NZ732171B2 - Human alpha-galactosidase variants - Google Patents
Human alpha-galactosidase variants Download PDFInfo
- Publication number
- NZ732171B2 NZ732171B2 NZ732171A NZ73217115A NZ732171B2 NZ 732171 B2 NZ732171 B2 NZ 732171B2 NZ 732171 A NZ732171 A NZ 732171A NZ 73217115 A NZ73217115 A NZ 73217115A NZ 732171 B2 NZ732171 B2 NZ 732171B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- gla
- sequence
- alpha galactosidase
- recombinant
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01022—Alpha-galactosidase (3.2.1.22)
Abstract
The present invention provides engineered human alpha-galactosidase A (GLA) polypeptides and compositions thereof, having improved stability and activity at high and low pH, and reduced immunogenicity.
Description
HUMAN GALACTOSIDASE VARIANTS
The present application claims priority to US Prov. Pat. Appln. Ser. No. 62/095313, filed
December 22, 2014, and US Prov. Pat. Appln. Ser. No. 62/216452, filed ber 10, 2015, both of
which are hereby incorporated by reference in their entireties for all purposes.
FIELD OF THE INVENTION
The present invention provides engineered human galactosidase polypeptides and
itions thereof. The engineered human alpha-galactosidase polypeptides have been optimized
to provide improved stability under both acidic (pH <45) and basic (pH >7) conditions. The invention
also relates to the use of the compositions sing the engineered human alpha-galactosidase
polypeptides for therapeutic es.
NCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
The official copy of the Sequence Listing is submitted concurrently with the specification as
an ASCII formatted text file via b, with a file name of “CX7-147WO2_ST25.txt”, a creation
date ofNovember 30, 2015, and a size of 2,545,851 bytes. The Sequence Listing filed via EFS-Web
is part of the specification and is incorporated in its ty by reference herein.
BACKGROUND OF THE INVENTION
Human alpha galactosidase (“GLA”; EC 3.2.1.22) is a lysosomal glycoprotein responsible for
hydrolyzing terminal alpha galactosyl moieties from glycolipids and glycoproteins. It works on many
substrates present in a range of human tissues. Fabry disease (also ed to as angiokeratoma
corporis diffusum, on-Fabry disease, hereditary dystopic lipidosis, alpha-galactosidase A
deficiency, GLA deficiency, and ceramide trihexosidase deficiency) is an X-linked inborn error of
glycosphingolipid catabolism that results from deficient or absent activity of alpha-galactosidase A.
Patients affected with Fabry disease accumulate globotriosylceramide (Gb3) and related
glycosphingolipids in the plasma and cellular lysosomes of blood vessels, tissue and organs (See e.g.,
Nance et al., Arch. Neurol., 63:453-457 [2006]). As the patient ages, the blood vessels become
progressively narrowed, due to the accumulation of these lipids, ing in decreased blood flow and
nourishment to the tissues, particularly in the skin, kidneys, heart, brain, and nervous system. Thus,
Fabry disease is a systemic disorder that manifests as renal failure, cardiac disease, cerebrovascular
disease, fiber peripheral neuropathy, and skin lesions, as well as other ers (See e. g.,
Schiffinann, Pharm. Ther., 122:65-77 [2009]). Affected patients exhibit symptoms such as painful
hands and feet, clusters of small, dark red spots on their skin, the decreased ability to sweat, corneal
opacity, gastrointestinal issues, tinnitus, and hearing loss. Potentially life-threatening cations
include progressive renal damage, heart attacks, and stroke. This disease affects an estimated 1 in
40,000-60,000 males, but also occurs in females. Indeed, heterozygous women with Fabry disease
experience significant life-threatening conditions requiring medical treatment, including nervous
system abnormalities, chronic pain, e, high blood pressure, heart e, kidney failure, and
stroke (See e.g., Want et al., Genet. Med., 13:457-484 [2011]). Signs of Fabry disease can start any
time from infancy on, with signs usually beginning to show between ages 4 and 8, although some
patients exhibit a milder, late-onset disease. Treatment is generally supportive and there is no cure for
Fabry disease, thus there remains a need for a safe and effective treatment.
SUMMARY OF THE INVENTION
The present invention provides engineered human alpha-galactosidase polypeptides and
compositions thereof. The engineered human alpha-galactosidase polypeptides have been zed
to provide ed stability under both acidic (pH <45) and basic (pH >7) conditions. The invention
also relates to the use of the compositions comprising the engineered human alpha-galactosidase
ptides for therapeutic purposes.
The present invention provides recombinant alpha galactosidase A and/or biologically active
recombinant alpha galactosidase A fragment comprising an amino acid sequence comprising at least
about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least
about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID
NO:5. In some embodiments, the alpha osidase A comprises at least one mutation in at least
one on as provided in Tables 2.1, 2.2, 2.4, and/or 2.5, wherein the positions are numbered with
reference to SEQ ID NO:5. In some embodiments, the alpha galactosidase A comprises at least one
mutation in at least one position as provided in Table 2.3, n the positions are numbered with
reference to SEQ ID NO:10. In some additional embodiments, the recombinant alpha galactosidase A
is derived from a human alpha galactosidase A. In some further embodiments, the inant alpha
galactosidase A comprises the polypeptide sequence of SEQ ID NO:15, 13, 10, or 18. In still some
onal embodiments, the recombinant alpha galactosidase A is more thermostable than the alpha
galactosidase A of SEQ ID NO:5. In some further embodiments, the recombinant alpha galactosidase
A is more stable at pH 7.4 than the alpha galactosidase A of SEQ ID NO:5, while in additional
embodiments, the recombinant alpha osidase A is more stable at pH 4.3 than the alpha
galactosidase A of SEQ ID NO:5. In some embodiments the recombinant alpha galactosidase A is
more stable at pH 7.4 and pH 4.3 than the alpha galactosidase A of SEQ ID NO:5. In still some
further embodiments, the recombinant alpha galactosidase A is a deimmunized alpha galactosidase A.
In some embodiments, the inant alpha galactosidase A is a nized alpha galactosidase
A provided in Table 7.1. In still some additional embodiments, the recombinant alpha galactosidase
A is purified. In some ments, the inant alpha galactosidase A exhibits at least one
improved property selected from: i) ed catalytic activity; ii) increased tolerance to pH 7.4; iii)
increased tolerance to pH 4.3; or iv) reduced immunogenicity; or a combination of any of i), ii), iii),
or iv), as compared to a reference ce. In some embodiments, the reference sequence is SEQ
ID NO:5, while in some alternative embodiments, the reference sequence is SEQ ID NO:10.
The present invention also provides recombinant polynucleotide sequences encoding at least
one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or
Table 7.1). In some embodiments, the recombinant polynucleotide sequence is codon-optimized.
The present invention also provides expression vectors comprising the recombinant
polynucleotide sequence encoding at least one recombinant alpha galactosidase A as provided herein
(e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the recombinant
polynucleotide sequence is operably linked to a control sequence. In some additional embodiments,
the control sequence is a promoter. In some further embodiments, the promoter is a heterologous
promoter. In some embodiments, the expression vector further comprises a signal sequence, as
provided herein.
The present invention also provides host cells comprising at least one sion vector as
provided herein. In some embodiments, the host cell comprises an expression vector comprising the
recombinant polynucleotide sequence encoding at least one recombinant alpha galactosidase A as
provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the
host cell is eukaryotic.
The present invention also provides methods of producing an alpha galactosidase A variant,
comprising culturing a host cell provided , under conditions that the alpha osidase A
encoded by the recombinant polynucleotide is ed. In some embodiments, the methods further
comprise the step of recovering alpha galactosidase A. In some further embodiments, the methods
further comprise the step of purifying the alpha galactosidase A.
The present invention also provides compositions sing at least one recombinant alpha
galactosidase A as ed herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some
embodiments, the present invention provides pharmaceutical compositions. In some additional
embodiments, the present invention provides pharmaceutical compositions for the ent of Fabry
e, comprising an enzyme ition ed herein. In some ments, the
pharmaceutical compositions, further comprise a pharmaceutically acceptable carrier and/or excipient.
In some additional embodiments, the pharmaceutical composition is suitable for parenteral injection
or on to a human.
The present invention also provides methods for treating and/or preventing the ms of
Fabry disease in a subject, sing ing a subject having Fabry disease, and providing at least
one pharmaceutical ition compositions comprising at least one recombinant alpha
galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1), and
administering the pharmaceutical composition to the t. In some embodiments, the symptoms of
Fabry disease are ameliorated in the t. In some additional embodiments, the subject to Whom
2015/063329
the pharmaceutical composition of the present invention has been administered is able to eat a diet
that is less restricted in its fat t than diets ed by subjects exhibiting the symptoms of
Fabry disease. In some embodiments, the subject is an infant or child, while in some alternative
embodiments, the subject is an adult or young adult.
The present invention also es for the use of the compositions provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides a graph g the relative activity of different GLA constructs in S.
cerevisiae after 2-5 days of ing.
Figure 2 provides graphs g the Absolute (Panel A) and relative (Panel B) activity of
GLA variants after incubation at various pHs.
Figure 3 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of
GLA variants after incubation at various temperatures.
Figure 4 provides graphs g the absolute (Panel A&B) and relative (Panel C&D)
activity of GLA variants after challenge with buffers that contain increasing s of serum.
Figure 5 provides a graph showing the relative activity of GLA variants expressed in
HEK293Tcells.
Figure 6 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of
GLA ts expressed in HEK293T cells, normalized for activity, and incubated at various pHs.
Figure 7 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of
GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various
atures.
Figure 8 provides graphs showing GLA variant activity remaining after incubation in acidic
(Panel A) or basic (Panel B) solutions.
Figure 9 provides a graph showing the GLA activity recovered in rat serum following
administration of GLA variants.
PTION OF THE INVENTION
The present invention provides engineered human alpha-galactosidase polypeptides and
compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized
to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention
also relates to the use of the compositions comprising the engineered human alpha-galactosidase
polypeptides for therapeutic purposes.
In some embodiments, the engineered human alpha-galactosidase ptides have been
optimized to provide improved stability at s levels. The invention also relates to the use of the
compositions comprising the engineered human galactosidase polypeptides for therapeutic
purposes.
Enzyme replacement therapy for treatment of Fabry disease (e.g,. Fabrazyme® agalsidase
beta; Genzyme) is available and is considered for eligible individuals. Currently used enzyme
replacements therapies are recombinantly expressed forms of the wild-type human GLA. It is known
that intravenously administered GLA circulates, becomes endocytosed, and travels to the
endosomes/lysosomes of target , where it reduces the accumulation of Gb3. These drugs do not
completely relieve patient symptoms, as neuropathic pain and transient ischemic attacks continue to
occur at reduced rates. In addition, the uptake of GLA by most target organs is poor in comparison to
the liver, and the enzyme is le at the pH of blood and lysosomes. Thus, issues remain with
available treatments. In addition, patients may develop an immune se (IgG and IgE antibodies
targeting the administered drug), and suffer severe allergic (anaphylactic) reactions, severe infusion
reactions, and even death. The present invention is intended to provide more stable enzymes suitable
for treatment of Fabry disease, yet with reduced side effects and improved outcomes, as compared to
currently available ents. Indeed, the present ion is intended to provide recombinant GLA
enzymes that have sed stability in blood (pH 7.4), which the enzyme encounters upon injection
into the bloodstream. In addition, the enzyme has increased stability at the pH of the lysosome (pH
4.3), the location where the enzyme is active during therapy. Thus, directed evolution of
recombinantly expressed human GLA in Saccharomyces cerevisiae, employing high throughput
ing of diverse enzyme variant libraries, was used to provide novel GLA variants with desired
stability properties. In addition, variant enzymes were screened and their amino acid sequence
determined in order to identify novel GLA variants with a predicted reduced immunogenicity. By
providing GLA variants with increased pH stability and reduced immunogenicity, the present
invention provides compositions and methods suitable for use in patients by increasing patient
tolerance of treatment and providing flexibility in dosing and formulation for improved t
outcomes.
Abbreviations and Definitions:
Unless defined otherwise, all cal and scientific terms used herein generally have the
same meaning as commonly understood by one of ordinary skill in the art to which this invention
ns. Generally, the lature used herein and the laboratory procedures of cell culture,
lar genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and c
acid chemistry described below are those well-known and commonly employed in the art. Such
techniques are well-known and described in numerous texts and reference works well known to those
of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and
chemical analyses. All patents, patent applications, articles and publications mentioned herein, both
supra and infra, are hereby expressly incorporated herein by reference.
Although any suitable s and als similar or equivalent to those bed herein
find use in the practice of the present ion, some methods and materials are bed herein. It is
to be understood that this invention is not limited to the particular methodology, protocols, and
reagents described, as these may vary, depending upon the context they are used by those of skill in
the art. Accordingly, the terms defined immediately below are more fully described by reference to
the application as a whole. All patents, patent applications, articles and publications mentioned herein,
both supra and infra, are hereby expressly incorporated herein by nce.
Also, as used herein, the ar ”a”, ”an,” and ”the” include the plural references, unless the
context clearly indicates otherwise.
Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range
disclosed herein is intended to encompass every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is
also intended that every maximum (or minimum) numerical limitation disclosed herein includes every
lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were
expressly written herein.
The term “about” means an able error for a particular value. In some instances “about”
means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means
within 1, 2, 3, or 4 standard ions of a given value.
Furthermore, the headings ed herein are not limitations of the various aspects or
embodiments of the invention which can be had by reference to the application as a whole.
Accordingly, the terms defined immediately below are more fully defined by reference to the
ation as a whole. Nonetheless, in order to facilitate tanding of the invention, a number of
terms are defined below.
Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' ation; amino
acid sequences are written left to right in amino to carboxy orientation, respectively.
As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e.,
equivalent to the term “including” and its corresponding cognates).
“EC” number refers to the Enzyme lature of the Nomenclature Committee of the
International Union of Biochemistry and Molecular y BMB). The IUBMB biochemical
fication is a cal classification system for s based on the chemical reactions they
catalyze.
“ATCC” refers to the American Type Culture Collection whose biorepository collection
includes genes and strains.
“NCBI” refers to National Center for ical Information and the sequence databases
provided therein.
“Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer
of at least two amino acids covalently linked by an amide bond, regardless of length or post-
translational modification (e.g., glycosylation or phosphorylation).
“Amino acids” are referred to herein by either their commonly known three-letter symbols or
by the one-letter symbols recommended by IUPAC-IUB Biochemical lature Commission.
Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.
The term “engineered,” “recombinant,39 ccnon-naturally occurring,” and “variant,” when used
with reference to a cell, a polynucleotide or a polypeptide refers to a material or a material
corresponding to the natural or native form of the material that has been modified in a manner that
would not otherwise exist in nature or is cal thereto but produced or derived from synthetic
materials and/or by lation using inant techniques.
As used herein, “wild-type” and “naturally-occurring” refer to the form found in nature. For
example a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that
can be isolated from a source in nature and which has not been intentionally modified by human
manipulation.
unized” as used herein, refers to the manipulation of a protein sequence to create a
variant that is predicted to be not as immunogenic as the wild-type or reference protein. In some
embodiments, the predicted deimmunization is complete, in that the variant n is predicted to not
stimulate an immune response in patients to whom the variant protein is administered. This response
can be measured by various methods including but not limited to, the presence or abundance of anti-
drug antibodies, the ce or abundance of lizing antibodies, the presence of an anaphylactic
response, peptide presentation on major histocompatibility complex-II (MHC-II) proteins, or the
prevalence or intensity of cytokine release upon administration of the protein. In some embodiments,
the variant protein is less immunogenic than the wild-type or reference n. In some
embodiments, deimmunization involves modifications to subsequences of proteins (e.g., epitopes) that
are recognized by human yte antigen (HLA) receptors. In some embodiments, these epitopes
are removed by changing their amino acid sequences to e a deimmunized variant protein in
which such subsequences are no longer ized by the HLA receptors. In some other
embodiments, these epitopes retain binding y to HLA receptors, but are not presented. In some
embodiments, the deimmunized protein shows lower levels of response in biochemical and cell-
biological predictors of human immunological responses including dendritic-cell T-cell activation
assays, or (HLA) peptide binding assays. In some embodiments, these es are removed by
changing their amino acid ce to produce a deimmunized variant protein in which the epitopes
are no longer recognized by T-cell receptors. In still other embodiments the deimmunized protein
induces anergy in its corresponding T-cells, activates T regulatory cells, or results in clonal deletion of
recognizing s.
“Coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino
acid sequence of a protein.
The term “percent (%) sequence identity” is used herein to refer to comparisons among
polynucleotides and ptides, and are determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the
comparison window may comprise additions or deletions (i.e., gaps) as compared to the nce
sequence for optimal ent of the two sequences. The percentage may be calculated by
determining the number of positions at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions, dividing the number of matched
ons by the total number of positions in the window of ison and multiplying the result by
100 to yield the percentage of sequence ty. atively, the percentage may be calculated by
determining the number of positions at which either the identical c acid base or amino acid
e occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to
yield the number of matched positions, dividing the number of matched positions by the total number
of positions in the window of comparison and multiplying the result by 100 to yield the percentage of
sequence identity. Those of skill in the art appreciate that there are many established algorithms
ble to align two sequences. Optimal alignment of ces for comparison can be conducted,
e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl.
Math., 2:482 [1981]), by the homology alignment thm ofNeedleman and Wunsch (Needleman
and Wunsch, J. Mol. Biol., 48:443 [1970), by the search for similarity method of Pearson and Lipman
(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 852444 [1988]), by computerized entations
of these algorithms (e. g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software
Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity e, but are not limited to the
BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J.
Mol. Biol., 215: 403-410 [1990]; and Altschul et al., 1977, Nucleic Acids Res., 3389-3402 [1977],
respectively). Software for performing BLAST analyses is publicly available through the National
Center for Biotechnology Information e. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either
match or satisfy some positive-valued threshold score T when aligned with a word of the same length
in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et
al, . These initial neighborhood word hits act as seeds for initiating searches to find longer
HSPs containing them. The word hits are then extended in both directions along each sequence for as
far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the ters M (reward score for a pair of matching es; always >0) and
N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is
used to calculate the cumulative score. Extension of the word hits in each direction are halted when:
the cumulative alignment score falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring
e alignments; or the end of either sequence is reached. The BLAST thm parameters W, T,
and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide
ces) uses as defaults a ngth (W) of l 1, an expectation (E) of 10, M=5, N=-4, and a
comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff
and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]). Exemplary determination of sequence
alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG
Wisconsin Software package (Accelrys, n WI), using default parameters provided.
“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison.
A nce sequence may be a subset of a larger sequence, for example, a segment of a full-length
gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid
residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in
length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides
may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between
the two sequences, and (2) may further comprise a sequence that is divergent between the two
sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically
performed by comparing sequences of the two polynucleotides or ptides over a rison
window” to identify and compare local regions of sequence similarity. In some embodiments, a
“reference ce” can be based on a primary amino acid ce, where the nce sequence
is a sequence that can have one or more s in the primary sequence. “Comparison window”
refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids
residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous
nucleotides or amino acids and n the portion of the sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 t or less as compared to the reference sequence
(which does not se additions or deletions) for optimal alignment of the two sequences. The
ison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50,
100, or longer windows.
“Corresponding to”, “reference to” or “relative to” when used in the context of the numbering
of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a
ed reference sequence when the given amino acid or polynucleotide sequence is compared to
the reference sequence. In other words, the residue number or residue position of a given polymer is
designated with respect to the reference sequence rather than by the actual numerical position of the
residue within the given amino acid or polynucleotide sequence. For example, a given amino acid
ce, such as that of an ered GLA, can be aligned to a reference sequence by introducing
gaps to optimize residue matches between the two sequences. In these cases, although the gaps are
present, the numbering of the residue in the given amino acid or polynucleotide sequence is made
with t to the reference sequence to which it has been aligned.
“Amino acid difference” or “residue difference” refers to a difference in the amino acid
residue at a position of a polypeptide ce relative to the amino acid residue at a corresponding
position in a reference ce. The positions of amino acid differences generally are referred to
herein as “Xn,” Where n refers to the corresponding position in the nce sequence upon Which the
residue difference is based. For e, a “residue difference at position X93 as compared to SEQ
ID NO:2” refers to a ence of the amino acid residue at the polypeptide position corresponding to
position 93 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a serine at
position 93, then a “residue ence at position X93 as compared to SEQ ID NO:2” an amino acid
substitution of any residue other than serine at the position of the polypeptide corresponding to
position 93 of SEQ ID NO:2. In most instances herein, the specific amino acid e difference at a
position is indicated as “XnY” Where “Xn” specified the corresponding position as described above,
and “Y” is the single letter identifier of the amino acid found in the engineered ptide (i.e., the
different residue than in the reference polypeptide). In some instances (e.g., in Tables 2.1, 2.2, 2.3,
2.4, 2.5, and 6.1), the present disclosure also provides specific amino acid differences denoted by the
conventional notation “AnB”, Where A is the single letter identifier of the residue in the reference
sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter
identifier of the residue substitution in the sequence of the engineered polypeptide. In some ces,
a polypeptide of the present disclosure can include one or more amino acid residue differences
relative to a nce sequence, Which is indicated by a list of the specified positions Where residue
differences are present relative to the reference sequence. In some embodiments, Where more than
one amino acid can be used in a specific residue position of a polypeptide, the various amino acid
residues that can be used are separated by a “/” (e.g., X307H/X307P or X307H/P). In some
ments, the enzyme variants comprise more than one substitution. These substitutions are
separated by a slash for ease in g (e. g., Cl43A/K206A). The present application includes
engineered polypeptide sequences sing one or more amino acid differences that include
/or both conservative and non-conservative amino acid substitutions.
“Conservative amino acid substitution” refers to a substitution of a residue with a different
residue having a similar side chain, and thus typically involves substitution of the amino acid in the
polypeptide With amino acids Within the same or similar defined class of amino acids. By way of
example and not limitation, an amino acid With an aliphatic side chain may be substituted With
another aliphatic amino acid (e. g., alanine, valine, leucine, and isoleucine); an amino acid With
hydroxyl side chain is substituted With another amino acid With a yl side chain (e.g., serine and
threonine); an amino acids having aromatic side chains is substituted With another amino acid having
an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid With a
basic side chain is substituted With another amino acid With a basis side chain (e.g., lysine and
ne); an amino acid With an acidic side chain is substituted With another amino acid With an
acidic side chain (e. g., aspartic acid or glutamic acid); and/or a hydrophobic or hilic amino acid
is replaced with another hydrophobic or hydrophilic amino acid, respectively.
WO 05889
“Non-conservative substitution” refers to substitution of an amino acid in the polypeptide
with an amino acid with significantly differing side chain properties. Non-conservative substitutions
may use amino acids between, rather than within, the defined groups and affects (a) the structure of
the peptide backbone in the area of the substitution (e.g, proline for glycine) (b) the charge or
hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary
non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino
acid; an ic amino acid substituted with a small amino acid; and a hydrophilic amino acid
substituted with a hydrophobic amino acid.
“Deletion” refers to modification to the polypeptide by removal of one or more amino acids
from the reference polypeptide. ons can comprise removal of l or more amino acids, 2 or more
amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more
amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino
acids making up the nce enzyme while retaining enzymatic ty and/or retaining the
improved ties of an engineered enzyme. Deletions can be directed to the internal portions
and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a
continuous segment or can be discontinuous.
“Insertion” refers to modification to the polypeptide by addition of one or more amino acids
from the nce polypeptide. Insertions can be in the internal portions of the polypeptide, or to the
carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art.
The insertion can be a contiguous segment of amino acids or separated by one or more of the amino
acids in the lly occurring polypeptide.
A “functional fragment” or a “biologically active fragment” used interchangeably herein
refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal
deletions, but where the remaining amino acid sequence is identical to the corresponding positions in
the sequence to which it is being compared (e. g., a full-length engineered GLA of the present
invention) and that retains substantially all of the activity of the full-length polypeptide.
“Isolated polypeptide” refers to a ptide which is substantially separated from other
inants that lly accompany it, e.g., protein, lipids, and polynucleotides. The term
embraces polypeptides which have been d or purified from their naturally-occurring
environment or expression system (e.g., host cell or in vitro sis). The recombinant GLA
polypeptides may be t within a cell, present in the cellular medium, or prepared in various
forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant GLA
polypeptides can be an isolated polypeptide.
“Substantially pure polypeptide” refers to a composition in which the polypeptide species is
the predominant species present (i.e., on a molar or weight basis it is more abundant than any other
individual macromolecular s in the composition), and is generally a substantially purified
composition when the object s comprises at least about 50 percent of the macromolecular
species present by mole or % weight. Generally, a substantially pure GLA composition comprises
about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more,
and about 98% or more of all macromolecular species by mole or % weight t in the
wmmfimnhmmmm®mhmMammMmmmdwmmmmdmamthmmgMMme
contaminant species cannot be detected in the composition by conventional detection methods)
wherein the composition consists essentially of a single macromolecular species. Solvent species,
small molecules (<500 Daltons), and elemental ion species are not considered macromolecular
species. In some embodiments, the isolated recombinant GLA polypeptides are substantially pure
ptide compositions.
