US 20090305978A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0305978A1 Zane (43) Pub. Date: Dec. 10, 2009

(54) METHODS FOR INCREASING THE SIZE OF Related U.S. Application Data ANMALS USING NEEDLELESS DELIVERY CONSTRUCTS (60) Provisional application No. 60/783,534, filed on Mar. 16, 2006. (75) Inventor: Doris Tham Zane, San Mateo, CA (US) Publication Classification Correspondence Address: (51) Int. Cl. JONES DAY A638/27 (2006.01) 222 EAST 41ST ST A6IP 43/00 (2006.01) NEW YORK, NY 10017 (US) (52) U.S. Cl...... S14/12 (73) Assignee: TRINITY BIOSYSTEMS, INC., Menlo Park, CA (US) (57) ABSTRACT The present invention relates, in part, to methods for increas (21) Appl. No.: 12/282,853 ing the size of a Subject by administering a delivery construct (22) PCT Fled: Mar. 15, 2007 comprising growth to a Subject. In one aspect, the method for increasing the size of a subject by at least about (86) PCT NO.: PCT/US2007/006590 12% comprises contacting an apical Surface of a polarized epithelial cell of the subject with an amount of a delivery S371 (c)(1), construct comprising growth hormone that is effective to (2), (4) Date: Apr. 20, 2009 increase the size of the subject by at least about 12%. Patent Application Publication Dec. 10, 2009 Sheet 1 of 19 US 2009/0305978A1

Figure 1A

Met Ala Giu. Glu Ala Phe Asp Leu Trp Asn. Glu. Cys Ala Iys Ala Cys S O s Val Lieu. Asp Lleu Lys Asp Gly Wall Arg Ser Ser Arg Met Ser Wall Asp 2O 25 30 Pro Ala Ile Aia Asp Thr Asn. Gly Gin Gly Val Lieu. His Tyr Ser Met 35 40 45 Val Lieu. Glul Gilly Gly ASI Asp Alia Leu Lys Lell Ala Ile Asp ASI Ala SO 55 60 Leu Ser Ile Thr Ser Asp Gly Lieu. Thir Ile Arg Lieu. Glu Gly Gly Wall 5S 70 75 8O Glu Pro Asn Llys Pro Val Arg Tyr Ser Tyr Thr Arg Glin Ala Arg Gly 85 SC 95 Ser Trp Ser Leu Asn. Trp Lieu Val Pro Ile Gly. His Glu. Llys Pro Ser OO OS 10 Asn. Ile Lys Val Phe Ile His Glu Lieu. Asn Ala Giy Asn. Glin Leu Ser 15 2O 25 His Met Ser Pro Ile Tyr Thir Ilie Glu Met Gly Asp Glu Lieu Lieu. Ala 13 O 35 40 Lys fell Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn 1.45 SO S5 60 Glu Met Glin. Pro Thr Lieu Ala Ile Ser His Ala Gly Val Ser Val Val - 65 7 O 17S Met Ala Glin Thr Glin Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala 18O 85 19 O Ser Gly Llys Val Lieu. Cys Lieu Lieu. Asp Pro Lieu Asp Gly Val Tyr Asn 195 2CO 2 OS Tyr Lieu Alia Glin Glin Arg Cys Asn Lieu. Asp Asp Thir Trp Gill. Gly Lys 2O 25 22 O Ilie Tyr Arg Wall Leu Ala Gly Asn. Pro Ala Lys His Asp Leu. Asp Ile 225 230 235 24 O Llys Pro Thr Val Ile Ser His Arg Feu. His Phe Pro Glu Gly Gly Ser 245 25 O 255 Teu Ala Ala Luell. Thir Ala. His Glin Ala Cys His Leu Pro Lieu. Glu. Thr 260 26S 27 O Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Glin Lieu. Glu Gln Cys 275 280 285 Gly Tyr Pro Val Glin Arg Lieu Val Ala Leu. Tyr Leu Ala Ala Arg Lieu 29 O 29S 3 OO Ser Trp Asn Gin Val Asp Gin Wall Ile Arg Asn Ala Lieu. A la Ser Pro 3 OS 310 3.5 320 Gly Ser Gly Gly Asp Lieu. Gly Glu Ala Ile Arg Glu Gin Pro Glu Gln 3.25 33 O 3.35 Ala Arg Lieu Ala Lieu. Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe val 34 O 34S 350 Arg Glin Gly Thr Gly Asn. Asp Glu Ala Gly Ala Ala Asn Lieu Glin Gly 3.SS 360 365 Gly Lieu. Arg Glin Pro Arg Phe Pro Thir Ilie Pro Leu Ser Arg Leu Phe 370 375 38O Patent Application Publication Dec. 10, 2009 Sheet 2 of 19 US 2009/0305978A1

Figure 1B

Asp Asn Ala Met Lieu. Arg Ala His Arg Lieu. His Gln Leu Ala Phe Asp 385 39 O. 395 4 OO Thir Tyr Glin Glu Phe Glu Glu Ala Tyr Ile Pro Llys Glu Glin Lys Tyr 4 O 5 410 45 Ser Phe Lieu. Glin Asn Pro Glin Thr Ser Lieu. Cys Phe Ser Glu Ser Ile 42O 42S 43 O. Pro Thr Pro Ser Asn. Arg Glu Glu. Thr Glin Glin Lys Ser Asn Lieu. Glu 435 440 445 Lieu Lleu. Arg Ile Ser Leu Lleu. Leu le Glin Ser Trp Lieu. Glu Pro Val 450 4S5 46O Glin Phe Leu Arg Ser Val Phe Ala Asn. Ser Leu Val Tyr Gly Ala Ser 46S 470 475 48O Asp Ser Asn Val Tyr Asp I.eu. Lieu Lys Asp Leu Glu Glu Giy Ile Glin 48S 490 495 Thr Lieu Met Gly Arg Lieu. Glu Asp Gly Ser Pro Arg Thr Gly Glin Ile 5 OO SO 5 5. O Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr Asn. Ser His Asm Asp Asp 55 52O 525 Ala Lieu. Leu Lys Asn. Tyr Gly Lieu. Lieu. Tyr Cys Phe Arg Lys Asp Met S30 S35 S4 O Asp Llys Val Glu Thir Phe Lieu. Arg Ile Val Glin Cys Arg Ser Val Glu S45 550 SS5 S60 Gly Ser Cys Gly Phe 565 Patent Application Publication Dec. 10, 2009 Sheet 3 of 19 US 2009/0305978 A1

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METHODS FOR INCREASING THE SIZE OF CD86, TNFC receptor, TOLL receptor, M-CSF receptor, ANMALS USING NEEDLELESS DELIVERY GM-CSF receptor, scavenger receptor, and VEGF receptor. CONSTRUCTS 0007. In certain embodiments, the transcytosis domain is selected from the group consisting of transcytosis domains 1. FIELD OF THE INVENTION from Pseudomonas exotoxin A, botulinum toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero 0001. The present invention relates, in part, to methods for toxin, shiga toxin, and shiga-like toxin. increasing the size of a subject by administering a delivery 0008. In certain embodiments, the cleavable linker is construct comprising a growth hormone to a subject. In one cleavable by an that is selected from the group con aspect, the method comprises contacting an apical Surface of sisting of GI, I, I, a polarized epithelial cell of the subject with an amount of a AI, Subtilisin AII, I, and I. In certain delivery construct comprising growth hormone that is effec embodiments, the cleavable linker comprises an amino acid tive to increase the size of the subject by at least about 12%. sequence that is selected from the group consisting of Ala Ala-Pro-Phe (SEQID NO.1), Oly-Gly-Phe (SEQID NO.:2), 2. BACKGROUND Ala-Ala-Pro-Val (SEQ ID NO.:3), Gly-Gly-Leu (SEQ ID 0002 Advances in biochemistry and molecular biology NO.4), Ala-Ala-Leu (SEQID NO.:5), Phe-Val-Arg (SEQID have resulted identification and characterization of many NO.:6), Val-Gly-Arg (SEQID NO.:7). therapeutic macromolecules, including, for example, growth 0009. In certain embodiments, the epithelial cell is hormone (GH). Administration of GH can result in drastic selected from the group consisting of nasal epithelial cells, improvements in quality of life for subjects afflict with a wide oral epithelial cells, intestinal epithelial cells, rectal epithelial range of ailments. cells, vaginal epithelial cells, and pulmonary epithelial cells. 0003. However, administration of GH remains problem In certain embodiments, the epithelial cell is a nasal epithelial atic. Currently, GH is typically administered by injection. cell. In certain embodiments, the epithelial cell is an intestinal Such injections require penetration of the Subject's skin and epithelial cell. tissues and are associated with pain. Further, penetration of 0010. In certain embodiments, the subject is a mouse or the skin breaches one effective nonspecific mechanism of rat. In certain embodiments, the Subject is a human. protection against infection, and thus can lead to potentially 0011. In certain embodiments, the delivery construct con serious infection. In addition, injection of GH appears to be tacts the apical membrane of the epithelial cell. associated with induction of potentially adverse immune 0012. In certain embodiments, the size of the subject is responses relative to other routes of administration. increased by at least about 13%. In certain embodiments, the 0004. Accordingly, efforts have been made to obtain size of said subject is increased by at least about 14%. In methods and compositions that can be used to administer GH certain embodiments, the size of said Subject is increased by to subjects withoutbreaching the skin of the subject. See U.S. at least about 15%. In certain embodiments, the size of said application Ser. No. 1 1/244,349. However, additional meth subject is increased by at least about 16%. In certain embodi ods and compositions are needed to optimize the therapeutic ments, the size of said Subject is increased by at least about effects of such administration. 17%. In certain embodiments, the size of said subject is increased by at least about 18%. 3. SUMMARY OF THE INVENTION 0013. In certain embodiments, the size of said subject that is increased is a weight of said Subject. In certain embodi 0005. In certain aspects, the invention provides a method ments, the size of said Subject that is increased is a height of for increasing the size of a subject by at least about 12%, said Subject. In certain embodiments, the size of said subject comprising contacting an apical Surface of a polarized epi that is increased is a length of said Subject. thelial cell of the subject with an amount of a delivery con 0014. In certain embodiments, the GH is human growth struct effective to increase the size of the subject by at least hormone (hGH). In certain embodiments, the hCGH has an about 12%, wherein said delivery construct comprises a amino acid sequence that is SEQID NO.:8. receptor binding domain, a transcytosis domain, a cleavable 0015. In certain embodiments, the methods further com linker, and growth hormone (GH), wherein the transcytosis prise performing a method of the invention a second time domain transcytoses the GH to and through the basal-lateral about 1 day after the method of the invention is performed the membrane of said epithelial cell, and wherein cleavage at said first time. In certain embodiments, the methods further com cleavable linker separates said GH from the remainder of said prise performing a method of the invention a second time construct, thereby delivering the GH to the subject in an about 2 days after the method of the invention is performed amount effective to increase the size of the subject by at least the first time. In certain embodiments, the methods further about 12%. comprise performing a method of the invention a second time 0006. In certain embodiments, the receptor binding about 3 days after the method of the invention is performed domain is selected from the group consisting of receptor the first time. binding domains from Pseudomonas exotoxin A, cholera toxin, diptheria toxin, Shiga toxin, or shiga-like toxin; mono 4. BRIEF DESCRIPTION OF THE DRAWINGS clonal antibodies; polyclonal antibodies; single-chain anti bodies; TGF C.; EGF: IGF-I; IGF-II; IGF-III; IL-1, IL-2: 0016 FIGS. 1A and 1B present the amino acid sequence IL-3, IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In certain of HGH Delivery Construct (SEQ ID NO:9), an exemplary embodiments, the receptor binding domain binds to a cell Delivery Construct for delivering hCH. Surface receptor selected from the group consisting of 0017 FIG. 2 presents a graphical comparison of weight C.2-macroglobulin receptor, EGFR, IGFR, transferrin recep gain in lit/litmice administered 30 ughOH subcutaneously or tor, chemokine receptor, CD25, CD11B, CD11C, CD80, intranasally with the HGH Delivery Construct. US 2009/0305978 A1 Dec. 10, 2009

