ENCAPSULATED CELL THERAPY CARTRIDGE

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No.

61/876,638, filed on September 1 1, 2013, the contents of which are herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named "NETE-062_001WO_ST25.txt", which was created on September 10, 2014 and is 96, 100 bytes in size, are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of encapsulated cell therapy.

BACKGROUND OF THE INVENTION

Encapsulated cell technology or ECT is a delivery system that uses live cells to secret a therapeutic agent. This is usually achieved by genetically engineering a specific type of cell to overexpress a particular agent. The engineered cells are then encapsulated in semi- permeable polymer capsules. The capsule is then implanted into the target surgical site. The semi-permeable membrane allows the free diffusion of nutrients and therapeutic molecules yet prevents the direct contact of the host immune systems cells with the cells within the device. However, current encapsulated cell delivery devices used in treatment of retinitis pigmentosa or geographic atrophy are limited in their capability to achieve microgram production levels of encapsulated cell produced protein drug.

Therefore, there is a need for an encapsulated implant design capable of allowing increased cell encapsulation volumes while maintaining cell viability and maximizing protein drug production.

SUMMARY OF THE INVENTION

Provided herein are multi-chamber implantable cell culture devices containing two or more individual chambers for the delivery " ^f ° ^^"^cally active molecule to a specific target region. For example, the biologically active molecule can be produced by a cell line containing one or more genetically engineered cells (e.g., ARPE-19 cells).

Cell lines (such as, but not limited to, ARPE-19 cells) can be genetically engineered to produce a therapeutic amount of one or more biologically active molecules. For example, the one or more biologically active molecule(s) can include anti-angiogenic antibodies and molecules, anti-angiogenic antibody scaffolds, soluble receptors, agents targeting and inhibiting or modulating immunologic pathway molecules, growth factor inhibitors, , cytokines, growth factors, neurotrophic factors, angiogenic factors, neurotransmitters, hormones, enzymes, anti-inflammatory factors, therapeutic proteins, gene transfer vectors, antibodies and antibody fragments, antigens, or any combination thereof.

In various embodiments, such molecules can include, but are not limited to, C3a inhibitors, C3b inhibitors, other agents targeting and inhibiting or modulating immunologic pathway molecules, brain derived neurotrophic factor (BDNF), NT-4, ciliary neurotrophic factor (CNTF), Axokine, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF I), insulin-like growth factor II (IGF II), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGF a), transforming growth factor β (TGF β), nerve growth factor (NGF), platelet derived growth factor (PDGF), glia-derived neurotrophic factor (GDNF), Midkine, phorbol 12-myristate 13 -acetate, tryophotin, activin, thyrotropin releasing hormone, interleukins, bone morphogenic protein, macrophage inflammatory proteins, heparin sulfate, amphiregulin, retinoic acid, tumor necrosis factor a, fibroblast growth factor receptor, epidermal growth factor receptor (EGFR), PEDF, LEDGF, NTN, Neublastin, neurotrophins, lymphokines, VEGF inhibitors, PDGF inhibitors, placental growth factor (PIGF) inhibitors, Tie2, CD55, C59, a bispecific molecule that simultaneously binds VEGF and PDGF, and other agents expected to have therapeutically useful effects on potential target tissues. Such cell lines can be encapsulated in encapsulation cell therapy (ECT) devices using any method(s) known in the art.

Described herein are implantable cell culture devices containing two or more (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) individual chambers. Each individual chamber contains a core that contains a therapeutically effective amount of one or more biologically active molecules and a semi-permeable membrane surrounding the core, wherein the membrane permits the diffusion of biologically active molecule(s) there through it.

For example, the one or more biologically active molecules in the core of each chamber can be produced by one or more genetically engineered cell lines contained within the core(s) of the individual chambers of the device. For example, the one or more cell lines contain one or more genetically engineered ARPE- 19 cells that are contained within the cores of the individual chambers.

For example, the one or more biologically active molecules can be introduced into the one or more genetically engineered cell lines (for example, ARPE- 19 cells) by an iterative transfection process, wherein the iterative transfection process comprises one, two, three or more transfections (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transfections). Those skilled in the art will recognize that the number of transfections in the iterative transfection process will determine the number of the (same or different) biologically active molecule(s) produced by the resulting cell line. The iterative transfection process can be used to introduce multiple copies of the same or different biologically active molecule(s) into the cells (e.g., ARPE- 19 cells).

In some embodiments, the device is cryopreserved. The core of each chamber within such cryopreserved devices may also contain a cryoprotectant agent, which can be added to the cell culture media contained within the core.

Any cryopreservation method(s) known in the art can be employed. By way of non- limiting example, encapsulated cell therapy devices can be placed in cryogenic storage vials, frozen under controlled rate freezing (e.g., to a temperature of -80° C), and finally stored in vapor phase liquid nitrogen (e.g., -190° C) conditions.

Cryopreserved devices can be transported under vapor phase liquid nitrogen (e.g., -190 ° C) conditions and/or under dry ice (e.g., -70° C) conditions.

Cryopreserved devices can be thawed using any method(s) known in the art prior to use.

In some embodiments, the device contains 2-20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12,

13, 14, 15, 16, 17, 18, 19 or 20) individual chambers. The inner diameter of each chamber can be between 100 microns and 900 microns (e.g., 100, 200, 300, 400, 500, 600, 700, 800 or 900 μιη) and bundled in numbers between 2 and 20 chambers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20). The wall thicknesses of each chamber should be manufactured to create the minimal diffusion distance yet achieve adequate column strength. The nominal ratio of inner diameter to wall thickness of each chamber is about 5: 1 to 20: 1 in scale. For example, the ratio is 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13 : 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, or 20: 1 in scale. For example, the ratio is 10: 1 in scale. The overall size of the device will vary depending upon the number and size of the individual chambers used in the assembly. For example, the range of diameter of the device is between 0.5 mm and 5.0 mm (e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 mm) with lengths varying from a minimum of 0.4 mm to a maximum of 11 mm (e.g., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 or 1 1 mm). For example, the internal volume of the device is between 2 microliters and 100 microliters (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μΐ).

The device may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) access ports.

In some embodiments, the core of each chamber contains between 1.0 x 10 ⁴ cells and 7.5 xlO ⁵ cells (e.g., 1.0 x 10 ⁴ , 5.0 x 10 ⁴ , 1.0 x 10 ⁵ , 3.0 x 10 ⁵ , 5.0 x 10 ⁵ or 7.5 x 10 ⁵ cells). Those skilled in the art will recognize that the exact cell number in each chamber can depend both upon the growth rate of the cell/cell line encapsulated and/or the volume of the individual chambers used to construct the device.

The core of each chamber may additionally contain a matrix disposed within the semi-permeable membrane. In some embodiments, the matrix includes a plurality of monofilaments, wherein the monofilaments are twisted into a yarn or woven into a mesh or are twisted into a yarn that is in non-woven strands, and wherein the cells or tissue are distributed thereon. Those skilled in the art will recognize that the monofilaments can be made from a biocompatible material selected from acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene terephthalate, nylon, polyamides,

polyurethanes, polybutester, silk, cotton, chitin, carbon, and/or biocompatible metals. For example, the monofilaments are polyethylene terephthalate (PET) fibers that comprises between 1-85% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%) of the internal volume of each chamber of the device.

The cell encapsulation devices described herein can also have a tether anchor. For example, the tether anchor may be an anchor loop that is adapted for anchoring the device to a structure within a target region (such as an ocular structure).

Any of the devices described herein can be implanted into (or are for implantation in) the eye or another target region of the body, such as, for example, the spleen, ear, heart, colon, liver, kidney, breast, joint, bone marrow, subcutaneous, and/or peritoneal spaces. By way of non-limiting example, the devices can be implanted into (or are for implantation in) the vitreous, the aqueous humor, the Subtenon's space, the periocular space, the posterior chamber, and/or the anterior chamber of the eye.

In some illustrative embodiments, the semi-permeable membranes in the core of each chamber of the devices described herein are made from a permselective, immunoisolatory membrane. For example, the semi-permeable membranes are made from an ultrafiltration membrane or a microfiltration membrane. Those skilled in the art will recognize that an ultrafiltration membrane typically has a pore size of 1-100 nm, whereas a microfiltration membrane typically has a pore size of 0.1-10 μιη. In other embodiments, the semi-permeable membranes may be formed into a porous structure. Those skilled in the art will recognize that a semi-permeable membrane typically has a median pore size of about 1-500 nm (e.g., 1, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm).

In still other embodiments, the semi-permeable membrane may be made from any biocompatible material selected from the group consisting of polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), and derivatives, copolymers and mixtures thereof.

In any of the devices described herein, the nominal molecule weight cutoff (MWCO) of the semi-permeable membrane is between 10 and 1000 KD (e.g., 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650,

700, 750, 800, 850, 900, 950 or 1000 KD). The semi-permeable membrane may be between about 10- 200 μιη (e.g. 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 μιη) thick. The length of the device can be between about 1 mm - 20 mm (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the device has an internal diameter of between about 0.1 mm - 2.0 mm (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0).

In one embodiment, the ends of the device are sealed using methyl methacrylate or any other medical-grade, biocompatible material formulated or manufactured to form a hermetic seal integrating all components of the device at each end of the device.

Also provided are uses of any of the implantable cell culture devices to deliver an appropriate therapeutic dose of any biologically active molecule(s) to a target region of a subject. For example, the therapeutic dose is at least 0.1 pg/day (e.g., at least 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, lxlO ⁶ , lxlO ⁷ , lxlO ⁸ , lxlO ⁹ , lxlO ¹⁰ or more pg/day).

Also provided herein are methods for treating a disorder by implanting any of the implantable cell culture devices into a target region of a patient, and allowing soluble receptors or anti-angiogenic antibodies and molecules to diffuse from the device and bind to VEGF and/or PDGF in the target region, thereby treating the disorder. For example, cell lines (i.e., any of the cell lines described herein) are provided for use in treating a disorder, wherein the cell lines are incorporated in an implantable cell culture device, wherein the devices are implanted into a target region of a patient, and wherein, one or more soluble receptors or anti- angiogenic antibodies and molecules to diffuse from the device and bind to VEGF and/or PDGF in the target region, thereby treating the disorder.

Also provided are methods for treating a disorder by implanting any of the implantable cell culture devices into a target region of a patient, and allowing one or more biologically active molecules to diffuse from the device in the target region, thereby treating the disorder. For example, also provided are cell lines (i.e., any of the cell lines described herein) for use in treating a disorder, wherein the cell lines are incorporated in an implantable cell culture device, wherein the devices are implanted into a target region of a patient, and wherein one or more biologically active molecules diffuse from the device in the target region, thereby treating the disorder.

The skilled artisan could readily determine which disorder(s) can be treated by the device. Exemplary disorders that can be treated by any of the devices include, but are not limited to, ophthalmic disorders, endothelial cell proliferation or vascularization related disorders, cancer, infectious disorders, inflammatory disorders, immunologic disorders, digestive disorders, vascular disorders, lung disorders, oral disorders, blood disorders, liver disorders, skin disorders, prostate disorders, kidney disorders, metabolic disorders, endocrine disorders, neurologic disorders, and/or neurodegenerative disorders.

For example, the ophthalmic disorders to be treated are associated with the general disease groups angiogenesis, inflammation or degeneration and include, but are not limited to, branch or central retinal vein occlusion (BRVO or CRVO), uveitis, macular telangiectasia, retinopathy of prematurity, diabetic macular edema, diabetic retinopathy, age-related macular degeneration (e.g. wet form age-related macular degeneration or atrophic AMD (also called the dry form of AMD)), glaucoma, retinitis pigmentosa, cataract formation, retinoblastoma and retinal ischemia. In some embodiments, age-related macular degeneration is wet form age-related macular degeneration. In other embodiments, the ophthalmic disorder is BRVO or CRVO.

For example, the cell proliferation disorder may be selected from hematologic disorders, atherosclerosis, inflammation, increased vascular permeability and/or malignancy.

In such methods, a therapeutically effective amount (for example, between 0.1 pg and 10 mg per patient per day) of the soluble receptors or anti-angiogenic antibodies and molecules diffuse into the target region, wherein the soluble receptor is a soluble VEGF receptor or a soluble PDGF receptor.

Alternatively, a therapeutically effective amount (for example, between 0.1 pg and 10 mg per patient per day) of the biologically active molecules diffuses into the target region.

Those skilled in the art will recognize that any of the devices described herein can also be used to treat a variety of non-ocular disorders. For non-ocular disorders, the design of the devices will have to be modified. Modification of the device design is within the routine level of skill in the art.

Also provided are methods of delivering one or more biologically active molecules to a recipient host by implanting any of the implantable cell culture devices described herein into a target region of the recipient host, wherein the one or more encapsulated cells or cell lines (e.g., ARPE-19 cells) secrete the biologically active molecules at the target region.

In any method(s) described herein, preferred target regions can include, but are not limited to, the aqueous and vitreous humors of the eye, spleen, ear, heart, colon, liver, kidney, breast, joint, bone marrow, subcutaneous, and/or peritoneal spaces. Other target regions may include, but are not limited to, the whole body for systemic delivery and/or localized target sites within or near organs in the body such as breast, colon, spleen, ovary, testicle, and/or bone marrow. In such methods, a therapeutically effective amount per patient per day of the biologically active molecules diffuses into the target region.

Those skilled in the art will recognize that in any of the methods and uses described herein with regard to ocular implantation and/or disorders, between 0.1 pg and 10,000 μg per patient per day of biologically active molecule(s) can diffuse from the implantable cell culture devices. For systemic implantation into other target regions of the body, the therapeutically effective amount could be upwards of 1000 mg per patient per day. For such systemic indications, those skilled in the art will recognize that far larger ECT devices would have to be employed.

Also provided are methods for making the implantable cell culture devices. For example, the device can be made by a method including the steps of genetically engineering at least one cell to secrete one or more biologically active molecules; producing an individual chamber; and assembling two or more individual chambers to form the device. The method of making a device may also include a step of encapsulating the genetically engineered cells within a semi-permeable membrane. In some embodiments, the step of encapsulating the genetically engineered cells is performed for each individual chamber before the assembling step. In some embodiments, the step of encapsulating the genetically engineered cells is performed for all the chambers at one time after the assembling step.

Also provided are methods of making the ECT cartridge devices where the two or more individual chambers are formed prior to the addition of the genetically engineered at least one ARPE-19 cell.

