US009102763B2
(12) United States Patent (10) Patent No.: US 9,102,763 B2 MacKay et al. (45) Date of Patent: Aug. 11, 2015
(54) CONTROLLED RELEASE OF OCULAR 2009,028O190 A1 11/2009 Folkman et al. BOPHARMACEUTICALS USING 2010/0233095 A1 9/2010 Duan et al. 2010. 0233242 A1 9/2010 Tirrell et al. BORESPONSIVE PROTEIN POLYMERS 2011/OO39776 A1 2/2011 Chilkoti (75) Inventors: John Andrew MacKay, Pasadena, CA (US); Wan Wang, Los Angeles, CA FOREIGN PATENT DOCUMENTS (US) WO 2013O16578 A2 1, 2013 (73) Assignee: University of Southern California, Los OTHER PUBLICATIONS Angeles, CA (US) MacEwan et al., Elastin-Like Polypeptides: Biomedical Applications (*) Notice: Subject to any disclaimer, the term of this of Tunable Biopolymers, Jan. 20, 2010, Peptide Science 94(1):60 patent is extended or adjusted under 35 77.3 U.S.C. 154(b) by 0 days. Samudre et al., Lacritin, a Novel Human Tear Glycoprotein, Pro motes Sustained Basal Tearing and Is Well Tolerated, Aug. 2011, (21) Appl. No.: 13/559,053 Investigative Ophthalmology & Visual Science 52(9):6265-6270.* PCT/US2012/048404 International Search Report dated Jan. 22, (22) Filed: Jul. 26, 2012 2013. PCT/US2012/048404 Written Opinion dated Jan. 22, 2013. (65) Prior Publication Data Floss et al. Elastin-like polypeptides revolutionize recombinant pro tein expression and their biomedical application. Trends in US 2013/O196926A1 Aug. 1, 2013 Biotechnology (2010). 28(1): 37-45. Shamji et al. Treatment of neuroinflammation by soluble tumor Related U.S. Application Data necrosis factor receptor Type II fused to a thermally responsive carrier: Laboratory Investifgation. J Neurosurg Spine (2008). (60) Provisional application No. 61/511,928, filed on Jul. 9(2): 221-228. 26, 2011. * cited by examiner (51) Int. Cl. A6 IK 47/48 (2006.01) Primary Examiner – John Ulm C07K 4/78 (2006.01) (74) Attorney, Agent, or Firm — Stephen W. Chen; Nixon A6 IK38/8 (2006.01) Peabody LLP C07K I4/47 (2006.01) A6 IK9/00 (2006.01) (57) ABSTRACT CO7K I 4/475 (2006.01) Described herein are bioresponsive protein polymers for (52) U.S. Cl. therapeutic applications, including delivery to physiologi CPC ...... C07K 14/78 (2013.01); A61 K9/0048 cally demanding environments, such as the eye surface. Bio (2013.01); A61 K38/18 (2013.01); A61 K responsive protein polymers can be fused with biopharma 47/48292 (2013.01); A61K 47/48338 (2013.01); ceuticals using genetic engineering techniques for enhanced C07K 14/47 (2013.01); C07K 14/475 therapeutic activity. In certain embodiments, the unique tem (2013.01); C07K 2319/00 (2013.01) perature-sensitive phase separation properties of biorespon (58) Field of Classification Search sive protein polymers, allows generation of therapeutics None resistant to ocular clearance. Such fusion proteins containing See application file for complete search history. bioresponsive protein polymers and biopharmaceuticals allow retention of drugs in the eye for much longer periods of (56) References Cited time. Improved biostability and bioavailability improves drug efficacy, while reducing cost and eliminating the need for U.S. PATENT DOCUMENTS repeated drug Application. 2004/0081984 A1* 4/2004 Laurie et al...... 435/6 2007/02O7522 A1 9, 2007 Laurie et al. 15 Claims, 23 Drawing Sheets U.S. Patent Aug. 11, 2015 Sheet 1 of 23 US 9,102,763 B2
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9|,eun61-I 3 US 9,102,763 B2 1. 2 CONTROLLED RELEASE OF OCULAR strategy allows for retention of drugs in the eye for much BOPHARMACEUTICALS USING longer periods of time, on the order of days to weeks, thereby BORESPONSIVE PROTEIN POLYMERS improving drug efficacy, while reducing cost and eliminating the need for repeated drug application. CROSS REFERENCE TO RELATED APPLICATIONS SUMMARY OF THE INVENTION This application includes a claim of priority under 35 The present invention provides, in one embodiment, an U.S.C. S 119(e) to U.S. Patent Application No. 61/51 1,928, isolated fusion protein including a bioresponse protein poly filed Jul. 26, 2011. 10 mer, and a therapeutic protein conjugated to the bioresponse protein polymer. In another embodiment, the bioresponse GOVERNMENT RIGHTS protein polymer is an elastin-like polypeptide (ELP). In another embodiment, the ELP includes amino acid motif This invention was made with government Support under (Val-Pro-Gly-X-Gly), (i.e., n number of SEQID NO.19), Contract No. R21EB012281-01 awarded by the National 15 where n includes 10 to 300 units and X is a natural or Institutes of Health. The government has certain rights in the synthetic amino acid. In another embodiment, n is 96 and X, invention. is serine, Valine, or isoleucine. In another embodiment, thera peutic protein is lacritin, a functional equivalent or active FIELD OF INVENTION fragment thereof. In another embodiment, lacritin, functional equivalentor active fragment thereof includes human lacritin. This invention relates to the use of protein polymers to In another embodiment, the lacritin, functional equivalent or improve drug delivery to various organs, particularly the eye, active fragment thereof includes amino acid sequence: SEQ and other related uses. IDNO:3, SEQIDNO:4, SEQID NO:5, SEQID NO:6, SEQ ID NO: 7, or SEQ ID NO: 8. In another embodiment, the BACKGROUND 25 bioresponse protein polymer and therapeutic protein are con jugated via a linker peptide. In another embodiment, the All publications herein are incorporated by reference to the linker peptide includes amino acid sequence: SEQID NO: 9. same extent as if each individual publication or patent appli In another embodiment, the bioresponse protein polymer is cation was specifically and individually indicated to be incor ELP, the therapeutic protein is lacritin, and the ELP is conju porated by reference. The following description includes 30 gated to the lacritin via a linker peptide. In another embodi information that may be useful in understanding the present ment, the fusion protein includes an ELP including amino invention. It is not an admission that any of the information acid motif (Val-Pro-Gly-X-Gly), (i.e., n number of SEQ provided herein is prior art or relevant to the presently ID NO.19), n is 96 and X is valine, conjugated to a lacritin, claimed invention, or that any publication specifically or functional equivalent or active fragment thereof including implicitly referenced is prior art. 35 amino acid sequence: SEQID NO:3, SEQID NO: 4, SEQID Affecting over 3.2 million Americans, dry eye syndrome is NO: 5, SEQID NO: 6, SEQID NO: 7, or SEQID NO: 8, via a common disorder of the tear film characterized by a linker peptide including SEQID NO:9. decreased tear production. This disease is prevalent among Another aspect of the present invention provides, in one the elderly and is particularly common in postmenopausal embodiment, an isolated nucleotide encoding a fusion protein women. Typically, dry eye is treated using conventional drops 40 including a bioresponse protein polymer, and a therapeutic containing Small molecule drugs, although recombinant tear protein conjugated to the bioresponse protein polymer. In protein rescue for dry eye syndrome has become a possibility another embodiment, the bioresponse protein polymer with the discovery of a novel human tear glycoprotein, lac includes an elastin-like polypeptide (ELP). In another ritin. Lacritin is capable of promoting basal tear peroxidase embodiment, the ELP includes amino acid motif (Val-Pro secretion by rat lacrimalacinar cells in vitro, basal tear secre 45 Gly-X-Gly), (i.e., n number of SEQID NO. 19), where n tion by rabbit in vivo and possibly triggers downstream sig includes 10 to 300 repeat units and X is a natural or syn naling pathway through tyrosine phosphorylation and cal thetic amino acid. In another embodiment, the therapeutic cium release. However, this option has found limited practical protein includes lacritin, a functional equivalent or active application as tears wash drugs away from the eye within fragment thereof. In another embodiment, the isolated nucle minutes, and less than 2% of the medication is absorbed. As 50 otide of is constructed using recursive directional ligation. In tears rapidly wash away both Small and large molecule drugs, another embodiment, the isolated nucleotide encodes for a this has prevented the development of numerous protein fusion protein including an ELP including amino acid motif based drugs. By contrast, protein drugs administered to other (Val-Pro-Gly-X-Gly), (i.e., n number of SEQID NO.19), sites in the body have continued to be developed into effective n is 96 and X is valine, conjugated to a lacritin, functional therapies. Thus, there is a clear unmet need to develop effec 55 equivalent or active fragment thereof including amino acid tive delivery strategies that for administration and retention of sequence: SEQ ID NO:3, SEQ ID NO: 4, SEQID NO: 5, biopharmaceuticals in a target organ, Such as the Surface of SEQID NO: 6, SEQID NO:7, or SEQID NO: 8, via a linker the eye. peptide including SEQID NO:9. Accordingly, the inventive compositions and methods dis Another aspect of the present invention provides, in one closed herein establish new and improved techniques for 60 embodiment, a method of treating a disease and/or condition improving drug delivery to organs that are presently limited in a human Subject, include providing a quantity of a compo by biochemical and biomechanical environments due to pH, sition, wherein the composition includes a fusion protein, the temperature, hydrodynamic flow, mechanical/structure fea fusion protein including a bioresponse protein polymer and a tures, among others. To overcome these obstacles, the inven therapeutic protein conjugated to the bioresponse protein tors have developed temperature sensitive protein polymers 65 polymer; and treating a human Subject by administering a and fused these polymers directly to a biopharmaceutical therapeutically effective dosage of the composition to the with enhanced therapeutic activity at the eye surface. This Subject, thereby treating the Subject. In another embodiment, US 9,102,763 B2 3 4 the human Subject is in need of treatment for an eye disease one sharp phase transition at 18.7°C. *m/Z (Expected M.W.) and/or condition selected from the group consisting of acan is calculated by DNAStar Lasergene Editseq; M+H+(Ob thamoeba keratitis, allergies, amblyopia, Bell’s palsy, ble served M.W.) is measured by MALDI-TOF: *T, is character pharitis, cataracts, chalazion, color blindness, corneal ulcer, ized at 25uM in PBS. detached retina, dry eye syndrome, keratoconjunctivitis FIG.3. Construction of bacterial expression vector encod sicca, eye occlusions, eye twitching, macular hole, nystag ing for a lacritin ELP fusion protein. (A): A gene encoding the mus, ocular migraine, ocular rosacea, optic neuritis, optic lacritin between the Ndel and BamHI sites was ligated into a neuropathy, photophobia, pinguecula, pterygium, ptosis, pET25B(+) vector. A technique used by our group called Sjogren's syndrome, Strabismus, stye, Subconjunctival hem Recursive Directional Ligation was used to create ELP genes orrhage, uveitis, CMV retinitis, conjunctivitis, diabetic retin 10 that can be extracted by cleavage at a BseRI and the BamHI opathy, eye herpes, glaucoma, karatoconus, macular degen restriction site. Fusion of the appropriate digested vectors, eration, macular dystrophy, ocular hypertension, retinitis yields a bacterial expression plasmid that encodes for an in pigmentosa, and/or Stargardt’s disease. In another embodi frame fusion protein consisting of an amino terminal lacritin ment, the bioresponse protein polymer includes an elastin and a carboxy terminal ELP. The two domains are linked by a like polypeptide (ELP), the therapeutic protein includes lac 15 thrombin cleavage recognition site to enable proteolytic ritin, and the ELP is conjugated to the lacritin via a linker cleavage and purification of the free lacritin. (B): Size exclu peptide. sion chromatograph of Lac-V96 purification. Another aspect of the present invention provides, in one FIG. 4. Purity of a lacritin-ELP fusion protein and mass embodiment, a pharmaceutical composition including a bio spectrometry analysis. (A): SDS PAGE of copper chloride response protein polymer, atherapeutic protein conjugated to stained purified lacritin-ELP fusion protein, Lac-V96. Also the bioresponse protein polymer, and a pharmaceutically included are purified ELP alone, V96, and the purified lacritin acceptable carrier. In another embodiment, the bioresponse protein (Lac). (B): Matrix assisted laser desorption ion time protein polymer includes an elastin-like polypeptide (ELP). of flight (MALDITOF) was used to confirm the exact masses the therapeutic protein includes lacritin, and the ELP is con of the ELP and Lac-V96 fusion proteins. Results of this study jugated to the lacritin via a linker peptide. 