0018 FIG. 3 presents a graphical comparison of weight struct, 30 ughOH administered intranasally with the HGH gain in lit/litmice administered 60 ughOH subcutaneously or delivery construct, 300 ughOH administered orally with the intranasally with the HGH Delivery Construct. HGH Delivery Construct, 60 ughOH administered intrana 0019 FIG. 4 presents a graphical comparison of weight sally with the HGH delivery construct, and 60 ughOH admin gain in lit/lit mice administered either 30 ughGH or 60 ug istered subcutaneously. hGH subcutaneously. 0032 FIG. 17 presents a graphical representation of the 0020 FIG. 5 presents a graphical comparison of weight pharmacokinetic profile of hGH serum concentration follow gain in lit/lit mice administered either 30 ughCH or 60 jig ing administration of 30 ughGH intranasally with the HGH hGH intranasally with the HGH Delivery Construct. Delivery Construct to BALB/c mice. 0021 FIG. 6 presents a table showing growth of lit/litmice 0033 FIG. 18 presents a table showing amounts of hCH administered 30 ug hCGH or 60 ug hCH subcutaneously or observed in the serum of BALB/c mice at various time points intranasally with the HGH Delivery Construct normalized to following intranasal administration. growth of mice not administered any hCGH. 0022 FIG. 7 presents a graphical representation of 5. DETAILED DESCRIPTION OF THE amounts of hCGH observed in the serum of mice administered INVENTION 30 ughCH subcutaneously, 300 ughCH orally with the HGH 0034 5.1. Definitions Delivery Construct, and 60 lug hCH intranasally with the 0035. Unless defined otherwise, all technical and scien HGH Delivery Construct. tific terms used herein have the meaning commonly under 0023 FIG. 8 presents a graphical representation of stood by a person skilled in the art to which this invention amounts of bioactive hCH observed in the serum of mice belongs. As used herein, the following terms have the mean administered 30 ughGH subcutaneously, 300 ughOH orally ings ascribed to them unless specified otherwise. with the HGH Delivery Construct, and 60 lug hCH intrana 0036. A “ligand is a compound that specifically binds to sally with the HGH Delivery Construct. a target molecule. Exemplary ligands include, but are not 0024 FIG. 9 presents a graphical representation of limited to, an antibody, a cytokine, a Substrate, a signaling amounts of IGF-1 observed in the serum of mice administered molecule, and the like. 30 ughCH subcutaneously, 300 ughCH orally with the HGH 0037. A “receptor is compound that specifically binds to Delivery Construct, and 60 lug hCH intranasally with the a ligand. HGH Delivery Construct. 0038 A ligand or a receptor (e.g., an antibody) “specifi 0025 FIG. 10 presents a graphical representation of cally binds to or “is specifically immunoreactive with amounts of IGF1-BP3 observed in the serum of mice admin another molecule when the ligand or receptor functions in a istered 30 ughGH subcutaneously, 300 ughCH orally with binding reaction that indicates the presence of the molecule in the HGH Delivery Construct, and 60 lug hCH intranasally a sample of heterogeneous compounds. Thus, under desig with the HGH Delivery Construct. nated assay (e.g., immunoassay) conditions, the ligand or 0026 FIG. 11 presents a graphical representation of receptor binds preferentially to a particular compound and amounts of anti-hGH IgG antibodies observed in the serum of does not bind in a significant amount to other compounds mice administered 30 g. hCH subcutaneously, 300 ughCH present in the sample. For example, a polynucleotide specifi orally with the HGH Delivery Construct, and 60 lug hCH cally binds under hybridization conditions to another poly intranasally with the HGH Delivery Construct. nucleotide comprising a complementary sequence and an 0027 FIG. 12 presents a graphical representation of antibody specifically binds under immunoassay conditions to amounts of anti-ntPEIgG antibodies observed in the serum of an antigen bearing an epitope used to induce the antibody. mice administered 30 g. hCH subcutaneously, 300 ughCH 0039 “Immunoassay” refers to a method of detecting an orally with the HGH Delivery Construct, and 60 lug hCH analyte in a sample involving contacting the sample with an intranasally with the HGH Delivery Construct. antibody that specifically binds to the analyte and detecting 0028 FIG. 13 presents a graphical representation of binding between the antibody and the analyte. A variety of amounts of corticosterone observed in the serum of mice immunoassay formats may be used to select antibodies spe administered 30 ughGH subcutaneously, 300 ughOH orally cifically immunoreactive with a particular protein. For with the HGH Delivery Construct, and 60 lug hCH intrana example, Solid-phase ELISA immunoassays are routinely sally with the HGH Delivery Construct. used to select monoclonal antibodies specifically immunore 0029 FIG. 14 presents a graphical representation of active with a protein. See Harlow and Lane (1988) Antibod amounts of leptin observed in the serum of mice administered ies, A Laboratory Manual, Cold Spring Harbor Publications, 30 ughCH subcutaneously, 300 ughCH orally with the HGH New York, for a description of immunoassay formats and Delivery Construct, and 60 lug hCH intranasally with the conditions that can be used to determine specific immunore HGH Delivery Construct. activity. In one example, an antibody that binds a particular 0030 FIG. 15 presents a graphical representation of antigen with an affinity (K) of about 10 uM specifically amounts of observed in the serum of mice adminis binds the antigen. tered 30 ughGH subcutaneously, 300 lighCH orally with the 0040 “Linker refers to a molecule that joins two other HGH Delivery Construct, and 60 ughGH intranasally with molecules, either covalently, or through ionic, van der Waals the HGH Delivery Construct. or hydrogen bonds, e.g., a nucleic acid molecule that hybrid 0031 FIG. 16 presents a table summarizing amounts of izes to one complementary sequence at the 5' end and to hCH, bioactive hCH, IGF-1, IGF1-BP3, anti-hGH IgG anti another complementary sequence at the 3' end, thus joining bodies, anti-ntPEIgG antibodies, corticosterone, leptin, insu two non-complementary sequences. A “cleavable linker” lin, and ntPE observed in mouse serum 30 minutes following refers to a linker that can be degraded or otherwise severed to the day 10 administration of 30 ughGH administered SC, 30 separate the two components connected by the cleavable ug hCH administered orally with the HGH Delivery Con linker. Cleavable linkers are generally cleaved by , US 2009/0305978 A1 Dec. 10, 2009 typically peptidases, proteases, nucleases, lipases, and the aeruginosa after its synthesis. See, e.g., Vouloux et al., 2000, like. Cleavable linkers may also be cleaved by environmental J. Bacterol. 182:4051-8. Domain Ib has no known function cues. Such as, for example, changes in temperature, pH, salt and spans amino acids 365-399. Domain III mediates cyto concentration, etc. when there is such a change in environ toxicity of PE and includes an endoplasmic reticulum reten ment following transcytosis of the delivery construct across a tion sequence. PE cytotoxicity is believed to result from ADP polarized epithelial membrane. ribosylation of elongation factor 2, which inactivates protein 0041. “Pharmaceutical composition” refers to a composi synthesis. Domain III spans amino acids 400-613 of PE. tion Suitable for pharmaceutical use in an animal. A pharma Deleting amino acid E553 (“AE553) from domain III elimi ceutical composition comprises a pharmacologically effec nates EF2 ADP ribosylation activity and detoxifies PE. PE tive amount of an active agent and a pharmaceutically having the mutation AE553 is referred to herein as acceptable carrier. “Pharmacologically effective amount “PEAE553. Genetically modified forms of PE are described refers to that amount of an agent effective to produce the in, e.g., U.S. Pat. Nos. 5,602,095; 5.512,658 and 5,458.878 intended pharmacological result. “Pharmaceutically accept Pseudomonas exotoxin, as used herein, also includes geneti able carrier refers to any of the standard pharmaceutical cally modified, allelic, and chemically inactivated forms of carriers, vehicles, buffers, and excipients, such as a phosphate PE within this definition. See, e.g., Vasil et al., 1986, Infect. buffered saline solution, 5% aqueous solution of dextrose, Immunol. 52:538-48. Further, reference to the various and emulsions, such as an oil/water or water/oil emulsion, and domains of PE is made herein to the reference PE sequence various types of wetting agents and/or adjuvants. Suitable presented as FIG. 3. However, one or more domain from pharmaceutical carriers and formulations are described in modified PE, e.g., genetically or chemically modified PE, or Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack a portion of Such domains, can also be used in the chimeric Publishing Co., Easton. A “pharmaceutically acceptable salt immunogens of the invention so long as the domains retain is a salt that can be formulated into a compound for pharma functional activity. One of skill in the art can readily identify ceutical use including, e.g., metal salts (sodium, potassium, such domains of such modified PE based on, for example, magnesium, calcium, etc.) and salts of ammonia or organic homology to the PE sequence exemplified in FIG.3 and test amines. for functional activity using, for example, the assays 0042 Preferred pharmaceutical carriers depend upon the described below. intended mode of administration of the active agent. Typical 0047 "Polynucleotide' refers to a polymer composed of modes of administration include enteral (e.g., oral, intranasal, nucleotide units. Polynucleotides include naturally occurring rectal, or vaginal) or parenteral (e.g., subcutaneous, intramus nucleic acids, such as deoxyribonucleic acid (“DNA) and cular, intravenous or intraperitoneal injection; or topical (e.g., ribonucleic acid (“RNA) as well as nucleic acid analogs. transderrnal, or transmucosal administration). Nucleic acid analogs include those which include non-natu 0043 “Small organic molecule' refers to organic mol rally occurring bases, nucleotides that engage in linkages ecules of a size comparable to those organic molecules gen with other nucleotides other than the naturally occurring erally used in pharmaceuticals. The term excludes organic phosphodiester bond or which include bases attached through biopolymers (e.g., proteins, nucleic acids, etc.). Preferred linkages other than phosphodiester bonds. Thus, nucleotide Small organic molecules range in size up to about 5000 Da, up analogs include, for example and without limitation, phos to about 2000 Da, or up to about 1000 Da. phorothioates, phosphorodithioates, phosphorotriesters, 0044. A “subject of diagnosis, treatment, or administra phosphoramidates, boranophosphates, methylphosphonates, tion is a human or non-human animal, including a mammal or chiral-methyl phosphonates, 2-O-methyl ribonucleotides, a primate, and preferably a human. peptide-nucleic acids (PNAS), and the like. Such polynucle 0045 “Treatment” refers to prophylactic treatment or otides can be synthesized, for example, using an automated therapeutic treatment. A "prophylactic treatment is a treat DNA synthesizer. The term “nucleic acid' typically refers to ment administered to a subject who does not exhibit signs of large polynucleotides. The term "oligonucleotide' typically a disease or exhibits only early signs for the purpose of refers to short polynucleotides, generally no greater than decreasing the risk of developing pathology. A “therapeutic' about 50 nucleotides. It will be understood that when a treatment is a treatment administered to a subject who exhib nucleotide'sequence is represented by a DNA sequence (i.e., its signs of pathology for the purpose of diminishing or elimi A, T, G, C), this also includes an RNA sequence (i.e., A., U.G., nating those signs. C) in which “U” replaces “T” 0046 “Pseudomonas exotoxin A' or “PE is secreted by 0048 Conventional notation is used herein to describe Pseudomonas aeruginosa as a 67 kD protein composed of polynucleotide sequences: the left-hand end of a single three prominent globular domains (Ia, II, and III) and one stranded polynucleotide sequence is the 5'-end; the left-hand small subdomain (Ib) that connects domains II and III. See A. direction of a double-stranded polynucleotide sequence is S. Allured et al., 1986, Proc. Natl. Acad. Sci. 83:1320-1324. referred to as the 5'-direction. Without intending to be bound to any particular theory or 0049. The direction of 5' to 3' addition of nucleotides to mechanism of action, domain Ia of PE is believed to mediate nascent RNA transcripts is referred to as the transcription cell binding because domain Ia specifically binds to the low direction. The DNA strand having the same sequence as an density lipoprotein receptor-related protein (“LRP), also mRNA is referred to as the “coding strand': sequences on the known as the C.2-macroglobulin receptor (“O2-MR) and DNA strand having the same sequence as an mRNA tran CD-91. See M. Z. Kounnas et al., 1992, J. Biol. Chem. 267: scribed from that DNA and which are located 5' to the 5'-end 12420-23. Domain Ia spans amino acids 1-252. Domain II of of the RNA transcript are referred to as “upstream PE is believed to mediate transcytosis to the interior of a cell sequences'; Sequences on the DNA strand having the same following binding of domain Ia to the C2-MR. Domain II sequence as the RNA and which are 3' to the 3' end of the spans amino acids 253-364. Certain portions of this domain coding RNA transcript are referred to as “downstream may be required for secretion of PE from Pseudomonas sequences US 2009/0305978 A1 Dec. 10, 2009

0050 “Complementary” refers to the topological compat 0055 “Polar Amino Acid' refers to a hydrophilic amino ibility or matching together of interacting Surfaces of two acid having a side chain that is uncharged at physiological pH, polynucleotides. Thus, the two molecules can be described as but which has at least one bond in which the pair of electrons complementary, and furthermore, the contact surface charac shared in common by two atoms is held more closely by one teristics are complementary to each other. A first polynucle of the atoms. Genetically encoded polar amino acids include otide is complementary to a second polynucleotide if the Asn (N), Gin (Q) Ser (S) and Thr(T). nucleotide sequence of the first polynucleotide is Substan 0056 “Nonpolar Amino Acid' refers to a hydrophobic tially identical to the nucleotide sequence of the polynucle amino acid having a side chain that is uncharged at physi otide binding partner of the second polynucleotide, or if the ological pH and which has bonds in which the pair of elec first polynucleotide can hybridize to the second polynucle trons shared in common by two atoms is generally held otide under Stringent hybridization conditions. Thus, the equally by each of the two atoms (i.e., the side chain is not polynucleotide whose sequence 5'-TATAC-3' is complemen polar). Genetically encoded nonpolar amino acids include tary to a polynucleotide whose sequence is 5'-GTATA-3'. Ala (A), Gly (G), Ile (I), Leu (L), Met (M) and Val (V). 0051. The term “96 sequence identity” is used inter 0057 “Hydrophilic Amino Acid refers to an amino acid changeably herein with the term"/6 identity” and refers to the exhibiting a hydrophobicity of less than Zero according to the level of amino acid sequence identity between two or more normalized consensus hydrophobicity scale of Eisenberg et peptide sequences or the level of nucleotide sequence identity al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded between two or more nucleotide sequences, when aligned hydrophilic amino acids include Arg (R), Asn (N), Asp (D), using a sequence alignment program. For example, as used Glu (E), Gln (Q). His (H), Lys (K), Ser (S) and Thr (T). herein, 80% identity means the same thing as 80% sequence 0.058 “Hydrophobic Amino Acid' refers to an amino acid identity determined by a defined algorithm, and means that a exhibiting a hydrophobicity of greater than Zero according to given sequence is at least 80% identical to another length of the normalized consensus hydrophobicity Scale of Eisenberg another sequence. Exemplary levels of sequence identity et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded include, but are not limited to, 60, 70, 80, 85,90, 95, 98% or hydrophobic amino acids include Ala (A), Gly (G), Ile (I), more sequence identity to a given sequence. Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr (Y) and Val 0052. The term “96 ” is used inter (V). changeably herein with the term “96 homology” and refers to 0059 "Acidic Amino Acid' refers to a hydrophilic amino the level of amino acid sequence homology between two or acid having a side chain pK value of less than 7. Acidic amino more peptide sequences or the level of nucleotide sequence acids typically have negatively charged side chains at physi homology between two or more nucleotide sequences, when ological pH due to loss of a hydrogen ion. Genetically aligned using a sequence alignment program. For example, as encoded acidic amino acids include Asp (D) and Glu (E). used herein, 80% homology means the same thing as 80% 0060 “Basic Amino Acid' refers to a hydrophilic amino sequence homology determined by a defined algorithm, and acid having a side chain pK value of greater than 7. Basic accordingly a homologue of a given sequence has greater than amino acids typically have positively charged side chains at 80% sequence homology over a length of the given sequence. physiological pH due to association with a hydrogen ion. Exemplary levels of sequence homology include, but are not Genetically encoded basic amino acids include Arg (R). His limited to, 60, 70, 80, 85,90, 95, 98% or more sequence (H) and Lys (K). homology to a given sequence. 0061 “Encoding refers to the inherent property of spe 0053 Exemplary computer programs which can be used cific sequences of nucleotides in a polynucleotide. Such as a to determine identity between two sequences include, but are , a cDNA, or an mRNA, to serve as templates for Syn not limited to, the suite of BLAST programs, e.g., BLASTN, thesis of other polymers and macromolecules in biological BLASTX, and TBLASTX, BLASTP and TBLASTN, pub processes having either a defined sequence of nucleotides licly available on the Internet at the NCBI website. See also (i.e., rRNA, tRNA and mRNA) or a defined sequence of Altschulet al., 1990, J. Mol. Biol. 215:403-10 (with special amino acids and the biological properties resulting therefrom. reference to the published default setting, i.e., parameters Thus, a gene encodes a protein if transcription and translation w=4, t-17) and Altschul et al., 1997, Nucleic Acids Res., of mRNA produced by that gene produces the protein in a cell 25:3389-3402. Sequence searches are typically carried out or other biological system. Both the coding Strand, the nucle using the BLASTP program when evaluating a given amino otide sequence of which is identical to the mRNA sequence acid sequence relative to amino acid sequences in the Gen and is usually provided in sequence listings, and non-coding Bank Protein Sequences and other public databases. The Strand, used as the template for transcription, of a gene or BLASTX program is preferred for searching nucleic acid cDNA can be referred to as encoding the protein or other sequences that have been translated in all reading frames product of that gene or cDNA. Unless otherwise specified, a against amino acid sequences in the GenBank Protein “nucleotide sequence encoding an amino acid sequence' Sequences and other public databases. Both BLASTP and includes all nucleotide sequences that are degenerate versions BLASTX are run using default parameters of an open gap of each other and that encode the same amino acid sequence. penalty of 11.0, and an extended gap penalty of 1.0, and Nucleotide sequences that encode proteins and RNA may utilize the BLOSUM-62 matrix. Seeid. include introns. 0054) A preferred alignment of selected sequences in 0062 “Amplification” refers to any means by which a order to determine “96 identity” between two or more polynucleotide sequence is copied and thus expanded into a sequences, is performed using for example, the larger number of polynucleotide molecules, e.g., by reverse CLUSTAL-W program in MacVector version 6.5, operated transcription, polymerase chain reaction, chain reac with default parameters, including an open gap penalty of tion, and the like. 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 0063 “Primer' refers to a polynucleotide that is capable of similarity matrix. specifically hybridizing to a designated polynucleotide tem US 2009/0305978 A1 Dec. 10, 2009

plate and providing a point of initiation for synthesis of a Southern or northern blot is 50% formalin with 1 mg of complementary polynucleotide. Such synthesis occurs when heparin at 42°C., with the hybridization being carried out the polynucleotide primer is placed under conditions in which overnight. An example of highly stringent wash conditions is synthesis is induced, i.e., in the presence of nucleotides, a 0.15 M NaCl at 72° C. for about 15 minutes. An example of complementary polynucleotide template, and an agent for stringent wash conditions is a 0.2xSSC wash at 65° C. for 15 polymerization Such as DNA polymerase. A primer is typi minutes. See Sambrook et al. for a description of SSC buffer. cally single-stranded, but may be double-stranded. Primers A high Stringency wash can be preceded by a low stringency are typically deoxyribonucleic acids, but a wide variety of wash to remove background probe signal. An exemplary synthetic and naturally occurring primers are useful for many medium stringency wash for a duplex of e.g., more than applications. A primer is complementary to the template to about 100 nucleotides, is 1xSSC at 45° C. for 15 minutes. An which it is designed to hybridize to serve as a site for the exemplary low Stringency wash for a duplex of, e.g., more initiation of synthesis, but need not reflect the exact sequence than about 100 nucleotides, is 4-6xSSC at 40° C. for 15 of the template. In such a case, specific hybridization of the minutes. In general, a signal to noise ratio of 2x (or higher) primer to the template depends on the stringency of the than that observed for an unrelated probe in the particular hybridization conditions. Primers can be labeled with, e.g., hybridization assay indicates detection of a specific hybrid chromogenic, radioactive, or fluorescent moieties and used as ization. detectable moieties. 0069. “Polypeptide' refers to a polymer composed of 0064 “Probe,” when used in reference to a polynucle amino acid residues, related naturally occurring structural otide, refers to a polynucleotide that is capable of specifically variants, and synthetic non-naturally occurring analogs hybridizing to a designated sequence of another polynucle thereof linked via peptide bonds, related naturally occurring otide. A probe specifically hybridizes to a target complemen structural variants, and synthetic non-naturally occurring tary polynucleotide, but need not reflect the exact comple analogs thereof. Synthetic polypeptides can be synthesized, mentary sequence of the template. In Such a case, specific for example, using an automated polypeptide synthesizer. hybridization of the probe to the target depends on the strin Conventional notation is used herein to portray polypeptide gency of the hybridization conditions. Probes can be labeled sequences; the beginning of a polypeptide sequence is the with, e.g., chromogenic, radioactive, or fluorescent moieties amino-terminus, while the end of a polypeptide sequence is and used as detectable moieties. In instances where a probe the carboxyl-terminus. provides a point of initiation for synthesis of a complemen 0070 The term “protein' typically refers to large polypep tary polynucleotide, a probe can also be a primer. tides, for example, polypeptides comprising more than about 0065. “Hybridizing specifically to” or “specific hybridiza 50 amino acids. The term “protein’ can also refer to dimers, tion” or “selectively hybridize to’, refers to the binding, trimers, and multimers that comprise more than one polypep duplexing, or hybridizing of a nucleic acid molecule prefer tide. entially to a particular nucleotide sequence under stringent (0071 "Conservative substitution” refers to the substitu conditions when that sequence is present in a complex mix tion in a polypeptide of an amino acid with a functionally ture (e.g., total cellular) DNA or RNA. similar amino acid. The following six groups each contain 0066. The term “stringent conditions’ refers to conditions amino acids that are conservative Substitutions for one under which a probe will hybridize preferentially to its target another: Subsequence, and to a lesser extent to, or not at all to, other 0.072 Alanine (A), Serine (S), and Threonine (T) sequences. “Stringent hybridization' and “stringent hybrid 0.073 Aspartic acid (D) and Glutamnic acid (E) ization wash conditions” in the context of nucleic acid hybrid 0.074 Asparagine (N) and Glutamine (Q) ization experiments such as Southern and northern hybridiza 0075 Arginine (R) and Lysine (K) tions are sequence dependent, and are different under 0.076 Isoleucine (I), Leucine (L), Methionine (M), and different environmental parameters. An extensive guide to the Valine (V) hybridization of nucleic acids can be found in Tijssen, 1993, 0077. Phenylalanine (F), Tyrosine (Y), and Tryptophan Laboratory Techniques in Biochemistry and Molecular Biol (W). ogy—Hybridization with Nucleic Acid Probes, part I, chapter 0078. The term “about as used herein, unless otherwise 2. “Overview of principles of hybridization and the strategy indicated, refers to a value that is no more than 10% above or of nucleic acid probe assays, Elsevier, N.Y.; Sambrooket al., below the value being modified by the term. For example, the 2001, Molecular Cloning: A Laboratory Manual, Cold term “about 5 g/kg' means a range of from 4.5 ug/kg to 5.5 Spring Harbor Laboratory, 3" ed., NY; and Ausubel et al., ug/kg. As another example, "about 1 hour” means a range of eds., Current Edition, Current Protocols in Molecular Biol from 48 minutes to 72 minutes. ogy, Greene Publishing Associates and Wiley Interscience, (0079 5.2. Methods for Increasing the Size of a Subject NY. 0080. In certain aspects, the invention provides methods 0067 Generally, highly stringent hybridization and wash for increasing the size of a subject by at least about 12%. conditions are selected to be about 5° C. lower than the These methods generally comprise administering an amount thermal melting point (Tm) for the specific sequence at a of a delivery construct comprising a growth hormone that is defined ionic strength and pH. The Tm is the temperature effective to increase the size of a Subject to a mucous mem (under defined ionic strength and pH) at which 50% of the brane of the subject to whom the GH is delivered. The deliv target sequence hybridizes to a perfectly matched probe. Very ery construct is typically administered in the form of a phar stringent conditions are selected to be equal to the Tm for a maceutical composition, as described below. particular probe. I0081. Thus, in certain aspects, the invention provides a 0068. One example of stringent hybridization conditions method for increasing the size of a subject by at least about for hybridization of complementary nucleic acids which have 12%, comprising contacting an apical Surface of a polarized more than about 100 complementary residues on a filter in a epithelial cell of the subject with an amount of a delivery US 2009/0305978 A1 Dec. 10, 2009