Preferably, any method s of making the ECT cartridge devices described herein involves degassing and prewetting step(s) to insure that all chambers of the cartridge device are filled. For example, the degassing/prewetting step is performed prior to the addition of the genetically engineered at least one ARPE-19 cell.

In any devices, the two or more individual chambers each may contain genetically engineered cells that secrete the same one or more biologically active molecules.

Alternatively, the two or more individual chambers each may contain genetically engineered cells that secrete different one or more biologically active molecules.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a series of cartridge configurations with increasing number of individual chambers.

Figure 2 shows a prototype cartridge with eight individual chambers each created from 400 micron inner diameter membranes with 50 micron wall thickness.

Figure 3 is a cartoon describing the single access port allowing encapsulation of one cell line or multiple mixed cell lines in all the chambers.

Figure 4 is a cartoon describing the multiple access ports allowing encapsulation of individual cell line in each chamber. This configuration allows delivery of two, three or more distinct and therapeutically different drug products from a single intraocular device.

Figure 5 is a histological cross-section of a 1.3 mm inner diameter, single chamber device showing cell necrosis occurred after a two-week intraocular implant in the rabbit.

Figure 6 is a histological cross-section of a cartridge implant with 7 individual chambers following a two-week intraocular implant in the rabbit. In contrast to the single implant with equivalent cell volume (Figure 5), the cartridge configuration with reduced diffusion distance for each individually cell encapsulated chamber provides improved nutrient access and improved cell viability.

Figure 7 is a graph showing the expression level of VEGF antagonist over time for each group with different device configuration, cell density and/or cell volume.

Figure 8 is a graph showing an efficiency of VEGF antagonist expression as a function of chamber inner diameter.

Figure 9 is a bar graph showing VEGFR release from devices with different configurations.

Figure 10 is a schematic of the NT-503-3 (Generation 3) Multi-Chamber Implant. Figure 11 shows VEGF binding of ECT produced VEGF-R.

Figure 12 shows measurement of the ability of the ECT produced VEGF-R to neutralize the bioactivity of rhVEGF on human umbilical vein endothelial cell proliferation. HUVEC cells.

Figures 13A and 13B show the efficacy observed following intraocular delivery of VEGF-R over course of 12 months in human patients with wet-AMD. NT-503 is currently being studied in wet AMD patients receiving two early generation (NT-503 -2) implant devices in one eye. Results observed thus far in the double-implant NT 503-2 study have been encouraging. The top panel (A) shows a study patient with marked reduction in intraretinal fluid and subretinal hyper-reflective material and improvement in visual acuity as early as one month. Improvements in OCT and VA thickness continue and remain at month 12. The bottom panel (B) graphically demonstrates robust reductions in central macular thickness occurring within the first month and persisting out to at least 12 months. As the study is on-going, the data presented in the figure are a "snap shot" of OCT response as not all patients have completed 12 months of follow-up.

Figures 14A and 14B show representative histologic (H&E) section of encapsulated NT-503 cells arranged in 5-chamber cartridge format following 2-weeks incubation in culture media (A). VEGF-R concentration quantified in rabbit vitreous following 1 -month intraocular implant comparing single NT-503-3 and double earlier Generation NT-503-2 (B).

Figure 15 shows protein profile secretion of ubiquitous proteins from multiple lots of Generation 3 cartridge devices. All lots demonstrate a correlation coefficient of greater than 90%. The purity index of therapeutic proteins was greater than 80% relative to non- therapeutic or excipient protein.

Figure 16 shows the NT-503-3 shelf life comparison for Generation 2 and Generation

3 ECT devices.

Figure 17 shows NT-503-3 cartridge shelf life and recovery profiles.

Figure 18 shows NT-503-3 explant expression and corresponding vitreous concentrations following 4 week hold in packaging and 1 month implantation in rabbits.

Figure 19 shows NT-503-3 explant expression and corresponding vitreous concentrations following 6 week hold in packaging and 1 month implantation in rabbits.

Figure 20 shows in vivo encapsulated cell histology for 1 month explants

Figure 21 shows representative examples of cartridge devices without degassing/pre- wetting procedure (shown are histologic cross-sections, cells stained with Eosin and

Hemotoxylin). Lack of complete filling is evident in as many as all five of the cartridge chambers when cells filled without a pre-wetting and degassing step.

Figure 22 shows representative examples of cartridge devices following

implementation of a degassing/pre-wetting (shown are histologic cross-sections, cells stained with Eosin and Hemotoxylin). Complete filling evident in all five chambers of each representative cartridge device in following a degassing and pre-wetting step.

Figure 23 shows Generation 3 cartridge device protein levels following encapsulation of PDGFR, VEGFR and their combination at 50:50 ratio. DETAILED DESCRIPTION OF THE INVENTION

Current encapsulated cell delivery devices used in treatment of retinitis pigmentosa or geographic atrophy (e.g., single implants of NT-503 Second Generation or NT-503-2 devices) are limited in their capability to achieve microgram production levels of encapsulated cell produced biologically active molecule (e.g., protein drug). With the assumption that the per-cell production or PCD (pictogram per cell per day) of the encapsulated cells has been optimized for maximum production, the amount of cells that can be encapsulated and the efficiency of those cells within a single chamber device are constrained. Constraints to higher production of protein are related to both viable cell number and the efficiency of protein production of the cells in the chamber. A desirable alternative would be an encapsulated implant design capable of improving cell viability allowing more cells to populate a device and, in addition, improving the per cell capability to produce the biologically active molecule following encapsulation and intraocular implant.

One potential modification to the single-chamber design of the second generation ECT devices is to increase the diameter and or length, thereby increasing the internal volume of the chamber and allowing more cells to be encapsulated and more biologically active molecule to be produced from the device. Another potential modification to the single- chamber design is to create flat-sheet devices to increase volume and importantly increasing surface-to-volume ratio. Other devices have also been conceptualized such as complex geometric designs that also would increase volume and surface to volume relationships. However, volume increases leading to cell number increases in a single chamber

encapsulated device have significant limitations.

In studies conducted with single-chamber second generation devices with increasing internal diameter greater than 1 mm, the results were inversely proportional to increasing diameter and confirmed that there exists a maximum diffusion distance within which the population of cells must remain in order to maintain both viability and maximum cell production rate. Moreover, exceeding the length of current encapsulated cell implant devices are not practical in applications associated with eye due to anatomical volume constraints, while construction materials currently used in manufacture of cell encapsulation devices preclude complex geometric configurations (e.g., star-shaped, or other similar designs).

Therefore, there is a need for an encapsulated implant device capable of allowing increased cell encapsulation volumes while maintaining cell viability and maximizing biologically active molecule production. Accordingly, multi-chamber devices are provided that have superior properties compared to single-chamber devices known in the art. Particularly, these devices overcome the issue of increasing cell mass and diffusion distance constraints between the encapsulated cells and the nutrient source (e.g., human vitreous) by combining multiple, smaller inner diameter chambers into a cohesive, single implant cartridge format. For example, as shown in Figures 5 and 6, the multi-chamber configuration (Figure 6) provides better nutrient access, thereby improving cell viability, when compared to a single-chamber device with equivalent cell volume (Figure 5).

Hence, provided herein is an implantable cell culture device (also called a cartridge) that contains two or more (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) individual chambers. Each individual chamber includes a core that contains a therapeutically effective amount of one or more biologically active molecules from a cellular source and/or from a non-cellular source, and a semi-permeable membrane surrounding the core that permits the diffusion of the biologically active molecules there through. (See, e.g., Figures 1 and 2).

The one or more biologically active molecules can be isolated and purified from cells or tissue sources by any known standard protein purification techniques in the art.

Alternatively, the one or more biologically active molecules can be produced by any recombinant DNA techniques known in the art. The one or more biologically active molecules can also be synthesized chemically using standard peptide synthesis techniques available in the art.

In some embodiments, the one or more biologically active molecules are produced by one or more genetically engineered cell lines, such as, a cell line comprising one or more genetically engineered ARPE-19 cells. However, those skilled in the art will recognize that any other suitable cell line can also be utilized in these devices.

In some embodiments, the cells are genetically engineered using any suitable techniques known in the art. In other embodiments, the one or more biologically active molecules can be introduced into the cells (e.g., APRE-19 cells) by an iterative transfection process that comprises one, two, three or more transfections (e.g., 4, 5, 6, 7, 8, 9, 10, or more). The iterative transfection process can be used to introduce multiple copies of the same or different biologically active molecule(s) into the cells (e.g., ARPE-19 cells). Each transfection can be carried out by any methods known in the art. The iterative transfection process is also described in WO 2012/075184, which is incorporated herein by reference. A gene of interest (i.e., a gene that encodes a given biologically active molecule) can be inserted into a cloning site of a suitable expression vector using standard techniques known in the art.

A wide variety of host/expression vector combinations may be used to express the gene encoding the biologically active molecule(s) of interest. Long-term, stable in vivo expression is achieved using expression vectors (i.e., recombinant DNA molecules) in which the gene of interest is operatively linked to a promoter that is not subject to down regulation upon implantation in vivo in a mammalian host. Suitable promoters include, for example, strong constitutive mammalian promoters, such as beta-actin, eIF4Al, GAPDH, etc. Stress- inducible promoters, such as the metallothionein 1 (MT-1) or VEGF promoter may also be suitable. Additionally, hybrid promoters containing a core promoter and custom 5 ' UTR or enhancer elements may be used. Other known non-retroviral promoters capable of controlling gene expression, such as CMV or the early and late promoters of SV40 or adenovirus are suitable. Enhancer elements may also be place to confer additional gene expression under stress environments, such as low (¾. One example is the erythropoietin enhancer which confers up-regulation of associated gene elements upon hypoxic induction.

The expression vectors containing the gene of interest may then be used to transfect the desired cell line. Standard transfection techniques such as liposomal, calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized.

Commercially available mammalian transfection kits, such as Fugene6 (Roche Applied Sciences), may be purchased. Additionally, viral vectors may be used to transducer the desired cell line. An example of a suitable viral vector is the commercially available pLenti family of viral vectors (Invitrogen). Human mammalian cells can be used. In all cases, it is important that the cells or tissue contained in the device are not contaminated or adulterated. For antibody scaffold proteins requiring heavy and light chain components, dual constructs, each encoding a relevant antibody heavy or light chain, can be co-transfected simultaneously, thereby yielding cell lines expressing functional bivalent Fab and tetravalent full antibody molecules.

Exemplary promoters include the SV40 promoter and the CMV/EF 1 alpha promoter. Other useful expression vectors, for example, may consist of segments of

chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., pUC, pBlueScript™ plasmids from E. coli including pBR322, pCRl, pMB9 and their derivatives. Expression vectors containing the geneticin (G418), hygromycin or blasticidin drug selection genes (Southern, P. J., In Vitro, 18, p. 315 (1981), Southern, P. J. and Berg, P., J. Mol. Appl. Genet, 1, p. 327 (1982)) are also useful. These vectors can employ a variety of different enhancer/promoter regions to drive the expression of both a biologic gene of interest and/or a gene conferring resistance to selection with toxin such as G418, hygromycin B, or blasticidin. A variety of different mammalian promoters can be employed to direct the expression of the genes for G418 and hygromycin B and/or the biologic gene of interest. The G418 resistance gene codes for aminoglycoside phosphotransferase (APH) which enzymatically inactivates G418 (100-1000 μg/μl) added to the culture medium. Only those cells expressing the APH gene will survive drug selection usually resulting in the expression of the second biologic gene as well. The hygromycin B phosphotransferase (HPH) gene codes for an enzyme which specifically modifies hygromycin toxin and inactivates it. Genes co-transfected with or contained on the same plasmid as the hygromycin B phosphotransferase gene will be preferentially expressed in the presence of hygromycin B at 50-200 μg/ml concentrations.

Examples of expression vectors that can be employed include, but are not limited to, the commercially available pRC/CMV (Invitrogen), pRC/RSV (Invitrogen), pCDNAlNEO (Invitrogen), pCI-Neo (Promega), pcDNA3.3 (Invitrogen) and GS vector system (Lonza Group, Switzerland). Other suitable commercially available vectors include pBlast, pMono, or pVitro. In some embodiments, the expression vector system is the pCpGfree-vitro expression vectors available with neomycin (G418), hygromycin, and blasticidin resistance genes(InvivoGen, San Diego, CA).

In some embodiments, the pNUT expression vector, which contains the cDNA of the mutant DHFR and the entire pUC18 sequence including the polylinker, can be used. See, e.g., Aebischer, P., et al, Transplantation, 58, pp. 1275-1277 (1994); Baetge et al., PNAS, 83, pp. 5454-58 (1986). The pNUT expression vector can be modified such that the DHFR coding sequence is replaced by the coding sequence for G418 or hygromycin drug resistance. The SV40 promoter within the pNUT expression vector can also be replaced with any suitable constitutively expressed mammalian promoter, such as those discussed above.

Those skilled in the art will recognize that any other suitable, commercially available expression vectors (e.g., pcDNA family (Invitrogen), pBlast, pMono, pVitro, or pCpG-vitro (Invivogen)) can also be used. Principal elements regulating expression are typically found in the expression cassette. These elements include the promoter, 5' untranslated region (5' UTR) and 3 ' untranslated region (3 ' UTR). Other elements of a suitable expression vector may be critical to plasmid integration or expression but may not be readily apparent. The skilled artisan will be able to design and construct suitable expression vectors for use in the claimed invention. The choice, design, and/or construction of a suitable vector is well within the routine level of skill in the art.

The sequences suitable biologically active molecule(s) that can be used have also been published and/or are known in the art. Other genes encoding the biologically active molecules that are not publicly available may be obtained using standard recombinant DNA methods such as PCR amplification, genomic and cDNA library screening with

oligonucleotide probes.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or

electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al„ MOLECULAR CLONING: A LABORATORY MANUAL, 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989), and other laboratory manuals.

The cell of choice is the ARPE-19 cell line, a spontaneously arising continuous human retinal pigmented epithelial cell line. However, those skilled in the art will recognize that other suitable cells, including but not limited to CHO cells, BHK cells, RPE (primary cells or immortalized cells), can also be used. The choice of cell depends upon the intended application. The encapsulated cells may be chosen for secretion of a biologically active molecule. Cells can also be employed which synthesize and secrete agonists, analogs, derivatives or fragments of the construct, which are active. Those skilled in the art will recognize that other suitable cell types may also be genetically engineered to secrete biologically active molecule(s).