“Improved enzyme property” refers to an engineered GLA polypeptide that exhibits an
improvement in any enzyme property as compared to a nce GLA polypeptide and/or as a wild-
type GLA polypeptide or another engineered GLA polypeptide. Improved properties include but are
not limited to such properties as increased protein expression, increased thermoactivity, increased
thermostability, increased pH activity, increased stability, sed enzymatic activity, increased
substrate specificity or affinity, increased specific activity, increased ance to substrate or end-
t inhibition, increased chemical stability, ed chemoselectivity, improved solvent
stability, increased tolerance to acidic or basic pH, increased tolerance to proteolytic activity (i.e.,
reduced sensitivity to proteolysis), reduced aggregation, sed solubility, reduced
immunogenicity, improved post-translational modification (e. g., glycosylation), and d
ature profile.
“Increased enzymatic activity” or ced catalytic activity” refers to an improved property
of the ered GLA polypeptides, which can be represented by an se in specific activity
(e. g. , product produced/time/weight protein) or an increase in percent conversion of the substrate to
the product (e. g., percent conversion of starting amount of substrate to product in a specified time
period using a specified amount of GLA) as ed to the reference GLA enzyme. Exemplary
methods to determine enzyme activity are provided in the Examples. Any property ng to enzyme
activity may be affected, including the classical enzyme properties of Km, Vmax or km, changes of
which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about
1.] fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold, 5-fold,
lO-fifld,20-fifld,25-fifld,50-fifld,75-fifld,lOO-fifld,l50-fifld,200-fifld(n1noreenzwnaficacfivfiy
than the naturally occurring GLA or another ered GLA from which the GLA polypeptides were
In some embodiments, the engineered GLA ptides have a km, of at least 0.1/sec, at least
0.5/sec, at least l.0/sec, at least c, at least 10.0/sec and in some preferred embodiments greater
than 10.0/sec. In some embodiments, the Km is in the range of about luM to about 5mM; in the range
ofabout5uh4toabout2nflfl,intherangeofaboufll)uhdtoabouthflfl;orintherangeofabout
10uM to about lmM. In some specific embodiments, the engineered GLA enzyme exhibits improved
enzymatic ty after exposure to certain conditions in the range of 1.5 to 10 fold, 1.5 to 25 fold,
1.5 to 50 fold, 1.5 to 100 fold or r than that of a reference GLA enzyme (e. g., a wild-type GLA
or any other reference GLA). GLA activity can be measured by any suitable method known in the art
(e.g., standard assays, such as monitoring changes in spectrophotometric properties of reactants or
ts). In some embodiments, the amount of products produced can be measured by High-
Performance Liquid Chromatography (HPLC) separation combined with UV absorbance or
fluorescent detection directly or following o-phthaldialdehyde (OPA) derivatization. Comparisons of
enzyme activities are made using a defined preparation of enzyme, a defined assay under a set
condition, and one or more defined substrates, as further bed in detail herein. Generally, when
lysates are compared, the s of cells and the amount of protein assayed are determined as well
as use of identical expression systems and identical host cells to minimize variations in amount of
enzyme produced by the host cells and present in the s.
The term “improved tolerance to acidic pH” means that a inant GLA according to the
invention will have increased stability (higher retained activity at about pH 4.8 after exposure to
acidic pH for a specified period of time (1 hour, up to 24 hours)) as compared to a reference GLA or
another enzyme.
“Physiological pH” as used herein means the pH range generally found in a subject’s (e.g.,
human) blood.
The term “basic pH” (e. g., used with reference to improved stability to basic pH conditions or
increased tolerance to basic pH) means a pH range of about 7 to 11.
The term c pH” (e.g., used with reference to improved stability to acidic pH ions
or sed tolerance to acidic pH) means a pH range of about 1.5 to 4.5.
“Conversion” refers to the enzymatic sion (or biotransformation) of a substrate(s) to
the corresponding product(s). “Percent conversion” refers to the t of the substrate that is
converted to the product within a period of time under ed conditions. Thus, the atic
activity” or ity” of a GLA polypeptide can be expressed as “percent conversion” of the substrate
to the product in a c period of time.
“Hybridization stringency” relates to hybridization conditions, such as washing conditions, in
the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions
of lower stringency, followed by washes of varying but higher stringency. The term ately
stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic
acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target
DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent
conditions are conditions equivalent to hybridization in 50% formamide, 5>< Denhart's solution,
><SSPE, 0.2% SDS at 42°C, followed by washing in O.2><SSPE, 0.2% SDS, at 42°C. “High
stringency hybridization” refers generally to conditions that are about 10°C or less from the thermal
melting temperature Tm as determined under the solution condition for a defined polynucleotide
sequence. In some embodiments, a high stringency condition refers to conditions that permit
hybridization of only those c acid sequences that form stable hybrids in 0.018M NaCl at 65°C
(i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency
conditions, as contemplated herein). High stringency conditions can be provided, for example, by
hybridization in conditions equivalent to 50% formamide, 5>< t's solution, 5><SSPE, 0.2% SDS
at 42°C, followed by washing in 0.1 ><SSPE, and 0.1% SDS at 65°C. Another high stringency
condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v)
SDS at 65°C and g in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency
hybridization conditions, as well as moderately stringent conditions, are described in the references
cited above.
“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein
to those preferentially used in a ular organism such that the encoded protein is more efficiently
expressed in the organism of interest. gh the genetic code is degenerate in that most amino
acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well
known that codon usage by particular organisms is nonrandom and biased towards particular codon
triplets. This codon usage bias may be higher in reference to a given gene, genes of common function
or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate
n coding regions of an organism's genome. In some embodiments, the polynucleotides ng
the GLA enzymes may be codon optimized for optimal production from the host organism selected
for expression.
“Control sequence” refers herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide and/or polypeptide of the present application.
Each control sequence may be native or foreign to the nucleic acid sequence encoding the
ptide. Such control sequences include, but are not d to, a leader, polyadenylation
sequence, propeptide sequence, promoter ce, signal peptide sequence, initiation sequence and
ription terminator. At a minimum, the l sequences include a promoter, and transcriptional
and translational stop signals. The control sequences may be provided with linkers for the purpose of
introducing specific restriction sites facilitating ligation of the control sequences with the coding
region of the nucleic acid ce ng a polypeptide.
“Operably linked” is defined herein as a configuration in which a control ce is
appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of
interest such that the control sequence directs or regulates the expression of the cleotide and/or
polypeptide of interest.
“Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for
expression of a polynucleotide of interest, such as a coding ce. The promoter sequence contains
transcriptional l sequences, which mediate the expression of a polynucleotide of interest. The
promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of
choice including , truncated, and hybrid promoters, and may be obtained from genes encoding
extracellular or ellular polypeptides either homologous or heterologous to the host cell.
“Suitable reaction ions” refers to those conditions in the enzymatic sion reaction
solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, vents, etc.)
under which a GLA polypeptide of the present application is capable of converting a substrate to the
desired product compound, Exemplary “suitable reaction conditions” are provided in the present
application and illustrated by the Examples. “Loading”, such as in und loading” or “enzyme
loading” refers to the concentration or amount of a component in a reaction mixture at the start of the
reaction. “Substrate” in the t of an enzymatic conversion reaction process refers to the
compound or molecule acted on by the GLA polypeptide. “Product” in the context of an enzymatic
conversion process refers to the compound or molecule ing from the action of the GLA
polypeptide on a substrate.
As used herein the term “culturing” refers to the growing of a population of microbial cells
under any le conditions (e. g., using a liquid, gel or solid medium).
Recombinant polypeptides can be produced using any suitable methods known the art. Genes
encoding the wild-type polypeptide of st can be cloned in vectors, such as plasmids, and
expressed in desired hosts, such as E. coli, S. cerevisiae, etc. Variants of recombinant polypeptides
can be generated by various methods known in the art. Indeed, there is a wide variety of different
mutagenesis techniques well known to those skilled in the art. In addition, nesis kits are also
available from many commercial molecular biology ers. Methods are available to make
specific substitutions at defined amino acids (site-directed), specific or random mutations in a
zed region of the gene (regio-specific), or random mutagenesis over the entire gene (e.g.,
saturation mutagenesis). us suitable methods are known to those in the art to generate
enzyme variants, including but not limited to site-directed mutagenesis of single-stranded DNA or
double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, prone PCR, shuffling,
and chemical saturation mutagenesis, or any other suitable method known in the art. miting
examples of methods used for DNA and protein engineering are provided in the following s: US
Pat. No. 6,117,679; US Pat. No. 6,420,175; US Pat. No. 6,376,246; US Pat. No. 182; US Pat.
No. 7,747,391; US Pat. No. 7,747,393; US Pat. No. 7,783,428; and US Pat. No. 8,383,346. After the
variants are produced, they can be screened for any desired property (e.g., high or increased activity,
or low or d activity, increased thermal activity, increased thermal stability, and/or acidic pH
stability, etc.). In some ments, “recombinant GLA polypeptides” (also referred to herein as
“engineered GLA polypeptides,39 ccvariant GLA enzymes,” and “GLA variants”) find use.
As used herein, a ”vector” is a DNA construct for introducing a DNA sequence into a cell. In
some embodiments, the vector is an expression vector that is operably linked to a suitable control
sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the DNA
ce. In some embodiments, an ”expression vector” has a promoter sequence operably linked to
2015/063329
the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also
comprises a transcription terminator sequence.
As used herein, the term ssion” includes any step involved in the production of the
polypeptide including, but not limited to, transcription, ranscriptional modification, translation,
and post-translational modification. In some embodiments, the term also encompasses secretion of
the polypeptide from a cell.
As used herein, the term “produces” refers to the production of proteins and/or other
compounds by cells. It is ed that the term encompass any step involved in the production of
polypeptides ing, but not limited to, transcription, post-transcriptional modification, translation,
and post-translational modification. In some embodiments, the term also asses secretion of the
polypeptide from a cell.
As used , an amino acid or nucleotide sequence (e.g., a promoter sequence, signal
peptide, terminator sequence, etc.) is ”heterologous” to another sequence with which it is operably
linked if the two sequences are not associated in nature.
As used , the terms “host cell” and “host strain” refer to suitable hosts for expression
vectors comprising DNA provided herein (e. g., the polynucleotides encoding the GLA variants). In
some ments, the host cells are prokaryotic or otic cells that have been transformed or
transfected with vectors constructed using recombinant DNA techniques as known in the art.
The term “analogue” means a polypeptide having more than 70% sequence identity but less
than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some
embodiments, analogues means polypeptides that contain one or more turally occurring amino
acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally
ing amino acids. In some embodiments, analogues also include one or more D-amino acid
residues and non-peptide linkages between two or more amino acid residues.
The term peutic” refers to a compound administered to a subject who shows signs or
symptoms of pathology having beneficial or desirable medical effects.
The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical
use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of an
engineered GLA polypeptide encompassed by the invention and an acceptable carrier.
The term “effective amount” means an amount ent to produce the desired . One of
general skill in the art may determine what the effective amount by using routine experimentation.
The terms “isolated” and “purified” are used to refer to a molecule (e. g., an isolated nucleic
acid, polypeptide, etc.) or other component that is removed from at least one other component with
which it is naturally associated. The term “purified” does not require absolute purity, rather it is
intended as a relative definition.
-l6-
The term “subject” encompasses mammals such as humans, non-human primates, livestock,
companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the
term encompass females as well as males.
As used herein, the term “patient” means any subject that is being assessed for, treated for, or
is experiencing disease.
The term “infant” refers to a child in the period of the first month after birth to approximately
one (1) year of age. As used herein, the term rn” refers to child in the period from birth to the
28th day of life. The term “premature infant” refers to an infant born after the twentieth completed
week of gestation, yet before full term, generally weighing ~500 to ~2499 grams at birth. A “very
low birth weight infant” is an infant weighing less than 1500 g at birth.
As used , the term “child” refers to a person who has not attained the legal age for
consent to treatment or ch procedures. In some embodiments, the term refers to a person
between the time of birth and adolescence.
As used herein, the term “adult” refers to a person who has ed legal age for the nt
iction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any
fully grown, mature organism. In some embodiments, the term “young adult” refers to a person less
than 18 years of age, but who has reached sexual maturity.
As used herein, “composition” and lation” encompass ts comprising at least one
engineered GLA of the present ion, intended for any suitable use (e.g., pharmaceutical
compositions, dietary/nutritional supplements, feed, etc.).
The terms “administration” and “administering” a composition mean providing a composition
of the present ion to a subject (e.g., to a person suffering from the s of Fabry disease).
The term “carrier” when used in reference to a pharmaceutical composition means any of the
standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and
nts.
The term “pharmaceutically acceptable” means a material that can be administered to a
subject without causing any undesirable biological s or interacting in a deleterious manner with
any of the components in which it is contained and that possesses the desired biological activity.
As used herein, the term “excipient” refers to any pharmaceutically acceptable additive,
carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API;
e.g., the engineered GLA ptides of the present invention). Excipients are typically included for
formulation and/or administration purposes.
The term “therapeutically effective amount” when used in reference to symptoms of
disease/condition refers to the amount and/or tration of a compound (e. g., engineered GLA
polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease/condition
or prevents or delays the onset of symptom(s).
The term “therapeutically effective ” when used in nce to a disease/condition
refers to the amount and/or concentration of a composition (e.g., engineered GLA polypeptides) that
ameliorates, attenuates, or eliminates the disease/condition. In some embodiments, the term is use in
reference to the amount of a composition that s the biological (e. g., medical) response by a
tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other
clinician.
It is intended that the terms “treating,” “treat” and “treatment” encompass preventative (e.g.,
prophylactic), as well as tive treatment.
Engineered GLA Expression and Activity:
Two strategies for secreted GLA expression were utilized, using the yeast MFG signal peptide
(MF-SP) or a longer leader ce of 83 amino acids (MF-leader) to drive ion of a yeast
codon-optimized mature human GLA. Clones were expressed from a pYT-72 vector in S. cerevisiae
strain INVScl. Both approaches provided supernatants with measurable activity on the fluorogenic
substrate 4-methylumbelliferyl (x-D-galactopyranoside (4-MuGal). However, the construct with the
yeast MFG signal peptide provided 3-fold higher activities and was used as the starting ce for
directed ion.
To identify mutational diversity, a l3-position conserved “homolog” combinatorial library
and a l92-position site saturation mutagenesis library were constructed. Equivalent volumes of
supernatant were screened in an unchallenged condition (no incubation, pH 4.8) or following a one-
hour tion in a low pH .2) or high pH (7.1- 8.2) environment. GLA ts with
increased activity due to increased GLA sion or GLA specific activity were identified based on
their fold improvement over the parent GLA. GLA variants with increased stability were identified
by dividing the fold-improvement observed under challenged conditions by the fold-improvement
observed under unchallenged conditions. This approach reduces the bias towards selecting variants
based on increased expression but without changes in specific ty at pH extremes. Composite
activity scores (the product of fold-improvements for all three ions) and stability (the product of
stability scores) were used to rank mutations in improved variants for inclusion in subsequent GLA
libraries.
Engineered GLA:
In some embodiments the engineered GLA which exhibits an improved property has at least
about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or at about 100% amino acid ce identity with SEQ ID N05,
and an amino acid residue difference as compared to SEQ ID NO:5, at one or more amino acid
positions (such as at l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 14, 15, 20 or more amino acid positions
2015/063329
compared to SEQ ID N05, or a sequence having at least 85%, at least 88%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or greater amino acid sequence identity with SEQ ID NO:5). In some embodiment the
residue difference as compared to SEQ ID NO:5, at one or more positions will include at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the
engineered GLA ptide is a ptide listed in Table 2.1, 2.2, 2.4, 2.5, or Table 7.1.
In some embodiments the engineered GLA which exhibits an improved property has at least
about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or at about 100% amino acid ce identity with SEQ ID NO:10,
and an amino acid residue difference as ed to SEQ ID NO:10, at one or more amino acid
ons (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions
compared to SEQ ID NO:10, or a sequence having at least 85%, at least 88%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or greater amino acid sequence identity with SEQ ID NO:10). In some embodiment the
residue difference as compared to SEQ ID NO:10, at one or more positions will include at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the
engineered GLA polypeptide is a polypeptide listed in Table 2.3.
In some embodiments the engineered GLA which exhibits an improved property has at least
85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID
NO:5. In some embodiments the engineered GLA which exhibits an improved property has at least
85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with SEQ ID
NO: 10.
In some embodiments, the engineered GLA polypeptide is selected from SEQ ID NOS:15,
13,10, and 18.
In some embodiments, the engineered GLA ptide comprises a functional fragment of
an engineered GLA polypeptide encompassed by the invention. Functional nts have at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% of the activity of the engineered GLA polypeptide from which is was derived
(i.e., the parent engineered GLA). A functional fragment comprises at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even 99%
of the parent sequence of the engineered GLA. In some embodiments the functional nt is
truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35,
less than 40, less than 45, and less than 50 amino acids.
2015/063329
Polynucleotides Encoding Engineered ptides, Expression Vectors and Host Cells:
The present invention provides polynucleotides encoding the engineered GLA polypeptides
described herein. In some embodiments, the polynucleotides are operatively linked to one or more
heterologous regulatory sequences that control gene expression to create a recombinant
polynucleotide e of expressing the polypeptide. Expression constructs ning a
heterologous polynucleotide encoding the engineered GLA polypeptides can be introduced into
appropriate host cells to express the corresponding GLA polypeptide.
As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge
of the codons corresponding to the various amino acids provide a description of all the
polynucleotides e of encoding the subject polypeptides. The degeneracy of the genetic code,
where the same amino acids are encoded by alternative or synonymous codons, allows an ely
large number of nucleic acids to be made, all of which encode the engineered GLA polypeptide. Thus,
having knowledge of a particular amino acid sequence, those skilled in the art could make any number
of ent nucleic acids by simply modifying the sequence of one or more codons in a way which
does not change the amino acid sequence of the protein. In this regard, the present invention
specifically contemplates each and every possible variation of polynucleotides that could be made
encoding the polypeptides described herein by selecting combinations based on the possible codon
s, and all such variations are to be considered specifically disclosed for any polypeptide
described herein, including the variants provided in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1.
In s embodiments, the codons are preferably selected to fit the host cell in which the
n is being produced. For example, preferred codons used in ia are used for expression in
bacteria. Consequently, codon optimized cleotides encoding the engineered GLA polypeptides
contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions
of the full length coding region.
In some embodiments, as described above, the polynucleotide encodes an engineered
polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide
comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference
sequence selected from SEQ ID NOS:5, and/or 10, or the amino acid sequence of any variant as
disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, and one or more residue differences as compared to
the reference polypeptide of SEQ ID NOS:5, and/or 10, or the amino acid sequence of any variant as
disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, (for e 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
amino acid residue positions). In some ments, the reference sequence is selected from SEQ ID
NO:5 and/or 10. In some embodiments, the polynucleotide s an engineered polypeptide
having GLA activity with the properties sed herein, wherein the polypeptide ses an
amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence
SEQ ID N05, and one or more e differences as compared to SEQ ID NO:5, at residue positions
selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, or 6.1, when optimally aligned with the
polypeptide of SEQ ID N05.
In some embodiments, the cleotide encodes an engineered polypeptide having GLA
ty with the properties disclosed herein, n the polypeptide comprises an amino acid
sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID
NO:10, and one or more residue differences as compared to SEQ ID NO:10, at residue positions
selected from those provided in Tables 2.3, when optimally aligned with the polypeptide of SEQ ID
NO: 10.
In some embodiments, the polynucleotide encoding the engineered GLA polypeptides
comprises a polynucleotide ce selected from a polynucleotide sequence ng SEQ ID
NOS:10, 13, 15, 18, 21, and 24. In some embodiments, the polynucleotide encoding an ered
GLA polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 93%,
95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOS: 8, 9, 11, 12, 14, 16, 17, 19,
, 22, and/or 23. In some embodiments, the polynucleotide encoding the engineered GLA
polypeptides comprises a polynucleotide sequence selected from SEQ ID NOS:8, 9, 11, 12, 14, 16,
17, 19, 20, 22, and 23.
In some embodiments, the polynucleotides are capable of izing under highly stringent
conditions to a reference polynucleotide sequence selected from SEQ ID NOS: 8, 9, 11, 12, 14, 16,
17, 19, 20, 22, and 23, or a complement thereof, or a polynucleotide sequence encoding any of the
variant GLA polypeptides provided herein. In some embodiments, the polynucleotide capable of
hybridizing under highly stringent conditions s a GLA polypeptide comprising an amino acid
sequence that has one or more residue differences as compared to SEQ ID NO:5 and/or 10, at residue
positions selected from any positions as set forth in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or 6.1.
In some embodiments, an isolated polynucleotide encoding any of the engineered GLA
ptides provided herein is manipulated in a variety of ways to provide for expression of the
polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are ed as
expression vectors where one or more control sequences is present to regulate the expression of the
polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its ion
into a vector may be desirable or necessary depending on the sion vector. The techniques for
modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well
known in the art.
In some ments, the control sequences include among other sequences, promoters,
leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and
transcription ators. As known in the art, suitable promoters can be ed based on the host
cells used. Exemplary promoters for filamentous fungal host cells, include ers obtained from
the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus
niger l alpha-amylase, illus niger acid stable alpha-amylase, Aspergillus niger or
Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans idase, and
Fusarium oxysporum trypsin-like protease (See e. g., WO 96/00787), as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus
oryzae triose phosphate isomerase), and mutant, ted, and hybrid promoters thereof. Exemplary
yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae
enolase (ENO-l), Saccharomyces cerevisiae galactokinase (GALl), Saccharomyces cerevisiae
alcohol ogenase/glyceraldehydephosphate dehydrogenase (ADHZ/GAP), and
romyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are
known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]). Exemplary promoters for use in
mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian
vacuolating Virus 40 (SV40), from Homo sapiens phosphorglycerate kinase, beta actin, tion
factor-la or glyceraldehydephosphate dehydrogenase, or from Gallus gallus ’ B-actin.
In some ments, the control sequence is a suitable transcription terminator sequence, a
sequence recognized by a host cell to terminate transcription. The terminator sequence is operably
linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which
is functional in the host cell of choice finds use in the t invention. For example, exemplary
ription terminators for filamentous fungal host cells can be obtained from the genes for
Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, illus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
Exemplary terminators for yeast host cells can be obtained from the genes for romyces
cerevisiae e, Saccharomyces cerevisiae rome C (CYCl), and Saccharomyces cerevisiae
glyceraldehydephosphate ogenase. Other useful terminators for yeast host cells are known
in the art (See e. g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but
are not limited to those from cytomegalovirus (CMV), Simian ating Virus 40 (SV40), or from
Homo sapiens growth hormone.
In some embodiments, the control sequence is a le leader ce, a non-translated
region of an mRNA that is important for translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleic acid ce encoding the polypeptide. Any leader sequence
that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans
triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to those
obtained from the genes for Saccharomyces cerevisiae enolase (ENO-l), Saccharomyces cerevisiae 3-
phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae
alcohol dehydrogenase/glyceraldehydephosphate dehydrogenase (ADHZ/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to
the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence
which is onal in the host cell of choice may be used in the present invention. Exemplary
polyadenylation sequences for filamentous fungal host cells include, but are not d to those from
the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger mylase, Aspergillus
nidulans nilate synthase, Fusarium oxysporum trypsin-like se, and Aspergillus niger
alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art
(See e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).
In some embodiments, the control sequence is a signal peptide coding region that codes for an
amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded
ptide into the cell's secretory pathway. The 5' end of the coding sequence of the c acid
sequence may inherently contain a signal peptide coding region naturally linked in translation reading
frame with the segment of the coding region that encodes the ed polypeptide. Alternatively, the
' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding
sequence. Any signal peptide coding region that directs the expressed ptide into the secretory
pathway of a host cell of choice finds use for expression of the ered GLA polypeptides
provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but
are not limited to the signal peptide coding s obtained from the genes for Aspergillus oryzae
TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful
signal es for yeast host cells include, but are not limited to those from the genes for
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal
peptides for mammalian host cells include but are not limited to those from the genes for
immunoglobulin gamma (IgG).
In some embodiments, the control sequence is a propeptide coding region that codes for an
amino acid sequence positioned at the amino terminus of a polypeptide. The ant polypeptide is
referred to as a “proenzyme,39 ccpropolypeptide,” or “zymogen,” in some cases). A propolypeptide
can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the
propeptide from the propolypeptide.
In another aspect, the present invention also es a recombinant expression vector
comprising a polynucleotide encoding an engineered GLA polypeptide, and one or more expression
regulating regions such as a er and a ator, a replication origin, etc., depending on the
type of hosts into which they are to be introduced. in some embodiments, the various nucleic acid and
control sequences described above are joined together to produce a inant expression vector
which includes one or more convenient ction sites to allow for insertion or substitution of the
nucleic acid sequence encoding the variant GLA polypeptide at such sites. Alternatively, the
polynucleotide sequence(s) of the present invention are expressed by inserting the polynucleotide
sequence or a c acid construct comprising the polynucleotide sequence into an appropriate
vector for expression. In creating the expression vector, the coding sequence is located in the vector
so that the coding sequence is operably linked with the appropriate l sequences for expression.
The recombinant expression vector may be any vector (e.g. a plasmid or , that can be
conveniently subjected to recombinant DNA procedures and can result in the expression of the variant
GLA polynucleotide sequence. The choice of the vector will typically depend on the compatibility of
the vector with the host cell into which the vector is to be introduced. The s may be linear or
closed circular plasmids.