construct effective to increase the size of the subject by at increased by at least about 30%. In certain embodiments, the least about 12%, wherein said delivery construct comprises a size of said subject is increased by at least about 32%. In receptor binding domain, a transcytosis domain, a cleavable certain embodiments, the size of said Subject is increased by linker, and growth hormone (GH), wherein the transcytosis at least about 35%. In certain embodiments, the size of said domain transcytoses the GH to and through the basal-lateral subject is increased by at least about 37%. In certain embodi membrane of said epithelial cell, and wherein cleavage at said ments, the size of said Subject is increased by at least about cleavable linker separates said GH from the remainder of said 40%. In certain embodiments, the size of said subject is construct, thereby delivering the OH to the subject in an increased by at least about 42%. In certain embodiments, the amount effective to increase the size of the subject by at least size of said subject is increased by at least about 45%. In about 12%. certain embodiments, the size of said Subject is increased by 0082 In certain embodiments, the receptor binding at least about 47%. In certain embodiments, the size of said domain is selected from the group consisting of receptor subject is increased by at least about 50%. binding domains from Pseudomonas exotoxin A, cholera I0089. In certain embodiments, the size of said subject is toxin, diptheria toxin, Shiga toxin, or shiga-like toxin; mono increased by between about 12% and about 18%. In certain clonal antibodies; polygonal antibodies; single-chain anti embodiments, the size of said subject is increased by between bodies; TGF C.; EGF: IGF-I; IGF-41; IGF-III; IL-1, IL-2: about 2% and about 50%. In certain embodiments, the size of IL-3, IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In certain said subject is increased by between about 2% and about embodiments, the receptor binding domain binds to a cell 40%. In certain embodiments, the size of said subject is Surface receptor selected from the group consisting of increased by between about 2% and about 30%. In certain C.2-macroglobulin receptor, EGFR, IGFR, transferrin recep embodiments, the size of said subject is increased by between tor, chemokine receptor, CD25, CD11B, CD11C, CD80, about 2% and about 20%. In certain embodiments, the size of CD86, TNFC receptor, TOLL receptor, M-CSF receptor, said subject is increased by between about 8% and about GM-CSF receptor, scavenger receptor, and VEGF receptor. 50%. In certain embodiments, the size of said subject is 0083. In certain embodiments, the transcytosis domain is increased by between about 8% and about 40%. In certain selected from the group consisting of transcytosis domains embodiments, the size of said subject is increased by between from Pseudomonas exotoxin A, botulinum toxin, diptheria about 8% and about 30%. In certain embodiments, the size of toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero said subject is increased by between about 8% and about toxin, shiga toxin, and shiga-like toxin. 20%. In certain embodiments, the size of said subject is 0084. In certain embodiments, the cleavable linker is increased by between about 12% and about 50%. In certain cleavable by an enzyme that is selected from the group con embodiments, the size of said subject is increased by between sisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin about 12% and about 40%. In certain embodiments, the size AI, Subtilisin AII, Thrombin I, and Urokinase I. In certain of said subject is increased by between about 12% and about embodiments, the cleavable linker comprises an amino acid 30%. In certain embodiments, the size of said subject is sequence that is selected from the group consisting of Ala increased by between about 12% and about 20%. Ala-Pro-Phe(SEQID NO.1), Gly-Gly-Phe (SEQID NO.:2), 0090. In certain embodiments, the size of said subject that Ala-Ala-Pro-Val (SEQ ID NO.:3), Gly-Gly-Leu (SEQ ID is increased is a weight of said Subject. In certain embodi NO.4), Ala-Ala-Leu (SEQID NO.:5), Phe-Val-Arg (SEQID ments, the size of said Subject that is increased is a height of NO.:6), Val-Gly-Arg (SEQID NO.:7). said Subject. In certain embodiments, the size of said subject 0085. In certain embodiments, the epithelial cell is that is increased is a length of said Subject. selected from the group consisting of nasal epithelial cells, 0091. In certain embodiments, the GH is human growth oral epithelial cells, intestinal epithelial cells, rectal epithelial hormone (hGH). In certain embodiments, the hCGH has an cells, vaginal epithelial cells, and pulmonary epithelial cells. amino acid sequence that is SEQID NO.:8. In certain embodiments, the epithelial cell is a nasal epithelial 0092. In certain embodiments, the methods further com cell. In certain embodiments, the epithelial cell is an intestinal prise performing a method of the invention a second time epithelial cell. about 1 day after the method of the invention is performed the I0086. In certain embodiments, the subject is a mouse or first time. In certain embodiments, the methods further com rat. In certain embodiments, the Subject is a human. prise performing a method of the invention a second time 0087. In certain embodiments, the delivery construct con about 2 days after the method of the invention is performed tacts the apical membrane of the epithelial cell. the first time. In certain embodiments, the methods further 0088. In certain embodiments, the size of the subject is comprise performing a method of the invention a second time increased by at least about 13%. In certain embodiments, the about 3 days after the method of the invention is performed size of said subject is increased by at least about 14%. In the first time. certain embodiments, the size of said Subject is increased by 0093. In certain embodiments, the invention provides a at least about 15%. In certain embodiments, the size of said method for delivering a GH to the bloodstream of a subject subject is increased by at least about 16%. In certain embodi that results in at least about 30% bioavailability of the GH, ments, the size of said Subject is increased by at least about comprising administering a delivery construct comprising the 17%. In certain embodiments, the size of said subject is GH to the subject, thereby delivering at least about 30% of the increased by at least about 18%. In certain embodiments, the total GH administered to the blood of the subject in a bio size of said subject is increased by at least about 20%. In available form of the GH. In certain embodiments, at least certain embodiments, the size of said Subject is increased by about 10% of the total GH administered is bioavailable to the at least about 22%. In certain embodiments, the size of said subject. In certain embodiments, at least about 15% of the subject is increased by at least about 25%. In certain embodi total GH administered is bioavailable to the subject. In certain ments, the size of said Subject is increased by at least about embodiments, at least about 20% of the total GH adminis 27%. In certain embodiments, the size of said subject is tered is bioavailable to the subject. In certain embodiments, at US 2009/0305978 A1 Dec. 10, 2009

least about 25% of the total GH administered is bioavailable GH in the subject are achieved about 60 minutes after admin to the subject. In certain embodiments, at least about 35% of istration. In certain embodiments, peak plasma concentra the total GH administered is bioavailable to the subject. In tions of the delivered GH in the subject are achieved about 90 certain embodiments, at least about 40% of the total GH minutes after administration. In certain embodiments, peak administered is bioavailable to the subject. In certain embodi plasma concentrations of the delivered GH in the subject are ments, at least about 45% of the total GH administered is achieved about 120 minutes after administration. bioavailable to the subject. In certain embodiments, at least 0095. In certain embodiments, the peak plasma concentra about 50% of the total GH administered is bioavailable to the tion of the delivered GH is between about 0.01 ng/ml plasma subject. In certain embodiments, at least about 55% of the and about 10 g/ml plasma. In certain embodiments, the peak total GH administered is bioavailable to the subject. In certain plasma concentration of the delivered GH is between about embodiments, at least about 60% of the total GH adminis 0.01 ng/ml plasma and about 1 lug/ml plasma. In certain tered is bioavailable to the subject. In certain embodiments, at embodiments, the peak plasma concentration of the delivered least about 65% of the total GH administered is bioavailable GH is between about 0.01 ng/ml plasma and about 0.1 g/ml to the subject. In certain embodiments, at least about 70% of plasma. In certain embodiments, the peak plasma concentra the total GH administered is bioavailable to the subject. In tion of the delivered GH is between about 0.01 ng/ml plasma certain embodiments, at least about 75% of the total GH and about 10 ng/ml plasma. In certain embodiments, the peak administered is bioavailable to the subject. In certain embodi plasma concentration of the delivered GH is between about 1 ments, at least about 80% of the total GH administered is ng/ml plasma and about 10 g/ml plasma. In certain embodi bioavailable to the subject. In certain embodiments, at least ments, the peak plasma concentration of the delivered GH is about 85% of the total GH administered is bioavailable to the between about 1 ng/ml plasma and about 1 g/ml plasma. In subject. In certain embodiments, at least about 90% of the certain embodiments, the peak plasma concentration of the total GH administered is bioavailable to the subject. In certain delivered GH is between about 1 ng/ml plasma and about 0.5 embodiments, at least about 95% of the total GH adminis ug/ml plasma. In certain embodiments, the peak plasma con tered is bioavailable to the subject. In certain embodiments, centration of the delivered GH is between about 1 ng/ml the percentage of bioavailability of the GH is determined by plasma and about 0.1 g/ml plasma. In certain embodiments, comparing the amount of GH present in a Subject's blood the peak plasma concentration of the delivered GH is between following administration of a delivery construct comprising about 10 ng/ml plasma and about 1 g/ml plasma. In certain the GH to the amount of GH present in a subject's blood embodiments, the peak plasma concentration of the delivered following administration of the GH through another route of GH is between about 10 ng/ml plasma and about 0.5 g/ml administration. In certain embodiments, the other route of plasma. administration is injection, e.g., Subcutaneous injection, 0096. In certain embodiments, the peak plasma concentra intravenous injection, intra-arterial injection, etc. In other tion of the delivered GH is at least about 10 ug/ml plasma. In embodiments, the percentage of bioavailability of the GH is certain embodiments, the peak plasma concentration of the determined by comparing the amount of GH present in a delivered GH is at least about 5 lug/ml plasma. In certain subject’s blood following administration of a delivery con embodiments, the peak plasma concentration of the delivered struct comprising the GH to the total amount of GH admin GH is at least about 1 ug/ml plasma. In certain embodiments, istered as part of the delivery construct. the peak plasma concentration of the delivered GH is at least 0094. In certain embodiments, peak plasma concentra about 500 ng/ml plasma. In certain embodiments, the peak tions of the delivered GH in the subject are achieved about 10 plasma concentration of the delivered GH is at least about 250 minutes after administration. In certain embodiments, peak ng/ml plasma. In certain embodiments, the peak plasma con plasma concentrations of the delivered GH in the subject are centration of the delivered GH is at least about 100 ng/ml achieved about 15 minutes after administration. In certain plasma. In certain embodiments, the peak plasma concentra embodiments, peak plasma concentrations of the delivered tion of the delivered GH is at least about 50 ng/ml plasma. In GH in the subject are achieved about 5 minutes after admin certain embodiments, the peak plasma concentration of the istration. In certain embodiments, peak plasma concentra delivered GH is at least about 10 ng/ml plasma. In certain tions of the delivered GH in the subject are achieved about 20 embodiments, the peak plasma concentration of the delivered minutes after administration. In certain embodiments, peak GH is at least about 5 ng/ml plasma. In certain embodiments, plasma concentrations of the delivered GH in the subject are the peak plasma concentration of the delivered GH is at least achieved about 25 minutes after administration. In certain about 1 ng/ml plasma. In certain embodiments, the peak embodiments, peak plasma concentrations of the delivered plasma concentration of the delivered GH is at least about 0.1 GH in the subject are achieved about 30 minutes after admin ng/ml plasma. istration. In certain embodiments, peak plasma concentra 0097. In certain embodiments, the subject is a mammal. In tions of the delivered GH in the subject are achieved about 35 further embodiments, the Subject is a rodent, a lagomorph, or minutes after administration. In certain embodiments, peak a primate. In yet further embodiments, the rodent is a mouse plasma concentrations of the delivered GH in the subject are or rat. In other embodiments, the lagomorph is a rabbit. In still achieved about 40 minutes after administration. In certain other embodiments, the primate is a human, monkey, or ape. embodiments, peak plasma concentrations of the delivered In a preferred embodiment, the Subject is a human. GH in the subject are achieved about 45 minutes after admin (0098 5.3. Delivery Constructs istration. In certain embodiments, peak plasma concentra 0099 Generally, the delivery constructs of the present tions of the delivered GH in the subject are achieved about 50 invention are polypeptides that have structural domains cor minutes after administration. In certain embodiments, peak responding to domains Ia and II of PE. These structural plasma concentrations of the delivered GH in the subject are domains perform certain functions, including, but not limited achieved about 55 minutes after administration. In certain to, cell recognition and transcytosis, that correspond to the embodiments, peak plasma concentrations of the delivered functions of the domains of PE. US 2009/0305978 A1 Dec. 10, 2009

0100. In addition to the portions of the molecule that cor group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:1), Gly respond to PE functional domains, the delivery constructs Gly-Phe (SEQID NO.:2), Ala-Ala-Pro-Val (SEQID NO.:3), useful in the methods of this invention further comprise a Gly-Gly-Leu (SEQID NO.4), Ala-Ala-Leu (SEQID NO.:5), growth hormone for delivery to a biological compartment of Phe-Val-Arg (SEQID NO.:6), Val-Gly-Arg (SEQID NO.:7) a subject. The GH can be introduced into any portion of the and is cleavable by an enzyme that exhibits higher activity on delivery construct that does not disrupt a cell-binding or tran the basal-lateral side of a polarized epithelial cell than it does scytosis activity. The GH is connected with the remainder of on the apical side of the polarized epithelial cell. In certain the delivery construct with a cleavable linker. embodiments, the first and/or the second cleavable linker 0101. Accordingly, the delivery constructs of the inven comprises an amino acid sequence that is selected from the tion generally comprise the following structural elements, group consisting of Ala-Ala-Pro-Phe (SEQ ID NO.:1), Gly each element imparting particular functions to the delivery Gly-Phe (SEQID NO.:2), Ala-Ala-Pro-Val (SEQID NO.:3), construct: (1) a “receptor binding domain that functions as a Gly-Gly-Leu (SEQID NO.4), Ala-Ala-Leu (SEQID NO.:5), ligand for a cell Surface receptor and that mediates binding of Phe-Val-Arg (SEQID NO.:6), Val-Gly-Arg (SEQID NO.:7) the construct to a cell; (2) a “transcytosis domain' that medi and is cleavable by an enzyme that exhibits higher activity in ates transcytosis from alumen bordering the apical Surface of the plasma than it does on the apical side of a polarized a mucous membrane to the basal-lateral side of a mucous epithelial cell. membrane; (3) the growth hormone; and (4) a cleavable linker 0107. In certain embodiments, the enzyme that is present that connects the GH to the remainder of the delivery con at a basal-lateral membrane of a polarized epithelial cell is Struct. selected from the group consisting of Cathepsin GI. Chymot 0102 The delivery constructs of the invention offer sev rypsin I, Elastase I, Subtilisin AI, Subtilisin AII, Thrombin I, eral advantages over conventional techniques for local or and Urokinase I. systemic delivery of GH to a subject. Foremost among Such 0108. In certain embodiments, the receptor binding advantages is the ability to deliver the GH without using a domain is selected from the group consisting of receptor needle to puncture the skin of the subject. Many subjects binding domains from Pseudomonas exotoxin A, cholera require repeated, regular doses of GH. Such subjects’ quality toxin, botulinum toxin, diptheria toxin, Shiga toxin, or shiga of life would be greatly improved if the delivery of GH could like toxin; monoclonal antibodies; polyclonal antibodies; be accomplished without injection, by avoiding pain or single-chain antibodies: TGFC: EGF; IGF-I; IGF-II; IGF-III: potential complications associated therewith. IL-1, IL-2, IL-3, IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. In (0103. In addition, connection of the GH to the remainder certain embodiments, the receptor binding domain binds to a of the delivery construct with a linker that is cleaved by an cell-surface receptor that is selected from the group consist enzyme present at a basal-lateral membrane of an epithelial ing of C2-macroglobulin receptor, epidermal growth factor cell allows the GH to be liberated from the delivery construct receptor, transferrin receptor, chemokine receptor, CD25, and released from the remainder of the delivery construct CD11B, CD11C, CD80, CD86, TNFC receptor, TOLL recep Soon after transcytosis across the epithelial membrane. Such tor, M-CSF receptor, GM-CSF receptor, scavenger receptor, liberation reduces the probability of induction of an immune and VEGF receptor. In further embodiments, the receptor response against the GH. It also allows the GH to interact with binding domain of Pseudomonas exotoxin A is Domain Ia of its target free from the remainder of the delivery construct. Pseudomonas exotoxin A. In yet further embodiments, the 0104. Other advantages of the delivery constructs of the receptor binding domain of Pseudomonas exotoxin A has an invention will be apparent to those of skill in the art. amino acid sequence that is SEQID NO.:9. 0105. In certain embodiments, the invention provides a 0109. In certain embodiments, the transcytosis domain is delivery construct that comprises a receptor binding domain, selected from the group consisting of transcytosis domains a transcytosis domain, a growth hormone to be delivered to a from Pseudomonas exotoxin A, botulinum toxin, diptheria subject, and a cleavable linker. Cleavage at the cleavable toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero linker separates the GH from the remainder of the construct. toxin, Shiga toxin, and Shiga-like toxin. In further embodi The cleavable linker can be cleavable by an enzyme that is ments, the transcytosis domain is Pseudomonas exotoxin A present at a basal-lateral membrane of a polarized epithelial transcytosis domain. In still further embodiments, the cell of the subject or in the plasma of the subject. In certain Pseudomonas eXotoxin A transcytosis domain has an amino embodiments, the enzyme that is at a basal-lateral membrane acid sequence that is SEQID NO.10. of a polarized epithelial cell exhibits higher activity on the 0110 5.3.1. Receptor Binding Domain basal-lateral side of a polarized epithelial cell than it does on 0111. The delivery constructs of the invention generally the apical side of the polarized epithelial cell. In certain comprise a receptor binding domain. The receptor binding embodiments, the enzyme that is in the plasma of the Subject domain can be any receptor binding domain known to one of exhibits higher activity in the plasma than it does on the apical skill in the art without limitation to bind to a cell surface side of a polarized epithelial cell. receptor that is present on the apical membrane of an epithe 0106. In certain embodiments, the delivery construct fur lial cell. Preferably, the receptor binding domain binds spe ther comprises a second cleavable linker. In certain embodi cifically to the cell surface receptor. The receptor binding ments, the first and/or the second cleavable linker comprises domain should bind to the cell surface receptor with sufficient an amino acid sequence that is selected from the group con affinity to allow endocytosis of the delivery construct. sisting of Ala-Ala-Pro-Phe (SEQ ID NO.:1), Gly-Gly-Phe 0112. In certain embodiments, the receptor binding (SEQIDNO.:2), Ala-Ala-Pro-Val (SEQID NO.:3), Gly-Gly domain can comprise a peptide, a polypeptide, a protein, a Leu (SEQID NO.:4), Ala-Ala-Leu (SEQIDNO.:5), Phe-Val lipid, a carbohydrate, or a small organic molecule, or a com Arg (SEQID NO.:6), Val-Gly-Arg (SEQ ID NO.:7). In cer bination thereof. Examples of each of these molecules that tain embodiments, the first and/or the second cleavable linker bind to cell Surface receptors present on the apical membrane comprises an amino acid sequence that is selected from the of epithelial cells are well known to those of skill in the art. US 2009/0305978 A1 Dec. 10, 2009