25 are indicated in Table 3; however, these results demonstrate Another aspect of the present invention provides, in one that the correct protein has been expressed and purified using embodiment, a a method of using an ELP in a purification our methods. Lac-ELP fusion gene was biosynthesized using process, including: a) providing a sample including a ELP pET25b(+) vector. After expression in BLR(DE3) cells, ELPs construct, b) inducing phase transition in the sample by add and Lac-ELP fusion proteins were purified using Inverse ing 0 to 20 MNaCl and heating to temperatures up to about 30 phase transition cycling (ITC) and size exclusion chromatog 10, 20, 30, 35, 37, 40, or 45°C., b) centrifuging the sample at raphy. Free lacritin was released by thrombin cleavage. 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 g, c) discarding FIG.5. Further characterization of purity of a Lacririn-ELP the Supernatant, and d) cooling remaining pelletto about, 1,2, fusion protein and mass spectrometry analysis. (A): Another 3, 4, 5, or 6-10° C. SDS PAGE of copper chloride stained purified lacritin-ELP 35 fusion protein. Lanes from L to Rare 1.7: Marker. 2: Lac-V96 BRIEF DESCRIPTION OF THE FIGURES After ITC purification, 3: Lac-V96 After size exclusion col umn purification, 4: V96 After ITC purification, 5: Lac. After Exemplary embodiments are illustrated in referenced fig thrombin cleavage Before hot spin, 6: Lac. After thrombin ures. It is intended that the embodiments and figures disclosed cleavage After hot spin. (B): Another MALDITOF confirm herein are to be considered illustrative rather than restrictive. 40 ing the exact masses of the ELP and Lac-V96 fusion proteins, FIG. 1 ELP protein polymers. (A): Cartoon showing Lac demonstrating correct expression of the protein purified using ELP fusion protein stimulates primary rabbit lacrimal gland our methods. acinar cells (LGACs) secretion. (B): Elastin-like polypep FIG. 6. Phase diagrams for lacritin ELP fusion proteins. tides (ELPs) phase transition. (C): Cartoon showing drug The fusion protein between lacritin and the ELPV96 displays delivery to the eye via topical administration or lacrimal 45 ELP phase transition behavior. (A): T, characterization of gland injection. V96. (B): T characterization of Lac-V96. (C): Concentration FIG. 2. Examples of Lac-ELPs. (A): Cartoon showing dependent T, of V96 and Lac-V96. (D): Above the line structure of a typical Lac-ELP fusion protein. Lacritin and depicted for Lac-V96, the fusion protein undergoes phase ELP tag are at the N and C terminus of the fusion construct separation, around 30°C. This fusion construct would there accordingly. A thrombin recognition site (GLVPRIGS) is 50 fore be soluble at room temperature and undergo phase sepa designed between the two moieties for releasing free lacritin. ration at the temperature of the ocular surface, which is >32 Four types of ELP tags are chosen: I96 SEQID NO: 11, V96 C. The fusion of lacritin has a detectable, but minimal effect SEQID NO: 17, S96 SEQID NO: 12 and S48 I48 (SEQ on the transition temperature compared to unmodified ELP. ID NO: 13). (B): Sequence, M.W. and Phase Transition Tem FIG. 7. Lacritin undergoes degradation at 37°C. (A): Time perature (T,) characterization of Lac-ELP library. At T
*Molecular weight estimated for expressed gene product, as confirmed using MALDI-TOF mass spectrometry ** Assembly temperature determined using optical density at 350 nm on a temperature gradient of 1° C. min ***Radii determined using dynamic light scattering at 25uMELP in phosphate buffered saline.
TABL E 2
Lacritin-ELP constructs evaluated
Expected Protein Approximate *T, ocular Name * * lacritin-ELP amino acid sequence MW (kD) (° C.) clearance
Lac-V96 MEDASSDSTGADPAOEAGTSKPNEEISGPAEPASPPETTTTAOETSAA 52.5 26.8 slow SEO ID AWOGTAKWTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPG NO: 14 GKQFIENGSEFAQKLLKKFSLLKPWA-GLVPRGS- GVPGVGoY
Lac-I96 MEDASSDSTGADPAOEAGTSKPNEEISGPAEPASPPETTTTAOETSAA SO 10 fast SEO ID AWOGTAKWTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPG NO: 15 GKQFIENGSEFAQKLLKKFSLLKPWA-GLVPRIGS- GVPGIGY
Lac-S96 MEDASSDSTGADPAOEAGTSKPNEEISGPAEPASPPETTTTAOETSAA SO 6 O fast SEO ID AWOGTAKWTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPG NO: 16 GKQFIENGSEFAQKLLKKFSLLKPWA-GLVPRGS- GVPGSGoY
* As observed for Lac V96 and expected based on the approximate transition temperature for I96 and S96. ** Amino-terminal lacritin with the Signal peptide removed followed by an underlined thrombin Cleavage site, followed by a carboxy terminal ELP. US 9,102,763 B2 15 16 Example 2 temperature can be induced using a mild combination of 0 to 3 MNaCl and heating to temperatures up to about 37°C. This Characterization of Lacritin-ELP Conjugate causes the phase separation of ELPs, which are centrifuged Assembly and Bioactivity under about 10,000 g. The supernatant, which includes bac terial contaminants, is discarded. Next, the ELP pellet is The inventors cloned (FIG. 3A) and purified lacritin-ELP cooled to 4°C. and agitated gently until it returns to solution. conjugates (Table 2). One of these conjugates, Lac-V96, has The redissolved ELP is again centrifuged to discard any been extensively characterized through proteolytic cleavage remaining protein contaminants. As needed, this cycle of hot via thrombin. A purified ELP fusion proteins (Lac-V96), a and cold centrifugation is repeated between 3 and 6 times to purified ELP (V96), and the purified cleaved lacritin (Lac) are 10 produce pure fusion constructs. all depicted (FIG. 4A, 5A). The molecular mass for these constructs was confirmed using mass spectrometry (FIG. 4B, Example 5 5B, Table 3). The ELP lacritin fusion protein Lac-V96 was able to undergo temperature dependent phase separation Lac-ELPs and ELPs are Purified by Inverse (FIG. 6); furthermore, Lac-V96 is soluble at room tempera 15 Transition Cycling (ITC) ture and phase separates above 30° C. More importantly, the inventors have applied this lacritin fusion protein to Success In another example of the versatility of bioresponse poly fully induce expression of an enzyme marker associated with mers, ELPs can used as purification tag to extract soluble secretion, beta hexosaminidase, in primary cells isolated fusion proteins from E. coli debris due to their unique revers from rabbit lacrimal gland (LGACs). ible inverse phase transition behavior. Lacritin has been pre viously purified using intein system, which requires multiple Example 3 steps of chromatography. The inventors herein describe an alternative purification approach using ELPs. With IPTG Constructing Genes Encoding for Lacritin-ELPs induction, Lac-I96, Lac-V96, Lac-S96 and Lac-S4848 can 25 achieve satisfactory yield of more than 40 mg/L. MALDI Genetic engineering can be used to prepare repetitive TOF analysis and western blotting with anti-lacritin antisera polypeptides of specific chain length. In this approach, oli further confirmed successful construction of Lac-ELPs (Fig gonucleotide cassettes encoding the monomer gene for the ure FIG.15C). Interestingly, after ITC purification, the inven pentapeptide ELP is ligated into a specially modified cloning tors observed two major bands on SDS-PAGE of purified vector, Such as pet25b+. Restriction enzymes are selected 30 Lac-ELPs (FIG. 5A), upper band around 52 kD and lower RE1 and RE2, which cleave the monomer genes as demon band around 40 kD, which Suggested there was pre-mature strated in FIG. 10B. The cloning vector with the monomer cleavage of lacritin from the fusion construct. Further degra gene is digested with both RE1 and RE2 to produce an insert, dation study confirmed this observation (FIG. 16A). The which is gel purified and ligated into a separate preparation of inventors utilized Superose size exclusion column to remove vector linearized by digestion with only RE1. This approach 35 free ELP tags (FIG. 3B). Internal lacritin control was liber leads to the controlled dimerization of the synthetic gene. ated from ELP tag via cleavage at designed thrombin recog This technique can be repeated until an ELP of desired chain nition site encoded between the two moieties (FIGS. 1A and length is formed. After formation of a library of synthetic 2A). Similar to previous reports, lacritin ran higher on SDS genes encoding ELPs of different lengths and guest residues PAGE than expected M.W. 12 kD (FIGS. 4A and 5A). (X), selected ELPs are transferred onto a plasmid at an RE1 40 Using above method, Lac-ELPs, ELPs and lacritin can cut site to the carboxy terminus of a lacritingene (FIG. 10B). reach more than 95% purity on SDS-PAGE stained with Following every ligation, bacterial colonies are grown up in a coomassie blue (FIGS. 4A and 5A) The inventors also noticed 4 mL culture and harvested for plasmid DNA. The DNA is several smaller MW bands (around 10 kD) in purified lac then screened by diagnostic digestion using Xbaland BamHI ELP and lacritin products (FIGS. 4A, 5A, and 8C). Analysis for the correct insertion of both the lacritin and ELP genes. 45 of MALDI-TOF data indicated that these difference size frag Positive plasmids are then sent for DNA sequencing from the ments released from lac-ELPs are multiple sites within the T7 promoter and terminator sequences, which is used to lacritin-thrombin region of the fusion protein may be suscep confirm the presence of a ribosome binding sequence, a start tible to enzymatic attack by proteases or act as a protease codon, an inframe lacritingene, an in frame thrombin cleav itself (FIGS. 16D&E). age site, an inframe ELP, and an in frame stop codon. Suc 50 More specifically, ELPs I96, V96, S96 and S48I48 were cessfully sequenced plasmids are then moved to expression expressed in BLR(DE3) E. coli cells (Novagen Inc., Milwau bacterial cultures. kee, Wis.). Briefly, after overnight start culture, protein was expressed for 24h in an orbital shaker at 37° C. at 250 rpm. Example 4 Cell culture were harvested and re-suspended in phosphate 55 buffer saline (PBS). After sonication and removing insoluble Purification of Lacritin-ELP Fusion Proteins cell debris and nucleic, ELPs were purified from clarified cell Supernatant by inverse transition cycling (ITC) as previous Purified plasmids (FIG.3A) are transferred into an expres reported until ELP purity was determined to be approxi sion host optimized for the production of repetitive mately 99% pure by SDS-PAGEgels stained with CuCl. sequences, the BLR(DE3)TM (Novagen). The inventors iden 60 Lac-ELPs were expressed in BLR (DE3) E. coli cells using tified a critical production stage, whereby reducing the incu IPTG induction. Briefly, after overnight starter culture, cell bation temperature to 30 degrees Celsius for 24 hours is pellet was inoculated into 1 LTB medium and grew at 37°C. important to prevent premature cleavage of ELPs and lacritin. until OD600 nm reached 0.5. 