In some embodiments, the expression vector is an autonomously replicating vector (i.e., a
vector that exists as an extra-chromosomal entity, the replication of which is independent of
chromosomal replication, such as a plasmid, an chromosomal element, a minichromosome, or
an artificial chromosome). The vector may contain any means for assuring self-replication. In some
alternative embodiments, the vector may be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the chromosome(s) into which it has been
integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together
contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
In some embodiments, the expression vector preferably contains one or more selectable
markers, which permit easy ion of transformed cells. A table marker” is a gene the product
of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to
auxotrophs, and the like. Suitable markers for yeast host cells e, but are not limited to ADE2,
HIS3, LEU2, LYS2, MET3, TRPl, and URA3. Selectable markers for use in a filamentous fungal
host cell e, but are not limited to, amdS midase), argB (omithine carbamoyltransferases),
bar (phosphinothricin acetyltransferase), hph (hygromycin otransferase), niaD (nitrate
reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC
(anthranilate synthase), as well as equivalents thereof.In another aspect, the present invention
provides a host cell sing a polynucleotide encoding at least one engineered GLA polypeptide
of the present application, the polynucleotide being operatively linked to one or more control
sequences for expression of the engineered GLA enzyme(s) in the host cell. Host cells for use in
expressing the polypeptides encoded by the expression vectors of the present invention are well
known in the art and include but are not limited to, fungal cells, such as yeast cells (e.g.,
Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC Accession No. 201178]); insect cells (e.g.,
hila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e. g., CHO, COS, and BHK), and
human cells (e.g., HEK293T, human fibroblast, THP-l Jurkat and Bowes melanoma cell lines).
Accordingly, in another , the t invention provides methods for producing the
engineered GLA polypeptides, where the methods comprise culturing a host cell e of
expressing a polynucleotide encoding the engineered GLA polypeptide under conditions suitable for
WO 05889
expression of the polypeptide. In some embodiments, the methods further comprise the steps of
isolating and/or purifying the GLA polypeptides, as bed .
riate culture media and growth conditions for the above-described host cells are well
known in the art. Polynucleotides for expression of the GLA polypeptides may be introduced into
cells by various methods known in the art. ques include, among others, electroporation,
biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and
lasthsion.
The ered GLA with the properties disclosed herein can be obtained by subjecting the
polynucleotide encoding the naturally occurring or engineered GLA ptide to mutagenesis
and/or directed evolution methods known in the art, and as described herein. An exemplary directed
evolution technique is mutagenesis and/or DNA shuffling (See e. g., Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO
00/42651; WO 01/75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be
used include, among others, staggered extension process (StEP), in vitro recombination (See e. g.,
Zhao et al., Nat. Biotechnol., 16:258—261 [1998]), mutagenic PCR (See e.g., Caldwell et al., PCR
Methods Appl., 3:Sl36-Sl40 [1994]), and cassette mutagenesis (See e.g., Black et al., Proc. Natl.
Acad. Sci. USA 93:3525-3529 [1996]).
nufle,nnnagenefisand(firmnedevohnnninxfihodscanbereadflyapphedto
polynucleotides to generate variant libraries that can be expressed, screened, and assayed.
Mutagenesis and directed evolution methods are well known in the art (See e.g., US Patent Nos.
,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 679, 6,132,970,
6,165,793, 6,180,406, 674, 638, 6,287,861, 862, 242, 6,297,053, 6,303,344,
6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859,
6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964,
@381702,@39lfi52,@39L640,6§95fi47,@406£55,6406910,@413745,641&774,@42&175
@423542,@426224,@436675,@444A68,@451253,6479652,@482647,6489J46,@50@602
6,506,603, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 098, 6,576,467, 6,579,678, 6,586,182,
6,602,986, 6,613,514, 6,653,072, 6,716,631, 296, 6,961,664, 6,995,017, 7,024,312, 7,058,515,
1101291 7J48fi54,1288375,Z42L347,Z430A77,1534564,Z620500,Z620§02,Z62%170
Z702A64,Z747391,Z741393,Z75L986,Z776fi98,7J83A28,Z791030,Z853A10,Z86&138
Z873A99,Z904249,'L957912,&383346,&504A98,&849fi75,&876fl66,&768£71,andafl
related non-US counterparts; Ling et al., Anal. Biochem, 254(2):l57-78 [1997]; Dale et al., Meth.
Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet, 19:423-462 [1985]; Botstein et al., Science,
229:1193-1201 [1985]; , Biochem. J., 7 [1986]; Kramer et al., Cell, 38:879-887 ;
Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];
Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391 :288-291 [1998];
Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. USA,
4-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature,
9-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; US Pat. Appln.
Publn. Nos. 2008/0220990, US 2009/0312196, US2014/0005057, /0214391,
US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and all related non-US
counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO 00/42651, WO
01/75767, and WC 2009/152336; all of which are incorporated herein by nce).
In some ments, the enzyme variants obtained following mutagenesis treatment are
screened by ting the enzyme variants to a defined temperature (or other assay conditions) and
measuring the amount of enzyme activity remaining after heat treatments or other assay conditions.
DNA containing the polynucleotide encoding the GLA polypeptide is then isolated from the host cell,
sequenced to fy the nucleotide sequence changes (if any), and used to express the enzyme in a
different or the same host cell. Measuring enzyme activity from the expression libraries can be
performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as
HPLC analysis).
For engineered polypeptides ofknown sequence, the polynucleotides encoding the enzyme
can be prepared by standard solid-phase methods, according to known tic methods. In some
embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e. g.,
by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any d
continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be
prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al.,
Tetra. Lett., 22:1859-69 [1981]; and Matthes et al., EMBO J., 3:801-05 [1984]), as it is typically
practiced in automated synthetic methods. ing to the phosphoramidite ,
oligonucleotides are synthesized (e. g., in an automatic DNA synthesizer), purified, annealed, ligated
and cloned in appropriate vectors.
Accordingly, in some ments, a method for preparing the engineered GLA polypeptide
can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid
sequence selected from the amino acid sequence of any t provided in Table 2.1, 2.2, 2.3, 2.4,
2.5, and/or 6.1, as well as SEQ ID NOS:10, 13, 15, 18, 21, and/or 24, and (b) expressing the GLA
polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid
ce encoded by the polynucleotide can ally have one or l (e.g., up to 3, 4, 5, or up to
) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino
acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23,
1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or
substitutions. In some ments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9,
,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid
residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has
optionally 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,18, 20, 21, 22, 23, 24, or 25 amino acid
residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be
conservative or non-conservative substitutions.
The expressed ered GLA polypeptide can be assessed for any desired improved
property (e.g., ty, selectivity, stability, acid tolerance, protease sensitivity, etc.), using any
suitable assay known in the art, including but not d to the assays and conditions described
herein.
In some embodiments, any of the engineered GLA polypeptides expressed in a host cell are
recovered from the cells and/or the culture medium using any one or more of the well-known
ques for protein purification, including, among others, lysozyme treatment, sonication,
filtration, salting-out, ultra-centrifugation, and chromatography.
tographic techniques for isolation of the GLA polypeptides include, among others,
reverse phase chromatography high performance liquid chromatography, ion exchange
chromatography, hydrophobic interaction chromatography, gel electrophoresis, and y
chromatography. Conditions for purifying a particular enzyme depends, in part, on factors such as net
charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be nt
to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the
improved variant GLA enzymes. In some embodiments utilizing affinity chromatography purification,
any antibody which specifically binds the variant GLA polypeptide finds use. In some embodiments
utilizing affinity chromatography ation, proteins that bind to the glycans covalently attached to
GLA find use. In still other embodiments utilizing affinity-chromatography purifications, any small
molecule that binds to the GLA active site finds use. For the production of antibodies, various host
animals, including but not d to rabbits, mice, rats, etc., are immunized by ion with a GLA
polypeptide (e.g., a GLA variant), or a fragment thereof in some embodiments, the GLA polypeptide
or nt is attached to a suitable carrier, such as BSA, by means of a side chain functional group
or linkers attached to a side chain functional group.
In some embodiments, the engineered GLA polypeptide is produced in a host cell by a
method comprising ing a host cell (e.g., S. cerevisiae, Daucus carota, Nicotiana tabacum, I-I.
sapiens (e. g., HEK293T), or Cricez‘ulus griseus (e.g., (71:10)) comprising a polynucleotide sequence
encoding an engineered GLA polypeptide as described herein under conditions conducive to the
production of the ered GLA polypeptide and ring the engineered GLA polypeptide from
the cells and/or culture medium.
In some embodiments, the invention asses a method of ing an engineered GLA
polypeptide comprising culturing a inant eukaryotic cell comprising a polynucleotide sequence
encoding an engineered GLA polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to reference sequences
SEQ ID NOS:5 and/or 10, and one or more amino acid residue differences as compared to SEQ ID
NO:5 and/or 10, selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, and/or 6.1, and/or
2015/063329
combinations thereof when optimally aligned with the amino acid sequence of SEQ ID NO:5 and/or
, under suitable culture conditions to allow the production of the engineered GLA polypeptide and
optionally recovering the engineered GLA polypeptide from the culture and/or cultured bacterial cells.
In some embodiments, once the engineered GLA polypeptides are recovered from the
recombinant host cells or cell culture medium, they are further d by any suitable method(s)
known in the art. In some additional embodiments, the purified GLA polypeptides are combined with
other ingredients and compounds to provide compositions and formulations comprising the
engineered GLA polypeptide as appropriate for different ations and uses (e.g., pharmaceutical
compositions). In some additional embodiments, the purified GLA polypeptides, or the formulated
GLA polypeptides are lyophilized.
Compositions:
The present invention provides various compositions and formats, including but not limited to
those described below. In some embodiments, the present invention provides ered GLA
polypeptides le for use in pharmaceutical and other compositions, such as dietary/nutritional
supplements.
Depending on the mode of administration, these compositions comprising a therapeutically
effective amount of an engineered GLA according to the ion are in the form of a solid, semi-
solid, or liquid. In some embodiments, the itions include other pharmaceutically acceptable
components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers,
and other ingredients. s on techniques for formulation and administration are well known in the
art and described in the literature.
In some embodiments, the engineered GLA polypeptides are formulated for use in
pharmaceutical compositions. Any suitable format for use in delivering the engineered GLA
polypeptides find use in the present invention, including but not limited to pills, tablets, gel tabs,
capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, s, sprays,
ointments, liniments, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks,
solutions, suspensions (including but not limited to oil-based sions, oil-in water emulsions,
etc.), slurries, syrups, controlled release formulations, suppositories, etc. In some embodiments, the
ered GLA polypeptides are provided in a format suitable for injection or infusion (i.e., in an
inj e ation). In some embodiments, the engineered GLA polypeptides are provided in
biocompatible matrices such as sol-gels, ing -based (e. g., ane) sol-gels. In some
embodiments, the engineered GLA polypeptides are encapsulated. In some alternative embodiments,
the engineered GLA polypeptides are ulated in nanostructures (e.g., nanotubes, nanotubules,
nanocapsules, or microcapsules, microspheres, liposomes, etc.). Indeed, it is not ed that the
present invention be limited to any particular delivery formulation and/or means of delivery. It is
intended that the engineered GLA polypeptides be administered by any suitable means known in the
art, including but not limited to parenteral, oral, topical, transdermal, intranasal, intraocular,
intrathecal, via implants, etc.
In some ments, the engineered GLA polypeptides are chemically modified by
glycosylation, chemical crosslinking reagents, pegylation (i.e., modified with polyethylene glycol
[PEG] or activated PEG, etc.) or other compounds (See e. g., Ikeda, Amino Acids 29:283-287 ;
US Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,53,653; US Pat. Appln. Publ. Nos.
2013/0039898, 177722, etc.). Indeed, it is not intended that the present invention be limited to
any particular delivery method and/or mechanism.
In some onal embodiments, the engineered GLA polypeptides are provided in
formulations comprising matrix-stabilized enzyme crystals. In some embodiments, the formulation
comprises a cross-linked crystalline ered GLA enzyme and a polymer with a ve moiety
that adheres to the enzyme crystals. The present invention also provides engineered GLA
polypeptides in rs.
In some ments, compositions comprising the engineered GLA polypeptides of the
t invention include one or more commonly used carrier compounds, ing but not d to
sugars (e. g., lactose, sucrose, mannitol, and/or sorbitol), starches (e. g., corn, wheat, rice, potato, or
other plant ), cellulose (e. g., methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy-
methylcellulose), gums (e. g., arabic, tragacanth, guar, etc.), and/or proteins (e. g., gelatin, collagen,
etc.).
In some embodiments, the present invention provides engineered GLA polypeptides suitable
for use in decreasing the concentration of glycolipids in fluids such as blood, cerebrospinal fluid, etc.
The dosage of engineered GLA polypeptide(s) administered depends upon the condition or disease,
the general condition of the t, and other factors known to those in the art. In some
embodiments, the compositions are ed for single or multiple administrations. In some
embodiments, it is contemplated that the concentration of engineered GLA polypeptide(s) in the
composition(s) administered to a human with Fabry e is sufficient to effectively treat, and/or
ameliorate disease (e. g., Fabry disease). In some embodiments, the engineered GLA polypeptides are
administered in combination with other pharmaceutical and/or dietary compositions.
EXPERIMENTAL
The following Examples, including experiments and results achieved, are provided for
illustrative purposes only and are not to be construed as limiting the t invention.
In the experimental disclosure below, the following abbreviations apply: ppm (parts per
million); M (molar); mM (millimolar), uM and HM (micromolar); nM (nanomolar); mol (moles); gm
and g (gram); mg (milligrams); ug and ug (micrograms); L and 1 (liter); ml and mL (milliliter); cm
meters); mm (millimeters); um and um (micrometers); sec. (seconds); min(s) (minute(s)); h(s)
and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); °C (degrees
WO 05889 2015/063329
Centigrade); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli
W31 10 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center
[CGSC], New Haven, CT); HPLC (high pressure liquid chromatography); MWCO ular weight
cut-off); SDS-PAGE (sodium l sulfate polyacrylamide gel electrophoresis);
PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl B-D-l-
lactopyranoside); PMBS (polymyXin B sulfate); NADPH (nicotinamide e dinucleotide
phosphate); GIDH (glutamate dehydrogenase); FIOPC (fold improvements over positive control);
PBMC heral blood mononuclear cells); LB (Luria broth); MeOH (methanol); Athens Research
(Athens Research Technology, Athens, GA); ProSpec (ProSpec Tany Technogene, East Brunswick,
NJ); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Ram Scientific (Ram Scientific, Inc., s,
NY); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore pore, Corp., Billerica MA); Difco
(Difco tories, BD Diagnostic Systems, Detroit, MI); Molecular Devices (Molecular Devices,
LLC, Sunnyvale, CA); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Axygen (Axygen, Inc.,
Union City, CA); Toronto Research Chemicals (Toronto Research Chemicals Inc., Toronto, Ontario,
Canada); Cambridge Isotope Laboratories, (Cambridge e Laboratories, Inc., Tewksbury, MA);
Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, NY),
Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (part of Thermo Fisher
Scientific, Waltham, MA); Corning (Corning, Inc., Palo Alto, CA); Megazyme (Megazyme
International, Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, NY); GE Healthcare
(GE Healthcare Bio-Sciences, Piscataway, NJ); Pierce (Pierce Biotechnology (now part of Thermo
Fisher Scientific), rd, IL); LI-COR (LI-COR Biotechnology, Lincoln, NE); Amicus (Amicus
eutics, Cranbury, NJ); PhenomeneX (PhenomeneX, Inc., Torrance, CA); Optimal (Optimal
Biotech Group, Belmont, CA); and d (Bio-Rad Laboratories, Hercules, CA).
The following polynucleotide and polypeptide sequences find use in the present invention. In
some cases (as shown below), the polynucleotide sequence is followed by the encoded polypeptide.
Polynucleotide sequence of full length human GLA cDNA (SEQ ID NO.1):
ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCC
CTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCT
ACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCC
AGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAG
GCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAA
GAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGC
ATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAAT
AAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTT
GCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAAT
TTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTG
TACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATC
CGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATA
AAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCA
2015/063329
GGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAG
CAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACC
TCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCA
ACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTG
TGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATT
GGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAAT
CCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGG
ACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAAT
ACAATGCAGATGTCATTAAAAGACTTACTTTAG (SEQ ID NO: 1)
Polypeptide sequence of full length human GLA:
iMQLRNPELHLGCALALRFLALVSWHHPGARALDNGLARTPTNKRNLHWERFMCNLDCQEEP
DSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLA
‘NYVHSKGLKILHYADVGNKTCAGFPGSFGYYTHDAQTFALRVGVDLLKFDGCYCDSLENLAD
GYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILD
WTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHIS
PQAKALLQDKDVMINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSY
TLAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKD
LL (SEQ ID NO:2)
Polynucleotide sequence of mature yeast optimized (yCDS) human GLA:
AACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
AAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:3)
Polynucleotide sequence of mature human GLA (native hCDS):
CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
2015/063329
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
T (SEQ ID NO:4)
Polypeptide sequence of mature Human GLA (SEQ ID No.5):
RTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF
YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:5)
cleotide sequence of pCKl 10900i E. coli expression :
TCGAGTTAATTAAGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC
ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA
ACAATTTCACACAGGAAACGGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTAC
AATCTAGAGGCCAGCCTGGCCATAAGGAGATATACATATGAGTATTCAACATTTCCGTGT
TATTCCCTTTTCTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG
GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA
TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAGCGTTTTCCAATGATGAG
CACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCA
ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA
AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA
GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC
GTTTTTTTGCACACCATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTG
AATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAAC
GTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA
CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG
GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT
GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA
CTATGGATGAACGTAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGG
GGCCAAACTGGCCACCATCACCATCACCATTAGGGAAGAGCAGATGGGCAAGCTTGACC
TGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTTGAATTCTACGTAAAAAGC
CGCCGATACATCGGCTGCTTTTTTTTTGATAGAGGTTCAAACTTGTGGTATAATGAAATA
AGATCACTCCGGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAA
ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGA
ACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGA
TATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTAT
TCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGG
TGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGA
AACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATA
TTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA
GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTG
ATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGC
GACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCAT
GTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTA
ACTGCAGGAGCTCAAACAGCAGCCTGTATTCAGGCTGCTTTTTTCGTTTTGGTCTGCGCGT
AATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTG
AGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTG
TCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTAC
CAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGT
ATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTG
GAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATA
ACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCC
GCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGA
GCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCG
CGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGT
TCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGA
GGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCT
CCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGAACCACCGCTGGTAGCGGTGG
TTTTTTTAGGCCTATGGCCTTTTTTTTTTGTGGGAAACCTTTCGCGGTATGGTATTAAAGC
GCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTC
GCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCAC
GTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCC
CAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCT
CCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATC
AACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAA
GCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTG
GATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTT
GATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGA
CTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCC
ATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAA
TCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAAC
AAACCATGCAAATGCTGAATGAGGGCATCGTTTCCACTGCGATGCTGGTTGCCAACGATC
AGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGAC
ATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACC
ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTC
TCTCAGGGCCAGGCGGTTAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAA
AACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT
GCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGGTACCCGATAAAA
GCGGCTTCCTGACAGGAGGCCGTTTTGTTTC($flQHDNOfi)
cleotide sequence of pYT-72Bg1 secreted yeast expression vector:
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACMM¥ATATCATA
AduykkAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGA
CTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCT
CTGCATCTTCAATGGCTTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACAT
AGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGC
GGAACCATGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCA
AGGACGCAGATGGCAACAAACCCAAGGAGCCTGGGATAACGGAGGCTTCATCGGAGAT
GATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAAC
TAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACTTTCAATGTAGGGAATTCGTT
CTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTA
TCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCT
TGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCA
TCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTAACAACAACGAAGT
CAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCA
AAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTT
GTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACG
GCATCAGCCTTTTTGGAGGCTTCCAGCGCCTCATTTGGAAGTGGAACACCTGTAGCATCG
ATAGCAGCCCCCCCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCA
GAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCT
GGCAAAACGACGATTTTTTTAGGGGCAGACATTACAATGGTATATCCTTGAAATATATAT
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCAGCTTCTCAATGATATTCGAATAC
GCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTT
GTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATAGTA
TATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATFF
CGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGG
TTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGT
GCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCT
GAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAA
TCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAA
GAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAAC
AAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAA
CAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAA
CTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTT
CCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTG
CATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACT
TTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTT
TCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGA
TTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAA
CATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGT
AGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTT
AGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTT
GTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTT
CTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCC
GAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCG
TGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATG
CGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTA
TCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATG
CTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGA
TCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGA
TGAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACA
TTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATG
TGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTT
TATTGTACGATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGA
GGTCCTATTTACTCGTTTTAGGACTGGTTCAGAATTCCTCAAAAATTCATCCAAA
TATACAAGTGGATCGATGATAAGCTGTCAAACATGAGAATTCTTGAAGACGAAAGGGCC
TACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGG
TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA
AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGG
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC
TTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG
GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC
GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT
TATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG
ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA
GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA
ACGATCGGAGGACCGAAGGAGCTAACCGCTFUjTGCACAACATGGGGGATCATGTAAC
TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA
CCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT
ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA
GCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT
CTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTG
AGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATAC
TTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA
TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT
AffidthGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA
AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC
TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT
AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC
TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT
GATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA
CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG
AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG
GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG
TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG
CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG
CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCG
CCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG
AGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATT
CGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAG
TATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACAC
CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGA
CCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGC
AGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCAT
CCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGG
CCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCT
GTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTG
ATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATG
CGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGT
AGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGC
AGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCAT
GTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATC
GGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGAC
AGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGT
GCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATT
CACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAG
GTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGC
GGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGC
TCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTG
GTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGAC
GCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGG
CATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCG
CCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACG
AAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGT
CGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTC
CTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGC
GCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGGATCTGG
GCAAAACGTAGGGGCAAACAAACGGAAAAATCGTTTCTCAAATTTTCTGATGCCAAGAA
CTCTAACCAGTCTTATCTAAAAATTGCCTTATGATCCGTCTCTCCGGTTACAGCCTGTGTA
ACTGATTAATCCTGCCTTTCTAATCACCATTCTAATGTTTTAATTAAGGGATTTTGTCTTC
WO 05889
ATTAACGGCTTTCGCTCATAAAAATGTTATGACGTTTTGCCCGCAGGCGGGAAACCATCC
ACTTCACGAGACTGATCTCCTCTGCCGGAACACCGGGCATCTCCAACTTATAAGTTGGAG
AAATAAGAGAATTTCAGATTGAGAGAATGAAAAAAAAAAAAAAAAAAAGGCAGAGGAG
AGCATAGAAATGGGGTTCACTTTTTGGTAAAGCTATAGCATGCCTATCACATATAAATAG
AGTGCCAGTAGCGACTTTTTTCACACTCGAAATACTCTTACTACTGCTCTCTTGTTGTTTT
TTCTTGTTTCTTCTTGGTAAATAGAATATCAAGCTACAAAAAGCATACAATCAA
CTATCAACTATTAACTATATCGTAATACACAGGATCCACCATGAAGGCTGCTGCGCTTTC
CTGCCTCTTCGGCAGTACCCTTGCCGTTGCAGGCGCCATTGAATCGAGAAAGGTTCACCA
GAAGCCCCTCGCGAGATCTGAACCTTTTTACCCGTCGCCATGGATGAATCCCAACGCCAT
CGGCTGGGCGGAGGCCTATGCCCAGGCCAAGTCCTTTGTCTCCCAAATGACTCTGCTAGA
GAAGGTCAACTTGACCACGGGAGTCGGCTGGGGGGAGGAGCAGTGCGTCGGCAACGTG
GGCGCGATCCCTCGCCTTGGACTTCGCAGTCTGTGCATGCATGACTCCCCTCTCGGCGTG
CGAGGAACCGACTACAACTCAGCGTTCCCCTCTGGCCAGACCGTTGCTGCTACCTGGGAT
CGCGGTCTGATGTACCGTCGCGGCTACGCAATGGGCCAGGAGGCCAAAGGCAAGGGCAT
CAATGTCCTTCTCGGACCAGTCGCCGGCCCCCTTGGCCGCATGCCCGAGGGCGGTCGTAA
CTGGGAAGGCTTCGCTCCGGATCCCGTCCTTACCGGCATCGGCATGTCCGAGACGATCAA
GGGCATTCAGGATGCTGGCGTCATCGCTTGTGCGAAGCACTTTATTGGAAACGAGCAGG
AGCACTTCAGACAGGTGCCAGAAGCCCAGGGATACGGTTACAACATCAGCGAAACCCTC
TCCTCCAACATTGACGACAAGACCATGCACGAGCTCTACCTTTGGCCGTTTGCCGATGCC
GTCCGGGCCGGCGTCGGCTCTGTCATGTGCTCGTACAACCAGGGCAACAACTCGTACGCC
TGCCAGAACTCGAAGCTGCTGAACGACCTCCTCAAGAACGAGCTTGGGTTTCAGGGCTTC
GTCATGAGCGACTGGTGGGCACAGCACACTGGCGCAGCAAGCGCCGTGGCTGGTCTCGA
TATGTCCATGCCGGGCGACACCATGGTCAACACTGGCGTCAGTTTCTGGGGCGCCAATCT
CACCCTCGCCGTCCTCAACGGCACAGTCCCTGCCTACCGTCTCGACGACATGTGCATGCG
CATCATGGCCGCCCTCTTCAAGGTCACCAAGACCACCGACCTGGAACCGATCAACTTCTC
CTTCTGGACCCGCGACACTTATGGCCCGATCCACTGGGCCGCCAAGCAGGGCTACCAGG
AGATTAATTCCCACGTTGACGTCCGCGCCGACCACGGCAACCTCATCCGGAACATTGCCG
GTACGGTGCTGCTGAAGAATACCGGCTCTCTACCCCTGAACAAGCCAAAGTTC
GTGGCCGTCATCGGCGAGGATGCTGGGCCGAGCCCCAACGGGCCCAACGGCTGCAGCGA
CCGCGGCTGTAACGAAGGCACGCTCGCCATGGGCTGGGGATCCGGCACAGCCAACTATC
CGTACCTCGTTTCCCCCGACGCCGCGCTCCAGGCGCGGGCCATCCAGGACGGCACGAGG
AGCGTCCTGTCCAACTACGCCGAGGAAAATACAAAGGCTCTGGTCTCGCAGGC
CAATGCAACCGCCATCGTCTTCGTCAATGCCGACTCAGGCGAGGGCTACATCAACGTGG
ACGGTAACGAGGGCGACCGTAAGAACCTGACTCTCTGGAACAACGGTGATACTCTGGTC
AAGAACGTCTCGAGCTGGTGCAGCAACACCATCGTCGTCATCCACTCGGTCGGCCCGGTC
CTCCTGACCGATTGGTACGACAACCCCAACATCACGGCCATTCTCTGGGCTGGTCTTCCG
GGCCAGGAGTCGGGCAACTCCATCACCGACGTGCTTTACGGCAAGGTCAACCCCGCCGC
CCGCTCGCCCTTCACTTGGGGCAAGACCCGCGAAAGCTATGGCGCGGACGTCCTGTACA
AGCCGAATAATGGCAATTGGGCGCCCCAACAGGACTTCACCGAGGGCGTCTTCATCGAC
TACTTCGACAAGGTTGACGATGACTCGGTCATCTACGAGTTCGGCCACGGCCTG
AGCTACACCACCTTCGAGTACAGCAACATCCGCGTCGTCAAGTCCAACGTCAGCGAGTA
CCGGCCCACGACGGGCACCACGATTCAGGCCCCGACGTTTGGCAACTTCTCCACCGACCT
CGAGGACTATCTCTTCCCCAAGGACGAGTTCCCCTACATCCCGCAGTACATCTACCCGTA
CCTCAACACGACCGACCCCCGGAGGGCCTCGGGCGATCCCCACTACGGCCAGACCGCCG
AGGAGTTCCTCCCGCCCCACGCCACCGATGACGACCCCCAGCCGCTCCTCCGGTCCTCGG
GCGGAAACTCCCCCGGCGGCAACCGCCAGCTGTACGACATTGTCTACACAATCACGGCC
GACATCACGAATACGGGCTCCGTTGTAGGCGAGGAGGTACCGCAGCTCTACGTCTCGCT
GGGCGGTCCCGAGGATCCCAAGGTGCAGCTGCGCGACTTTGACAGGATGCGGATCGAAC
CCGGCGAGACGAGGCAGTTCACCGGCCGCCTGACGCGCAGAGATCTGAGCAACTGGGAC
GTCACGGTGCAGGACTGGGTCATCAGCAGGTATCCCAAGACGGCATATGTTGGGAGGAG
CAGCCGGAAGTTGGATCTCAAGATTGAGCTTCCTTGATAAGTCGACCTCGACTTTGTTCC
CACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGT
TTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATAT
AGCTATTCACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATT
TCAATTTGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGA
ATCGAATCCTAAGAGAATTGGATCTGATCCACAGGACGGGTGTGGTCGCCATGATCGCG
TAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTC
GGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCA
GCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGC
AGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGAC
GATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGA
TAAACTACCGCATTAAAGCTTTTTCTTTCCAATTTTTTTTTTTTCGTCATTATAAAAATCAT
TACGACCGAGATTCCCGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGT
TTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGGCCGCATCTTCTCAA
ATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCCCTTCCCTTTG
CAAATAGTCCTCTTCCAACAATAATAATGTCAGATCCTGTAGAGACCACATCATCCACGG
TTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAAACCCACACCGGGTGTCATAAT
CAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAAC
AAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAG
GGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCT
TCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCCCACCACACCGTGTGCATTCGTA
ATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGAGTACTGCAATTTGACTGTATTAC
CAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAAATTGTACTTGGCGGATAATGCCT
GCTTAACTGTGCCCTCCATGGAAAAATCAGTCAAGATATCCACATGTGTTTTTA
GTAAACAAATTTTGGGACCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAA
CATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTGCATGATATTAAATAGCTTGGCAG
CAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATATGTAGCTTTCGACATGATTTATC
TTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGT
TTCTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCT
TGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAA
AGAATAAAAAAAAAATGATGAATTGAAAAGCTTATCGATCCTACCCCTTGCGCTAAAGA
AGTATATGTGCCTACTAACGCTTGTCTTTGTCTCTGTCACTAAACACTGGATTATTACTCC
CAGATACTTATTTTGGACTAATTTAAATGATTTCGGATCAACGTTCTTAATATCGCTGAAT
CTTCCACAATTGATGAAAGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTA
AATCTCGAAGTATACTCAAACGAATTTAGTATTTTCTCAGTGATCTCCCAGATGCTTTCAC
CCTCACTTAGAAGTGCTTTAAGCATTTTTTTACTGTGGCTATTTCCCTTATCTGCTTCTTCC
GATGATTCGAACTGTAATTGCAAACTACTTACAATATCAGTGATATCAGATTGATGTTTT
TGTCCATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAATCAATTTCTTTAATGAGGCT
TCCAGAATTGTTGCTTTTTGCGTCTTGTATTTAAACTGGAGTGATTTATTGACAATATCGA
GCGAATTGCTTATGATAGTATTATAGCTCATGAATGTGGCTCTCTTGATTGCTGT
TCCGTTATGTGTAATCATCCAACATAAATAGGTTAGTTCAGCAGCACATAATGCTATTTT
CTCACCTGAAGGTCTTTCAAACCTTTCCACAAACTGACGAACAAGCACCTTAGGTGGTGT
TTTACATAATATATCAAATTGTGGCATGCTTAGCGCCGATCTTGTGTGCAATTGATATCTA
ACTACTCTATTTATCTTGTATCTTGCAGTATTCAAACACGCTAACTCGAAAAACT
AACTTTAATTGTCCTGTTTGTCTCGCGTTCTTTCGAAAAATGCACCGGCCGCGCATTATTT
GTACTGCGAAAATAATTGGTACTGCGGTATCTTCATTTCATATTTTAAAAATGCACCTTTG
CTGCTTTTCCTTAATTTTTAGACGGCCCGCAGGTTCGTTTTGCGGTACTATCTTGTGATAA
AAAGTTGTTTTGACATGTGATCTGCACAGATTTTATAATGTAATAAGCAAGAATACATTA
GAACAATACTGGTAAAAGAAAACCAAAATGGACGACATTGAAACAGCCAAGA
ATCTGACGGTAAAAGCACGTACAGCTTATAGCGTCTGGGATGTATGTCGGCTGTTTATTG
AAATGATTGCTCCTGATGTAGATATTGATATAGAGAGTAAACGTAAGTCTGATGAGCTAC
TCTTTCCAGGATATGTCATAAGGCCCATGGAATCTCTCACAACCGGTAGGCCGTATGGTC
TTGATTCTAGCGCAGAAGATTCCAGCGTATCTTCTGACTCCAGTGCTGAGGTAATTTTGC
CTGCTGCGAAGATGGTTAAGGAAAGGTTTGATTCGATTGGAAATGGTATGCTCTCTTCAC
AAGAAGCAAGTCAGGCTGCCATAGATTTGATGCTACAGAATAACAAGCTGTTAGACAAT
AGAAAGCAACTATACAAATCTATTGCTATAATAATAGGAAGATTGCCCGAGAAAGACAA
GAAGAGAGCTACCGAAATGCTCATGAGAAAAATGGATTGTACACAGTTATTAGTCCCAC
CAGCTCCAACGGAAGAAGATGTTATGAAGCTCGTAAGCGTCGTTACCCAATTGCTTACTT
TAGTTCCACCAGATCGTCAAGCTGCTTTAATAGGTGATTTATTCATCCCGGAATCTCTAA
AGGATATATTCAATAGTTTCAATGAACTGGCGGCAGAGAATCGTTTACAGCAAAAAAAG
AGTGAGTTGGAAGGAAGGACTGAAGTGAACCATGCTAATACAAATGAAGAAGTTCCCTC
CAGGCGAACAAGAAGTAGAGACACAAATGCAAGAGGAGCATATAAATTACAAAACACC
ATCACTGAGGGCCCTAAAGCGGTTCCCACGAAAAAAAGGAGAGTAGCAACGAGGGTAA
GGGGCAGAAAATCACGTAATACTTCTAGGGTATGATCCAATATCAAAGGAAATGATAGC
ATTGAAGGATGAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTA
GTGCTGAAGGAAGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTA
GACTACCTTTCATCCTACATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGC
ACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACG
TGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGA
AACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAG
CGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCG
GACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTA
TCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCC
TTGCATCTAAACTCGACCTCTACATCAACAGGCTTCCAATGCTCTTCAAATTTTA
CTGTCAAGTAGACCCATACGGCTGTAATATGCTGCTCTTCATAATGTAAGCTTATCTTTAT
CGAATCGTGTGAAAAACTACTACCGCGATAAACCTTTACGGTTCCCTGAGATTGAATTAG
TTCCTTTAGTATATGATACAAGACACTTTTGAACTTTGTACGACGAATTTTGAGGTTCGCC
ATCCTCTGGCTATTTCCAATTATCCTGTCGGCTATTATCTCCGCCTCAGTTTGATCTTCCGC
TTCAGACTGCCATTTTTCACATAATGAATCTATTTCACCCCACAATCCTTCATCCGCCTCC
GCATCTTGTTCCGTTAAACTATTGACTTCATGTTGTACATTGTTTAGTTCACGAGAAGGGT
CCTCTTCAGGCGGTAGCTCCTGATCTCCTATATGACCTTTATCCTGTTCTCTTTCCACAAA
CTTAGAAATGTATTCATGAATTATGGAGCACCTAATAACATTCTTCAAGGCGGAGAAGTT
TGGGCCAGATGCCCAATATGCTTGACATGAAAACGTGAGAATGAATTTAGTATTATTGTG
ATATTCTGAGGCAATTTTATTATAATCTCGAAGATAAGAGAAGAATGCAGTGACCTTTGT
ATTGACAAATGGAGATTCCATGTATCTAAAAAATACGCCTTTAGGCCTTCTGATACCCTT
TCCCCTGCGGTTTAGCGTGCCTTTTACATTAATATCTAAACCCTCTCCGATGGTGGCCTTT
CTAATAAATGCAACCGATATAAACTGTGATAATTCTGGGTGATTTATGATTCGA
TCGACAATTGTATTGTACACTAGTGCAGGATCAGGCCAATCCAGTTCTTTTTCAATTACC
GGTGTGTCGTCTGTATTCAGTACATGTCCAACAAATGCAAATGCTAACGTTTTGTATTTCT
TATAATTGTCAGGAACTGGAAAAGTCCCCCTTGTCGTCTCGATTACACACCTACTTTCATC
GTACACCATAGGTTGGAAGTGCTGCATAATACATTGCTTAATACAAGCAAGCAGTCTCTC
GCCATTCATATTTCAGTTATTTTCCATTACAGCTGATGTCATTGTATATCAGCGCTGTAAA
AATCTATCTGTTACAGAAGGTTTTCGCGGTTTTTATAAACAAAACTTTCGTTACGAAATC
GAGCAATCACCCCAGCTGCGTATTTGGAAATTCGGGAAAAAGTAGAGCAACGCGAGTTG
CATTTTTTACACCATAATGCATGATTAACTTCGAGAAGGGATTAAGGCTAATTTCACTAG
TCAAAAACCTCAATCTGTCCATTGAATGCCTTATAAAACAGCTATAGATTGCAT
AGAAGAGTTAGCTACTCAATGCTTTTTGTCAAAGCTTACTGATGATGATGTGTCTACTTTC
AGGCGGGTCTGTAGTAAGGAGAATGACATTATAAAGCTGGCACTTAGAATTCCACGGAC
TATAGACTATACTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCC
GAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTG
ATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAA
AAGGCACTTCTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCA (SEQ
ID NO:7)
Polynucleotide sequence of Variant N0. 73 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
AGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:8)
Polynucleotide sequence of Variant N0. 73:
CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
GACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
T (SEQ ID NO:9)
Polypeptide sequence of Variant N0. 73:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF
YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:10)
Polynucleotide sequence of Variant N0. 218 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
TGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
ATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGTTGGGTTTCTATAACTGGACATCTAGGCTAAAAAGTCACATTAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
@EQHDNOAD
Polynucleotide sequence of Variant N0. 218 hCDS:
CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTGTAGCTATTATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAAAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
'rmEQHDNOAm
Polypeptide sequence of Variant N0. 218:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
PQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
2015/063329
LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAIINRQEIGGPRSYTLAVASLGKGVACNPACFITQLLPVKRKLGFY
NWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:13)
Polynucleotide sequence of Variant N0. 326 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
GACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT
GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT
ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA
ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG
CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG
CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT
TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC
TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
@EQHDNOAQ
Polypeptide sequence of Variant N0. 326:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI
DDCWHAAPQRDSEGRLQADPQRFPHIHRQLANYVHSKGLKLGTLADVGNKTCAGFPGSFGYY
IMDAQTFADWKRIHLKFDGCYCDSLENLADGYKHMSLALNRTGR$VYSCEWTLYMWWFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF
GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK
GDNFEVWERPLa31AWAVAHNRQEKKWRSYTmVAsLGKGVACNPACFHQLLPVKRQLGF
YNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:15)
Polynucleotide sequence of t N0. 206 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
TGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
ATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGTTGGGTTTCTATAATTGGACCTCTAGGCTAAGAAGTCACATCAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:16)
Polynucleotide sequence of Variant N0. 206 hCDS:
AATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
CTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
AATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAGAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
T (SEQ ID NO:17)
Polypeptide sequence of Variant N0. 206:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF
YNWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:18)
Polynucleotide sequence of Variant N0. 205 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
ATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
CCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGAGAAAGTTGGGTTTCTATGATTGGGACTCTAGGCTAAGAAGTCACATCAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:19)
Polynucleotide sequence of t N0. 205 hCDS:
CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
CTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATGATTGGGATTCAAGGTTAAGAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
T (SEQ ID NO:20)
Polypeptide sequence of Variant N0. 205:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
LSWNQQVTQMALVflUMAAPLHMSNDLRHEPQAKALLQDKDVDUNQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF
YDWDSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID N021)
Polynucleotide sequence of Variant N0. 76 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
TGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGAGGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG
GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA
CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA
TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC
TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC
CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT
AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT
GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID N022)
Polynucleotide sequence of t N0. 76 hCDS:
CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT
GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG
AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC
ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC
TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA
GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG
ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA
TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC
CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC
CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC
TGATGATTCCTGGCGTAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA
GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA
TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA
TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT
GGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC
GACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG
GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT
GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT
GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC
CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT
T (SEQ ID NO:23)
Polypeptide sequence of Variant N0. 76:
RTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG
LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG
DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF
YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID N024)
Polynucleotide sequence of Mfalpha signal peptide:
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT (SEQ
ID N025)
Polypeptide sequence of Mfalpha signal peptide:
MRFPSIFTAVLFAASSALA (SEQ ID NO:26)
Polynucleotide sequence of MMO435:
ttaactatatcgtaatacacaggatccaccATGAGATTTCCTTCAATTTTTACTG (SEQ ID NO:27)
Polynucleotide ce of MMO439:
AGTAGGTGTACGGGCTAACCCGTTATCCAAAGCTAATGCGGAGGATGC (SEQ ID NO:28)
Polynucleotide sequence of MM0514:
TGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGGATAACGGGTTAGCCCG
(SEQ ID N029)
Polynucleotide sequence of MMO481:
GAGCTAAAAGTACAGTGGGAACAAAGTCGAGGTCGACTTATAACAAATCTTTCAAAGAC
A (SEQ ID NO:30)
Polynucleotide ce of Synthetic mammalian signal peptide:
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCC (SEQ
ID NO:31)
Polynucleotide sequence of LAKE FW:
CGATCGAAGCTTCGCCACCA (SEQ ID No.32)
Polynucleotide sequence of Br reverse:
CTTGCCAATCCATTGTCCAGGGAGTGGACACCAGTCGTTA (SEQ ID NO:33)
Polynucleotide sequence of Br FW:
TAACGACTGGTGTCCACTCCCTGGACAATGGATTGGCAAG (SEQ ID NO:34)
Polynucleotide sequence othLA RV:
CGATCGGCGGCCGCTCAAAGTAAGTCTTTTAATGACA (SEQ ID NO:35)
Polynucleotide sequence of SP-GLA (yCDS):
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGG
ATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTA
ATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATG
GCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGA
TGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGA
GATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTA
GGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTAC
TATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGA
TGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTA
AACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTT
CAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGA
CATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGA
AAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGG
GAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGC
CGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACT
TCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATT
GAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTG
TTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCT
CTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAA
GTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGG
TACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA (SEQ
ID NO:36)
Polynucleotide Sequence 0fMF1eader—GLA(yCDS):
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC
CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT
TACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT
AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA
TCTTTGGATAAAAGATTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCAC
TGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGA
GAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTT
ATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGT
TACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACA
GCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCC
CAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATT
TGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAAC
ACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCT
TGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATT
GGCGTAACTTTGCTGACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGA
CTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTG
ATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTT
CGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCC
AAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTA
AACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCT
GGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTA
CACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTAC
ACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAG
TCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTT
GAAAGATTTGTTA (SEQ ID NO:37)
ptide Sequence of MFleader:
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLL
FINTTIASIAAKEEGVSLDKR (SEQ ID N038)
Polynucleotide sequence of Variant N0. 395 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
TGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT
GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT
ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA
ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG
CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG
CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT
TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC
TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:39)
Polypeptide sequence of Variant N0. 395:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY
FADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF
GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK
WERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF
YNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:40)
Polynucleotide sequence of Variant N0. 402 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT
GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT
ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA
AAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG
CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG
CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT
ACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC
TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA
(SEQ ID NO:41)
Polypeptide sequence of Variant N0. 402:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI
PQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF
GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK
GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF
YNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO:42)
Polynucleotide sequence of Variant N0. 625 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA
GATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
ATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG
GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT
GGCCGCACCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTT
ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA
ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG
CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG
CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT
TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC
TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAAAGATTTGTTA
(SEQ ID NO:43)
Polypeptide sequence of Variant N0. 625:
LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVTEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF
GLSWDQQVTQMALWAIMAAPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRK
GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF
YNWTSRLKSHINPTGTVLLQLENTMQTSLKDLL (SEQ ID NO:44)
Polynucleotide sequence of Variant N0. 648 yCDS:
TTGGATAACGGGTTAGCCCGTACACCTCCGATGGGTTGGCTTCACTGGGAAAGATTCATG
TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCGAAGA
GATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA
TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC
AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG
TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT
TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT
GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT
CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG
TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT
GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG
ATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA
GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT
GGCCGGCCCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTT
ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA
ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG
CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG
TGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT
TAAGAGACAATTGGGTTTCTATAACGCAACCTCTAGGCTAAAAAGTCACATTAATCCTAC
TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAAAGATTTGTTA
(SEQ ID NO:45)
Polypeptide ce of Variant N0. 648:
LDNGLARTPPMGWLHWERFMCNLDCQEEPDSCISEKLFEEMAERMVTEGWKDAGYEYLCI
DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY
DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ
KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF
GLSWDQQVTQMALWAIMAGPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRK
GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF
YNATSRLKSHINPTGTVLLQLENTMQTSLKDLL (SEQ ID NO:46)
EXAMPLE 1
GLA Gene Acquisition and Construction of Expression Vectors
A synthetic gene coding for a WT human GLA was designed for optimized gene expression
in Saccharomyces siae (SEQ ID NO:3) and subcloned into the E. coli expression
, assembled,
vector pCKlOO900i (SEQ ID NO:6).
A ic GLA expression construct encoding a 19 amino acid S. cerevisae MFalpha signal
peptide fused to the mature form of yeast-optimized GLA was generated in a yeast expression vector
designed for secreted sion, as follows. A fragment coding for the MFalpha signal e (SEQ
ID NO:25) was amplified by PCR using the oligonucleotides MMO435 (SEQ ID NO:27)and
MMO439 (SEQ ID NO:28) from S288C genomic DNA, and a fragment coding for a synthetic GLA
(SEQ ID NO:3) was amplified using primers MM0514 (SEQ ID N029) and MMO48l (SEQ ID
NO:30). onal sequence at the 5’ ends of these oligonucleotides provide homology for yeast
recombination g when cotransformed with linearized plasmid pYT-72Bgl (SEQ ID NO:7). In
the resulting vector, the expression of fusion n SP-GLA (SEQ ID NO:36) is driven by the
ADH2 promoter. A fusion construct encoding a fusion of an 83 amino acid MFalpha leader e
(SEQ ID NO:38) N—terminally fused to GLA (SEQ ID NO:37) was cloned using the same techniques.
Recombination cloning and gene expression were performed in S. cerevisiae strain INVScl. Directed
evolution techniques generally known by those skilled in the art were used to generate ies of
gene variants from this plasmid construct (See e.g., US Pat. No. 8,383,346 and W02010/144103).
A chimeric GLA expression construct encoding a synthetic signal peptide fused to a synthetic
gene coding for the mature human GLA coding sequence for secreted expression in transient
transfections was generated as follows. Oligonucleotides PLEVl l3Fw (SEQ ID NO:32) and
SPGLARV (SEQ ID NO:33) were used to amplify a fragment coding for a tic signal peptide
(SEQ ID NO:3 1) using PCR. A second fragment coding for the native human coding sequence for the
mature form of GLA (SEQ ID NO:4) was amplified using oligonucleotides w (SEQ ID
NO:34) and GLARv (SEQ ID NO:35). ng by Overlap Extension PCR was used to recombine
these fragments, and the resulting chimeric nt was ligated into the HindIII/Not I linearized
mammalian expression vector pLEVl l3. Directed evolution techniques generally known by those
skilled in the art were used to generate specific gene ts from this plasmid construct.
EXAMPLE 2
High-Throughput Growth and Assays
High-Throughput [HTP] Growth of GLA and GLA Variants
Yeast (INVScl) cells transformed with vectors sing GLA and GLA variants using the
lithium acetate method were selected on SD-Ura agar plates. After 72 h incubation at 30 °C colonies
were placed into the wells of Axygen® 1.1 ml 96-well deep well plates filled with 200 ul/well SD-Ura
broth (2 g/L SD-Ura, 6.8 g/L yeast nitrogen base without amino acids [Sigma h]), 3.06 g/L
sodium dihydrogen ate, 0.804 g/L disodium hydrogen phosphate, pH 6.0 supplemented with
6% glucose. The cells were allowed to grow for 20-24 hours in a Kuhner shaker (250 rpm, 30 °C, and
85% relative humidity). Overnight culture s (20 [LL) were erred into Corning ® 96-
well deep plates filled with 380[LL of SD-ura broth supplemented with 2% glucose. The plates were
incubated for 66-84 h in a Kuhner shaker (250 rpm, 30 °C, and 85% ve humidity). The cells
were then pelleted (4000 rpm X 20 min), and the supematants isolated and stored at 4 °C prior to
analysis.
HTP-Analysis of Supernatants
GLA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl 0L-
D-galactopyranoside (MUGal). For this assay, 5-50 [LL of yeast culture supernatant produced as
described above, was mixed with 0-45 [LL of Mcllvaine Buffer (Mcllvaine, J. Biol. Chem., 49:183-
186 [1921]), pH 4.8 and 50 [LL of2 mM MUGal in 50 mM citrate, 200 mM KCl, pH 4.6 in a 96-well,
black, opaque bottom plate. The reactions were mixed briefly and incubated at 37 °C for 30-180
minutes, prior to quenching with 100 [LL of l M sodium carbonate. Hydrolysis was analyzed using a
SpectraMax® M2 microplate reader monitoring cence (Ex. 355 nm, Em. 448 nm).
HTP-Analysis of Supernatants Pretreated with Acid
GLA variants were challenged with acidic buffer to simulate the extreme pHs that the variants
may encounter in lysosomes. First, 50 [LL of yeast culture supernatant and 50 uL of Mcllvaine buffer
(pH 3.3-4.3) were added to the wells of a 96-well round bottom plate. The plates were sealed with a
PlateLoc Thermal Microplate Sealer (Agilent) and ted at 37 °C for 1-3 h. For the assay, 10-50
[LL of acid-pH-challenged sample was mixed with 0-40 [LL of Mcllvaine buffer pH 4.8, 25 [LL of l M
e buffer pH 4.3 and 25 [LL of 4 mM MUGal in Mcllvaine buffer pH 4.8. The reactions were
mixed briefly and incubated at 37 °C for 30-] 80 minutes, prior to quenching with 100 [LL of l M
sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring
fluorescence (Ex. 355 nm, Em. 448 nm).
HTP-Analysis of atants Pretreated with Base
GLA ts were challenged with basic (neutral) buffer to simulate the pHs that the variants
encounter in the blood following their administration to a patient. First, 50 [LL of yeast culture
supernatant and 50 uL of ine buffer (pH 7.0-8.2) or 200 mM sodium bicarbonate (pH 9.1-9.7)
were added to the wells of a 96-well round bottom plate. The plates were sealed and incubated at 37
°C for 1-18 h. For the assay, 10-50 [LL of basic-pH-challenged sample was mixed with 0-40 [LL of
Mcllvaine buffer pH 4.8, 25 [LL of l M citrate buffer pH 4.3 and 25 [LL of 4 mM MUGal in Mcllvaine
buffer pH 4.8. The reactions were mixed briefly and incubated at 37 °C for 30-180 minutes, prior to
quenching with 100 uL of l M sodium carbonate. Hydrolysis was analyzed using a aMax® M2
late reader monitoring fluorescence (EX. 355 nm, Em. 448 nm).