Suitable peptides or polypeptides include, but are not limited tor binding domains that have this kind of expression pattern to, bacterial toxin receptor binding domains, such as the include, but are not limited to, TGFC., EGF, IGF-I, IGF-II, and receptor binding domains from PE, cholera toxin, botulinum IGF-III. toxin, diptheria toxin, Shiga toxin, Shiga-like toxin, etc.; anti 0116. In certain embodiments, the delivery constructs of bodies, including monoclonal, polyclonal, and single-chain the invention comprise more than one domain that can func antibodies, or derivatives thereof, growth factors, such as tion as a receptor binding domain. For example, the delivery EGF, IGF-I, IGF-II, IGF-III etc.: cytokines, such as IL-1, construct can comprise PE domain Ia in addition to another IL-2, IL-3, IL-6, etc; chemokines, such as MIP-1a, MIP-1b, receptor binding domain. MCAF, IL-8, etc.; and other ligands, such as CD4, cell adhe 0117 The receptor binding domain can be attached to the sion molecules from the immunoglobulin Superfamily, inte remainder of the delivery construct by any method or means grins, ligands specific for the IgA receptor, etc. See, e.g., known by one of skill in the art to be useful for attaching such Pastan et al., 1992, Annu. Rev. Biochem. 61:331-54; and U.S. molecules, without limitation. In certain embodiments, the Pat. Nos. 5,668,255, 5,696,237, 5,863,745, 5,965,406, 6,022, receptor binding domain is expressed together with the 950, 6,051,405, 6,251,392, 6,440,419, and 6,488,926. The remainder of the delivery construct as a fusion protein. Such skilled artisan can select the appropriate receptor binding embodiments are particularly usefull when the receptor bind domain based upon the expression pattern of the receptor to ing domain and the remainder of the construct are formed which the receptor binding domain binds. from peptides or polypeptides. 0113 Lipids suitable for receptor binding domains 0118. In other embodiments, the receptor binding domain include, but are not limited to, lipids that themselves bind cell is connected with the remainder of the delivery construct with Surface receptors, such as sphingosine-1-phosphate, lyso a linker. In yet other embodiments, the receptor binding phosphatidic acid, sphingosylphosphorylcholine, retinoic domain is connected with the remainder of the delivery con acid, etc.; lipoproteins such as apolipoprotein E, apolipopro struct without a linker. Either of these embodiments are useful tein A, etc., and glycolipids such as lipopolysaccharide, etc.; when the receptor binding domain comprises a peptide, glycosphingolipids such as globotriaosylceramide and gala polypeptide, protein, lipid, carbohydrate, nucleic acid, or biosylceramide; and the like. Carbohydrates suitable for Small organic molecule. receptor binding domains include, but are not limited to, 0119. In certain embodiments, the linker can form a cova monosaccharides, disaccharides, and polysaccharides that lent bond between the receptor binding domain and the comprise simple Sugars such as glucose, fructose, galactose, remainder of the delivery construct. In certain embodiments, etc.; and glycoproteins such as mucins, selecting, and the like. the covalent bond can be a peptide bond. In other embodi Suitable Small organic molecules for receptor binding ments, the linker can link the receptor binding domain to the domains include, but are not limited to, vitamins, such as remainder of the delivery construct with one or more non vitamin A, B, B. B. B. B., B, C, D, E, and K, amino covalent interactions of sufficient affinity. One of skill in the acids, and other Small molecules that are recognized and/or art can readily recognize linkers that interact with each other taken up by receptors present on the apical Surface of epithe with sufficient affinity to be useful in the delivery constructs lial cells. U.S. Pat. No. 5,807,832 provides an example of of the invention. For example, biotin can be attached to the Such small organic molecule receptor binding domains, Vita receptor binding domain, and streptavidin can be attached to min B12. the remainder of the molecule. In certain embodiments, the 0114. In certain embodiments, the receptor binding linker can directly link the receptor binding domain to the domain can bind to a receptor found on an epithelial cell. In remainder of the molecule. In other embodiments, the linker further embodiments, the receptor binding domain can bind itself comprises two or more molecules that associate in order to a receptor found on the apical membrane of an epithelial to link the receptor binding domain to the remainder of the cell. The receptor binding domain can bind to any receptor molecule. Exemplary linkers include, but are not limited to, known to be present on the apical membrane of an epithelial straight or branched-chain carbon linkers, heterocyclic car cell by one of skill in the art without limitation. For example, bon linkers, Substituted carbon linkers, unsaturated carbon the receptor binding domain can bind to C2-MR, EGFR, or linkers, aromatic carbon linkers, peptide linkers, etc. IGFR. An example of a receptor binding domain that can bind 0.120. In embodiments where a linker is used to connect to C.2-MR is domain Ia of PE. Accordingly, in certain embodi the receptor binding domain to the remainder of the delivery ments, the receptor binding domain is domain Ia of PE. In construct, the linkers can be attached to the receptor binding other embodiments, the receptor binding domain is a portion domain and/or the remainder of the delivery construct by any of domain Ia of PE that can bind to C2-MR. Exemplary means or method known by one of skill in the art without receptor binding domains that can bind to EGFR include, but limitation. For example, the linker can be attached to the are riot limited to, EGF and TGFC. Examples of receptor receptor binding domain and/or the remainder of the delivery binding domains that can bind to IGFR include, but are not construct with an ether, ester, thioether, thioester, amide, limited to, IGF-I, IGF-II, or IGF-III. Thus, in certain embodi imide, disulfide, peptide, or other suitable moiety. The skilled ments, the receptor binding domain is EGF, IGF-I, IGF-II, or artisan can select the appropriate linker and method for IGF-III. In other embodiments, the receptor binding domain attaching the linker based on the physical and chemical prop is a portion of EGF, IGF-I, IGF-II, or IGF-III that can bind to erties of the chosen receptor binding domain and the linker. the EGF or IGF receptor. The linker can be attached to any Suitable functional group on 0115. In certain embodiments, the receptor binding the receptor binding domain or the remainder of the molecule. domain binds to a receptor that is highly expressed on the For example, the linker can be attached to sulfhydryl ( S), apical membrane of a polarized epithelial cell but is not carboxylic acid (COOH) or free amine ( NH2) groups, expressed or expressed at low levels on antigen presenting which are available for reaction with a suitable functional cells, such as, for example, dendritic cells. Exemplary recep group on a linker. These groups can also be used to connect US 2009/0305978 A1 Dec. 10, 2009

the receptor binding domain directly connected with the native PE, which spans residues 253-364 of PE. For example, remainder of the molecule in the absence of a linker. the transcytosis domain can comprise a portion of PE that 0121 Further, the receptor binding domain and/or the spans residues 280-344 of domain II of PE. The amino acids remainder of the delivery construct can be derivatized in order at positions 339 and 343 appear to be necessary for transcy to facilitate attachment of a linker to these moieties. For tosis. See Siegallet al., 1991, Biochemistry 30:7154-59. Fur example, Such derivatization can be accomplished by attach ther, conservative or nonconservative substitutions can be ing suitable derivative such as those available from Pierce made to the amino acid sequence of the transcytosis domain, Chemical Company, Rockford, Ill. Alternatively, derivatiza as long as transcytosis activity is not substantially eliminated. tion may involve chemical treatment of the receptor binding A representative assay that can routinely be used by one of domain and/or the remainder of the molecule. For example, skill in the art to determine whether a transcytosis domain has glycol cleavage of the Sugar moiety of a carbohydrate or transcytosis activity is described below. glycoprotein receptor binding domain with periodate gener I0127. Without intending to be limited to any particular ates free aldehyde groups. These free aldehyde groups may be theory or mechanism of action, the transcytosis domain is reacted with free amine or hydrazine groups on the remainder believed to permit the trafficking of the delivery construct of the molecule in order to connect these portions of the through a polarized epithelial cell after the construct binds to molecule. See, e.g., U.S. Pat. No. 4,671,958. Further, the a receptor present on the apical Surface of the polarized epi skilled artisan can generate free Sulfhydryl groups on proteins thelial cell. Such trafficking through a polarized epithelial cell to provide a reactive moiety for making a disulfide, thioether, is referred to herein as “transcytosis.” This trafficking permits thioester, etc. linkage. See, e.g., U.S. Pat. No. 4,659,839. the release of the delivery construct from the basal-lateral 0122) Any of these methods for attaching a linker to a membrane of the polarized epithelial cell. receptor binding domain and/or the remainder of a delivery I0128 5.3.3. Growth for Delivery construct can also be used to connect a receptor binding I0129. The delivery constructs of the invention also com domain with the remainder of the delivery construct in the prise a growth hormone. The GH can be attached to the absence of a linker. In such embodiments, the receptor bind remainder of the delivery construct by any method known by ing domain is coupled with the remainder of the construct one of skill in the art, without limitation. In certain embodi using a method suitable for the particular receptor binding ments, the GH is expressed together with the remainder of the domain. Thus, any method Suitable for connecting a protein, delivery construct as a fusion protein. In Such embodiments, peptide, polypeptide, nucleic acid, carbohydrate, lipid, or the GH can be inserted into or attached to any portion of the Small organic molecule to the remainder of the delivery con delivery construct, so long as the receptor binding domain, struct known to one of skill in the art, without limitation, can the transcytosis domain, and GH retain their activities. The be used to connect the receptor binding domainto the remain GH is connected with the remainder of the construct with a der of the construct. In addition to the methods for attaching cleavable linker, or a combination of cleavable linkers, as a linker to a receptor binding domain or the remainder of a described below. delivery construct, as described above, the receptor binding 0.130. In native PE, the Ib loop (domain Ib) spans amino domain can be connected with the remainder of the construct acids 365 to 399, and is structurally characterized by a disul as described, for example, in U.S. Pat. Nos. 6,673,905; 6,585, fide bond between two cysteines at positions 372 and 379. 973; 6,596,475; 5,856,090; 5,663,312; 5,391,723; 6,171,614; This portion of PE is not essential for any known activity of 5,366,958; and 5,614,503. PE, including cell binding, transcytosis, ER retention or ADP 0123. In certain embodiments, the receptor binding ribosylation activity. Accordingly, domain Ib can be deleted domain can be a monoclonal antibody. In some of these entirely, or modified to contain GH. embodiments, the chimeric immunogen is expressed as a 0131 Thus, in certain embodiments, the GH can be fusion protein that comprises an immunoglobulin heavy inserted into domain Ib. If desirable, the GH can be inserted chain from an immunoglobulin specific for a receptor on a into domain Ib wherein the cysteines at positions 372 and 379 cell to which the chimeric immunogen is intended to bind. are not cross-linked. This can be accomplished by reducing The light chain of the immunoglobulin then can be co-ex the disulfide linkage between the cysteines, by deleting the pressed with the chimeric immunogen, thereby forming a cysteines entirely from the Ib domain, by mutating the cys light chain-heavy chain dimer. In other embodiments, the teines to other residues, such as, for example, serine, or by antibody can be expressed and assembled separately from the other similar techniques. Alternatively, the OH can be remainder of the chimeric immunogen and chemically linked inserted into the Ib loop between the cysteines at positions thereto. 372 and 379. In such embodiments, the disulfide linkage 0.124 5.3.2. Transcytosis Domain between the cysteines can be used to constrain the GH if 0.125. The delivery constructs of the invention also com desirable. In any event, in embodiments where the GH is prise a transcytosis domain. The transcytosis domain can be inserted into domain Ib of PE, or into any other portion of the any transcytosis domain known by one of skill in the art to delivery construct, the GH should be flanked by cleavable effect transcytosis of chimeric proteins that have bound to a linkers such that cleavage at the cleavable linkers liberates the cell Surface receptor present on the apical membrane of an GH from the remainder of the construct. epithelial cell. In certain embodiments, the transcytosis (0132. In other embodiments, the GH can be connected domain is a transcytosis domain from PE, diptheria toxin, with the N-terminal or C-terminal end of a polypeptide por pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, tion of the delivery construct. In such embodiments, the shiga toxin, or shiga-like toxin. See, for example, U.S. Pat. method of connection should be designed to avoid interfer Nos. 5,965,406, and 6,022,950. In preferred embodiments, ence with other functions of the delivery construct, such as the transcytosis domain is domain II of PE. receptor binding or transcytosis. In yet other embodiments, 0126 The transcytosis domain need not, though it may, the GH can be connected with a side chain of an amino acid of comprise the entire amino acid sequence of domain II of the delivery construct. The GH is connected with the remain US 2009/0305978 A1 Dec. 10, 2009

der of the delivery construct with a cleavable linker, as across the mucous membrane and release from the epithelial described below. In such embodiments, the GH to be deliv cell into the cellular matrix on the basal-lateral side of the ered can be connected with the remainder of the delivery membrane. Further, cleaving enzymes could be used that are construct with one or more cleavable linkers such that cleav present inside the epithelial cell, such that the cleavable linker age at the cleavable linker(s) separates the GH from the is cleaved prior to release of the delivery construct from the remainder of the delivery construct. It should be noted that, in basal-lateral membrane, so long as the cleaving enzyme does certain embodiments, the GH of interest can also comprise a not cleave the delivery construct before the delivery construct short (1-20 amino acids, preferably 1-10 amino acids, and enters the trafficking pathway in the polarized epithelial cell more preferably 1-5 amino acids) leaderpeptide in addition to that results in release of the delivery construct and GH from the GH of interest that remains attached to the GH following the basal-lateral membrane of the cell. cleavage of the cleavable linker. Preferably, this leader pep 0.139. In certain embodiments, the cleaving enzyme is a tide does not affect the activity or immunogenicity of the GH. peptidase. In other embodiments, the cleaving enzyme is an Even more preferably, the cleavable linker is selected such RNAse. In yet other embodiments, the cleaving enzyme can that cleavage of the cleavable linker releases the GH in its cleave carbohydrates. Preferred peptidases include, but are mature, native, active form without any amino acids present not limited to, Cathepsin GI. Chymotrypsin I, Elastase I, in the released GH that are not present in endogenously pro Subtilisin AI, Subtilisin AII, Thrombin I, and Urokinase I. duced mature GH. Table 1 presents these enzymes together with an amino acid 0133. In embodiments where the GH is expressed together sequence that is recognized and cleaved by the particular with another portion of the delivery construct as a fusion peptidase. protein, the GH can be can be inserted into the delivery construct by any method known to one of skill in the art TABLE 1. without limitation. For example, amino acids corresponding to the GH can be inserted directly into the delivery construct, Peptidases Present Near Basal-Lateral Mucous with or without deletion of native amino acid sequences. In embranes certain embodiments, all or part of the Ib domain of PE can be Amino Acid Sequence Recognized and deleted and replaced with the GH. In certain embodiments, Peptidase Cleaved the cysteine residues of the Ib loop are deleted so that the GH Cathepsin GI Ala-Ala-Pro-Phe (SEQ ID NO. : 1) remains unconstrained. In other embodiments, the cysteine residues of the Ib loop are linked with a disulfide bond and Chymotrypsin I Gly-Gly-Phe (SEQ ID NO. : 2) constrain the GH. 0134. The GH can be any GH that is desired to be intro Elastase I Ala-Ala-Pro-Wall (SEQ ID NO. : 3) duced into a subject. Thus, the GH can be a human GH, a Subtilisin AI Gly-Gly-Lieu. (SEQ ID NO. : 4) mouse GH, a rat GH, and the like. Preferably, the GH is matched to the subject to whom the GH is to be administered, Subtilisin AII Ala-Ala-Lieu. (SEO ID NO. : 5) for example, if the GH is to be administered to a human, the Thrombin I Phe-Val-Arg (SEQ ID NO. : 6) GH is preferably human GH. 0135. In certain embodiments, the GH can be selected to Urokinase I Wal-Gly-Arg (SEO ID NO. : 7) not be cleavable by an enzyme present at the basal-lateral membrane of an epithelial cell. For example, the assays 0140. In certain embodiments, the delivery construct can described in the examples can be used to routinely test comprise more than one cleavable linker, wherein cleavage at whether such a cleaving enzyme can cleave the GH to be either cleavable linker can separate the GH to be delivered delivered. If so, the GH can be routinely altered to eliminate from the delivery construct. In certain embodiments, the the offending amino acid sequence recognized by the cleav cleavable linker can be selected to avoid the use of cleavable ing enzyme. The altered GH can then be tested to ensure that linkers that comprise sequences present in the GH to be it retains activity using methods routine in the art. delivered. For example, if the GH comprises AAL, the cleav 0.136 5.3.4. Cleavable Linkers able linker can be selected to be cleaved by an enzyme that 0.137 In the delivery constructs of the invention, the GH to does not recognize this sequence. be delivered to the subject is connected with the remainder of 0141 Further, the cleavable linker preferably exhibits a the delivery construct with one or more cleavable linkers. The greater propensity for cleavage than the remainder of the number of cleavable linkers present in the construct depends, delivery construct. As one skilled in the art is aware, many at least in part, on the location of the OH in relation to the peptide and polypeptide sequences can be cleaved by pepti remainder of the delivery construct and the nature of the GH. dases and proteases. In certain embodiments, the cleavable When the GH is inserted into the delivery construct, the GH linker is selected to be preferentially cleaved relative to other can be flanked by cleavable linkers, such that cleavage at both amino acid sequences present in the delivery construct during linkers separates the GH. The flanking cleavable linkers can administration of the delivery construct. In certain embodi be the same or different from each other. When the GH can be ments, the receptor binding domain is Substantially (e.g., separated from the remainder of the delivery construct with about 99%, about 95%, about 90%, about 85%, about 80, or cleavage at a single linker, the delivery constructs can com about 75%) intact following delivery of the delivery construct prise a single cleavable linker. to the bloodstream of the subject. In certain embodiments, the 0.138. The cleavable linkers are generally cleavable by a translocation domain is Substantially (e.g., about 99%, about cleaving enzyme that is present at or near the basal-lateral 95%, about 90%, about 85%, about 80, or about 75%) intact membrane of an epithelial cell. By selecting the cleavable following delivery of the delivery construct to the blood linker to be cleaved by such enzymes, the GH can be liberated stream of the subject. In certain embodiments, the GH is from the remainder of the construct following transcytosis substantially (e.g., about 99%, about 95%, about 90%, about US 2009/0305978 A1 Dec. 10, 2009

