATAGTCTTGTAAATGCGGGCCAGGATCTGCACACTG GTATTTCGG I I I I I GGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCG CGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGC ACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTCCAGGG GGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAGGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCT TTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG I I I IATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTT GATGTAATTCTCCTTGGAATTTGCCC I I I I I GAGTTTGGATCTTGGTTCATT CTCAAGCCTCAGACAGTGGTTCAAAG I I I I I I I CTTCCATTTCAGGTGTCG TGAACACGCTACCGGTCATGTCTCCCGCCCCAAGACCCCCCCGTTGTCTCC TGCTCCCCCTGCTCACGCTCGGCACCGCGCTCGCCTCCCTCGGCTCGGCCC AAAGCAGCAGCTTCAGCCCCGAAGCCTGGCTACAGCAATATGGCTACCTG CCTCCCGGGGACCTACGTACCCACACACAGCGCTCACCCCAGTCACTCTCA GCGGCCATCGCTGCCATGCAGAAG I I I IACGGCTTGCAAGTAACAGGCAA AGCTGATGCAGACACCATGAAGGCCATGAGGCGCCCCCGATGTGGTGTTC CAGACAAGTTTGGGGCTGAGATCAAGGCCAATGTTCGAAGGAAGCGCTA CGCCATCCAGGGTCTCAAATGGCAACATAATGAAATCACTTTCTGCATCCA GAATTACACCCCCAAGGTGGGCGAGTATGCCACATACGAGGCCATTCGCA AGGCGTTCCGCGTGTGGGAGAGTGCCACACCACTGCGCTTCCGCGAGGT GCCCTATGCCTACATCCGTGAGGGCCATGAGAAGCAGGCCGACATCATGAT CTTCTTTGCCGAGGGCTTCCATGGCGACAGCACGCCCTTCGATGGTGAGG GCGGCTTCCTGGCCCATGCCTACTTCCCAGGCCCCAACATTGGAGGAGAC ACCCACTTTGACTCTGCCGAGCCTTGGACTGTCAGGAATGAGGATCTGAAT GGAAATGACATCTTCCTGGTGGCTGTGCACGAGCTGGGCCATGCCCTGGG GCTCGAGCATTCCAGTGACCCCTCGGCCATCATGGCACCC I I I IACCAGTG GATGGACACGGAGAAI I I I GTGCTGCCCGATGATGACCGCCGGGGCATCC AGCAACTTTATGGGGGTGAGTCAGGGTTCCCCACCAAGATGCCCCCTCAA CCCAGGACTACCTCCCGGCCTTCTGTTCCTGATAAACCCAAAAACCCCACC TATGGGCCCAACATCTGTGACGGGAACTTTGACACCGTGGCCATGCTCCG AGGGGAGATGTTTGTCTTCAAGGAGCGCTGGTTCTGGCGGGTGAGGAAT AACCAAGTGATGGATGGATACCCAATGCCCATTGGCCAGTTCTGGCGGGG CCTGCCTGCGTCCATCAACACTGCCTACGAGAGGAAGGATGGCAAATTCG TCTTCTTCAAAGGAGACAAGCATTGGGTGTTTGATGAGGCGTCCCTGGAA CCTGGCTACCCCAAGCACATTAAGGAGCTGGGCCGAGGGCTGCCTACCGA C AAG ATTG ATG CTGCTCTCTTCTG G ATG CCCAATG G AAAG ACCTACTTCTTC CGTGGAAACAAGTACTACCGTTTCAACGAAGAGCTCAGGGCAGTGGATAGCGAGTACCCCAAGAACATCAAAGTCTGGGAAGGGATCCCTGAGTCTCCCADocket No. 33794 / 70815GAGGGTCATTCATGGGCAGCGATGAAGTCTTCACTTACTTCTACAAGGGG AACAAATACTGGAAATTCAACAACCAGAAGCTGAAGGTAGAACCGGGCTA CCCCAAGTCAGCCCTGAGGGACTGGATGGGCTGCCCATCGGGAGGCCGG CCGGATGAGGGGACTGAGGAGGAGACGGAGGTGATCATCATTGAGGTG GACGAGGAGGGCGGCGGGGCGGTGAGCGCGGCTGCCGTGGTGCTGCCC GTGCTGCTGCTGCTCCTGGTGCTGGCGGTGGGCCTTGCAGTCTTCTTCTTC AGACGCCATGGGACCCCCAGGCGACTGCTCTACTGCCAGCGTTCCCTGCT GGACAAGGTCTGA FAP GCGGATCCTCTAGAGTTAACATCGAGGGATCAAGCTTATCGATAATCAACC TCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CC I I I IACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCAI I I I CTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCA CTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGC TCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCC TTTGGGCCGCCTCCCCGCATCGATACCGTCGAGACCTAGAAAAACATGGA G CAATCACAAGTAG CAATACAGC AG CTACCAATG CTG ATTGTGCCTGG CTA GAAGCACAAGAGGAGGAGGAGGTGGG I I I I CCAGTCACACCTCAGGTAC CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCAC I I I I IAA AAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCA GAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGT GCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGA AGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATG ACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTT CATCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCT GTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCAGTGTG GAAAATCTCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC CCTGG AAG GTG CC ACTCCC ACTGTCCTTTCCTAATAAAATG AG G AAATTG C ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTC TAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG TG GTG GTTACG CGC AG CGTG ACCG CTACACTTGCC AGCG CCCTAG CGCCC G CTCCTTTCG CTTTCTTCCCTTCCTTTCTCG CC ACGTTCG CCG GCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGG I I I I I CGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG G ACTCTTGTTCC AAACTG G AACAACACTCAACCCTATCTCG GDocket No. 33794 / 70815TCTATTC I I I I GATTTATAAGGGAI I I I GCCGATTTCGGCCTATTGGTTAAAA AATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAG G CTCCCC AG C AGG CAG AAGTATG CAAAGC ATGC ATCTCAATTAGTC AG CA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTT CCGCCCATTCTCCGCCCCATGGCTGACTAAI I I I I I I IATTTATGCAGAGGC CGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC I l l i TTGGAGGCCTAGGC I l l i GCAAAAAGCTCCCGGGAGCTTGTATATCCATTT TCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGC ATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGT GCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGC CGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACC AGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA CGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGAC GCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGG GAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCG AGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTC TATGAAAGGTTGGGCTTCGGAATCG I I I I CCGGGACGCCGGCTGGATGAT CCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA AATAAAGCAI I I I I I I CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA ATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGA GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG GTAATACG GTTATCCACAG AATC AG GG G ATAACG CAG G AAAG AACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCG I I I I I CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT TTCCCCCTG G AAG CTCCCTCGTG CGCTCTCCTGTTCCG ACCCTG CCG CTTAC CG G ATACCTGTCCG CCTTTCTCCCTTCGG G AAG CGTG G CGCTTTCTC ATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTG AGTCC A ACCCG GTA AG AC ACG ACTTATCG CC ACTG G C AG C AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG GTATCTG CG CTCTG CTG AAG CC AGTTACCTTCG G A A A A AG AGTTG GTAG C TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG I I I I I I I GTTTGC AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT C I I I I CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA I I I I GG I CAI GAGAI I Al CAAAAAGGAI C I I CACC I AGAI CC I I I IAAATTAAAAATGAAG I I I IAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACDocket No. 33794 / 70815AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC G CAG AAGTG GTCCTG CAACTTTATCCG CCTCC ATCC AGTCTATTAATTGTTG CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG TTG CC ATTG CTAC AG G C ATCGTG GTGTC ACG CTCGTCGTTTG GTATG G CTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGC I I I I CTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC I I I IACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG CCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC TTCC I I I I I CAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGAT CCCCTATG GTG CACTCTCAGTAC AATCTG CTCTG ATG CCG CATAGTTAAGCC AGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGC AAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAA TCTGCTTAGGGTTAGGCG I I I I GCGCTGCTTCGCGATGTACGGGCCAGATA TACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTGATGCGG I I I I GGCAGTACATCA ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTG I I I I GGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCG CCCC ATTG ACG CAAATG G GCG GTAG GCGTGT ACGGTGGGAGGTCTATATAAGCAGCGCG I I I I GCCTGTACTGGGTCTCTCT GGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCA CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC I I I IAGTCA GTGTG G AAAATCTCTAGCAGTG GCG CCCG AAC AG G G ACTTG AAAGCG AA AGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG CGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATT TTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTA TTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCC AGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGG AGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAADocket No. 33794 / 70815GAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAG GATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAG C AAAAC AAAAGTAAG ACC ACCG CAC AG CAAG GG CCGCTG ATCTTCAG AC CTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATA AAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGA AGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCC TTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGAC GCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGA ACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACA GTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCA TTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG AACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAAC AATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAA GAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTG GAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGA TAGTAGGAGGCTTGGTAGGTTTAAGAATAG I I I I I GCTGTACTTTCTATAGT GAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCC AACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTACAAATG GCAGTATTCATCCACAAI I I IAAAAGAAAAGGGGGGATTGGGGGGTACAG TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAG AATTACAAAAACAAATTACAAAAATTCAAAAI I I I CGGGTTTATTACAGGG ACAGCAGAAATTCACTTTGAATTAATTCAAGCTTCGTGAGGCTCCGGTGCC CGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGG GAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC I I I I I CCCGAGGGTGGG GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC I I I I I CGCAAC GGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC CTG G CCTCTTTACG G GTTATG G CCCTTG CGTG CCTTG A ATTACTTCC ACCTG GCTCCAGTACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTG CGCTTTAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAAI I I I I GATGACCTGCTGCGACGC I I I I I I I CTGGCAAGATAGTCTTGTAAATGCGGGCCAGGATCTGCACACTG GTATTTCGG I I I I I GGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCG CGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGC ACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTCCAGGG GGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAGGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCT TTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGG I I I IATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTT GATGTAATTCTCCTTGGAATTTGCCC I I I I I GAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG I I I I I I I CTTCCATTTCAGGTGTCGDocket No. 33794 / 70815TGAACACGCTACCGGTCACCCTCGAAAGGCCTCTGATGAAGACTTGGGTA AAAATCGTATTTGG AGTTG CC ACCTCTG CTGTG CTTG CCTTATTGGTG ATGT GCATTGTCTTACGCCCTTCAAGAGTTCATAACTCTGAAGAAAATACAATGA GAGCACTCACACTGAAGGATAI I I IAAAI GGAACAI I I I CTTATAAAACATTI l l i CCAAACTGGATTTCAGGACAAGAATATCTTCATCAATCTGCAGATAAC AATATAGTACTTTATAATATTGAAACAGGACAATCATATACCAI I I I GAGTAA TAGAACCATGAAAAGTGTGAATGCTTCAAATTACGGCTTATCACCTGATCG GCAATTTGTATATCTAGAAAGTGATTATTCAAAGCTTTGGAGATACTCTTAC ACAGCAACATATTACATCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGGTCGCCTGTTGGGAGTA AATTAGCATATGTCTATCAAAACAATATCTATTTGAAACAAAGACCAGGAGA TCCACC I I I I CAAATAACATTTAATGGAAGAGAAAATAAAATATTTAATGGA ATCCCAGACTGGGTTTATGAAGAGGAAATGCTTGCTACAAAATATGCTCTC TGGTGGTCTCCTAATGGAAAAI I I I I GGCATATGCGGAATTTAATGATACGG ATATACCAGTTATTGCCTATTCCTATTATGGCGATGAACAATATCCTAGAACA ATAAATATTCCATACCCAAAGGCTGGAGCTAAGAATCCCGTTGTTCGGATAT TTATTATCGATACCACTTACCCTGCGTATGTAGGTCCCCAGGAAGTGCCTGT TCCAGCAATGATAGCCTCAAGTGATTATTATTTCAGTTGGCTCACGTGGGTT ACTGATGAACGAGTATGTTTGCAGTGGCTAAAAAGAGTCCAGAATGTTTC GGTCCTGTCTATATGTGACTTCAGGGAAGACTGGCAGACATGGGATTGTCC AAAGACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGA TTCTTTGTTTCAACACCAG I I I I CAGCTATGATGCCATTTCGTACTACAAAAT ATTTAGTGACAAGGATGGCTACAAACATATTCACTATATCAAAGACACTGTG GAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATAAATATATTC AGAGTAACACAGGATTCACTG I I I IATTCTAGCAATGAATTTGAAGAATACC CTG G AAG AAG AAACATCTACAG AATTAG CATTG G AAG