HTP-Analysis 0f Supernatants Pretreated with Bovine Serum
GLA variants were challenged with bovine serum to simulate the conditions the variants
encounter ing infusion into a patient. First, 20 uL of yeast culture supernatant and 80 uL of
bovine serum were added to the wells of a 96-well round bottom plate. The plates were sealed and
incubated at 37 °C for l h. For the assay, 50 uL of serum-challenged sample was mixed with 25 uL
of l M citrate buffer pH 4.3 and 25 uL of 4 mM MUGal in ine buffer pH 4.8. The reactions
were mixed briefly and incubated at 37 °C for 180 minutes, prior to quenching with 100 uL of 1 M
sodium ate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring
fluorescence (EX. 355 nm, Em. 448 nm).
Table 2.1 Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated le’2
Variant pH pH ID
# NC 4.3 7.0 Amino Acid Differences Relative to SEQ ID N0:5 N0:
1 A337S 47
2 E43D 48
4 E43D/E48D/I208V/N247D/Q2991UQ302K/R373K/I376V 50
: : l l E43D/E48D/I208V/R373K 51
6 E43D/E48D/I208V/R373K/I376V 52
-_: : E43D/E48D/N247D/Q302K/R373K 54
_—55
12 —— ++ 58
14 E43D/R373K/I376V 6O
16 : : E48D/R373K/I376V 62
18 F217S 64
19 == 65
21 I208V/N247D/R373K/I376V 67
22 l 1208V/Q2991UI376V 68
23 I208V/Q302K/R373K/I376V 69
Lb] ‘7’
Table 2.1 Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated le’2
Variant ID
# . Amino Acid Differences Relative to SEQ ID N0:5 N0:
Q299IUM322V/R373K 73
Q302K/I376V 76
R373K 77
R373K/I376V 78
1. Relative activity was calculated as activity of the variant/activity of WT GLA (SEQ ID NO:5
(encoded by SEQ ID NO:3).
2. -- = 0.5 to 1.5 relative ty over WT GLA (SEQ ID NO:5);
— >15 to 2.5 relative activity over WT GLA (SEQ ID NO:5); and
i — >2.5 relative activity over WT GLA (SEQ ID NO:5).
Table 2.2 - Relative Activity of GLA ts After N0 Challenge (NC)
0r Challenge at the Indicated le’z’3
t pH w:m ID
# NC 4.2 7.1 Amino Acid Differences Relative to SEQ ID N0:5 NO :
33 + + A199H/E367S
34 A337P OO\l 00
A339S 00 p—A
36 A350G
37 D105A 00 U)
38 D105S oo 4;
D124N/E147G/N161K/R162Q/T163V/R165A/Il67S/V168
U) 0 I/Y169V/S170-/M177S/F217E 00 U]
-— D396R
D396T 00 \l
E367N 00 00
E367T
E387K
E387Q
Table 2.2 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated le’z’3
Variant SEQ
NC Amino Acid Differences ve to SEQ ID NO: 5
_F217D
U1 U) -_ F217R
U1 U1 F352V/F3651 ._1._1 OO l—‘O
1—1 0N
U1 \l F365K
1—1 0 U]
01O )—k 0 O1
01 )—k
G303Q/R373V 1—1 O 00
O\ U)
0101 U1-l> H155L 1—1 ’—‘ O
-_ 1—1 1—1 1—1
01 \l -- ++
-_ll 02L ._1._1._1._1 ._1._1._1._1 N
\lO -_ ll 02L/L394V 1—1 1—1 01
71 I-
72 ll67V
\l U)
\l .5 K206M ._1._1._1._1 1—101—11—1 0OO\]
\] U] 1—1 0
[\D #N \l O\ K206R
\l \l 1—1 N 1—1
\l 00 I- ._1._1 [\D[\J LAN \l0 K343G
00 O
00 1—1 K362R 1—1 N U]
00 [\D
00 U) K36E )—k 5) \l
oo .5 -_
00 U1 -_ ._1._1 NN 000
-_ 1—1 L») O
00 \1 -_
00 00 -_ K3958
K395T ._1._1._1 WWW UJNH
0O K961 )—k L» .5
01—1 I- K96L
2015/063329
Table 2.2 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated le’z’3
t SEQ
HI:U) NC Amino Acid Differences Relative to SEQ ID NO: 5
K96R/L397V
0U1 L158A ._1._1._1 WWW 000“
0\l L158M
0 00
L316E ._1 J> O\
L386V ._1 J> 00
)—k .5 0
L397* ._1._1._1._1 U1U1U1U1 UJNl—‘O
L397D b—k U1 .5
U1 U1
L3971 ._1._1 U1 0
L397R ._1 U1 00
L398E
L398Q ._1._1._1._1 0000 UJNb—to
L44R/L3 84F ._1 O\ U1
VIZOD/Q302K )—k 0 \l
._1._1 00 000
)—k \l O
\/[39ZG ._1._1._1 \l\l\l UJNH
VB92F )—k \l .5
V1392$ 1—1 \1 U]
Table 2.2 - Relative Activity of GLA Variants After N0 nge (NC)
0r Challenge at the Indicated le’z’3
Variant SEQ
NC Amino Acid Differences Relative to SEQ ID NO: 5
_M39Y 176
-_N388R
Q19OS/T369D ._1 \l\l\l 000“
Q302A
Q8OA ._1 00 O\
Q8OV ._1 00 00
-_ p—A 00 0
-_Q88S ._1._1._1._1 @000 WNl—‘O
)—k 0 4;
I-R221K/A350G b—kb—A 00 0U}
R3011/K362T ._1 O 00
R3718
I-R87P/L398R NNNN 0000 WNl—‘O
Sl66H [\JO U1
S31D [\JO \l
—_ NN CO 000
-_ N p—A O
-_S374M
S374T NNN ._1._1._1 WNb—A
I-S393E N )—k 4;
S393G
Table 2.2 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated le’z’3
CI)HU(c:
Variant pH ’Um p—1
# NC 4.2 7.1 Amino Acid Differences Relative to SEQ ID N0:5 N0:
172 - S393H [\D ._i O\
173 S393P
174 I S471
175 S47R NNN ._l 000“
176 S47T [\D [\D O
177 S95D [\D [\D ._l
185 - [\D [\D 0
186 - U) O
187 - U) ._l
188 - U) N
189 - U) U)
190 || + NL» 4;
191 - [\D U) U]
1. ve activity was ated as activity of the variant/activity of WT GLA (SEQ ID NO:5
(encoded by SEQ ID NO:3).
2. Variant # 73 (Rd2BB) has the polynucleotide sequence of SEQ ID NO:8 and polypeptide
sequence of SEQ ID N0:10.
3. =
- <O.5 relative activity to WT GLA (SEQ ID NO:5);
-- = 0.5 to 1.5 relative activity over WT GLA (SEQ ID NO:5);
— >15 to 2.5 relative activity over WT GLA (SEQ ID NO:5); and
i — >2.5 relative activity over WT GLA (SEQ ID NO:5).
Table 2.3 - Relative Activity of GLA ts After N0 Challenge (NC)
0r Challenge at the Indicated pH
Variant pH pH Amino Acid Differences Relative to 81%?)
# NC 4.2 7.6 SEQ ID N0:10
—-U) 0
197 : : A350G/K362Q/T369A 24 1
198 : : A35OG/T369D 242
Table 2.3 - Relative ty of GLA Variants After N0 nge (NC)
0r Challenge at the Indicated pH
Variant in:NE «asam Amino Acid ences Relative to
SEQ ID NO: 10
—A350G/T369S [\D35
C143A
C143T
C59A
203 : : E367A/T369D
E367D
205 : : E367D/T369D
E367N »—n[\.) 00...
E367N/R373K 249
E367N/R373K/I376V [\J U1 O
E367P/T369D [\J U] ._l
F365L/E367N
—F365L/E367N/I376V iiUIUILAN
F365L/E367N/R373K/I376V [\D U] .b
H15Q/ [\D U1 U1
K343D/F365L/E367N
K343G
K343G/F365L/E367N/R373K
L316D
M322I/E367N/R373K p—A L»)
M322I/R373K
M322V/R373K/I376V ii00l—‘O
M3901
P228Q/T369D
Q302K/A337P/A350G/K362Q
Q302K/M322V/E367N
R1658
R221T/F365L [\J O\ \l
R325H
R373K
R373K/I376V iN\l\ll—‘O
[\D \l [\D
\l U)
1. ve activity was calculated as activity of the variant/activity 0f Rd2BB (SEQ ID NO:10)
2. Variant # 218 (Rd3BB) has the polynucleotide sequence of SEQ ID NO:11 and polypeptide
sequence of SEQ ID NO:13.
3. =
- <O.5 relative activity to Rd2BB (SEQ ID NO:10);
-- = 0.5 to 1.5 relative activity over Rd2BB (SEQ ID NO:10);
— >15 to 2.5 relative activity over Rd2BB (SEQ ID NO:10); and
i — >2.5 relative activity over Rd2BB (SEQ ID NO:10).
Table 2.4 - Relative Activity of GLA Variants After N0 Challenge (NC) 0r
Challenge at the Indicated pH 0r Conditionl’
Varian Amino Acid ences Relative to
t# 5 SEQ ID N0:5 (WT GLA)
K206A/F2 l 7R/N247D/L3 l6D/A35OG/E367D/T369D 274
K206A/F2l7R/N247D/Q302K/A35OG/E367D/T369D 2
K206A/F2 l 7R/N247D/Q302K/L3 l 6D/A337P/A350G
/E367D/T369D [\J
K206A/F2l7lUQ302K/E367D/T369D [\J \l \l
K206A/F2 l 71VQ302K/L3 37P/A350G/E367D
/T369D
K206A/I208V/M322V/K343G/F365L/R373K/I376V i'\l\]000
K206A/I208V/R22lK/N247D/M322I/K343D/F365L/
R373K/I376V 280
K206A/L269I/P349L/R373K
K206A/N247D/M322V/K343D/R373K/I376V 282
K206A/N247D/M322V/K343G/F365L/R373K [\D 00 U)
K206A/N247D/Q302K/A337P/K343G/A350G 28
K206A/N247D/Q3O2K/L3 l 6D/A35OG
K206A/N247D/Q302K/M322V/F365L/R373K/I376V NM 0000 GUI-b
Q302K/L3l6D/A337P [\D 00 \]
K96I/K206A/F217R/N247D/Q302K/L316D/A337P/E -
367D/T369D 294
L100F/K206A 295
K206A/I208V/R221K/N247D/Q302K/M322I/
K343D/F365L/I376V 296
L1OOF/K206A/I208V/R22lK/N247D/Q302K/M322V -
/K343D/F365L/I376V 297
L1OOF/K206A/I208V/R221T/N247D/K343D/F365L/I -
376V 298
L1OOF/K206A/I208V/R221T/Q302K/M322I/K343D/I
376V
L1OOF/K206A/M322V/F365L/R373K/I376V
260 --
__ K206A/N247D/F365L/R373K/I376V 301
261 ++ ++ ++ —— L1OOF/K206A/N247D/M322V/K343D/I376V 302
Table 2.4 - Relative Activity of GLA Variants After N0 Challenge (NC) or
Challenge at the Indicated pH or Conditionl’
Varian Seru Amino Ac1d Differences Relative to
H 41: . m SEQ ID N0:5 (WT GLA)
LlOOF/K206A/R22lK/N247D/Q302K/M322V/F365L
/R373K/l376V BEL»
Ll OOF/K206A/R22 l D/Q302K/M322V/l376V 304
L1 OOF/K206A/R221K/N247D/Q3OZK/M322V/K343
D/R373K/l376V U) O U]
LlOOF/K206A/R221K/R373K/l376V 3 O6
LlOOF/K206A/R22lT/M3221/K343E/F365L/R373K 3 O7
LlOOF/K206A/R22lT/N247D/Q302K/K343D/F365L/
R373K 8
LlOOF/K206A/R373K/l376V WU) OO C
206A/R221K/N247D/M3221/R373K 310
L44R/Cl43Y/K206A/A337P/A350G U) ’—‘ ’—‘
L44R/El 87G/K206A/A337P/A350G
L441UK206A WW ._l._l LAN
L44R/K206A/E367D/T369D
L441UK206A/F2171UA350G
++ ++ L441UK206A/F217R/N247D/A337P WWW ._l._l._l GUI-P
L44R/K206A/F217R/N247D/L316D/A337P/A350G/
E367D/T369D L») p—A \]
L441UK206A/F217R/N247D/L316D/A337P/E367D/T
369D U) ._i 00
L44R/K206A/F217R/N247D/L316D/A350G/E367D/
T369D p—A 0
L44R/K206A/F217R/N247D/Q302K/A350G 320
L44R/K206A/F2171VQ302K/E367D/T369D U) l—l
L441VK206A/1208V/R221K/M322V/K343D/F365L/
R373K U) [\J
L441UK206A/N247D/A337P U) L»)
L44R/K206A/N247D/Q3OZK/A337P/A350G/E367D/
T369D 324
L44R/K206A/R22lT/N247D/M3221/K343D/F365L/l
376V 325
96l/K206A
L44R/K96l/K206A/F217R/N247D U) [\J \l
96l/K206A/F217R/N247D/Q302K/A337P/A3
50G U) N 00
L44R/K96l/K206A/F217R/N247D/Q3OZK/A337P/K3
43D/A350G/E367D/T369D U) [\D O
++ L44lUK96l/K206A/F2171UQ3OZK/A350G U) U) 0
L44R/K96l/K206A/N247D/L316D/A337P/A350G/E3
67D/T369D
L44lULlOOF/K206A/F365L
Table 2.4 - Relative Activity of GLA Variants After N0 Challenge (NC) 0r
nge at the Indicated pH 0r Conditionl’
Varian Seru Amino Acid Differences Relative to
NC SE?
t # . . m SEQ ID N0:5 (WT GLA)
L44R/LlOOF/K206A/I208V/Q2l9H/N247D/Q302K/
+++ ++
292 M322V/K343D/R373K/I376V
L44R/Ll OOF/K206A/1208V/R22 1 D/Q3O2K/
M322V/F365L/I376V
L44IUL1OOF/K206A/1208V/R22lT/N247D/M322V/I
376V
L44IUL1OOF/K206A/1208V/R22lT/N247D/Q302K/
M322I/K343D/F365L/R373K/I376V
1. Relative activity was calculated as ty of the variant/activity of Rd2BB (SEQ ID NO:lO
(encoded by SEQ ID NO:8).
2. =
- <O.5 relative activity to Rd2BB (SEQ ID NO:10);
-- = 0.5 to 1.5 relative activity over Rd2BB (SEQ ID NO:10);
— >15 to 2.5 relative activity over Rd2BB (SEQ ID NO:10); and
i — >2.5 relative activity over Rd2BB (SEQ ID NO: 10).
Table 2.5 - Relative Activity of GLA Variants After N0 Challenge (NC)
1’ 2’ 3
0r Challenge at the Indicated pH 0r Condition
Amino Acid Differences Relative to SEQ
SEQ ID NO: 5 (WT GLA)
A66T/K206A/F217IUL316D/M322I/A337
G/A35OG/E367N/R373K
F217R/G23OV/N247D/Q302K/M3
221/E367N/T369S/R373K
K206A/F217R/N247D/L3 l6D/M322I/A33
7P/A35OG/K362Q/E367N/R373K
K206A/F217R/N247D/Q249H/Q302K/M3
221/K343G/A350G/E367T/R373K/L397F
K206A/I208V/R221T/N247D/M322V/K3
43G/E367N/R373K
K206A/M322I/E367N/R373K
K206A/M322V/K343G/E367N/R373K
K206A/N247D/M322I/A337E/K343D/F36
5L/E367N/R373K/I376V 344
K206A/Q302K/L316D/M3221/A337P/A35
OG/K362Q/E367N/T369S/R373K 345
Q302K/L316D/M3221/A337P/K34
3D/E367N/T369S/R373K 346
2015/063329
Table 2.5 - ve Activity of GLA Variants After N0 Challenge (NC)
1’ 2’ 3
0r Challenge at the Indicated pH 0r Condition
'Variant Amino Acid Differences Relative to
NC '5m 4; c Serum SEQ
0 SEQ ID NO: 5 (WT GLA)
K206A/R221K/N247D/Q302K/M3221/E3
67N/R373K
K206A/R22lK/Q302K/M3221/K343G/E3
+ +
67N/R373K/I376V 348
K96l/K206A/F217R/M3221/E367N/T369S
/R373K 349
K96I/K206A/F217R/N247D/Q302K/M322
I/A337P/K343G/A350G/E367N/R373K 350
K96l/K206A/N247D/M3221/A350G/E367
N/T369S/R373K
206A/N247D/Q302K/L3l6D/M32
21/A337P/A350G/E367N/T369S/R373K
206A/N247D/Q302K/L316D/M32
21/A337P/A350G/K362Q/E367N/T369S/R
373K
L100F/A125S/K206A/1208V/R221K/Q30
2K/M3221/K343G/E367N/R373K
L1OOF/K206A/1208V/N247D/Q302K/M32
2V/K343D/E367N/R373K/I376V 355
L1OOF/K206A/1208V/Q302K/M322V/F36
5L/E367N/R373K/I376V 356
L1OOF/K206A/1208V/R221K/M322V/K34
3D/E367N/R373K 357
L1OOF/K206A/1208V/R22lK/M322V/K34
3D/F365L/E367N/R373K
L1 OOF/K206A/1208V/R221T/M322V/E36
7N/R373K/l376V
LlOOF/K206A/M3221/E367N/R373K/l376
K206A/N247D/Q302K/M3221/E36
7N/R373K
Ll OOF/K206A/R22 l K/N247D/M3221/K34
3G/E367N/R373K
L1OOF/K206A/R221T/Q302K/M3221/K34
3D/E367N/R373K
L1OOF/Ll6OI/K206A/R221K/M322V/E36
7N/R373K
L23 S/K206A/M3221/E367N/R373K
L441VK206A/F217R/N247D/L316D/M32
21/A337P/K343G/K362Q/E367N/R373K 366
Table 2.5 - Relative Activity of GLA Variants After N0 Challenge (NC)
1’ 2’ 3
0r Challenge at the Indicated pH 0r ion
'Variant Amino Acid Differences Relative to S?Q
SEQ ID N0:5 (WT GLA) N3,
L441VK206A/F217R/N247D/Q302K/L316
1/A337P/K362Q/E367N/R373K 367
L441VK206A/F217R/N247D/Q302K/L316
D/M3221/K343D/A350G/K362Q/E367N/
R373K
L44R/K206A/F2l7lUQ302K/M3221/A337
P/A350G/E367N/T369S/R373K
L44R/K206A/1208V/N247D/Q302K/M32
21/K343D/E367N/R373K 370
L441VK206A/1208V/R22lK/M3221/K343
N/R373K 371
L441VK206A/1208V/R221K/N247D/Q302
K/M3221/K343D/E367N/R373K/I376V 372
L441UK206A/1208V/R221T/Q302K/M322
I/K343G/F365L/E367N/R373K/I376V 373
L441UK206A/L316D/M3221/A337P/A350
G/E367N/T369S/R373K 374
L44R/K206A/N247D/L316D/M3221/A350
G/K362Q/E367N/T369S/R373K 375
L441UK206A/N247D/Q302K/L316D/M32
21/A337P/K343G/A350G/K362Q/E367N/
T369S/R373K 376
L44R/K206A/N247D/Q3OZK/M3221/A35
OG/E367N/T369S/R373K
L44R/K206A/N247D/Q302K/M3221/K34
7N/R373K
L44lUK96l/K206A/F217R/N247D/L316D
/A337P/A350G/K362Q/E367N/R3
L44R/K96l/K206A/F217R/\247D/M3221/
A350G/K362Q/E367N/R373K
L441VK96l/K206A/F2 1 7R/\247D/M3221/
A350G/K362Q/E367N/T369S/R373K
L441VK96l/K206A/F217R/\247D/M3221/
E367N/T369S/R373K
L44R/K96l/K206A/F2 1 7R/\247D/Q302K
/L3 1 6D/M3221/A337P/E367N/R373K
L44lUK96l/K206A/F217RA247D/Q302K
/M3221/E367N/T369S/R373K
L44R/K96I/K206A/F217R/\247D/Q302K
344 -- -- -- ++
/M3221/K362Q/E367N/R373K 335
WO 05889
Table 2.5 - Relative Activity of GLA Variants After No Challenge (NC)
1’ 2’ 3
or Challenge at the ted pH or Condition
Variant Amino Acid ences Relative to
NC 1°H 4 0
# °
. SEQ ID N0:5 (WT GLA)
L44IUK96I/K206A/F217MQ219P/N247D/
M253K/S266F/D284E/Q29OP/L293F/Q3O
2K/V308G/S314F/M3221/A337P/K343E/E
367N/R373K
L44R/K96I/K206A/F217IUQ3OZK/M3221/
346 +
A350G/K362Q/E367N/T369S/R373K
L44R/K96I/K206A/M3221/A337P/E367N/
T369S/R373K
L44lULlOOF/K206A/1208V/R221K/M322
I/K343G/F365L/E367N/R373K
L44R/L1OOF/K206A/1208V/R221T/N247
D/M3221/F365L/E367N/R373K
L44R/L1OOF/K206A/1208V/R221T/N247
D/M322V/E367N/R373K/I376V 391
L44R/L1OOF/K206A/1208V/R221T/Q302
K/M3221/E367N/R373K/I376V
L44R/L1OOF/K206A/Q302K/M3221/E367
N/R373K/I376V
L44R/L1OOF/K206A/R221K/M3221/F365
L/E367N/R373K/I376V 394
L44R/Ll OOF/K206A/R22 l T/M3221/F365
L/E367N/R373K
L44lULlOOF/K206A/R221T/N247D/M322
I/K343D/E367N/R373K/I376V
L44IUL1OOF/K206A/R221T/N247D/Q302
K/M322I/E367N/R373K
L44IUL1OOF/K206A/R221T/N247D/Q302
K/M322V/E367N/R373K/I376V
L44lULlOOF/K206A/R221T/Q302K/M322
I/E367N/R373K
L44R/Ll OOF/Ql 8 l L/K206A/R221T/N247
K/M322V/E367N/R373K/I376V
1. Relative activity was calculated as activity of the variant/activity 0f Rd3BB (SEQ ID I\O: l 3
(encoded by SEQ ID NO:11).
2. Variant # 326 (Rd4BB) has the cleotide sequence of SEQ ID NO:14 and polypeptide
sequence of SEQ ID NO:15.
3. =
- <O.5 relative activity to Rd3BB (SEQ ID NO:13);
-- = 0.5 to 1.5 relative activity over Rd3BB (SEQ ID NO:13);
— >l.5 to 2.5 relative ty over Rd3BB (SEQ ID NO:13); and
i — >2.5 relative activity over Rd3BB (SEQ ID NO:13).