85%, about 80, or about 75%) intact following delivery of the delivery construct to the bloodstream of the subject. In certain TABLE 2 - continued embodiments, the cleavable linkeris Substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about Plasma Peptidases 75%) cleaved following delivery of the delivery construct to Amino Acid Sequence the bloodstream of the subject. Peptidase Recognized and Cleaved 0142. In other embodiments, the cleavable linker is Neprilysin in combi – Xaa-Phe-Xaa-Xaa cleaved by a cleaving enzyme found in the plasma of the nation with dipep- (SEQ ID NO. : 2O) tidyl-peptidase IV Xaa-Tyr-Xaa-Xaa Subject. Any cleaving enzyme known by one of skill in the art (SEQ ID NO. : 21) to be present in the plasma of the subject can be used to cleave Xaa-Trp-Xaa-Xaa the cleavable linker. Use of such enzymes to cleave the cleav (SEQ ID NO. : 22) able linkers is less preferred than use of cleaving enzymes Renin in combination Asp-Arg-Tyr-Ile-Pro-Phe-His found near the basal-lateral membrane of a polarized epithe with dipeptidyl- Leu-Leu- (Wall, Ala or Pro) - lial cell because it is believed that more efficient cleavage will peptidase IV Tyr- (Ser, Pro, or Ala) occur in near the basal-lateral membrane. However, if the (SEQ ID NO. :23) skilled artisan determines that cleavage mediated by a plasma enzyme is sufficiently efficient to allow cleavage of a suffi 0143. Thus, in certain more preferred embodiments, the cient fraction of the delivery constructs to avoid adverse cleavable linker can be any cleavable linker known by one of effects, such plasma cleaving enzymes can be used to cleave skill in the art to be cleavable by an enzyme that is present at the delivery constructs. Accordingly, in certain embodiments, the basal-lateral membrane of an epithelial cell. In certain the cleavable linker can be cleaved with an enzyme that is embodiments, the cleavable linker comprises a peptide. In selected from the group consisting of caspase-1, caspase-3, other embodiments, the cleavable linker comprises a nucleic 1, , proprotein acid, such as RNA or DNA. In still other embodiments, the convertase 4, proprotein convertase 4 PACE 4, prolyl oli cleavable linker comprises a carbohydrate. Such as a disac gopeptidase, endothelin cleaving enzyme, dipeptidyl-pepti charide or a trisaccharide. In certain embodiments, the cleav dase IV, , neprilysin, renin, and esterase. See, able linker is a peptide that comprises an amino acid sequence e.g., U.S. Pat. No. 6,673,574. Table 2 presents these enzymes that is selected from the group consisting of Ala-Ala-Pro-Phe together with an amino acid sequence(s) recognized by the (SEQ ID NO.:1), Gly-Gly-Phe (SEQ ID NO.:2), Ala-Ala particular peptidase. The peptidase cleaves a peptide com Pro-Val (SEQ ID NO.:3), Gly-Gly-Leu (SEQ ID NO.4), Ala-Ala-Leu (SEQID NO.:5), Phe-Val-Arg (SEQID NO.:6), prising these sequences at the N-terminal side of the amino Val-Gly-Arg (SEQID NO.:7). acid identified with an asterisk. 0144. Alternatively, in less preferred embodiments, the cleavable linker can be any cleavable linker known by one of TABLE 2 skill in the art to be cleavable by an enzyme that is present in Plasma Peptidases the plasma of the subject to whom the delivery construct is administered. In certain embodiments, the cleavable linker Amino Acid Sequence comprises a peptide. In other embodiments, the cleavable Peptidase Recognized and Cleaved linker comprises a nucleic acid, such as RNA or DNA. In still Caspase-1 Tyr-Val-Ala-Asp-Xaak other embodiments, the cleavable linker comprises a carbo (SEQ ID NO. : 1) hydrate, Such as a disaccharide or a trisaccharide. In certain Caspase-3 Asp-Xaa-Xaa-Asp-Xaa embodiments, the cleavable linker is a peptide that comprises (SEQ ID NO. : 12) an amino acid sequence that is selected from the group con sisting of amino acid sequences presented in Table 2. Proprotein Arg- (Xaa) n-Arg-Xaar; 0145. In certain embodiments, the delivery construct com convertase 1 n = 0, 2, 4 or 6 prises more than one cleavable linker. In certain embodi (SEQ ID NO. : 13) ments, cleavage at any of the cleavable linkers will separate Proprotein Lys - (Xaa) n-Arg-Xaa; the GH to be delivered from the remainder of the delivery convertase 2 n = 0, 2, 4, or 6 construct. In certain embodiments, the delivery construct (SEQ ID NO. : 14) comprises a cleavable linker cleavable by an enzyme present Proprotein Glp-Arg-Thr-Lys-Arg-Xaa at the basal-lateral side of a polarized epithelial membrane convertase 4 (SEQ ID NO. : 5) and a cleavable linkers cleavable by an enzyme that is present in the plasma of the subject to whom the delivery construct is Proprotein Arg-Val-Arg-Arg-Xaa administered. convertase 4 PACE 4 (SEQ ID NO. : 6) Decanoyl-Arg-Val-Arg-Arg-Xaak 0146 In other embodiments, the cleavable linker can be a (SEO ID NO. : 17) cleavable linker that is cleaved following a change in the Prolyloligopeptidase Pro-Xaa-Trp-Val-Pro-Xaa environment of the delivery construct. For example, the Endothelin cleaving (SEQ ID NO. : 8) cleavable linker can be a cleavable linker that is pH sensitive enzyme in combination and is cleaved by a change in pH that is experienced when the with dipeptidyl delivery construct is released from the basal-lateral mem peptidase IV brane of a polarized epithelial cell. For instance, the intestinal Signal peptidase Trp-Val* - Ala-Xaa lumen is strongly alkaline, while plasma is essentially neu (SEQ ID NO. : 19) tral. Thus, a cleavable linker can be a moiety that is cleaved upon a shift from alkaline to neutral pH. The change in the environment of the delivery construct that cleaves the cleav US 2009/0305978 A1 Dec. 10, 2009

able linker can be any environmental change that that is about 24 hours, by about 1 day to about 3 days, by about 1 day experienced when the delivery construct is released from the to about 1 week, by about 1 week to about 2 weeks, by about basal-lateral membrane of a polarized epithelial cell known 2 weeks to about 1 month, by about 4 to about 8 weeks, by by one of skill in the art, without limitation. about 1 to about 3 months, or by about 1 to about 6 months. 0147 5.4. Methods of Administration 0154 The growth hormones to be delivered are generally 0148. The delivery constructs of the invention can be growth hormones for which a large amount of knowledge administered to a subject by any method known to one of skill regarding dosage, frequency of administration, and methods in the art. In certain embodiments, the delivery constructs are for assessing effective concentrations in Subjects has accu contacted to a mucosal membrane of the Subject. For mulated. Such knowledge can be used to assess efficiency of example, the mucosal membrane can be present in the eye, delivery, effective concentration of the growth hormone in the nose, mouth, trachea, lungs, esophagus, Stomach, Small intes Subject, and frequency of administration. Thus, the knowl tine, large intestine, rectum, anus, Sweat glands, Vulva, edge of those skilled in the art can be used to determine vagina, or penis of the Subject. Preferably, the mucosal mem whether, for example, the amount of GH delivered to the brane is a mucosal membrane present in the digestive tract of subject is an amount effective to increase the size of the the Subject, such as a mucosal membrane in the mouth, subject by at least about 12%, the dosage should be increased esophagus, stomach, Small intestine, large intestine, or rec or decreased to achieve this result, the subject should be tum of the Subject. Nasal mucosal membranes are equally administered the delivery construct more or less frequently to preferred. achieve this result, and the like. 0149. In embodiments where the mucosal membrane is in 0155 5.4.2. Determining Amounts of Growth Hormone the digestive tract of the subject, the delivery constructs are Delivered preferably administered to the subject orally. Thus, the deliv 0156 The methods of the invention can be used to deliver, ery construct can be formulated to protect the delivery con either locally or systemically, a pharmaceutically effective struct from degradation in the acid environment of the stom amount of a GH to a subject. The skilled artisan can determine ach, if necessary. For example, many embodiments of the whether the methods result in delivery of such a pharmaceu delivery constructs of the invention comprise polypeptide tically effective amount of the GH. The exact methods will domains with defined activities. Unless such delivery con depend on the GH that is delivered, but generally will rely on structs are protected from acid and/or enzymatic hydrolysis in either determining the concentration of the GH in the blood of the stomach, the constructs will generally be digested before the subject or in the biological compartment of the subject delivery of substantial amounts of the GH to be delivered. where the GH exerts its effects. Alternatively or additionally, Accordingly, composition formulations that protect the deliv the effects of the GH on the subject can be monitored. ery construct from degradation can be used in administration 0157 For example, the skilled artisan can determine of these delivery constructs. whether a pharmaceutically effective amount of GH had been 0150 5.4.1. Dosage delivered to the Subject by, for example, taking a plasma 0151. Generally, an amount of the delivery construct com sample from the Subject and determining the concentration of prising a GH effective to increase the size of a subject by at GH therein. One exemplary method for determining the con least about 12% is administered to a subject. The skilled centration of GH is by performing an ELISA assay, but any artisan can readily determine if the dosage of the delivery other Suitable assay known to the skilled artisan can be used. construct is sufficient to deliver an effective amount of the 0158 Alternatively, one of skill in the art can determine if GH, as described below. In certain embodiments, between an effective amount of GH had been delivered to the subject about 1 Lugandabout 1 g of delivery construct is administered. by monitoring any effect of a GH known by one of skill in the In other embodiments, between about 10 ug and about 500 art, without limitation, can be assessed in determining mg of delivery construct is administered. In still other whether an effective amount of the GH has been adminis embodiments, between about 10 ug and about 100 mg of tered. Exemplary effects include, but are not limited to, recep delivery construct is administered. In yet other embodiments, tor binding, receptor activation, downstream effects of recep between about 10 ug and about 1000 ug of delivery construct tor binding, downstream effects of receptor activation, is administered. In still other embodiments, between about 10 coordination of compounds, effective blood clotting, bone ug and about 250 g of delivery construct is administered. In growth, wound healing, cellular proliferation, weight yet other embodiments, between about 10 ug and about 100 increase, etc. ug of delivery construct is administered. Preferably, between 0159 5.5. Polynucleotides Encoding Delivery Constructs about 10 ug and about 50 ug of delivery construct is admin 0160. In another aspect, the invention provides polynucle istered. otides comprising a nucleotide sequence encoding the deliv 0152 The volume of a composition comprising the deliv ery constructs. These polynucleotides are useful, for ery construct that is administered will generally depend on example, for making the delivery constructs. In yet another the concentration of delivery construct and the formulation of aspect, the invention provides an expression system that com the composition. In certain embodiments, a unit dose of the prises a recombinant polynucleotide sequence encoding a delivery construct composition is between about 0.05 ml and receptor binding domain, a transcytosis domain, and a about 1 ml, preferably about 0.5 ml. The delivery construct polylinkerinsertion site for a polynucleotide sequence encod compositions can be prepared in dosage forms containing ing a GH. The polylinkerinsertion site can be anywhere in the between 1 and 50 doses (e.g., 0.5 ml to 25 ml), more usually polynucleotide sequence so long as the polylinker insertion between 1 and 10 doses (e.g., 0.5 ml to 5 ml) does not disrupt the receptor binding domain or the transcy 0153. The delivery construct compositions of the inven tosis domain. The polylinker insertion site should be oriented tion can be administered in one dose or in multiple doses. A near a polynucleotide sequence that encodes a cleavable dose can be followed by one or more doses spaced by about 1 linker so that cleavage at the cleavable linker separates a GH to about 6 hours, by about 6 to about 12 hours, by about 12 to encoded by a nucleic acid inserted into the polylinker inser US 2009/0305978 A1 Dec. 10, 2009

tion site from the remainder of the encoded delivery con recognized and cleaved by PstI. In Such examples, a poly struct. Thus, in embodiments where the polylinker insertion nucleotide encoding GH that is flanked by PstI sequences can site is at an end of the encoded construct, the polynucleotide be inserted into the vector. comprises one nucleotide sequence encoding a cleavable 0166 Further, the polynucleotides can also encode a linker between the polylinker insertion site and the remainder secretory sequence at the amino terminus of the encoded of the polynucleotide. In embodiments where the polylinker delivery construct. Such constructs are useful for producing insertion site is not at the end of the encoded construct, the the delivery constructs in mammalian cells as they simplify polylinker insertion site can be flanked by nucleotide isolation of the immunogen. sequences that each encode a cleavable linker. 0.167 Furthermore, the polynucleotides of the invention 0161 In certain embodiments, the recombinant poly also encompass derivative versions of polynucleotides nucleotides are based on polynucleotides encoding PE, or encoding a delivery construct. Such derivatives can be made portions or derivatives thereof. In other embodiments, the by any method known by one of skill in the art without recombinant polynucleotides are based on polynucleotides limitation. For example, derivatives can be made by site that hybridize to a polynucleotide that encodes PE under specific mutagenesis, including Substitution, insertion, or stringent hybridization conditions. A nucleotide sequence deletion of one, two, three, five, ten or more nucleotides, of encoding PE is presented as SEQID NO.:24. This sequence polynucleotides encoding the delivery construct. Alterna can be used to prepare PCR primers for isolating a nucleic tively, derivatives can be made by random mutagenesis. One acid that encodes any portion of this sequence that is desired. method for randomly mutagenizing a nucleic acid comprises For example, PCR can be used to isolate a nucleic acid that amplifying the nucleic acid in a PCR reaction in the presence encodes one or more of the functional domains of PE. A of 0.1 mM MnCl and unbalanced nucleotide concentrations. nucleic acid so isolated can then be joined to nucleic acids These conditions increase the misincorporation rate of the encoding other functional domains of the delivery constructs polymerase used in the PCR reaction and result in random using standard recombinant techniques. mutagenesis of the amplified nucleic acid. 0162. Other in vitro methods that can be used to prepare a 0.168. Several site-specific mutations and deletions in chi polynucleotide encoding PE, PE domains, or any other func meric molecules derived from PE have been made and char tional domain useful in the delivery constructs of the inven acterized. For example, deletion of nucleotides encoding tion include, but are not limited to, reverse transcription, the amino acids 1-252 of PE yields a construct referred to as polymerase chain reaction (PCR), the ligase chain reaction “PE40-” Deleting nucleotides encoding amino acids 1-279 of (LCR), the transcription-based amplification system (TAS), PE yields a construct referred to as “PE37. See U.S. Pat. No. the self-sustained sequence replication system (3SR) and the 5,602,095. In both of these constructs, the receptor binding QP replicase amplification system (QB). Any Such technique domain of PE, i.e., domain Ia, has been deleted. Nucleic acids known by one of skill in the art to be useful in construction of encoding a receptor binding domain can be ligated to these recombinant nucleic acids can be used. For example, a poly constructs to produce delivery constructs that are targeted to nucleotide encoding the protein or a portion thereof can be the cell Surface receptor recognized by the receptor binding isolated by polymerase chain reaction of cDNA using primers domain. Of course, these recombinant polynucleotides are based on the DNA sequence of PE or a nucleotide encoding a particularly useful for expressing delivery constructs that receptor binding domain. have a receptor binding domain that is not domain Ia of PE. 0163 Guidance for using these cloning and in vitro ampli The recombinant polynucleotides can optionally encode an fication methodologies are described in, for example, U.S. amino-terminal methionine to assist in expression of the con Pat. No. 4,683, 195; Mullis et al., 1987, Cold Spring Harbor struct. In certain embodiments, the receptor binding domain Symp. Ouant. Biol.51:263; and Erlich, ed., 1989, PCR Tech can be ligated to the 5' end of the polynucleotide encoding the nology, Stockton Press, NY. Polynucleotides encoding a transcytosis domain. delivery constructor a portion thereof also can be isolated by 0169. Other nucleic acids encoding mutant forms of PE screening genomic or cDNA libraries with probes selected that can be used as a source of nucleic acids for constructing from the sequences of the desired polynucleotide under Strin the delivery constructs of the invention include, but are not gent, moderately stringent, or highly stringent hybridization limited to, PEA553 and those described in U.S. Pat. Nos. conditions. 5,602,095; 5,512,658 and 5,458.878, and in Vasil et al., 1986, 0164 Construction of nucleic acids encoding the delivery Infect. Immunol. 52:538-48. constructs of the invention can be facilitated by introducing 0170 Accordingly, in certain embodiments, the invention an insertion site for a nucleic acid encoding the GH into the provides a polynucleotide that encodes a delivery construct. construct. In certain embodiments, an insertion site for the The delivery construct comprises a receptor binding domain, GH can be introduced between the nucleotides encoding the a transcytosis domain, a GH to be delivered to a subject, and cysteine residues of domain Ib. In other embodiments, the a cleavable linker. Cleavage at the cleavable linker can sepa insertion site can be introduced anywhere in the nucleic acid rate the GH from the remainder of the construct. The cleav encoding the construct so long as the insertion does not dis able linker can be cleaved by an enzyme that is present at a rupt the functional domains encoded thereby. In certain basal-lateral membrane of a polarized epithelial cell of the embodiments, the insertion site can be in the ER retention Subject or in the plasma of the Subject. domain. 0171 In certain embodiments, the polynucleotide hybrid 0.165. In more specific embodiments, a nucleotide izes under stringent hybridization conditions to any poly sequence encoding a portion of the Ib domain between the nucleotide of this invention. In further embodiments, the cysteine-encoding residues can be removed and replaced with polynucleotide hybridizes under Stringent conditions to a a nucleotide sequence that includes a cloning site cleaved by nucleic acid that encodes any delivery construct of the inven a restriction enzyme. For example, the cloning site can be tion. US 2009/0305978 A1 Dec. 10, 2009

