# Patent

## Title
Lysosomal targeting peptides and uses thereof

## Abstract

The present invention provides further improved compositions and methods for efficient lysosomal targeting based on the GILT technology. Among other things, the present invention provides methods and compositions for targeting lysosomal enzymes to lysosomes using furin-resistant lysosomal targeting peptides. The present invention also provides methods and compositions for targeting lysosomal enzymes to lysosomes using a lysosomal targeting peptide that has reduced or diminished binding affinity for the insulin receptor.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation of U.S. patent application Ser. No. 12/991,104 filed Apr. 25, 2011, which is the National Stage Entry of PCT/US2009/43207 filed May 7, 2009, which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/051,336 filed May 7, 2008 and U.S. Provisional Patent Application No. 61/144,106 filed Jan. 12, 2009, the contents of each of which are hereby incorporated by reference in their entireties.


This application contains, as a separate part of the disclosure, a sequence listing in computer-readable form (Filename: 40017B_SeqListing.txt; Size: 96,281 bytes; Created: Nov. 5, 2014), which is incorporated by reference in its entirety.
BACKGROUND
Normally, mammalian lysosomal enzymes are synthesized in the cytosol and traverse the ER where they are glycosylated with N-linked, high mannose type carbohydrate. In the golgi, the high mannose carbohydrate is modified on lysosomal proteins by the addition of mannose-6-phosphate (M6P) which targets these proteins to the lysosome. The M6P-modified proteins are delivered to the lysosome via interaction with either of two M6P receptors. The most favorable form of modification is when two M6Ps are added to a high mannose carbohydrate.
More than forty lysosomal storage diseases (LSDs) are caused, directly or indirectly, by the absence of one or more lysosomal enzymes in the lysosome. Enzyme replacement therapy for LSDs is being actively pursued. Therapy generally requires that LSD proteins be taken up and delivered to the lysosomes of a variety of cell types in an M6P-dependent fashion. One possible approach involves purifying an LSD protein and modifying it to incorporate a carbohydrate moiety with M6P. This modified material may be taken up by the cells more efficiently than unmodified LSD proteins due to interaction with M6P receptors on the cell surface.
The inventors of the present application have previously developed a peptide-based targeting technology that allows more efficient delivery of therapeutic enzymes to the lysosomes. This proprietary technology is termed Glycosylation Independent Lysosomal Targeting (GILT) because a peptide tag replaces M6P as the moiety targeting the lysosomes. Details of the GILT technology are described in U.S. Application Publication Nos. 2003-0082176, 2004-0006008, 2003-0072761, 2005-0281805, 2005-0244400, and international publications WO 03/032913, WO 03/032727, WO 02/087510, WO 03/102583, WO 2005/078077, the disclosures of all of which are hereby incorporated by reference.
SUMMARY OF THE INVENTION
The present invention provides further improved compositions and methods for efficient lysosomal targeting based on the GILT technology. Among other things, the present invention provides methods and compositions for targeting lysosomal enzymes to lysosomes using furin-resistant lysosomal targeting peptides. The present invention also provides methods and compositions for targeting lysosomal enzymes to lysosomes using a lysosomal targeting peptide that has reduced or diminished binding affinity for the insulin receptor. The present invention encompasses unexpected discovery that furin-resistant lysosomal targeting peptides according to the invention have reduced binding affinity for the insulin receptor.
In some embodiments, the present invention provides a furin-resistant IGF-II mutein. In some embodiments, the present invention provides a furin-resistant IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1) and a mutation that abolishes at least one furin protease cleavage site.
In some embodiments, the present invention provides an IGF-II mutein comprising an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1) and a mutation that reduces or diminishes the binding affinity for the insulin receptor as compared to the wild-type human IGF-II.
In some embodiments, the furin-resistant IGF-II mutein has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor.
In some embodiments, the present invention provides a targeted therapeutic fusion protein containing a lysosomal enzyme; and an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), wherein the IGF-II mutein is resistant to furin cleavage and binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.
In some embodiments, the present invention provides a targeted therapeutic fusion protein containing a lysosomal enzyme; and an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), and having diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor; wherein the IGF-II mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.
In some embodiments, the present invention provides a targeted therapeutic fusion protein containing a lysosomal enzyme; and an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), and having diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor; wherein the IGF-II mutein is resistant to furin cleavage and binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.
In some embodiments, an IGF-II mutein suitable for the invention includes a mutation within a region corresponding to amino acids 30-40 of SEQ ID NO:1. In some embodiments, an IGF-II mutein suitable for the invention includes a mutation within a region corresponding to amino acids 34-40 of SEQ ID NO:1 such that the mutation abolishes at least one furin protease cleavage site. In some embodiments, a suitable mutation is an amino acid substitution, deletion and/or insertion. In some embodiments, the mutation is an amino acid substitution at a position corresponding to Arg37 or Arg40 of SEQ ID NO:1. In some embodiments, the amino acid substitution is a Lys or Ala substitution.
In some embodiments, a suitable mutation is a deletion or replacement of amino acid residues corresponding to positions selected from the group consisting of 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40 of SEQ ID NO:1, and combinations thereof.
In some embodiments, an IGF-II mutein according to the invention further contains a deletion or a replacement of amino acids corresponding to positions 2-7 of SEQ ID NO:1. In some embodiments, an IGF-II mutein according to the invention further includes a deletion or a replacement of amino acids corresponding to positions 1-7 of SEQ ID NO:1. In some embodiments, an IGF-II mutein according to the invention further contains a deletion or a replacement of amino acids corresponding to positions 62-67 of SEQ ID NO:1. In some embodiments, an IGF-II mutein according to the invention further contains an amino acid substitution at a position corresponding to Tyr27, Leu43, or Ser26 of SEQ ID NO:1. In some embodiments, an IGF-II mutein according to the invention contains at least an amino acid substitution selected from the group consisting of Tyr27Leu, Leu43Val, Ser26Phe and combinations thereof. In some embodiments, an IGF-II mutein according to the invention contains amino acids corresponding to positions 48-55 of SEQ ID NO:1. In some embodiments, an IGF-II mutein according to the invention contains at least three amino acids selected from the group consisting of amino acids corresponding to positions 8, 48, 49, 50, 54, and 55 of SEQ ID NO:1. In some embodiments, an IGF-11 mutein of the invention contains, at positions corresponding to positions 54 and 55 of SEQ ID NO:1, amino acids each of which is uncharged or negatively charged at pH 7.4. In some embodiments, the IGF-II mutein has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor.
In some embodiments, a lysosomal enzyme suitable for the invention is human acid alpha-glucosidase (GAA), or a functional variant thereof. In some embodiments, a lysosomal enzyme suitable for the invention includes amino acids 70-952 of human GAA.
In some embodiments, a targeted therapeutic fusion protein of the invention further includes a spacer between the lysosomal enzyme and the furin-resistant IGF-II mutein. In some embodiments, the spacer contains an amino acid sequence Gly-Ala-Pro.
The present invention also provides nucleic acids encoding the IGF-II mutein or the targeted therapeutic fusion protein as described in various embodiments above. The present invention further provides various cells containing the nucleic acid of the invention.
The present invention provides pharmaceutical compositions suitable for treating lysosomal storage disease containing a therapeutically effective amount of a targeted therapeutic fusion protein of the invention. The invention further provides methods of treating lysosomal storage diseases comprising administering to a subject in need of treatment a targeted therapeutic fusion protein according to the invention. In some embodiments, the lysosomal storage disease is Pompe Disease. In some embodiments, the lysosomal storage disease is Fabry Disease. In some embodiments, the lysosomal storage disease is Gaucher Disease.
In another aspect, the present invention provides a method of producing a targeted therapeutic fusion protein including a step of culturing mammalian cells in a cell culture medium, wherein the mammalian cells carry the nucleic acid of the invention, in particular, as described in various embodiments herein; and the culturing is performed under conditions that permit expression of the targeted therapeutic fusion protein.
In yet another aspect, the present invention provides a method of producing a targeted therapeutic fusion protein including a step of culturing furin-deficient cells (e.g., furin-deficient mammalian cells) in a cell culture medium, wherein the furin-deficient cells carry a nucleic acid encoding a fusion protein comprising a lysosomal enzyme and an IGF-II mutein having an amino acid sequence at least 70% identical to mature human IGF-II (SEQ ID NO:1), wherein the IGF-II mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and wherein the culturing is performed under conditions that permit expression of the targeted therapeutic fusion protein.
Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are for illustration purposes only, not for limitation.
FIG. 1 illustrates a map of N-terminus of ZC-701. Two amino acid residues boxed are sites of cleavage events. The first is the site of signal peptide cleavage, the second is the site of a furin cleavage.
FIG. 2 illustrates an exemplary SDS-PAGE analysis of ZC-701 after treatment with PNGase F. The lane on the right has been additionally treated with furin.
FIG. 3 Left: Schematic illustration of exemplary ZC-701 mutants in which furin cleavage site is modified. Center: Exemplary SDS-PAGE analysis of PNGase treated mutants after 3-7 days of cell culture. Right: Exemplary SDS-PAGE analysis of PNGase-treated mutants treated with furin.
FIG. 4 illustrates exemplary competitive IGF-II receptor binding results.
FIG. 5 illustrates additional exemplary competitive IGF-II receptor binding results.
FIG. 6 illustrates exemplary insulin receptor competition assay results.
FIG. 7 illustrates exemplary IGF-I receptor competition assay results.
FIG. 8 illustrates exemplary results of certain insulin receptor binding assay.
FIG. 9 illustrates exemplary results of certain insulin receptor binding assay.
FIG. 10 illustrates exemplary analysis of partially purified GILT-tagged GAA from transient transfections. HEK293 cells were transfected with constructs 1479, 1487 or ZC-701. After harvest, culture supernatants were partially purified by Hydrophobic Interaction Chromatography (HIC). All samples were treated with PNGase prior to electrophoresis. Left panels: SDS-PAGE of partially purified proteins. Purified ZC-701 B12 is shown as a control. Right panels: Immunoblot analysis of the partially purified proteins. The indicated primary antibody was used. Bottom panels were additionally treated with exogenous furin. The protein encoded by construct 1487 is identical in sequence to that encoded by construct 1461 (R37A). The protein encoded by construct 1479 is identical to that encoded by construct 1459 (R37K).
FIG. 11 illustrates exemplary uptake results of exemplary furin resistant GILT-tagged GAA into rat L6 myoblasts. Kuptakes for protein 1479, 1487, ZC-701, and purified ZC-701 are 4.5 nM, 4.4 nM, 5.0 nM and 2.6 nM respectively. The protein encoded by construct 1487 is identical in sequence to that encoded by construct 1461 in FIG. 3 (R37A). The protein encoded by construct 1479 is identical to that encoded by construct 1459 in FIG. 3 (R37K).



DEFINITIONS
Amelioration: As used herein, the term “amelioration” is meant the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not require complete recovery or complete prevention of a disease condition. In some embodiments, amelioration includes reduction of accumulated materials inside lysosomes of relevant diseases tissues.
Furin-resistant IGF-II mutein: As used herein, the term “furin-resistant IGF-II mutein” refers to an IGF-II-based peptide containing an altered amino acid sequence that abolishes at least one native furin protease cleavage site or changes a sequence close or adjacent to a native furin protease cleavage site such that the furin cleavage is prevented, inhibited, reduced, or slowed down as compared to a wild-type human IGF-II peptide. As used herein, a furin-resistant IGF-II mutein is also referred to as an IGF-II mutein that is resistant to furin.
Furin protease cleavage site: As used herein, the term “furin protease cleavage site” (also referred to as “furin cleavage site” or “furin cleavage sequence”) refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by furin or furin-like proteases. Typically, a furin protease cleavage site has a consensus sequence Arg-X-X-Arg (SEQ ID NO: 2), X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence. In some embodiments, a furin cleavage site may have a consensus sequence Lys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 3), X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence.
Furin: As used herein, the term “furin” refers to any protease that can recognize and cleave the furin protease cleavage site as defined herein, including furin or furin-like protease. Furin is also known as paired basic amino acid cleaving enzyme (PACE). Furin belongs to the subtilisin-like proprotein convertase family. The gene encoding furin was known as FUR (FES Upstream Region).
Furin-deficient cells: As used herein, the term “furin-deficient cells” refers to any cells whose furin protease activity is inhibited, reduced or eliminated. Furin-deficient cells include both mammalian and non-mammalian cells that do not produce furin or produce reduced amount of furin or defective furin protease.
Glycosylation Independent Lysosomal Targeting: As used herein, the term “glycosylation independent lysosomal targeting” (also referred to as “GILT”) refer to lysosomal targeting that is mannose-6-phosphate-independent.
Human acid alpha-glucosidase: As used herein, the term “human acid alpha-glucosidase” (also referred to as “GAA”) refers to precursor wild-type form of human GAA or a functional variant that is capable of reducing glycogen levels in mammalian lysosomes or that can rescue or ameliorate one or more Pompe disease symptoms.
Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A “control individual” is an individual afflicted with the same form of lysosomal storage disease (e.g., Pompe disease) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).
Individual, subject, patient: As used herein, the terms “subject,” “individual” or “patient” refer to a human or a non-human mammalian subject. The individual (also referred to as “patient” or “subject”) being treated is an individual (fetus, infant, child, adolescent, or adult human) suffering from a lysosomal storage disease, for example, Pompe disease (i.e., either infantile-, juvenile-, or adult-onset Pompe disease) or having the potential to develop a lysosomal storage disease (e.g., Pompe disease).
Lysosomal storage diseases: As used herein, “lysosomal storage diseases” refer to a group of genetic disorders that result from deficiency in at least one of the enzymes (e.g., acid hydrolases) that are required to break macromolecules down to peptides, amino acids, monosaccharides, nucleic acids and fatty acids in lysosomes. As a result, individuals suffering from lysosomal storage diseases have accumulated materials in lysosomes. Exemplary lysosomal storage diseases are listed in Table 1.
Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers to any enzyme that is capable of reducing accumulated materials in mammalian lysosomes or that can rescue or ameliorate one or more lysosomal storage disease symptoms. Lysosomal enzymes suitable for the invention include both wild-type or modified lysosomal enzymes and can be produced using recombinant and synthetic methods or purified from nature sources. Exemplary lysosomal enzymes are listed in Table 1.
Spacer: As used herein, the term “spacer” (also referred to as “linker”) refers to a peptide sequence between two protein moieties in a fusion protein. A spacer is generally designed to be flexible or to interpose a structure, such as an alpha-helix, between the two protein moieties. A spacer can be relatively short, such as the sequence Gly-Ala-Pro (SEQ ID NO: 4) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO: 5), or can be longer, such as, for example, 10-25 amino acids in length.
Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to an amount of a targeted therapeutic fusion protein which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of a therapeutic fusion protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic fusion protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.
Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a therapeutic fusion protein that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. For example, treatment can refer to improvement of cardiac status (e.g., increase of end-diastolic and/or end-systolic volumes, or reduction, amelioration or prevention of the progressive cardiomyopathy that is typically found in Pompe disease) or of pulmonary function (e.g., increase in crying vital capacity over baseline capacity, and/or normalization of oxygen desaturation during crying); improvement in neurodevelopment and/or motor skills (e.g., increase in AIMS score); reduction of glycogen levels in tissue of the individual affected by the disease; or any combination of these effects. In some embodiments, treatment includes improvement of glycogen clearance, particularly in reduction or prevention of Pompe disease-associated cardiomyopathy.
As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides improved methods and compositions for targeting lysosomal enzymes based on the glycosylation-independent lysosomal targeting (GILT) technology. Among other things, the present invention provides IGF-II muteins that are resistant to furin and/or has reduced or diminished binding affinity for the insulin receptor and targeted therapeutic fusion proteins containing an IGF-II mutein of the invention. The present invention also provides methods of making and using the same.
Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise.
Lysosomal Enzymes
A lysosomal enzyme suitable for the invention includes any enzyme that is capable of reducing accumulated materials in mammalian lysosomes or that can rescue or ameliorate one or more lysosomal storage disease symptoms. Suitable lysosomal enzymes include both wild-type or modified lysosomal enzymes and can be produced using recombinant or synthetic methods or purified from nature sources. Exemplary lysosomal enzymes are listed in Table 1.







TABLE 1







Lysosomal Storage Diseases and associated enzyme defects









Disease Name
Enzyme Defect
Substance Stored










A. Glycogenosis Disorders









Pompe Disease
Acid-a1, 4-
Glycogen α1-4 linked



Glucosidase
Oligosaccharides







B. Glycolipidosis Disorders









GM1 Gangliodsidosis
β-Galactosidase
GM1 Gangliosides


Tay-Sachs Disease
β-Hexosaminidase A
GM2 Ganglioside


GM2 Gangliosidosis:
GM2 Activator
GM2 Ganglioside


AB Variant
Protein


Sandhoff Disease
β-Hexosaminidase
GM2 Ganglioside



A & B


Fabry Disease
α-Galactosidase A
Globosides


Gaucher Disease
Glucocerebrosidase
Glucosylceramide


Metachromatic
Arylsulfatase A
Sulphatides


Leukodystrophy


Krabbe Disease
Galactosylceramidase
Galactocerebroside


Niemann-Pick, Types
Acid
Sphingomyelin


A and B
Sphingomyelinase


Niemann-Pick, Type C
Cholesterol
Sphingomyelin



Esterification Defect


Niemann-Pick, Type D
Unknown
Sphingomyelin


Farber Disease
Acid Ceramidase
Ceramide


Wolman Disease
Acid Lipase
Cholesteryl Esters







C. Mucopolysaccharide Disorders









Hurler Syndrome
α-L-Iduronidase
Heparan &


(MPS IH)

Dermatan Sulfates


Scheie Syndrome
α-L-Iduronidase
Heparan &


(MPS IS)

Dermatan, Sulfates


Hurler-Scheie
α-L-Iduronidase
Heparan &


(MPS IH/S)

Dermatan Sulfates


Hunter Syndrome
Iduronate Sulfatase
Heparan &


(MPS II)

Dermatan Sulfates


Sanfilippo A
Heparan N-Sulfatase
Heparan Sulfate


(MPS IIIA)


Sanfilippo B
α-N-
Heparan Sulfate


(MPS IIIB)
Acetylglucosaminidase


Sanfilippo C
Acetyl-CoA-
Heparan Sulfate


(MPS IIIC)
Glucosaminide



Acetyltransferase


Sanfilippo D
N-Acetylglucosamine-
Heparan Sulfate


(MPS IIID)
6-Sulfatase


Morquio A
Galactosamine-6-
Keratan Sulfate


(MPS IVA)
Sulfatase


Morquio B
β-Galactosidase
Keratan Sulfate


(MPS IVB)


Maroteaux-Lamy
Arylsulfatase B
Dermatan Sulfate


(MPS VI)


Sly Syndrome
β-Glucuronidase


(MPS VII)







D. Oligosaccharide/Glycoprotein Disorders









α-Mannosidosis
α-Mannosidase
Mannose/




Oligosaccharides


β-Mannosidosis
β-Mannosidase
Mannose/




Oligosaccharides


Fucosidosis
α-L-Fucosidase
Fucosyl




Oligosaccharides


Aspartylglucosaminuria
N-Aspartyl-β-
Aspartylglucosamine



Glucosaminidase
Asparagines


Sialidosis
α-Neuraminidase
Sialyloligosaccharides


(Mucolipidosis I)