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge(NC)
0r Challen_ e at the Indicated H or C0nditi0n1’2’3’4
Variant Amino acid differences relative to SEQ ID NO: 5
° ° (WT GLA)
206A/F217R/N247D/Q302K/L316D/M3221/
A337P/K362Q/E367N/R373K
L44R/S47R/K206A/F2 1 7R/N247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K
L44C/K206A/F217R/N247D/Q302K/L316D/M3221/
U) 0 [\D A337P/K362Q/E367N/R373K
47D/K206A/F2 1 7R/N247D/Q3OZK/L316D/
VI3221/A337P/K362Q/E367\/R373K
M39H/L44R/K206A/F2 1 7R/N247D/Q302K/L316D/
VI3221/A337P/K362Q/E367\/R373K
L441US47N/K206A/F2 1 7R/N247D/Q3OZK/L316D/
VI3221/A337P/K362Q/E367\/R373K .bO O\
L441US47V/K206A/F2 1 7R/N247D/Q3OZK/L316D/
V13221/A337P/K362Q/E367\/R373K -l> O \]
M391VL44R/K206A/F2 1 7R/N247D/Q3OZK/L316D/
V13221/A337P/K362Q/E367\/R373K -l> O 00
L44A/K206A/F2 1 7R/N247D/Q302K/L316D/M3221/
A337P/K362Q/E367N/R373K -l> O0
L44S/K206A/F217R/N247D/Q302K/L316D/M3221/
A337P/K362Q/E367N/R373K -l> ’—‘ O
L44Q/K206A/F217R/N247D/Q302K/L316D/M3221/
A337P/K362Q/E367N/R373K
L44W/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3221
/A337P/K362Q/E367N/R373K
L44V/K206A/F2 1 7R/N247D/Q302K/L316D/M3221/
K362Q/E367N/R373K
M411UL44R/K206A/F217R/N247D/Q302K/L316D/
A337P/K362Q/E367N/R373K
L44M/K206A/F217R/N247D/Q302K/L316D/M3221
/A337P/K362Q/E367N/R373K
L441US47I/K206A/F2 1 7R/N247D/Q3OZK/L316D/M
3221/A337P/K362Q/E367N/R373K
M41P/L441UK206A/F217R/N247D/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K
M39T/L44R/K206A/F2 1 7R/N247D/Q3OZK/L316D/
A337P/K362Q/E367N/R373K J> ._1 00
L44T/K206A/F217R/N247D/Q302K/L316D/M3221/
A337P/K362Q/E367N/R373K J> ._1 0
L441US47T/K206A/F2 1 7R/N247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K
Table 2.6 - Relative ty of GLA Variants After N0 Challenge(NC)
0r Challen_ e at the Indicated H or C0nditi0n1’2’3’4
Variant Amino acid differences ve to SEQ ID NO: 5
(WT GLA)
L44lUY92K/K206A/F2 1 7R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44lUY9ZS/K206A/F2 1 7R/N247D/Q3OZK/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K
L441UH94N/K206A/F217R/\247D/Q3OZK/L316D/
U) 00 [\D Vl3221/A337P/K362Q/E367\/R373K
L44R/Y92C/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L441VY92V/K206A/F21 7R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44lUY92A/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K .b [\D O\
L44R/H94lUK206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K -l> [\J \l
L44R/V93T/K206A/F217R/\247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K -l> [\J 00
L44R/V93L/K206A/F217R/\247D/Q302K/L316D/
/A337P/K362Q/E367\/R373K -l> [\J 0
L44R/V93S/K206A/F217R/\247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K -l> L») O
L44lUY92Q/K206A/F2 1 7R/N247D/Q3OZK/L316D/
Vl3221/A337P/K362Q/E367\/R373K 431
L441UY92W/K206A/F2 1 7R/N247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K/L397S 432
92T/K206A/F21 7R/l\247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K 43 3
L441VY92G/K206A/F217R/h247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K 434
L44R/Y921UK206A/F217R/h247D/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K 43 5
L441UY92H/K206A/F217R/h247D/Q3OZK/L316D/
A337P/K362Q/E367N/R373K -l> 0
L44lULl58M/K206A/F217R/\1247D/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K -l> U) \l
L44R/Ll58R/K206A/F217R/N247D/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K -l> U) 00
L44R/Al59S/K206A/F217R/N247D/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K -l> U) 0
L44lURl65K/K206A/F217R/N247D/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge (NC)
1’ 2’3’ 4
0r Challen_e at the Indicated H or ion
t Amino acid differences relative to SEQ ID NO: 5
(WT GLA)
L44R/Ll58C/K206A/F217R/\247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K
L44lUT l 63 S/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44R/Sl66P/K206A/F217R/\247D/Q302K/L316D/
.bO [\D Vl3221/A337P/K362Q/E367\/R373K
L44R/Sl66G/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44R/Sl66F/K206A/F217R/\247D/Q302K/L316D/
4;O4; Vl3221/A337P/K362Q/E367\/R373K
158E/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K .b .b O\
L441UR162K/K206A/F217R/N247D/Q3OZK/L316D/
Vl3221/A337P/K362Q/E367\/R373K 4; 4; \1
L44R/Ll58H/K206A/F217R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K 4; 4; oo
L44lUS l 66R/K206A/F21 7R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K 4; 4; \o
L44lURl65H/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K 4;m o
L441UR162H/K206A/F217R/\247D/Q3OZK/L316D/
/A337P/K362Q/E367\/R373K 451
L44R/Sl66A/K206A/F217R/\247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K 452
L44R/Sl66H/K206A/F217R/\247D/Q302K/L316D/
/A337P/K362Q/E367\/R373K 453
L44R/Tl 63 */K206A/F217R/\247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K 454
L44R/Ll58Q/K206A/F217R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K 455
L44R/Sl66D/K206A/F217R/N247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K
L44lURl62G/K206A/F217R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44lURl6ZS/K206A/F217R/N247D/Q302K/L316D/
Vl3221/A337P/K362Q/E367\/R373K
L44R/l\ l6lE/K206A/F217R/N247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K .b U1 0
L44lUS l 66E/K206A/F2 1 7R/N247D/Q3OZK/L316D/
Vl3221/A337P/K362Q/E367\/R373K .b O\O
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge(NC)
0r Challen_ e at the Indicated H or C0nditi0n1’2’3’4
t Amino acid differences relative to SEQ ID NO: 5
# ° ° (WT GLA)
L441US 1 66T/K206A/F2 1 7R/N247D/Q3OZK/L316D/
VI3221/A337P/K362Q/E367\/R373K
L441VR162Q/K206A/F217R/N247D/Q302K/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K
L44R/L158G/K206A/F217R/N247D/Q302K/L316D/
.b [\D [\D VI3ZZI/A337P/K362Q/E367\/R373K
L441UR162A/K206A/F217R/N247D/Q3OZK/L316D/
VI3ZZI/A337P/K362Q/E367\/R373K
L441UK206A/F217R/\247D/L255E/Q302K/L316D/
.b [\D .b VI322UA337P/K362Q/E367\/R373K
L44R/K206A/F217R/\247D/H271E/Q302K/L316D/
VI322UA337P/K362Q/E367\/R373K .b O\ O\
L441UK206A/F217R/\247D/M259E/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K -l> O\ \]
206A/F217R/\247D/L263G/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K -l> O\ 00
L441UK206A/F217R/\247D/M259S/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K -l> O\ 0
L44R/K206A/F217R/\247D/L255C/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K -l> \] O
L44R/K206A/F217R/\247D/H271T/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K 471
206A/F217R/\247D/R270G/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K 472
L44R/K206A/F217R/\247D/L255V/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K 473
L441UK206A/F217R/\247D/H271Q/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K 474
L441UK206A/F2 1 7R/N247D/R27OD/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K 475
L441UK206A/F2 1 7R/N247D/1258L/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K .b \l O\
L441UK206A/F217R/N247D/H271G/Q3OZK/L316D
/M3221/A337P/K362Q/E367N/R373K -l> \l \l
L441UK206A/F2 1 7R/N247D/L263E/Q3OZK/L316D/
M3221/A337P/K362Q/E367N/R373K -l> \] 00
L44R/K206A/F217R/N247D/L255*/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K 479
206A/F217R/N247D/H271A/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K 480
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge(NC)
0r Challen_ e at the Indicated H or C0nditi0n1’2’3’4
Variant Amino acid differences relative to SEQ ID NO: 5
° ° (WT GLA)
206A/F2 1 7R/N247D/L263C/Q3O2K/L316D/
M322I/A337P/K362Q/E367N/R373K
L441UK206A/F217R/N247D/H271V/Q302K/L316D
/M322I/A337P/K362Q/E367N/R373K
L44R/K206A/F2 1 7R/N247D/L255A/Q3O2K/L316D/
.b .b [\D M322I/A337P/K362Q/E367N/R373K
L441UK206A/F2 1 7R/N247D/L255 8/Q3O2K/L316D/
M322I/A337P/K362Q/E367N/R373K
L44R/K206A/F217R/N247D/M259W/Q302K/L316
.b .b .b D/M322I/A337P/K362Q/E367N/R373K
L441UK206A/F2 1 7R/N247D/L263F/Q3O2K/L316D/
M322I/A337P/K362Q/E367N/R373K .b
L44R/K206A/F2 1 7R/N247D/M259A/Q3O2K/L316D
/M322I/A337P/K362Q/E367N/R373K -l> 00 \]
L44R/K206A/F217R/N247D/L263W/Q302K/L316D
/M322I/A337P/K362Q/E367N/R373K -l> 00 00
206A/F217R/N247D/R27OQ/Q302K/L316D/
M322I/A337P/K362Q/E367N/R373K -l> 00 0
L441UK206A/F217R/\247D/L255T/Q3O2K/L316D/
M322I/A337P/K362Q/E367N/R373K -l> 0O
L441VK206A/F217R/\247D/I258M/Q302K/L316D/
M322I/A337P/K362Q/E367N/R373K 491
L44R/K206A/F217R/\247D/M259V/Q302K/L316D
/M322I/A337P/K362Q/E367N/R373K 492
L441VK206A/F217R/\247D/H271R/Q302K/L316D/
M322I/A337P/K362Q/E367N/R373K 493
L44R/K206A/F217R/\247D/R270L/Q302K/L316D/
M322I/A337P/K362Q/E367N/R373K 494
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390P 495
206A/F217R/\247D/Q302K/L316D/VI322I/
K362Q/E367N/R373K/M392D
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/T389M
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392A
206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390* .b00
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390H 500
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge (NC)
1’ 2’3’ 4
0r Challen_e at the Indicated H or Condition
Variant Amino acid differences relative to SEQ ID NO: 5
(WT GLA)
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/L3 86T
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392Q
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
.b 01 [\D A337P/K362Q/E367N/R373K/Q3 85L
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390T
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
4; O\ 4; A337P/K362Q/E367N/R373K/M392*
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390Q U1 0 O1
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392E U1 O \l
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/T3 89S U1 O 00
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/T389Q U1 O0
206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/Q3851 U1 1—‘ O
206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392R 511
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/T389W 512
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392K 513
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392L 514
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
K362Q/E367N/R373K/L386F 515
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
K362Q/E367N/R373K/T389D 516
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390E
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/L384W U] p—A 00
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392S U] p—A 0
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
.b \l0 A337P/K362Q/E367N/R373K/M392F 520
Table 2.6 - Relative Activity of GLA Variants After N0 Challenge (NC)
1’ 2’3’ 4
0r Challen_e at the Indicated H or Condition
Variant Amino acid differences relative to SEQ ID NO: 5
(WT GLA)
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390R
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390G
L44lUK206A/F2 1 7D/Q3O2K/L3 1 22I/
J; 00 [\D A337P/K362Q/E367N/R373K/Q385G
206A/F2 1 7D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M392C
L44lUK206A/F2 1 7D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M392V
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M392W U1 [\D 0\
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390C U1N \l
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/T389G U1N 00
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/T389N U1N0
L44lUK206A/F2 1 7D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/T3 891 U1 U) O
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390D 531
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390W 532
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/T389C 533
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M392P 534
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M390F 535
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/T389F 536
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 22I/
A337P/K362Q/E367N/R373K/M390V U1 U) \l
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
A337P/K362Q/E367N/R373K/M39OK U1 U) 00
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 22I/
A337P/K362Q/E367N/R373K/M3921 U1 U) 0
L44lUK206A/F2 1 7R/\247D/Q3O2K/L3 1 6D/VI322I/
.b00 A337P/K362Q/E367N/R373K/T389L 540
2015/063329
Table 2.6 - Relative Activity of GLA Variants After No Challenge (NC)
1’ 2’3’ 4
or Challen_e at the Indicated H or Condition
Variant Amino acid differences relative to SEQ ID NO: 5
(WT GLA)
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M390A
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/M392G
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0 [\D A337P/K362Q/E367N/R373K/L3 86S
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0 U) A337P/K362Q/E367N/R373K/Q385C
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0.b A337P/K362Q/E367N/R373K/M390S
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0 U1 A337P/K362Q/E367N/R373K/M392N U1 .b 01
206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0 O1 A337P/K362Q/E367N/R373K/Q385W U1 4; \1
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 O \l A337P/K362Q/E367N/R373K/M392T U1 4; oo
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
U1 0 00 A337P/K362Q/E367N/R373K/L384A U1 4; \o
-- L441UK206A/F217R/\247D/Q3O2K/L316D/VI322I/
+ K362Q/E367N/R373K/Q385T U1 ()1 o
L441UA199G/K206A/F217R/N247D/Q302K/L316D
- /M322I/A337P/K362Q/E367N/R373K/M392R 551
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/L397*
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/K395*
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/D396*
206A/F217R/\247D/Q302K/L316D/VI322I/
A337P/K362Q/E367N/R373K/S393 *
L441UK206A/F217R/\247D/Q302K/L316D/VI322I/
K362Q/E367N/R373K/L394*
1. Relative activity was calculated as activity of the variant/activity 0f Rd4BB (SEQ ID NO:15
(encoded by SEQ ID NO: 14).
2. Variant # 395 ) has the polynucleotide sequence of SEQ ID NO:39 and polypeptide
sequence of SEQ ID NO:40.
3. Variant # 402 (Rd6BB) has the polynucleotide sequence of SEQ ID NO:41 and polypeptide
sequence of SEQ ID NO:42
4. =
- <O.5 relative activity to Rd4BB (SEQ ID NO:15);
-- = 0.5 to 1.5 relative activity over Rd4BB (SEQ ID NO:15);
— >15 to 2.5 relative activity over Rd4BB (SEQ ID NO:15); and
i — >2.5 relative activity over Rd4BB (SEQ ID NO: 15).
2015/063329
Table 2.7 - Relative ty of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated pH 0r Condition
I.Variant Ill-lllllllllll-lllllllflO Ill-IIIIIIIIIIIIH-IIIIHH (I)
'1: '1:m
Amino acid differences relative to SEQ ID NO: 5 (WT GLA) ID
u p\1
D2E/L44R/Y92H/K206A/F2 l 7R/N247D/Q3OZK/L3 l6D/M3221
/Q326G/A337P/K362Q/E367N/R373K U1
D2Q/L44lUY92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3221
/A337P/K362Q/E367N/R373K U1
44lVY92H/K206A/F217R/N247D/Q302K/L316D/M32 -
—II/A337P/K362Q/E367N/R373K 560
L441UA77S/Y92H/K206A/F217R/N247D/Q302K/L316D/M322 -
—I21/A337P/K362Q/E367N/R373K 562
L441VE56K/Y92H/K206A/F217R/N247D/Q302K/L316D/M32 -
—I21/A337P/K362Q/E367N/R373K 564
91V/Y92H/K206A/F217R/N247D/Q302K/L316D/M32 -
76H/Y92H/K206A/F217R/N247D/Q302K/L316D/M32 -
L441VR74H/Y92H/K206A/F217R/N247D/Q302K/L316D/M32
21/A337P/K362Q/E367N/R373K U1 O\ \l
L44lUY92E/K206A/F2 1 7R/N247D/Q3OZK/L316D/M3221/A3 3
7P/K3 62Q/E367N/R373K U1 O\ 00
L44R/Y92H/D l 3OQ/K206A/F2 1 7R/N247D/Q3OZK/L3 1 6D/M3
8 221/A337P/K362Q/E367\/R373K U1 0\0
L44R/Y92H/Kl82A/K206A/F217R/N247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K U1 \lO
II L44lUY92H/Kl82E/K206A/F217R/N247D/Q302K/L316D/M3
37P/K362Q/E367\/R373K U1 \l ._l
L44R/Y92H/Kl82H/K206A/F217R/N247D/Q302K/L316D/M3
U) 221/A337P/K362Q/E367\/R373K U1 \lN
L44lUY92H/Kl82M/K206A/F217R/N247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K U1 \1 U)
L44R/Y92H/Kl82Q/K206A/F217R/N247D/Q302K/L316D/VI3
221/A337P/K362Q/E367\/R373K U1 \l J>
L44lUY92H/Kl821VK206A/F217R/N247D/Q302K/L316D/VI3
221/A337P/K362Q/E367\/R373K U1 \l U1
L44lUY92H/Kl 82T/K206A/F2 l 7R/N247D/Q3OZK/L3 1 6D/V13
221/A337P/K362Q/E367\/R373K U1 \l 01
L44R/Y92H/Kl 82V/K206A/F217R/N247D/Q302K/L3 16D/VI3
221/A337P/K362Q/E367\/R373K U1 \l \l
L44R/Y92H/Kl82Y/K206A/F217R/N247D/Q302K/L316D/VI3
221/A337P/K362Q/E367\/R373K U1 \1 00
L44lUY92H/K206A/F2 1 7R/N247D/A287C/Q3OZK/L3 1 6D/V13 570
Table 2.7 - Relative Activity of GLA Variants After N0 Challenge (NC)
or Challenge at the Indicated pH or Condition
I. (/2HO
Varlant pH “3
Amino acid differences relative to SEQ ID NO: 5 (WT GLA) ID
|92 221/A337P/K362Q/E367\/R373K
II 92H/K206A/F217R/\247D/A287H/Q3OZK/L3 1 6D/M3
221/A337P/K362Q/E367\/R373K 00 O
L44lUY92H/K206A/F217R/\247D/A287M/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K
L44R/Y92H/K206A/F217R/\247D/K283A/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K II0000N._i
L44R/Y92H/K206A/F217R/\247D/K283G/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K U1 00 U)
L44lUY92H/K206A/F217R/\247D/K283M/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K U1 00 J>
L44R/Y92H/K206A/F217R/\247D/K283V/Q3OZK/L3 1 6D/V13
37P/K362Q/E367\/R373K U1 00 U1
L44R/Y92H/K206A/F217R/\247D/K295A/Q302K/L316D/VI3
U1 U1 221/A337P/K362Q/E367\/R373K U1 00 0
II L44lUY92H/K206A/F2 l 7R/\247D/K295E/Q3OZK/L3 1 6D/V13
221/A337P/K362Q/E367\/R373K U1 00 \l
II L44lUY92H/K206A/F217R/\247D/K295L/Q302K/L316D/VI3
221/A337P/K362Q/E367\/R373K U1 00 00
L44R/Y92H/K206A/F217R/\247D/K295N/Q3OZK/L3 1 6D/V13
U1 00 37P/K362Q/E367\/R373K U1 00 O
L44R/Y92H/K206A/F217R/\247D/K295Q/Q302K/L316D/VI3
221/A337P/K362Q/E367\/R373K U1 0O
L44lUY92H/K206A/F217R/N247D/K29SS/Q302K/L316D/M32
21/A337P/K362Q/E367N/R373K U1 0 ._i
L44lUY92H/K206A/F2 l 7R/\247D/K295T/Q3OZK/L3 1 6D/M3
U1 U1 37P/K362Q/E367\/R373K U1 0N
L44R/Y92H/K206A/F217R/\247D/Q3OZK/L3 1 6D/A3 1 7D/M3
U1 U1 221/A337P/K362Q/E367\/R373K U1 0 U)
L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/A317Q/M3
U1 U1 U) 221/A337P/K362Q/E367\/R373K U1 0.b
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/A346G/K362Q/E367\/R373K U1 0 U1
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/G344A/K362Q/E367\/R373K U1 0 0\
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/G344D/K362Q/E367\/R373K U1 0 \l
II L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
+ + 7P/G344S/K362Q/E367N/R373K U1 0 00
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
+ 3L/K362Q/E367N/R373K U1 00
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/K362Q/E367N/L372W/R373K
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/K362Q/E367N/W368A/R373K 001—‘
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
7P/K362Q/E367N/W368L/R373K 602
2015/063329
Table 2.7 - Relative Activity of GLA ts After N0 Challenge (NC)
0r Challenge at the Indicated pH 0r Condition
I. SEQ
Variant PH PH
Amino acid differences relative to SEQ ID NO: 5 (WT GLA) ID
L441UY92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
7P/K362Q/E367N/W368N/R373K IOU)
L441UY92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
7P/K362Q/E367N/W3681VR373K O\O.b
L441UY92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
7P/K362Q/E367N/W368V/R373K 01O U1
L441UY92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
8E/K362Q/E367N/R373K
92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
7P/N348M/K362Q/E367N/R373K O1O \l
L441UY92H/K206A/F217R/\247D/Q302K/L316D/Vl3221/A33
7P/N348Q/K362Q/E367N/R373K 01O 00
L441VY92H/K206A/F21 7R/\247D/Q3OZK/L3 1 6D/Vl3221/A33
00 7P/N348R/K362Q/E367N/R373K
L441VY92H/K206A/F21 7R/\247D/Q3OZK/L3 1 6D/Vl3221/A33
7P/N348W/K362Q/E367N/R373K O\ 1—1 0
L44lUY92H/K206A/F2 1 7R/\247D/Q302K/L316D/VI3221/A3 3
U1 7P/T354S/K362Q/E367N/R373K O\ 1—1 ’—‘
L44R/Y92H/K206A/F217R/\247D/Q302K/N305K/L316D/M3
221/A337P/K362Q/E367N/R373K O\ ._1 [\D
-III5 L44lUY92H/K206A/F2 l 7D/Q3OZK/N3OSL/L3 1 6D/M3
221/A337P/K362Q/E367N/R373K O\ 1—1 U3
-III L44lUY92H/K206A/F2 1 7R/N247D/Q302K/S3 14A/L3 1 6D/M3221/A337P/K3 62Q/E367N/R373K O\ 1—1 Jk
L44lUY92H/K206A/F2 1 7R/N247D/Q302K/S3 14H/L3 1 6D/M32
U1 21/A337P/K362Q/E367N/R373K O\ 1—1 U1
L44lUY92H/K206A/F2 1 7R/N247D/Q302K/S3 14N/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K 01 )—k 01
L44lUY92H/K206A/F2 1 7R/N247D/Q302K/S3 14Y/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O\ 1—1 \l
L44R/Y92H/K206A/F2 1 7WW246A/N247D/Q3OZK/L3 1 6D/M3
221/A337P/K362Q/E367N/R373K O\ 1—1 00
L44R/Y92H/K206A/F2 1 7WW246I/N247D/Q3OZK/L3 1 6D/M32
00 21/A337P/K362Q/E367N/R373K O\ 1—1 0
L44R/Y92H/K206A/F2 1 7MW246P/N247D/Q3OZK/L3 1 6D/M3
221/A337P/K362Q/E367N/R373K 01 [\J O
L44lUY92H/K206A/F2 1 7WW246R/N247D/Q3OZK/L3 1 6D/M3
221/A337P/K362Q/E367N/R373K I.01 1—1 L44R/Y92H/K206A/F2 1 7WW246S/N247D/Q3OZK/L3 1 6D/M3
221/A337P/K362Q/E367N/R373K O1 N
L44lUY92H/K206A/8210A/F217R/N247D/Q3OZK/L3 1 6D/M32
7P/A350T/K362Q/E367N/R373K 01 [\D U)
L44lUY92H/K206A/8210A/F217R/N247D/Q3OZK/L3 1 6D/M32
7P/K362Q/E367N/R373K 01 [\J -l>
L44lUY92H/K206A/821OE/F217R/N247D/Q3OZK/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O1 [\J U1
--- L441UY92H/K206A/SZ 1 OK/F2 l 7R/N247D/Q3OZK/L3 1 6D/M32 626
Table 2.7 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated pH 0r Condition
IIIIIIIIIVariant PH PH
Amino acid differences relative to SEQ ID NO: 5 (WT GLA) ID
—-21/A337P/K362Q/E367N/R373K
III L44lUY92H/K206A/821ON/F217R/N247D/Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K 01 [Q \J
L44R/Y92H/K206A/8210lUF217R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K IIIIIIIIII01 M
L44lUY92H/K96A/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O\ 0
L44lUY92H/K96W/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K 01 U) C)
L44R/Y92H/Pl 79M/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3
37P/K362Q/E367\I/R373K 01 DJ hi
L44lUY92H/Rl89K/K206A/F217R/N247D/Q302K/L316D/M3
221/A337P/K362Q/E367\I/R373K O\ DJ [0
L44lUY92H/Rl89V/K206A/F217R/N247D/Q302K/L316D/M3
U1 221/A337P/K362Q/E367\I/R373K O\ DJ U)
92H/S95A/K206A/F2 l 7R/N247D/Q302K/L3 l6D/M322
l/A337P/K362Q/E367l\/R373K O\ m .5
L44R/Y92H/S95E/K206A/F217R/N247D/Q302K/L316D/M322
U1 l/A337P/K362Q/E367l\/R373K 01 (x U1
92H/Tl 86A]K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3
U1 U1 221/A337P/K362Q/E367\I/R373K O\ U) 0\
L44lUY92H/Tl 06A/F2 1 7D/Q302K/L3 1 6D/M3
III 221/A337P/K362Q/E367\I/R373K O\ DJ N
IIIL44lUY92H/Tl 06A/F2 1 7R/N247D/Q302K/L3 1 6D/M3 221/A337P/K362Q/E367\I/R373K 01 DJ M)
L44R/Y92H/Y120H/K206A/F217R/N247D/Q302K/L3 1 6D/M3
U1 a) 221/A337P/K362Q/E367\I/R373K 0\ U)
L44lUY92H/Yl ZOS/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
U1 21/A337P/K362Q/E367N/R373K IIIIII!IIIO\#
III L44lUY92H/Yl ZOS/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3221/A337P/L34 1 F/K362Q/E367N/R373K O\ .5 ._.
M39C/L441UY92H/K206A/F2 1 7D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K 01 -b IQ
M39E/L44lUY92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O\ #5 (N
M391VL44lUY92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O\ -b h.
M39V/L44R/Y92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
21/A337P/K362Q/E367N/R373K O1 .5 U1
TlOP/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322
l/A337P/K362Q/E367N/R373K 0-h 01
TlOP;L44R;Y92H;R189L;K206A;F217R;N247D;Q302K;L3l6
1;A337P;K362Q;E367N;R373K O\ #5 \d
T8L;L44R;Y92H;K206A;F217R;N247D;Q302K;L316D;M3221
;A337P;K362Q;E367N;R373K 0$5 a)
T8Q;L44R;Y92H;K206A;F217R;N247D;Q302K;L316D;M3221
;A337P;K362Q;E367N;R373K O\ .5 ‘C
1. Relative ty was calculated as activity of the variant/activity of Rd3BB (SEQ ID
NO:13) (encoded by SEQ ID NO:11).
2. =
- <1.5 relative ty to Rd3BB (SEQ ID NO:13);
-- = 1.5 to 5 relative actiVity over Rd3BB (SEQ ID NO:13);
— >5 to 10 relative actiVity over Rd3BB (SEQ ID NO:13); and
1 — >10 relative activity over Rd3BB (SEQ ID NO:13).