500 ul of 1 MIPTG Stock Solu ELPs and lacritin-ELPs are purified using inverse phase tran tion was added into cell culture and temperature was sition cycling. Briefly, this method makes use of the ELP 65 decreased to 25°C. to optimize protein expression. After 5h phase separation to induce selected precipitation of ELP induction, cells were harvested and purified using ITC. Due to fusion proteins using centrifugation. The phase transition fast degradation of Lac-ELP, fusion protein was further pol US 9,102,763 B2 17 18 ished by using Superose size exclusion column at 4°C. After derivative of the optical density at 350 nm. ELP transition balancing the column with PBS (Ph 7.4), 100 mg Lac-ELP temperatures are functions of the logarithm of concentration; was then loaded onto the column and washed out by isocratic therefore, a range of sample concentrations from 5 to 100 uM flow of PBS at 0.5 ml/min. Peak 1 was collected and concen ELP are typically observed and fit to the following equation: trated using Amicon Ultra 10 Kd. Free lacritin is released by thrombin cleavage of Lac-ELP fusion protein. Briefly, 300 ul Tim Log1of CEIPI--b thrombin beads slurry (Sigma-Aldrich) was added into 200 Where C (uM) is the ELP concentration, m is the slope mg ITC purified Lac-ELP and incubated at room temperature (C. per Logou.M., and b (C.) is the transition temperature for overnight. After pelleting down thrombin beads at 2,500 10 at 1 uM. The transition temperatures for an ELP with and rpm, solution was warmed up to 37° C. and centrifuged at without fusion to a lacritin protein domain are indicated, 4,000 rpm for 10 minto remove phase transitioned ELP tag. which demonstrate that there is a measurable, but minimal Supernatant after spin was concentrated using Amicon Ultra decrease in the transition temperature (FIG. 8A-D). Further, it 3 Kd. Protein concentrations were determined by UV-visible spectroscopy at 280 nm (ÖELP-1285M cm, eLac is observed that lacritin undergoes degradation at 37°C. (FIG. ELP=6990M cm, eLac=5500M cm). Protein 15 7A-C). molecular weight is further confirmed by MALDI-TOF Typically, PBS solutions of protein sample (100 uM, 50 analysis. uM, 25 uM, 10 uM and 5 uM) were heated at 1° C./min between 10° C. and 85°C. T. under each concentration is Example 6 defined as the maximum first derivative of turbidity change. Particle size distribution of Lacritin, ELP and Lac-ELP Assessing the Purity and Molecular Weight for ELP fusions was measured as a function of temperature increase Fusion Proteins by dynamic light scattering (DLS). Briefly, protein samples were prepared at 25uM in PBS and filtered through a 20 nm Purified proteins were further characterized by SDS-PAGE filter (Whatman Anodisc) at 4°C. Autocorrelation functions and MALDI-TOF analysis. The temperature-dependent 25 were collected using a DynaPro-LSR dynamic light scatter phase transition behavior of both ELPs and Lac-ELPs was ing Wyatt Plate Reader (Wyatt Technology, Santa Barbara, characterized by measuring the optical density at 350 nm Calif.). Light scattering data were collected at regular tem (OD350 nm) as a function of solution temperatures between perature intervals (1°C.) as solutions were heated from 5 to 15 and 85°C. in a DU800 UV-visible spectrophotometer. 60° C. The results were analyzed using a Rayleigh sphere More specifically, ELP and ELP fusion proteins, are run on 30 model and fitted into either a regularization or cumulant algo a standard SDS-PAGE apparatus. ELPs on PAGE can be rithm based on the Sum-of-squares value. Critical micelle stained and imaged using copper chloride. In addition to temperature (CMT) for each protein construct was defined as purified ELP and ELP-lacritin, thrombin can be added to the the lowest temperature at which the Rhis significantly greater fusion proteins. After incubation under standard conditions, than the average monomer Rh. this process liberates the free lacritin (FIGS. 1A, 2A, 4A and 35 5A). Although lacritin is approximately 12 kDa, it is has been Example 8 reported to run on PAGE as a band around 18 kDa, which was observed by the inventors. The resulting gel depicts the purity Lacritin Moiety Influences Phase Transition of the peptides purified using ELP phase separation (FIGS. Behavior of Parent ELPs 4A and 5A). 40 Further confirming the identity of these gene products, the As ELP fusion protein, Lac-ELP would be expected to specific protein spots are isolated and characterized using undergo phase transition like parent ELPs, one can charac matrix assisted laser desorption ion time of flight mass spec terize the influence of fusion lacritin moiety on Tt of attached trometry (MALDI-TOF) (FIGS. 4B and 5B). The resulting ELPs. FIG. 2B and FIG. 8 showed inverse phase transition 45 characterization of all expressed ELPs and lac-ELP fusion masses are consistent with the expected protein mass to a high proteins at a concentration of 5, 10, 25, 50 and 100 uM in degree of accuracy (Table 3). phosphate buffered saline (PBS) over the experimentally accessible temperature range of 10-90° C. As described, Tt of TABLE 3 ELPs could be predicted by a simple equation, which MALDI-TOF analysis of purified ELP and lacritin fusion proteins 50 accounts for the ELP concentration and its length: TtmLog 10CELP)+b, where CELP (uM) is the ELP concentration, m Expected Mass MALDI-TOF is the slope (C. per Log 10 uMI), and b ( C.) is the Protein (kD) result (kD) transition temperature at 1 uM. Comparison of lacritin, Lac Lac-V96 52.52 52.29 V96 and V96 phase transition curve at 25uM (FIGS. 8A and V96 39.55 39.21 55 8B) demonstrated maintenance of the phase transition behav Lac 12.85 12.73 ior of parent ELPs. However, a 5° C. decrease in Tt at 25uM was observed for the fusion protein. Interestingly, ATt of Lac-S48I48 and Lac Example 7 S96 was much more dramatic compared to S48I48 and S96 60 (FIGS. 8C and 8D). While S48I48 exhibited one smooth Measurement of Phase Transition Temperature for micelle formation and one sharp bulk phase transition, only ELP Fusion Proteins one sharp phase transition around 15° C. was noticed with Lac-S48I48, which was 60° C. lower than bulk phase transi ELP transition temperatures are determined on a Beckman tion temperature of S48I48 (FIG. 8D). On the contrary, Lac DU800 UV-VIS spectrophotometer under a temperature gra 65 S96 completely abolished phase transition behavior of parent dient of 1° C. per minute in PBS. The transition temperature, S96 within accessible temperature range of 10-90° C. (FIG. by turbidometric analysis, is defined as the maximum first 8C). US 9,102,763 B2 19 20 Example 9 bachol (CCh.--, CCh-) (FIGS. 10A and 12). The results show that ELP-lacritin retains biological activity in an physiologi Lac-ELPs Pre-Assemble into Nano-Sized Particles cally relevant cell type, as demonstrated by changes in secre tion of proteins delivered via Ad-Syn-GFP or LifeAct-RFP Phase transition characterization results suggested the 5 reporter constructs transfection into LGACs (FIG. 13). fusion lacritin moiety may interact with each other in a ran Changes in luminal regions and secretory vesicles was dom/organized pattern other rather simply staying as mono observed following ELP-lacritin and LAC administration. mers. One can further characterize the self-assembly property These results were further confirmed via direct measurement of purified Lac-ELPs using Dynamic Light Scattering (DLS). of protein secretion assays of rabbit LGACs, following ELP As shown in FIG.9A, free lacritin exhibited as 2-3 mm mono- 10 lacritin and LAC stimulation (FIG. 12). mers between 5° C. and 60°. While S96 stayed stable as 2-3 nm monomers within experimental temperature range, Lac Example 11 S96 pre-assembled as 30-40 nm particles (FIG.9C). Lac-V96 and Lac-I96 similarly exhibited as 30-40 nm pre-assembled Lac-ELPs and Lacritin Stimulate B-Hexosaminidase particles until bulk phase transition above Tt (Figure (B). 15 Secretion in Rabbit LGACs in a Time and Dose S48I48 was chosen as our micelle scaffold. Remarkably, as Dependent Manner different from sharp phase transition of I96 and V96 below 37°C., S4848 assembled from 2-3 mm monomers into 20 nm. In another example, a well-established in vitro rabbit micelles above its critical micelle temperature (CMT) at LGAC secretion model to evaluate prosecretory function of around 26° C. (FIG. 9D). Same as other Lac-ELPs, Lac- 20 lacritin and its Lac-ELP fusion constructs. FIG. 11 Summa S48I48 preassemble into 30-40 nm particles even at 5°C. As rizes the B-hexosaminidase secretion results of acute Lac temperature was raised, Lac-S48I48 aggregated into 140-150 ELPs and lacritin stimulation on rabbit LGACs. As internal nm mono-dispersed particles above its Tt (FIG.9D). Interest positive control, 100 uM carbachol significantly stimulated ingly, native ELPs by themselves do not preassemble as their secretion during the 4 h treatment time range. Compared to fusion constructs. 25 V96, Lac-V96 and lacritin significantly stimulated secretion The DLS observations were further supported by high at a concentration of 10 uM (p<0.01) and 20 uM (p<0.001) resolution TEM and Cryo-TEM images of the corresponding (FIG. 11A); apparent effects at 104 and 0.1 uM were not nanoparticles. While S48I48 formed perfect micelle structure statistically significant. When treating LGACs with Lac-V96 (FIGS. 9E&H), Lac-S48I48 presented a much larger size in and lacritin for 0-4 hat a concentration of 10 M, significant its nanostructure, which was around 60-70 nm in diameter 30 secretion effect was observed after 30 min treatment and (FIGS. 9F&I). Discrepancy of micelles diameter measured reached the peak at 1 h (p<0.0001); after 1 h, B-hexosamini using three techniques may come from the hydrophobicity of dase secretion slower down (FIG. 11B). The inventors further the fusion protein. As DLS measures hydrodynamic radius of compared LGACs response to Lac-ELPs with different the particle, lacritin moiety may exist in its most extended hydrophobicity and conformation. While difference between conformation in the solution and thus gave a 130-140 nm Rh 35 Lac-I96 and Lac-V96 groups were marginal, Lac-S96 group reading. Both TEM and Cryo-TEM measured dry samples so exhibited a slight higher B-hexosaminidase secretion level that only the most hydrophobic core was shown in the figures. and Lac-S48I48 group showed lowest response. This differ Due to fast degradation of Lac-S48I48, both TEM and Cryo ence may come from the NGSEFAQKLL residues of lacritin TEM images of Lac-S48I48 also showed partially degraded sequence which is required for Syndecan-1 binding, which is product: S48I48 micelles. 