Galactosialidosis
Lysosomal Protective
Sialyloligosaccharides


(Goldberg Syndrome)
Protein Deficiency


Schindler Disease
α-N-Acetyl-



Galactosaminidase







E. Lysosomal Enzyme Transport Disorders









Mucolipidosis II
N-Acetylglucosamine-
Heparan Sulfate


(I-Cell Disease)
1-Phosphotransferase


Mucolipidosis III
Same as ML II


(Pseudo-Hurler


Polydystrophy)







F. Lysosomal Membrane Transport Disorders









Cystinosis
Cystine Transport
Free Cystine



Protein


Salla Disease
Sialic Acid Transport
Free Sialic Acid and



Protein
Glucuronic Acid


Infantile Sialic Acid
Sialic Acid Transport
Free Sialic Acid and


Storage Disease
Protein
Glucuronic Acid







G. Other









Batten Disease
Unknown
Lipofuscins


(Juvenile Neuronal


Ceroid Lipofuscinosis)


Infantile Neuronal
Palmitoyl-Protein
Lipofuscins


Ceroid Lipofuscinosis
Thioesterase


Mucolipidosis IV
Unknown
Gangliosides &




Hyaluronic Acid


Prosaposin
Saposins A, B, C or D









In some embodiments, a lysosomal enzyme suitable for the invention includes a polypeptide sequence having 50-100%, including 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%, sequence identity to the naturally-occurring polynucleotide sequence of a human enzyme shown in Tables 1, while still encoding a protein that is capable of reducing accumulated materials in mammalian lysosomes or that can rescue or ameliorate one or more lysosomal storage disease symptoms.
“Percent (%) amino acid sequence identity” with respect to the lysosomal enzyme sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the naturally-occurring human enzyme sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology 266, 460-480 (1996); http://blast.wust1/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, world threshold (T)=11. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.
Pompe Disease
One exemplary lysosomal storage disease is Pompe disease. Pompe disease is a rare genetic disorder caused by a deficiency in the enzyme acid alpha-glucosidase (GAA), which is needed to break down glycogen, a stored form of sugar used for energy. Pompe disease is also known as glycogen storage disease type II, GSD II, type II glycogen storage disease, glycogenosis type II, acid maltase deficiency, alpha-1,4-glucosidase deficiency, cardiomegalia glycogenic diffusa, and cardiac form of generalized glycogenosis. The build-up of glycogen causes progressive muscle weakness (myopathy) throughout the body and affects various body tissues, particularly in the heart, skeletal muscles, liver, respiratory and nervous system.
The presenting clinical manifestations of Pompe disease can vary widely depending on the age of disease onset and residual GAA activity. Residual GAA activity correlates with both the amount and tissue distribution of glycogen accumulation as well as the severity of the disease. Infantile-onset Pompe disease (less than 1% of normal GAA activity) is the most severe form and is characterized by hypotonia, generalized muscle weakness, and hypertrophic cardiomyopathy, and massive glycogen accumulation in cardiac and other muscle tissues. Death usually occurs within one year of birth due to cardiorespiratory failure. Hirschhorn et al. (2001) “Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency,” in Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, 8th Ed., New York: McGraw-Hill, 3389-3420. Juvenile-onset (1-10% of normal GAA activity) and adult-onset (10-40% of normal GAA activity) Pompe disease are more clinically heterogeneous, with greater variation in age of onset, clinical presentation, and disease progression. Juvenile- and adult-onset Pompe disease are generally characterized by lack of severe cardiac involvement, later age of onset, and slower disease progression, but eventual respiratory or limb muscle involvement results in significant morbidity and mortality. While life expectancy can vary, death generally occurs due to respiratory failure. Hirschhorn et al. (2001) “Glycogen Storage Disease Type II: Acid Alpha-glucosidase (Acid Maltase) Deficiency,” in Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, 8th Ed., New York: McGraw-Hill, 3389-3420.
A GAA enzyme suitable for treating Pompe disease includes a wild-type human GAA, or a fragment or sequence variant thereof which retains the ability to cleave a 1-4 linkages in linear oligosaccharides.
Enzyme Replacement Therapy
Enzyme replacement therapy (ERT) is a therapeutic strategy to correct an enzyme deficiency by infusing the missing enzyme into the bloodstream. As the blood perfuses patient tissues, enzyme is taken up by cells and transported to the lysosome, where the enzyme acts to eliminate material that has accumulated in the lysosomes due to the enzyme deficiency. For lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme must be delivered to lysosomes in the appropriate cells in tissues where the storage defect is manifest. Conventional lysosomal enzyme replacement therapeutics are delivered using carbohydrates naturally attached to the protein to engage specific receptors on the surface of the target cells. One receptor, the cation-independent M6P receptor (CI-MPR), is particularly useful for targeting replacement lysosomal enzymes because the CI-MPR is present on the surface of most cell types.
The terms “cation-independent mannose-6-phosphate receptor (CI-MPR),” “M6P/IGF-II receptor,” “CI-MPR/IGF-II receptor,” “IGF-II receptor” or “IGF2 Receptor,” or abbreviations thereof, are used interchangeably herein, referring to the cellular receptor which binds both M6P and IGF-II.
Glycosylation Independent Lysosomal Targeting
We have developed a Glycosylation Independent Lysosomal Targeting (GILT) technology to target therapeutic enzymes to lysosomes. Specifically, the GILT technology uses a peptide tag instead of M6P to engage the CI-MPR for lysosomal targeting. Typically, a GILT tag is a protein, peptide, or other moiety that binds the CI-MPR in a mannose-6-phosphate-independent manner. Advantageously, this technology mimics the normal biological mechanism for uptake of lysosomal enzymes, yet does so in a manner independent of mannose-6-phosphate.
A preferred GILT tag is derived from human insulin-like growth factor II (IGF-II). Human IGF-II is a high affinity ligand for the CI-MPR, which is also referred to as IGF-II receptor. Binding of GILT-tagged therapeutic enzymes to the M6P/IGF-II receptor targets the protein to the lysosome via the endocytic pathway. This method has numerous advantages over methods involving glycosylation including simplicity and cost effectiveness, because once the protein is isolated, no further modifications need be made.
Detailed description of the GILT technology and GILT tag can be found in U.S. Publication Nos. 20030082176, 20040006008, 20040005309, and 20050281805, the teachings of all of which are hereby incorporated by references in their entireties.
Furin-Resistant GILT Tag
During the course of development of GILT-tagged lysosomal enzymes for treating lysosomal storage disease, it has become apparent that the IGF-II derived GILT tag may be subjected to proteolytic cleavage by furin during production in mammalian cells (see the examples section). Furin protease typically recognizes and cleaves a cleavage site having a consensus sequence Arg-X-X-Arg (SEQ ID NO: 2), X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence. In some embodiments, a furin cleavage site has a consensus sequence Lys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 3), X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence. As used herein, the term “furin” refers to any protease that can recognize and cleave the furin protease cleavage site as defined herein, including furin or furin-like protease. Furin is also known as paired basic amino acid cleaving enzyme (PACE). Furin belongs to the subtilisin-like proprotein convertase family that includes PC3, a protease responsible for maturation of proinsulin in pancreatic islet cells. The gene encoding furin was known as FUR (FES Upstream Region).
The mature human IGF-II peptide sequence is shown below.







(SEQ ID NO: 1)


                                    ↓  ↓


AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRS


 


CDLALLETYCATPAKSE






As can be seen, the mature human IGF-II contains two potential overlapping furin cleavage sites between residues 34-40 (bolded and underlined). Arrows point to two potential furin cleavage positions.
We have developed modified GILT tags that are resistant to cleavage by furin and still retain ability to bind to the CI-MPR in a mannose-6-phosphate-independent manner. Specifically, furin-resistant GILT tags can be designed by mutating the amino acid sequence at one or more furin cleavage sites such that the mutation abolishes at least one furin cleavage site. Thus, in some embodiments, a furin-resistant GILT tag is a furin-resistant IGF-II mutein containing a mutation that abolishes at least one furin protease cleavage site or changes a sequence adjacent to the furin protease cleavage site such that the furin cleavage is prevented, inhibited, reduced or slowed down as compared to a wild-type IGF-II peptide (e.g., wild-type human mature IGF-II). Typically, a suitable mutation does not impact the ability of the furin-resistant GILT tag to bind to the human cation-independent mannose-6-phosphate receptor. In particular, a furin-resistant IGF-II mutein suitable for the invention binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner with a dissociation constant of 10−7 M or less (e.g., 10−8, 10−9, 10−10, 10−11, or less) at pH 7.4. In some embodiments, a furin-resistant IGF-II mutein contains a mutation within a region corresponding to amino acids 30-40 (e.g., 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40) of SEQ ID NO:1. In some embodiments, a suitable mutation abolishes at least one furin protease cleavage site. A mutation can be amino acid substitutions, deletions, insertions. For example, any one amino acid within the region corresponding to residues 30-40 (e.g., 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 32-39, 33-39, 34-39, 35-39, 36-39, 37-40, 34-40) of SEQ ID NO:1 can be substituted with any other amino acid or deleted. For example, substitutions at position 34 may affect furin recognition of the first cleavage site. Insertion of one or more additional amino acids within each recognition site may abolish one or both furin cleavage sites. Deletion of one or more of the residues in the degenerate positions may also abolish both furin cleavage sites.
In some embodiments, a furin-resistant IGF-II mutein contains amino acid substitutions at positions corresponding to Arg37 or Arg40 of SEQ ID NO:1. In some embodiments, a furin-resistant IGF-II mutein contains a Lys or Ala substitution at positions Arg37 or Arg40. Other substitutions are possible, including combinations of Lys and/or Ala mutations at both positions 37 and 40, or substitutions of amino acids other than Lys or Ala.
In some embodiments, the furin-resistant IGF-II mutein suitable for the invention may contain additional mutations. For example, up to 30% or more of the residues of SEQ ID NO:1 may be changed (e.g., up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more residues may be changed). Thus, a furin-resistant IGF-II mutein suitable for the invention may have an amino acid sequence at least 70%, including at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, identical to SEQ ID NO:1.
In some embodiments, a furin-resistant IGF-II mutein suitable for the invention is targeted specifically to the CI-MPR. Particularly useful are mutations in the IGF-II polypeptide that result in a protein that binds the CI-MPR with high affinity (e.g., with a dissociation constant of 10−7M or less at pH 7.4) while binding other receptors known to be bound by IGF-II with reduced affinity relative to native IGF-II. For example, a furin-resistant IGF-II mutein suitable for the invention can be modified to have diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor. For example, substitution of IGF-II residues Tyr 27 with Leu, Leu 43 with Val or Ser 26 with Phe diminishes the affinity of IGF-II for the IGF-I receptor by 94-, 56-, and 4-fold respectively (Torres et al. (1995) J. Mol. Biol. 248(2):385-401). Deletion of residues 1-7 of human IGF-II resulted in a 30-fold decrease in affinity for the human IGF-I receptor and a concomitant 12 fold increase in affinity for the rat IGF-II receptor (Hashimoto et al. (1995) J. Biol. Chem. 270(30):18013-8). The NMR structure of IGF-II shows that Thr 7 is located near residues 48 Phe and 50 Ser as well as near the 9 Cys-47 Cys disulfide bridge. It is thought that interaction of Thr 7 with these residues can stabilize the flexible N-terminal hexapeptide required for IGF-I receptor binding (Terasawa et al. (1994) EMBO J. 13(23)5590-7). At the same time this interaction can modulate binding to the IGF-II receptor. Truncation of the C-terminus of IGF-II (residues 62-67) also appear to lower the affinity of IGF-II for the IGF-I receptor by 5 fold (Roth et al. (1991) Biochem. Biophys. Res. Commun. 181(2):907-14).
The binding surfaces for the IGF-I and cation-independent M6P receptors are on separate faces of IGF-II. Based on structural and mutational data, functional cation-independent M6P binding domains can be constructed that are substantially smaller than human IGF-II. For example, the amino terminal amino acids (e.g., 1-7 or 2-7) and/or the carboxy terminal residues 62-67 can be deleted or replaced. Additionally, amino acids 29-40 can likely be eliminated or replaced without altering the folding of the remainder of the polypeptide or binding to the cation-independent M6P receptor. Thus, a targeting moiety including amino acids 8-28 and 41-61 can be constructed. These stretches of amino acids could perhaps be joined directly or separated by a linker. Alternatively, amino acids 8-28 and 41-61 can be provided on separate polypeptide chains. Comparable domains of insulin, which is homologous to IGF-II and has a tertiary structure closely related to the structure of IGF-II, have sufficient structural information to permit proper refolding into the appropriate tertiary structure, even when present in separate polypeptide chains (Wang et al. (1991) Trends Biochem. Sci. 279-281). Thus, for example, amino acids 8-28, or a conservative substitution variant thereof, could be fused to a lysosomal enzyme; the resulting fusion protein could be admixed with amino acids 41-61, or a conservative substitution variant thereof, and administered to a patient.
IGF-II can also be modified to minimize binding to serum TGF-binding proteins (Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76) to avoid sequestration of IGF-II/GILT constructs. A number of studies have localized residues in IGF-II necessary for binding to IGF-binding proteins. Constructs with mutations at these residues can be screened for retention of high affinity binding to the M6P/IGF-II receptor and for reduced affinity for IGF-binding proteins. For example, replacing Phe 26 of IGF-II with Ser is reported to reduce affinity of IGF-II for IGFBP-1 and -6 with no effect on binding to the M6P/IGF-II receptor (Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Other substitutions, such as Lys for Glu 9, can also be advantageous. The analogous mutations, separately or in combination, in a region of IGF-I that is highly conserved with IGF-II result in large decreases in IGF-BP binding (Magee et al. (1999) Biochemistry 38(48):15863-70).
An alternate approach is to identify minimal regions of IGF-II that can bind with high affinity to the M6P/IGF-II receptor. The residues that have been implicated in IGF-II binding to the M6P/IGF-II receptor mostly cluster on one face of IGF-II (Terasawa et al. (1994) EMBO J. 13(23):5590-7). Although IGF-II tertiary structure is normally maintained by three intramolecular disulfide bonds, a peptide incorporating the amino acid sequence on the M6P/IGF-II receptor binding surface of IGF-II can be designed to fold properly and have binding activity. Such a minimal binding peptide is a highly preferred lysosomal targeting domain. For example, a preferred lysosomal targeting domain is amino acids 8-67 of human IGF-II. Designed peptides, based on the region around amino acids 48-55, which bind to the M6P/IGF-II receptor, are also desirable lysosomal targeting domains. Alternatively, a random library of peptides can be screened for the ability to bind the M6P/IGF-II receptor either via a yeast two hybrid assay, or via a phage display type assay.
Binding Affinity for the Insulin Receptor
The inventors of the present application discovered unexpectedly that many furin-resistant IGF-II muteins described herein have reduced or diminished binding affinity for the insulin receptor. Thus, in some embodiments, a peptide tag suitable for the invention has reduced or diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor. In some embodiments, peptide tags with reduced or diminished binding affinity for the insulin receptor suitable for the invention include peptide tags having a binding affinity for the insulin receptor that is more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 50-fold, 100-fold less than that of the wild-type mature human IGF-II. The binding affinity for the insulin receptor can be measured using various in vitro and in vivo assays known in the art. Exemplary binding assays are described in the Examples section.
Mutagenesis
IGF-II muteins can be prepared by introducing appropriate nucleotide changes into the IGF-II DNA, or by synthesis of the desired IGF-II polypeptide. Variations in the IGF-II sequence can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding IGF-II that results in a change in the amino acid sequence of IGF-II as compared with a naturally-occurring sequence of mature human IGF-II. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Amino acid substitutions can also be the result of replacing one amino acid with another amino acid having dis-similar structural and/or chemical properties, i.e., non-conservative amino acid replacements. Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity in the in vivo or in vitro assays known in the art (such as binding assays to the CI-MPR or furin cleavage assays).
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce IGF-II muteins.
Spacer
A furin-resistant GILT tag can be fused to the N-terminus or C-terminus of a polypeptide encoding a lysosomal enzyme. The GILT tag can be fused directly to the lysosomal enzyme polypeptide or can be separated from the lysosomal enzyme polypeptide by a linker or a spacer. An amino acid linker or spacer is generally designed to be flexible or to interpose a structure, such as an alpha-helix, between the two protein moieties. A linker or spacer can be relatively short, such as the sequence Gly-Ala-Pro (SEQ ID NO: 4) or Gly-Gly-Gly-Gly-Gly-Pro (SEQ ID NO: 5), or can be longer, such as, for example, 10-25 amino acids in length. The site of a fusion junction should be selected with care to promote proper folding and activity of both fusion partners and to prevent premature separation of a peptide tag from a GAA polypeptide. In a preferred embodiment, the linker sequence is Gly-Ala-Pro (SEQ ID NO: 4).
Additional constructs of GILT-tagged GAA proteins that can be used in the methods and compositions of the present invention were described in detail in U.S. Publication No. 20050244400, the entire disclosure of which is incorporated herein by reference.
Cells
Any mammalian cell or cell type susceptible to cell culture, and to expression of polypeptides, may be utilized in accordance with the present invention, such as, for example, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), monkey kidney (COS), HT1080, C10, HeLa, baby hamster kidney (BHK), 3T3, C127, CV-1, HaK, NS/O, and L-929 cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include, but are not limited to, BALB/c mouse myeloma line (NSO/I, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells+/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some embodiments, the fusion protein of the present invention is produced from CHO cell lines.
The fusion protein of the invention can also be expressed in a variety of non-mammalian host cells such as, for example, insect (e.g., Sf-9, Sf-21, Hi5), plant (e.g., Leguminosa, cereal, or tobacco), yeast (e.g., S. cerivisae, P. pastoris), prokaryote (e.g., E. Coli, B. subtilis and other Bacillus spp., Pseudomonas spp., Streptomyces spp), or fungus.
In some embodiments, a fusion protein with or without a furin-resistant GILT tag can be produced in furin-deficient cells. As used herein, the term “furin-deficient cells” refers to any cells whose furin protease activity is inhibited, reduced or eliminated. Furin-deficient cells include both mammalian and non-mammalian cells that do not produce furin or produce reduced amount or defective furin protease. Exemplary furin deficient cells that are known and available to the skilled artisan, including but not limited to FD11 cells (Gordon et al (1997) Infection and Immunity 65(8):3370 3375), and those mutant cells described in Moebring and Moehring (1983) Infection and Immunity 41(3):998 1009. Alternatively, a furin deficient cell may be obtained by exposing the above-described mammalian and non-mammalian cells to mutagenesis treatment, e.g., irradiation, ethidium bromide, bromidated uridine (BrdU) and others, preferably chemical mutagenesis, and more preferred ethyl methane sulfonate mutagenesis, recovering the cells which survive the treatment and selecting for those cells which are found to be resistant to the toxicity of Pseudomonas exotoxin A (see Moehring and Moehrin (1983) Infection and Immunity 41(3):998 1009).
Underglycosylation
Targeted therapeutic proteins of the invention can be underglycosylated, that is, one or more carbohydrate structures that would normally be present on a naturally-occurring human protein is preferably omitted, removed, modified, or masked. Without wishing to be bound by any theories, it is contemplated that an underglycosylated protein may extend the half-life of the protein in a mammal. Underglycosylation can be achieved in many ways. In some embodiments, the targeted fusion protein of the invention can be produced using a secretory signal peptide to facilitate secretion of the fusion protein. For example, the fusion protein can be produced using an IGF-II signal peptide. In general, the fusion protein produced using an IGF-II signal peptide has reduced mannose-6-phosphate (M6P) level on the surface of the protein compared to wild-type enzyme. In some embodiments, a protein may be completely underglycosylated (as when synthesized in E. coli), partially unglycosylated (as when synthesized in a mammalian system after disruption of one or more glycosylation sites by site-directed mutagenesis), or may have a non-mammalian glycosylation pattern. For example, underglycosylated fusion proteins may be generated by modifying, substituting or eliminating one or more glycosylation sites by site-directed mutagenesis. For example, wild-type GAA typically have seven sites that match the canonical recognition sequence for N-linked glycosylation, Asn-Xaa-Thr/Ser (SEQ ID NO: 7) (Xaa can be any residue except Pro), namely, Asn-140, -233, -390, -470, -652, -882 and -925 (Hoefsloot et al., 1988; Martiniuk et al., 1990b). One or more Asn at the above described positions may be changed or eliminated to generated underglycosylated GAA. In some embodiments, Asn may be changed to Gln.
In some embodiments, a therapeutic fusion protein can be deglycosylated after synthesis. For example, deglycosylation can be through chemical or enzymatic treatments, and may lead to complete deglycosylation or, if only a portion of the carbohydrate structure is removed, partial deglycosylation.
In some embodiments, glycosylation of a lysosomal enzyme is modified, e.g., by oxidation and reduction, to reduce clearance of the therapeutic protein from the blood. For example, a lysosomal enzyme can be deglycosylated by periodate treatment. In particular, treatment with periodate and a reducing agent such as sodium borohydride is effective to modify the carbohydrate structure of most glycoproteins. Periodate treatment oxidizes vicinal diols, cleaving the carbon-carbon bond and replacing the hydroxyl groups with aldehyde groups; borohydride reduces the aldehydes to hydroxyls. For example, at 1 mM concentration, periodate exclusively oxidizes sialic acid groups and at or above 10 mM all available vicinal diols are converted to aldehydes (Hermanson, G. T. 1996, Bioconjugate techniques. Academic press). Once formed, aldehyde groups are highly reactive and may form Schiff's base linkages with primary amino groups in the protein resulting intramolecular linkages. Therefore, aldehyde groups formed ought to be reduced to alcohol groups. A commonly used reducing agent is NaBH4 and the reaction is best run under alkaline conditions. Many sugar residues including vicinal diols, therefore, are cleaved by this treatment. Nevertheless, while this treatment converts cyclic carbohydrates into linear carbohydrates, it does not completely remove the carbohydrate, minimizing risks of exposing potentially protease-sensitive or antigenic polypeptide sites.
Grubb, J. H., et al (Grubb et al, 2008, PNAS 105:2616) report treatment of human β-glucuronidase with sodium metaperiodate followed by sodium borohydride reduction. The modified beta-glucuronidase retained 90% of activity, but lost both mannose and mannose-6-phosphate dependent receptor uptake activity. The alkaline pH condition used in the reduction due to sodium borohydride reagent as described by Grubb et al is not suitable for all lysosomal enzymes, many of which are labile under alkaline conditions.
Therefore, in some embodiments, sodium cyanoborohydride is used as reducing agent. While the rate of reduction of aldehydes by cyanoborohydride is negligible at neutral pH and above, the rate of reaction becomes rapid at acidic pH (Borch, et al. 1971, JACS 93:2897). For example, regimens using sodium metaperiodate and cyanoborohydride at pH 3.5-4 can be used.
For example, treatment of GAA or alpha galactosidase A, the enzymes deficient in Pompe and Fabry diseases respectively, with periodate and cyanoborohydride at pH 5.6 resulted in good recovery of enzyme activity. Enzyme was incubated with equal volume mixture containing 20 mM sodium metaperiodate and 40 mM sodium cyanoborohydride in 0.1 M Na acetate, pH 5.6 for 60 min on ice. The unreacted periodate was quenched with glycerol (10% final concentration) for 15 min on ice. The proteins were finally exchanged into phosphate buffered saline, pH 6.2 by diafiltration using Amicon centrifugal filter devices. Other reducing reagents for example, dimethylamine borane, may also be useful to reduce aldehydes generated by sodium metaperiodate oxidation of glycoproteins such as GAA under acidic conditions.
Thus, in some embodiments, the reduction of sodium metaperiodate treated GAA involves use of sodium cyanoborohydride at acidic pH from pH 3.0 to pH 6. Optimal conditions for the chemical modification can be readily determined by using two assays: loss of binding to ConA sepharose, and diminished uptake into J774E macrophage.
For example, the ability of periodate/borohydride modified β-glucuronidase to bind to ConA-sepharose was compared to that of untreated β-glucuronidase. The enzymes were incubated with 50 μl ConA beads in 20 mM Tris-HCl, pH 6.8, 0.5 M NaCl for 15 min at room temperature. Beads were centrifuged at maximum speed for 15 sec. Supernatant (flow through) was carefully withdrawn, assayed for GUS activity and analyzed by SDS/PAGE. When we treated GUS exactly as reported in Grubb et al., 60% ConA binding activity was lost and unbound GUS was present only in the flow through of periodate treated and subsequently sodium borohydride reduced sample.
Administration of Therapeutic Proteins
In accordance of the invention, a therapeutic protein of the invention is typically administered to the individual alone, or in compositions or medicaments comprising the therapeutic protein (e.g., in the manufacture of a medicament for the treatment of the disease), as described herein. The compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration.
Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.
The composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
The composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in a preferred embodiment, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The therapeutic protein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
A therapeutic protein (or a composition or medicament containing a therapeutic protein) is administered by any appropriate route. In a preferred embodiment, a therapeutic protein is administered intravenously. In other embodiments, a therapeutic protein is administered by direct administration to a target tissue, such as heart or muscle (e.g., intramuscular), or nervous system (e.g., direct injection into the brain; intraventricularly; intrathecally). Alternatively, a therapeutic protein (or a composition or medicament containing a therapeutic protein) can be administered parenterally, transdermally, or transmucosally (e.g., orally or nasally). More than one route can be used concurrently, if desired.
A therapeutic protein (or a composition or medicament containing a therapeutic protein) can be administered alone, or in conjunction with other agents, such as antihistamines (e.g., diphenhydramine) or immunosuppressants or other immunotherapeutic agents which counteract anti-GILT-tagged lysosomal enzyme antibodies. The term, “in conjunction with,” indicates that the agent is administered prior to, at about the same time as, or following the therapeutic protein (or a composition or medicament containing the therapeutic protein). For example, the agent can be mixed into a composition containing the therapeutic protein, and thereby administered contemporaneously with the therapeutic protein; alternatively, the agent can be administered contemporaneously, without mixing (e.g., by “piggybacking” delivery of the agent on the intravenous line by which the therapeutic protein is also administered, or vice versa). In another example, the agent can be administered separately (e.g., not admixed), but within a short time frame (e.g., within 24 hours) of administration of the therapeutic protein.
The therapeutic protein (or composition or medicament containing the therapeutic protein) is administered in a therapeutically effective amount (i.e., a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease, as described above). The dose which will be therapeutically effective for the treatment of the disease will depend on the nature and extent of the disease's effects, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges using methods known in the art. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The therapeutically effective dosage amount can be, for example, about 0.1-1 mg/kg, about 1-5 mg/kg, about 5-20 mg/kg, about 20-50 mg/kg, or 20-100 mg/kg. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if disease symptoms worsen, the dosage amount can be increased.
The therapeutically effective amount of the therapeutic protein (or composition or medicament containing the therapeutic protein) is administered at regular intervals, depending on the nature and extent of the disease's effects, and on an ongoing basis. Administration at an “interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In some embodiments, the therapeutic protein is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, or daily. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if disease symptoms worsen, the interval between doses can be decreased.
As used herein, the term “bimonthly” means administration once per two months (i.e., once every two months); the term “monthly” means administration once per month; the term “triweekly” means administration once per three weeks (i.e., once every three weeks); the term “biweekly” means administration once per two weeks (i.e., once every two weeks); the term “weekly” means administration once per week; and the term “daily” means administration once per day.
The invention additionally pertains to a pharmaceutical composition comprising a therapeutic protein, as described herein, in a container (e.g., a vial, bottle, bag for intravenous administration, syringe, etc.) with a label containing instructions for administration of the composition for treatment of Pompe disease, such as by the methods described herein.
The invention will be further and more specifically described by the following examples. Examples, however, are included for illustration purposes, not for limitation.
EXAMPLES
Example 1
Furin Cleaves an IGF-II Based GILT Tag
ZC-701 has been developed for the treatment of Pompe disease. ZC-701 is a chimeric protein that contains an N-terminal IGF-II based GILT tag fused via a three amino acid spacer to residues 70-952 of human acid-α-glucosidase (hGAA). Specifically, ZC-701 includes amino acids 1 and 8-67 of human IGF-II (i.e., Δ2-7 of mature human IGF-II), the spacer sequence Gly-Ala-Pro, and amino acids 70-952 of human GAA. The full length amino acid sequence is shown below. The spacer sequence is bolded. The sequence N-terminal to the spacer sequence reflects amino acids 1 and 8-67 of human IGF-II and the sequence C-terminal to the spacer sequence reflects amino acids 70-952 of human GAA. The two potential overlapping furin cleavage sites within the IGF-II tag sequence is bolded and underlined. Arrows point to two potential furin cleavage positions.