0172. In certain embodiments, the polynucleotide encodes sone-inducible MMTV promoter, a SV40 promoter, a MRP a delivery construct that further comprises a second cleavable pol III promoter, a constitutive MPSV promoter, a tetracy linker. In certain embodiments, the first and/or second cleav cline-inducible CMV promoter (such as the human immedi able linker comprises an amino acid sequence that is selected ate-early CMV promoter), and a constitutive CMV promoter. from the group consisting of Ala-Ala-Pro-Phe (SEQID NO.: 0179 The expression vectors should contain expression 1), Gly-Gly-Phe (SEQID NO.:2), Ala-Ala-Pro-Val (SEQ ID and replication signals compatible with the cell in which the NO.:3), Gly-Gly-Leu (SEQID NO.:4), Ala-Ala-Leu (SEQID delivery constructs are expressed. Expression vectors useful NO.:5), Phe-Val-Arg (SEQID NO.:6), Val-Gly-Arg (SEQID for expressing delivery constructs include viral vectors such NO.:7). In certain embodiments, the first and/or second cleav as retroviruses, adenoviruses and adenoassociated viruses, able linker encoded by the polynucleotide is cleavable by an plasmid vectors, cosmids, and the like. Viral and plasmid enzyme that is selected from the group consisting of Cathe vectors are preferred for transfecting the expression vectors psin GI. Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin into mammalian cells. For example, the expression vector AII, Thrombin I, and Urokinase I. pcDNA 1 (Invitrogen, San Diego, Calif.), in which the expres 0173. In certain embodiments, the receptor binding sion control sequence comprises the CMV promoter, pro domain encoded by the polynucleotide is selected from the vides good rates of transfection and expression into Such group consisting of receptor binding domains from cells. Pseudomonas exotoxin A, cholera toxin, diptheria toxin, 0180. The expression vectors can be introduced into the shiga toxin, or shiga-like toxin; monoclonal antibodies; poly cell for expression of the delivery constructs by any method clonal antibodies; single-chain antibodies; TGF C.: EGF: known to one of skill in the art without limitation. Such IGF-I; IGF-II; IGF-III; IL-1, IL-2: IL-3; IL-6; MIP-1a: MIP methods include, but are not limited to, e.g., direct uptake of 1b; MCAF; and IL-8. In certain embodiments, the receptor the molecule by a cell from solution; facilitated uptake binding domain encoded by the polynucleotide binds to a through lipofection using, e.g., liposomes or immunolipo cell-surface receptor that is selected from the group consist Somes; particle-mediated transfection; etc. See, e.g., U.S. Pat. ing of C2-macroglobulin receptor, EGFR, IGFR, transferrin No. 5.272,065: Goeddelet al., eds, 1990, Methods in Enzy receptor, chemokine receptor, CD25, CD11B, CD11C, mology, Vol. 185, Academic Press, Inc., CA; Krieger, 1990, CD80, CD86, TNFC receptor, TOLL receptor, M-CSF recep Gene Transfer and Expression—A Laboratory Manual. tor, GM-CSF receptor, scavenger receptor, and VEGF recep Stockton Press, NY; Sambrook et al., 1989, Molecular Clon tor. In further embodiments, the receptor binding domain ing A Laboratory Manual, Cold Spring Harbor Laboratory, encoded by the polynucleotide is Domain Ia of Pseudomonas N.Y.; and Ausubel et al., eds., Current Edition, Current Pro exotoxin A. In yet further embodiments, the receptor binding tocols in Molecular Biology, Greene Publishing Associates domain encoded by the polynucleotide has an amino acid and Wiley Interscience, NY. sequence that is SEQID NO.:9. 0181. The expression vectors can also contain a purifica 0.174. In certain embodiments, the transcytosis domain tion moiety that simplifies isolation of the delivery construct. encoded by the polynucleotide is selected from the group For example, a polyhistidine moiety of e.g., six histidine consisting of transcytosis domains from Pseudomonas exo residues, can be incorporated at the amino terminal end of the toxin A, diptheria toxin, pertussis toxin, cholera toxin, heat protein. The polyhistidine moiety allows convenientisolation labile E. coli enterotoxin, shiga toxin, and shiga-like toxin. In of the protein in a single step by nickel-chelate chromatogra further embodiments, the transcytosis domain is Pseudomo phy. In certain embodiments, the purification moiety can be nas exotoxin A transcytosis domain. In still further embodi cleaved from the remainder of the delivery construct follow ments, the Pseudomonas exotoxin A transcytosis domain has ing purification. In other embodiments, the moiety does not an amino acid sequence that is SEQID NO.10. interfere with the function of the functional domains of the 0.175. In certain embodiments, the GH is human growth delivery construct and thus need not be cleaved. hormone. 0182 5.7. Cell for Expressing a Delivery Construct (0176 5.6. Expression Vectors 0183 In yet another aspect, the invention provides a cell 0177. In still another aspect, the invention provides comprising an expression vector for expression of the deliv expression vectors for expressing the delivery constructs. ery constructs, or portions thereof. The cell is preferably Generally, expression vectors are recombinant polynucle selected for its ability to express high concentrations of the otide molecules comprising expression control sequences delivery construct to facilitate purification of the protein. In operatively linked to a nucleotide sequence encoding a certain embodiments, the cell is a prokaryotic cell, for polypeptide. Expression vectors can readily be adapted for example, E. coli. As described in the examples, the delivery function in prokaryotes or eukaryotes by inclusion of appro constructs are properly folded and comprise the appropriate priate promoters, replication sequences, selectable markers, disulfide linkages when expressed in E. coli. etc. to result in stable transcription and translation of mRNA. 0184. In other embodiments, the cell is a eukaryotic cell. Techniques for construction of expression vectors and Useful eukaryotic cells include yeast and mammalian cells. expression of in cells comprising the expression vec Any mammalian cell known by one of skill in the art to be tors are well known in the art. See, e.g., Sambrook et al., 2001, useful for expressing a recombinant polypeptide, without Molecular Cloning A Laboratory Manual, 3" edition, Cold limitation, can be used to express the delivery constructs. For Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and example, Chinese hamster ovary (CHO) cells can be used to Ausubel et al., eds., Current Edition, Current Protocols in express the delivery constructs. Molecular Biology, Greene Publishing Associates and Wiley 0185. 5.8. Compositions Comprising Delivery Constructs Interscience, NY. 0186 The delivery constructs of the invention can be for 0.178 Useful promoters for use in expression vectors mulated as compositions. The compositions are generally include, but are not limited to, a metallothionein promoter, a formulated appropriately for the immediate use intended for constitutive adenovirus major late promoter, a dexametha the delivery construct. For example, if the delivery construct US 2009/0305978 A1 Dec. 10, 2009 is not to be administered immediately, the delivery construct described in DeYoung, 1989, IntJ Pancreatol. 5 Suppl:31-6, can be formulated in a composition Suitable for storage. One and the methods provided in U.S. Pat. Nos. 6,613,332, 6,174. Such composition is a lyophilized preparation of the delivery 529, 6,086,918, 5,922,680, and 5,807,832. construct together with a suitable stabilizer. Alternatively, the 5.8.1. Kits Comprising Compositions delivery construct composition can be formulated for storage (0191) in a solution with one or more suitable stabilizers. Any such 0.192 In yet another aspect, the invention provides a kit stabilizer known to one of skill in the art without limitation that comprises a composition of the invention. In certain can be used. For example, stabilizers suitable for lyophilized embodiments, the kit further comprises instructions that preparations include, but are not limited to, Sugars, salts, direct administration of the composition to a mucous mem Surfactants, proteins, chaotropic agents, lipids, and amino brane of the subject to whom the composition is administered. acids. Stabilizers suitable for liquid preparations include, but In certain embodiments, the kit further comprises instructions are not limited to, Sugars, salts, Surfactants, proteins, chao that directoral administration of the composition to the sub tropic agents, lipids, and amino acids. Specific stabilizers ject to whom the composition is administered. than can be used in the compositions include, but are not 0193 In certain embodiments, the kit comprises a compo limited to, trehalose, serum albumin, phosphatidylcholine, sition of the invention in more or more containers. In certain lecithin, and arginine. Other compounds, compositions, and embodiments, the composition can be in a unit dosage form, methods for stabilizing a lyophilized or liquid preparation of e.g., a tablet, lozenge, capsule, etc. In certain embodiments, the delivery constructs may be found, for example, in U.S. the composition can be provided in or with a device for Pat. Nos. 6,573,237, 6,525,102, 6,391,296, 6,255,284, 6,133, administering the composition, Such as, for example, a device 229, 6,007,791, 5,997.856, and 5,917,021. configured to administer a single-unit dose of the composi 0187 Further, the delivery construct compositions of the tion, e.g., an inhaler. invention can be formulated for administration to a subject. 0.194 5.9. Making and Testing Delivery Constructs Such vaccine compositions generally comprise one or more 0.195 The delivery constructs of the invention are prefer delivery constructs of the invention and a pharmaceutically ably produced recombinantly, as described below. However, acceptable excipient, diluent, carrier, or vehicle. Any Such the delivery constructs may also be produced by chemical pharmaceutically acceptable excipient, diluent, carrier, or synthesis using methods known to those of skill in the art. vehicle known to one of skill in the art without limitation can be used. Examples of a Suitable excipient, diluent, carrier, or (0196) 5.9.1. Manufacture of Delivery Constructs vehicle can be found in Remington's Pharmaceutical Sci 0.197 Methods for expressing and purifying the delivery ences, 21st Ed. 2005, Mack Publishing Co., Easton. constructs of the invention are described extensively in the 0188 In certain embodiments, the delivery construct com examples below. Generally, the methods rely on introduction positions are formulated for oral administration. In Such of an expression vector encoding the delivery construct to a embodiments, the compositions are formulated to protect the cell that can express the delivery construct from the vector. delivery construct from acid and/or enzymatic degradation in The delivery construct can then be purified for administration the stomach. Upon passage to the neutral to alkaline environ to a subject. ment of the duodenum, the delivery construct then contacts a (0198 5.9.2. Testing Delivery Constructs mucous membrane and is transported across the polarized 0199 Having selected the domains of the delivery con epithelial membrane. The delivery constructs may be formu struct, the function of these domains, and of the delivery lated in Such compositions by any method known by one of constructs as a whole, can be routinely tested to ensure that skill in the art, without limitation. the constructs can deliver a GH across mucous membranes of 0189 In certain embodiments, the oral formulation com a subject free from the remainder of the construct. For prises a delivery construct and one or more compounds that example, the delivery constructs can be tested for cell recog can protect the delivery construct while it is in the stomach. nition, transcytosis and cleavage using routine assays. The For example, the protective compound should be able to entire chimeric protein can be tested, or, the function of vari prevent acid and/or enzymatic hydrolysis of the delivery con ous domains can be tested by Substituting them for native struct. In certain embodiments, the oral formulation com domains of the wild-type toxin. prises a delivery construct and one or more compounds that (0200 5.9.2.1. Receptor Binding/Cell Recognition can facilitate transit of the construct from the stomach to the 0201 Receptor binding domain function can be tested by Small intestine. In certain embodiments, the one or more monitoring the delivery construct’s ability to bind to the target compounds that can protect the delivery construct from deg receptor. Such testing can be accomplished using cell-based radation in the stomach can also facilitate transit of the con assays, with the target receptor present on a cell Surface, or in struct from the stomach to the small intestine. Preferably, the cell-free assays. For example, delivery construct binding to a oral formulation comprises one or more compounds that can target can be assessed with affinity chromatography. The protect the delivery construct from degradation in the stom construct can be attached to a matrix in an affinity column, ach and facilitate transit of the construct from the stomach to and binding of the receptor to the matrix detected, or vice the Small intestine. For example, inclusion of sodium bicar versa. Alternatively, if antibodies have been identified that bonate can be useful in facilitating the rapid movement of bind to either the receptor binding domain or its cognate intra-gastric delivered materials from the stomach to the receptor, the antibodies can be used, for example, to detect the duodenum as described in Mrsny et al., 1999, Vaccine receptor binding domain in the delivery construct by immu 17:1425-1433. noassay, or in a competition assay for the cognate receptor. An 0190. Other methods for formulating compositions so that exemplary cell-based assay that detects delivery construct the delivery constructs can pass through the stomach and binding to receptors on cells comprises labeling the construct contact polarized epithelial membranes in the Small intestine and detecting its binding to cells by, e.g., fluorescent cell include, but are not limited to, enteric-coating technologies as Sorting, autoradiography, etc. US 2009/0305978 A1 Dec. 10, 2009

(0202 5.9.2.2. Transcytosis example, Coco-2 cells, under conditions that permit cleavage 0203 The function of the transcytosis domain can be of the linker. Cleavage can be detected by detecting the pres tested as a function of the delivery construct’s ability to pass ence or absence of the label using a reagent that specifically through an epithelial membrane. Because transcytosis first binds the delivery construct, or portion thereof. For example, requires binding to the cell, these assays can also be used to an antibody specific for the delivery construct can be used to assess the function of the cell recognition domain. bind a delivery construct comprising a label distal to the 0204 The delivery construct’s transcytosis activity can be cleavable linker in relation to the portion of the delivery tested by any method known by one of skill in the art, without construct bound by the antibody. Cleavage can then be limitation. In certain embodiments, transcytosis activity can assessed by detecting the presence of the label on molecules be tested by assessing the ability of a delivery construct to bound to the antibody. If cleavage has occurred, little or no enter a non-polarized cell to which it binds. Without intending label should be observed on the molecules bound to the anti to be bound to any particular theory or mechanism of action, body. By performing Such experiments, enzymes that prefer it is believed that the same property that allows a transcytosis entially cleave at the basolateral membrane rather than the domain to pass through a polarized epithelial cell also allows apical membrane can be identified, and, further, the ability of molecules bearing the transcytosis domain to enter non-po such enzymes to cleave the cleavable linker in a delivery larized cells. Thus, the delivery construct’s ability to enter the construct can be confirmed. cell can be assessed, for example, by detecting the physical 0210. Further, cleavage can also be tested using a fluores presence of the construct in the interior of the cell. For cence reporter assay as described in U.S. Pat. No. 6,759,207. example, the delivery construct can be labeled with, for Briefly, in Such assays, the fluorescence reporter is contacted example, a fluorescent marker, and the delivery construct to the basolateral side of a monolayer of suitable epithelial exposed to the cell. Then, the cells can be washed, removing cells under conditions that allow the cleaving enzyme to any delivery construct that has not entered the cell, and the cleave the reporter. Cleavage of the reporter changes the amount of label remaining determined. Detecting the label in structure of the fluorescence reporter, changing it from a this fraction indicates that the delivery construct has entered non-fluorescent configuration to a fluorescent configuration. the cell. The amount of fluorescence observed indicates the activity of 0205. In other embodiments, the delivery construct’s tran the cleaving enzyme present at the basolateral membrane. Scytosis ability can be tested by assessing the delivery con 0211 Further, cleavage can also be tested using an intra struct’s ability to pass through a polarized epithelial cell. For molecularly quenched molecular probe. Such as those example, the delivery construct can be labeled with, for described in U.S. Pat. No. 6,592,847. Such probes generally example, a fluorescent marker and contacted to the apical comprise a fluorescent moiety that emits photons when membranes of a layer of epithelial cells. Fluorescence excited with light of appropriate wavelength and a quencher detected on the basal-lateral side of the membrane formed by moiety that absorbs such photons when in close proximity to the epithelial cells indicates that the transcytosis domain is the fluorescent moiety. Cleavage of the probe separates the functioning properly. quenching moiety from the fluorescent moiety, Such that fluo 0206 5.9.2.3. Cleavable Linker Cleavage rescence can be detected, thereby indicating that cleavage has 0207. The function of the cleavable linker can generally be occurred. Thus, Such probes can be used to identify and assess tested in a cleavage assay. Any suitable cleavage assay known cleavage by particular cleaving enzymes by contacting the by one of skill in the art, without limitation, can be used to test basolateral side of a monolayer of suitable epithelial cells the cleavable linkers. Both cell-based and cell-free assays can with the probe under conditions that allow the cleaving be used to test the ability of an enzyme to cleave the cleavable enzyme to cleave the probe. The amount of fluorescence linkers. observed indicates the activity of the cleaving enzyme being 0208. An exemplary cell-free assay for testing cleavage of tested. cleavable linkers comprises preparing extracts of polarized epithelial cells and exposing a labeled delivery construct 6. EXAMPLES bearing a cleavable linker to the fraction of the extract that 0212. The following examples merely illustrate the inven corresponds to membrane-associated enzymes. In Such tion, and are not intended to limit the invention in any way. assays, the label can be attached to either the GH to be 0213 6.1. Construction of a Delivery Construct delivered or to the remainder of the delivery construct. 0214. An exemplary delivery construct expression vector Among these enzymes are cleavage enzymes found near the for delivering human growth hormone (hGH) was con basal-lateral membrane of a polarized epithelial cell, as structed according to the following protocol. First, the hCH described above. Cleavage can be detected, for example, by gene was amplified by PCR, incorporating restriction binding the delivery construct with, for example, an antibody enzymes recognition sites at two ends of the PCR products. and washing off unbound molecules. If label is attached to the After restriction enzyme digestion, the PCR products were GH to be delivered, then little or no label should be observed cloned into pPE64-PstI-A553, which was digested with the on the molecule bound to the antibodies. Alternatively, the corresponding restriction enzyme pairs. These constructs binding agent used in the assay can be specific for the GH, and thus comprise sequences encoding Domains I and II of ntPE the remainder of the construct can be labeled. In either case, (amino acids 26-372 as shown in FIG.1) and hCH (Accession cleavage can be assessed. No. AAA72260; see Ikehara et al., 1984, Proc. Natl. Acad. Sci. 0209 Cleavage can also be tested using cell-based assays U.S.A. 81:5956-5960), and are also tagged with a 6-His motif that test cleavage by polarized epithelial cells assembled into at the N-terminus of the polypeptide to facilitate purification. membranes. For example, a labeled delivery construct, or The final plasmids were verified by restriction enzyme diges portion of a delivery construct comprising the cleavable tions and DNA sequencing. linker, can be contacted to either the apical or basolateral side 0215 Next, an expression vector for expressing a delivery of a monolayer of suitable epithelial cells, such as, for construct comprising a cleavable linker between the hCH US 2009/0305978 A1 Dec. 10, 2009