40 believed to be crucial in downstream signal transduction. Since Lac-S96 is in its most soluble and extended conforma Example 10 tion at 37°C. (FIG. 8C), no downstream protein-protein bind ing was blocked by the ELP tag. Due to phase transition at 37° Measurement of Secretion of Beta Hexosaminidase C., Lac-I96 and Lac-V96 formed micron-sized coacervates from Primary Lacrimal Gland Cells 45 (FIG. 8B), so that binding of lacritin to Syndecan was not as efficient. The most interesting result comes from Lac-S48I48. Primary rabbit lacrimal glandacinar cells (LGAC) are used Though it did form micelle with lacritin on the corona as for in vitro Secretion assay. Lacrimalacini were isolated and designed (FIG. 8D & FIG. 9), this construct showed the least cultured for 2-3 days. Cells prepared in this way aggregate activity. It is possible that NGSEFAQKLL residues at the into acinus-like structures; individual cells within these struc- 50 C-terminus of lacritin were buried in the inter-section of tures display distinct apical and basolateral domains and micelle, which made them difficult to be recognized by the maintain a robust secretory response. Total protein is quanti receptors. fied by Biorad assay and secreted protein is quantified by 3-hexosaminidase assay. Briefly, primary rabbit acinar cells Example 12 are seeded in 12-well plate 2 days before test. 2 hours before 55 testing, old medium is replaced by 600 ul fresh PCM. Lac-ELPs and Lacritin Stimulate Chronic F-Actin Medium and cells are incubated at 37° C. for 2 hours. After Remodeling Around LGAC Lumen and Enhanced collecting a sample of medium before secretion, ELP or lac Secretory Vesicle Formation ritin ELP in PBS is added into each well and incubated at 37° C. for 30 min. All the samples are aggressively vortexed and 60 In response to secretagogues, LGACs exocytose the con centrifuged at 4°C., 12,000 rpm for 5 min. For Bioradassay, tents of mature secretory vesicles containing tear proteins at before and after secretion media samples are tested on their apical membranes into lumen area. Spurred by under 96-well plates in triplicate. For measurement of the B-hex standing the cellular mechanism of Lac-ELP and lacritin osaminidase activity, 4-methylumbelliferyl N-acetyl-3-D- triggered secretion, the inventors utilized live LGACs time glucosaminide is used as a Substrate in triplicate. Carbachol is 65 lapse confocal fluorescence microscopy imaging to investi used as a positive control for secretion; furthermore, specific gate changes of actin filaments located beneath the apical secretion is normalized to the controls with and without car membrane during exocytosis evoked by Lac-ELP and lacritin US 9,102,763 B2 21 22 (20 uM). For live cell imaging, rabbit lacrimalacini seeded on in conjunction with calmodulin, activates specific protein Matrigel-covered glass-bottomed round 35 mm dishes (Mat kinases that may be involved in Secretion. As a positive con Tek, Ashland Mass.) at a density of 4x10° cells per dish for 2 trol used in this study, carbachol triggered intracellular Ca2+ days were co-transduced with Ad-Syn-GFP and Ad-Life Act wave in LGACs at an optimal concentration of 10-100 uM RFP at MOI of 6 for each for 2 hours. Cells were then rinsed 5 (FIG. 15A1). Supramaximal concentrations of carbachol and cultured in fresh medium for overnight to allow protein caused a decreased response (data not shown). Ca2+ intensity expression. Dual transduction efficiency (as measured by elevated around 50-100%, with 50% of total acini responded RFP-actin expression) ranged from 80-90% in each experi to the stimuli. Although Small intra-acini variance was ment. On day 3, lacrimalacini were analyzed by time-lapse observed, reflecting as a different Ca2+ intensity change, all confocal fluorescence and DIC microscopy using Zeiss Mul 10 acini responded to exogenous carbachol simultaneously in a tiple Time Series V3.2 software modules. Live cell analyses twinkling Scintillation pattern. On the contrast, neither Lac were performed at 37° C. For time-lapse analysis, acini of ELPs nor Epidermal growth factor (EGF) was capable of similar size (4-6 cells arranged around a central lumen) were evoking the same Ca2+ reaction in LGACs, which Suggested chosen. DIC images and RFP, GFP fluorescence were Lac-ELPs and lacritin may trigger different signal transduc acquired simultaneously using the 488 line of the Argon 15 tion pathway other than utilizing second messenger Ca2+. It Laser. is possible that intracellular Ca2+ change was too low to be Similar to other epithelial cells, actin filaments in LGACs detected, on the basis that Lac-ELPs/lacritin caused lower are primarily enriched beneath the apical plasma membrane B-hexosaminidase secretion in LGACs than carbachol (FIG. and less abundant beneath basolateral membranes. Here, the 11). inventors transduced LGACs with high efficiency (80-90%) replication-defective adenovirus (Ad) encoding RFP-actin Example 14 (Ad-Life Act-RFP) to label the actin filament array in lacrimal acini and measured its dynamics change during stimuli. Application of Lac-ELPs to Human Corneal Cells Adenovirus encoding cytosolic protein Syn-GFP (Ad-Syn GFP) was double transduced. Images obtained for different 25 Described herein is application of Lac-ELPs to corneal treatments were shown for plain PCM medium/CCh- (FIG. cells. SV40-immortalized HCE-T cells were grown to 80% 14B), carbachol (100 uM) and Lac-V96 (20 uM). Image confluent on glass bottom 35-mm dish in keratinocyte-SFM acquisition of treated acini was initiated 30-60 seconds after media (Life Technologies, Rockville, Md.) containing bovine treatment addition, due to the time required to refocus. In the pituitary extract (50 ug/ml), EGF (5 ng/ml) and penicillin/ absence of any treatment, there was little global remodeling 30 streptomycin. To optimize cell responsiveness to EGF and of apical or basolateral actin filaments; only subtle basal lacritin-ELPs, cells were starved with EGF and BPE free release of a few SVs at the apical membrane were detected medium for 24 hours before experimentation. (FIG.14BCCh-). While positive control carbachol (100 uM) The inventors observed the same Ca2+ wave pattern in acutely (0-15 min) increased significant apical actin filament HCE-T cells treated with Lac-ELPs and lacritin (FIGS. turnover and promoted transient actin assembly around 35 15B3&B4). Interestingly, second messenger Ca2+ wrote a apparent fusion intermediates (FIG. 14B CCh.--); Lac-V96 different code in HCE-T cells: instead of simultaneous twin (20 uM) exhibited a milder and more chronic effect on LGAC kling Scintillation, an obvious propagation wave of "brighten morphology change (FIG. 14B Lac-V96 (20 uM). After a lag up' was observed across the cell sheet. time for about 20 min, 2 types of significant cellular changes Compared to LGACs, elevation of intracellular Ca2+ con were observed in LGACs perfused by Lac-V96: (1) increased 40 centration in HCE-T cells was sharper and decreased more irregularity in the continuity of apical actin filaments and Smoothly, with a maximum 5 fold fluorescence increase in formation of actin-coated structures beneath the apical and lacritin (10 uM) and Lac-S48I48 (40 uM) treating groups. also basal membrane (purple arrows). Percentage of total responding cells was depending on lac ritin/Lac-ELPs concentration (FIG. 15C). Moreover, HCE-T Example 13 45 cells appeared to have “memory” for exogenous Lac/Lac ELPs treatment, as treating the same group of cells for the Lac-ELPs and Lacritin Triggers Transient Cytosolic second time with the same concentration of proteins, Ca2+ Ca2+Wave in HCE-T Cells but not LGACs influx was higher (FIG. 15B4). The same Ca2+ wave pattern was observed in carbachol (FIG. 15A2) and EGF treatment The ability of actin filaments to remodel rapidly in 50 groups (FIG. 15B2). While carbachol exhibited a concentra response to changes in intracellular signaling is essential for tion dependent effect; reaction of HCE-T cells to EGF was their participation in exocytosis. Results from B-hexosamini more biphasic, with maximum response recorded at 10 ng/ml dase secretion and confocal imaging studies show that Lac (FIG. 15C). Ca2+ elevation in HCE-T cells depended on ELP and lacritin trigger different cellular response compared extracellular-cytosol Ca2+ gradient, as cells bathed in W/o to carbachol (FIGS. 11 and 14). Moreover, our B-hexosamini 55 Ca2+ solution did not respond to the same stimuli. dase secretion result shows the minimal therapeutic concen tration of lacritin is 10 uM, which is more than 100 fold of Example 15 previous reported 10-100 nM dose required for peroxidase secretion. Without being bound by any particular theory, dif Biostability of Lacritin ferent receptor expression levels on rabbit and rat LGACs, 60 may display differences in early signal transduction pathway Lacritin crystals have been developed but are not yet suit participating in Lac-ELPs/lacritin triggered secretion. In the able for X-ray diffraction. Without signal peptide, lacritin lacrimal gland, cholinergic agonists stimulate protein secre protein sequence itself has a calculated isoelectric point (pl) tion by activating phospholipase C to break down phosphati of 5.1715. Using ExPASy amino sequence composition dylinositol bisphosphate into 14.5-inositol trisphosphate 65 analysis, composition of lacritin contains 10.9% Ser, 8.4% (1,4,5-IP3) and diacylglycerol (DAG). 14.5-IP3 causes Thr, 9.2% Lys. With 10% Lys content, lacritin could easily be release of Ca2+ from intracellular stores. This Ca2+, perhaps a serine protease target. At the same time, lacritin contains US 9,102,763 B2 23 24 one His, thirteen Ser and three Asp, it may also exhibit autoly containing 10% fetal bovine serum for 66 hours until com sis property similar to trypsin. The described in vitro degra pletely detached from the flask surface. The Adeno-XTM virus dation results of purified lacritin show that half-life of lacritin purification kit was used for virus purification and the Adeno is only 24 hours (FIG. 16A), which makes its purification a XTM rapid titer kit for viral titration. challenge. 