To be a platform cell line for an encapsulated cell based delivery system, the cell line should have as many of the following characteristics as possible: (1) the cells should be hardy under stringent conditions (the encapsulated cells should be functional in the avascular tissue cavities such as in the eye, especially in the intra-ocular environment); (2) the cells should be able to be genetically modified (the desired therapeutic factors needed to be engineered into the cells); (3) the cells should have a relatively long life span (the cells should produce sufficient progenies to be banked, characterized, engineered, safety tested and clinical lot manufactured); (4) the cells should be of human origin (which increases compatibility between the encapsulated cells and the host); (5) the cells should exhibit greater than 80% viability for a period of more than one month in vivo in device (which ensures long-term delivery); (6) the encapsulated cells should deliver an efficacious quantity of a useful biological product (which ensures effectiveness of the treatment); (7) the cells should have a low level of host immune reaction (which ensures the longevity of the graft); and (8) the cells should be nontumorigenic (to provide added safety to the host, in case of device leakage).

The ARPE-19 cell line (see Dunn et al, 62 Exp. Eye Res. 155-69 (1996), Dunn et al, 39 Invest. Ophthalmol. Vis. Sci. 2744-9 (1998), Finnemann et al, 94 Proc. Natl. Acad. Sci. USA 12932-7 (1997), Handa et al, 66 Exp. Eye. 41 1-9 (1998), Holtkamp et al, 1 12 Clin. Exp. Immunol. 34-43 (1998), Maidji et al, 70 J. Virol. 8402-10 (1996); United States Patent No. 6,361,771) demonstrates all of the characteristics of a successful platform cell for an encapsulated cell-based delivery system. The ARPE-19 cell line is available from the American Type Culture Collection (ATCC Number CRL-2302). ARPE-19 cells are normal retinal pigmented epithelial (RPE) cells and express the retinal pigmentary epithelial cell- specific markers CRALBP and RPE-65. ARPE-19 cells form stable monolayers, which exhibit morphological and functional polarity.

The inner diameters of each individual chamber and the number of individual chambers can be optimized to create a cartridge with the maximum encapsulated cell efficiency and biologically active molecule production. The inner diameter of each individual chamber can be between 100 microns and 900 microns (i.e., 100, 200, 300, 400, 500, 600, 700, 800 or 900 μιη) and bundled in numbers between 2 and 20 chambers (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). The wall thicknesses of each chamber should be manufactured to create the minimal diffusion distance yet achieve adequate column strength. The nominal ratio of inner diameter to wall thickness of each chamber is about 5: 1 to 20: 1 in scale. For example, the ratio is 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1 or 20: 1 in scale. In one example, the ratio is 10: 1 in scale.

The overall size of the cartridge implant will vary depending upon the number and size of the individual chambers used in the assembly. The range of diameter of the cartridge can be between 0.5 mm and 5.0 mm (i.e., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mm) with lengths varying from a minimum of 0.4 mm to a maximum of 11 mm (i.e., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 or 11 mm). The internal volumes have a respective range depending upon the cartridge format and could vary from a minimum of 2 microliters to a maximum of 100 microliters (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μΐ).

The cartridge may further contain one or more access ports located on either end or at both ends of the device. Each individual chamber can be accessed individually through one access port (see, Figure 4). Alternatively, two or more chambers within a cartridge can be accessed through a single central access port (see, Figure 3).

Thus, when some or all the individual chambers of the device share a single port, a single suspension of one or more biologically active molecules is encapsulated within those chambers that share a single port. If the one or more biologically active molecules are produced by one or more genetically engineered cell lines, a single suspension of cells (one type of genetically engineered cells or a mixture of genetically engineered cells) that secretes the same one or more biologically active molecules is encapsulated within those chambers that share the single port. The increased number of individual chambers and decreased diffusion distance to the nutrient source will increase efficiency of the encapsulated cells and will allow either improved secretion levels of a protein drug compared to a single chamber device containing equivalent cell volume or can allow for viable support of a greater volume of cells and greater secretion of protein drug levels compared to a single chamber or equivalent internal volume.

Alternatively, when each individual chamber has its own access port, different suspension of one or more biologically active molecules can be encapsulated within each individual chamber. If the one or more biologically active molecules are produced by one or more genetically engineered cell lines, different cell suspensions are encapsulated within each individual chamber. Each encapsulated cell line produces a specific protein drug or drugs (i.e., one or more biologically active molecules) at a level consistent with the respective encapsulated cell line volume yet remains isolated within the individual chambers and/or groups of chambers. This configuration allows delivery of two, three, four, five, six, seven or more (i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or n, where n is the total number of chambers) distinct and therapeutically different biologically active molecules from a single intraocular cartridge device. The core of each individual chamber can function as a reservoir for biologically active molecules. In some embodiments, the core of each individual chamber may further contain a matrix disposed within the semi-permeable membrane. For example, the matrix may comprise a plurality of monofilaments that are either twisted into a yarn or woven into a mesh or twisted into a yarn that is in non-woven stands where the encapsulated cells are distributed. Materials useful in making monofilaments include any biocompatible materials that are able to be formed into fibers such as, for example, acrylic, polyester, polyethylene, polypropylene, polyacrylonitrile, polyethylene terephthalate, nylon, polyamides, polyurethanes, polybutester, or natural fibers such as cotton, silk, chitin or carbon or biocompatible metals. These monofilaments may prevent cells from aggregating and improve cellular distribution within each chamber. (See PCT Publication No. WO 96/02646, incorporated herein by reference).

In some embodiments, the monofilaments is made from polyethylene terephthalate (PET) fibers that comprises 1-85% (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%) of the internal volume of each chamber of the device.

Various polymers and polymer blends can be used to manufacture the surrounding semi-permeable membrane in each chamber, including polysulfone (including polyether sulfones), polyvinyl pyroldone, polyacrylates (including acrylic copolymers),

polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polyphosphazenes, polyacrylonitriles,

poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof. For example, the surrounding semi-permeable membrane is made from polysulfone or polyvinyl pyroldone. Preferably, the surrounding semipermeable membrane is a

biocompatible semipermeable hollow fiber membrane. Such membranes and methods of making them are disclosed by U.S. Pat. Nos. 5,284,761 and 5, 158,881, incorporated by reference.

In some embodiments, the surrounding semi-permeable membrane is made from a polyether sulfone hollow fiber, such as those described by U.S. Pat. No. 4,976,859 or U.S. Pat. No. 4,968,733, incorporated by reference.

In some embodiments, the surrounding semi-permeable membrane is a perms elective, immunoprotective membrane. That is, it protects cells in the core of the chamber from the immune system of the individual in whom the device is implanted. It does so (1) by preventing harmful substances of the individual's body from entering the core, (2) by minimizing contact between the individual and inflammatory, antigenic, or otherwise harmful materials which may be present in the core and (3) by providing a spatial and physical barrier sufficient to prevent immunological contact between the isolated moiety and detrimental portions of the individual's immune system.

In some embodiments, the surrounding semi-permeable membrane is ultrafiltration membrane or a microfiltration membrane. Those skilled in the art will recognize that ultrafiltration membranes are those having a pore size range of from about 1 to about 100 nanometers while a microporous membrane has a range of between about 1 to about 10 microns.

To be permselective, the membrane has a nominal molecular weight cutoff (MWCO) range appropriate both to the type and extent of immunological reaction anticipated to be encountered after the device is implanted and to the molecular size of the largest substance whose passage into and out of the device into the eye is desirable. The surrounding semipermeable membrane has nominal MWCO values from 10 kD up to 1000 kD. For example, the MWCO is between 50-700 kD or between 50-500 kD and ideally approximately 300 kD. In some embodiments, the MWCO is 500 kD. The median pore size of the membrane has a median pore size of approximately 1-500 nm (i.e., 1, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 nm).

The thickness of this physical barrier (i.e., semi-permeable membrane) can vary, but it will always be sufficiently thick to prevent direct contact between the cells and/or substances on either side of the barrier. The thickness of this region generally ranges between 5 and 200 microns. For example, thicknesses of 10 to 100 microns or of 20 to 50 or 20 to 75 microns can be used. In some embodiments, the semi-permeable membrane is between 90 and 120 μιη thick. Types of immunological attack which can be prevented or minimized by the use of the instant device include attack by macrophages, neutrophils, cellular immune responses (e.g., natural killer cells and antibody-dependent T cell-mediated cytolysis (ADCC)), and humoral response (e.g., antibody-dependent complement mediated cytolysis).

The surrounding semi-permeable membrane is produced in such a manner that it is free of isolated cells, and completely surrounds (i.e., isolates) the core, thereby preventing contact between any cells in the core and the recipient's body. The surrounding semipermeable membrane is formed into a porous structure in each chamber of the device.

The device may have a tether that aids in maintaining device placement during implant, and aids in retrieval. Such a tether may have any suitable shape that is adapted to secure the cartridge in place. For example, the tether may be a loop, a disk, or a suture. In some embodiments, the tether is shaped like an eyelet, so that a suture may be used to secure the tether (and thus the device) to the sclera, or other suitable ocular structure. In another embodiment, the tether is continuous with the cartridge at one end, and forms a pre-threaded suture needle at the other end. In one embodiment, the tether is an anchor loop that is adapted for anchoring the cartridge to a structure within the target region (for example, an ocular structure). The tether may be constructed of a shape memory metal and/or any other suitable medical grade material known in the art.

Any suitable method of sealing the capsules know in the art may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing. In addition, any suitable "dry" sealing method can also be used. In such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced. Subsequent to filling, the capsule is sealed. Such methods are described in, e.g., United States Patent Nos. 5,653,688; 5,713,887; 5,738,673; 6,653,687; 5,932,460; and 6, 123,700, which are herein incorporated by reference. In some embodiments, the ends of the device are sealed using methyl methacrylate. Additional suitable sealants include any other medical-grade, biocompatible material formulated or manufactured to form a hermetic seal integrating all components of the device at each end of the device.

Devices may be manufactured, formed and/or assembled by any suitable method known in the art. (See, e.g., United States Patent Nos. 6,361,771 ; 5,639,275; 5,653,975; 4,892,538; 5, 156,844; 5,283, 138; and 5,550,050, each of which is incorporated herein by reference). For example, the device can be made by a method including steps of genetically engineering at least one cell to secrete one or more biologically active molecules; producing an individual chamber; and assembling two or more individual chambers to form the device. The method of making a device may also include a step of encapsulating the genetically engineered cells within a semi-permeable membrane of the individual camber. In some embodiments, the step of encapsulating the genetically engineered cells is performed for each individual chamber before the assembling step (e.g., the cells are encapsulated in the individual chambers before the device is assembled). In other embodiments, the step of encapsulating the genetically engineered cells is performed for all the chambers after the assembling step (e.g., the device is assembled first and then the cells are added to individual chambers). Alternatively, for some individual chambers, the genetically engineered cells are encapsulated prior to the assembling step and for other individual chambers, the genetically engineered cells are encapsulated after the assembling step.

Cell filling (i.e., encapsulation) of cartridges containing 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) individual chambers requires a degassing and pre-wetting stage to ensure optimal distribution of cell mass within all chambers of the cartridge. Failure to include this step during encapsulation results in unacceptable variability of cell filling in all chambers and potentially no filling in some chambers. (See Example 8, infra).

In single ECT device loading, the air initially inside the device is expelled through pores as the cell suspension liquid pushes the air into and out of the dry pores of the device membrane. Cells accumulate within the internal space of the single ECT device during this step as the liquid medium of the cell suspension ultrafiltrates across and out of the membrane pores.

However, in the cartridge configuration, this process is complicated due to several factors including an orientation of the cartridge device which is not equally vertical for all chambers resulting in preferential ultrafiltrate of liquid from some chambers relative to others and resulting in contact and accumulation of that ultrafiltrate liquid on the outer surface of the remaining gas-filled chambers of the cartridge. The pores in those chamber membranes with liquid contact on outer surface prior to "degassing" are then are blocked, essentially creating a barrier to air passage and to subsequent liquid ultrafiltration.

The back pressure created due to the blocked chambers directs the liquid cell suspension through those remaining "degassed" chambers, which remain open to

ultafiltration, ultimately resulting in overfilling or cell packing. Those chambers unfilled or partially filled will not repopulate and, therefore, it was critical to develop and employ a system to equilibrate filling of all chambers of a cartridge in the multi-chamber filling process of an ECT cartridge device.

Figure 21 shows representative examples of cartridge devices without this degassing/pre-wetting procedure.

To ensure that all chambers would be equally filled and distributed with encapsulated cells, all air was evacuated from the internal volume of the device and the filling port (e.g., vacuum degassing), and importantly, all surfaces, particularly each chamber membrane surface and interconnecting pores, were filled with a wetting liquid (such as Hanks Balanced Salt Solution or other isotonic solutions (e.g., saline, DMEM, etc.). Following degassing and liquid filling of the cartridge and cell-filling system, the cartridge device can be successfully loaded with cells per any established encapsulation methods known in the art.

Figure 22 shows representative examples of cartridge devices after implementation of a degassing/pre-wetting step. Those skilled in the art will recognize that any devices may be cryopreserved following manufacture and prior to administration and/or implementation. Cryopreservation, if successful, helps to improve the shelf-life of the devices, which, in turn, would improve device storage and/or simplify device manufacturing.

Any suitable cryopreservation known in the art can be used to cryopreserve any of the devices described herein.

For example, cryopreservation in vapor phase liquid nitrogen is an established method for long term storage of living cells, and is dependent on appropriate cryoprotectant agents and the ability of cells to survive ultra-low temperature conditions. Once optimal conditions are met for cryopreservation, cells may be stored nearly indefinitely within vapor phase liquid nitrogen. By way of non-limiting example, using a cryopreservation system, any of the devices can be filled with cells formulated with cryoprotectant agent (e.g., 10% glycerol), placed in cryogenic storage vials, frozen under controlled rate freezing (e.g., to -80° C), and finally stored in vapor phase liquid nitrogen (e.g., -190° C) conditions. However, any other cryopreservation method(s) known in the art can also be used. Determination of the appropriate cryopreservation method(s) is within the routine level of skill in the art.

In addition, because the entire supply chain is simplified, any of the devices can be transported under vapor phase liquid nitrogen (-190° C) conditions and/or dry ice (-70° C) conditions (or any combination(s) thereof).

Cryopreserved devices can be thawed using any suitable method or protocol known in the art prior to use.

Additional cryopreservation agents and processes are described in WO 2013/181424, the contents of which are incorporated herein by reference in their entireties.