CTATCCTCC AAG CA AGAAGTGTGTTACTTGCCATCTAAGGAAAGAAAGGTGCCAATATTACACA GCAAGTTTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTACGGCCCA GGCATCCCCATTTCCACCCTTCATGATGGACGCACTGATCAAGAAATTAAA ATCCTGGAAGAAAACAAGGAATTGGAAAATGCTTTGAAAAATATCCAGCT GCCTAAAGAGGAAATTAAGAAACTTGAAGTAGATGAAATTACTTTATGGTA CAAGATGATTCTTCCTCCTCAATTTGACAGATCAAAGAAGTATCCCTTGCTA ATTCAAGTGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCTGTATTTGCT GTTAATTGGATATCTTATCTTGCAAGTAAGGAAGGGATGGTCATTGCCTTG GTGGATGGTCGAGGAACAGCTTTCCAAGGTGACAAACTCCTCTATGCAGT GTATCGAAAGCTGGGTGTTTATGAAGTTGAAGACCAGATTACAGCTGTCA GAAAATTCATAGAAATGGGTTTCATTGATGAAAAAAGAATAGCCATATGGG GCTGGTCCTATGGAGGATACGTTTCATCACTGGCCCTTGCATCTGGAACTG GTC I I I I CAAATGTGGTATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATTA CGCGTCTGTCTACACAGAGAGATTCATGGGTCTCCCAACAAAGGATGATAA TCTTGAGCACTATAAGAATTCAACTGTGATGGCAAGAGCAGAATATTTCAG AAATGTAG ACTATCTTCTCATCC ACG G AAC AG CAG ATG ATAATGTG CACTTT CAAAACTCAGCACAGATTGCTAAAGCTCTGGTTAATGCACAAGTGGATTTC 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Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer. J. Transl. Med. 13, 277 (2015).14. Liu, E. et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N. Engl. J. Med. 382, 545-553 (2020).15. Fayette, J. et al. Results of a phase II study evaluating monalizumab in combination with cetuximab in previously treated recurrent or metastatic squamous cell carcinoma of the head and neck (R / M SCCHN). Ann. Oncol. 29, viii374 (2018).16. Sarhan, D. et al. 161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS. Blood Adv. 2, 1459-1469 (2018).17. Felices, M. et al. IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer. Gynecol. Oncol. 145, 453—461 (2017).18. Wennerberg, E., Kremer, V., Childs, R. & Lundqvist, A. CXCL10-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo. Cancer Immunol. Immunother. 64, 225-235 (2014).19. Kremer, V. et al. Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma. J. Immunother. Cancer 5, 73 (2017).20. Kameritsch, P. & Renkawitz, J. Principles of Leukocyte Migration Strategies. Trends in Cell Biology vol. 30818-832 (2020).21. Oda, K. et al. Conversion of secretory proteins into membrane proteins by fusing with a glycophosphatidylinositol anchor signal of alkaline phosphatase. Biochem J. 301, 577-583 (1994).22. Klein, T. & Bischoff, R. Physiology and pathophysiology of matrix metalloproteases. Amino Acids 41 : 271 -290 (2011 ).23. Itoh, Y. Membrane-type matrix metalloproteinases: their functions and regulations. Matrix Biol. 44-46: 207-223 (2015).24. Kessenbrock, K., Plaks, V., Werb, Z. 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Mmp-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases. Molecular Cancer Research, 4(5), 293-302 (2006).39. Notary, K., Allen, E., Pindzola, J., & Yoon, C. "Regulation of Cell Invasion and Morphogenesis in a Three-Dimensional Type I Collagen Matrix by Membrane-Type Matrix Metalloproteinases 1, 2, and 3." The Journal of Cell Biology, 149(6), 1309-1323 (2000). 40. Radisky, D.C. Epithelial-mesenchymal transition. J Cell Sci. 118(19): 4325-4326 (2005).41. Roy, R., Yang, J., Moses, M.A. Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol. 27: 5287-5297 (2009).42. Murashige, M. et al. Enhanced expression of tissue inhibitors of metalloproteinases in human colorectal tumors. Jpn J Clin Oncol. 26: 303-309 (1996).43. Young, T.N. et al. Characterization of gelatinases linked to extracellular matrix invasion in ovarian adenocarcinoma: purification of matrix metalloproteinase 2. 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M., Ohyashiki, K., Pandolfi, P. P., & Broxmeyer, H. E. Stromal cell-derived factor-1alpha / CXCL12-induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1. Blood, 105(2), 474-480 (2005).56. Oda, K. et al. Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphate. Biochem J., 301(2): 577-583 (1994).