5 While carbachol triggered immediate intense F-actin As thermo-responsive biopolymer, ELPs show unique remodeling beneath both apical and basal membrane of potential as a polypeptide “tag” for protein purification and as LGAC luminal region, Lac-ELPs and lacritin demonstrated a a carrier for therapeutic protein cargo. Compared with tradi milder and more chronic effect, which suggests Lac-ELPs tional His-tag and intein system, ELP system shows a more and lacritin signaling may involve other pathways other than economic purification budget with satisfactory yield. In addi 10 activating muscarinic type 3 acetylcholine receptors (M3R). tion, Scale-up of this purification method is easy because it is The inventors investigated signal transduction pathways trig not limited by resin capacity. The method Inverse transition gered by Lac-ELP/lacritin stimuli by recording cytosolic cycling (ITC) exploits the observation that proteins or pep Ca2+ wave change in LGACs and SV-40 transduced human tides that are fused to a stimulus responsive ELP retain this corneal epithelial cells (HCE-Ts) stained with intracellular behavior in the complex milieu of contaminating cellular 15 calcium indicator Fluo-4AM. While 10 uM carbachol treat components. In the described results, all control ELPs (V96, ment significantly elevated cytosolic Ca2+ level in LGACs, S96, I96, S48I48) show a yield of 50-100 mg/L (FIG. 5A). Ca2+ change in LGACs in regards to lacritin and Lac-ELPs Due to fast degradation and pre-mature cleavage of ELP tag, treatments were hardly detected. Interestingly, Ca2+ wave a size-exclusion polishing step is required for lac-ELPs. But propagation in HCE-T cells can be triggered by 10 uM lac yield of the fusion protein is still over 30 mg/L. Removing ritin/Lac-ELPs. Taken together, the prosecretory and elevat ELP tag to release free lacritin is as simple as to trigger ELP ing cytosolic Ca2+ activities of Lac-ELPs Supported our phase transition and centrifuge it out of Solution after throm hypothesis that ELPs are promising as an ocular drug delivery bin cleavage (FIG.5A). The inventors used thrombin kit from carrier. Sigma with thrombin attached to the agarose beads, thus A better understanding of the complex spatiotemporal during low speed centrifugation, thrombin was also removed 25 Ca2+ signal pattern in LGACs and HCE-T cells might there from the Supernatant, leaving only soluble lacritin. fore shed light on intracellular processes influencing lacritin and Lac-ELPs activity. The Ca2+ signaling results show that Example 16 lacritin/Lac-ELPs did not exhibit the same Ca2+ oscillation pattern in LGACs as carbachol. Without being bound by any Signalling Pathways Involved in Prosecretory 30 particular theory, this suggests a different downstream signal Activity ing pathway may be involved in its prosecretory activity. As a key regulator in maintaining corneal epithelial cell prolifera There are many therapeutics with great potential for oph tion and migration, EGF was included in the Ca2+ signaling thalmology but cannot be delivered in sufficiently high con study, which acts in a paracrine fashion on epithelial cells centrations into the eye at the site of required action because 35 proliferation via orchestrated calcium influx from intracellu of their improper size. The inventors have shown that fusion lar calcium stores and extracellular space. EGF, carbachol of lacritin with different ELPs, can improve in vitro pharma and lacritin/Lac-ELPs exhibited similar Ca2+ wave pattern cokinetics and thus enhance therapeutic efficacy. Three types on HCE-T cells, which promptus start wondering which cell of ELP tags have been chosen to fulfill this aim: In the sim receptors were involved in carbachol and EGF signaling. It is plest form, S96 tag is fused to lacritin as a soluble macromo 40 well known that carbachol stimulates tear fluid production lecular carrier. I96 and V96 as two hydrophobic tags with through the activation of muscarinic receptors. different Tt under 37° C. so that fusion protein forms a vis Compared to conventional synthetic low molecular weight cous coacervate, which may be used as insoluble drug depot drugs, proteins are more unstable during their storage and when locally delivered at desired site. In a more sophisticated administration and Susceptible to denaturation during the design, nanoscale self-assembly S48I48 block copolymer is 45 drug production process. Our degradation study of lacritin is chosen to assist Lac-ELP assemble into spherical micelles. an example of this concept. Peroxidase secretion by rat lacrimal gland has been widely used for measuring protein secretion in vitro. However, it is Example 17 not secreted by rabbit lacrimal gland, the most widely used animal model in vivo for evaluating secretion. B-hexosamini 50 Discussion dase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary To achieve minimal invasive means for the delivery of culture on stimulation with secretagogs. In this study, the therapeutic proteins to treat dry eye disease is one of the inventors utilized this model to quantitatively evaluate challenges of ophthalmology. One obstacle of ocular drug prosecretory activity of Lac-ELPs and lacritin. Optimal rabbit 55 delivery is the anatomical and physiological barriers in the LGACs response was observed after 1 hour Lac-ELP or lac eye and low bioavailability of present medications. Biode ritin treatment in a dose dependent manner, both showing gradable polymer-based drug delivery systems show consid 30-40% response compared to carbachol group. erable promise for the treatment of ocular diseases by provid To identify the impact of exogenous recombinant lac-ELP ing a Sustained-release platform. and lacritin on morphology change and mature secretion 60 The results described herein demonstrate successful bio vesicle formation of LGACs, the inventors double transduced construction of lac-ELPs with versatile ELP tags and allowed LGACs with adenovirus Ad-Life Act-RFP (F-actin marker) us to potentially modulate in vitro and in vivo pharmacoki and Ad-Syn-GFP (secretion protein marker) to observe cell netic profile of native lacritin. B-hexosaminidase secretion response using confocal microscope. Ad-syncollin-GFP was results from our in vitro rabbit model also show promising generated and for amplification, QB1 cells, a derivative of 65 therapeutic potential of lacritin-ELPs. Hydrophobicity of HEK293 cells, were infected with Ad-syncollin-GFP and ELP tag did not show a significant impact on B-hexosamini grown at 37° C. and 5% CO2 in DMEM (high glucose) dase secretion results. However, multivalent presentation of US 9,102,763 B2 25 26 lacritin on the corona of ELP micelle decreased efficiency of in Some embodiments, the numerical parameters set forth in lacritin proseretory activity. Lacritin-ELPs and lacritin the written description and attached claims are approxima induce chronic F-actin remodeling aroundacinar lumen and tions that can vary depending upon the desired properties elevated mature secretion vesicle formation. Lac-ELPs and sought to be obtained by a particular embodiment. In some lacritin triggers transient Ca2+ waves in SV40-transduced embodiments, the numerical parameters should be construed Human Epithelial Cells (HCE-Ts). in light of the number of reported significant digits and by The results herein described demonstrate construction of a applying ordinary rounding techniques. Notwithstanding that lacritin-ELP fusion protein with biocompatible phase transi the numerical ranges and parameters setting forth the broad tion behavior without retardation of effective biological activ Scope of some embodiments of the invention are approxima ity. The fusion protein imparts the thermo-responsive prop 10 tions, the numerical values set forth in the specific examples erty of the ELP and prosecretory function of lacritin, which are reported as precisely as practicable. The numerical values has great potential for controlling ocular bioavailability. This presented in some embodiments of the invention may contain use of ELPs for constructing thermo-responsive ophthalmic certain errors necessarily resulting from the standard devia drugs opens new possibilities for the treatment of dry eye tion found in their respective testing measurements. disease. 15 In some embodiments, the terms “a” and “an and “the The various methods and techniques described above pro and similar references used in the context of describing a vide a number of ways to carry out the invention. Of course, particular embodiment of the invention (especially in the it is to be understood that not necessarily all objectives or context of certain of the following claims) can be construed to advantages described may be achieved in accordance with cover both the singular and the plural. The recitation of ranges any particular embodiment described herein. Thus, for of values herein is merely intended to serve as a shorthand example, those skilled in the art will recognize that the meth methodofreferring individually to each separate value falling ods can be performed in a manner that achieves or optimizes within the range. Unless otherwise indicated herein, each one advantage or group of advantages as taught herein with individual value is incorporated into the specification as if it out necessarily achieving other objectives or advantages as were individually recited herein. All methods described may be taught or Suggested herein. A variety of advantageous 25 herein can be performed in any suitable order unless other and disadvantageous alternatives are mentioned herein. It is wise indicated herein or otherwise clearly contradicted by to be understood that some preferred embodiments specifi context. The use of any and all examples, or exemplary lan cally include one, another, or several advantageous features, guage (e.g. "Such as') provided with respect to certain while others specifically exclude one, another, or several dis embodiments herein is intended merely to better illuminate advantageous features, while still others specifically mitigate 30 the invention and does not pose a limitation on the scope of a present disadvantageous feature by inclusion of one, the invention otherwise claimed. No language in the specifi another, or several advantageous features. cation should be construed as indicating any non-claimed Furthermore, the skilled artisan will recognize the applica element essential to the practice of the invention. bility of various features from different embodiments. Simi Groupings of alternative elements or embodiments of the larly, the various elements, features and steps discussed 35 invention disclosed herein are not to be construed as limita above, as well as other known equivalents for each Such tions. Each group member can be referred to and claimed element, feature or step, can be mixed and matched by one of individually or in any combination with other members of the ordinary skill in this art to perform methods in accordance group or other elements found herein. One or more members with principles described herein. Among the various ele of a group can be included in, or deleted from, a group for ments, features, and steps some will be specifically included 40 reasons of convenience and/or patentability. When any Such and others specifically excluded in diverse embodiments. inclusion or deletion occurs, the specification is herein Although the invention has been disclosed in the context of deemed to contain the group as modified thus fulfilling the certain embodiments and examples, it will be understood by written description of all Markush groups used in the those skilled in the art that the embodiments of the invention appended claims. extend beyond the specifically disclosed embodiments to 45 Preferred embodiments of this invention are described other alternative embodiments and/or uses and modifications herein, including the best mode known to the inventor for and equivalents thereof. carrying out the invention. Variations on those preferred Many variations and alternative elements have been dis embodiments will become apparent to those of ordinary skill closed in embodiments of the present invention. Still further in the art upon reading the foregoing description. It is con variations and alternate elements will be apparent to one of 50 templated that skilled artisans can employ such variations as skill in the art. Among these variations, without limitation, are appropriate, and the invention can be practiced otherwise than the methods of preparing, isolating, or purifying fusion pro specifically described herein. Accordingly, many embodi teins containing bioresponse