(SEQ ID NO: 8)


                               ↓  ↓


AALCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLAL


 


LETYCATPAKSEGAPAHPGRPRAVPTQCDVPPNSRFDCAPDKAITQEQCE


 


ARGCCYIPAKQGLQGAQMGQPWCFFPPSYPSYKLENLSSSEMGYTATLTR


 


TTPTFFPKDILTLRLDVMMETENRLHFTIKDPANRRYEVPLETPRVHSRA


 


PSPLYSVEFSEEPFGVIVHRQLDGRVLLNTTVAPLFFADQFLQLSTSLPS


 


QYITGLAEHLSPLMLSTSWTRITLWNRDLAPTPGANLYGSHPFYLALEDG


 


GSAHGVFLLNSNAMDVVLQPSPALSWRSTGGILDVYIFLGPEPKSVVQQY


 


LDVVGYPFMPPYWGLGFHLCRWGYSSTAITRQVVENMTRAHFPLDVQWND


 


LDYMDSRRDFTFNKDGFRDFPAMVQELHQGGRRYMMIVDPAISSSGPAGS


 


YRPYDEGLRRGVFITNETGQPLIGKVWPGSTAFPDFTNPTALAWWEDMVA


 


EFHDQVPFDGMWIDMNEPSNFIRGSEDGCPNNELENPPYVPGVVGGTLQA


 


ATICASSHQFLSTHYNLHNLYGLTEAIASHRALVKARGTRPFVISRSTFA


 


GHGRYAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVCGFLGNTS


 


EELCVRWTQLGAFYPFMRNHNSLLSLPQEPYSFSEPAQQAMRKALTLRYA


 


LLPHLYTLFHQAHVAGETVARPLFLEFPKDSSTWTVDHQLLWGEALLITP


 


VLQAGKAEVTGYFPLGTWYDLQTVPIEALGSLPPPPAAPREPAIHSEGQW


 


VTLPAPLDTINVHLRAGYITPLQGPGLTTTESRQQPMALAVALTKGGEAR


 


GELFWDDGESLEVLERGAYTQVIFLARNNTIVNELVRVTSEGAGLQLQKV


 


TVLGVATAPQQVLSNGVPVSNFTYSPDTKVLDICVSLLMGEQFLVSWC






During the course of development of ZC-701, it has become apparent that the IGF-II derived GILT tag on a fraction of the ZC-701 molecules is subjected to proteolytic cleavage by furin during production in CHO cells. N-terminal analysis of ZC-701 batch 10-2-F45-54 revealed the presence of two n-terminal sequences. One conformed to the predicted n-terminus of ZC-701 indicating the presence of the predicted ZC-701 protein. The other n-terminal sequence aligned with sequence within the tag portion of ZC-701 indicating the presence of a derivative of ZC-701 consistent with an endoproteolytic cleavage at amino acid residue 34 of ZC-701. Based on the estimated molar ratios of the two n-termini, this batch of ZC-701 was found to have about a 1:1 ratio of intact and cleaved species.
Upon receipt of this result, each of the other batches of ZC-701 were subjected to n-terminal sequencing. All of the batches displayed the same two n-termini with the cleaved species ranging from 20-50% of the total compound. One batch, previously shown to have low uptake activity, displayed a set of n-termini indicative of additional proteolysis. We concluded that the proteolytic event responsible for the second species in all of our batches of ZC-701 was perpetrated by furin or a furin-like protease.
FIG. 1 shows a map of the amino terminus of ZC-701. The two amino acid boxed residues are the sites of n-termini mapped in all of the ZC-701 batches. The first of the N-termini is the site of signal peptide cleavage, which yields the predicted n-terminus of ZC-701. The second boxed residue is the site of an undesired proteolytic cleavage event. The amino acid sequence proximal to the cleavage site is Arg-Arg-Ser-Arg (SEQ ID NO: 9). This matches the canonical cleavage site of a protease present in CHO cells called furin, which cleaves after Arg-X-X-Arg (SEQ ID NO: 10). Furin is a member of a family of prohormone convertases that includes PC3, a protease responsible for maturation of proinsulin in pancreatic islet cells. In fact the PC3 cleavage site in proinsulin is conserved and identical to the site at which furin cleaves the IGF-II tag.
The Furin cleaved ZC-701 differs in molecular weight from intact ZC-701 by about 3000 daltons, which represents less than a 3% difference in molecular weight. Due to the heterogeneity of the oligosaccharide in the protein, the presence of the cleaved ZC-701 was not previously detected by SDS-PAGE. However, if ZC-701 is first deglycosylated by treatment with Peptide N-Glycosidase F (PNGase F), then the cleaved protein can be resolved from the intact ZC-701 by SDS-PAGE.
As shown in FIG. 2, lane 1 of the SDS-PAGE gel shows the electrophoretic pattern of deglycosylated purified ZC-701. Two bands are evident. The upper band is believed to be intact ZC-701 and the lower band is believed to be furin cleaved ZC-701. To prove that the lower band is indeed Furin cleaved ZC-701, same proteins loaded in lane 1 were first treated with furin and then loaded in lane 2. As shown in FIG. 2, all of the proteins in lane 2 co-migrates with the lower band in lane 1 indicating that the lower band is in fact furin cleaved ZC-701.
We have estimated the proportion of ZC-701 that has been cleaved with furin in a number of batches of ZC-701 by quantification of the band intensity in SDS-PAGE and by quantification of amino acids released in N-terminal sequencing experiments. As discussed above, the fraction of cleaved ZC-701 has ranged from 20% to 50% in different batches.
Example 2
Targeted Fusion Proteins Containing a Furin-Resistant IGF-II Based GILT Tag
We can design around the problem of furin cleavage by altering the amino acid sequence of IGF-II such that the amino acid alteration abolishes at least one furin cleavage site. A series of mutant versions of ZC-701 were generated and assayed for resistance to cleavage by furin. Exemplary mutant versions of ZC-701 were generated as described below.
ZC-701
The GILTΔ2-7-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7-GAA70-952 (Plasmid p701). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70.







(SEQ ID NO: 11)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGC


 


AGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCT


 


CCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcac


 


accccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaac


 


agccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcga


 


ggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagccc


 


agatggggcagccctggtgcttcttcccacccagctaccccagctacaag


 


ctggagaacctgagctcctctgaaatgggctacacggccaccctgacccg


 


taccacccccaccttcttccccaaggacatcctgaccctgcggctggacg


 


tgatgatggagactgagaaccgcctccacttcacgatcaaagatccagct


 


aacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggc


 


accgtccccactctacagcgtggagttctctgaggagccatcggggtgat


 


cgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgc


 


ccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcg


 


cagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcac


 


cagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccg


 


gtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggc


 


gggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggt


 


cctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctgg


 


atgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtac


 


ctggacgagtgggatacccgttcatgccgccatactggggcctgggcttc


 


cacctgtgccgctggggctactcctccaccgctatcacccgccaggtggt


 


ggagaacatgaccagggcccacttccccctggacgtccaatggaacgacc


 


tggactacatggactcccggagggacttcacgttcaacaaggatggcttc


 


cgggacttcccggccatggtgcaggagctgcaccagggcggccggcgcta


 


catgatgatcgtggatcctgccatcagcagctcgggccctgccgggagct


 


acaggccctacgacgagggtctgcggaggggggttttcatcaccaacgag


 


accggccagccgctgattgggaaggtatggcccgggtccactgccttccc


 


cgacttcaccaaccccacagccctggcctggtgggaggacatggtggctg


 


agttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgag


 


ccttccaacttcatcaggggctctgaggacggctgccccaacaatgagct


 


ggagaacccaccctacgtgcctggggtggttggggggaccctccaggcgg


 


caaccatctgtgcctccagccaccagtactctccacacactacaacctgc


 


acaacctctacggcctgaccgaagccatcgcctcccacagggcgctggtg


 


aaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctgg


 


ccacggccgatacgccggccactggacgggggacgtgtggagctcctggg


 


agcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgggg


 


gtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcaga


 


ggagctgtgtgtgcgctggacccagctgggggccttctaccccttcatgc


 


ggaaccacaacagcctgctcagtctgccccaggagccgtacagatcagcg


 


agccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactc


 


ctcccccacctctacacgctgttccaccaggcccacgtcgcgggggagac


 


cgtggcccggcccctcttcctggagttccccaaggactctagcacctgga


 


ctgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtg


 


ctccaggccgggaaggccgaagtgactggctacttccccttgggcacatg


 


gtacgacctgcagacggtgccaatagaggcccttggcagcctcccacccc


 


cacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtg


 


acgctgccggcccccctggacaccatcaacgtccacctccgggctgggta


 


catcatccccctgcagggccctggcctcacaaccacagagtcccgccagc


 


agcccatggccctggctgtggccctgaccaagggtggagaggcccgaggg


 


gagctgttctgggacgatggagagagcctggaagtgctggagcgaggggc


 


ctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagc


 


tggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgact


 


gtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccc


 


tgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtg


 


tctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctaga


 


gcttgctagcggccgc






Construct 1459

The GILTΔ2-7/K37-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7/K37-GAA70-952 (Plasmid p1459). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/K37 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7/K37 cassette contains an Arg to Lys substitution at amino acid 37 of the human IGF-II sequence (uppercase bold).