Table 2.8 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated pH 0r Condition
. SEQ
vamnt pH pH
NC Amino acid differences relative to SEQ ID NO: 5 (WT GLA) ID
# 3.3 9.7
L44R/Sl66P/K206A/F217R/N247D/Q302K/L316D/M3221/A337
609 __ ++ ++
P/K362Q/E367N/R373K 650
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/M259E/Q302K/
610 _
M3221/A337P/K362Q/E367N/R373K/M390Q 651
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/M3221
61 1
/A337P/K362Q/E367N/R373K/M390Q 652
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/M3221
612 -
/K362Q/E367N/R373K/M392T 653
L441US47N/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
613 -
L316D/M3221/A337P/K362Q/E367N/R373K/M390Q 654
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/Q302K/L316D/
M3221/A337P/K362Q/E367N/R373K 655
L44R/S47N/Y92H/Sl66P/K206A/F217R/N247D/Q302K/L316D/
61 5
M3221/A337P/K362Q/E367N/R373K/M390H 656
L441VS47T/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271A
/Q302K/L316D/M3221/A337P/K362Q/E367N/R373K/M39OQ 657
L441UY92H/L136V/Sl66P/K206A/F217R/N247D/M259A/Q302
K/L316D/M3221/A337P/K362Q/E367N/R373K/M390Q 658
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
618 -
M3221/A337P/K362Q/E367N/R373K 659
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
619 -
L316D/M3221/A337P/K362Q/E367N/R373K/M390H 660
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
620 _ :
L316D/M3221/A337P/K362Q/E367N/R373K/M390Q 661
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/M259E/H271A/
621 __ ++ _
L316D/M3221/A337P/K362Q/E367N/R373K/M390Q 662
L44R/S47N/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271A
622 /Q302K/L316D/M3221/A337P/K362Q/E367N/R373K/M390Q/M
392T 663
L441US47N/Sl66P/K206A/F217R/N247D/H271A/A276S/Q302K
+ /L316D/M3221/A337P/K362Q/E367N/R373K/M392T 664
L44R/S47N/S166P/K206A/F217R/N247D/H271A/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K/M390Q 665
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
L316D/M3221/A337P/K362Q/E367N/R373K/M392T 44
L441UY92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/L316
D/M3221/A337P/K362Q/E367N/R373K/M390Q 666
Table 2.8 - Relative Activity of GLA Variants After No nge (NC)
or Challen_e at the Indicated H or Condition
variant *3fl pH
NC Amino acid ences relative to SEQ ID NO: 5 (WT GLA) ID
# 3.3 9.7
L44R/S47N/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271A
627 /Q302K/L316D/M3221/A337P/K362Q/E367N/R373K/M39OH/M
392T 667
L441US47N/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/
L316D/M3221/A337P/K362Q/E367N/R373K/M390H 668
47T/S166P/K206A/F217R/N247D/H271A/Q302K/L316D
/M3221/A337P/K362Q/E367N/R373K/M390Q 669
L44IUS47T/Y92H/S166P/K206A/F217R/N247D/M259W/H271A
O\ L») o
/Q3OZK/L3 16D/M3221/A337P/K362Q/E367N/R373K/M390H 670
L44R/S47T/A53 S/Y92H/S166P/K206A/F217R/N247D/H271A/Q
302K/L316D/M3221/A337P/K362Q/E367N/R373K/M390Q 671
L441US47N/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302K/
VI3221/A337P/K362Q/E367\/R373K/M392T 672
E43D/L441VY9ZS/Sl66P/K206A/F217R/\247D/Q302K/L316D/
\/I3221/A337P/K362Q/E367N/R373K 673
44R/Y92E/Sl66P/K206A/F217R/\247D/Q302K/L316D/
\/I3221/A337P/K362Q/E367N/R373K 674
E43D/L44mY92H/s166P/K206A/F217R/\247D/Q302K/L3 1 6D/
63 5
VI3221/A337P/K362Q/E367\/R373K 675
E43D/L44lUY92N/Sl66P/K206A/F217R/\247D/Q302K/L316D/
VI3221/A337P/K362Q/E367\/R373K 676
E43Q/L44R/Y92E/S166P/K206A/F217R/N247D/Q302K/L316D/
63 7
VI3221/A337P/K362Q/E367\/R373K 677
1. Relative activity was calculated as ty of the variant/activity of Rd3BB (SEQ ID
NO:13) (encoded by SEQ ID NO:11).
2. Variant # 625 (Rd7BB) has the polynucleotide sequence of SEQ ID NO:43 and
polypeptide sequence of SEQ ID NO:44.
3. = <1.5 relative activity to Rd3BB (SEQ ID NO: 13);
-- = 1.5 to 5 relative activity over Rd3BB (SEQ ID NO:13);
— >5 to 10 ve activity over Rd3BB (SEQ ID NO:13); and
— >10 relative activity over Rd3BB (SEQ ID NO: 13).
Table 2.9 - Relative Activity of GLA Variants After No Challenge (NC)
or Challenge at the Indicated pH or Condition
Amino acid differences relative to SEQ ID NO: 5 (WT GLA)
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
H271A/Q302K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247Y/H271A
/Q3OZK/L3 16D/M322I/A337P/K362Q/E367N/R373K/M392T
T1OP/M39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N2
640 + + 47D/H271A/Q302K/L3 1 6D/M322I/A337P/K362Q/E367N/R37
3K/M392T 680
641 - - - T1OP/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/S 681
Table 2.9 - Relative Activity of GLA Variants After N0 Challenge (NC)
0r Challenge at the Indicated pH 0r Condition
Variant
Amino acid differences relative to SEQ ID NO: 5 (WT GLA)
266P/H271A/Q3O2K/L3 1 6D/M322l/A337P/K362Q/E367N/R37
3K/M392T
T l OP/E43D/L44lUS47T/Y92H/S l 66P/K206A/F21 7R/N247D/A
O\ 4;N 261 A/Q302K/N305L/L3 1 2l/A337P/K362Q/E3
67N/W368A/R373K/M392T O\ 00 [\D
TlOP/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
-- Q3O2K/L3 1 6D/M322l/R325S/A337P/K362Q/E367N/R373K/M
392T 00 U)
/Q3O2K/L3 16D/M322l/A337P/K362Q/E367N/R373K/M392T 684
IIHIIIIHII _
/Q3O2K/L3 16D/M322l/A337P/K362Q/E367N/R373K/M392T 685
L441US47T/Y92H/G1 13C/S166P/K206A/F217R/N247D/H27l
A]Q3O2K/L3 1 6D/M322l/A337P/K362Q/E367N/R373K/M392T 686
Ll4F/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
Q3O2K/L3 1 6D/M322l/A337P/K362Q/E367N/R373K/M392T 687
T l OP/M39E/L44R/S47T/Y92H/S l 66P/K206A/F2 1 7R/N247D/A
261 G/H27lA/Q302K/L3 16D/M322l/A337P/K362Q/E367N/W3
68A/R373K/M392T
Tl OP/M39E/L44lUS47T/Y92H/S 1 06A/F21 71VW246P/
N247D/H271A/Q302K/L3 l 6D/M322l/A337P/K362Q/E367N/R
373K/M392T
R7H/Tl OP/L44R/S47T/Y92H/S l 66P/K206A/F2 1 7R/N247D/H2
O2K/L3 1 6D/M322l/A337P/K362Q/E367N/R373K/M39
44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
Q3O2K/L3 1 6D/M322l/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/S l 66P/K206A/F2 l 7WW246P/N247D/A261
G/H271A/Q302K/N305L/L316D/M322l/A337P/K362Q/E367N
/R373K/M392T
T 1 OP/L44R/S47T/Y92H/S1 66P/K206A/F2 1 7R/W246P/N247D/
H271A/Q302K/L3 l 6D/M322l/A337P/K362Q/E367N/R373K/M
392T
4lUS47T/Y92H/S 1 06A/F2 l 7R/N247D/H271A/Q
302K/L3 16D/M322l/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/S l 66P/K206A/F2 l 7WW246P/N247D/A261
G/H271A/Q302K/N305L/L316D/M322l/A337P/K362Q/E367N
/W368A/R373K/M392T
T 1 OP/L44R/S47T/Y92H/S1 66P/K206A/F2 1 7R/W246P/N247D/
0U1 0 A261 G/H27lA/Q302K/L3 1 6D/M322l/A337P/K362Q/E367N/R
373K/M392T
47T/P67T/Y92H/Sl66P/Kl 82N/K206A/F217R/N247D/
H271A/Q302K/L3 l 6D/M322l/A337P/K362Q/E367N/R373K/M
392T
M39E/L44R/S47T/Y92H/S 1 66P/K206A/F2 l 7MW246P/N247D
/H27lA/Q302K/L316D/M322l/A337P/K362Q/E367N/R373K/
M392T
L441US47T/W64L/Y92H/Sl66P/K206A/F217R/N247D/H271A
659 - - -
/Q3O2K/L3 16D/M322l/A337P/K362Q/E367N/R373K/M392T 699
Table 2.9 - Relative ty of GLA Variants After N0 Challenge (NC)
0r Challenge at the ted pH 0r Condition
Amino acid differences ve to SEQ ID NO: 5 (WT GLA)
M39E/L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7R/N247D/A26 1 G
/H271A/Q302K/N305L/L3 1 6D/M3221/A337P/K362Q/E367N/R
373K/M392T
A]Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T 701
L441VS47T/Y92H/Sl66P/K206A/F217R/V23 81/N247D/H271A
/Q302K/L3 16D/M3221/A337P/K362Q/E367N/R373K/M392T 702
44lVS47T/Y92H/Sl66P/K206A/F217R/N247D/A261G/
H271A/Q302K/L316D/M3221/A337P/K362Q/E367N/W368A/
M392T
T10P/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/Q252H/
M2531VA254E/A261G/H271A/Q302K/L316D/M3221/A337P/K
362Q/E367N/R373K/M392T
R7C/L441VS47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/W368A/R373K/
M392T 705
L44R/S47T/Y92H/Sl66P/K206A/F2171VP228L/N247D/H271A
/Q302K/L3 16D/M3221/A337P/K362Q/E367N/R373K/M392T 706
D30G/L44lUS47T/Y92H/Sl 66P/K206A/F2 1 7R/N247D/H271A/
Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T 707
M39E/L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7D/H271A
/Q302K/L3 16D/M3221/A337P/K362Q/E367N/R373K/M392T 708
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/P26ZS/H271A
/Q302K/L3 16D/M3221/A337P/K362Q/E367N/R373K/M392T 709
L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7R/N247D/H271A/Q302
K/N305L/L316D/M3221/A337P/K362Q/E367N/R373K/M392T 710
T10P/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
++ ++ Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/W368A/R373K/
M392T 71 1
L441VS47T/Y92H/D144Y/Sl66P/K206A/F217R/N247D/H271
A]Q302K/L3 1 21/A337P/K362Q/E367N/R373K/M392T 712
L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7R/N247D/H271A/Q302
K/L316D/M3221/A337P/K362Q/E367N/R373K/N377Y/M392T 713
A]Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T 714
L441VS47T/M65V/Y92H/Sl66P/K206A/F217R/N247D/H271A
/Q302K/L3 16D/M3221/A337P/K362Q/E367N/R373K/M392T 715
M39E/L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7R/N247D/H271A
/Q302K/L316D/M3221/A337P/K362Q/E367N/W368A/R373K/
M392T 716
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/M253W/H271
D/P274S/K277R/Q302K/L316D/M3221/A337P/K362Q/
E367N/R373K/M392T 717
L441VS47T/Y92H/Sl66P/K206A/F217R/N247D/M253W/A257
G/H271A/K277R/Q281L/Q302K/L316D/A319D/M3221/A337P
/K362Q/E367N/R373K/M392T 718
T10P/M39E/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H
679 + + + 271A/Q302K/N305L/L316D/M3221/A337P/K362Q/E367N/R37
3K/M392T 719
Table 2.9 - Relative Activity of GLA Variants After No Challenge (NC)
or Challenge at the Indicated pH or ion
Amino acid differences relative to SEQ ID NO: 5 (WT GLA)
T1OP/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H
271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M3
4lUS47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q
302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 721
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302
Y/M322I/A337P/K362Q/E367N/R373K/M392T 722
M39E/E43D/L44R/S47T/Y92H/S 1 66P/K206A/F21 71VW246P/
N247D/M253W/H271A/S273D/Q3O2K/L3 1 6D/M322I/A337P/
K362Q/E367N/W368A/R373K/M392T 723
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302
K/N3OSL/L3 16D/M322I/A337P/K362Q/E367N/W368A/R373K
/M392T
E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W
/A257G/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/
E367N/W368A/R373K/M392T
T1OP/El7G/L44lUS47T/Y92H/S166P/K206A/F217R/N247D/H
271A]Q3O2K/L3 1 2I/A337P/K362Q/E367N/R373K/M3
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q290
R/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/W368A/R373K
/M392T
L44R/S47T/Y92H/S166P/K206A/F2171VP228Q/N247D/H271
K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302
K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T
T1OP/L44R/S47T/Y92H/M156V/S166P/K206A/F217R/N247D/
H271A/Q302K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T
T1OP/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/S166P/K206A/F217R/N247D/W256L/H271
A]Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302
K/L3 1 6D/M322I/A337P/K362Q/E367N/W368A/R373K/M392
1. Relative activity was calculated as activity of the variant/activity 0f Rd7BB (SEQ ID NO:44)
(encoded by SEQ ID NO:43).
2. =
- <O.5 relative ty to Rd7BB (SEQ ID NO:44);
-- = >O.5 to 1.5 relative activity over Rd7BB (SEQ ID NO:44); and
--+ = >1.5 relative activity over Rd7BB (SEQ ID NO:44);
EXAMPLE 3
In vitro Characterization of GLA Variants
tion of GLA in Yeast
In order to produce GLA-containing supernatant, replica HTP-cultures of GLA were grown as
described in Example 2. Supernatants from a es (n = 12-36) were combined prior to
further analysis.
Production of GLA in HEK293T Cells
Secreted expression of GLA variants in mammalian cells was med by transient
ection of HEK293 cells. Cells were transfected with GLA variants (SEQ ID NOS:3, 4, 9, 12, 17,
, 23, and 41) fused to an N—terminal tic mammalian signal peptide and subcloned into the
mammalian sion vector pLEV113 as bed in Example 1. HEK293 cells were transfected
with plasmid DNA and grown in suspension for 4 days using techniques known to those skilled in the
art. Supernatants were collected and stored at 4 °C.
EXAMPLE 4
Purification of GLA Variants
Purification of GLA Variants From Mammalian Cell Supernatants
GLA variants were purified from mammalian culture supernatant essentially as known in the
art (See, Yasuda et al., Prot. Exp. Pur,. 37, 499-506 [2004]). Concanavalin A resin (Sigma Aldrich)
was equilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgC12, CaClg, and MnClz pH 6.0
(Con A binding buffer). Supernatant was diluted 1:1 with binding buffer and loaded onto the
column. The column was washed with 15 volumes of Concanavalin A binding buffer, and s
were eluted by the addition of Concanavalin A binding buffer including 0.9 M methyl-(x-D-
mannopyranoside and 0.9 M methyl-(x-D-glucopyranoside. Eluted protein was concentrated and
buffer exchanged three times using a con® Plus-20 filtration unit with a 10 kDa lar
weight cut off (Millipore) into ThioGal binding buffer (25 mM citrate-phosphate, 0.1 M NaCl, pH
4.8). Buffer exchanged samples were loaded onto a Immobilized-D-galactose resin (Pierce)
equilibrated with ThioGal binding buffer. The resin was washed with six volumes of ThioGal binding
buffer and eluted with 25 mM citrate phosphate, 0.1 M NaCl, 0.1 M D-galactose, pH 5.5. Eluted
samples were concentrated using a Centricon® Plus-20 filtration unit with a 10 kDa molecular weight
cut off Purification resulted in n 24-10 ug of purified protein/ml of culture supernatant based
on Bradford quantitation.
SDS-PAGE Analysis of GLA Variants
Samples of yeast e supernatant and mammalian cell culture supernatant and purified
GLA were analyzed by SDS-PAGE. In the yeast supematants, GLA levels were too low to be
detected via this method. Bands corresponding to the ~49 kDa predicted GLA molecular weight were
found in both mammalian cell culture supematants and purified GLA s.
Immunoblot Analysis of GLA Variants
Samples of yeast supernatant and ian cell culture supernatant were analyzed by
immunoblot. Briefly, s were separated via SDS-PAGE. Protein was transferred to a PVDF
membrane using an iBlot dry blot system (Life logies). The membrane was blocked with
Odyssey blocking buffer (TBS) (LI-COR) for 1 h at RT and probed with a 1:250 on of rabbit (X-
GLA IgG (Thermo-Fischer) in Odyssey blocking buffer with 0.2% Tween® 20 for 14 h at 4 °C. The
membrane was washed 4 X 5 min with Tris-buffered saline + 0.1% Tween® 20 and probed with a
1:5000 dilution of IRDye800CW donkey (x-rabbit IgG R) in y blocking buffer with
0.2% Tween® 20 and 0.01% SDS for 1 hr at RT. The membrane was washed 4 X 5 min with Tris-
buffered saline + 0.1% Tween® 20, and analyzed using an Odyssey Imager (LI-COR). Bands
corresponding to the ~49 kDa predicted GLA molecular weight were found in both the mammalian
cell culture and yeast supematants. In S. cerevisiae expressed samples, mutants containing the
mutation E367N ran at a slightly higher MW. This on introduces a canonical NXT N—linked
glycosylation site (where X is any amino acid except P) and the possible introduction of an additional
N—linked glycan may account for the higher MW.
EXAMPLE 5
In vitro Characterization of GLA Variants
Optimization of Signal Peptide for Secreted Expression of GLA by S. cerevisiae
S. cerevisiae transformed with Mfleader-GLA (SEQ ID NO:7), SP-GLA (SEQ ID NO:36) or
a vector control were grown in HTP as described in Example 2. Cultures were grown for 48-120 h
prior to harvest of the supernatant and analysis (n = 6) as described in Example 2. Figure 1 provides a
graph showing the relative ty of different GLA constructs in S. cerevisiae after 2-5 days of
culturing. As indicated in this Figure, SP-GLA (SEQ ID NO:36) produced a high level of active
enzyme that saturated after three days of growth.
pH Stability of GLA Variants Expressed in S. cerevisiae
GLA variants were challenged with different buffers to assess the overall ity of the
enzyme. First, 50 uL of supernatant from a GLA variant yeast culture and 50 uL of McIlvaine buffer
(pH 2.86-9.27) or 200 mM sodium carbonate (pH 9.69) were added to the wells of a 96-well round
2015/063329
bottom plate (Costar #3798, Corning). The plates were sealed and incubated at 37 °C for 1h. For the
assay, 50 [AL of challenged supernatant was mixed with 25 [LL of l M citrate buffer pH 4.3 and 25 [LL
of 4 mM MUGal in Mcllvaine buffer pH 4.8. The reactions were mixed briefly and ted at 37
°C for 60-180 minutes, prior to quenching with 100 [LL of l M sodium ate. Hydrolysis was
ed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448
nm). Figure 2 provides graphs showing the absolute (Panel A) and relative (Panel B) ty of GLA
variants after incubation at various pHs.
Thermostabili of GLA Variants Ex ressed in S. cerevisiae
GLA variants were challenged at various temperatures in the presence and absence of 1 uM
l-deoxygalactonojirimycin (Migalastat; Toronto Research Chemicals) to assess the overall stability of
the enzyme. First, 50 [AL of atant from a GLA variant yeast culture and 50 uL of Mcllvaine
buffer (pH 7.65) +/- 2 mM ygalactonojirimycin were added to the wells of a 96-well PCR
plate (Biorad, HSP-960l). The plates were sealed and incubated at 30-54 °C for 1h using the gradient
program of a cycler. For the assay, 50 [AL of challenged supernatant was mixed with 25 [LL of
l M citrate buffer pH 4.3 and 25 [LL of 4 mM MUGal in Mcllvaine buffer pH 4.8. The reactions were
mixed briefly and incubated at 37 °C for 90 minutes, prior to quenching with 100 [LL of l M sodium
carbonate. ysis was analyzed using a SpectraMax® M2 microplate reader monitoring
fluorescence (Ex. 355 nm, Em. 448 nm). Figure 3 provides graphs showing the absolute (Panel A)
and relative (Panel B) actiVity of GLA variants after incubation at various temperatures.
Serum Stability of GLA Variants Expressed in S. cerevisiae
To assess the relative stability of variants in the presence of blood, samples were exposed to
serum. First, 20 [AL of supernatant from a GLA variant yeast culture and 0-80 [LL of water and 0-80
[LL of bovine serum were added to the wells of a 96-well round bottom plate (Costar #3798, Corning).
The plates were sealed and incubated at 37 °C for 1h. For the assay, 50 [AL of challenged supernatant
was mixed with 25 [LL of l M citrate buffer pH 4.3 and 25 [LL of 4 mM MUGal in Mcllvaine buffer
pH 4.8. The reactions were mixed briefly and incubated at 37 °C for 90 minutes, prior to ing
with 100 [LL of l M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate
reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). Figure 4 provides graphs showing the
absolute s A and B) and relative (Panels C and D) actiVity of GLA variants after challenge with
various tages of serum.
ve Activities of GLA Variants Expressed in HEK293T Cells
Supematants from GLA variants expressed in HEKT293T cells were serially diluted 2x with
supernatant from an non GLA expressing yeast culture. Dilutions (5O uL) were mixed with 25 [AL of
4 mM MUGal in ine Buffer pH 4.8 and 25 [LL of l M citrate buffer pH 4.3 in a Corning® 96-
well, black, opaque bottom plate. The reactions were mixed briefly and incubated at 37 °C for 60
s, prior to ing with 100 [LL of l M sodium carbonate. Hydrolysis was analyzed using a
SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). Figure 5
provides a graph showing the relative activity of GLA variants expressed in HEK293T cells.
Supernatants from cells transfected with variant GLA enzymes showed markedly higher hydrolase
activities compared to the WT enzymes, and much more activity per volume than was seen in S.
siae expression.
pH Stability of GLA Variants Expressed in HEK293T Cells
GLA variants were challenged with different buffers to assess their overall stability.
Supernatants from mammalian cell cultures were normalized to equal activities by dilution with
supernatant from a non GLA expressing culture. Normalized supernatants (50 [LL) and 50 uL of
Mcllvaine buffer (pH 4.06-8.14) were added to the wells of a 96-well round bottom plate (Costar
#3798, ). The plates were sealed and ted at 37 °C for 3 h. For the assay, 50 [LL of
challenged supernatant was mixed with 25 [LL of l M e buffer pH 4.3 and 25 [LL of 4 mM
MUGal in Mcllvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37 °C for 3 h,
prior to quenching with 100 [LL of l M sodium carbonate. Hydrolysis was analyzed using a
SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). Figure 6
provides graphs g the absolute (Panel A) and relative (Panel B) ty of GLA variants
expressed in HEK293T cells, normalized for activity, and incubated at various pHs.
All enzymes were found to be more stable versus pH challenges when compared to WT GLA
expressed in S. cerevisiae (compare with Figure 2). This difference is ly due to differential
glycosylation between expression hosts. However, it is not intended that the t invention be
limited to any particular mechanism or theory. Mutant enzymes had broader pH stability profiles
ed to the WT enzyme expressed in T.
Thermostability of GLA Variants Expressed in HEK293T cells
GLA variants were challenged at various temperatures in the presence and absence of 1 [LM
l-deoxygalactonojirimycin (Migalastat) to assess their l ity. Supernatants from
mammalian cell cultures were normalized to approximately equal activities by dilution with
supernatant from a non GLA expressing culture. Diluted supernatants were added to the wells of a
96-well PCR plate (Biorad, HSP-960l). The plates were sealed and ted at 30-54 °C for 1h
using the gradient program of a thermocycler. For the assay, 20 [LL of challenged supernatant was
mixed with 30 [LL of l M citrate buffer pH 4.3 and 50 [LL of 4 mM MUGal in Mcllvaine buffer pH
4.8. The reactions were mixed briefly and incubated at 37 °C for 90 minutes, prior to quenching with
100 [LL of l M sodium ate. Hydrolysis was analyzed using a SpectraMax® M2 microplate
reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). Figure 7 provides graphs showing the
te (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells,
normalized for activity, and incubated at various temperatures. As shown in this Figure, all of the
enzymes were more stable after temperature challenges when ed to WT GLA expressed in S.
cerevisiae (compare with Figure 2), likely due to differential glycosylation between expression hosts.
In the GLA variants (SEQ ID NOS:10 and 13) the TIn of the enzyme was increased by 2 and 4°C
tively. Addition of Migalastat increased the Tm by 5.5 °C, r at a 0.2 uM final
tration in the assay, activity in the Migalastat treated sample was reduced by ~60%.
Activity ofWT GLA and GLA Variants on an Alternative Substrate
To confirm that improved activity in MUGal hydrolysis corresponded to more native
substrates, mammalian cell-expressed GLA variants were assayed using N—Dodecanoyl-NBD-
ceramide trihexoside (NBD-GB3) as ate. HEK293T culture supernatant (10 uL), 100 mM
sodium citrate pH 4.8 (80 uL), and NBD-GB3 (0.1 mg/ml) in 10% ethanol (10 uL) were added to
microcentrifuge tubes. Samples were inverted to mix, and incubated at 37 °C for 1 h. The reaction
was quenched Via addition of 50 uL methanol, diluted with 100 uL chloroform, ed and the
organic layer was isolated for analysis. The organic phase (10 uL) was spotted onto a silica plate and
analyzed by thin layer chromatography (chloroform:methanol:water, 100:42:6), detecting the starting
material and product using a 365 nm UV lamp. Significant conversion was observed only with SEQ
ID NO:13, confirming that the variant exhibits improved actiVity, as compared to the WT GLA.