portion of the delivery construct and the remainder of the by cardiac puncture. Specific tissues (lymph nodes, trachea, molecule was constructed. For this exemplary delivery con brain, spleen liver, GI tract) are removed, briefly rinsed in struct, referred to herein as "HGH Delivery Construct, the PBS to remove any residual blood and frozen in OCT. Sec cleavable linker sequence introduced was GGLRQPR. To do tions (5 microns thick) are placed onto slides. Slides are fixed So, oligonucleotides that encode the specified amino acid in acetone for 10 min and rinsed with PBS. Slides are incu sequence flanked by appropriate restriction sites and one of bated with 3% peroxidase for 5 min. Slides are then blocked the following amino acid sequences were synthesized, then with protein for an additional 5 min. Primary anti-human ligated into an expression vector prepared as described above growth hormone antibody is incubated onto slides for 30 min between the ntlE sequences and the hCH sequences. at a 1:100 dilution followed by PBS washes. Biotin-labeled 0216) To separate hCH from remainder of the molecule in secondary antibody is then incubated for approximately 15 the event, for example, that the fusion protein is taken up by minutes followed by PBS washes. Streptavidin HRP label is antigen presenting cells, a protease site was also inserted incubated onto slides for 15 min followed by PBS washes. between the cleavable linker and rGH. To do so, constructs HRP Chromagen is applied for 5 min followed by several containing a sequence encoding the furin site in combination rinses in distilled H2O. Finally, the slides are counterstained with the cleavable linker was made. Oligonucleotide with hematoxylin for 1 min, coverslipped, and examined for sequences for the five cleavable linkers and a furin clip site are the presence of GH. shown in Table 3, below. The final construct was confirmed by 0223 6.5. Delivery of an Exemplary Growth Hormone in restriction enzyme digestion and DNA sequencing. an In Vivo System 0224. This example describes use of exemplary HGH TABLE 3 Delivery Construct in a mouse model, showing effective transport and cleavage of the delivery construct in Vivo, the Oligonucleotides Encoding Cleavable Linkers bioactivity of the GH delivered by HGH Delivery Construct, AACTGCAGGGAGGCTTACGCCAGCCTCGACTGCAGAA hGH, and the effects of hCH on mouse growth. (SEQ ID NO: 25) 0225 6.5.1. Administration of a Delivery Construct Com TTCTGCAGTCGAGGCTGGCGTAAGCCTCCCTGCAGTT prising Rat Growth Hormone (SEQ ID NO: 26) 0226 Female mice of a genetically growth-deficient strain known as Little (lit/lit) or Lit=C57BL/6J-Ghrhrzlitz/J (The Jackson Laboratory, Bar Harbor, Me.) were weighed for three 0217 6.2. Expression of Delivery Constructs consecutive days to establish a stable baseline weight (veri 0218 E. coli BL21 (DE3) plysS competent cells fying growth restriction as well as general health) and ran (Novagen, Madison, Wis.) were transformed using a standard domly sorted to a treatment group. Treatment groups included heat-shock method in the presence of the appropriate plasmid mice administered an amount of HGH Delivery Construct to generatentPE-human Growth Hormone (hGH) expression corresponding to 30 ug or 60 ughCH, mice administered 30 cells, selected on amplicillin-containing media, and isolated ug or 60 ug recombinanthCH (rhGH), and mice administered and grown in Luria-Bertani broth (Difico; Becton Dickinson, either PBS subcutaneously or left untreated as a negative Franklin Lakes, N.J.) with antibiotic, then induced for protein control. expression by the addition of 1 mM isopropyl-D-thiogalac 0227. The HGH Delivery Construct was administered as topyranoside (IPTG) at OD 0.6. Two hours following IPTG follows. Mice were dosed intranasally by diluting the HGH induction, cells were harvested by centrifugation at 5,000 Delivery Construct in phosphate buffered saline (pH 7.4) to rpm for 10 min. Inclusion bodies were isolated following cell the proper concentration to deliver a desired dose in 40 ul (20 lysis and proteins were solubilized in the buffer containing ul/nares) using a positive displacement pipette. Dosing was 100mM Tris-HCl (pH 8.0), 2 mM EDTA, 6Mguanidine HCl, performed under mild anesthesia induced by isoflurane. and 65 mM dithiothreitol. Solubilized His ntPE-rCH is 0228 Mice administered rhGH received subcutaneous refolded in the presence of 0.1 M Tris, pH=7.4, 500 mM injections of rhGH (QED Bioscience, Inc.; San Diego, Calif.) L-arginine, 0.9 mM GSSG, 2 mM EDTA. The refolded pro in an injection volume of 100 ul following reconstitution of teins were purified by Q Sepharose Ion Exchange and Super the lyophilized preparation with water according to the manu dex 200 Gel Filtration chromatography (Amersham Bio facturer's instructions. Dosing was performed under mild Sciences, Inc., Sweden). The purity of proteins was assessed anesthesia induced by isoflurane. by SDS-PAGE and analytic HPLC (Agilent, Inc. Palo Alto, 0229. In addition, to compare the pharmacokinetic and Calif.). pharmacodynamic properties of oral and intranasal adminis 0219. 6.3. Characterization of a Delivery Construct tration of HGH Delivery Construct, HGH Delivery Construct 0220. One or more of the following procedures are used to was also orally administered to lit/lit mice according to the assess proper refolding of a delivery construct. The protein following procedure. An amount of HGH Delivery Construct refolding process is monitored by measuring, e.g., Delivery corresponding to either 30 ug or 300 ughCH (in 250 ul total Construct 1 binding activity with ntPE binding receptor, CD Volume) was administered orally using an animal feeding 91 receptors, and hCGH binding proteins on a Biacore SPR needle to mice. The HGH Delivery Construct was diluted in 1 instrument (Biacore, Sweden) according to the manufactur mg/ml bovine serum albumin (BSA) and phosphate buffered er's instructions. saline (PBS) prior to administration. 0221 6.4. Detection of Growth Hormone Protein in Tissue 0230 All groups (oral, intranasal, and Subcutaneous by Histological Examination administration) were dosed daily and weighed daily for ten 0222. This example describes histological detection in tis days. All weights were determined using a scale calibrated sues of a representative GH for delivery, human growth hor just prior to each weighing session. On day 10 of administra mone. Following administration of a delivery construct, ani tion, mice from all groups were euthanized by CO asphyxi mals are euthanized by CO asphyxiation and exanguinated ation and exsanguinated 30 minutes after the final adminis US 2009/0305978 A1 Dec. 10, 2009

tration of hCGH. Serum concentrations of hCGH, bioactive hCH, IGF-1, IGF1-BP3, anti-hGH IgG antibodies, anti-ntPE TABLE 4-continued IgG antibodies, corticosterone, leptin, and insulin were deter Effect on Growth Rate mined using commercial ELISA assays for mice from each (% weight change? group. Amount and Route of hCGHAdministration day ghgH) 0231 6.5.2. Growth of Mice Administered HGH Delivery 30 lighGH DC 6.5 x 10 Construct 60 lighOH DC 3.1 x 10' 0232. The results of the growth experiments conducted as 0234. As shown in Table 4, administration of 30 ughCH described in Section 6.5.1, above, are presented in FIGS. 2-6. and 60 ughGH intranasally with the HGH Delivery Construct FIG. 2 compares the weight gain observed for mice adminis resulted in essentially identical effects on the rate of growth of tered 30 ughCH subcutaneously or 30 ughGH intranasally the mice. In contrast, 60 ug rhGH administered subcutane with the HGH Delivery Construct. FIG. 3 compares the ously did not increase the rate of growth of the mice beyond weight gain observed for mice administered 60 ughGH sub that observed for mice administered 30 ug rhGH subcutane cutaneously or 60 ughGH intranasally with the HGH Deliv ously. Thus, these experiments demonstrate that a higher effective dose of hCGH can be effectively administered intra ery Construct. FIG. 4 compares the weight gain observed for nasally with the HGH Delivery Construct than can be admin mice administered either 30 ughGH or 60 ughOH subcuta istered subcutaneously. neously. FIG. 5 compares the weight gain observed for mice 0235 6.5.3. Pharmacokinetics of an Exemplary Growth administered either 30 g. hCGH or 60 lug hCH intranasally Hormone Administered with a Delivery Construct in an In with the HGH Delivery Construct. FIG. 6 presents a table of Vivo System 0236. To assess effects of administration of rhGH admin the underlying data used to construct the graphs of FIGS. 2-5. istered subcutaneously and hCGH administered orally or intra No data are presented for mice administered the HGH Deliv nasally with HGH Delivery Construct, serum concentrations ery Construct orally as such mice did not significantly of several molecules were determined with ELISA assays. In increase in weight. As shown in FIGS. 5 and 6, administration particular, serum concentrations of hCGH, bioactive hCH, of 60 ughGH intranasally with the HGH Delivery Construct IGF-1, IGF1-BP3, anti-hGH IgG antibodies, anti-ntPE IgG resulted in a weight gain of 13% by day 5 of administration antibodies, corticosterone, leptin, insulin, and ntlE were and a weight gain of 18% by day 10 of administration. measured in lit/lit mouse serum 30 minutes following the day 10 administration of hCGH. Serum concentrations of at least 0233. The slopes of the best-fit lines were calculated for some of these molecules were determined for 30 lug hCH each of the different experimental groups shown in FIGS. 2-5 administered subcutaneously, 30 ughCH administered orally and used to calculate the effects of the hOH administered on with the HGH Delivery Construct, 30 ughOH administered growth rate. Results of these calculations are shown in Table intranasally with the HGH delivery construct, 300 ughCH 4, below. administered orally with the HGH Delivery Construct, 60 ug hGH administered intranasally with the HGH delivery con TABLE 4 struct, and 60 ug hCH administered Subcutaneously. Data thus obtained is presented in FIGS. 7-15 and summarized in Effect on Growth Rate the Table presented as FIG. 16. (% weight change? 0237 Serum concentrations of hCGH, bioactive hCH, IGF Amount and Route of hCGH Administration day ghgH) 1, IGF1-BP3, corticosterone, leptin, and insulin were deter 30 lighGH IN with HGH Delivery Construct 4.6 x 10' mined using commercially available kits for performing 60 lighCH IN with HGH Delivery Construct 4.8 x 10' ELISA assays. The exact kit, its supplier, and the molecule measured with the kit are presented in Table 5, below.

TABLE 5

Molecule Supplier Catalog # or Protocol #

GH Diagnostic Systems Laboratories DSL-10-19100, Ultra-Sensitive Human (Webster, TX) Growth Hormone ELISA Bioactive hCGH Diagnostic Systems Laboratories DSL-10-11 100, Bioactive GH ELISA (Webster, TX) IGF-1 R& D Systems MG100, Mouse IGF-1 Immunoassay (Minneapolis, MN) IGF-1 BP3 R& D Systems DY775, Mouse IGFBP-3 ELISA (Minneapolis, MN) Development System Corticosterone Neogen Corporation Product # 402810, Corticosterone ELISA (Lansing, MI) Leptin R& D Systems MOB00, Mouse Leptin Immunoassay (Minneapolis, MN) Insulin ALPCO Diagnostics 10-1150-01, Mercodia Ultrasensitive (Salem, NH) Mouse Insulin ELISA US 2009/0305978 A1 Dec. 10, 2009 20

0238 ELISA assays to determine the concentration of Cat. No.31460) at 1:4000 dilutions and incubated for 1 h. All anti-hGH IgG antibodies, anti-ntPEIgG antibodies, and ntPE incubation and coating steps were performed at room tem were performed as follows: perature on a shaker at 6 RPM. The HRP substrate, TMB 0239. To measure mouse anti-hGH IgG antibodies, Costar (3.3'5.5"tetramethylbenzidine), used to quantify bound anti 9018 E.I.A./R.I.A. 96-well plates were coated overnight with body, was measured at 450 nm. 100 ng?well of rhGH (QED, Cat. No. 20901) in 0.2M 0242 ELISA results are reported as the averages of the NaHCO, NaCO, pH 9.4. Each 96-well plate was washed triplicate OD (450 nm) value of each sample. Concentrations four times with PBS containing 0.05% Tween 20-0.01% were determined by the exceeding mean value plus three thimerosal (wash buffer); blocked for 1 h with 200 ul/well of times the standard error of the mean (SEM) of the appropriate PBS/Tween 20 containing 0.5% BSA-0.01% thimerosal (as control value. say buffer). Each plate was washed again and serum samples 0243 The results of the ELISA assays are presented in were diluted at 1:20 in assay buffer. Samples were loaded in FIGS. 7-15. FIG. 7 shows Serum concentrations of hCGH 30 100 ul/well triplicates onto a 96-well plate, and incubated 1 h minutes following administration of either 30 ug rhGH SC, to detect specific mouse serum IgG. Each 96-well plate was 300 ughCH orally with HGH Delivery Construct, or 60 ug then washed four times with wash buffer, and added 100 hGH with HGH delivery construct. FIG.8 shows the amounts ul/well of horseradish peroxidase (HRP) conjugated goat of bioactive hCH observed in the serum of mice administered anti-mouse IgG (Pierce, Cat. No.31430) at 1:6000 dilutions 30 ughCH subcutaneously, 300 ughCH orally with the HGH and incubated for 1 h. All incubation and coating steps were Delivery Construct, and 60 ug hCH intranasally with the performed at room temperature on a shaker at 6 RPM. The HGH Delivery Construct. HRP substrate, TMB (3.3'5.5"tetramethylbenzidine), used to 0244 FIG.9 shows the amounts of IGF-1 observed in the quantify bound antibody, was measured at 450 nm. serum of mice administered 30 ughGH subcutaneously, 300 0240. To measure mouse anti-ntPEIgG antibodies, Costar ughGH orally with the HGH Delivery Construct, and 60 ug 9018 E.I.A./R.I.A. 96-well plates were coated overnight with hCH intranasally with the HGH Delivery Construct. FIG. 10 200 ng/well of ntlE in 0.2M NaHCO, NaCO, pH 9.4. shows the amounts of IGF1-BP3 observed in the serum of Each 96-well plate was washed four times with PBS contain mice administered 30 g. hCH subcutaneously, 300 ughCH ing 0.05% Tween 20-0.01% thimerosal (wash buffer): orally with the HGH Delivery Construct, and 60 lug hCH blocked for 1 h with 200ul/well of PBS/Tween 20 containing intranasally with the HGH Delivery Construct. 0.5% BSA-0.01% thimerosal (assay buffer). Each plate was 0245 FIG. 11 shows the amounts of anti-hGH IgG anti washed again and serum samples were diluted at 1:20 in assay bodies observed in the serum of mice administered 30 Jug buffer. Samples were loaded in 100 ul/well triplicates onto a hGH subcutaneously, 300 ug hCH orally with the HGH 96-well plate, and incubated 1 h to detect specific mouse Delivery Construct, and 60 ug hCH intranasally with the serum IgG. Each 96-well plate was then washed four times HGH Delivery Construct. FIG. 12 shows the amounts of with wash buffer, and added 100 ul/well of horseradish per anti-ntPE IgG antibodies observed in the serum of mice oxidase (HRP) conjugated goat anti-mouse IgG (Pierce, Cat. administered 30 ughGH subcutaneously, 300 ughOH orally No.31430) at 1:6000 dilutions and incubated for 1 h. All with the HGH Delivery Construct, and 60 lug hCH intrana incubation and coating steps were performed at room tem sally with the HGH Delivery Construct. perature on a shaker at 6 RPM. The HRP substrate, TMB 0246 FIG. 13 shows amounts of corticosterone observed (3.3'5.5"tetramethylbenzidine), used to quantify bound anti in the serum of mice administered 30 ug hCH subcutane body, was measured at 450 nm. ously, 300 lighCH orally with the HGH Delivery Construct, 0241. To measure intPE concentrations in serum samples, and 60 ug hCGH intranasally with the HGH Delivery Con Costar 9018 E.I.A./R.I.A. 96-well plates were coated over struct. FIG. 14 shows the amounts of leptin observed in the night with 200 ng/well of M40-1 mAb (specific for ntPE) in serum of mice administered 30 ughGH subcutaneously, 300 0.2M NaHCO, NaCOs, pH 9.4. Each 96-well plate was ughGH orally with the HGH Delivery Construct, and 60 ug washed four times with PBS containing 0.05% Tween 20-0. hCH intranasally with the HGH Delivery Construct. FIG. 15 01% thimerosal (wash buffer); blocked for 1 h with 200 shows the amounts of insulin observed in the serum of mice ul/well of PBS/Tween 20 containing 0.5% BSA-0.01% administered 30 ughGH subcutaneously, 300 ughOH orally thimerosal (assay buffer). Purified intPE diluted in assay with the HGH Delivery Construct, and 60 lug hCH intrana buffer was used as the standard curve. Standard curve was sally with the HGH Delivery Construct. prepared by adding 5ul of the 10 mg/ml intPE to 10 ml assay 0247 Taken together, FIGS. 7-15 show that both oral and buffer (1:2000), mixing well and moving 50 ul to 950 ul assay intranasal administration of the HGH delivery construct are buffer (1:20). This solution was used as the first point for the able to exert downstream effects similar to those caused by standard curve. For each plate, 0.5 ml was moved to 0.5 ml subcutaneous administration of hCGH, especially relative to assay buffer, and did a 1:2 serial dilution. The 10 points are of the negative control. Interestingly, administration of rhGH the standard curve were: 25, 12.5, 6.25, 3.125, 1.56, 0.78, Subcutaneously appears to result in a larger serum concentra 0.39, 0.195, 0.098, and 0.049 ng/well. Each plate was washed tion of bioactive hCGH relative to administration orally or again and serum samples were diluted at 1:10 in assay buffer. intranasally with HGH Delivery Construct (FIG. 8), but Standard curve and samples were loaded in 100 ul/well trip downstream effects (e.g., IGF-1 and IGF1-BP3 expression) licates onto a 96-well plate, and incubated 3 h to detect total are comparable for the three routes of administration (FIGS. ntPE protein in serum samples. Each 96-well plate was then 9 and 10). washed four times with washbuffer, and added 100 ul/well of 0248 Mouse anti-hGH IgG antibody induction was com rabbit anti-ntPE polyclonal antibody at 1:4000 dilutions and parable for each route of administration, though it should be incubated for 2 h. Each 96-well plate was then washed four noted that much more hCGH (300 ug oral, 60 ug intranasal) was times with wash buffer, and added 100 ul/well of horseradish administered with the HGH Delivery Construct relative to the peroxidase (HRP) conjugated goat anti-rabbit IgG (Pierce, subcutaneous administration (30 ug) (FIG. 11). Oral admin US 2009/0305978 A1 Dec. 10, 2009