(SEQ ID NO: 12)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCAAGCGC


 


AGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCT


 


CCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcac


 


accccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaac


 


agccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcga


 


ggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagccc


 


agatggggcagccctggtgcttcttcccacccagctaccccagctacaag


 


ctggagaacctgagctcctctgaaatgggctacacggccaccctgacccg


 


taccacccccaccttcttccccaaggacatcctgaccctgcggctggacg


 


tgatgatggagactgagaaccgcctccacttcacgatcaaagatccagct


 


aacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggc


 


accgtccccactctacagcgtggagttctctgaggagcccttcggggtga


 


tcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcg


 


cccctgttctttgcggaccagttccttcagctgtccacctcgctgccctc


 


gcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagca


 


ccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgccc


 


ggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacgg


 


cgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtgg


 


tcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctg


 


gatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagta


 


cctggacgttgtgggatacccgttcatgccgccatactggggcctgggct


 


tccacctgtgccgctggggctactcctccaccgctatcacccgccaggtg


 


gtggagaacatgaccagggcccacttccccctggacgtccaatggaacga


 


cctggactacatggactcccggagggacttcacgttcaacaaggatggct


 


tccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgc


 


tacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggag


 


ctacaggccctagacgagggtctgcggaggggggttttcatcaccaacga


 


gaccggccagccgtgattgggaaggtatggcccgggtccactgccttccc


 


cgacttcaccaaccccacagccctggcctggtgggaggacatggtggctg


 


agttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgag


 


ccttccaacttcatcaggggctctgaggacggctgccccaacaatgagct


 


ggagaacccaccctacgtgcctggggtggttggggggaccctccaggcgg


 


caaccatctgtgcctccagccaccagtttctctccacacactacaacctg


 


cacaacctctacggcctgaccgaagccatcgcctcccacagggcgctggt


 


gaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgctg


 


gccacggccgatacgccggccactggacgggggacgtgtggagctcctgg


 


gagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctggg


 


ggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcag


 


aggagctgtgtgtgcgctggacccagctggggccttctaccccttcatgc


 


ggaaccacaacagcctgctcagtctgccccaggagccgtacagcttcagc


 


gagccggcccagcaggccatgaggaaggccctcaccctgcgctacgcact


 


cctcccccacctctacacgctgttccaccaggcccacgtcgcgggggaga


 


ccgtggcccggcccctcttcctggagttccccaaggactctagcacctgg


 


actgtggaccaccagctcctgtggggggaggccctgctcatcaccccagt


 


gctccaggccgggaaggccgaagtgactggctacttccccttgggcacat


 


ggtacgacctgcagacggtgccaatagaggcccttggcagcctcccaccc


 


ccacctgcagctccccgtgagccagccatccacagcgaggggcagtgggt


 


gacgctgccggcccccctggacaccatcaacgtccacctccgggctgggt


 


acatcatccccctgcagggccctggcctcacaaccacagagtcccgccag


 


cagcccatggccctggctgtggccctgaccaagggtggagaggcccgagg


 


ggagctgttctgggacgatggagagagcctggaagtgctggagcgagggg


 


cctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgag


 


ctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgat


 


gtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccc


 


tgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtg


 


tctcgctgttgatgggagagcagtttctcgtcagctggtgttagtctaga


 


gcttgctagcggccgc






Construct 1460

The GILTΔ2-7/K40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7/K40-GAA70-952 (Plasmid p1460). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/K40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7/K40 cassette contains an Arg to Lys substitution at amino acid 40 of the human IGF-II sequence (uppercase bold).







(SEQ ID NO: 13)


ggtaccagctgctagcaagctattcacaccaATGGGAATCCCAATGGGGA


 


AGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATT


 


GCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGG


 


GGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCA


 


GCAAGGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTC


 


CTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcaca


 


ccccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaaca


 


gccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcgag


 


gcccgcggctgctgctacatccctgcaaagcaggggctgcagggagccca


 


gatggggcagccctggtgcttcttcccacccagctaccccagctacaagc


 


tggagaacctgagctcctctgaaatgggctacacggccaccctgacccgt


 


accacccccaccttcttccccaaggacatcctgaccctgcggctggacgt


 


gatgatggagactgagaaccgcctccacttcacgatcaaagatccagcta


 


acaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggca


 


ccgtccccactctacagcgtggagttctctgaggagcccttcggggtgat


 


cgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcgc


 


ccctgttctttgcggaccagttccttcagctgtccacctcgctgccctcg


 


cagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagcac


 


cagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgcccg


 


gtgcgaacctctacgggtctcaccctttctacctggcgctggaggacggc


 


gggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtggt


 


cctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctgg


 


atgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagtac


 


ctggacgttgtgggatacccgttcatgccgccatactggggcctgggctt


 


ccacctgtgccgctggggctactcctccaccgctatcacccgccaggtgg


 


tggagaacatgaccagggcccacttccccctggacgtccaatggaacgac


 


ctggatacatggatcccggagggacttcacgttcaacaaggatggcttcc


 


gggacttcccggccatggtgcaggagctgcaccagggcggccggcgctac


 


atgatgatcgtggatcctgccatcagcagctcgggccctgccgggagcta


 


caggccctacgacgagggtctgcggagggggttttcatcaccaacgagac


 


cggccagccgctgattgggaaggtatggcccgggtccactgccttccccg


 


acttcaccaaccccacagccctggcctggtgggaggacatggtggctgag


 


ttccatgaccaggtgcccttcgacggcatgtggattgacatgaacgagcc


 


ttccaacttcatcaggggctctgaggacggctgccccaacaatgagctgg


 


agaacccaccctacgtgcctggggtggttggggggaccctccaggcggca


 


accatctgtgcctccagccaccagtttctctccacacactacaacctgca


 


caacctctacggcctgaccgaagccatcgcctcccacagggcgctggtga


 


aggctcgggggacacgcccatttgtgatctcccgctcgacctttgctggc


 


cacggccgatacgccggccactggacgggggacgtgtggagctcctggga


 


gcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctggggg


 


tgcctctggtcggggccgacgtctgcggcttcctgggcaacacctcagag


 


gagctgtgtgtgcgctggacccagctggggccttctaccccttcatgcgg


 


aaccacaacagcctgctcagtctgccccaggagccgtacagcttcagcga


 


gccggcccagcaggccatgaggaaggccctcaccctgcgctacgcactcc


 


tcccccacctctacacgctgttccaccaggcccacgtcgcgggggagacc


 


gtggcccggcccctcttcctggagttccccaaggactctagcacctggac


 


tgtggaccaccagctcctgtggggggaggccctgctcatcaccccagtgc


 


tccaggccgggaaggccgaagtgactggctacttccccttgggcacatgg


 


tacgacctgcvagacggtgccaatagaggcccttggcagcctcccacccc


 


cacctgcagctccccgtgagccagccatccacagcgaggggcagtgggtg


 


acgctgccggcccccctggacaccatcaacgtccacctccgggctgggta


 


catcatccccctgcagggccctggcctcacaaccacagagtcccgccagc


 


agcccatggccctggctgtggccctgaccaagggtggagaggcccgaggg


 


gagctgttctgggacgatggagagagcctggaagtgctggagcgaggggc


 


ctacacacaggtcatcttcctggccaggaataacacgatcgtgaatgagc


 


tggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtgact


 


gtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgtccc


 


tgtctccaacttcacctacagccccgacaccaaggtcctggacatctgtg


 


tctcgctgttgatgggagagcagtttctcgtcagctggtggttagtctag


 


agcttgctagcggccgc






Construct 1461

The GILTΔ2-7/A37-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7/A37-GAA70-952 (Plasmid p1461). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/A37 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7/A37 cassette contains an Arg to Ala substitution at amino acid 37 of the human IGF-II sequence (uppercase bold).







(SEQ ID NO: 14)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCGCTCGC


 


AGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCT


 


CCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcac


 


accccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaac


 


agccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcga


 


ggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagccc


 


agatggggcagccctggtgcttcttcccacccagctaccccagctacaag


 


ctggagaacctgagctcctctgaaatgggctacacggccaccctgacccg


 


taccacccccaccttcttccccaaggacatcctgaccctgcggctggacg


 


tgatgatggagactgagaaccgcctccacttcacgatcaaagatccagct


 


aacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggc


 


accgtccccactctacagcgtggagttctctgaggagcccttcggggtga


 


tcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcg


 


cccctgttctttgcggaccagttccttcagctgtccacctcgctgccctc


 


gcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagca


 


ccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgccc


 


ggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacgg


 


cgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtgg


 


tcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctg


 


gatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagta


 


cctggacgttgtgggatacccgttcatgccgccatactggggcctgggct


 


tccacctgtgccgctggggctactcctccaccgctatcacccgccaggtg


 


gtggagaacatgaccagggcccacttccccctggacgtccaatggaacga


 


cctggactacatggactcccggagggacttcacgttcaacaaggatggct


 


tccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgc


 


tacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggag


 


ctacaggccctacgacgagggtctgcggaggggggttttcatcaccaacg


 


agaccggccagccgctgattgggaaggtatggcccgggtccactgccttc


 


cccgacttcaccaaccccacagccctggcctggtgggaggacatggtggc


 


tgagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacg


 


agccttccaacttcatcaggggctctgaggacggctgccccaacaatgag


 


ctggagaacccaccctacgtgcctggggtggttggggggaccctccaggc


 


ggcaaccatctgtgcctccagccaccagtttctctccacacactacaacc


 


tgcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctg


 


gtgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgc


 


tggccacggccgatacgccggccactggacgggggacgtgtggagctcct


 


gggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctg


 


ggggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctc


 


agaggagctgtgtgtgcgctggacccagctgggggccttctaccccttca


 


tgcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttc


 


agcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgc


 


actcctcccccacctctacacgctgttccaccaggcccacgtcgcggggg


 


agaccgtggcccggcccctcttcctggagttccccaaggactctagcacc


 


tggactgtggaccaccagctcctgtggggggaggccctgctcatcacccc


 


agtgctccaggccgggaaggccgaagtgactggctacttccccttgggca


 


catggtacgacctgcagacggtgccaatagaggcccttggcagcctccca


 


cccccacctgcagctccccgtgagccagccatccacagcgaggggcagtg


 


ggtgacgctgccggcccccctggacaccatcaacgtccacctccgggctg


 


ggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgc


 


cagcagcccatggccctggctgtggccctgaccaagggtggagaggcccg


 


aggggagctgttctgggacgatggagagagcctggaagtgctggagcgag


 


gggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaat


 


gagctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggt


 


gactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtg


 


tccctgtctccaacttcacctacagccccgacaccaaggtcctggacatc


 


tgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtc


 


tagagcttgctagcggccgc






Construct 1463

The GILTΔ2-7/A40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7/A40-GAA70-952 (Plasmid p1463). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7/A40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7/A40 cassette contains an Arg to Ala substitution at amino acid 40 of the human IGF2 sequence (uppercase bold).







(SEQ ID NO: 15)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGC


 


AGCGCTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCT


 


CCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcac


 


accccggccgtcccagagcagtgcccacacagtgcgacgtcccccccaac


 


agccgcttcgattgcgcccctgacaaggccatcacccaggaacagtgcga


 


ggcccgcggctgctgctacatccctgcaaagcaggggctgcagggagccc


 


agatggggcagccctggtgcttcttcccacccagctaccccagctacaag


 


ctggagaacctgagctcctctgaaatgggctacacggccaccctgacccg


 


taccacccccaccttcttccccaaggacatcctgaccctgcggctggacg


 


tgatgatggagactgagaaccgcctccacttcacgatcaaagatccagct


 


aacaggcgctacgaggtgcccttggagaccccgcgtgtccacagccgggc


 


accgtccccactctacagcgtggagttctctgaggagcccttcggggtga


 


tcgtgcaccggcagctggacggccgcgtgctgctgaacacgacggtggcg


 


cccctgttctttgcggaccagttccttcagctgtccacctcgctgccctc


 


gcagtatatcacaggcctcgccgagcacctcagtcccctgatgctcagca


 


ccagctggaccaggatcaccctgtggaaccgggaccttgcgcccacgccc


 


ggtgcgaacctctacgggtctcaccctttctacctggcgctggaggacgg


 


cgggtcggcacacggggtgttcctgctaaacagcaatgccatggatgtgg


 


tcctgcagccgagccctgcccttagctggaggtcgacaggtgggatcctg


 


gatgtctacatcttcctgggcccagagcccaagagcgtggtgcagcagta


 


cctggacgttgtgggatacccgttcatgccgccatactggggcctgggct


 


tccacctgtgccgctggggctactcctccaccgctatcacccgccaggtg


 


gtggagaacatgaccagggcccacttccccctggacgtccaatggaacga


 


cctggactacatggactcccggagggacttcacgttcaacaaggatggct


 


tccgggacttcccggccatggtgcaggagctgcaccagggcggccggcgc


 


tacatgatgatcgtggatcctgccatcagcagctcgggccctgccgggag


 


ctacaggccctacgacgagggtctgcggaggggggttttcataccaacga


 


gaccggccagccgctgattgggaaggtatggcccgggtccactgccttcc


 


ccgacttcaccaaccccacagccctggcctggtgggaggacatggtggct


 


gagttccatgaccaggtgcccttcgacggcatgtggattgacatgaacga


 


gccttccaacttcatcaggggctctgaggacggctgccccaacaatgagc


 


tggagaacccaccctacgtgcctggggtggttggggggaccctccaggcg


 


gcaaccatctgtgcctccagccaccagtttctctccacacactacaacct


 


gcacaacctctacggcctgaccgaagccatcgcctcccacagggcgctgg


 


tgaaggctcgggggacacgcccatttgtgatctcccgctcgacctttgct


 


ggccacggccgatacgccggccactggacgggggacgtgtggagctcctg


 


ggagcagctcgcctcctccgtgccagaaatcctgcagtttaacctgctgg


 


gggtgcctctggtcggggccgacgtctgcggcttcctgggcaacacctca


 


gaggagctgtgtgtgcgctggacccagctgggggccttctaccccttcat


 


gcggaaccacaacagcctgctcagtctgccccaggagccgtacagcttca


 


gcgagccggcccagcaggccatgaggaaggccctcaccctgcgctacgca


 


ctcctcccccacctctacacgctgttccaccaggcccacgtcgcggggga


 


gaccgtggcccggcccctcttcctggagttccccaaggactctagcacct


 


ggactgtggaccaccagctcctgtggggggaggccctgctcatcacccca


 


gtgctccaggccgggaaggccgaagtgactggctacttccccttgggcac


 


atggtacgacctgcagacggtgccaatagaggcccttggcagcctcccac


 


ccccacctgcagctccccgtgagccagccatccacagcgaggggcagtgg


 


gtgacgctgccggcccccctggacaccatcaacgtccacctccgggctgg


 


gtacatcatccccctgcagggccctggcctcacaaccacagagtcccgcc


 


agcagcccatggccctggctgtggccctgaccaagggtggagaggcccga


 


ggggagctgttctgggacgatggagagagcctggaagtgctggagcgagg


 


ggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaatg


 


agctggtacgtgtgaccagtgagggagctggcctgcagctgcagaaggtg


 


actgtcctgggcgtggccacggcgccccagcaggtcctctccaacggtgt


 


ccctgtctccaacttcacctacagccccgacaccaaggtcctggacatct


 


gtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagtct


 


agagcttgctagcggccgc






Construct 1479

The GILTΔ2-7M1/K37-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7M1/K37-GAA70-952 (Plasmid p1479). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/K37 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7M1/K37 cassette contains an Arg to Lys substitution at amino acid 37 of the human IGF-II sequence (uppercase bold).







(SEQ ID NO: 16)


ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCT


 


GCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCG


 


GGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCTAC


 


TTCAGCAGACCCGCAAGCCGTGTGAGTAAGCGCAGCCGTGGCATTGTTGA


 


GGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCG


 


CTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccaga


 


gcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgc


 


ccctgacaggccatcacccaggaacagtgcgaggccgcggctgctgctac


 


atccctgcaaagcaggggctgcagggagcccagatggggcagccctggtg


 


cttcttcccacccagctaccccagctacaagctggagaacctgagctcct


 


ctgaaatgggctacacggccaccctgacccgtaccacccccaccttcttc


 


cccaaggacatcctgaccctgcggctggacgtgatgatggagactgagaa


 


ccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgc


 


ccttggagaccccgcgtgtccacagccgggcaccgtccccactctacagc


 


gtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctgga


 


cggccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggacc


 


agttccttcagctgtccacctcgctgccctcgcagtatatcacaggcctc


 


gccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcac


 


cctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggt


 


ctcaccctttctacctggcgctggaggacggcgggtcggcacacggggtg


 


ttcctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgc


 


catagaggaggtcgacaggtgggatcctggatgtctacatcttcctgggc


 


ccagagcccaagagcgtggtgcagcagtacctggacgttgtgggataccc


 


gttcatgccgccatactggggcctgggcttccacctgtgccgctggggct


 


actcctccaccgctatcacccgccaggtggtggagaacatgaccagggcc


 


cacttccccctggacgtccaatggaacgacctggactacatggactcccg


 


gagggacttcacgttcaacaaggatggcttccgggacttcccggccatgg


 


tgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcct


 


gccatcagcagctcgggccagccgggagctacaggccctacgacgagggt


 


ctgcggaggggggttttcatcaccaacgagaccggccagccgctgattgg


 


gaaggtatggcccgggtccactgccttccccgacttcaccaaccccacag


 


ccaggcctggtgggaggacatggtggctgagttccatgaccaggtgccct


 


tcgacggcatgtggattgacatgaacgagccttccaacttcatcaggggc


 


tctgaggacggctgccccaacaatgagaggagaacccaccctacgtgcct


 


ggggtggttggggggaccctccaggcggcaaccatctgtgcctccagcca


 


ccagtttctctccacacactacaacctgcacaacctctacggcctgaccg


 


aagccatcgcctcccacagggcgaggtgaaggctcgggggacacgcccat


 


ttgtgatctcccgctcgacctttgaggccacggccgatacgccggccact


 


ggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgcca


 


gaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgt


 


ctgcggcttcctgggcaacacctcagaggagagtgtgtgcgctggaccca


 


gctgggggccttctaccccttcatgcggaaccacaacagcctgctcagta


 


gccccaggagccgtacagcttcagcgagccggcccagcaggccatgagga


 


aggccctcaccctgcgctacgcactcctcccccacctctacacgctgttc


 


caccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctgga


 


gttccccaaggactctagcacctggactgtggaccaccagctcctgtggg


 


gggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtg


 


actggctacttccccttgggcacatggtacgacctgcagacggtgccaat


 


agaggcccttggcagcctcccacccccacctgcagctccccgtgagccag


 


ccatccacagcgaggggcagtgggtgacgctgccggcccccctggacacc


 


atcaacgtccacctccgggctgggtacatcatccccctgcagggccaggc


 


ctcacaaccacagagtcccgccagcagcccatggccctggctgtggccct


 


gaccaagggtggagaggcccgaggggagagttctgggacgatggagagag


 


cctggaagtgctggagcgaggggcctacacacaggtcatcttcctggcca


 


ggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagct


 


ggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgcccca


 


gcaggtcctctccaacggtgtccctgtctccaacttcacctacagccccg


 


acaccaaggtcctggacatctgtgtctcgctgttgatgggagagcagttt


 


ctcgtcagctggtgttagtctagagcttgctagcggccgc






Construct 1487

The GILTΔ2-7M1/A37-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7M1/A37-GAA70-952 (Plasmid p1487). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/A37 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7M1/A37 cassette contains an Arg to Ala substitution at amino acid 37 of the human IGF-II sequence (uppercase bold).