proteins polymers and/or thera ments of this invention include all modifications and equiva peutic proteins, functional equivalents, and/or active frag lents of the subject matter recited in the claims appended ments thereof, methods of treating various disease and/or 55 hereto as permitted by applicable law. Moreover, any combi conditions using fusion proteins, including types of diseases, nation of the above-described elements in all possible varia conditions and/or target organ(s) that relate to the teachings of tions thereof is encompassed by the invention unless other the invention, techniques and composition and use of solu wise indicated herein or otherwise clearly contradicted by tions used therein, and the particular use of the products COInteXt. created through the teachings of the invention. Various 60 Furthermore, numerous references have been made to pat embodiments of the invention can specifically include or ents and printed publications throughout this specification. exclude any of these variations or elements. Each of the above cited references and printed publications In some embodiments, the numbers expressing quantities are herein individually incorporated by reference in their of ingredients, properties such as concentration, reaction con entirety. ditions, and so forth, used to describe and claim certain 65 In closing, it is to be understood that the embodiments of embodiments of the invention are to be understood as being the invention disclosed herein are illustrative of the principles modified in some instances by the term “about.” Accordingly, of the present invention. Other modifications that can be US 9,102,763 B2 27 28 employed can be within the scope of the invention. Thus, by with the teachings herein. Accordingly, embodiments of the way of example, but not of limitation, alternative configura- present invention are not limited to that precisely as shown tions of the present invention can be utilized in accordance and described.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 24
<21 Os SEQ ID NO 1 &211s LENGTH: 406 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 catatggaag acgcttctt C tact ct acc ggtgctgacc cqgct Cagga agctgg tacc 60 totaalaccga acgaagaaat citctggit cog gctgaaccgg Cttct CCCC ggaalaccacc 12O
accaccgctic aggaaacct c togctgctgct gttcagggta cc.gctaaagt tacct cittct 18O cgt caggaac togaac cc.gct galaatctatic gttgaaaaat citat cotgct gaccgaacag 24 O
gct ctggcta aagctggtaa agg tatgcac ggtggtgttc C9ggtggtaa acagttcatc 3 OO gaaaacggitt Ctgaatt.cgc tic agaaactg. Ctgaaaaaat tdtctctgct galaaccgtgg 360
gctggtctgg tt cogcogtgg ttctggittac tat ct cct C ggat.cc 4 O6
<21 Os SEQ ID NO 2 &211s LENGTH: 508 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens
<4 OOs SEQUENCE: 2 gcagattctg toggttatact cactic ct cat cocaaagaat galaatttacc actict c ct ct 60 tottggcagc tigtag caggg gC cctggtct atgctgaaga tigcct cctot gact cacgg 12O gtgctgatcc ticcicaggaia gctgggacct ctaa.gc.ctaa taagagat C to aggt ccag 18O
Cagaaccagc tit caccc.cca gagacalacca caa.ca.gc.cca ggagactitcg gCdgcagcag 24 O
ttcaggggac agc.calaggt C acct Caagca ggcaggaact aaac cc cctgaaatccatag 3 OO tggagaaaag tatct tact a acagaacaag cc cttgcaaa agcaggaaaa ggaatgcacg 360
gaggcgtgcc aggtggaaaa caatt catcg aaaatggaag taatttgca caaaaattac 42O tgaagaaatt cagtictatta aalaccatggg catgaga agc tigaaaagaat gggat cattg 48O
gacittaaagc cittaaat acc cttgtagc 508
<21 Os SEQ ID NO 3 &211s LENGTH: 138 212s. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs SEQUENCE: 3 Met Llys Phe Thir Thr Lieu. Lieu. Phe Lieu Ala Ala Val Ala Gly Ala Lieu. 1. 5 1O 15 Val Tyr Ala Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala 2O 25 3 O
Gln Glu Ala Gly Thr Ser Llys Pro Asn Glu Glu Ile Ser Gly Pro Ala 35 4 O 45
Glu Pro Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu Thir Ser SO 55 60
Ala Ala Ala Val Glin Gly Thr Ala Llys Val Thir Ser Ser Arg Glin Glu 65 70 7s 8O
Lieu. Asn Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu US 9,102,763 B2 29 30 - Continued
85 90 95 Glin Ala Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly 1OO 105 11 O
Gly Lys Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu 115 12 O 125
Llys Llys Phe Ser Lieu Lleu Lys Pro Trp Ala 13 O 135
<210s, SEQ ID NO 4 &211s LENGTH: 118 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 4
Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin Glu Ala Gly 1. 5 15
Thir Ser Llys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu Pro Ala Ser 2O 25
Pro Pro Gu Thir Thir Thir Thir Ala Glin Glu Thir Ser Ala Ala Ala Wall 35 4 O 45
Gln Gly Thr Ala Lys Val Thir Ser Ser Arg Glin Glu Lell Asn Pro Luell SO 55 6 O
Llys Ser Ile Val Glu Lys Ser Ile Luell Luell Thir Glu Glin Ala Luell Ala 65 70
Lys Ala Gly Lys Gly Met His Gly Gly Wall Pro Gly Gly Glin Phe 85 90 95
Ile Glu Asn Gly Ser Glu Phe Ala Glin Luell Lell Lys Phe Ser 1OO 105 11 O Lieu. Lieu Lys Pro Trp Ala 115
<210s, SEQ ID NO 5 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 5
Met Glu Asp Ala Ser Ser Asp Ser Thir Gly Ala Asp Pro Ala Glin Glu 1. 5 15
Ala Gly. Thir Ser Llys Pro Asn. Glu Glu Ile Ser Gly Pro Ala Glu Pro 2O 25
Ala Ser Pro Pro Glu. Thir Thir Thr Thir Ala Glin Glu Thir Ser Ala Ala 35 4 O 45
Ala Val Glin Gly Thr Ala Lys Val Thir Ser Ser Arg Glin Glu Luell Asn SO 55 6 O
Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Luell Lell Thir Glu Glin Ala 65 70 7s 8O
Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Wall Pro Gly Gly 85 90 95
Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Lell Luell 1OO 105 11 O
Phe Ser Lieu. Lieu Lys Pro Trp Ala 115 12 O
<210s, SEQ ID NO 6 &211s LENGTH: 121 212. TYPE: PRT US 9,102,763 B2 31 - Continued <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 6 Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 5 1O 15 Glu Ala Gly. Thir Ser Llys Pro Asn. Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45 Ala Ala Val Glin Gly. Thir Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu SO 55 6 O Asn Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O Ala Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95 Lys Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O Llys Phe Ser Lieu Lleu Lys Pro Trp Ala 115 12 O
<210s, SEQ ID NO 7 &211s LENGTH: 117 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OO > SEQUENCE: 7 Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 5 1O 15 Glu Ala Gly. Thir Ser Llys Pro Asn. Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45 Ala Ala Val Glin Gly. Thir Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu SO 55 6 O Asn Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O Ala Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95 Lys Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O Llys Phe Ser Lieu. Lieu 115
<210s, SEQ ID NO 8 &211s LENGTH: 112 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 8 Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 5 1O 15
Glu Ala Gly. Thir Ser Llys Pro Asn. Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45
Ala Ala Val Glin Gly. Thir Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu SO 55 6 O US 9,102,763 B2 33 34 - Continued
Asn Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O Ala Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95 Lys Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O
<210s, SEQ ID NO 9 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 9 Gly Lieu Val Pro Arg Gly Ser Gly 1. 5
<210s, SEQ ID NO 10 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION : THROMBIN RECOGNITION SITE
<4 OOs, SEQUENCE: 10 Gly Lieu Val Pro Arg Gly Ser 1. 5
<210s, SEQ ID NO 11 & 211 LENGTH: 482 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: I96 SEQUENCE
<4 OOs, SEQUENCE: 11 Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 1. 5 1O 15 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 2O 25 3O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 35 4 O 45 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly SO 55 6 O Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 65 70 7s 8O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 85 90 95 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 1OO 105 11 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 115 12 O 125
Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 13 O 135 14 O
Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 145 150 155 160
Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 1.65 17O 17s
Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 18O 185 19 O US 9,102,763 B2 35 36 - Continued
Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 195 2OO 2O5 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 21 O 215 22O Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 225 23 O 235 24 O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 245 250 255 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 26 O 265 27 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 27s 28O 285 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 29 O 295 3 OO Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 3. OS 310 315 32O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 3.25 330 335 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 34 O 345 35. O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 355 360 365 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 37 O 375 38O Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 385 390 395 4 OO Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 4 OS 41O 415 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 42O 425 43 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 435 44 O 445 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 450 45.5 460 Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 465 470 47s 48O Gly Tyr
<210s, SEQ ID NO 12 &211s LENGTH: 482 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: S96 SEQUENCE
<4 OOs, SEQUENCE: 12 Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 1. 5 1O 15
Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 2O 25 3O
Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 35 4 O 45
Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly SO 55 6 O US 9,102,763 B2 37 38 - Continued Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 65 70 7s 8O Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 85 90 95 Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 1OO 105 11 O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 115 12 O 125 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 13 O 135 14 O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 145 150 155 160 Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 1.65 17O 17s Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 18O 185 19 O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 195 2OO 2O5 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 21 O 215 22O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 225 23 O 235 24 O Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 245 250 255 Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 26 O 265 27 O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 27s 28O 285 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 29 O 295 3 OO Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 3. OS 310 315 32O Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 3.25 330 335 Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 34 O 345 35. O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 355 360 365 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 37 O 375 38O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 385 390 395 4 OO Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 4 OS 41O 415
Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 42O 425 43 O
Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 435 44 O 445
Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 450 45.5 460
Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 465 470 47s 48O Gly Tyr US 9,102,763 B2 39 40 - Continued
<210s, SEQ ID NO 13 &211s LENGTH: 482 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: S48I48 SEQUENCE
<4 OOs, SEQUENCE: 13 Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 1. 5 1O 15 Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 2O 25 3O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 35 4 O 45 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly SO 55 6 O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 65 70 7s 8O Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 85 90 95 Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 1OO 105 11 O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 115 12 O 125 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 13 O 135 14 O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 145 150 155 160 Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 1.65 17O 17s Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 18O 185 19 O Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 195 2OO 2O5 Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly 21 O 215 22O Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser 225 23 O 235 24 O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 245 250 255 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 26 O 265 27 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 27s 28O 285 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 29 O 295 3 OO
Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 3. OS 310 315 32O
Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 3.25 330 335
Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 34 O 345 35. O
Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 355 360 365 US 9,102,763 B2 41 - Continued
Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 37 O 375 38O Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 385 390 395 4 OO Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 4 OS 41O 415 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 42O 425 43 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 435 44 O 445 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 450 45.5 460 Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 465 470 47s 48O Gly Tyr
<210s, SEQ ID NO 14 &211s LENGTH: 609 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: LAC-W96
<4 OOs, SEQUENCE: 14 Met Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin Glu 1. 5 1O 15 Ala Gly. Thir Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu Pro 2O 25 3O
Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala Ala 35 4 O 45 Ala Val Glin Gly Thr Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu. Asn SO 55 6 O Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin Ala 65 70 7s 8O Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly Lys 85 90 95 Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys Llys 1OO 105 11 O Phe Ser Lieu. Lieu Lys Pro Trp Ala Gly Lieu Val Pro Arg Gly Ser Gly 115 12 O 125 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 13 O 135 14 O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 145 150 155 160 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1.65 17O 17s
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 18O 185 19 O
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 195 2OO 2O5
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 21 O 215 22O
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 225 23 O 235 24 O US 9,102,763 B2 43 44 - Continued Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 245 250 255 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 26 O 265 27 O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 27s 28O 285 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 29 O 295 3 OO Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 3. OS 310 315 32O Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 3.25 330 335 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 34 O 345 35. O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 355 360 365 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 37 O 375 38O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 385 390 395 4 OO Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 4 OS 41O 415 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 42O 425 43 O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 435 44 O 445 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 450 45.5 460 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 465 470 47s 48O Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 485 490 495 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val SOO 505 51O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 515 52O 525 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 53 O 535 54 O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 5.45 550 555 560 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 565 st O sts Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 58O 585 59 O
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 595 6OO 605
<210s, SEQ ID NO 15 &211s LENGTH: 609 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: LAC-IS 6 US 9,102,763 B2 45 46 - Continued
<4 OOs, SEQUENCE: 15
Met Glu Asp Ala Ser Ser Asp Ser Thir Gly Ala Asp Pro Ala Glin Glu 1. 15
Ala Gly Thir Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu Pro 25 3O
Ala Ser Pro Pro Glu Thir Thir Thir Thir Ala Glin Glu Thir Ser Ala Ala 35 4 O 45
Ala Wall Glin Gly Thir Ala Lys Wall Thir Ser Ser Arg Glin Luell Asn SO 55 6 O
Pro Luell Ser Ile Wall Glu Ser Ile Luell Lell Thir Glin Ala 65 70 8O
Lell Ala Ala Gly Gly Met His Gly Gly Wall Pro Gly 85 90 95
Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Lell 105
Phe Ser Luell Luell Lys Pro Trp Ala Gly Luell Wall Pro Arg Ser Gly 115 12 O 125
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Gly Wall 13 O 135 14 O
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Wall Pro 145 150 155 160
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 1.65 17O 17s
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile 18O 185 19 O
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 195
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 21 O 215 22O
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 225 23 O 235 24 O
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 245 250 255
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile 26 O 265 27 O
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 285
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 29 O 295 3 OO
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 3. OS 310 315
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 3.25 330 335
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile 34 O 345 35. O
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 355 360 365
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 37 O 375 38O
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 385 390 395 4 OO
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 4 OS 41O 415 US 9,102,763 B2 47 48 - Continued
Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 42O 425 43 O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 435 44 O 445 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 450 45.5 460 Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 465 470 47s 48O Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 485 490 495 Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile SOO 505 51O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 515 52O 525 Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val 53 O 535 54 O Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro 5.45 550 555 560 Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly 565 st O sts Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile 58O 585 59 O Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly Val Pro Gly Ile Gly 595 6OO 605 Tyr
<210s, SEQ ID NO 16 &211s LENGTH: 609 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: LAC-S96
<4 OOs, SEQUENCE: 16 Met Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin Glu 1. 5 1O 15 Ala Gly. Thir Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu Pro 2O 25 3O
Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala Ala 35 4 O 45 Ala Val Glin Gly Thr Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu. Asn SO 55 6 O Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin Ala 65 70 7s 8O Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly Lys 85 90 95
Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys Llys 1OO 105 11 O
Phe Ser Lieu. Lieu Lys Pro Trp Ala Gly Lieu Val Pro Arg Gly Ser Gly 115 12 O 125
Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val 13 O 135 14 O
Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro 145 150 155 160 US 9,102,763 B2 49 50 - Continued
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 1.65 17O 17s
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 18O 185 19 O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 195
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 21 O 215 22O
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 225 23 O 235 24 O
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 245 250 255
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 26 O 265 27 O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 285
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 29 O 295 3 OO
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 3. OS 310 315
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 3.25 330 335
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 34 O 345 35. O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 355 360 365
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 37 O 375
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 385 390 395 4 OO
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 4 OS 415
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 425 43 O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 435 44 O 445
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 450 45.5 460
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 465 470
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 485 490 495
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser SOO 505
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 515 525
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 53 O 535 54 O
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 5.45 550 555 560
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 565 st O sts
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser US 9,102,763 B2 51 - Continued
58O 585 59 O Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly Val Pro Gly Ser Gly 595 6OO 605 Tyr
<210s, SEQ ID NO 17 &211s LENGTH: 484 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: W96