In any devices or methods, the one or more biologically active molecules are selected from the group consisting of anti-angiogenic antibodies and molecules, anti-angiogenic antibody scaffolds, soluble receptors, agents targeting and inhibiting or modulating immunologic pathway molecules, growth factor inhibitors, cytokines (interleukins, lymphokines), growth factors, neurotrophic factors (neurotrophins), angiogenic factors, neurotransmitters, hormones, enzymes, anti-inflammatory factors, therapeutic proteins, gene transfer vectors, antibodies and antibody fragments, antigens, and any combination thereof. Anti-angiogenic antibody-scaffolds and anti-angiogenic molecules that can be used are described in WO 2012/075184, which is herein incorporated by reference.

For example, the anti-angiogenic antibody scaffolds and/or anti-angiogenic molecules may include one or more of the following:

1) p834 (VEGFR-Fc#l, [RS-VEGF Receptor 1, Domain 2 and VEGF Receptor 2, Domain 3 (RlD2-R2D3)]-EFEPKSC-hIgGl Fc)

2) p838 (VEGFR-Fc#2, [VEGF Receptor 2, Domains 1, 2, and 3 (R2D1-R2D2- R2D3)])

3) p876 (VEGF antibody ScFv#l, with His-tag)

4) p913 (VEGF antibody ScFv#2, without His-tag)

5) p873 (Aflibercept, VEGFR-Fc#3, VEGF Receptor 1, Domain 2 and VEGF Receptor 2, Domain 3 (R1D2-R2D3) hlgGl Fc)

6) p874/p875 (Bevacizumab, VEGF full antibody #1, heavy chain/light chain)

7) p915/p914 (Ranibizumab, VEGF antibody Fab, heavy chain fragment/light chain)

8) p916/p914 (Ranibizumab, VEGF full antibody #2, heavy chain/light chain)

9) p917 (VEGFR-Fc#l, [RS-VEGF Receptor 1, Domain 2 and VEGF Receptor 2, Domain 3 (RlD2-R2D3)]-hIgGl Fc)

10) p964 (PDGFR-Beta domains 1-5 receptor-IgG4 Fc fusion)

1 1) p963 (PDGFR-Beta domains 1-5 receptor-IgGl Fc fusion)

12) p974 (PDGFR-Beta domains 1-3 receptor-IgGl Fc fusion)

13) p978 (PDGFR-Beta domains 1-5 receptor )

14) p977 (PDGFR-Beta domains 1-5 receptor plus His6 tag) p834

atggtcagctactgggacaccggggtcctgctgtgcgcgctgctcagctgtctgctt ctcacaggatc tagttcaggttcgcgaagtgatacaggtagacctttcgtagagatgtacagtgaaatccc cgaaatta tacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcacctaacatca ctgttact ttaaaaaagtttccacttgacactttgatccctgatggaaaacgcataatctgggacagt agaaaggg cttcatcatatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaacagt caatgggc atttgtataagacaaactatctcacacatcgacaaaccaatacaatcatcgatgtggttc tgagtccg tctcatggaattgaactatctgttggagaaaagcttgtcttaaattgtacagcaagaact gaactaaa tgtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgt aaaccgag acctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatg gtgtaacc cggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaac agcacatt tgtcagggtccatgaaaaagaattcgagcccaaatcttgtgacaaaactcacacatgccc accgtgcc cagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggaca ccctcatg atctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgag gtcaagtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaaca gcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagg agtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa gggcagcc ccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggt cagcctga cctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggc agccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc aagctcac cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggc tctgcaca accactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO:l)

mvsywdtgvllcallscllltgsssgsrsdtgrpfvemyseipeiihmtegrelvip crvtspnitvt lkkfpldtlipdgkriiwdsrkgfiisnatykeiglltceatvnghlyktnylthrqtnt iidvvlsp shgielsvgeklvlnctartelnvgidfnweypsskhqhkklvnrdlktqsgsemkkfls tltidgvt rsdqglytcaassglmtkknstfvrvhekefepkscdkthtcppcpapellggpsvflfp pkpkdtlm isrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqd wlngkeyk ckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiave wesngqpe nnykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk ( SEQ ID NO: 2)

p838

atggagagcaaggtgctgctggccgtcgccctgtggctctgcgtggagacccgggcc gcctctgtggg tttgcctagtgtttctcttgatctgcccaggctcagcatacaaaaagacatacttacaat taaggcta atacaactcttcaaattacttgcaggggacagagggacttggactggctttggcccaata atcagagt ggcagtgagcaaagggtggaggtgactgagtgcagcgatggcctcttctgtaagacactc acaattcc aaaagtgatcggaaatgacactggagcctacaagtgcttctaccgggaaactgacttggc ctcggtca tttatgtctatgttcaagattacagatctccatttattgcttctgttagtgaccaacatg gagtcgtg tacattactgagaacaaaaacaaaactgtggtgattccatgtctcgggtccatttcaaat ctcaacgt gtcactttgtgcaagatacccagaaaagagatttgttcctgatggtaacagaatttcctg ggacagca agaagggctttactattcccagctacatgatcagctatgctggcatggtcttctgtgaag caaaaatt aatgatgaaagttaccagtctattatgtacatagttgtcgttgtagggtataggatttat gatgtggt tctgagtccgtctcatggaattgaactatctgttggagaaaagcttgtcttaaattgtac agcaagaa ctgaactaaatgtggggattgacttcaactgggaatacccttcttcgaagcatcagcata agaaactt gtaaaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcacctta actataga tggtgtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgac caagaaga acagcacatttgtcagggtccatgaaaaaccttttgttgcttttggaagtggcgaattcg agcccaaa tcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccg tcagtctt cctcttccccccaaaacccaaggacacccteatgatctcccggacccctgaggtcacatg cgtggtgg tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagg tgcataat gccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctc accgtcct gcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctccc agccccca tcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc ccccatcc cgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccc agcgacat cgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgt gctggact ccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagg ggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcc ctgtctcc gggtaaa (SEQ ID NO:3)

meskvllavalwlcvetraasvglpsvsldlprlsiqkdiltikanttlqitcrgqr dldwlwpnnqs gseqrvevtecsdglfcktltipkvigndtgaykcfyretdlasviyvyvqdyrspfias vsdqhgvv yitenknktvvipclgsisnlnvslcarypekrfvpdgnriswdskkgftipsymisyag mvfceaki ndesyqsimyivvvvgyriydvvlspshgielsvgeklvlnctartelnvgidfnweyps skhqhkkl vnrdlktqsgsemkkfIstitidgvtrsdqglytcaassglmtkknstfvrvhekpfvaf gsgefepk scdkthtcppcpapellggpsvfIfppkpkdtlmisrtpevtcvvvdvshedpevkfnwy vdgvevhn aktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprep qvytlpps rdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdk srwqqgnv fscsvmhealhnhytqkslslspgk (SEQ ID NO: 4)

p876

atggacatgcgggtgccagctcagctgctgggactgctgctgctgtggctgcccggc accagatgcga catccagctgacccagtccccctccagcctgtccgcctctgtgggcgacagagtgaccat cacctgtt ccgcctcccaggacatcagcaactacctgaactggtatcagcagaagcccggcaaggccc ccaaggtg ctgatctacttcaccagcagcctgcactccggcgtgccctcccggttctccggctccggc tccggcac cgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactactgcca gcagtaca gcaccgtgccctggaccttcggccagggcaccaaggtggaaatcaagggaggtggaggaa gcggtgga ggaggtagcggaggcggcggcagcgaggtgcagctggtggaatccggcggaggactggtg cagcctgg cggctccctgagactgtcttgcgccgcctccggctacgacttcacccactacggcatgaa ctgggtcc gacaggcccctggcaagggactggaatgggtgggctggatcaacacctacaccggcgagc ccacctac gccgccgacttcaagcggcggttcaccttcagcctggacaccagcaagagcaccgcctac ctgcagat gaactccctgcgggccgaggacaccgccgtgtactactgcgccaagtacccctactacta cggcacca gccactggtacttcgacgtgtggggccagggcaccctggtcaccgtctcctcacaccatc accaccac cac (SEQ ID NO:5)

mdmrvpaqllgllllwlpgtrcdiqltqspsslsasvgdrvtitcsasqdisnylnw yqqkpgkapkv liyftsslhsgvpsrfsgsgsgtdftltisslqpedfatyycqqystvpwtfgqgtkvei kggggsgg ggsggggsevqlvesggglvqpggslrlscaasgydfthygmnwvrqapgkglewvgwin tytgepty aadfkrrftfsldtskstaylqmnslraedtavyycakypyyygtshwyfdvwgqgtlvt vsshhhhh h (SEQ ID NO: 6)

p913

atggacatgcgggtgccagctcagctgctgggactgctgctgctgtggctgcccggc accagatgcga catccagctgacccagtccccctccagcctgtccgcctctgtgggcgacagagtgaccat cacctgtt ccgcctcccaggacatcagcaactacctgaactggtatcagcagaagcccggcaaggccc ccaaggtg ctgatctacttcaccagcagcctgcactccggcgtgccctcccggttctccggctccggc tccggcac cgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactactgcca gcagtaca gcaccgtgccctggaccttcggccagggcaccaaggtggaaatcaagggaggtggaggaa gcggtgga ggaggtagcggaggcggcggcagcgaggtgcagctggtggaatccggcggaggactggtg cagcctgg cggctccctgagactgtcttgcgccgcctccggctacgacttcacccactacggcatgaa ctgggtcc gacaggcccctggcaagggactggaatgggtgggctggatcaacacctacaccggcgagc ccacctac gccgccgacttcaagcggcggttcaccttcagcctggacaccagcaagagcaccgcctac ctgcagat gaactccctgcgggccgaggacaccgccgtgtactactgcgccaagtacccctactacta cggcacca gccactggtacttcgacgtgtggggccagggcaccctggtcaccgtctcctca (SEQ ID NO: 19) mdmrvpaqllgllllwlpgtrcdiqltqspsslsasvgdrvtitcsasqdisnylnwyqq kpgkapkv liyftsslhsgvpsrfsgsgsgtdftltisslqpedfatyycqqystvpwtfgqgtkvei kggggsgg ggsggggsevqlvesggglvqpggslrlscaasgydfthygmnwvrqapgkglewvgwin tytgepty aadfkrrftfsldtskstaylqmnslraedtavyycakypyyygtshwyfdvwgqgtlvt vss ( SEQ ID NO:20)

p873

atggtcagctactgggacaccggggtcctgctgtgcgcgctgctcagctgtctgctt ctcacaggatc tagttcaggtagtgatacaggtagacctttcgtagagatgtacagtgaaatccccgaaat tatacaca tgactgaaggaagggagctcgtcattccctgccgggttacgtcacctaacatcactgtta ctttaaaa aagtttccacttgacactttgatccctgatggaaaacgcataatctgggacagtagaaag ggcttcat catatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaacagtcaatgg gcatttgt ataagacaaactatctcacacatcgacaaaccaatacaatcatcgatgtggttctgagtc cgtctcat ggaattgaactatctgttggagaaaagcttgtcttaaattgtacagcaagaactgaacta aatgtggg gattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgtaaaccg agacctaa aaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatggtgtaa cccggagt gaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaacagcaca tttgtcag ggtccatgaaaaagacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg gggaccgt cagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg tcacatgc gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc gtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcag cgtcctca ccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctccca gcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac accctgcc cccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggctt ctatccca gcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc ctcccgtg ctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg cagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccc tgtctccgggt (SEQ ID NO: 7)

mvsywdtgvllcallscllltgsssgsdtgrpfvemyseipeiihmtegrelvipcr vtspnitvtlk kfpldtlipdgkriiwdsrkgfiisnatykeiglltceatvnghlyktnylthrqtntii dvvlspsh gielsvgeklvlnctartelnvgidfnweypsskhqhkklvnrdlktqsgsemkkflstl tidgvtrs dqglytcaassglmtkknstfvrvhekdkthtcppcpapellggpsvfIfppkpkdtlmi srtpevtc vvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykc kvsnkalp apiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpen nykttppv ldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg (SEQ ID NO: 8)

p874

atggactggacctggtctatcctgttcctggtggccgctgcaaccggcacctactcc gaggtgcagct ggtggaatccggcggaggactggtgcagcctggcggctccctgagactgtcttgcgccgc ctccggct acaccttcaccaactacggcatgaactgggtccgacaggcccctggcaagggactggaat gggtgggc tggatcaacacctacaccggcgagcccacctacgccgccgacttcaagcggcggttcacc ttcagcct ggacaccagcaagagcaccgcctacctgcagatgaactccctgcgggccgaggacaccgc cgtgtact actgcgccaagtacccccactactacggcagcagccactggtacttcgacgtgtggggcc agggcacc ctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcc tccaagag cacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggt gacggtgt cgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcct caggactc tactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatc tgcaacgt gaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaa aactcaca catgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccc caaaaccc aaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc cacgaaga ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaa gccgcggg aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact ggctgaat ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc atctccaa agccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagt gggagagc aatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcc ttcttcct ctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctc cgtgatgc atgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID

NO: 9)

mdwtwsilflvaaatgtysevqlvesggglvqpggslrlscaasgytftnygmnwvr qapgkglewvg wintytgeptyaadfkrrftfsldtskstaylqmnslraedtavyycakyphyygsshwy fdvwgqgt lvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssgl yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggps vfIfppkp kdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvlt vlhqdwln gkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyps diavewes ngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl spgk (SEQ ID NO:10)

p875

atggacatgcgggtgccagctcagctgctgggactgctgctgctgtggctgcccggc accagatgcga catccagatgacccagtccccctccagcctgtccgcctctgtgggcgacagagtgaccat cacctgtt ccgcctcccaggacatcagcaactacctgaactggtatcagcagaagcccggcaaggccc ccaaggtg ctgatctacttcaccagcagcctgcactccggcgtgccctcccggttctccggctccggc tccggcac cgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactactgcca gcagtaca gcaccgtgccctggaccttcggccagggcaccaaggtggaaatcaagcggaccgtggccg ctccctcc gtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtctgc ctgctgaa caacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccgg caactccc aggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctga ccctgtcc aaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtcc agccccgt gaccaagtccttcaaccggggcgagtgc (SEQ ID NO: 11)

mdmrvpaqllgllllwlpgtrcdiqmtqspsslsasvgdrvtitcsasqdisnylnw yqqkpgkapkv liyftsslhsgvpsrfsgsgsgtdftltisslqpedfatyycqqystvpwtfgqgtkvei krtvaaps vfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstys lsstltls kadyekhkvyacevthqglsspvtksfnrgec (SEQ ID NO: 12)

p915

atggactggacctggtctatcctgttcctggtggccgctgcaaccggcacctactcc gaggtgcagct ggtggaatccggcggaggactggtgcagcctggcggctccctgagactgtcttgcgccgc ctccggct acgacttcacccactacggcatgaactgggtccgacaggcccctggcaagggactggaat gggtgggc tggatcaacacctacaccggcgagcccacctacgccgccgacttcaagcggcggttcacc ttcagcct ggacaccagcaagagcaccgcctacctgcagatgaactccctgcgggccgaggacaccgc cgtgtact actgcgccaagtacccctactactacggcaccagccactggtacttcgacgtgtggggcc agggcacc ctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcc tccaagag cacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggt gacggtgt cgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcct caggactc tactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatc tgcaacgt gaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaa aactcacc tg (SEQ ID NO: 13)

mdwtwsilflvaaatgtysevqlvesggglvqpggslrlscaasgydfthygmnwvr qapgkglewvg wintytgeptyaadfkrrftfsldtskstaylqmnslraedtavyycakypyyygtshwy fdvwgqgt lvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssgl yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthl (SEQ ID NO: 14) p914

atggacatgcgggtgccagctcagctgctgggactgctgctgctgtggctgcccggc accagatgcga catccagctgacccagtccccctccagcctgtccgcctctgtgggcgacagagtgaccat cacctgtt ccgcctcccaggacatcagcaactacctgaactggtatcagcagaagcccggcaaggccc ccaaggtg ctgatctacttcaccagcagcctgcactccggcgtgccctcccggttctccggctccggc tccggcac cgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactactgcca gcagtaca gcaccgtgccctggaccttcggccagggcaccaaggtggaaatcaagcggaccgtggccg ctccctcc gtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtctgc ctgctgaa caacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccgg caactccc aggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctga ccctgtcc aaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtcc agccccgt gaccaagtccttcaaccggggcgagtgc (SEQ ID NO: 15)

mdmrvpaqllgllllwlpgtrcdiqltqspsslsasvgdrvtitcsasqdisnylnw yqqkpgkapkv liyftsslhsgvpsrfsgsgsgtdftltisslqpedfatyycqqystvpwtfgqgtkvei krtvaaps vfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstys lsstltls kadyekhkvyacevthqglsspvtksfnrgec (SEQ ID NO: 16)

p916

atggactggacctggtctatcctgttcctggtggccgctgcaaccggcacctactcc gaggtgcagct ggtggaatccggcggaggactggtgcagcctggcggctccctgagactgtcttgcgccgc ctccggct acgacttcacccactacggcatgaactgggtccgacaggcccctggcaagggactggaat gggtgggc tggatcaacacctacaccggcgagcccacctacgccgccgacttcaagcggcggttcacc ttcagcct ggacaccagcaagagcaccgcctacctgcagatgaactccctgcgggccgaggacaccgc cgtgtact actgcgccaagtacccctactactacggcaccagccactggtacttcgacgtgtggggcc agggcacc ctggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggcaccctcc tccaagag cacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggt gacggtgt cgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcct caggactc tactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatc tgcaacgt gaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaa aactcaca catgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccc caaaaccc aaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc cacgaaga ccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaa gccgcggg aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact ggctgaat ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc atctccaa agccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagt gggagagc aatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcc ttcttcct ctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctc cgtgatgc atgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 17)

mdwtwsilflvaaatgtysevqlvesggglvqpggslrlscaasgydfthygmnwvr qapgkglewvg wintytgeptyaadfkrrftfsldtskstaylqmnslraedtavyycakypyyygtshwy fdvwgqgt lvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssgl yslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggps vfIfppkp kdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvlt vlhqdwln gkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyps diavewes ngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl spgk (SEQ ID NO:18)

p917

atggtcagctactgggacaccggggtcctgctgtgcgcgctgctcagctgtctgctt ctcacaggatc tagttcaggttcgcgaagtgatacaggtagacctttcgtagagatgtacagtgaaatccc cgaaatta tacacatgactgaaggaagggagctcgtcattccctgccgggttacgtcacctaacatca ctgttact ttaaaaaagtttccacttgacactttgatccctgatggaaaacgcataatctgggacagt agaaaggg cttcatcatatcaaatgcaacgtacaaagaaatagggcttctgacctgtgaagcaacagt caatgggc atttgtataagacaaactatctcacacatcgacaaaccaatacaatcatcgatgtggttc tgagtccg tctcatggaattgaactatctgttggagaaaagcttgtcttaaattgtacagcaagaact gaactaaa tgtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgt aaaccgag acctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatg gtgtaacc cggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaac agcacatt tgtcagggtccatgaaaaagacaaaactcacacatgcccaccgtgcccagcacctgaact cctggggg gaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtc acatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg gacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcg tcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctcca acaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacac cctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaa aggcttct atcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga ccacgcct cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc aggtggca gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgca gaagagcc tctccctgtctccgggt (SEQ ID NO: 21) mvsywdtgvllcallscllltgsssgsrsdtgrpfvemyseipeiihmtegrelvipcrv tspnitvt lkkfpldtlipdgkriiwdsrkgfiisnatykeiglltceatvnghlyktnylthrqtnt iidvvlsp shgielsvgeklvlnctartelnvgidfnweypsskhqhkklvnrdlktqsgsemkkfls tltidgvt rsdqglytcaassglmtkknstfvrvhekdkthtcppcpapellggpsvfIfppkpkdtl misrtpev tcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkey kckvsnka lpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqp ennykttp pvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg ( SEQ ID NO : 22 ) p964

atgcggcttccgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctg tctctcctgtt acttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgt cctcaatg tctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaacggatgt cccaggag cccccacaggaaatggccaaggcccaggatggcaccttctccagcgtgctcacactgacc aacctcac tgggctagacacgggagaatacttttgcacccacaatgactcccgtggactggagaccga tgagcgga aacggctctacatctttgtgccagatcccaccgtgggcttcctccctaatgatgccgagg aactattc atctttctcacggaaataactgagatcaccattccatgccgagtaacagacccacagctg gtggtgac actgcacgagaagaaaggggacgttgcactgcctgtcccctatgatcaccaacgtggctt ttctggta tctttgaggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctg atgcctac tatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactgtg gtccgcca gggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtcaacttcgagtg gacatacc cccgcaaagaaagtgggcggctggtggagccggtgactgacttcctcttggatatgcctt accacatc cgctccatcctgcacatccccagtgccgagttagaagactcggggacctacacctgcaat gtgacgga gagtgtgaatgaccatcaggatgaaaaggccatcaacatcaccgtggttgagagcggcta cgtgcggc tcctgggagaggtgggcacactacaatttgetgagetgcatcggagccggacactgcagg tagtgttc gaggcctacccaccgcccactgtcctgtggttcaaagacaaccgcaccctgggcgactcc agcgctgg cgaaatcgccctgtccacgcgcaacgtgtcggagacccggtatgtgtcagagctgacact ggttcgcg tgaaggtggcagaggctggccactacaccatgcgggccttccatgaggatgctgaggtcc agctctcc ttccagctacagatcaatgtccctgtccgagtgctggagctaagtgagagccaccctgac agtgggga acagacagtccgctgtcgtggccggggcatgccccagccgaacatcatctggtctgcctg cagagacc tcaaaaggtgtccacgtgagctgccgcccacgctgctggggaacagttccgaagaggaga gccagctg gagactaacgtgacgtactgggaggaggagcaggagtttgaggtggtgagcacactgcgt ctgcagca cgtggatcggccactgtcggtgcgctgcacgctgcgcaacgctgtgggccaggacacgca ggaggtca tcgtggtgccacactccttgcccttcaagcccccatgcccatcatgcccagcacctgagt tcctgggg ggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacc cctgaggt cacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgt ggatggcg tggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtg tggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctcc aacaaagg cctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccaca ggtgtaca ccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtca aaggcttc taccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaag accacgcc tcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagag caggtggc aggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacac agaagagc ctctccctgtctctgggtaaa

(SEQ ID NO:23) mrlpgampalalkgellllslllllepqisqglvvtppgpelvlnvsstfvltcsgsapv vwermsqe ppqemakaqdgtfssvltltnltgldtgeyfcthndsrgletderkrlyifvpdptvgfI pndaeelf iflteiteitipcrvtdpqlvvtlhekkgdvalpvpydhqrgfsgifedrsyickttigd revdsday yvyrlqvssinvsvnavqtvvrqgenitlmcivignevvnfewtyprkesgrlvepvtdf lldmpyhi rsilhipsaeledsgtytcnvtesvndhqdekainitvvesgyvrllgevgtlqfaelhr srtlqvvf eayppptvlwfkdnrtlgdssageialstrnvsetryvseltlvrvkvaeaghytmrafh edaevqls fqlqinvpvrvlelseshpdsgeqtvrcrgrgmpqpniiwsacrdlkrcprelpptllgn sseeesql etnvtyweeeqefevvstlrlqhvdrplsvrctlrnavgqdtqevivvphslpfkppcps cpapeflg gpsvfIfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqf nstyrvvs vltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvs ltclvkgf ypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmheal hnhytqks lslslgk (SEQ ID NO:24) p963

atgcggcttccgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctg tctctcctgtt acttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgt cctcaatg tctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaacggatgt cccaggag cccccacaggaaatggccaaggcccaggatggcaccttctccagcgtgctcacactgacc aacctcac tgggctagacacgggagaatacttttgcacccacaatgactcccgtggactggagaccga tgagcgga aacggctctacatctttgtgccagatcccaccgtgggcttcctccctaatgatgccgagg aactattc atctttctcacggaaataactgagatcaccattccatgccgagtaacagacccacagctg gtggtgac actgcacgagaagaaaggggacgttgcactgcctgtcccctatgatcaccaacgtggctt ttctggta tctttgaggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctg atgcctac tatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactgtg gtccgcca gggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtcaacttcgagtg gacatacc cccgcaaagaaagtgggcggctggtggagccggtgactgacttcctcttggatatgcctt accacatc cgctccatcctgcacatccccagtgccgagttagaagactcggggacctacacctgcaat gtgacgga gagtgtgaatgaccatcaggatgaaaaggccatcaacatcaccgtggttgagagcggcta cgtgcggc tcctgggagaggtgggcacactacaatttgetgagetgcatcggagccggacactgcagg tagtgttc gaggcctacccaccgcccactgtcctgtggttcaaagacaaccgcaccctgggcgactcc agcgctgg cgaaatcgccctgtccacgcgcaacgtgtcggagacccggtatgtgtcagagctgacact ggttcgcg tgaaggtggcagaggctggccactacaccatgcgggccttccatgaggatgctgaggtcc agctctcc ttccagctacagatcaatgtccctgtccgagtgctggagctaagtgagagccaccctgac agtgggga acagacagtccgctgtcgtggccggggcatgccccagccgaacatcatctggtctgcctg cagagacc tcaaaaggtgtccacgtgagctgccgcccacgctgctggggaacagttccgaagaggaga gccagctg gagactaacgtgacgtactgggaggaggagcaggagtttgaggtggtgagcacactgcgt ctgcagca cgtggatcggccactgtcggtgcgctgcacgctgcgcaacgctgtgggccaggacacgca ggaggtca tcgtggtgccacactccttgcccttcaaggaccccgagcccaaatcttgtgacaaaactc acacatgc ccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaa cccaagga cacccteatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacga agaccctg aggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgc gggaggag cagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctg aatggcaa ggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacca agaaccag gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggag agcaatgg gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttctt cctctaca gcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga tgcatgag gctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 25) mrlpgampalalkgellllslllllepqisqglvvtppgpelvlnvsstfvltcsgsapv vwermsqe ppqemakaqdgtfssvltltnltgldtgeyfcthndsrgletderkrlyifvpdptvgfI pndaeelf iflteiteitipcrvtdpqlvvtlhekkgdvalpvpydhqrgfsgifedrsyickttigd revdsday yvyrlqvssinvsvnavqtvvrqgenitlmcivignevvnfewtyprkesgrlvepvtdf lldmpyhi rsilhipsaeledsgtytcnvtesvndhqdekainitvvesgyvrllgevgtlqfaelhr srtlqvvf eayppptvlwfkdnrtlgdssageialstrnvsetryvseltlvrvkvaeaghytmrafh edaevqls fqlqinvpvrvlelseshpdsgeqtvrcrgrgmpqpniiwsacrdlkrcprelpptllgn sseeesql etnvtyweeeqefevvstlrlqhvdrplsvrctlrnavgqdtqevivvphslpfkdpepk scdkthtc ppcpapellggpsvfIfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhn aktkpree qynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlpps rdeltknq vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnv fscsvmhe alhnhytqkslslspgk (SEQ ID NO: 26)

p974

atgcggcttccgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctg tctctcctgtt acttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgt cctcaatg tctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaacggatgt cccaggag cccccacaggaaatggccaaggcccaggatggcaccttctccagcgtgctcacactgacc aacctcac tgggctagacacgggagaatacttttgcacccacaatgactcccgtggactggagaccga tgagcgga aacggctctacatctttgtgccagatcccaccgtgggcttcctccctaatgatgccgagg aactattc atctttctcacggaaataactgagatcaccattccatgccgagtaacagacccacagctg gtggtgac actgcacgagaagaaaggggacgttgcactgcctgtcccctatgatcaccaacgtggctt ttctggta tctttgaggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctg atgcctac tatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactgtg gtccgcca gggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtcaacttcgagtg gacatacc cccgcaaagaaagtgggcggctggtggagccggtgactgacttcctcttggatatgcctt accacatc cgctccatcctgcacatccccagtgccgagttagaagactcggggacctacacctgcaat gtgacgga gagtgtgaatgaccatcaggatgaaaaggccatcaacatcaccgtggttgagagcggcta cgtgcggc tcctgggagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctg aactcctg gggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgg acccctga ggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta cgtggacg gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtacc gtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtc tccaacaa agccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc acaggtgt acaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctgg tcaaaggc ttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactac aagaccac gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaa gagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactaca cgcagaag agcctctccctgtctccgggtaaa (SEQ ID NO: 27)