istration of the HGH Delivery Construct appeared to induce a assay as described above. The results of the analysis are lower titer of antibodies against the ntPE portion of the deliv presented in tabular format as FIG. 18. The pharmacokinetic ery construct relative to intranasal administration. (FIG. 12). profile of hCH serum concentration is presented in graphical format as FIG. 17. 0249 6.6. Pharmacokinetics of Intranasal Administration 0251. As shown in FIG. 17, peak serum concentrations of of hCGH in BALB/C Mice hGH were achieved 60 minutes following intranasal admin 0250 In this example, the pharmacokinetics of intranasal istration. administration of 30 ughGH with HGH Delivery Construct 0252 All publications and patent documents cited in this were monitored as follows. Four BALB/C mice pertime point application are incorporated by reference in their entirety for were intranasally administered an amount of HGH Delivery all purposes to the same extent as if each individual publica Construct corresponding to 30 ughCH. Mice were sacrificed tion or patent document were so individually denoted. Cita by CO asphyxiation, exsanguinated, and the hCH, bioactive tion of these documents is not an admission that any particular hGH, and ntlE serum concentrations determined by ELISA reference is “prior art” to this invention.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 26

<210 SEQ ID NO 1 <211 LENGTH: 4 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <223> OTHER INFORMATION: Cleavable linker

<4 OO SEQUENCE: 1

Ala Ala Pro Phe 1.

<210 SEQ ID NO 2 <211 LENGTH: 3 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa <4 OO SEQUENCE: 2 Gly Gly Phe 1.

<210 SEQ ID NO 3 <211 LENGTH: 4 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa <4 OO SEQUENCE: 3

Ala Ala Pro Wall 1.

<210 SEQ ID NO 4 <211 LENGTH: 3 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa <4 OO SEQUENCE: 4 Gly Gly Lieu. 1.

<210 SEQ ID NO 5 <211 LENGTH: 3 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa

<4 OO SEQUENCE: 5

Ala Ala Lieu. US 2009/0305978 A1 Dec. 10, 2009 22

- Continued

<210 SEQ ID NO 6 <211 LENGTH: 3 &212> TYPE : PRT <213> ORGANISM: Pseudomonas aeruginosa

<4 OO SEQUENCE: 6 Phe Val Arg 1.

<210 SEQ ID NO 7 <211 LENGTH: 3 &212> TYPE : PRT <213> ORGANISM: Pseudomonas aeruginosa

<4 OO SEQUENCE: 7 Val Gly Arg 1.

<210 SEQ ID NO 8 <211 LENGTH: 192 &212> TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 8

Met Phe Pro Thir Ile Pro Lell Ser Arg Luell Phe Asp Asn Ala Met Luell 1. 5 1O 15

Arg Ala His Arg Lell His Glin Luell Ala Phe Asp Thir Glin Glu Phe 25

Glu Glu Ala Tyr Ile Pro Glu Glin Tyr Ser Phe Luell Glin Asn 35 4 O 45

Pro Glin Thir Ser Lell Ser Glu Ser Ile Pro Thir Pro Ser Asn SO 6 O

Arg Glu Glu Thir Glin Glin Ser Asn Luell Glu Lell Lell Arg Ile Ser 65 70

Lell Luell Luell Ile Glin Ser Trp Luell Glu Pro Wall Glin Phe Luell Arg Ser 85 90 95

Wall Phe Ala Asn Ser Lell Wall Gly Ala Ser Asp Ser Asn Wall Tyr 105 11 O

Asp Luell Luell Asp Lell Glu Glu Gly Ile Glin Thir Lell Met Gly Arg 115 12 O 125

Lell Glu Asp Gly Ser Pro Arg Thir Gly Glin Ile Phe Glin Thir Tyr 13 O 135 14 O

Ser Phe Asp Thir Asn Ser His Asn Asp Asp Ala Lell Luell Asn 145 150 155 160

Gly Luell Luell Tyr Phe Arg Asp Met Asp Wall Glu Thir 1.65 17O 17s

Phe Luell Arg Ile Wall Glin Arg Ser Wall Glu Gly Ser Cys Gly Phe 18O 185 19 O

SEO ID NO 9 LENGTH: 266 TYPE : PRT ORGANISM: Pseudomonas aeruginosa

SEQUENCE: US 2009/0305978 A1 Dec. 10, 2009 23

- Continued Met His Lieu. Ile Pro His Trp Ile Pro Leu Val Ala Ser Lieu. Gly Lieu. 1. 5 1O 15 Lieu Ala Gly Gly Ser Ser Ala Ser Ala Ala Glu Glu Ala Phe Asp Lieu. 2O 25 3O Trp Asn. Glu. Cys Ala Lys Ala Cys Val Lieu. Asp Lieu Lys Asp Gly Val 35 4 O 45 Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly SO 55 6 O Glin Gly Val Lieu. His Tyr Ser Met Val Lieu. Glu Gly Gly Asn Asp Ala 65 70 7s 8O Lieu Lys Lieu Ala Ile Asp Asn Ala Lieu. Ser Ile Thir Ser Asp Gly Lieu. 85 90 95 Thir Ile Arg Lieu. Glu Gly Gly Val Glu Pro Asn Llys Pro Val Arg Tyr 1OO 105 11 O Ser Tyr Thr Arg Glin Ala Arg Gly Ser Trp Ser Lieu. Asn Trp Lieu Val 115 12 O 125 Pro Ile Gly His Glu Lys Pro Ser Asn Ile Llys Val Phe Ile His Glu 13 O 135 14 O Lieu. Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thir Ile 145 150 155 160 Glu Met Gly Asp Glu Lieu. Lieu Ala Lys Lieu Ala Arg Asp Ala Thr Phe 1.65 17O 17s Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Lieu. Ala Ile 18O 185 19 O Ser His Ala Gly Val Ser Val Val Met Ala Glin Thr Gln Pro Arg Arg 195 2OO 2O5 Glu Lys Arg Trip Ser Glu Trp Ala Ser Gly Llys Val Lieu. Cys Lieu. Lieu. 21 O 215 22O Asp Pro Lieu. Asp Gly Val Tyr Asn Tyr Lieu Ala Glin Glin Arg Cys Asn 225 23 O 235 24 O Lieu. Asp Asp Thir Trp Glu Gly Lys Ile Tyr Arg Val Lieu Ala Gly Asn 245 250 255 Pro Ala Lys His Asp Lieu. Asp Ile Llys Pro 26 O 265

<210 SEQ ID NO 10 <211 LENGTH: 153 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa <4 OO SEQUENCE: 10 Thr Val Ile Ser His Arg Lieu. His Phe Pro Glu Gly Gly Ser Leu Ala 1. 5 1O 15 Ala Lieu. Thir Ala His Glin Ala Cys His Leu Pro Leu Glu Thr Phe Thr 2O 25 3O Arg His Arg Glin Pro Arg Gly Trp Glu Glin Lieu. Glu Gln Cys Gly Tyr 35 4 O 45 Pro Val Glin Arg Lieu Val Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser Trp SO 55 6 O Asn Glin Val Asp Glin Val Ile Arg Asn Ala Lieu Ala Ser Pro Gly Ser 65 70 7s 8O Gly Gly Asp Lieu. Gly Glu Ala Ile Arg Glu Glin Pro Glu Glin Ala Arg 85 90 95 US 2009/0305978 A1 Dec. 10, 2009 24

- Continued

Lieu Ala Lieu. Thir Lieu. Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Glin 1OO 105 11 O

Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Wall Wal Ser 115 12 O 125 Lieu. Thir Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser 13 O 135 14 O Gly Asp Ala Lieu. Lieu. Glu Arg Asn Tyr 145 150

<210 SEQ ID NO 11 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 5 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 11 Tyr Val Ala Asp Xaa 1. 5

<210 SEQ ID NO 12 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 2, 3, 5 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 12 Asp Xaa Xala Asp Xaa 1. 5

<210 SEQ ID NO 13 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 2 <223> OTHER INFORMATION: Xaa = Any Amino Acid &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 3, 4, 5, 6, 7 <223> OTHER INFORMATION: Xaa = Any Amino Acid or absent &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 9 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 13 Arg Xaa Xala Xala Xaa Xaa Xaa Arg Xaa 1. 5

<210 SEQ ID NO 14 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 2 <223> OTHER INFORMATION: Xaa = Any Amino Acid &220s FEATURE: US 2009/0305978 A1 Dec. 10, 2009 25

- Continued <221 NAME/KEY: VARIANT <222> LOCATION: 3, 4, 5, 6, 7 &223> OTHER INFORMATION: Xaa Any Amino Acid or absent &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 9 &223> OTHER INFORMATION: Xaa Any Amino Acid <4 OO SEQUENCE: 14 Lys Xaa Xala Xala Xaa Xaa Xaa Arg Xaa 1. 5

<210 SEQ ID NO 15 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 6 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 15 Gly Arg Thir Lys Arg Xaa 1. 5

<210 SEQ ID NO 16 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 5 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 16 Arg Val Arg Arg Xaa 1. 5

<210 SEQ ID NO 17 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 6 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 17 Asp Arg Val Arg Arg Xaa 1. 5

<210 SEQ ID NO 18 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 2, 6 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 18 Pro Xaa Trp Val Pro Xaa 1. 5

<210 SEQ ID NO 19 <211 LENGTH: 4 US 2009/0305978 A1 Dec. 10, 2009 26

- Continued

TYPE PRT ORGANISM: Pseudomonas aeruginosa FEATURE: NAME/KEY: VARIANT LOCATION: 4 OTHER INFORMATION: Xaa = Any Amino Acid SEQUENCE: 19 Trp Val Ala Xala 1.

SEQ ID NO 2 O LENGTH: 4 TYPE PRT ORGANISM: Pseudomonas aeruginosa FEATURE: NAME/KEY: VARIANT LOCATION: 1, 3, 4 OTHER INFORMATION: Xaa = Any Amino Acid SEQUENCE: 2O

Xaa Phe Xaa Xala 1.

SEQ ID NO 21 LENGTH: 4 TYPE PRT ORGANISM: Pseudomonas aeruginosa FEATURE: NAME/KEY: VARIANT LOCATION: 1, 3, 4 OTHER INFORMATION: Xaa = Any Amino Acid SEQUENCE: 21 Xaa Tyr Xaa Xala 1.

SEQ ID NO 22 LENGTH: 4 TYPE PRT ORGANISM: Pseudomonas aeruginosa FEATURE: NAME/KEY: VARIANT LOCATION: 1, 3, 4 OTHER INFORMATION: Xaa = Any Amino Acid SEQUENCE: 22 Xaa Trp Xaa Xala 1.

SEQ ID NO 23 LENGTH: 10 TYPE PRT ORGANISM: Pseudomonas aeruginosa FEATURE: NAME/KEY: VARIANT LOCATION: 9, 10 OTHER INFORMATION: Xaa = Any Amino Acid SEQUENCE: 23 Asp Arg Tyr Ile Pro Phe His Lieu. Xaa Xala 1.

SEQ ID NO 24 LENGTH: 1839 TYPE: DNA

US 2009/0305978 A1 Dec. 10, 2009 28

- Continued

<210 SEQ ID NO 26 <211 LENGTH: 37 &212> TYPE: DNA <213> ORGANISM: Pseudomonas aeruginosa <4 OO SEQUENCE: 26 ttctgcagtic gaggctggcg taa.gc.ctic cc ticagtt 37

What is claimed is: 7. The method of claim 1, wherein the epithelial cell is 1. A method for increasing the size of a subject by at least selected from the group consisting of nasal epithelial cells, about 12%, comprising contacting an apical Surface of a oral epithelial cells, intestinal epithelial cells, rectal epithelial polarized epithelial cell of the subject with an amount of a cells, vaginal epithelial cells, and pulmonary epithelial cells. delivery construct effective to increase the size of the subject 8. The method of claim 1, wherein the epithelial cell is a by at least about 12%, wherein said delivery construct com nasal epithelial cell. prises a receptor binding domain, a transcytosis domain, a 9. The method of claim 1, wherein the epithelial cell is an intestinal epithelial cell. cleavable linker, and growth hormone (GH), wherein the 10. The method of claim 1, wherein said subject is a human. transcytosis domain transcytoses the GH to and through the 11. The method of claim 1, wherein said delivery construct basal-lateral membrane of said epithelial cell, and wherein contacts the apical membrane of the epithelial cell. cleavage at said cleavable linker separates said GH from the 12. The method of claim 1, wherein said size of said subject remainder of said construct, thereby delivering the GH to the is increased by at least about 13%. subject in an amount effective to increase the size of the 13. The method of claim 1, wherein said size of said subject subject by at least about 12%. is increased by at least about 14%. 2. The method of claim 1, wherein said receptor binding 14. The method of claim 1, wherein said size of said subject domain is selected from the group consisting of receptor is increased by at least about 15%. binding domains from Pseudomonas exotoxin A, cholera 15. The method of claim 1, wherein said size of said subject toxin, diptheria toxin, Shiga toxin, or shiga-like toxin; mono is increased by at least about 16%. clonal antibodies; polyclonal antibodies; single-chain anti 16. The method of claim 1, wherein said size of said subject bodies; TGF C.; EGF: IGF-I; IGF-II; IGF-III; IL-1, IL-2: is increased by at least about 17%. IL-3; IL-6; MIP-1a; MIP-1b; MCAF; and IL-8. 17. The method of claim 1, wherein said size of said subject 3. The method of claim 1, wherein said receptor binding is increased by at least about 18%. domain binds to a cell surface receptor selected from the 18. The method of claim 1, wherein said size of said subject group consisting of C2-macroglobulin receptor, EGFR, is a weight of said Subject. IGFR, transferrin receptor, chemokine receptor, CD25, 19. The method of claim 1, wherein said size of said subject CD11B, CD11C, CD80, CD86, TNFC receptor, TOLL recep is a length of said Subject. tor, M-CSF receptor, GM-CSF receptor, scavenger receptor, 20. The method of claim 1, wherein said size of said subject and VEGF receptor. is a height of said Subject. 4. The method of claim 1, wherein said transcytosis domain 21. The method of claim 1, wherein said GH is human is selected from the group consisting of transcytosis domains growth hormone (hGH). from Pseudomonas exotoxin A, botulinum toxin, diptheria 22. The method of claim 21, wherein said hCH has an toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero amino acid sequence that is SEQID NO.:8. toxin, shiga toxin, and shiga-like toxin. 23. The method of claim 1, further comprising performing 5. The method of claim 1, wherein said cleavable linker is the method of claim 1 a second time about 1 day after the cleavable by an enzyme that is selected from the group con method of claim 1 is performed the first time. sisting of Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin 24. The method of claim 1, further comprising performing AI, Subtilisin AII, Thrombin I, and Urokinase I. the method of claim 1 a second time about 2 days after the 6. The method of claim 1, wherein said cleavable linker method of claim 1 is performed the first time. comprises an amino acid sequence that is selected from the 25. The method of claim 1, further comprising performing group consisting of Ala-Ala-Pro-Phe (SEQID NO.:1), Gly the method of claim 1 a second time about 3 days after the Gly-Phe (SEQID NO.:2), Ala-Ala-Pro-Val (SEQID NO.:3), method of claim 1 is performed the first time. Gly-Gly-Leu (SEQID NO.:4), Ala-Ala-Leu (SEQID NO.:5), Phe-Val-Arg (SEQID NO.:6), Val-Gly-Arg (SEQID NO.:7). c c c c c