Claims

Docket No. 33794 / 70815What is claimed is:

1. A pharmaceutical composition comprising genetically modified immune cells, wherein the modified immune cells overexpress one or more membrane-bound proteases.

2. The pharmaceutical composition of claim 1 , wherein at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease.

3. The pharmaceutical composition of claim 1 or claim 2, wherein at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound.

4. The pharmaceutical composition of claim 3, wherein the soluble protease is engineered to:i) remove the secretory domain; andii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor .

5. The pharmaceutical composition of any one of claims 1 -4, wherein at least one of the one or more membrane-bound proteases is a matrix metalloproteinase.

6. The pharmaceutical composition of claim 5, wherein the membrane-bound matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP- 25, or a combination thereof.

7. The pharmaceutical composition of claim 3 or claim 4, wherein the soluble protease is a matrix metalloproteinase selected from MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP- 26, MMP-27, MMP-28, or a combination thereof.

8. The pharmaceutical composition of claim 7, wherein the secretory domain is a pro-MMP cleavage site.

9. The pharmaceutical composition of any one of claims 1 -8, wherein at least one of the one or more overexpressed membrane-bound proteases is a cathepsin.

10. The pharmaceutical composition of claim 9, wherein the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof.

11. The pharmaceutical composition of any one of claims 1 -10, wherein at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domain-containing protein (ADAM).Docket No. 33794 / 7081512. The pharmaceutical composition of claim 11 , wherein the a disintegrin and metalloproteinase domain-containing protein (ADAM) is ADAM10, ADAM17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof.

13. The pharmaceutical composition of any one of claims 1-12, wherein at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof.

14. The pharmaceutical composition of any one of claims 1-13, wherein at least one of the one or more overexpressed membrane-bound proteases is overexpressed by at least 5-fold when compared to an unmodified control cell.

15. The pharmaceutical composition of any one of claims 1 -14, wherein the one or more membrane-bound proteases are human proteases.

16. The pharmaceutical composition of any one of claims 1 -15, wherein the genetically modified immune cells are natural killer (NK) cells, T cells, or a combination thereof.

17. The pharmaceutical composition of claim 16, wherein the T cells are CD4 T cells, CD8 T cells, or a combination thereof.

18. The pharmaceutical composition of claim 16, wherein the natural killer (NK) cells are derived from NK92, NK92-GFP, NKL, YT, KHYG-1 , NK92-CD16V, or a combination thereof.

19. The pharmaceutical composition of claim 16, wherein the genetically modified natural killer (NK) cells are derived from a human donor, optionally wherein the human donor is a cancer patient.

20. The pharmaceutical composition of claim 19, wherein the genetically modified natural killer (NK) cells are autologous or allogenic.

21. The pharmaceutical composition of claim 19, wherein, prior to genetic modification, the natural killer cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof.

22. The pharmaceutical composition of claim 19, wherein the genetically modified natural killer cells are further modified to express a chimeric antigen receptor (CAR).

23. The pharmaceutical composition of claim 19, wherein the natural killer cells are further genetically modified to overexpress one or more chemokines or cytokines.

24. The pharmaceutical composition of claim 23, wherein the one or more chemokines or cytokines are CCL2, CCL5, CCL20, CXCL1 , CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof.Docket No. 33794 / 7081525. The pharmaceutical composition of claim 23 or claim 24, wherein the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

26. The pharmaceutical composition of any one of claims 1 -20, wherein the genetic modification of the immune cells is performed by transformation, transfection, or transduction.

27. The pharmaceutical composition of any one of claims 1 -21 , further comprising a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of any one of claims 1 -27, wherein the composition comprises the genetically modified immune cells in a therapeutically effective amount for infiltrating a solid cancer.

29. The pharmaceutical composition of claim 28, wherein infiltration is increased by at least 30% when compared to an unmodified immune cell.

30. The pharmaceutical composition of claim 28 or claim 29, wherein the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof.

31. The pharmaceutical composition of claim 30, wherein the cancer is pancreatic cancer.

32. The pharmaceutical composition of claim 31 , wherein the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC).

33. The pharmaceutical composition of any of claims 1 -32 formulated for administration to a human subject.

34. The pharmaceutical composition of any of claims 1 -33 further comprising a pharmaceutically acceptable excipient.

35. A pharmaceutical composition for use in treating a disease comprising administering to a subject in need thereof, a therapeutically effective amount of genetically modified immune cells, wherein the genetically modified immune cells overexpress one or more membrane-bound protease.

36. The pharmaceutical composition for use of claim 35, wherein at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease.

37. The pharmaceutical composition for use of claim 35 or 36, wherein at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound.Docket No. 33794 / 7081538. The pharmaceutical composition for use of claim 37, wherein the soluble protease is engineered to:i) remove the secretory domain; andii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor.

39. The pharmaceutical composition for use of any one of claims 35-38, wherein at least one of the one or more membrane-bound proteases is a matrix metalloproteinase.

40. The pharmaceutical composition for use of claim 39, wherein the matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP- 25, or a combination thereof.

41. The pharmaceutical composition for use of claim 37 or claim 38, wherein the soluble matrix metalloproteinase is selected from MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP- 26, MMP-27, MMP-28, or a combination thereof.

42. The pharmaceutical composition for use of claim 41 , wherein the secretory domain is a pro-MMP cleavage site.

43. The pharmaceutical composition for use of any one of claims 35-42, wherein at least one of the one or more overexpressed membrane-bound proteases is a cathepsin.

44. The pharmaceutical composition for use of claim 43, wherein the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof.