mrlpgampalalkgellllslllllepqisqglvvtppgpelvlnvsstfvltcsgs apvvwermsqe ppqemakaqdgtfssvltltnltgldtgeyfcthndsrgletderkrlyifvpdptvgfI pndaeelf iflteiteitipcrvtdpqlvvtlhekkgdvalpvpydhqrgfsgifedrsyickttigd revdsday yvyrlqvssinvsvnavqtvvrqgenitlmcivignevvnfewtyprkesgrlvepvtdf lldmpyhi rsilhipsaeledsgtytcnvtesvndhqdekainitvvesgyvrllgepkscdkthtcp pcpapell ggpsvfIfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeq ynstyrvv svltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqv sltclvkg fypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhea lhnhytqk slslspgk (SEQ ID NO:28) p978

atgcggcttccgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctg tctctcctgtt acttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgt cctcaatg tctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaacggatgt cccaggag cccccacaggaaatggccaaggcccaggatggcaccttctccagcgtgctcacactgacc aacctcac tgggctagacacgggagaatacttttgcacccacaatgactcccgtggactggagaccga tgagcgga aacggctctacatctttgtgccagatcccaccgtgggcttcctccctaatgatgccgagg aactattc atctttctcacggaaataactgagatcaccattccatgccgagtaacagacccacagctg gtggtgac actgcacgagaagaaaggggacgttgcactgcctgtcccctatgatcaccaacgtggctt ttctggta tctttgaggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctg atgcctac tatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactgtg gtccgcca gggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtcaacttcgagtg gacatacc cccgcaaagaaagtgggcggctggtggagccggtgactgacttcctcttggatatgcctt accacatc cgctccatcctgcacatccccagtgccgagttagaagactcggggacctacacctgcaat gtgacgga gagtgtgaatgaccatcaggatgaaaaggccatcaacatcaccgtggttgagagcggcta cgtgcggc tcctgggagaggtgggcacactacaatttgetgagetgcatcggagccggacactgcagg tagtgttc gaggcctacccaccgcccactgtcctgtggttcaaagacaaccgcaccctgggcgactcc agcgctgg cgaaatcgccctgtccacgcgcaacgtgtcggagacccggtatgtgtcagagctgacact ggttcgcg tgaaggtggcagaggctggccactacaccatgcgggccttccatgaggatgctgaggtcc agctctcc ttccagctacagatcaatgtccctgtccgagtgctggagctaagtgagagccaccctgac agtgggga acagacagtccgctgtcgtggccggggcatgccccagccgaacatcatctggtctgcctg cagagacc tcaaaaggtgtccacgtgagctgccgcccacgctgctggggaacagttccgaagaggaga gccagctg gagactaacgtgacgtactgggaggaggagcaggagtttgaggtggtgagcacactgcgt ctgcagca cgtggatcggccactgtcggtgcgctgcacgctgcgcaacgctgtgggccaggacacgca ggaggtca tcgtggtgccacactccttgcccttcaag (SEQ ID NO: 29)

mrlpgampalalkgellllslllllepqisqglvvtppgpelvlnvsstfvltcsgs apvvwermsqe ppqemakaqdgtfssvltltnltgldtgeyfcthndsrgletderkrlyifvpdptvgfl pndaeelf iflteiteitipcrvtdpqlvvtlhekkgdvalpvpydhqrgfsgifedrsyickttigd revdsday yvyrlqvssinvsvnavqtvvrqgenitlmcivignevvnfewtyprkesgrlvepvtdf lldmpyhi rsilhipsaeledsgtytcnvtesvndhqdekainitvvesgyvrllgevgtlqfaelhr srtlqvvf eayppptvlwfkdnrtlgdssageialstrnvsetryvseltlvrvkvaeaghytmrafh edaevqls fqlqinvpvrvlelseshpdsgeqtvrcrgrgmpqpniiwsacrdlkrcprelpptllgn sseeesql etnvtyweeeqefevvstlrlqhvdrplsvrctlrnavgqdtqevivvphslpfk (SEQ ID NO: 30)

p977

atggggcagtgcaggaaaagtggcactatgaaccctgcagccctagacaattgtact aaccttcttct ctttcctctcctgacaggttggtgtacagtagcttccaagtactccaccatgcggcttcc gggtgcga tgccagctctggccctcaaaggcgagctgctgttgctgtctctcctgttacttctggaac cacagatc tctcagggcctggtcgtcacacccccggggccagagcttgtcctcaatgtctccagcacc ttcgttct gacctgctcgggttcagctccggtggtgtgggaacggatgtcccaggagcccccacagga aatggcca aggcccaggatggcaccttctccagcgtgctcacactgaccaacctcactgggctagaca cgggagaa tacttttgcacccacaatgactcccgtggactggagaccgatgagcggaaacggctctac atctttgt gccagatcccaccgtgggcttcctccctaatgatgccgaggaactattcatctttctcac ggaaataa ctgagatcaecattccatgccgagtaacagacccacagctggtggtgacactgcacgaga agaaaggg gacgttgcactgcctgtcccctatgatcaccaacgtggcttttctggtatctttgaggac agaagcta catctgcaaaaccaccattggggacagggaggtggattctgatgcctactatgtctacag actccagg tgtcatccatcaacgtctctgtgaacgcagtgcagactgtggtccgccagggtgagaaca tcaccctc atgtgcattgtgatcgggaatgaggtggtcaacttcgagtggacatacccccgcaaagaa agtgggcg gctggtggagccggtgactgacttcctcttggatatgccttaccacatccgctccatcct gcacatcc ccagtgccgagttagaagactcggggacctacacctgcaatgtgacggagagtgtgaatg accatcag gatgaaaaggccatcaacatcaccgtggttgagagcggctacgtgcggctcctgggagag gtgggcac actacaatttgctgagctgcatcggagccggacactgcaggtagtgttcgaggcctaccc accgccca ctgtcctgtggttcaaagacaaccgcaccctgggcgactccagcgctggcgaaatcgccc tgtccacg cgcaacgtgtcggagacccggtatgtgtcagagctgacactggttcgcgtgaaggtggca gaggctgg ccactacaccatgcgggccttccatgaggatgctgaggtccagctctccttccagctaca gatcaatg tccctgtccgagtgctggagctaagtgagagccaccctgacagtggggaacagacagtcc gctgtcgt ggccggggcatgccccagccgaacatcatctggtctgcctgcagagacctcaaaaggtgt ccacgtga gctgccgcccacgctgctggggaacagttccgaagaggagagccagctggagactaacgt gacgtact gggaggaggagcaggagtttgaggtggtgagcacactgcgtctgcagcacgtggatcggc cactgtcg gtgcgctgcacgctgcgcaacgctgtgggccaggacacgcaggaggtcatcgtggtgcca cactcttt gcccttcaagcggggcagccaccaccaccaccaccac (SEQ ID NO: 31)

mgqcrksgtmnpaaldnctnlllfplltgwctvaskystmrlpgampalalkgelll lslllllepqi sqglvvtppgpelvlnvsstfvltcsgsapvvwermsqeppqemakaqdgtfssvltltn ltgldtge yfcthndsrgletderkrlyifvpdptvgflpndaeelfiflteiteitipcrvtdpqlv vtlhekkg dvalpvpydhqrgfsgifedrsyickttigdrevdsdayyvyrlqvssinvsvnavqtvv rqgenitl mcivignevvnfewtyprkesgrlvepvtdf11dmpyhirsilhipsaeledsgtytcnv tesvndhq dekainitvvesgyvrllgevgtlqfaelhrsrtlqvvfeayppptvlwfkdnrtlgdss ageialst rnvsetryvseltlvrvkvaeaghytmrafhedaevqlsfqlqinvpvrvlelseshpds geqtvrcr grgmpqpniiwsacrdlkrcprelpptllgnsseeesqletnvtyweeeqefevvstlrl qhvdrpls vrctlrnavgqdtqevivvphslpfkrgshhhhhh (SEQ ID NO: 32)

In other examples, the one or more biologically active molecules are selected from the group consisting of C3a inhibitors, C3b inhibitors, other agents targeting and inhibiting or modulating immunologic pathway molecules, brain derived neurotrophic factor (BDNF), NT- 4, ciliary neurotrophic factor (CNTF), Axokine, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF I), insulin-like growth factor II (IGF II), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGF a), transforming growth factor β (TGF β), nerve growth factor (NGF), platelet derived growth factor (PDGF), glia-derived neurotrophic factor (GDNF), Midkine, phorbol 12-myristate 13- acetate, tryophotin, activin, thyrotropin releasing hormone, interleukins, bone morphogenic protein, macrophage inflammatory proteins, heparin sulfate, amphiregulin, retinoic acid, tumor necrosis factor a, fibroblast growth factor receptor, epidermal growth factor receptor (EGFR), PEDF, LEDGF, NTN, Neublastin, neurotrophins, lymphokines, VEGF inhibitors, PDGF inhibitors, PIGF inhibitors, Tie2, CD55, C59, a bispecific molecule that

simultaneously binds VEGF and PDGF, and other agents expected to have therapeutically useful effects on potential target tissues.

Known anti-VEGF compounds can include, but are not limited to, anti-VEGF receptor fragments (i.e., Aflibercept) and/or anti-VEGF antibodies (or antigen binding fragments thereof) (i.e., Bevacizumab, DrugBank DB00112; or Ranibizumab DrugBank DB01270). The sequences of these known anti-VEGF compounds are known in the art. The one or more biologically active molecules can be C3a inhibitors, C3b inhibitors, VEGF inhibitors, PDGF inhibitors, or any combinations thereof.

The methods and devices are intended for use in a primate, for example, a human host, recipient, patient, subject or individual. A number of different implantation sites are contemplated for the devices and methods. Suitable implantation sites include, but are not limited to, eye, spleen, ear, heart, colon, liver, kidney, breast, joint, bone marrow, subcutaneous, and/or peritoneal spaces. For example, implantation sites include the aqueous and vitreous humors of the eye, the periocular space, the anterior chamber, the posterior chamber, and/or the Subtenon's capsule. Within the body, implantation sites may include subcutaneous or intraperitoneal. In addition, implantation may be directed at localized delivery at or near lesions requiring the desired biologic therapy. Example of such disease sites may be inflamed joints or sites of benign or malignant tumors. Access by the device to the circulatory system can further extend the range of potential disease sites within the body to distally affected organs and tissues.

Any of the devices can be used to deliver an appropriate therapeutic dose of the one or more biologically active molecules to an implantation site described herein.

The devices are also able to deliver an appropriate therapeutic dosage of the one or more biologically active molecules for at least 6 months (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months).

Early generation ECT products producing soluble VEGF-receptor (VEGF- R) (e.g., single chambered NT-503 second generation ECT devices) have been shown to deliver the biologically active molecule for at least 6 months (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months) and have demonstrated clinically meaningful improvements in BCVA and reductions in macular thickening in patients with active neovascular AMD for over 12 months. Thus, the second generation ECT devices exhibit extended duration delivery of the VEGF-R ECT product.

Likewise, higher dose levels (such as those produced by the ECT cartridge devices described herein) are also expected to achieve efficacy comparable or greater than standard- of-care treatments.

Accordingly, both NT-503 second generation ECT devices and third generation ECT cartridge devices are capable of extended duration (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months) delivery of ECT products.

The number of devices and device size should be sufficient to produce a therapeutic effect upon implantation and is determined by the amount of biological activity required for the particular application. In the case of secretory cells releasing therapeutic substances, standard dosage considerations and criteria known to the art will be used to determine the amount of secretory substance required. Factors to be considered include the size and weight of the recipient; the productivity or functional level of the cells; and, where appropriate, the normal productivity or metabolic activity of the organ or tissue whose function is being replaced or augmented. It is also important to consider that a fraction of the cells may not survive the immunoisolation and implantation procedures. Moreover, whether the recipient has a preexisting condition that can interfere with the efficacy of the implant must also be considered. Devices can easily be manufactured which contain many thousands of cells. For example, current ophthalmic clinical devices (e.g., the second generation ECT devices) contain between 200,000 and 750,000 cells, whereas micronized devices would contain between 10,000 and 100,000 cells. Other large scale devices (e.g., for systemic applications) may contain between 1,000,000 to 100,000,000 cells.

The therapeutically effective amount used in any devices (i.e., therapeutic dosages) may be between 0.1 pg and 10000 μg (e.g., between 0.1 pg and 5000 μg; between 0.1 pg and 2500 μg; between 0.1 pg and 1000 μg; between 0.1 pg and 500 μg; between 0.1 pg and 250 μg; between 0.1 pg and 100 μg; between 0.1 pg and 50 μg; between 0.1 pg and 25 μg;

between 0.1 pg and 10 μg; between 0.1 pg and 5 μg; between 0.1 pg and 100 ng; between 0.1 pg and 50 ng; between 0.1 pg and 25 ng; between 0.1 pg and 10 ng; or between 0.1 pg and 5 ng) per eye per patient per day.

The therapeutically effective amount used in any devices (i.e., therapeutic dosages) may be between 0.1 pg and 10000 μg (e.g., between 0.1 pg and 5000 μg; between 0.1 pg and 2500 μg; between 0.1 pg and 1000 μg; between 0.1 pg and 500 μg; between 0.1 pg and 250 μg; between 0.1 pg and 100 μg; between 0.1 pg and 50 μg; between 0.1 pg and 25 μg;

between 0.1 pg and 10 μg; between 0.1 pg and 5 μg; between 0.1 pg and 100 ng; between 0.1 pg and 50 ng; between 0.1 pg and 25 ng; between 0.1 pg and 10 ng; or between 0.1 pg and 5 ng) per patient per day.

In one non-limiting example, the therapeutic amount is at least 0.5-50 μg/ml steady state in the eye. Suitable therapeutic amounts may include, for example, 0.5 μg, 0.6 μg, 0.7 ug, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 1 1 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, 25 μg, 26 μg, 27 μg, 28 μg, 29 μg, 30 μg, 31 μg, 32 μg, 33 μg, 34 μg, 35 μg, 36 μg, 37 μg, 38 μg, 39 μg, 40 μg, 41 μ¾ 42 μg, 43 μ^ 44 μ _δ , 45 μg, 46 μ^ 47 μg, 48 μ¾ 49 μ¾ 50 μg, 51 μ¾ 52 μg, 53 μg, 54 μ¾ 55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 74 μg, 75 μg, 76 μg, 77 μg, 78 μ _δ , 79 μg, 80 μ¾ 81 μ _δ , 82 μg, 83 μ _δ , 84 μg, 85 μ _δ , 86 μ _δ , 87 μg, 88 μ¾ 89 μg, 90 μ¾ 91 μ _§ , 92 μ _§ , 93 μg, 94 μ _§ , 95 μ _§ , 96 μ _§ , 97 μ _§ , 98 μ _§ , 99 μ _§ , 100 μ _§ , 150 μg, 200 μ _§ , 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1500 μg, 2000 μg, 2500 μg, 3000 μg, 3500 μg, 4000 μg, 4500 μg, 5000 μg, 5500 μg, 6000 μg, 6500 μg, 7000 μg, 7500 μg, 8000 μg, 8500 μg, 9000 μg, 9500 μg or 10000 μg.