<4 OOs, SEQUENCE: 17 Gly Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1. 5 1O 15 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 2O 25 3O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 35 4 O 45 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val SO 55 6 O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 65 70 7s 8O Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 85 90 95 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 1OO 105 11 O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 115 12 O 125 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 13 O 135 14 O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 145 150 155 160 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 1.65 17O 17s Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 18O 185 19 O Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 195 2OO 2O5 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 21 O 215 22O Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 225 23 O 235 24 O Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 245 250 255
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 26 O 265 27 O
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 27s 28O 285
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 29 O 295 3 OO
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 3. OS 310 315 32O
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 3.25 330 335 US 9,102,763 B2 53 54 - Continued
Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Val Pro Gly Wall 34 O 345 35. O
Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly 355 360 365
Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall 37 O 375
Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro 385 390 395 4 OO
Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly 4 OS 415
Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall 425 43 O
Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly 435 44 O 445
Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall 450 45.5 460
Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro Gly Wall Gly Wall Pro 465 470 47s 48O
Gly Wall Gly Tyr
SEQ ID NO 18 LENGTH: 609 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: LAC-S48 I 48
< 4 OOs SEQUENCE: 18
Met Glu Asp Ala Ser Asp Ser Thir Gly Ala Asp Pro Ala Glin Glu 1. 15
Ala Gly Thir Ser Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu Pro 25
Ala Ser Pro Pro Glu Thir Thir Thir Thir Ala Glin Glu Thir Ser Ala Ala 35 4 O 45
Ala Wall Glin Gly Thir Ala Lys Wall Thir Ser Ser Arg Glin Glu Luell Asn SO 55 6 O
Pro Luell Ser Ile Wall Glu Ser Ile Luell Lell Thir Glu Glin Ala 65 70 8O
Lell Ala Ala Gly Gly Met His Gly Gly Wall Pro Gly Gly 85 90 95
Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Lell Luell 105 11 O
Phe Ser Luell Luell Pro Trp Ala Gly Luell Wall Pro Arg Gly Ser Gly 115 12 O 125
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 13 O 135 14 O
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 145 150 155 160
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 1.65 17O 17s
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 18O 185 19 O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 195 2OO 2O5 US 9,102,763 B2 55 56 - Continued
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 21 O 215 22O
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 225 23 O 235 24 O
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 245 250 255
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 26 O 265 27 O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 285
Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall 29 O 295 3 OO
Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro 3. OS 310 315
Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly 3.25 330 335
Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser 34 O 345 35. O
Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly Wall Pro Gly Ser Gly 355 360 365
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 37 O 375
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 385 390 395 4 OO
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 4 OS 415
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile 425 43 O
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 435 44 O 445
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 450 45.5 460
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 465 470
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 485 490 495
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile SOO 505
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 515 525
Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall 53 O 535 54 O
Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro 5.45 550 555 560
Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly 565 st O sts
Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile 58O 585 59 O
Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly Wall Pro Gly Ile Gly 595 6OO 605
Tyr
<210s, SEQ ID NO 19 US 9,102,763 B2 57 - Continued
&211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: GENERIC TAG 1 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 19 Val Pro Gly Xaa Gly 1. 5
<210s, SEQ ID NO 2 O &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: GENERIC TAG 2 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<4 OOs, SEQUENCE: 2O Val Pro Gly Xaa Gly Gly 1. 5
<210s, SEQ ID NO 21 &211s LENGTH: 126 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: LACRITIN PROTEASE PRODUCT 1.
<4 OOs, SEQUENCE: 21 Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 5 1O 15 Glu Ala Gly. Thir Ser Llys Pro Asn. Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Ala Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45 Ala Ala Val Glin Gly. Thir Ala Lys Val Thir Ser Ser Arg Glin Glu Lieu SO 55 6 O Asn Pro Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O Ala Lieu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95 Lys Glin Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O Llys Phe Ser Lieu Lleu Lys Pro Trp Ala Gly Lieu Val Pro Arg 115 12 O 125
<210s, SEQ ID NO 22 &211s LENGTH: 118 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: LACRITIN PROTEASE PRODUCT 2
<4 OOs, SEQUENCE: 22 Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 5 1O 15 US 9,102,763 B2 59 60 - Continued
Glu Al a Gly. Thir Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Al a Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45
Ala Al a Val Glin Gly Thr Ala Lys Val Thr Ser Ser Arg Glin Glu Lieu. SO 55 6 O
Asn P o Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O
Ala Le u Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95
Gl in Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O Phe Ser Lieu Lleu Lys 115
SEQ ID NO 23 LENGTH: 119 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: LACRITIN PROTEASE PRODUCT 3
<4 OOs, SEQUENCE: 23
Met Gly Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala Glin 1. 15
Glu Al a Gly. Thir Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala Glu 2O 25 3O
Pro Al a Ser Pro Pro Glu. Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala 35 4 O 45
Ala Al a Val Glin Gly Thr Ala Lys Val Thr Ser Ser Arg Glin Glu Lieu. SO 55 6 O
Asn P o Lieu Lys Ser Ile Val Glu Lys Ser Ile Lieu. Lieu. Thr Glu Glin 65 70 7s 8O
Ala Le u Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 85 90 95
Gl in Phe Ile Glu Asn Gly Ser Glu Phe Ala Glin Llys Lieu. Lieu Lys 1OO 105 11 O
Phe Ser Lieu Lleu Lys Llys 115
SEQ ID NO 24 LENGTH: 62 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: LACRITIN PROTEASE PRODUCT 4
SEQUENCE: 24
Pro As in Glu Glu Ile Ser Gly Pro Ala Glu Pro Ala Ser Pro Pro Glu 1. 5 1O 15
Thir Thir Thir Thir Ala Glin Glu. Thir Ser Ala Ala Ala Wall Glin Gly Thr
Ala Llys Val Thir Ser Ser Arg Glin Glu Lieu. ASn Pro Lieu Lys Ser Ile 35 4 O 45
Wall Gl u Lys Ser Ile Lieu. Lieu. Thr Glu Glin Ala Lieu Ala Lys SO 55 6 O US 9,102,763 B2 61 62 The invention claimed is: providing a quantity of a composition, wherein the com 1. An isolated fusion protein comprising: position comprises the fusion protein of claim 1; and a bioresponse protein polymer comprising an elastin-like enhancing ocular persistence of the therapeutic protein for polypeptide (ELP), and treating a human subject by administering a therapeuti a therapeutic protein conjugated via a linker peptide to the 5 cally effective dosage of the composition to the subject, bioresponse protein polymer, wherein the ELP com wherein ocular persistence of the therapeutic protein prises n number of amino acid motif SEQ ID NO: 19, conjugated to the bioresponse protein polymer is greater where n comprises 10 to 300 units, and X of SEQID than ocular persistence of the therapeutic protein in the NO:19 is serine, valine, or isoleucine. absence of conjugation to the bioresponse protein poly 2. The isolated fusion protein of claim 1, wherein n is 96. 10 3. The isolated fusion protein of claim 1, wherein the C. therapeutic protein comprises lacritin or a functional equiva 12. The method of claim 11, wherein the human subject is in need of treatment for an eye disease and/or condition lent. Selected from the group consisting of acanthamoeba kerati 4. The isolated fusion protein of claim 3, wherein the tis, allergies, amblyopia, Bell’s palsy, blepharitis, cataracts, lacritin is human lacritin. 15 5. The isolated fusion protein of claim 3, wherein the chalazion, colorblindness, corneal ulcer, detached retina, dry lacritin or functional equivalent comprises amino acid eye syndrome, keratoconjunctivitis sicca, eye occlusions, eye sequence: SEQID NO:3, SEQID NO: 4, SEQID NO: 5, twitching, macular hole, nystagmus, ocular migraine, ocular SEQID NO: 6, SEQID NO: 7, or SEQID NO:8. rosacea, optic neuritis, optic neuropathy, photophobia, 6. The isolated fusion protein of claim 1, wherein the linker pinguecula, pterygium, ptosis, Sjogren's syndrome, strabis peptide comprises amino acid sequence: SEQID NO: 9. mus, stye, Subconjunctival hemorrhage, uveitis, CMV retini 7. The isolated fusion protein of claim 1, wherein the tis, conjunctivitis, diabetic retinopathy, eye herpes, glau bioresponse protein polymer comprises an ELP, the therapeu coma, karatoconus, macular degeneration, macular tic protein is lacritin, and the ELP is conjugated to the lacritin dystrophy, ocular hypertension, retinitis pigmentosa, and/or via a linker peptide. Stargardt’s disease. 8. An isolated polynucleotide encoding a fusion protein 25 13. The method of claim 11, wherein the bioresponse pro comprising: tein polymer comprises an elastin-like polypeptide (ELP), the a bioresponse protein polymer comprising an elastin-like therapeutic protein comprises lacritin, and the ELP is conju polypeptide (ELP), and gated to the lacritin via a linker peptide. a therapeutic protein conjugated to the bioresponse protein 14. A pharmaceutical composition comprising: 30 a bioresponse protein polymer comprising an elastin-like polymer via a linker peptide), wherein the ELP com polypeptide (ELP), wherein the ELP comprises n num prises n number of amino acid motif SEQ ID NO: 19, ber of amino acid motif SEQID NO: 19, where n com where n comprises 10 to 300 units and X of SEQ ID prises 10 to 300 units, and X of SEQ ID NO:19 is NO:19 is serine, valine, or isoleucine. serine, Valine, or isoleucine; 9. The isolated polynucleotide of claim 8, wherein n is 96 35 units. a therapeutic protein conjugated to the bioresponse protein 10. A method of constructing the isolated polynucleotide of polymer via a linker peptide; and claim 8 using recursive directional ligation. a pharmaceutically acceptable carrier. 11. A method of enhancing ocular persistence of a thera 15. The pharmaceutical composition of claim 14, wherein peutic protein for treating a disease and/or condition in a the therapeutic protein comprises lacritin. human subject, comprising: ck ck ck ck ck