45. The pharmaceutical composition for use of any one of claims 35-44, wherein at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domain-containing protein (ADAM).

46. The pharmaceutical composition for use of claim 45, wherein the a disintegrin and metalloproteinase domain-containing protein (ADAM) is ADAM10, ADAM17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof.

47. The pharmaceutical composition for use of any one of claims 35-46, wherein at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof.

48. The pharmaceutical composition for use of any one of claims 35-47, at least one of the one or more overexpressed membrane-bound proteases is overexpressed by at least 5-fold when compared to an unmodified control cell.Docket No. 33794 / 7081549. The pharmaceutical composition for use of any one of claims 35-48, wherein the subject is a human subject.

50. The pharmaceutical composition for use of any one of claims 35-49, wherein the disease is cancer.

51. The pharmaceutical composition for use of claim 50, wherein the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof.

52. The pharmaceutical composition for use of claim 51 , wherein the cancer is pancreatic cancer.

53. The pharmaceutical composition for use of claim 52, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

54. The pharmaceutical composition for use of any one of claims 35-53, wherein the genetically modified immune cells are natural killer (NK) cells, T cells, or a combination thereof.

55. The pharmaceutical composition for use of claim 54, wherein the T cells are CD4 T cells, CD8 T cells, or a combination thereof.

56. The pharmaceutical composition for use of claim 54, wherein the natural killer (NK) cells are derived from a human donor, optionally wherein the human donor is a cancer patient.

57. The pharmaceutical composition for use of claim 56, wherein the genetically modified natural killer (NK) cells are autologous or allogenic.

58. The pharmaceutical composition for use of claim 54 wherein, prior to genetic modification, the natural killer (NK) cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof.

59. The pharmaceutical composition for use of claim 54, wherein the genetically modified natural killer (NK) cells are further modified to express a chimeric antigen receptor (CAR).

60. The pharmaceutical composition for use of claim 54, wherein the natural killer (NK) cells are genetically modified to overexpress one or more chemokines or cytokines.

61. The pharmaceutical composition for use of claim 60, wherein the one or more chemokines or cytokines are CCL2, CCL5, CCL20, CXCL1 , CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof.Docket No. 33794 / 7081562. The pharmaceutical composition for use of claim 60 or claim 61 , wherein the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

63. The pharmaceutical composition for use of any one of claims 35-62, wherein the genetic modification of the immune cells is performed by transformation, transfection, or transduction.

64. The pharmaceutical composition for use of any one of claims 35-63, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

65. The pharmaceutical composition for use of any one of claims 35-64 formulated for administration to a human subject.

66. The pharmaceutical composition for use of any one of claims 35-65, wherein the pharmaceutical composition is administered directly into a cancerous tumor.

67. The pharmaceutical composition for use of any one of claims 35-66, wherein the pharmaceutical composition is administered intravenously.

68. The pharmaceutical composition for use of any one of claims 35-67, wherein the genetically modified immune cells treat the disease by breaking down extracellular matrix (ECM).

69. The pharmaceutical composition for use of any one of claims 35-68, wherein the genetically modified immune cells treat the disease by targeting fibroblasts.

70. The pharmaceutical composition for use of claim 69, wherein the fibroblasts are cancer-associated fibroblasts (CAFs).

71. The pharmaceutical composition for use of any one of claims 35-70, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

72. A method of treating a disease in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of genetically modified immune cells, wherein the genetically modified immune cells overexpress one or more membrane-bound protease.

73. The method of claim 72, wherein at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease.

74. The method of claim 72 or 73, wherein at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound.

75. The method of claim 74, wherein the soluble protease is engineered to:Docket No. 33794 / 70815i) remove the secretory domain; andii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor.

76. The method of any one of claims 72-75, wherein at least one of the one or more membrane-bound proteases is a matrix metalloproteinase.

77. The method of claim 76, wherein the matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25, or a combination thereof.

78. The method claim 74 or claim 75, wherein the soluble matrix metalloproteinase is selected from MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP-26, MMP-27, MMP-28, or a combination thereof.

79. The method of claim 78, wherein the secretory domain is a pro-MMP cleavage site.

80. The method of any one of claims 72-79, wherein at least one of the one or more overexpressed membrane-bound proteases is a cathepsin.

81. The method of claim 80, wherein the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof.

82. The method of any one of claims 72-81 , wherein at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domaincontaining protein (ADAM).

83. The method of claim 82, wherein the a disintegrin and metalloproteinase domaincontaining protein (ADAM) is ADAM 10, ADAM 17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof.