(SEQ ID NO: 17)


ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCT


 


GCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCG


 


GGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCTAC


 


TTCAGCAGACCCGCAAGCCGTGTGAGTGCTCGCAGCCGTGGCATTGTTGA


 


GGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCG


 


CTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccaga


 


gcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgc


 


ccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgct


 


acatccctgcaaagcaggggctgcagggagcccagatggggcagccctgg


 


tgcttcttcccacccagctaccccagctacaagctggagaacctgagctc


 


ctctgaaatgggctacacggccaccctgacccgtaccacccccaccttct


 


tccccaaggacatcctgaccctgcggctggacgtgatgatggagactgag


 


aaccgcctccacttcacgatcaaagatccagctaacaggcgctacgaggt


 


gcccttggagaccccgcgtgtccacagccgggcaccgtccccactctaca


 


gcgtggagttctagaggagccatcggggtgatcgtgcaccggcagctgga


 


cggccgcgtgctgctgaacacgacggtggcgcccctgactttgcggacca


 


gaccttcagctgtccacctcgctgccacgcagtatatcacaggcctcgcc


 


gagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccag


 


tggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctca


 


ccctttctacctggcgctggaggacggcgggtcggcacacggggtgacct


 


gctaaacagcaatgccatggatgtggtcctgcagccgagccctgccctta


 


gaggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccag


 


agcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttc


 


atgccgccatactggggcctgggcttccacctgtgccgctggggctactc


 


accaccgctatcacccgccaggtggtggagaacatgaccagggcccactt


 


ccccctggacgtccaatggaacgacctggactacatggactcccggaggg


 


acttcacgttcaacaaggatggcttccgggacttcccggccatggtgcag


 


gagctgcaccagggcggccggcgctacatgatgatcgtggatcctgccat


 


cagcagacgggccctgccgggagctacaggccctacgacgagggtctgcg


 


gaggggggttttcatcaccaacgagaccggccagccgctgattgggaagg


 


tatggcccgggtccactgccttccccgacttcaccaaccccacagccctg


 


gcctggtgggaggacatggtggctgagaccatgaccaggtgccatcgacg


 


gcatgtggattgacatgaacgagccttccaacttcatcaggggctctgag


 


gacggctgccccaacaatgagctggagaacccaccctacgtgcctggggt


 


ggttggggggaccctccaggcggcaaccatctgtgcctccagccaccagt


 


ttactccacacactacaacctgcacaacctctacggcctgaccgaagcca


 


tcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtg


 


atctcccgctcgacctttgctggccacggccgatacgccggccactggac


 


gggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaa


 


tcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgc


 


ggcttcctgggcaacacctcagaggagagtgtgtgcgaggacccagaggg


 


ggccttctaccccttcatgcggaaccacaacagcctgctcagtctgcccc


 


aggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggcc


 


ctcaccagcgctacgcactcctcccccacctctacacgctgttccaccag


 


gcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttccc


 


caaggactctagcacctggactgtggaccaccagacctgtggggggaggc


 


cagctcatcaccccagtgctccaggccgggaaggccgaagtgactggcta


 


cttcccatgggcacatggtacgacctgcagacggtgccaatagaggccat


 


ggcagcctcccacccccacctgcagctccccgtgagccagccatccacag


 


cgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtcc


 


acctccgggctgggtacatcatccccctgcagggccaggcctcacaacca


 


cagagtcccgccagcagcccatggccaggctgtggccctgaccaagggtg


 


gagaggcccgaggggagctgttctgggacgatggagagagcctggaagtg


 


ctggagcgaggggcctacacacaggtcatatcctggccaggaataacacg


 


atcgtgaatgagaggtacgtgtgaccagtgagggagctggcctgcagctg


 


cagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctc


 


caacggtgtccagtaccaacttcacctacagccccgacaccaaggtcctg


 


gacatctgtgtacgctgttgatgggagagcagtttctcgtcagctggtgt


 


tagtctagagcttgctagcggccgc






As shown in FIG. 3, three exemplary mutants (i.e., constructs 1459, 1460 and 1461) in which alanine or lysine has been substituted for one of the canonical arginine residues were expressed without detectable cleavage by furin. As also shown in FIG. 3 (right panel), construct 1461 containing a R37A substitution is additionally resistant to addition of exogenous furin.
Construct 1726
The GILTΔ2-7Δ30-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ30-39-GAA70-952 (Plasmid 1726). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ30-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ30-39 cassette contains a deletion of amino acid residues 30-39 (Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 18)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCCGTGGCATCGTCCCCGCCAAGTCCGAG


 


GGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcga


 


cgtcccccccaacagccgcttcgattgcgccctgacaaggccatcaccca


 


ggaacagtgcgaggcccgcggctgctgctacatccctgcaaagcaggggc


 


tgcagggagcccagatggggcagccctggtgcttcttcccacccagctac


 


cccagctacaagctggagaacctgagctcctctgaaatgggcatacacgg


 


ccaccctgacccgtaccacccccaccttcttccccaggacatcctgaccc


 


tgcggctggacgtgatgatggagactgagaaccgcctccacttcacgatc


 


aaagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgt


 


ccacagccgggcaccgtcccactctacagcgtggagttctctgaggagcc


 


cttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaaca


 


cgacggtggcgcccctgttctttgcggaccagttccttcagctgtccacc


 


tcgtgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctg


 


atcgtcagcaccagctggaccaggatcaccctgtggaaccgggacttgcg


 


cccacgcccggtgcgaacctctacgggtctcaccctttctacctggaggt


 


cagacaggtgggatcctggatgtctacatcttcctgggcccagagcccaa


 


gagcgtggtgcagcagtacctggacgccttagctggaggtcgacaggtgg


 


gatcctggatgtctacatcttcctgggcccagagcccaagagcgtggtgc


 


agcagtacctggacgttgtgggatacccgttcatgccgccatactggggc


 


ctgggcttccacctgtgccgctggggctactcctccaccgctatcacccg


 


ccaggtggtggagaacatgaccagggcccacttccccctggacgtccaat


 


ggaacgacctggactacatggactcccggagggacttcacgttcaacaag


 


gatggcttccgggacttcccggccatggtgcaggagctgcaccagggcgg


 


ccggcgctacatgatgatcgtggatcctgccatcagcagctcgggccctg


 


ccgggagctacaggccctacgacgagggtctgcggaggggggttttcatc


 


accaacgagaccggccagccgctgattgggaggtatggcccgggtccact


 


gccttccccgacttcaccaaccccacagccctggcctggtgggaggacat


 


ggtggctgagttccatgaccaggtgcccttcgacggcatgtggattgaca


 


tgaacgagccttccaacttcatcaggggctctgaggacggctgccccaac


 


aatgagctggagaacccaccctacgtgcctggggtggtggggggaccctc


 


caggcggcaaccatctgtgcctccagccaccagtttctctccacacacta


 


caacctgcacaacctctacggcctgaccgaagccatcgcctcccacaggg


 


cgctggtgaaggctccgggggacacgcccatttgtgatctcccgctcgac


 


ctttgctggccacggccgatacgccggccactggacgggggacgtgtgga


 


gctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaac


 


ctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgggcaa


 


cacctcagaggagctgtgtgcgctggacccagctgggggccttctacccc


 


ttcatgcggaaccacaacagcctgctcagtgtgccccaggagccgtacag


 


cttcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgct


 


acgcactcctcccccacctctacacgctgttccaccaggcccacgtcgcg


 


ggggagaccgtggcccggcccctcttcctggagttccccaggactctagc


 


acctggactgtggaccaccagctcctgtggggggaggccctgctcatcac


 


cccagtgctccaggccgggaaggccgaagtgactggctacttcccctggg


 


cacatggtacgacctgcagacggtgccaatagaggcccttggcagcctcc


 


cacccccacctgcagctccccgtgagccagccatccacgcgaggggcagt


 


gggtgacgctgccgcccccctggacaccatcaacgtccacctccgggctg


 


ggtacatcatccccctgcagggccctggcctcacaaccacagagtcccgc


 


cagcagcccatggccctggctgtggccctgaccaagggtggagaggcccg


 


aggggagctgttctgggacgatggagagagcctggaagtgctggagcgag


 


gggcctacacacaggtcatcttcctggccaggaataacacgatcgtgaat


 


gagctggtacgtgtgaccagtgagggagctggccatgcagctgcagaagg


 


tgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacggt


 


gtccctgtctccaacttcacctacagccccgacaccaaggtcctggacat


 


ctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgttagt


 


ctagagcttgctagcggccgc






Construct 1749

The GILTΔ2-7Δ31-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ31-39-GAA70-952 (Plasmid 1749). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ31-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ31-39 cassette contains a deletion of amino acid residues 31-39 (Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 19)


ggtaccagcgctagcaagctaattcacaccaATGGGAATCCCAATGGGGA


 


AGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATT


 


GCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGG


 


GGACCGCGGCTTCTACTTCAGCAGGCGTGGCATCGTTGAGGAGTGCTGTT


 


TCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCC


 


AAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccac


 


acagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaagg


 


ccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgca


 


aagcaggggctgcagggagcccagatggggcagccctggtgcttatccca


 


cccagctaccccagctacaagaggagaacctgagctcctctgaaatgggc


 


tacacggccaccctgacccgtaccacccccaccttatccccaaggacatc


 


ctgaccctgcggctggacgtgatgatggagactgagaaccgcctccactt


 


cacgatcaaagatccagctaacaggcgctacgaggtgcccttggagaccc


 


cgcgtgtccacagccgggcaccgtccccactctacagcgtggagttctct


 


gaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgctg


 


ctgaacacgacggtggcgcccctgttctttgcggaccagttccttcagct


 


gtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacctca


 


gtcccctgatgacagcaccagctggaccaggatcaccagtggaaccggga


 


ccttgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacc


 


tggcgctggaggacggcgggtcggcacacggggtgttcctgctaaacagc


 


aatgccatggatgtggtcctgcagccgagccctgcccttagctggaggtc


 


gacaggtgggatcctggatgtctacatcttcctgggcccagagcccaaga


 


gcgtggtgcagcagtacctggacgagtgggatacccgttcatgccgccat


 


actggggcctgggcttccacctgtgccgctggggctactcctccaccgct


 


atcacccgccaggtggtggagaacatgaccagggcccacttccccctgga


 


cgtccaatggaacgacctggactacatggactcccggagggacttcacgt


 


tcaacaaggatggatccgggacttcccggccatggtgcaggagagcacca


 


gggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcgg


 


gccctgccgggagctacaggccctacgacgagggtctgcggaggggggtt


 


ttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccgg


 


gtccactgccttccccgacttcaccaaccccacagccctggcctggtggg


 


aggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtgg


 


attgacatgaacgagccttccaacttcatcaggggctctgaggacggctg


 


ccccaacaatgagaggagaacccaccctacgtgcctggggtggttggggg


 


gaccaccaggcggcaaccatctgtgcctccagccaccagtttactccaca


 


cactacaacctgcacaacctctacggcctgaccgaagccatcgcctccca


 


cagggcgctggtgaaggctcgggggacacgcccatttgtgatctcccgct


 


cgacctttgctggccacggccgatacgccggccactggacgggggacgtg


 


tggagctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtt


 


taacctgctgggggtgcctctggtcggggccgacgtctgcggcttcctgg


 


gcaacacctcagaggagctgtgtgtgcgctggacccagctgggggccttc


 


taccccttcatgcggaaccacaacagcctgctcagtctgccccaggagcc


 


gtacagcttcagcgagccggcccagcaggccatgaggaaggccacaccct


 


gcgctacgcactcctcccccacctctacacgctgttccaccaggcccacg


 


tcgcgggggagaccgtggcccggcccctcttcctggagttccccaaggac


 


tctagcacctggactgtggaccaccagctcctgtggggggaggccctgct


 


catcaccccagtgctccaggccgggaaggccgaagtgactggctacttcc


 


ccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggc


 


agcctcccacccccacctgcagctccccgtgagccagccatccacagcga


 


ggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacc


 


tccgggctgggtacatcatccccctgcagggccaggcctcacaaccacag


 


agtmcgccagcagcccatggccaggctgtggccctgaccaagggtggaga


 


ggcccgaggggagctgttctgggacgatggagagagcctggaagtgctgg


 


agcgaggggcctacacacaggtcatcttcctggccaggaataacacgatc


 


gtgaatgagaggtacgtgtgaccagtgagggagaggcctgcagctgcaga


 


aggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaac


 


ggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctgga


 


catctgtgtctcgctgttgatgggagagcagtttctcgtcagaggtgtta


 


gtctagagcttgctagcggccgc






Construct 1746

The GILTΔ2-7Δ32-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ32-39-GAA70-952 (Plasmid 1746). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ32-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ32-39 cassette contains a deletion of amino acid residues 32-39 (Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 20)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCCGTGGCATCGTTGAGGAGTGC


 


TGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCC


 


CGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgc


 


ccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgac


 


aaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccc


 


tgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttct


 


tcccacccagctaccccagctacaagctggagaacctgagctcctctgaa


 


atgggctacacggccaccctgacccgtaccacccccaccttcttccccaa


 


ggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcc


 


tccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttg


 


gagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtgga


 


gttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggcc


 


gcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagttc


 


cttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccga


 


gcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctgt


 


ggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcac


 


cctactacctggcgctggaggacggcgggtcggcacacggggtgttcctg


 


ctaaacagcaatgccatggatgtggtcctgcagccgagccctgccatagc


 


tggaggtcgacaggtgggatcctggatgtctacatcttcctgggcccaga


 


gcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttca


 


tgccgccatactggggcctgggcaccacctgtgccgctggggctactcct


 


ccaccgctatcacccgccaggtggtggagaacatgaccagggcccacttc


 


cccctggacgtccaatggaacgacctggactacatggactcccggaggga


 


cttcacgttcaacaaggatggcttccgggacttcccggccatggtgcagg


 


agctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatc


 


agcagctcgggccctgccgggagctacaggccctacgacgagggtctgcg


 


gaggggggttttcatcaccaacgagaccggccagccgctgattgggaagg


 


tatggcccgggtccactgccttccccgacttcaccaaccccacagccctg


 


gcctggtgggaggacatggtggctgagttccatgaccaggtgccatcgac


 


ggcatgtggattgacatgaacgagccttccaacttcatcaggggctctga


 


ggacggctgccccaacaatgagaggagaacccaccctacgtgcctggggt


 


ggaggggggaccctccaggcggcaaccatctgtgcctccagccaccagtt


 


tctctccacacactacaacctgcacaacctctacggcctgaccgaagcca


 


tcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtg


 


atctcccgctcgacctttgaggccacggccgatacgccggccactggacg


 


ggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaat


 


cctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcgg


 


cttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctggg


 


ggccttctaccccttcatgcggaaccacaacagcctgacagtctgcccca


 


ggagccgtacagatcagcgagccggcccagcaggccatgaggaaggccct


 


caccctgcgctacgcactcctcccccacctctacacgctgttccaccagg


 


cccacgtcgcgggggagaccgtggcccggcccctcttcctggagttcccc


 


aaggactctagcacctggactgtggaccaccagctcctgtggggggaggc


 


cctgacatcaccccagtgaccaggccgggaaggccgaagtgactggctac


 


ttccccttgggcacatggtacgacctgcagacggtgccaatagaggccct


 


tggcagcctcccacccccacctgcagctccccgtgagccagccatccaca


 


gcgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtc


 


cacctccgggagggtacatcatccccctgcagggccctggcctcacaacc


 


acagagtcccgccagcagcccatggccctggagtggccctgaccaagggt


 


ggagaggcccgaggggagagttctgggacgatggagagagcctggaagtg


 


ctggagcgaggggcctacacacaggtcatatcctggccaggaataacacg


 


atcgtgaatgagaggtacgtgtgaccagtgagggagaggcctgcagagca


 


gaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctcca


 


acggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctg


 


gacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtg


 


ttagtctagagcttgctagcggccgc






Construct 1747

The GILTΔ2-7Δ33-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ33-39-GAA70-952 (Plasmid 1747). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ33-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ33-39 cassette contains a deletion of amino acid residues 33-39 (Ser-Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 21)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCACGTGGCATCGTTGAGGAG


 


TGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTAC


 


CCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcag


 


tgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccct


 


gacaaggccatcacccaggaacagtgcgaggcccgcggctgctgctacat


 


ccctgcaagcaggggctgcagggagcccagatggggcagccctggtgctt


 


cttcccacccagctaccccagctacaagctggagaacctgagctcctctg


 


aaatgggctacacggccaccctgacccgtaccacccccaccttcttcccc


 


aaggacatcctgaccctgcggctggacgtgatgatggagactgagaaccg


 


cctccacttcacgatcaaagatccagctaacaggcgctacgaggtgccct


 


tggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgtg


 


gagttctctgaggagcccttcggggtgatcgtgcaccggcagctggacgg


 


ccgcgtgctgctgaacacgacggtggcgcccctgttctttgcggaccagt


 


tccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcgcc


 


gagcacctcagtcccctgatgctcagcaccagctggaccaggatcaccct


 


gtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctc


 


accctttctacctggcgctggaggacggcgggtcggcacacggggtgttc


 


ctgctaaacagcaatgccatggaatgtggtcctgcagccgagccctgccc


 


ttagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggc


 


ccagagcccaagagcgtggtgcagcagtacctggacgttgtgggataccc


 


gttcatgccgccatactggggcctgggcttccacctgtgccgctggggct


 


actcctccaccgctatcacccgccaggtggtggagaacatgaccagggcc


 


cacttccccctggacgtccaatggaacgacctggactacatggactcccg


 


gagggacttcacgttcaacaaggatggcttccgggacttcccggccatgg


 


tgcaggagctgcaccagggcggccggcgctacatgatgatcgtggatcct


 


gccatcagcagctcgggccctgccgggagctacaggccctacgacgaggg


 


tctgcggaggggggttttcatcaccaacgagaccggccagccgctgattg


 


ggaaggtatggcccgggtccactgccttccccgacttcaccaaccccaca


 


gccctggcctggtgggaggacatggtggctgagttccatgaccaggtgcc


 


cttcgacggcatgtggattgacatgaacgagccttccaacttcatcaggg


 


gctctgaggacggctgccccaacaatgagctggagaacccaccctacgtg


 


cctggggtggtggggggaccctccaggcggcaaccatctgtgcctccagc


 


caccagtttctctccacacactacaacctgcacaacctctacggcctgac


 


cgaagccatcgcctcccacaggggcgctggtgaaggctcgggggacacgc


 


ccatttctgatctcccgctcgacctttgctggccacggccgatacgccgg


 


ccactggacgggggacgtgtggagctcctgggagcagctcgcctcctccg


 


tgccagaaatcctgcagtttaacctgctgggggtgcctctggtcggggcc


 


gacgtctgcggcttcctgggcaacacctcagaggactgtgtgtgcgctgg


 


acccagctgggggccttctaccccttcatgcggaccacaacagcctgctc


 


agctggccccaggagccgtacagcttcagcgagccggcccagcaggccat


 


gaggaaggccctcaccctgcgctacgcactcctcccccacctctacacgc


 


tgttccaccaggcccacgtgcgggggagaccgtggcccggcccctcttcc


 


tggagttccccaaggactctagcacctggactgtggaccaccagctcctg


 


tggggggaggccctgctcatcaccccagtgctccaggccgggaaggccga


 


agtgactggctacttccccttgggcacatggtacgacctgcagacggtgc


 


caatagaggcccttggcagcctcccaccccacctgcagctccccgtgagc


 


cagccatccacagcgaggggcagtgggtgacgctgccggcccccctggac


 


accatcaacgtccacctccgggctgggtacatcatccccctgcagggccc


 


tggcctcacaaccacagagtcccgccagcagcccatggccctggctgtgg


 


ccctgaccaagggtggagaggcccgaggggagctgttctgggacgatgga


 


gagagcctggaagtgctggagcgaggggcctacacacaggtcatcttcct


 


ggccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgagg


 


gagctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcg


 


ccccagcaggtcctctccaacggtgtcccgtctccaacttcacctacagc


 


cccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagca


 


gtttctcgtcagctggtgttagtctagagcttgctagcggccgc






Construct 1758

The GILTΔ2-7Δ34-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ34-39-GAA70-952 (Plasmid 1758). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ34-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ34-39 cassette contains a deletion of amino acid residues 34-39 (Arg-Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 22)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGGCATCGTTGAG


 


GAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGC


 


TACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagag


 


cagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcc


 


cctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgcta


 


catccctgcaaagcaggggctgcagggagcccagatggggcagccctggt


 


gcttcttcccacccagctaccccagctacaagctggagaacctgagctcc


 


tctgaaatgggctacacggccaccctgacccgtaccacccccaccttctt


 


ccccaaggacatcctgaccctgcggctggacgtgatgatggagactgaga


 


accgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtg


 


cccttggagaccccgcgtgtccacagccgggcaccgtccccactctacag


 


cgtggagttctctgaggagcccttcggggtgatcgtgcaccggcagctgg


 


acggccgcgtgctgctgaacacgacggtggcgcccctgactttgcggacc


 


agaccttcagctgtccacctcgctgccctcgcagtatatcacaggcctcg


 


ccgagcacctcagtcccctgatgctcagcaccagctggaccaggatcacc


 


ctgtggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtc


 


tcaccattctacctggcgctggaggacggcgggtcggcacacggggtgtt


 


cctgctaaacagcaatgccatggatgtggtcctgcagccgagccctgcca


 


tagctggaggtcgacaggtgggatcctggatgtctacatcttcctgggcc


 


cagagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccg


 


ttcatgccgccatactggggcctgggcttccacctgtgccgctggggcta


 


ctcctccaccgctatcacccgccaggtggtggagaacatgaccagggccc


 


acttccccctggacgtccaatggaacgacctggactacatggactcccgg


 


agggacttcacgttcaacaaggatggatccgggacttcccggccatggtg


 


caggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgc


 


catcagcagctcgggccctgccgggagctacaggccctacgacgagggtc


 


tgcggaggggggttttcatcaccaacgagaccggccagccgctgattggg


 


aaggtatggcccgggtccactgccttccccgacttcaccaaccccacagc


 


cctggcctggtgggaggacatggtggctgagttccatgaccaggtgccat


 


cgacggcatgtggattgacatgaacgagccttccaacttcatcaggggct


 


ctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcct


 


ggggtggttggggggaccctccaggcggcaaccatctgtgcctccagcca


 


ccagtactctccacacactacaacctgcacaacctctacggcctgaccga


 


agccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccat


 


ttgtgatctcccgctcgacctttgctggccacggccgatacgccggccac


 


tggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgcc


 


agaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacg


 


tctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacc


 


cagctgggggccttctaccccttcatgcggaaccacaacagcctgctcag


 


tctgccccaggagccgtacagatcagcgagccggcccagcaggccatgag


 


gaaggccctcaccctgcgctacgcactcctcccccacctctacacgctgt


 


tccaccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctg


 


gagttccccaaggactctagcacctggactgtggaccaccagctcctgtg


 


gggggaggccctgctcatcaccccagtgctccaggccgggaaggccgaag


 


tgactggctacttccccttgggcacatggtacgacctgcagacggtgcca


 


atagaggccatggcagcctcccacccccacctgcagctccccgtgagcca


 


gccatccacagcgaggggcagtgggtgacgctgccggcccccctggacac


 


catcaacgtccacctccgggctgggtacatcatccccctgcagggccctg


 


gcctcacaaccacagagtcccgccagcagcccatggccctggctgtggcc


 


ctgaccaagggtggagaggcccgaggggagctgttctgggacgatggaga


 


gagcctggaagtgctggagcgaggggcctacacacaggtcatcttcctgg


 


ccaggaataacacgatcgtgaatgagctggtacgtgtgaccagtgaggga


 


gctggcctgcagctgcagaaggtgactgtcctgggcgtggccacggcgcc


 


ccagcaggtcctctccaacggtgtccctgtctccaacttcacctacagcc


 


ccgacaccaaggtcctggacatctgtgtctcgctgttgatgggagagcag


 


tttctcgtcagctggtgttagtctagagcttgctagcggccgc






Construct 1750

The GILTΔ2-7Δ35-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ35-39-GAA70-952 (Plasmid 1750). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ35-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ35-39 cassette contains a deletion of amino acid residues 35-39 (Val-Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 23)


ggtaccagagctagcaagctaattcacaccaATGGGAATCCCAATGGGGA


 


AGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCATT


 


GCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGG


 


GGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTCGTGGCATCGTTG


 


AGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGT


 


GCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccag


 


agcagtgcccacacagtgcgacgtcccccccaacagccgatcgattgcgc


 


ccagacaaggccatcacccaggaacagtgcgaggcccgcggctgctgcta


 


catccctgcaaagcaggggctgcagggagcccagatggggcagccctggt


 


gcttatcccacccagctaccccagctacaagctggagaacctgagctcct


 


agaaatgggctacacggccaccctgacccgtaccacccccaccttatccc


 


caaggacatcctgaccctgcggctggacgtgatgatggagactgagaacc


 


gcctccacttcacgatcaaagatccagctaacaggcgctacgaggtgccc


 


ttggagaccccgcgtgtccacagccgggcaccgtccccactctacagcgt


 


ggagactctgaggagcccttcggggtgatcgtgcaccggcagctggacgg


 


ccgcgtgctgctgaacacgacggtggcgcccctgttattgcggaccagtt


 


ccttcagagtccacctcgctgccctcgcagtatatcacaggcctcgccga


 


gcacctcagtcccctgatgacagcaccagaggaccaggatcaccctgtgg


 


aaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctcacca


 


ttctacctggcgctggaggacggcgggtcggcacacggggtgacctgcta


 


aacagcaatgccatggatgtggtcctgcagccgagccctgcccttagctg


 


gaggtcgacaggtgggatcctggatgtctacatatcctgggcccagagcc


 


caagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgc


 


cgccatactggggcctgggcttccacctgtgccgctggggctactcctcc


 


accgctatcacccgccaggtggtggagaacatgaccagggcccacttccc


 


cctggacgtccaatggaacgacctggactacatggactcccggagggact


 


tcacgttcaacaaggatggatccgggacttcccggccatggtgcaggaga


 


gcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagca


 


gctcgggccctgccgggagctacaggccctacgacgagggtctgcggagg


 


ggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatg


 


gcccgggtccactgccttccccgacttcaccaaccccacagccctggcct


 


ggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggc


 


atgtggattgacatgaacgagccttccaacttcatcaggggctctgagga


 


cggctgccccaacaatgagctggagaacccaccctacgtgcctggggtgg


 


ttggggggaccaccaggcggcaaccatagtgcctccagccaccagtttct


 


accacacactacaacctgcacaacctctacggcctgaccgaagccatcgc


 


ctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgatct


 


cccgctcgacctagctggccacggccgatacgccggccactggacggggg


 


acgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatcctg


 


cagtttaacctgctgggggtgcctaggtcggggccgacgtctgcggcttc


 


ctgggcaacacctcagaggagctgtgtgtgcgctggacccagagggggcc


 


ttctacccatcatgcggaaccacaacagcctgctcagtctgccccaggag


 


ccgtacagcttcagcgagccggcccagcaggccatgaggaaggccctcac


 


cctgcgctacgcactcctcccccacctctacacgctgttccaccaggccc


 


acgtcgcgggggagaccgtggcccggcccctcttcctggagttccccaag


 


gactctagcacctggactgtggaccaccagtcctgtggggggaggccagc


 


tcatcaccccagtgaccaggccgggaaggccgaagtgactggctacttcc


 


ccttgggcacatggtacgacctgcagacggtgccaatagaggcccttggc


 


agcctcccacccccacctgcagctccccgtgagccagccatccacagcga


 


ggggcagtgggtgacgctgccggcatccctggacaccatcaacgtccacc


 


tccgggctgggtacatcatccccctgcagggccctggcctcacaaccaca


 


gagtcccgccagcagcccatggccaggctgtggccctgaccaagggtgga


 


gaggcccgaggggagctgttctgggacgatggagagagcctggaagtgct


 


ggagcgaggggcctacacacaggtcatcttcctggccaggaataacacga


 


tcgtgaatgagaggtacgtgtgaccagtgagggagctggcctgcagctgc


 


agaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctctcc


 


aacggtgtccctgtctccaacttcacctacagcccgacaccaaggtcctg


 


gacatctgtgtctcgagttgatgggagagcagtttctcgtcagctggtgt


 


tagtctagagcttgctagcggccgc






Construct 1748

The GILTΔ2-7Δ36-39-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ36-39-GAA70-952 (Plasmid 1748). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ36-39 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ36-39 cassette contains a deletion of amino acid residues 36-39 (Ser-Arg-Arg-Ser) from the human IGF-II sequence.







(SEQ ID NO: 24)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGCGTGGCATC


 


GTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTA


 


CTGTGCTACCCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtc


 


ccagagcagtgcccacacagtgcgacgtcccccccaacagccgatcgatt


 


gcgcccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgc


 


tgctacatccctgcaaagcaggggctgcagggagcccagatggggcagcc


 


ctggtgatcttcccacccagctaccccagctacaagaggagaacctgagc


 


tcactgaaatgggctacacggccaccagacccgtaccacccccaccttct


 


tccccaaggacatcctgaccctgcggaggacgtgatgatggagactgaga


 


accgcctccacttcacgatcaaagatccagctaacaggcgctacgaggtg


 


cccttggagaccccgcgtgtccacagccgggcaccgtccccactctacag


 


cgtggagttactgaggagcccttcggggtgatcgtgcaccggcagctgga


 


cggccgcgtgagctgaacacgacggtggcgcccctgttattgcggaccag


 


ttcatcagagtccacctcgctgccctcgcagtatatcacaggcctcgccg


 


agcacctcagtcccctgatgctcagcaccagctggaccaggatcaccctg


 


tggaaccgggaccttgcgcccacgcccggtgcgaacctctacgggtctca


 


ccctttctacctggcgctggaggacggcgggtcggcacacggggtgttcc


 


tgctaaacagcaatgccatggatgtggtcctgcagccgagccagccctta


 


gctggaggtcgacaggtgggatcctggatgtctacatcttcctgggccca


 


gagcccaagagcgtggtgcagcagtacctggacgagtgggatacccgttc


 


atgccgccatactggggcctgggcttccacctgtgccgctggggctactc


 


ctccaccgctatcacccgccaggtggtggagaacatgaccagggcccact


 


tccccctggacgtccaatggaacgacctggactacatggactcccggagg


 


gacttcacgttcaacaaggatggcttccgggacttcccggccatggtgca


 


ggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgcca


 


tcagcagacgggccagccgggagctacaggccctacgacgagggtctgcg


 


gaggggggattcatcaccaacgagaccggccagccgctgattgggaaggt


 


atggcccgggtccactgccttccccgacttcaccaaccccacagccctgg


 


cctggtgggaggacatggtggctgagttccatgaccaggtgcccttcgac


 


ggcatgtggattgacatgaacgagccttccaacttcatcaggggctctga


 


ggacggctgccccaacaatgagctggagaacccaccctacgtgcctgggg


 


tggttggggggaccctccaggcggcaaccatagtgcctccagccaccagt


 


ttactccacacactacaacctgcacaacctctacggcctgaccgaagcca


 


tcgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtg


 


atctcccgctcgacctttgctggccacggccgatacgccggccactggac


 


gggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaa


 


tcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgc


 


ggcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctg


 


ggggccttctacccatcatgcggaaccacaacagcctgctcagtagcccc


 


aggagccgtacagatcagcgagccggcccagcaggccatgaggaaggcca


 


caccctgcgctacgcactcctcccccacctctacacgctgttccaccagg


 


cccacgtcgcgggggagaccgtggcccggcccctcttcctggagttcccc


 


aaggactctagcacctggactgtggaccaccagacctgtggggggaggcc


 


ctgctcatcaccccaggctccaggccgggaaggccgaagtgactggctac


 


ttccccttgggcacatggtacgacctgcagacggtgccaatagaggccat


 


ggcagcctcccacccccacctgcagctccccgtgagccagccatccacag


 


cgaggggcagtgggtgacgctgccggcccccctggacaccatcaacgtcc


 


acctccgggagggtacatcatccccctgcagggccctggcctcacaacca


 


cagagtcccgccagcagcccatggccctggctgtggccctgaccaagggt


 


ggagaggcccgaggggagagttctgggacgatggagagagcctggaagtg


 


aggagcgaggggcctacacacaggtcatcttcctggccaggaataacacg


 


atcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagct


 


gcagaaggtgactgtcctgggcgtggccacggcgccccagcaggtcctct


 


ccaacggtgtccctgtctccaacttcacctacagccccgacaccaaggtc


 


ctggacatctgtgtctcgctgttgatgggagagcagtttctcgtcagctg


 


gtgttagtctagagcttgctagcggccgc






Construct 1751

The GILTΔ2-7Δ29-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ29-40-GAA70-952 (Plasmid 1751). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ29-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ29-40 cassette contains a deletion of amino acid residues 29-40 (Ser-Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 25)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCGGCATCGTTGAGGAGTGCTGTTTCCGCAGC


 


TGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGA


 


GGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagtgcg


 


acgtcccccccaacagccgcttcgattgcgcccctgacaaggccatcacc


 


caggaacagtgcgaggcatgcggctgctgctacatccctgcaaagcaggg


 


gctgcagggagcccagatggggcagccaggtgcttcttcccacccagcta


 


ccccagctacaagctggagaacctgagctcctctgaaatgggctacacgg


 


ccaccctgacccgtaccacccccaccacttccccaaggacatcctgacca


 


gcggctggacgtgatgatggagactgagaaccgcctccacttcacgatca


 


aagatccagctaacaggcgctacgaggtgcccttggagaccccgcgtgtc


 


cacagccgggcaccgtccccactctacagcgtggagttctctgaggagcc


 


cttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctgaaca


 


cgacggtggcgcccctgttctttgcggaccagttccttcagagtccacct


 


cgctgccctcgcagtatatcacaggcctcgccgagcacctcagtcccctg


 


atgctcagcaccagctggaccaggatcaccctgtggaaccgggaccttgc


 


gcccacgcccggtgcgaacctctacgggtctcaccctttctacctggcgc


 


tggaggacggcgggtcggcacacggggtgttcctgctaaacagcaatgcc


 


atggatgtggtcctgcagccgagccctgcccttagctggaggtcgacagg


 


tgggatcctggatgtctacatcttcctgggcccagagcccaagagcgtgg


 


tgcagcagtacctggacgttgtgggatacccgttcatgccgccatactgg


 


ggcctgggcttccacctgtgccgctggggctactcctccaccgctatcac


 


ccgccaggtggtggagaacatgaccagggcccacttccccctggacgtcc


 


aatggaacgacctggactacatggactcccggagggacttcacgttcaac


 


aaggatggcttccgggacttcccggccatggtgcaggagctgcaccaggg


 


cggccggcgctacatgatgatcgtggatcctgccatcagcagctcgggcc


 


ctgccgggagctacaggccctacgacgagggtagcggaggggggttttca


 


tcaccaacgagaccggccagccgctgattgggaaggtatggcccgggtcc


 


actgccttccccgacttcaccaaccccacagccctggcctggtgggagga


 


catggtggctgagttccatgaccaggtgcccttcgacggcatgtggattg


 


acatgaacgagcatccaacttcatcaggggctctgaggacggctgcccca


 


acaatgagctggagaacccaccctacgtgcctggggtggttggggggacc


 


accaggcggcaaccatctgtgcctccagccaccagtttctaccacacact


 


acaacctgcacaacctctacggcctgaccgaagccatcgcctcccacagg


 


gcgctggtgaaggctcgggggacacgcccatttgtgatctcccgctcgac


 


attgctggccacggccgatacgccggccactggacgggggacgtgtggag


 


ctcctgggagcagctcgcctcctccgtgccagaaatcctgcagtttaacc


 


tgctgggggtgcctaggtcggggccgacgtctgcggcttcctgggcaaca


 


cctcagaggagctgtgtgtgcgctggacccagctgggggccttctacccc


 


ttcatgcggaaccacaacagcctgacagtagccccaggagccgtacagct


 


tcagcgagccggcccagcaggccatgaggaaggccctcaccctgcgctac


 


gcactcctcccccacctctacacgctgttccaccaggcccacgtcgcggg


 


ggagaccgtggcccggcccctatcctggagttccccaaggactctagcac


 


ctggactgtggaccaccagctcctgtggggggaggccagacatcacccca


 


gtgctccaggccgggaaggccgaagtgactggctacttcccatgggcaca


 


tggtacgacctgcagacggtgccaatagaggcccttggcagcctcccacc


 


cccacctgcagctccccgtgagccagccatccacagcgaggggcagtggg


 


tgacgctgccggcccccctggacaccatcaacgtccacctccgggctggg


 


tacatcatccccctgcagggccctggcctcacaaccacagagtcccgcca


 


gcagcccatggccctggctgtggccagaccaagggtggagaggcccgagg


 


ggagctgttctgggacgatggagagagcctggaagtgctggagcgagggg


 


cctacacacaggtcatatcctggccaggaataacacgatcgtgaatgagc


 


tggtacgtgtgaccagtgagggagaggcctgcagctgcagaaggtgactg


 


tcctgggcgtggccacggcgccccagcaggtcctaccaacggtgtccctg


 


tctccaacttcacctacagccccgacaccaaggtcctggacatctgtgta


 


cgctgttgatgggagagcagtttctcgtcagctggtgttagtctagagct


 


tgctagcggccgc






Construct 1752

The GILTΔ2-7Δ30-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ30-40-GAA70-952 (Plasmid 1752). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ30-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ30-40 cassette contains a deletion of amino acid residues 30-40 (Arg-Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 26)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCGGCATCGTTGAGGAGTGCTGTTTCCGC


 


AGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTC


 


CGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccacacagt


 


gcgacgtcccccccaacagccgcttcgattgcgcccctgacaaggccatc


 


acccaggaacagtgcgaggcccgcggctgctgctacatccctgcaaagca


 


ggggctgcagggagcccagatggggcagccctggtgcttcttcccaccca


 


gctaccccagctacaagctggagaacctgagctcctctgaaatgggctac


 


acggccaccctgacccgtaccacccccaccttcctccccaaggacatcct


 


gaccctgcggctggacgtgatgatggagactgagaaccgcctccacttca


 


cgatcaagatccagctaacaggcgctacgaggtgcccttggagaccccgc


 


gtgtccacagccgggcaccgtccccactcacagcgtggagttctctgagg


 


agcccttcggggtgatcgtgcaccggcagctggacggccgcgtgctgctg


 


aacacgacggtggcgcccctgttctttgcggaccagttccttcagctgtc


 


cacctcgctgccctcgcagtatatcacaggcctcgccgagcacctcagtc


 


cctgatgctcagcaccagctggaccaggatcacctgtggaaccgggacct


 


tgcgcccacgcccggtgcgaacctctacgggtctcaccctttctacctgg


 


cgctggaggtcgacaggtggatcctggatgtctacatcttcctgggccca


 


gagcccaagagcgtggtgcagcagtacctggacgttgtgggatacccgtt


 


catgccgccatactggggcctgggcttccacctgtgccgctggggctact


 


cctccaccgctatcacccgccaggtggtggagaacatgaccagggcccac


 


ttccccctggacgtccaatggaacgacctggactacatggactcccggag


 


ggacttcacgttcaacaaggatggcttccgggacttcccggccatggtgc


 


aggagctgcaccagggcggccggcgctacatgatgatcgtggatcctgcc


 


atcagcagctcgggccctgccgggagctacaggccctacgacgagggtct


 


gcggaggggggtttttcatcaccaacgagaccggccagccgctgattggg


 


aaggtatggcccgggtccactgccttccccgacttcaccaaccccacagc


 


cctggcctggtgggaggacatggtggctgagttccatgaccaggtgccct


 


tcgacggcatgtggattgacatgaacgagcctccaacttcatcaggggct


 


ctgaggacggctgccccaacaatgagctggagaacccaccctacgtgcct


 


ggggtggtggggggaccctccaggcggcaaccatctgtgcctccagccac


 


cagtttctctccacacactacaacctgcacaacctctacggcctgaccga


 


agccatcgcctcccacagggcgctggtgaaggctcgggggacacgcccat


 


ttgtgatctcccgctcgacctttgctggccacggccgatacgccggccac


 


tggacgggggacgtgtggagctcctgggagcagctcgcctcctccgtgcc


 


agaaatcctgcagtttaacctgctgggggtgcctctggtcggggccgacg


 


tctgcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacc


 


cagctgggggccttctaccccttcatgcggaaccacaacagcctgctcag


 


tctgccccaggagccgtacagcttcagcgagccggcccagcaggccatga


 


ggaaggccctcaccctgcgctacgcactcctcccccacctctacacgctg


 


ttccaggcccacgtcgcgggggagaccgtggcccggcccctcttcctgga


 


gttccccaaggactctagcacctggactgtggaccaccagctcctgtggg


 


gggaggccctgctcatcaccccagtgctccaggccgggaaggccgaagtg


 


actggctacttccccttgggcacatggtacgacctgcagacggtgccaat


 


agaggcccttggcagcctcccacccccacctgcagctccccgtgagccag


 


cctccacgcgaggggcagtgggtgacgctgccggccccctggacaccatc


 


aacgtccacctccgggctgggtacatcatccccctgcagggccctggcct


 


cacaaccacagagtcccgccagcagcccatggccctggctgtggccctga


 


ccaagggtggagaggcccgaggggagctgttctgggacgatggagagagc


 


ctggaagtgctggagcgaggggcctacacaggtcatgttcctggccagga


 


ataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggc


 


ctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagag


 


gtcctctccaacggtgtccctgtctccaacttcacctacagccccgacac


 


caaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcg


 


tcagctggtgttagtctagagcttgctagcggccgc






Construct 1753

The GILTΔ2-7Δ31-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ31-40-GAA70-952 (Plasmid 1753). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ31-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ31-40 cassette contains a deletion of amino acid residues 31-40 (Pro-Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 27)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCATTCTTGGCCTTCGCCTCGTGCTGCA


 


TTGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGT


 


GGGGACCGCGGCTTCTACTTCAGCAGGGGCATCGTTGAGGAGTGCTGTTT


 


CCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCA


 


AGTCCGAGGGCGCGCCGgcacaccccggccgtgggagagcagtgcccaca


 


cagtgcgacgtcccccccaacagccgcttcgattgcgccctgacaaggcc


 


atcacccaggaacaggacatcctgaccctgcggctggacgtgatgatgga


 


gactggggctgcagggagcccagatggggcagccctggtgcttcttccca


 


cccagctaccccagctacaagctggagaacctgagctcctctgaaatggg


 


ctacacggccaccctgacccgtaccacccccaccttcttccccaaggaca


 


tcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccac


 


ttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagac


 


cccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttct


 


ctgaggagcccttcggggtgatcgtgcaccggcagctggacggccgcgtg


 


ctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttca


 


gctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacc


 


tcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaac


 


cgggaccttgcgcccacgcccggtgcgaacctctacgggtctcacccttt


 


ctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaa


 


acagcaatgccatggatgtggtcctgcagccgagccctgcccttagctgg


 


aggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagcc


 


caagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgc


 


cgccatactggggcctgggcttccacctgtgccgctggggctactcctcc


 


accgctatcacccgccaggtggtggagaacatgaccagggcccacttccc


 


cctggacgtccaatggaacgacctggactacatggactcccggagggact


 


tcacgttcaacaaggatggcttccgggacttcccggccatggtgcaggag


 


ctgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcag


 


cagctcgggccctgccgggagctacaggccctacgacgagggtctgcgga


 


ggggggttttcatcaccaacgagaccggccagccgctgattgggaaggta


 


tggcccgggtccactgccttccccgacttcaccaacccacagccctggcc


 


tggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacgg


 


catgtggattgacatgaacgagccttccaacttcatcaggggctctgagg


 


acggctgccccaacaatgagctggagaacccaccctacgtgcctggggtg


 


gttggggggaccctccaggcggcaaccatctgtgcctccagccaccagtt


 


tctctccacacactacaacctgcacaacctctacggctgaccgaagccat


 


cgcctcccacagggcgctggtgaaggctcggggacacgcccatttgtgat


 


ctcccgctcgacctttgctggccacggccgatacgccggccggccactgg


 


acgggggacgtgtggagctcctgggagcagctcgcctcctccgtgccaga


 


aatcctgcagtttaacctgctgggggtgcctctggtcggggccgacgtct


 


gcggcttcctgggcaacacctcagaggagctgtgtgtgcgctggacccag


 


ctgggggccttctaccccttcatgcggaaccacaacagcctgctcagtct


 


gccccaggagccgtcagcttcagcgagccggcccagcaggccatgaggaa


 


ggccctcaccctcgcgtacgcactcctcccccacctctacacgctgttcc


 


accaggcccacgtcgcgggggagaccgtggcccggcccctcttcctggag


 


ttccccaaggactctagcacctggactgtggaccaccagctcctgtgggg


 


ggaggcctgctcatcaccccagtgctccaggccgggaaggccgaagtgac


 


tggctacttccccttgggcacatggtacgacctgcagacggtgccaatag


 


aggcccttggcagcctcccacccccacctgcagctccccgtgagccagcc


 


atccacagcgaggggcagtgggtgacgctgccggcccccctggacaccat


 


caacgtccacctccgggctgggtacatcatccccctgcagggccctggcc


 


tcacaaccacagagtcccgccagcagcccatggccctggctgtggccctg


 


accaagggtggagaggcccgaggggagctgttctgggacgatggagagag


 


cctggagtgctggagcgaggggcctacacaggtcatcttcctggccagga


 


ataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggc


 


ctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagca


 


ggtcctctccacggtgtccctgtctccaacttcacctacagccccgacac


 


caaggtcctggacatctgtgtctcgctggttgatgggagagcagtttctc


 


tgtcagctggtgttagtctagagcttgctagcggccgc






Construct 1754

The GILTΔ2-7Δ32-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ32-40-GAA70-952 (Plasmid 1754). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ32-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ32-40 cassette contains a deletion of amino acid residues 32-40 (Ala-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 28)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGGCATCGTTGAGGAGTGCTGT


 


TCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCC


 


AAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgcccac


 


acagtgcgacgtcccccccaacagccgcttcgattgcgcccctgacaagg


 


ccatcacccaggaacagtgcgaggcccgcggctgctgctacatccctgca


 


aagcaggggctgcagggagcccagatggggcagccctggtgatcttccca


 


cccagctaccccagctacaagctggagaacctgagctcctctgaaatggg


 


ctacacggccaccctgacccgtaccacccccaccttcttccccaaggaca


 


tcctgaccctgcggctggacgtgatgatggagactgagaaccgcctccac


 


ttcacgatcaaagatccagctaacaggcgctacgaggtgcccttggagac


 


cccgcgtgtccacagccgggcaccgtccccactctacagcgtggagttct


 


ctgaggagccatcggggtgatcgtgcaccggcagctggacggccgcgtgc


 


tgctgaacacgacggtggcgcccctgttctttgcggaccagttccttcag


 


ctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacct


 


cagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaacc


 


gggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattct


 


acctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaac


 


agcaatgccatggatgtggtcctgcagccgagccctgcccttagctggag


 


gtcgacaggtgggatcctggatgtctacatcttcctgggcccagagccca


 


agagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccg


 


ccatactggggcctgggcttccacctgtgccgctggggctactcctccac


 


cgctatcacccgccaggtggtggagaacatgaccagggcccacttccccc


 


tggacgtccaatggaacgacctggactacatggactcccggagggacttc


 


acgttcaacaaggatggcttccgggacttcccggccatggtgcaggagct


 


gcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagca


 


gctcgggccctgccgggagctacaggccctacgacgagggtctgcggagg


 


ggggttttcatcaccaacgagaccggccagccgctgattgggaaggtatg


 


gcccgggtccactgccttccccgacttcaccaaccccacagccctggcct


 


ggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggc


 


atgtggattgacatgaacgagccttccaacttcatcaggggctctgagga


 


cggctgccccaacaatgagctggagaacccaccctacgtgcctggggtgg


 


ttggggggaccctccaggcggcaaccatctgtgcctccagccaccagttt


 


ctctccacacactacaacctgcacaacctctacggcctgaccgaagccat


 


cgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtga


 


tctcccgctcgacctttgctggccacggccgatacgccggccactggacg


 


ggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaat


 


cctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcg


 


gcttcctgggcaacacctcagaggagagtgtgtgcgctggacccagctgg


 


gggccttctacccatcatgcggaaccacaacagcctgctcagtctgcccc


 


aggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggcc


 


ctcaccctgcgctacgcactcctcccccacctctacacgctgttccacca


 


ggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttcc


 


ccaaggactctagcacctggactgtggaccaccagctcctgtggggggag


 


gccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactgg


 


ctacttccccttgggcacatggtacgacctgcagacggtgccaatagagg


 


ccatggcagcctcccacccccacctgcagctccccgtgagccagccatcc


 


acagcgaggggcagtgggtgacgctgccggcccccctggacaccatcaac


 


gtccacctccgggctgggtacatcatccccctgcagggccctggcctcac


 


aaccacagagtcccgccagcagcccatggccctggctgtggccctgacca


 


agggtggagaggcccgaggggagctgttctgggacgatggagagagcctg


 


gaagtgctggagcgaggggcctacacacaggtcatcttcctggccaggaa


 


taacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggcc


 


tgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagcag


 


gtcctctccaacggtgtccctgtctccaacttcacctacagccccgacac


 


caaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctcg


 


tcagctggtgttagtctagagcttgctagcggccgc






Construct 1755

The GILTΔ2-7Δ33-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ33-40-GAA70-952 (Plasmid 1755). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ33-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ33-40 cassette contains a deletion of amino acid residues 33-40 (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 29)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAGGCATCGTTGAGGAGTGC


 


TGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCC


 


CGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcagtgc


 


ccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccctgac


 


aaggccatcacccaggaacagtgcgaggcccgcggctgctgctacatccc


 


tgcaaagcaggggctgcagggagcccagatggggcagccctggtgcttct


 


tcccacccagctaccccagctacaagctggagaacctgagctcctctgaa


 


atgggctacacggccaccctgacccgtaccacccccaccttcttccccaa


 


ggacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcc


 


tccacttcacgatcaaagatccagctaacaggcgctacgaggtgccatgg


 


agaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggag


 


ttctctgaggagcccttcggggtgatcgtgcaccggcagctggacggccg


 


cgtgctgctgaacacgacggtggcgcccctgttattgcggaccagttcct


 


tcagctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagc


 


acctcagtmcctgatgctcagcaccagctggaccaggatcaccctgtgga


 


accgggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccct


 


ttctacctggcgctggaggacggcgggtcggcacacggggtgttcctgct


 


aaacagcaatgccatggatgtggtcctgcagccgagccctgccatagctg


 


gaggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagc


 


ccaagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatg


 


ccgccatactggggcctgggatccacctgtgccgctggggctactcctcc


 


accgctatcacccgccaggtggtggagaacatgaccagggcccacttccc


 


cctggacgtccaatggaacgacctggactacatggactcccggagggact


 


tcacgttcaacaaggatggatccgggacttcccggccatggtgcaggagc


 


tgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagc


 


agctcgggccctgccgggagctacaggccctacgacgagggtctgcggag


 


gggggttttcatcaccaacgagaccggccagccgctgattgggaaggtat


 


ggcccgggtccactgccttccccgacttcaccaaccccacagccctggcc


 


tggtgggaggacatggtggctgagaccatgaccaggtgccatcgacggca


 


tgtggattgacatgaacgagccttccaacttcatcaggggctctgaggac


 


ggctgccccaacaatgagctggagaacccaccctacgtgcctggggtggt


 


tggggggaccctccaggcggcaaccatctgtgcctccagccaccagtttc


 


tctccacacactacaacctgcacaacctctacggcctgaccgaagccatc


 


gcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtgat


 


ctcccgctcgacctttgctggccacggccgatacgccggccactggacgg


 


gggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaatc


 


ctgcagtttaacctgctgggggtgcctctggtcggggccgacgtctgcgg


 


cttcctgggcaacacctcagaggagctgtgtgtgcgctggacccagctgg


 


gggccttctaccccttcatgcggaaccacaacagcctgctcagtctgccc


 


caggagccgtacagcttcagcgagccggcccagcaggccatgaggaaggc


 


cctcaccctgcgctacgcactcctcccccacctctacacgctgttccacc


 


aggcccacgtcgcgggggagaccgtggcccggcccctcttcctggagttc


 


cccaaggactctagcacctggactgtggaccaccagctcctgtgggggga


 


ggccctgctcatcaccccagtgctccaggccgggaaggccgaagtgactg


 


gctacttccccttgggcacatggtacgacctgcagacggtgccaatagag


 


gcccttggcagcctcccacccccacctgcagctccccgtgagccagccat


 


ccacagcgaggggcagtgggtgacgctgccggcccccctggacaccatca


 


acgtccacctccgggctgggtacatcatccccctgcagggccaggcctca


 


caaccacagagtcccgccagcagcccatggccctggctgtggccctgacc


 


aagggtggagaggcccgaggggagctgttctgggacgatggagagagcct


 


ggaagtgctggagcgaggggcctacacacaggtcatcttcctggccagga


 


ataacacgatcgtgaatgagctggtacgtgtgaccagtgagggagctggc


 


ctgcagctgcagaaggtgactgtcctgggcgtggccacggcgccccagca


 


ggtcctctccaacggtgtccctgtctccaacttcacctacagccccgaca


 


ccaaggtcctggacatctgtgtctcgctgttgatgggagagcagtttctc


 


gtcagaggtgttagtctagagcttgctagcggccgc






Construct 1756

The GILTΔ2-7Δ34-40-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7Δ34-40-GAA70-952 (Plasmid 1756). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7Δ34-40 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7Δ34-40 cassette contains a deletion of amino acid residues 34-40 (Arg-Val-Ser-Arg-Arg-Ser-Arg) from the human IGF-II sequence.







(SEQ ID NO: 30)


ggtaccagctgctagcaagctaattcacaccaATGGGAATCCCAATGGGG


 


AAGTCGATGCTGGTGCTTCTCACCTTCTTGGCCTTCGCCTCGTGCTGCAT


 


TGCTGCTCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTG


 


GGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCGGCATCGTTGAGGAG


 


TGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTAC


 


CCCCGCCAAGTCCGAGGGCGCGCCGgcacaccccggccgtcccagagcag


 


tgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgcccct


 


gacaaggccatcacccaggaacagtgcgaggcccgcggagctgctacatc


 


cctgcaaagcaggggctgcagggagcccagatggggcagccaggtgatat


 


cccacccagctaccccagctacaagctggagaacctgagctcctctgaaa


 


tgggctacacggccaccctgacccgtaccacccccaccttcttccccaag


 


gacatcctgaccctgcggctggacgtgatgatggagactgagaaccgcct


 


ccacttcacgatcaaagatccagctaacaggcgctacgaggtgccatgga


 


gaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggagt


 


tactgaggagccatcggggtgatcgtgcaccggcagaggacggccgcgtg


 


ctgctgaacacgacggtggcgcccctgttctttgcggaccagttccttca


 


gctgtccacctcgctgccctcgcagtatatcacaggcctcgccgagcacc


 


tcagtcccctgatgctcagcaccagctggaccaggatcaccctgtggaac


 


cgggaccttgcgcccacgcccggtgcgaacctctacgggtctcacccttt


 


ctacctggcgctggaggacggcgggtcggcacacggggtgttcctgctaa


 


acagcaatgccatggatgtggtcctgcagccgagccagcccttagctgga


 


ggtcgacaggtgggatcctggatgtctacatcttcctgggcccagagccc


 


aagagcgtggtgcagcagtacctggacgttgtgggatacccgttcatgcc


 


gccatactggggcctgggcttccacctgtgccgctggggctactcctcca


 


ccgctatcacccgccaggtggtggagaacatgaccagggcccacttcccc


 


ctggacgtccaatggaacgacctggactacatggactcccggagggactt


 


cacgttcaacaaggatggcttccgggacttcccggccatggtgcaggagc


 


tgcaccagggcggccggcgctacatgatgatcgtggatcctgccatcagc


 


agctcgggccctgccgggagctacaggccctacgacgagggtctgcggag


 


gggggttttcatcaccaacgagaccggccagccgctgattgggaaggtat


 


ggcccgggtccactgcatccccgacttcaccaaccccacagccctggcct


 


ggtgggaggacatggtggctgagttccatgaccaggtgcccttcgacggc


 


atgtggattgacatgaacgagccttccaacttcatcaggggctctgagga


 


cggctgccccaacaatgagctggagaacccaccctacgtgcctggggtgg


 


ttggggggaccctccaggcggcaaccatctgtgcctccagccaccagttt


 


ctctccacacactacaacctgcacaacctctacggcctgaccgaagccat


 


cgcctcccacagggcgctggtgaaggctcgggggacacgcccatttgtga


 


tctcccgctcgacctttgctggccacggccgatacgccggccactggacg


 


ggggacgtgtggagctcctgggagcagctcgcctcctccgtgccagaaat


 


cctgcagtttaacctgctgggggtgcctaggtcggggccgacgtctgcgg


 


cttcctgggcaacacctcagaggagagtgtgtgcgctggacccagagggg


 


gccttctaccccttcatgcggaaccacaacagcctgctcagtagccccag


 


gagccgtacagatcagcgagccggcccagcaggccatgaggaaggccaca


 


ccctgcgctacgcactcctcccccacctctacacgctgttccaccaggcc


 


cacgtcgcgggggagaccgtggcccggcccctatcctggagttccccaag


 


gactctagcacctggactgtggaccaccagctcagtggggggaggccctg


 


ctcatcaccccagtgctccaggccgggaaggccgaagtgactggctactt


 


cccatgggcacatggtacgacctgcagacggtgccaatagaggcccttgg


 


cagcctcccacccccacctgcagctccccgtgagccagccatccacagcg


 


aggggcagtgggtgacgctgccggcccccctggacaccatcaacgtccac


 


ctccgggctgggtacatcatccccctgcagggccctggcctcacaaccac


 


agagtcccgccagcagcccatggccaggctgtggccctgaccaagggtgg


 


agaggcccgaggggagagttctgggacgatggagagagcctggaagtgct


 


ggagcgaggggcctacacacaggtcatcttcctggccaggaataacacga


 


tcgtgaatgagctggtacgtgtgaccagtgagggagctggcctgcagagc


 


agaaggtgactgtcagggcgtggccacggcgccccagcaggtcctctcca


 


acggtgtccctgtctccaacttcacctacagccccgacaccaaggtcctg


 


gacatctgtgtctcgagttgatgggagagcagtttctcgtcagctggtgt


 


tagtctagagcttgctagcggccgc






Construct 1763

The GILTΔ2-7M1/L27A37-GAA70-952 cassette below was cloned using the Asp718 and NotI sites of the cassette and vector pCEP4 to produce pCEP-GILTΔ2-7M1/L27A37-GAA70-952 (Plasmid 1763). Restriction sites for cloning are in lowercase bold. The spacer amino acid sequence Gly, Ala, Pro (underlined sequence) separate the GAA gene and GILTΔ2-7M1/L27A37 tag (upper case sequence). The spacer and tag are placed upstream of GAA residue Ala70. The GILTΔ2-7M1/L27A37 cassette contains Y27L and R37A substitutions in the human IGFII sequence. The DNA sequence of the GILT cassette differs from the human DNA sequence at every 6th codon.