Ophthalmic disorders that may be treated by various embodiments of the present invention include, but are not limited to, branch or central retinal vein occlusion (BRVO or CRVO), uveitis, macular telangiectasia, diabetic retinopathies, diabetic macular edema, proliferative retinopathies, retinal vascular diseases, vascular anomalies, age-related macular degeneration and other acquired disorders, endophthalmitis, infectious diseases,

inflammatory but non-infectious diseases, AIDS-related disorders, ocular ischemia syndrome, pregnancy-related disorders, peripheral retinal degenerations, retinal degenerations, toxic retinopathies, retinal tumors, choroidal tumors, choroidal disorders, vitreous disorders, retinal detachment and proliferative vitreoretinopathy, non-penetrating trauma, penetrating trauma, post-cataract complications, and inflammatory optic neuropathies.

Those skilled in the art will recognize that age-related macular degeneration includes, but is not limited to, wet and dry age-related macular degeneration, exudative age-related macular degeneration, and myopic degeneration.

In some embodiments, the disorder to be treated is the wet form of age-related macular degeneration or BRVO or CRVO. The present devices may also be useful for the treatment of ocular neovascularization, a condition associated with many ocular diseases and disorders. For example, retinal ischemia-associated ocular neovascularization is a major cause of blindness in diabetes and many other diseases.

The devices may also be useful for inhibiting endothelial cell proliferation in hematologic disorders, atherosclerosis, inflammation, increased vascular permeability and malignancy.

The devices may also be useful for d treating a variety of disorders selected from the group consisting of ophthalmic disorders, endothelial cell proliferation or vascularization related disorders, cancer, infectious disorders, inflammatory disorders, immunologic disorders, digestive disorders, vascular disorders, lung disorders, oral disorders, blood disorders, liver disorders, skin disorders, prostate disorders, kidney disorders, metabolic disorders, endocrine disorders, neurologic disorders, and neurodegenerative disorders..

Determination of suitable therapeutic dosages for use in the treatment of these disorders is within the routine level of skill in the art.

As used herein, the terms "individual" or "recipient" or "host" are used

interchangeably to refer to a human or an animal subject.

As used herein, a "biologically active molecule" ("BAM") is any substance that is capable of exerting a biologically useful effect upon the body of an individual in whom a device is implanted. Anti-angiogenic antibody-scaffolds and anti-angiogenic antibodies and molecules are examples of BAMs. BAMs may include immunologic factors or targets, growth factor inhibitors, soluble receptors, anti-angiogenic antibodies and molecules, anti- angiogenic antibody scaffolds, cytokine, growth factors, neurotrophic factors, angiogenic factors, neurotransmitters, hormones, enzymes, anti-inflammatory factors, therapeutic proteins, gene transfer vectors, antibodies and antibody fragments, antigens, peptides, and any combination thereof. In various embodiments, such molecules can include, but are not limited to, C3a inhibitors, C3b inhibitors, other agents targeting and inhibiting or modulating immunologic pathway molecules, brain derived neurotrophic factor (BDNF), NT -4, ciliary neurotrophic factor (CNTF), Axokine, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF I), insulin-like growth factor II (IGF II), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGF a), transforming growth factor β (TGF β), nerve growth factor (NGF), platelet derived growth factor (PDGF), glia-derived neurotrophic factor (GDNF), Midkine, phorbol 12-myristate 13 -acetate, tryophotin, activin, thyrotropin releasing hormone, interleukins, bone morphogenic protein, macrophage inflammatory proteins, heparin sulfate, amphiregulin, retinoic acid, tumor necrosis factor a, fibroblast growth factor receptor, epidermal growth factor receptor (EGFR), PEDF, LEDGF, NTN, Neublastin, neurotrophins, lymphokines, VEGF inhibitors, PDGF inhibitors, PIGF inhibitors, Tie2, CD55, C59, bispecific molecules the simultaneously bind VEGF and PDGF, and other agents expected to have therapeutically useful effects on potential target tissues. As used herein, one or more biologically active molecules can target one or more (1, 2, 3 or more) same or different specific sites/targets. In some embodiments, the biologically active molecules can be bi-specific molecules, where one delivered molecule could potentially target two distinct sites/receptors.

The terms "cartridge device" and "cartridge" and "NT-503-3" and "third generation ECT device" and "third generation NT-503 ECT device" and the like are used

interchangeably herein to refer to the ECT devices described herein.

The terms "second generation ECT device" and "NT-503 -2" and "single chamber device" and the like are used interchangeably herein to refer to the "traditional" one- chambered ECT devices that secrete anti-angiogenic antibody scaffolds or molecules that are known in the relevant art.

Unless otherwise specified, the term "cells" means cells in any form, including but not limited to cells retained in tissue, cell clusters, and individually isolated cells.

As used herein, the term "NTC-203-910 cells" and the like refer to parental NTC-200 cells engineered to produce soluble VEGF-receptor protein

As used herein, the term "encapsulated NTC cells" refer to any engineered derivation of the parental NTC-200 (ARPE-19) cells engineered to produce a therapeutic molecule (i.e., NTC-201-6A, which are NTC-200 cells engineered to produce high expression of ciliary neurotrophic factor).

As used herein, the term "encapsulated cells" and the like refer to any therapeutic cell lines capable of encapsulation and survival in a "biocompatible capsule" or biocompatible device".

As used herein a "biocompatible capsule" or "biocompatible device" or

"biocompatible vehicle" means that the capsule or device or vehicle, upon implantation in an individual, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.

As used herein an "immunoisolatory capsule" or "immunoprotective capsule" or "immunoisolatory device" or "immunoprotective device" or "immunoisolatory vehicle" or "immunoprotective vehicle" means that the capsule upon implantation into an individual, favorably partitions the device cellular contents and minimizes the deleterious effects of the host's immune system on the cells within its core.

As used herein "long-term, stable expression of a biologically active molecule" means the continued production of a biologically active molecule at a level sufficient to maintain its useful biological activity for periods greater than one month, for example greater than three months or greater than six months. Implants of the devices and the contents thereof are able to retain functionality for greater than three months in vivo and in many cases for longer than a year, and in some cases longer than two years or more.

The term "internal scaffold" is one example of a "matrix" that can be used in the devices described herein.

The "semi-permeable" nature of the jacket membrane surrounding the core permits molecules produced by the cells (e.g., metabolites, nutrients and/or therapeutic substances) to diffuse from the device into the surrounding host eye tissue, but is sufficiently impermeable to protect the cells in the core from detrimental immunological attack by the host.

The terms "encapsulated cell therapy" or "ECT" are used interchangeably herein to refer to any device capable of isolating cells from the recipient host's immune system by surrounding the cells with a semipermeable biocompatible material before implantation within the host. Those skilled in the art will recognize that in any of the devices, methods, and/or uses presented herein, elements of any ECT devices known in the art can be employed.

The term "treatment" or "treating" refers to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, and/or preventing disease in a subject who is free therefrom. For a given subject, improvement in a symptom, its worsening, regression, or progression may be determined by any objective or subjective measure. Efficacy of the treatment may be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve for a selected population). Thus, effective treatment would include therapy of existing disease, control of disease by slowing or stopping its progression, prevention of disease occurrence, reduction in the number or severity of symptoms, or a combination thereof. The effect may be shown in a controlled study using one or more statistically significant criteria. For example, in some embodiments, treatment refers to inhibiting endothelial cell proliferation or vascularization.

EXAMPLE

Example 1: Multi-chamber cartridge compared to single encapsulation chamber of equivalent volume

Retinal pigment epithelial cells engineered to produce a soluble VEGF antagonist were cultured and prepared at a density of 50,000 per microliter in a serum- free media. Cells were encapsulated in a cartridge device manufactured with seven chambers each 400 microns inner diameter and 50 micron wall thickness and total length of 8.5 mm. Cells were also encapsulated in a 1.3 mm single chamber device, 8.5 mm in length with an approximate equivalent volume to the chambered device. Both device groups were encapsulated with equivalent cell number, cell volume and rate of cell infusion. Devices were conditioned in culture media for 1 week prior to implant in eyes of New Zealand White rabbits. Cohorts were evaluated prior to implant to determine VEGF antagonist expression using ELISA specific for the soluble VEGF antagonist and viability of the encapsulated cells following histological processing (plastic embedding followed by H&E staining). Both encapsulation device designs resulted in equivalent in vitro release kinetics and cell viability. Expression of VEGF antagonist was between 4000 and 5000 ng/device/day while viability was between 4 and 5 over a scale range of 0 to 5 (0 = poor viability and distribution, 5 = excellent viability and distribution).

Retinal pigment epithelial cells engineered to produce a soluble VEGF antagonist were cultured and prepared at a density of 50,000 per microliter in a serum- free media. Cells were encapsulated in a cartridge device manufactured with seven chambers each 400 microns inner diameter and 50 micron wall thickness and total length of 8.5 mm. Cells were also encapsulated in a 1.3 mm single chamber device, 8.5 mm in length with an approximate equivalent volume to the chambered device. Both device groups were encapsulated with equivalent cell number, cell volume and rate of cell infusion. Devices were conditioned in culture media for 1 week prior to implant in eyes of New Zealand White rabbits. Cohorts were evaluated prior to implant to determine VEGF antagonist expression using ELISA specific for the soluble VEGF antagonist and viability of the encapsulated cells following histological processing (plastic embedding followed by H&E staining). Both encapsulation device designs resulted in equivalent in vitro release kinetics and cell viability. Expression of VEGF antagonist was between 4000 and 5000 ng/device/day while viability was between 4 and 5 over a scale range of 0 to 5 (0 = poor viability and distribution, 5 = excellent viability and distribution).

Both groups were implanted over the course of two-week in the rabbit eye, explanted and evaluated again for VEGF antagonist expression and encapsulated cell viability. The results at two-week demonstrated the superiority of the cartridge implant design compared to the single increased diameter design. Expression of VEGF antagonist at 2-week is found in Table 1. Table 1. Expression of VEGF antagonist at 2-week

Cell viability for the single chamber device following 2-weeks intraocular implant was 2.5 while the rating for the cartridge implant was 5.0. Representative histological examples of the 2-week explanted cells for the single chamber or multi-chamber cartridge implants are shown in Figures 5 and 6, respectively.

Example 2: Cell efficiency as a function of decreasing diffusion distance

Individual chambers with varying internal diameters were cell encapsulated at the following volumes: 3, 4, 6 and 10 microliters. Devices were evaluated prior to implant at again following both 2 and 4 week implant periods in the rabbit vitreous. Expression of VEGF antagonist and histology was assessed for each group at the pre-implant and explant periods. Expression levels were compared and an efficiency of VEGF antagonist expression as a function of cell number was determined.

Figure 7 shows the expression level of VEGF antagonist over time for each group. It is apparent that the greatest change in expression occurs from the chamber device with the greatest internal diameter. As the diameters decrease the change in expression of VEGF antagonist decreases. An efficiency of VEGF antagonist expression as a function of chamber inner diameter is shown in Figure 8.

Example 3: Cartridge shelf-life stability

Cartridge devices were manufactured with 5 individual chambers each having an internal diameter of 600 microns. Devices were encapsulated at various conditions of cell density and volume (B, C, D group as shown in Figure 9) with ARPE-19 cell engineered to express VEGF antagonist and compared to a single chamber device (i.e., the control group in Figure 9). Following encapsulation devices were placed in packages containing serum-free culture media and sealed. Device expression of VEGF antagonist was evaluated over time and the results are found in Figure 7. Cartridge expression of VEGF antagonist was approximately 3-4 fold greater than the control group at all time points and irrespective of the individual loading conditions. Cartridge expression remained stable over the evaluation period.

Example 4: 5-chamber cartridge device

NTC-203-910 cells were encapsulated in a 5-chamber cartridge device (Generation 3) at a cell density of 50,000 cells per microliter and maintained in 37 milliliters of culture media without exchange for 2 weeks. Expression of soluble VEGFR-receptor was evaluated in culture media at 2-weeks post encapsulation and then a cohort of devices were implanted bilaterally in the temporal inferior quadrant of New Zealand White rabbits and compared to implants of two single encapsulation devices (Generation 2) placed in the temporal and nasal inferior quadrant.

In this preliminary evaluation of a single cartridge geometry compared to two encapsulation implants, which had previously demonstrated functional and structural preservation of function in human wet AMD patients, all devices were explanted at 1 -month to determine potential clinical efficacy.

Explant device VEGFR expression and histology as well as accumulated vitreous levels of VEGFR from both implant groups were evaluated. The results of device explants and vitreous levels are found in Table 2 below. Explanted devices were histologically sectioned by performing radial 4 micron cuts from the distal end of the device to the proximal end and the cells stained with eosin and hemotoxylin. Single devices implanted two per eye resulted in poor cell viability and distribution of cells particularly demonstrating cell death at the core of each device compared to cartridge devices, which resulted in a good distribution and viability of cells both at the periphery and core of each individual chamber of the cartridge. Presumably, the reduced diffusion distance and greater surface to volume ratio of each individual chamber compared to the single implant devices was a main contributor to improved cell viability. Table 2. Implant VEGFR levels comparing Generation 3 5 -Cartridge and double Generation 2 devices

Example 5: Design Considerations and Performance of a Next-Generation

Encapsulated Cell Technology (ECT) Intraocular Implant Delivering Soluble VEGF- Receptor

Objective

Early generation ECT products delivering soluble VEGF-receptor (VEGF- R) (e.g., single chambered ECT devices) for over 12 months have demonstrated clinically meaningful improvements in BCVA and reductions in macular thickening in patients with active neovascular AMD. Higher dose levels are expected to achieve efficacy comparable or greater than standard-of-care treatments.