84. The method of any one of claims 72-83, wherein at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof.

85. The method of any one of claims 72-84, wherein at least one of the one or more overexpressed membrane-bound proteases is overexpressed by at least 5-fold when compared to an unmodified control cell.

86. The method of any one of claims 72-85, wherein the subject is a human subject.

87. The method of any one of claims 72-86, wherein the disease is cancer.Docket No. 33794 / 7081588. The method of claim 87, wherein the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof.

89. The method of claim 88, wherein the cancer is pancreatic cancer.

90. The method of claim 89, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

91. The method of any one of claims 72-90, wherein the genetically modified immune cells are natural killer (NK) cells, T cells, or a combination thereof.

92. The method of claim 91 , wherein the T cells are CD4 T cells, CD8 T cells, or a combination thereof.

93. The method of claim 91 , wherein the natural killer (NK) cells are derived from a human donor, optionally wherein the human donor is a cancer patient.

94. The method of claim 93, wherein the genetically modified natural killer (NK) cells are autologous or allogenic.

95. The method of claim 91 wherein, prior to genetic modification, the natural killer (NK) cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof.

96. The method of claim 91 , wherein the genetically modified natural killer (NK) cells are further modified to express a chimeric antigen receptor (CAR).

97. The method of claim 91 , wherein the natural killer (NK) cells are genetically modified to overexpress one or more chemokines or cytokines.

98. The method of claim 97, wherein the one or more chemokines or cytokines are CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof.

99. The method of claim 97 or claim 98, wherein the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

100. The method of any one of claims 72-99, wherein the genetic modification of the immune cells is performed by transformation, transfection, or transduction.

101. The method of any one of claims 72-100, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

102. The method of any one of claims 72-101 formulated for administration to a human subject.Docket No. 33794 / 70815103. The method of any one of claims 72-102, wherein the pharmaceutical composition is administered directly into a cancerous tumor.

104. The method of any one of claims 72-103, wherein the pharmaceutical composition is administered intravenously.

105. The method of any one of claims 72-104, wherein the genetically modified immune cells treat the disease by breaking down extracellular matrix (ECM).

106. The method of any one of claims 72-105, wherein the genetically modified immune cells treat the disease by targeting fibroblasts.

107. The method of claim 106, wherein the fibroblasts are cancer-associated fibroblasts (CAFs).

108. The method of any one of claims 72-107, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

109. A method of enhancing the ability of immune cells to migrate into a tumor comprising genetically modifying the immune cells to overexpress a membrane-bound protease.

110. The method of claim 109, wherein at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease.

111. The method of claim 109 or 110, wherein at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound.

112. The method of claim 111, wherein the soluble protease is engineered to:i) remove the secretory domain; andii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor.

113. The method of any one of claims 109-112, wherein at least one of the one or more membrane-bound proteases is a matrix metalloproteinase.

114. The method of claim 113, wherein the matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25, or a combination thereof.

115. The method of claim 111 or claim 112, wherein the soluble matrix metalloproteinase is selected from MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11 , MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP-26, MMP-27, MMP-28, or a combination thereof.

116. The method of claim 115, wherein the secretory domain is a pro-MMP cleavage site.Docket No. 33794 / 70815117. The method of any one of claims 109-116, wherein at least one of the one or more overexpressed membrane-bound proteases is a cathepsin.

118. The method of claim 117, wherein the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof.

119. The method of any one of claims 109-118, wherein at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domaincontaining protein (ADAM).

120. The method of claim 119, wherein the a disintegrin and metalloproteinase domaincontaining protein (ADAM) is ADAM 10, ADAM 17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof.

121. The method of any one of claims 109-120, wherein at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof.

122. The method of any one of claims 109-121 , wherein at least one of the one or more overexpressed membrane-bound proteases is overexpressed by at least 5-fold when compared to an unmodified control cell.

123. The method of any one of claims 109-122, wherein the genetically modified immune cells are natural killer (NK) cells, T cells, or a combination thereof.

124. The method of claim 123, wherein the genetically modified natural killer cells are derived from a human donor, optionally wherein the human donor is a cancer patient.

125. The method of claim 124, wherein the genetically modified natural killer (NK) cells are autologous or allogenic.

126. The method of claim 123, wherein, prior to genetic modification, the natural killer cells are isolated from peripheral blood, pluripotent stem cells, or a combination thereof.

127. The method of claim 123, wherein the genetically modified natural killer cells are further modified to express a chimeric antigen receptor (CAR).

128. The method of claim 123, wherein the natural killer cells are genetically engineered to overexpress one or more chemokines or cytokines.