(SEQ ID NO: 31)


ggtaccaagcttgccATGGGAATCCCAATGGGCAAGTCGATGCTGGTGCT


 


GCTCACCTTCTTGGCCTTTGCCTCGTGCTGCATTGCCGCTCTGTGCGGCG


 


GGGAACTGGTGGACACCCTCCAATTCGTCTGTGGGGACCGGGGCTTCCTG


 


TTCAGCAGACCCGCAAGCCGTGTGAGTGCTCGCAGCCGTGGCATTGTTGA


 


GGAGTGCTGTTTTCGCAGCTGTGACCTGGCTCTCCTGGAGACGTACTGCG


 


CTACCCCCGCCAAGTCTGAGGGCGCGCCGgcacaccccggccgtcccaga


 


gcagtgcccacacagtgcgacgtcccccccaacagccgcttcgattgcgc


 


ccctgacaaggccatcacccaggaacagtgcgaggcccgcggctgctgct


 


acatccctgcaaagcaggggctgcagggagcccagatggggcagccaggt


 


gcttatcccacccagctaccccagctacaagaggagaacctgagacctag


 


aaatgggctacacggccaccagacccgtaccacccccaccttatccccaa


 


ggacatcctgaccctgcggaggacgtgatgatggagactgagaaccgcct


 


ccacttcacgatcaaagatccagctaacaggcgctacgaggtgcccttgg


 


agaccccgcgtgtccacagccgggcaccgtccccactctacagcgtggag


 


ttctctgaggagccatcggggtgatcgtgcaccggcagctggacggccgc


 


gtgctgctgaacacgacggtggcgcccagttctttgcggaccagttcctt


 


cagagtccacctcgctgccctcgcagtatatcacaggcctcgccgagcac


 


ctcagtcccctgatgctcagcaccagaggaccaggatcaccagtggaacc


 


gggaccttgcgcccacgcccggtgcgaacctctacgggtctcaccattct


 


acctggcgctggaggacggcgggtcggcacacggggtgttcctgctaaac


 


agcaatgccatggatgtggtcctgcagccgagccctgccatagaggaggt


 


cgacaggtgggatcctggatgtctacatatcctgggcccagagcccaaga


 


gcgtggtgcagcagtacctggacgttgtgggatacccgttcatgccgcca


 


tactggggcagggcttccacctgtgccgctggggctactcaccaccgcta


 


tcacccgccaggtggtggagaacatgaccagggcccacttccccctggac


 


gtccaatggaacgacctggactacatggactcccggagggacttcacgtt


 


caacaaggatggcttccgggacttcccggccatggtgcaggagctgcacc


 


agggcggccggcgctacatgatgatcgtggatcctgccatcagcagctcg


 


ggccctgccgggagctacaggccctacgacgagggtctgcggaggggggt


 


tttcatcaccaacgagaccggccagccgctgattgggaaggtatggcccg


 


ggtccactgccttccccgacttcaccaaccccacagccctggcctggtgg


 


gaggacatggtggctgagttccatgaccaggtgcccttcgacggcatgtg


 


gattgacatgaacgagccttccaacttcatcaggggctctgaggacggct


 


gccccaacaatgagctggagaacccaccctacgtgcctggggtggttggg


 


gggaccctccaggcggcaaccatctgtgcctccagccaccagtttctctc


 


cacacactacaacctgcacaacctctacggcctgaccgaagccatcgcct


 


cccacagggcgctggtgaaggctcgggggacacgcccatttgtgatctcc


 


cgctcgacctttgctggccacggccgatacgccggccactggacggggga


 


cgtgtggagctcctgggagcagctcgcctcaccgtgccagaaatcctgca


 


gtttaacctgctgggggtgcctctggtcggggccgacgtagcggcttcct


 


gggcaacacctcagaggagctgtgtgtgcgaggacccagctgggggcctt


 


ctaccccttcatgcggaaccacaacagcctgctcagtagccccaggagcc


 


gtacagcttcagcgagccggcccagcaggccatgaggaaggccctcaccc


 


tgcgctacgcactcctcccccacctctacacgctgttccaccaggcccac


 


gtcgcgggggagaccgtggcccggcccacttcctggagttccccaaggac


 


tctagcacctggactgtggaccaccagctcctgtggggggaggccagctc


 


atcaccccagtgctccaggccgggaaggccgaagtgactggctacttccc


 


cttgggcacatggtacgacctgcagacggtgccaatagaggcccttggca


 


gcctcccacccccacctgcagctccccgtgagccagccatccacagcgag


 


gggcagtgggtgacgctgccggcccccctggacaccatcaacgtccacct


 


ccgggctgggtacatcatccccagcagggccaggcctcacaaccacagag


 


tcccgccagcagcccatggccaggctgtggccctgaccaagggtggagag


 


gcccgaggggagctgttctgggacgatggagagagcctggaagtgctgga


 


gcgaggggcctacacacaggtcatcttcctggccaggaataacacgatcg


 


tgaatgagaggtacgtgtgaccagtgagggagctggcctgcagagcagaa


 


ggtgactgtcctgggcgtggccacggcgccccagcaggtcctctccaacg


 


gtgtccctgtctccaacttcacctacagccccgacaccaaggtcctggac


 


atctgtgtctcgctgttgatgggagagcagtttctcgtcagctggtgtta


 


gtctagagcttgctagcggccgc






Example 3
Expression and Purification of GILT-Tagged GAA Enzymes
Tissue Culture
GILT-tagged GAA plasmids were each transfected into suspension FreeStyle 293-F cells as described by the manufacturer (Invitrogen). Briefly, cells were grown in Opti-MEM I media (Invitrogen) in polycarbonate shaker flasks on an orbital shaker at 37° C. and 8% CO2. Cells were adjusted to a concentration of 1×106 cells/ml, then transfected with a 1:1:1 ratio of ml DNA:μl 293fectin. Culture aliquots were harvested 5-7 days post-transfection and centrifuged at 5,000×g for 5 minutes. Supernatants were stored frozen at −80° C.
Protein Purification and Concentration
Starting material was mammalian cell culture supernatant, as described above, thawed from storage at −80° C. Citric acid was added to reach pH 6.0, then ammonium sulfate was added to reach a final concentration of 1M. The material was passed through a 0.2 μm Supor-Mach filter (Nalgene).
The filtered material was loaded onto a Phenyl-SepharoseTM 6 Low-Sub Fast-Flow (GE Healthcare) column prepared with HIC Load Buffer (50 mM citrate pH 6.0, 1M AmSO4). The column was washed with 10 column volumes of HIC Wash Buffer (50 mM citrate pH 6.0, 0.8M AmSO4), and eluted with 5 column volumes of HIC Elution Buffer (50 mM citrate pH 6.0). Samples from the elution peaks were pooled and buffer was exchanged into phosphate buffered saline (145.15 mM NaCl, 2.33 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 6.2) using centricon spin concentrators (Amicon) and Bio-Spin-6 de-salting columns (Bio-Rad).
Enzyme Activity
GAA expression was determined by a para-nitrophenol (PNP) enzymatic assay. GAA enzyme was incubated in 50 μl reaction mixture containing 100 mM sodium acetate pH 4.2 and 10 mM Para-Nitrophenol (PNP) α-glucoside substrate (Sigma N1377). Reactions were incubated at 37° C. for 20 minutes and stopped with 300 μl of 100 mM sodium carbonate. Absorbance at 405 nm was measured in 96-well microtiter plates and compared to standard curves derived from p-nitrophenol (Sigma N7660). 1 GAA PNP unit is defined as 1 nmole PNP hydrolyzed/hour.
Example 4
Competitive Receptor Binding Assays
The affinity of GILT-tagged proteins for the IGF2 receptor (IGF2R), IGF1 receptor (IGF1R) and the insulin receptor (IR) was examined in competitive binding experiments performed in a 96-well plate format. Receptors were coated at room temperature overnight onto Reacti-bind white plates (Pierce, Cat#437111) in Coating Buffer (0.05M Carbonate buffer, pH 9.6) at a concentration of either 0.5 μg/well (IGF2R) or 1 μg/well (IGF1R, IR). Plates were washed with wash buffer (Phosphate Buffered Saline plus 0.05% Tween-20), then blocked in Super Blocking Buffer (Pierce, Cat#37516) for 1 hour. After another plate washing, biotinylated ligands (Cell Sciences) were added to wells; IGF2R wells received 8 nM IGF2-biotin, IGF1R wells received 30 nM IGF1-biotin, and IR wells received 20 nM insulin-biotin. Along with the biotinylated ligands, wells also contained serial dilutions of the GILT-tagged GAA protein samples or non-biotinylated control ligands to act as binding inhibitors for the biotinylated ligands. Following a two-hour rocking incubation, plates were washed and bound biotinylated ligands were detected with a streptavidin-HRP incubation (R&D, Cat#890803, 1:200 dilution in blocking buffer, 30 minutes), followed by a Super Elisa Pico Chemiluminescent Substrate incubation (Pierce, Cat#37070, 5 minutes). The chemiluminescent signal was measured at 425 nm.
The percent bound biotinylated ligand was calculated for each competitor concentration in the IGF2R binding competition assay and the IC50 values were determined (FIG. 4). Protein 1752 with a deletion of IGF2 residues 30-40 displayed a similar IC50 value as the GILT-tagged ZC-701 (FIG. 4), indicating that deletion of these residues in the IGF2 loop region does not appear to effect IGF2R binding. Protein 1751 with a deletion of IGF2 residues 29-40 displayed a higher IC50 value (FIG. 4), indicating that it does not compete as well for binding to the IGF2R.
On a separate IGF2R assay plate, comparison of ZC-701 and protein 1763 yielded IC50 values that differed by 35% (See FIG. 5).
In an assay measuring the competition of biotinylated insulin binding to plate-bound insulin, 1751 and 1752 proteins were not as effective as inhibitors compared to 701 or IGF-II (See FIG. 6). This indicates that the 1751 and 1752 proteins, with deletions in the loop region corresponding to amino acids 30-40 of the GILT tag, had a reduced affinity for the insulin receptor compared to the intact GILT tag on 701 or IGF-II.
In an assay measuring the competition of biotinylated IGF-I binding to plate-bound IGF1R, 1763 protein was not as effective as an inhibitor compared to 701, IGF-II or IGF-I (See FIG. 7). This indicates that the 1763 protein, with Δ2-7, Y27L and R37A mutations in the GILT tag, had a reduced affinity for the IGF1 receptor compared to ZC-701 or IGF-II.
Example 4
Additional Insulin Receptor Binding Assay
Protein ZC-1487 was tested for its binding affinity for the insulin receptor. Protein ZC-1487 contains the GILTD2-7M1/A37 cassette contains with and Arg to Ala substitution at amino acid 37 of the human IGF2 sequence and is resistant to proteolysis by furin. Two different batches of this protein purified from CHO cells, ZC-1487-B26 and ZC-1487-B28 were analyzed in an assay measuring the competition of biotinylated insulin binding to plate-bound insulin.
An insulin receptor binding assay was conducted by competing insulin, IGF-II, ZC710B20 and ZC1487B26 or ZC-1487-B28 with Biotinylated-insulin binding to the insulin receptor (Insulin-R).
Specifically, white Reacti-BindTM plates were coated with Insulin-R at a concentration of 1 ug/well/100 ul (38.4 nM). The coated plates were incubate over night at room temperature, then washed 3× with washing buffer (300 ul/well). The plates were then blocked with blocking buffer (300 ul/well) for 1 hour. The washing steps were repeated and any trace of solution in the plates was taken out.
Biotinylated-insulin was mixed at 20 nM with different concentrations of insulin, IGF-II, ZC701B20, B26 and B28 by serial dilutions (final concentrations are shown in Table 2). 100 ul of diluted Insulin, IGF-II, ZC710B20, ZC1487B26, and ZC1487B28 in 20 nM Insulin-biotin were added into the coated plates and the plates were incubated at room temperature for 2 hours. The plates were then washed 3 times with washing buffer. 100 ul of strepavidin-HRP working solution (50 ul strepavidin-HRP in 10 ml blocking buffer) was added into the plates and the plates were incubated at room temperature for 30 minutes. 100 ul of Elisa-Pico working solution containing Elisa-Pico chemiluminescent substrate was added and the chemiluminescence was measured at 425 nm. Exemplary results are shown in Table 2, FIG. 8, and FIG. 9. Both batches of ZC-1487 were not as effective as inhibitors compared to ZC-701 or the insulin control. As can be seen from Table 2 and FIG. 8, furin resistant peptide ZC-1487B26 binds to the insulin receptor more than 10-fold less avidly than does ZC-701 and more than 20-fold less than does the wild-type IGF-II
This indicates that the 1487 protein had a reduced affinity for the insulin receptor compared to the GILT tag on ZC-701.







TABLE 2





Insulin-Receptor Binding Activity - Chemiluminescence Intensity



























Insulin-B (nM)
2000
1000
500
250
125
62.5
31.25
15.625
7.8125
3.90625
1.95313
0










Insulin (nM)



















20 nM
38.00
43.00
66.00
102.00
243.00
479.00
750.00
780.00
503
1175
1046
2180


20 nM
13.00
25.00
57.00
141.00
229.00
517.00
517.00
885.00
1003
1344
1462
1694


ave
25.5
34.0
61.5
121.5
236.0
498.0
633.5
832.5
753.0
1259.5
1254.0
1937.0







IFG-II (nM)



















20 nM
70.00
268.00
356.00
644.00
828.00
991.00
1189.00
1492.00
1478
1478
1410
1874


20 nM
140.00
176.00
379.00
566.00
919.00
1224.00
1447.00
1377.00
1483
1370
1249
1959


ave
105.0
222.0
367.5
605.0
873.5
1107.5
1318.0
1434.5
1480.5
1424.0
1329.5
1916.5





Insulin-B (nM)
4000
2000
1000
500
250
125
62.5
31.25
15.625
7.8125
3.90625
0










ZC701B20 (nM)



















20 nM
191.00
387.00
526.00
715.00
800.00
1284.00
1116.00
1248.00
1474
1241
1450
1790


20 nM
250.00
329.00
483.00
774.00
767.00
1071.00
1024.00
968.00
1471
1118
1234
1886


ave
220.5
358.0
504.5
744.5
783.5
1177.5
1070.0
1108.0
1472.5
1179.5
1342.0
1838.0







ZC1487B26 (nM)



















20 nM
967.00
1190.00
1334.00
1210.00
1294.00
1462.00
1402.00
1281.00
1323
1612
1173
1952


20 nM
962.00
1189.00
1395.00
1379.00
1612.00
1396.00
1221.00
1013.00
1326
1182
1102
2069


ave
964.5
1189.5
1364.5
1294.5
1453.0
1429.0
1311.5
1147.0
1324.5
1397.0
1137.5
2010.5



2000
1000
500
250
125
62.5
31.25
5.625
7.8125
3.90625
1.95313
0



(4000)
(2000)
(1000)
(500)
(250)
(125)
(62.5)
(31.25)
(15.625)
(7.8125)
(3.90625)









Example 5
Uptake Assays
Some mutants were tested for retention of uptake activity. HEK293 cells were transfected with constructs 1479 (R37K), 1487 (R37A) or ZC-701. After harvest, culture supernatants were partially purified by HIC chromatography. All samples were treated with PNGase prior to electrophoresis.
FIG. 10 shows partially purified preparations of targeted fusion proteins containing a furin-resistant IGF-II mutein tag analyzed by SDS-PAGE and immunoblotting. As can be seen, the fusion protein encoded by construct 1487 containing R37A mutation is resistant to exogenous furin.
FIG. 11 illustrates exemplary uptake results of furin resistant GILT-tagged GAA into rat L6 myoblasts. As shown in FIG. 11, exemplary Kuptakes for proteins 1479, 1487, ZC-701, and purified ZC-701 are 4.5 nM, 4.4 nM, 5.0 nM and 2.6 nM, respectively, which indicates that the proteins encoded by constructs 1487 (R37A) and 1479 (R37K) retain the ability for efficient uptake into rat L6 myoblasts. The efficient uptake of fusion proteins containing a furin-resistant GILT tag also indicates that the furin-resistant tag retains high affinity for the CI-MPR.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. The articles “a”, “an”, and “the” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth herein. It should also be understood that any embodiment of the invention, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. Furthermore, where the claims recite a composition, the invention encompasses methods of using the composition and methods of making the composition. Where the claims recite a composition, it should be understood that the invention encompasses methods of using the composition and methods of making the composition.
INCORPORATION OF REFERENCES
All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.

## Claims

1. A targeted therapeutic fusion protein comprising:
a lysosomal enzyme which is α-N-Acetylglucosaminidase (Naglu);
an IGF-II mutein comprising amino acids 8-67 of SEQ ID NO: 1 and an Ala substitution at position Arg37 of SEQ ID NO:1, wherein the IGF-II mutein (i) has diminished binding affinity for the insulin receptor relative to the affinity of naturally-occurring human IGF-II for the insulin receptor, (ii) is resistant to furin cleavage and (iii) binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and
a spacer between the lysosomal enzyme and the IGF-II mutein, wherein the spacer comprises the amino acid sequence Gly-Ala-Pro.

2. A pharmaceutical composition suitable for treating lysosomal storage disease comprising a targeted therapeutic fusion protein of claim 1 and a physiologically acceptable carrier or excipient.

3. The targeted therapeutic fusion protein of claim 1, wherein the IGF-II mutein is fused via the spacer to the C-terminus of the lysosomal enzyme.

4. The targeted therapeutic fusion protein of claim 1, wherein the IGF-II mutein is fused via the spacer to the N-terminus of the lysosomal enzyme.

5. The targeted therapeutic fusion protein of claim 1, wherein the IGF-II mutein consists of amino acids 8-67 of SEQ ID NO:1 having an Ala substitution at position Arg37 of SEQ ID NO:1.