A new ECT device, NT-503-3, incorporating multiple (e.g., more than 1) optimized cell encapsulation chambers into a single cartridge implant was designed to substantially increase VEGF-R by increasing the total number of encapsulated cells, and by improving cell viability and protein expression efficiency. This design also supports combination therapy from a single device by allowing discrete encapsulations of different therapeutic cell lines in a single intraocular implant product. A schematic of the NT-503-3 (Generation 3) Multi- Chamber Implant is shown in Figure 10.

Methods and Materials

The performance of NT-503-3 was evaluated following encapsulation of a human RPE cell line transfected to produce VEGF-R. Dose levels of VEGF-R were characterized by ELISA. Binding efficiency and bioactivity were quantified by a VEGF -binding and HUVEC assay, respectively. GLP toxicology studies, which include clinical examination, ERG, IOP, ocular histopathology, clinical chemistry and detection of serum antibodies to VEGF-R and the encapsulated cell line are ongoing. Results

A single NT-503-3 implant increased VEGF-R dose 5-fold compared to the previous ECT single implant (NT-503-2), which had demonstrated clinical efficacy in wet-AMD patients when implanted with two devices. VEGF-R produced by NT-503-3 results in high binding affinity to VEGF with a Kd of 0.7 pM and inhibits VEGF with an IC50 of 20-30 pM. Intraocular implants investigated in rabbits through 3-months of a 9-month GLP toxicology study demonstrate that the NT-503-3 product is safe and well tolerated.

In Figure 11, VEGF binding of ECT produced VEGF-R was evaluated by the ability to neutralize recombinant human VEGF following co-incubation and detection of remaining free (unbound) VEGF using ELISA assay. Inhibitory activity of both purified VEGF-R protein and ECT device secreted VEGF-R (conditioned media, CM) indicate IC50 = 12 pM compared to Lucentis drug with IC50 = 250 pM. In a direct comparison to Lucentis, ECT secreted VEGF-R demonstrates a 20-fold increase in binding neutralization of human VEGF.

Figure 12 shows measurement of the ability of the ECT produced VEGF-R to neutralize the bioactivity of rhVEGF on human umbilical vein endothelial cell proliferation. HUVEC cells were incubated with various concentrations of ECT produced VEGF-R and compared to supernatant of non-transfected parental cells. The EC50 for the ECT produced VEGF R was approximately 40-50 pM with complete inhibition observed at 100 pM.

The efficacy observed following intraocular delivery of VEGF-R over the course of 12 months in human patients is shown in Figures 13A-B. A representative histologic section of encapsulated NT-503 cells arranged in a 5-chamber cartridge format is shown in Figures 14A-B.

Table 3 shows the ocular examination results following scheduled 6-month evaluation.

Table 3

Aqueous Lens:

Lens: Vitreous Haze Vitreous Ce8

Toxicsiogy Group Rare Ce« Catara Iniiarorosiory itmce - 4) {tee - 4-=

(O S - 4÷) Gels Posterior

0/8 m 0/8 Q/8 fmpianl 0/8 m 0 s 1 6 CN-)

Empty NT-5G3-3 Iropfarsi m m m m

Injected T-5Q3-3 ce«s ^■ m m 0/6 Compared to naive controls, no changes in body weights, body temperature, IOP, ERG, hematology, clinical chemistry parameters, organ weight or macroscopic changes were attributable to NT-503-3 single implant with or without encapsulated cells or NT-503-3 cells directly injected into the eye. No increases in antibody titers to the cell line or to VEGF-R have been detected in any group.

Conclusions

Clinically relevant VEGF-R expression and a safe toxicology profile have been achieved with the NT-503-3 design. A single, intraocular NT-503-3 implant is expected to provide equivalent or improved efficacy compared to standard-of- care therapy while eliminating the burden of frequent injections in patients with active neovascular AMD. The mu!ti-piatform cartridge design also supports ongoing combination therapy development.

Devices and hold media (Endo-SFM) from three lots of NT-503-3 Investigational Cartridge Product were tested by USP <85> for endotoxin inhibition/enhancement to qualify the product for endotoxin testing by the Gel-Clot method. Three devices from each of 3 manufactured lots were divided in half and placed into 10-mT , of endotoxin-free water (limulus amoebocyte lysate reagent water or LRW) for 1 hour at 37"C prior to testing. The water sample from the device was tested undiluted. The hold media From each device was tested directly, undiluted.

Each device and hold media sample was tested "as is" and also spiked to an endotoxin concentration of approximately 0-03 EU/mL. Each sample was titered to endpoint along with the spiked water control, An endotoxin value between 0.016 and 0,063 EU/mL indicates no inhibition or enhancement of the assay by the sample.

When NT-503-3 Investigational Product arid hold media were spiked with endotoxin control standard at the assay limit of detection, no enhancement or inhibition of the assay results were observed. The results from the devices are summarized below in Table 4 and the results for the corresponding device hold media (Endo-SFM) are found in Table 5.

Table 4 Endotoxin inhibition/enhancement qualification results for Cartridge device lots

VD-031814, P07-14-014DEV, and D07-14-015DEV

Table 5 Endotoxin inhibition/enhancement qualification results for hold media (Endo-SFM) from Cartridge device lots VD-031814, D07-14-014DEV, and D07-14-015DEV

Example 6: NT503 Analytical Results Including Establishment of Product Purity Specifications from Three Consecutive Manufacturing Lots

Preliminary product purity was established for all detectable proteins continuously secreted and analyzed over a 24 hour period from NT-503 cartridge devices. Proteomic mass spectroscopy analysis was used to evaluate the protein profile of 20 device samples from 2 engineering and 3 consistency lots establishing repeatable detection of the most abundant proteins, quantifying NT-503 VEGFR plus 49 additional proteins. A direct mass purity index of cell produced proteins ratio to total proteins was established and a Pearson's moment correlation of all detected proteins based upon the relative percentage of each individual protein mass to total mass established & separate standard purity profile reference, A combination of analysis specifications including a mass purity index plus a total protein correlation provides a complementary product profile for purity evaluation. Product acceptance specifications require manufactured clinical lot samples to demonstrate both a purity index relating NT-503 produced protein to all pre-defined proteins detected > 70% and a coefficient of determination, or R ² , greater than 0.70 correlations for all pre-defined 50 proteins in each tested sample compared to the reference standard. Acceptance criteria for both specifications for all samples from three subsequent manufacture lots of Generation 3 Cartridge devices meet or exceed the lower limit of specification for purity. Included in Table 1 are the results for EL1SA VEGFR release as well as metabolic cell activity expressed absorbance of converted tetrazolium salt to a formazen dye (CC -8 assay).

The majority of proteins other than the product NT-503 VEGFR are ubiquitous cytoskeleton, extracellular matrix or metabolic uma proteins consistent with hRPE and hRPE- 1 proteome MS profile.

Figure 15 shows protein profile secretion of ubiquitous proteins from multiple lots of Generation 3 cartridge devices. Table 6 shows analytical test results from three consecutive NT-503 cartridge device manufacturing lots.

Table 6

Example 7: Shelf Life and Shipping Qualification of NT-503-3 Implants

Purpose

The purpose of this study was to verify performance of Generation 3 NT-503 (NT- 503-3) devices following exposure to temperature ranges and durations representative of qualified shipping conditions. NT-503-3 devices contain VEGFR secreting cell line NTC- 203-910 encapsulated in 5 individual hollow fiber chambers assembled as a cartridge, and held in primary packages containing Endo-SFM culture media.

In vitro performance was evaluated weekly over the course of 6 weeks for devices held at standard 37°C incubation. Additionally, an extension of product shipment period from standard 1 week to 2 weeks was evaluated at temperature ranges between 16°C and

37°C representing potential clinical shipment using a qualified shipping system. Verification of shelf-life and shipment period stability was established for NT-503-3 devices following bilateral intraocular implants for 1 month in New Zealand white (NZW) rabbits. Summary

NT-503-3 implants were manufactured under development protocol for the evaluation of device expression and cell viability over the course of a 6 week shelf life period (post manufacture). Three lots of implants were manufactured utilizing the NTC-203 cell line, VEGFR-910(834- 10-5)-4-47, and sterile Generation 3 cartridge devices.

A critical component of the current study was the ability of NT-503-3 implants to

"recover" when removed from the packaging and placed into fresh Endo-SFM. It is characteristic for the NT-503-3 packaging system (primary jar with Endo), that expression of VEGFR-910 reduces over time as the environment within the jar becomes metabolized. This reduction in 910 has proven to be transient though, whereby aged devices will demonstrate a robust recovery in expression once returned to a fresh media environment. The ability of all devices at all time points, and at all temperatures, to recover in their expression of 910 provides support that when implanted, the NT-503-3 product will perform as intended.

Surprisingly the decline in VEGFR expression over time was less acute than that recorded previously with single Generation 2 devices over a similar shelf-life period. The percentage loss of VEGFR for the Cartridge devices was 84% at 6 weeks relative to the initial VEGFR levels compared to a percentage loss of 97% for Generation 2. The slower decline in VEGFR is likely a function of the improvement in the design and the increased surface to volume ratio and decreased diffusion distance of cells to a nutrient source due to the thinner individual chambers of the cartridge compared to the single wider Generation 2 device.

In addition, the current study evaluated NT-503-3 devices in vivo to further confirm optimal performance even when implanted at the end of product shelf-life, or after exposure to the limits of shipping temperatures. Following 4 and 6 week holds in primary packaging, at all temperatures tested in the range of 16-37°C, NT-503-3 devices were implanted in New Zealand White (NZW) rabbits for 1 month. At the 1 month time point, all animals were examined by a veterinary ophthalmologist, and all devices and eyes were sampled for their concentrations of VEGFR-910.

For both the in vitro and in vivo arms of the current study, NT-503-3 implants demonstrated the ability to express optimal levels of 910 when packaged for up to 6 weeks. Furthermore, the data confirmed that shipment for up to 2 weeks, within a maintained temperature range of 16-37°C, is acceptable for NT-503-3 implants. Key results in support of those conclusions include:

• The 37°C control arm of the current study met the NT-503-3 1 week release specification for VEGFR-910 expression.

• All NT-503-3 devices at all time points, and for all simulated shipping groups, demonstrated the ability to recover their expression of 910 once removed from packaging and returned to a fresh media environment. The down-regulation of 910 production is a known, transient phenomenon due to the gradual metabolization of packaging nutrients.

• The percentage of decline in VEGFR over a shelf-life period for the

Generation 3 cartridge device was 84% compared to a more severe decline of 97% for the earlier Generation 2 single device design.

• NT-503-3 devices implanted after 4 and 6 weeks post-manufacture, whether held at 37°C or in simulated shipping conditions, exhibited comparable levels of 910 in the vitreous and explant sampling confirming that no loss in functionality occurs over the course of the proposed NT-503-3 product shelf life.

• At all shelf-life and shipment conditions tested in the current study the subsequent 1 -month device explant and vitreous levels of VEGFR protein as well as encapsulated cell viability were equivalent or exceeded the 1 -month levels demonstrated in all previous NT-503 device evaluations.

The results of the Generation 2 and Generation 3 (i.e., NT-503-3) shelf life comparison are shown in Figure 16. NT-503-3 cartridge shelf life and recovery profiles are shown in Figure 17.

Figure 18 shows NT-503-3 explant expression and corresponding vitreous concentrations following 4 week hold in packaging and 1 month implantation in rabbits, and Figure 19 shows NT-503-3 explant expression and corresponding vitreous concentrations following 6 week hold in packaging and 1 month implantation in rabbits. Example 8: Methods for optimal cell filling of a cartridge device

Cell filling (encapsulation) of cartridges composed of 5 individual chambers and presumably any composition greater than 2 chambers requires a degassing and pre-wetting stage to ensure optimal distribution of cell mass within all chambers of the cartridge. Cell encapsulation without this step results in unacceptable variability of cell filling in all chambers and potentially no filling in some chambers.

Those skilled in the art will recognize that the sub-optimal cell-filling phenomenon may be explained by reference to the Laplace equation and bubble point method used to determine pore size of a membrane. The bubble point method utilizes the surface tension at the liquid-air interface of the membrane surface as means to prevent passage of air from the inside to outside of the membrane at a given pressure. Eventual penetration at increased pressure allows for pore size determination using the Laplace relationship.

The use of a system to equilibrate filling of all chambers of a cartridge is critical to develop and employ in the multi-chamber filling process for ECT cartridge devices.

Figure 21 shows representative examples of cartridge devices without degassing/pre- wetting procedure.

The solution to ensure all chambers were be equally filled and distributed with encapsulated cells was to evacuate all air from the internal volume of the device and filling port and, importantly, completely fill all surfaces, particularly each chamber membrane surface and interconnecting pores with a wetting liquid. The present devices utilized an enclosed aseptic vacuum system that removes entrapped air in a pre-filled multi-chamber ECT device at a vacuum range of between 18 and 29.5 in Hg-gauge but preferably at 28.5 in Hg-gauge. Following vacuum degassing, the inner surfaces of the entire cartridge and filling system are completely filled with a liquid such as Hanks Balanced Salt Solution or other isotonic solution (saline, DMEM, etc.) for a defined period of vacuum immediately following termination of the degassing stage but just prior to development of subsequent liquid boiling.

Following degassing and liquid filling of the cartridge and cell-filling system, the cartridge device can be successfully loaded with cells per established encapsulation methods.

Figure 22 shows representative examples of cartridge devices following

implementation of a degassing/pre-wetting step. Example 9: ECT Cartridge Devices Use to Evaluate a Combined PDGFR and VEGFR Product

Separate cell lines expressing VEGFR (NTC-203-910) and those expressing PDGFR (NTC-206-999) were individually formulated at densities of 50,000 cells/microliter and encapsulated as individual cell lines in a Generation 3 cartridge device or encapsulated as a mixed suspension in a 50:50 ratio. Generation 3 devices with either VEFGFR secreting cells alone, PDGFR alone or as a combination were maintained in Endothelial SFM culture media in a closed package for 2 weeks. Levels of protein from each device group were evaluated at 2 weeks. VEGFR and PDGFR protein expression was within the expected range of between 10 and 18 micrograms per day.

Interestingly and unexpectedly the ratio of the combined VEGFR/PDGFR cell lines encapsulated in the Generation 3 cartridge device did not significantly vary and continued to maintain a 50:50 protein secretion ratio consistent with the initial cell loading ratio. (See Figure 23).

EQUIVALENTS

The details of one or more embodiments of the invention are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated by reference.

The foregoing description has been presented only for the purposes of illustration and is not intended to limit the invention to the precise form disclosed, but by the claims appended hereto.