129. The method of claim 128, wherein the one or more chemokines or cytokines are CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL9, CXCL10, CXCL12, CXCL14, CXCL16, CXCL28, IL-2, IL-12, IL-15, IL-18, IL-23, IFN-y, IFN-a, or a combination thereof.Docket No. 33794 / 70815130. The method of claim 128 or claim 129, wherein the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

131. The method of any one of claims 109-130, wherein the genetic modifications of the immune cells are performed by transformation, transfection, or transduction.

132. The method of any one of claims 109-131 , further comprising administering the genetically modified immune cells to a patient suffering from cancer.

133. The method of claim 132, wherein the cancer is lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer, gastric cancer, kidney cancer, esophageal cancer, liver cancer, or a combination thereof.

134. The method of claim 133, wherein the cancer is pancreatic cancer.

135. The method of claim 134, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

136. The method of any one of claims 109-135, wherein the genetically modified immune cells have an enhanced ability to breakdown extracellular matrix (ECM).

137. The method of any one of claims 109-136, wherein the genetically modified immune cells have an enhanced ability to target fibroblasts.

138. The method of claim 137, wherein the fibroblasts are cancer-associated fibroblasts (CAFs).

139. The method of any one of claims 109-138, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

140. A method of preparing one or more genetically modified immune cells comprising: transfecting a vector containing a gene for one or more membrane-bound proteases into one or more immune cells in a medium; replicating the one or more immune cells transfected with the vector, and; isolating the one or more immune cells transfected with the vector that overexpress the one or more membrane-bound proteases.

141. The method of claim 140, wherein at least one of the one or more overexpressed membrane-bound proteases is a heterologous protease.

142. The method of claim 140 or 141 , wherein at least one of the one or more overexpressed membrane-bound proteases is a soluble protease engineered to be membrane-bound.

143. The method of claim 142, wherein the soluble protease is engineered to:i) remove the secretory domain;Docket No. 33794 / 70815ii) express a transmembrane domain or express a glycophosphatidylinositol (GPI) anchor.

144. The method of any one of claims 140-143, wherein at least one of the one or more overexpressed membrane-bound proteases is a matrix metalloproteinase.

145. The method of claim 144, wherein the matrix metalloproteinase is selected from MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25, or a combination thereof.

146. The method of claim 142 or claim 143, wherein the soluble matrix metalloproteinase is selected from MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11 , MMP-13, MMP-18, MMP-19, MMP-20, MMP-21, MMP-23A, MMP-23B, MMP-26, MMP-27, MMP-28, or a combination thereof.

147. The method of claim 146, wherein the secretory domain is a pro-MMP cleavage site.

148. The method of any one of claims 140-147, wherein at least one of the one or more overexpressed membrane-bound proteases is a cathepsin.

149. The method of claim 148, wherein the cathepsin is Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin H, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V, or a combination thereof.

150. The method of any one of claims 140-149, wherein at least one of the one or more overexpressed membrane-bound proteases is a disintegrin and metalloproteinase domaincontaining protein (ADAM).

151. The method of claim 150, wherein the a disintegrin and metalloproteinase domaincontaining protein (ADAM) is ADAM 10, ADAM 17, ADAMTS-2, ADAMTS-2, ADAMTS-3, ADAMTS-4, ADAMTS-5, ADAMTS-14, or a combination thereof.

152. The method of any one of claims 140-151 , wherein at least one of the one or more overexpressed membrane-bound proteases is neutrophil elastase, pancreatic elastase, plasmin, tissue plasminogen activator (tPA), or a combination thereof.

153. The method of any one of claims 140-152, wherein at least one of the one or more overexpressed membrane-bound proteases is overexpressed by at least 5-fold when compared to an unmodified control cell.

154. The method of any one of claims 140-153, wherein the vector is viral.

155. The method of any one of claims 140-154, wherein the one or more immune cells are natural killer (NK) cells, T cells, or a combination thereof.Docket No. 33794 / 70815156. The method of claim 155, wherein the natural killer cells are genetically modified to overexpress one or more chemokines or cytokines.

157. The method of claim 156, wherein the one or more chemokines or cytokines are overexpressed upon engagement with a cancer cell.

158. A kit comprising genetically modified immune cells, wherein the genetically modified immune cells overexpresses one or more membrane-bound proteases, and wherein the kit further comprises a sterile container, a syringe, and instructions for use.

159. The kit of claim 158, wherein the sterile container is able to withstand freezing at liquid nitrogen temperatures, and wherein the kit further comprises a cryopreservation medium.

160. The kit of claim 159, further comprising a transportation container that is able to withstand freezing at dry ice or liquid nitrogen temperatures.

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