Specific Activity of GLA ts
GLA variants purified as described in Example 4, were evaluated for their specific activity.
Between O-O.25 ng of d enzyme was added to 4 mM MUGal in McIlvaine buffer pH 4.8 (final
pH of 4.8). s were incubated for 60 min at 37 °C and quenched Via on of 100 uL of 1 M
sodium ate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring
cence (Ex. 355 nm, Em. 448 nm), and ated to absolute amounts of 4-methylumbelliferone
through the use of a standard curve.
pH Stability of Purified GLA Variants Over Time
To confirm that purified GLA variants show the desired pH stability observed after
expression in yeast, WT GLA (SEQ ID NO:5) and SEQ ID NO:42 were incubated in acidic or basic
buffers and analyzed for residual activity. GLA variants (200 ng) were added to McIlvaine buffer pH
4.1 or 7.5 and incubated for 0-24 h at 37 °C. Samples (50 uL) were added to a mixture of 25 uL 1M
citric acid pH 4.3 and 25 uL of 4 mM MUGal in McIlvaine buffer pH 4.8, and incubated at 37 °C for
1h. Samples were quenched with 100 uL of 1 M sodium carbonate, diluted 1:4 in 1 M sodium
carbonate and analyzed by fluorescence spectroscopy (Ex. 355, Em. 448). SEQ ID NO:42 was
erably more stable under both acidc and basic challenge conditions confirming that stability
advances ped in yeast ated to the protein expressed in mammalian cells (See Figure 8 for
graphs of the results).
Thermostability of Purified GLA Variants sed in HEK293T Cells
The stability of WT GLA (SEQ ID NO:5) and SEQ ID NO:42 were determined to
assess their overall stability. Purified enzyme as described in Example 4 was diluted to 20 [Lg/ml in
1x PBS with 1x Sypro Orange (Thermo Fischer Scientific), and added to a 96-well PCR plate (Biorad,
01). The plates were heated from 30 to 75 °C at 0.5 °C/min on a RT-PCR machine and Sypro
Orange fluorescence was monitored. Under these conditions WT GLA melted at 37 °C, while SEQ
ID NO:42 melted at 55 °C
EXAMPLE 6
In vivo Characterization of GLA Variants
Serum Pharmacokinetics of Purifed GLA Variants
Purified GLA ts produced as described in Example 4 were assessed for stability in the
serum of live rats. WT GLA (SEQ ID NO:5) or SEQ ID NO:42 at 1 mg/ml were administered
intravenously at 1 ml/kg to three na'1've jugular vein cannulated Sprague-Dawley rats (7-8 weeks old)
each. Prior to administration and at 5, 15, 30, 60, 120, and 240 minutes post-administration, 200 [AL
of blood was collected from each rat in an EDTA tube and centrifuged at 4°C and 6000 rpm to
te >80 [1L of serum per sample. Samples were frozen and stored on dry ice prior to analysis.
For analysis, serum (10 uL) was added to 40 [AL of 5 mM MUGal in McIlvaine buffer pH 4.4, and
incubated at 37 °C for 1h. Samples were quenched with 50 [AL of l M sodium carbonate, diluted
1:100 in l M sodium ate and ed by fluorescence spectroscopy (Ex. 355, Em. 448). Four
hours post-administration SEQ ID NO:42 retained 15.3% of maximal activity, while WT GLA
retained only 0.66% (See, Figure 9).
EXAMPLE 7
Deimmunization of GLA
In this Example, experiments conducted to identify diversity that would remove predicted T-
cell epitopes from GLA are described.
Identification of Deimmunizing ity:
To identify mutational diversity that would remove T-cell epitopes, computational methods
were used to identify GLA subsequences that were predicted to bind efficiently to representative HLA
receptors. In addition, experimental searches for amino acid mutations were conducted, particularly
for mutations that do not affect GLA activity (e.g., in the assays bed in Example 2). The amino
acid sequences of active variants were then analyzed for predicted genicity using
computational methods.
Computational Identification of Putative T-cell Epitopes in a WT GLA:
Putative T-cell epitopes in a WT GLA (SEQ ID NO:5) were identified using the Immune
Epitope Database (IEDB; Immune Epitope Database and is Resource website) tools, as known
in the art and proprietary statistical analysis tools (See e.g., iedb.org and Vita et al., Nucl. Acids Res.,
38(Database issue):D854-62 [2010]. Epub 2009 Nov 11]). The WT GLA was parsed into all possible
-mer analysis frames, with each frame overlapping the last by 14 amino acids. The 15-mer analysis
frames were evaluated for immunogenic potential by scoring their 9-mer core regions for predicted
binding to eight common class II HLA-DR alleles (DRB1*0101, 301, DRB1*0401,
DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) that collectively cover
nearly 95% ofthe human population (See e.g., Southwood et al., J. Immunol., 160:3363-3373 [1998]),
using methods recommended on the IEDB website. ial T-cell epitope clusters contained within
the enzyme (i.e., sub-regions ned within GLA which have an unusually high potential for
immunogenicity) were identified using tical analysis tools, as known in the art. The identified T-
cell epitope clusters were screened against the IEDB database of known epitopes. These screens
identified five putative T-cell epitopes in the WT enzyme. These epitopes are referred to as TCE-I, II,
III, IV, and V below.
Design of Deimmunizing Libraries:
First, the sequences of active GLA mutants identified in Example 2 are assessed for the
presence of T-cell epitopes. Mutations identified to potentially reduce binding to the HLA—DR alleles
are incorporated into a recombination library. Additional libraries are prepared using saturation
mutagenesis of every single amino acid within the five T-cell epitopes. Hits from these libraries are
subjected to further rounds of tion mutagenesis, HTP screening, and recombination to remove
all le T-cell epitopes.
uction and ing of nizing Libraries:
Combinatorial and saturation mutagenesis libraries designed as described above were
constructed by methods known in the art, and tested for activity in an unchallenged assay as described
in Example 2. Active variants were identified and sequenced. Their activities and mutations with
respect to WT GLA are ed in the table below.
Identification of nizing Diversity:
Active variants were analyzed for their levels of predicted immunogenicity by evaluating
their binding to the eight common Class II HLA-DR alleles as described above. The total
immunogenicity score and immunogenic hit count are shown in Table 7.1. The total immunogenicity
score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a
higher level of ted immunogenicity). The genic “hit count” (IHC) indicates the number
of lS-mer analysis frames with an unusually high potential for immunogenicity (i.e., a higher score
tes a higher potential for immunogenicity). Mutations ing in a lower total immunogenicity
score and/or an immunogenic hit count less than that of the reference sequence were considered to be
potential “deimmunizing mutations” A collection of the most deimmunizing mutations were
recombined to generate a number of variants that were active and predicted to be significantly less
immunogenic than WT GLA. In the following Table, total immunogenicity score (T18) and
immunogenic hit count (IHC) are provided.
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
# : Active Mutations
A339S L»)
A350G L»)
A66T/K206A/F2171UL3 l6D/M3221/A337P/K343G/A350G/E3
67N/R373K
L») 00 DlOSS L»)
00 U] D124N/El47G/Nl6lK/Rl62Q/Tl63V/Rl65A/ll 67S/Vl 68l/Yl
L») 0 69V/S l 70-/Ml 77S/F217E L»)
D2E/L441VY92H/K206A/F217R/N247D/Q302K/L316D/M3221.
/Q326G/A337P/K362Q/E367N/R373K [\J
D2Q/L44R/Y92H/K206A/F2 l 7R/N247D/Q302K/L3 221
/A337P/K362Q/E367N/R373K [\J
Q302K/L3 l 6D/M3221/A337P/K362Q/E367N/R373K/M392T
44 90 E387K 38
2015/063329
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
4# : Active ons
E387R U) 00
E4OD
2I/A337P/K362Q/E367N/R373K
E4OS/L44R/Y92H/K206A/F2 1 7R/N247D/Q3O2K/L3 1 6D/M322
I/A337P/K362Q/E367N/R373K 407
.b 00 E43D 450 LAN \lU1
[\D 0
[\D 00
.b 00
U1 48D/N247D/Q3O2K/R373K 442
U1 U1 E43D/E48D/Q302K/R373K/I376V 442
1—1 0 U] 0 E43D/I208V/N247D 435
U1 \] E43D/I208V/N247D/Q2991UR373K/I376V 435
12 58 E43D/I208V/Q299R/R373K/I376V 436
703 E43D/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/A261G/
H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/W368A/
R373K/M392T 315
725 E43D/L441US47T/Y92H/Sl66P/K206A/F217R/N247D/M253W
/A257G/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/
E367N/W368A/R373K/M392T
D/M322I/A337P/K362Q/E367N/R373K [\D
D/M322I/A337P/K362Q/E367N/R373K
441VY92N/S 1 66P/K206A/F2 1 7R/N247D/Q3O2K/L316
D/M322I/A337P/K362Q/E367N/R373K
E43D/L44R/Y92S/Sl66P/K206A/F217R/N247D/Q302K/L316
D/M322I/A337P/K362Q/E367N/R373K
E43D/N247D/R373K/I376V E!”U)44 E43D/R373K/I376V 44
377 E43Q/L44R/Y92E/S 1 66P/K206A/F2 1 7R/N247D/Q302K/L3 1 6
D/M322I/A337P/K362Q/E367N/R373K 370
E48D/I208V/Q299R/Q302K/R373K 437
O\ [\D E48D/R373K/I376V 443
p—A i.b
U1 i0
U1 U1 0
52 98 F217D 450 38
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
# : Active Mutations
Ult—‘Ul 450
16040 452
F3651
F365K
U]0 U) 00
G303Q/R373V
._i 0119 U1 ._i
O\ O U1 U1
O\ 111
._i ._i NfiUl CON
0\l 437
O\ 00 ._i._i ._i._i .bw 450
\]O\ 00 450
IlO2L/L394V 449
\] ._i 1123T/T369N 449
\][\D 1167V U) 00
[\D U) OOOUlUl
[\D 0
A350G 429
K206A/A350G/K362Q/T369A 413
K206A/A350G/T369D 426
K206A/A350G/T369S 429
K206A/E367A/T369D 439
K206A/E367D 427
O6A/F217R/G23OV/N247D/Q3O2K/M322I/E367N/T369S/R
373K
Table 7.1 Total Immunogenicity Score (TIS), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
_N- Active ons
K206A/F217R/N247D/L316D/A350G/E367D/T369D
:-. K206A/F217R/N247D/L316D/M3221/A337P/A350G/K362Q/E .I7298 373K 420 37
340 K206A/F217R/N247D/Q249H/Q302K/M3221/K343G/A350G/E
367T/R373K/L397F 4o
K206A/F217R/N247D/Q302K/L316D/A337P/A350G/E367D/T
369D
K206A/F217R/Q302K/L3 1 6D/A337P/A350G/E367D/T369D 411 37
I...211 K206A/F365L/E367N/I376V 40
_- K206A/F365L/E367N/R373K/I376V
_% K206A/1208V/M322V/K343G/F365L/R373K/I376V K206A/1208V/R221K/N247D/M3221/K343D/F365L/R373K/I3
K206A/1208V/R221T/N247D/M322V/K343G/E367N/R373K
K206A/K343D/F365L/E367N 41
K206A/K343G
_fl—&K206A/K343G/F365L/E367N/R373K 40
302 343 K206A/M322V/K343G/E367N/R373K 425 38
220 261 M322V/R373K/I376V 422 37
221 262 K206A/M3901 414 33
_-I303 76V 440 40
K206A/N247D/M322V/K343D/R373K/I376V
_- K206A/N247D/M322V/K343G/F365L/R373K -37
_% K206A/N247D/Q302K/A337P/K343G/A350G -
K206A/N247D/Q302K/L3 1 6D/A350G 38
K206A/N247D/Q302K/M322V/F365L/R373K/I376V 37
K206A/P228Q/T369D 38
K206A/Q302K/A337P/A350G/K362Q 38
K206A/Q302K/L316D/A337P 38
K206A/Q3OZK/L3 1 21/A337P/A350G/K362Q/E367N/T
369S/R373K 42
-_373K _-
n_247288 K206A/R221K/N247D/M322V/K343D/R373K .-
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
#6 ° Active Mutations
347. 441
348 436
267 427
289 418
U) \]
R325H
R373K
K206A/R373K/I376V U) \l
K206A/S374R
K206A/T369S
[\D 0
[\D 0
\1 U] 00
d 1—1 U1 0
\] U1 0
K206R 450
K206s 429
\l\l OO\] K206T/V359S 437
\]0 445
000 345
1—1 N .50
)—k [\D U1 [\D
1—1 N U] 0
U1 O
1—1 N L») N
1—1 N
L» .5 oo
00 \] 131451
00 00 132 450
000 133 448
90 134 K961 433
250 291 K96I/K206A/F217R 412
308 349 K96I/K206A/F2171VM3221/E367N/T369S/R373K 434 .5O
292 K96I/K206A/F217R/N247D 41 1
293 K96I/K206A/F217R/N247D/A350G/E367D/T369D 401
K96I/K206A/F217R/N247D/Q302K/L316D/A337P/E367D/T36
9D 393 35
K96I/K206A/F217R/N247D/Q3OZK/M3221/A337P/K343G/A35
0G/E367N/R373K 413 36
_-K96I/K206A/N247D/M3221/A350G/E367N/T369S/R373K
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
Active Mutations
K961/K206A/N247D/Q3O2K/L3 1 6D/M3221/A337P/A350G/E36
9S/R373K
K961/K206A/N247D/Q3O2K/L3 1 6D/M3221/A337P/A350G/K3
62Q/E367N/T369S/R373K
_35-4—--L1OOF/Al2SS/K206A/1208V/R221K/Q302K/M322I/K343G/E3
67N/R373K
L100F/K206A
L1OOF/K206A/I208V/N247D/Q302K/M322V/K343D/E367N/R
373K/1376V
L100F/K206A/1208V/Q302K/M322V/F365L/E367N/R373K/13
L1 OOF/K206A/I208V/R22 1 K/M322V/K343D/E367N/R373K
--L1OOF/K206A/1208V/R221K/M322V/K343D/F365L/E367N/R3 _-073K 40
L1OOF/K206A/I208V/R221K/N247D/Q302K/M3221/K343D/F3
65L/I376V
L1OOF/K206A/I208V/R221K/N247D/Q302K/M322V/K343D/F
365L/1376V
L1OOF/K206A/I208V/R221T/M322V/E367N/R373K/1376V I.
L1OOF/K206A/I208V/R221T/N247D/K343D/F365L/1376V
L100F/K206A/1208V/R221T/Q302K/M322I/K343D/1376V
L1OOF/K206A/M322I/E367N/R373K/1376V
_&—--L1OOF/K206A/M322V/F365L/R373K/1376V 37
_---—
303 L100F/K206A/R221K/N247D/Q302K/M322V/F365L/R373K/1
262 376V 411 37
263 304 K206A/R221K/N247D/Q302K/M322V/1376V 413 37
305 L1 OOF/K206A/R221K/N247D/Q302K/M322V/K343D/R373K/1
376V 37
L1OOF/K206A/R221T/M322I/K343E/F365L/R373K 37
L1 OOF/K206A/R22 1 T/N247D/Q3O2K/K343D/F365L/R373K 37
L1 OOF/K206A/R221T/Q3O2K/M3221/K343D/E367N/R373K 3 8
K206A/R373K/1376V 37
L1OOF/Ll6OI/K206A/R221K/M322V/E367N/R373K 42
44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/H271A/
Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T 8
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant ID
# N0° Active ons
140 L1581 458 42
97 1 4 1 L158M 450 40
142 L158R 431 35
143 L23M 450 3 8
324 365 L23 S/K206A/M3221/E367N/R373K 442 3 8
1 00 144 L23T 450 3 8
101 145 L316D 448
102 146 L316E 448
269 310 L37I/K206A/R221K/N247D/M3221/R373K 434
103 147 448
104 148
106 ._1._1 Ul-b WNHOO
109 ._1._1 UIUI Jk-lk-P-P-P Jk-lkUlUl-b NOOOO WWWUJUJ O\\]OOOO\]
110 154
111 155
112 156
113 157
114 158
115 159
116 1—1 0\ 0
117 161
119 1—11—1 00 LAN
120 ._. O\ .1; ##Jk-P-P #Ul-PUl-P ©0000 mmmmm \IOO\]OO\]
409 L44A/K206A/F217R/N247D/Q302K/L316D/M3221/A337P/K3
L» O\
403 L44C/K206A/F217R/N247D/Q302K/L316D/M3221/A337P/K3
362 67N/R373K L» N
_360 62Q/E367N/R373K ././. L» N
_374 62Q/E367N/R373K .UU. L» U)
411 L44Q/K206A/F217R/N247D/Q302K/L316D/M3221/A337P/K3
370 67N/R373K U) 0
43 9 L44R/A159S/K206A/F217R/N247D/Q3OZK/L316D/M3221/A3
398 37P/K362Q/E367N/R373K L» .1;
561 L44R/A77S/Y92H/K206A/F217R/N247D/Q302K/L316D/M322
520 I/A337P/K362Q/E367N/R373K 393
270 311 L441UC143Y/K206A/A337P/A350G 430 LAN
562 L44R/D52N/Y92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
521 21/A337P/K362Q/E367N/R373K 393 I“
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variant
_1:?(: Active Mutations
L44R/E187G/K206A/A337P/A350G
L44R/E56K/Y92H/K206A/F217R/N247D/Q302K/L316D/M32
522 2I/A337P/K362Q/E367N/R373K 393 24
423 L44R/H94N/K206A/F2 1 7D/Q3O2K/L316D/M322I/A3 3
7P/K362Q/E367N/R373K N D 3O
L441UH941UK206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3 3
7P/K362Q/E367N/R373K
L44R/K206A
273 314 L441UK206A/E367D/T369D ?2.-
274 315 L441UK206A/F2171VA350G
275 316 L441UK206A/F217R/N247D/A337P
480 L44R/K206A/F217R/\247D/H271A/Q302K/L316D/VI322I/A3
439 37P/K362Q/E367\/R373K
466 L44R/K206A/F217R/\247D/H271E/Q302K/L316D/VI322I/A3
425 37P/K362Q/E367\/R373K
477 206A/F217R/\247D/H271G/Q302K/L316D/VI322I/A3
436 37P/K362Q/E367\/R373K \. D. 36
474 L44R/K206A/F217R/\247D/H271Q/Q302K/L316D/VI322I/A3
433 37P/K362Q/E367\/R373K
493 L441UK206A/F217R/\247D/H2711UQ302K/L316D/VI322I/A3
452 37P/K362Q/E367\/R373K -,
471 L44R/K206A/F217R/\247D/H271T/Q302K/L316D/VI322I/A3
430 37P/K362Q/E367\/R373K
482 L44R/K206A/F217R/\247D/H271V/Q302K/L316D/VI322I/A3
441 37P/K362Q/E367\/R373K U) O\
476 L441UK206A/F217R/\247D/I258L/Q302K/L316D/M322I/A33
435 7P/K362Q/E367N/R373K U) 4;
L441UK206A/F217R/N247D/1258M/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K \.D. U) o
-_37P/K362Q/E367N/R373K U) 4;
/K362Q/E367N/R373KL441VK206A/F217R/N247D/L255E/Q3O2K/L316D/M3221/A3 U) 4;
37P/K362Q/E367N/R373K U) 4;
L44R/K206A/F217R/N247D/L255S/Q302K/L316D/M322I/A33
7P/K362Q/E367N/R373K U) m
L441UK206A/F2 1 7R/N247D/L255T/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K U) m
L44R/K206A/F2 1 7R/N247D/L255V/Q3O2K/L316D/M322I/A3
62Q/E367N/R373K \.D. U) o
II—37P/K362Q/E367N/R373K U) U)
II—37P/K362Q/E367N/R373KL44R/K206A/F217R/N247D/L263F/Q3O2K/L316D/M322I/A33 N\o
7P/K362Q/E367N/R373K 34
2015/063329
Table 7.1 Total Immunogenicity Score (T18), and genic Hit Count (IHC) for GLA
Active Mutations
L44R/K206A/F217R/N247D/L263G/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L441UK206A/F2 1 7R/N247D/L263W/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
206A/F217R/N247D/L316D/A337P/A350G/E367D/T3
L441VK206A/F217R/N247D/L316D/A337P/E367D/T369D
L441UK206A/F217R/N247D/L316D/A350G/E367D/T369D
L44R/K206A/F217R/N247D/L316D/M322I/A337P/K343G/K3
62Q/E367N/R373K
L441UK206A/F2 1 7R/N247D/M259A/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L44R/K206A/F2 1 7R/N247D/M259E/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
---37P/K362Q/E367N/R373K N D
-_-I37P/K362Q/E367N/R373K N D
L44R/K206A/F217R/N247D/M259W/Q302K/L316D/M322I/A
337P/K362Q/E367N/R373K N D
279 320 L44R/K206A/F217R/N247D/Q302K/A350G
367 L44R/K206A/F217R/\247D/Q302K/L316D/VI3221/A337P/K3
326 62Q/E367N/R373K 37
554 L44R/K206A/F217R/\247D/Q302K/L316D/VI3221/A337P/K3 42
513 62Q/E367N/R373K/D396* . . 32
553 L44R/K206A/F217R/\247D/Q302K/L316D/VI3221/A337P/K3 --
512 62Q/E367N/R373K/K395* \. D. 32
549 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
508 62Q/E367N/R373K/L384A \ D 30
518 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
477 62Q/E367N/R373K/L384W
515 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
474 62Q/E367N/R373K/L386F
543 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
502 62Q/E367N/R373K/L386S
501 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
460 62Q/E367N/R373K/L386T
556 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
515 62Q/E367N/R373K/L394*
552 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
511 62Q/E367N/R373K/L397*
541 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
500 67N/R373K/M390A
527 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
486 62Q/E367N/R373K/M390C
531 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
490 62Q/E367N/R373K/M390D
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390E
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390F
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390G
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390H
538 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390K
495 206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390P
506 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390Q
521 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390R
545 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M39OS
504 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
67N/R373K/M390T
537 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390V
532 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M390W
498 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392A
524 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
67N/R373K/M392C
496 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392D
507 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392E
520 206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392F
542 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392G
539 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392I
513 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392K
514 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392L
546 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392N
534 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
67N/R373K/M392P
502 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
461 62Q/E367N/R373K/M392Q
2015/063329
Table 7.1 Total Immunogenicity Score (TIS), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392S
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392T
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392V
L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/M392W
544 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/Q385C
523 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
67N/R373K/Q385G
510 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/Q3 851
503 206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/Q3 85L
550 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/Q3 85T
547 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/Q385W
555 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/S393*
533 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T3 89C
516 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T389D
528 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T389G
530 206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T3 891
540 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T3 89L
497 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T389M
529 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T389N
536 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T3 89P
509 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T389Q
508 L44R/K206A/F217R/\247D/Q302K/L316D/VI322I/A337P/K3
62Q/E367N/R373K/T3 89S
Q/E367N/R373K/T389W 30
----50G/K362Q/E367N/R373K
L441UK206A/F2 1 7R/N247D/R270D/Q302K/L316D/M3221/A3
37P/K362Q/E367N/R373K
Table 7.1 Total Immunogenicity Score (T18), and genic Hit Count (IHC) for GLA
Variants
Active Mutations
L441UK206A/F2 1 7R/N247D/R270G/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
206A/F2 1 7R/N247D/R27OL/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
206A/F2 1 7R/N247D/R27OQ/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K N. D.
L44R/K206A/F2171UQ302K/E367D/T369D 26
---9S/R373K
L441VK206A/I208V/N247D/Q3O2K/M322I/K343D/E367N/R3
L44R/K206A/I208V/R221K/M322I/K343D/E367N/R373K
L44R/K206A/I208V/R221K/M322V/K343D/F365L/R373K 37
-III67N/R373K/I376V
L44R/K206A/I208V/R221T/Q3O2K/M322I/K343G/F365L/E36
7N/R373K/I376V
L441VK206A/L316D/M322I/A337P/A350G/E367N/T369S/R37
_?—-O-42L441UK206A/N247D/L316D/M322I/A350G/K362Q/E367N/T3
69S/R373K
L44R/K206A/N247D/Q302K/A337P/A350G/E367D/T369D
L44R/K206A/N247D/Q3O2K/L3 1 6D/M322I/A337P/K343 G/A3
50G/K362Q/E367N/T369S/R373K 432
L44R/K206A/N247D/Q3O2K/M322I/A350G/E367N/T369S/R3
73K 457
_&L44R/K206A/N247D/Q3O2K/M322I/K343D/E367N/R373K 442
L44R/K206A/R221T/N247D/M3221/K343D/F365L/I376V 432
_- L441VK96I/K206A 419
_- L44R/K96I/K206A/F217R/N247D 418
-_II338 Q/E367N/R373K 410 35
-IIN/R373K
L441UK96I/K206A/F2 1 7R/N247D/M322I/A350G/K362Q/E367
N/T369S/R373K
L441VK96I/K206A/F2 1 7R/N247D/M322I/E367N/T369S/R373
I_IIOG/E367D/T369D 35
II—IIP/E367N/R373K
L441UK96I/K206A/F217R/N247D/Q3O2K/M322I/E367N/T369
S/R373K
344 385 L441UK96I/K206A/F2 1 7R/N247D/Q3O2K/M322I/K362Q/E367 418 35
2015/063329
Table 7.1 Total genicity Score (TIS), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
Active Mutations
—_—-
--E/Q29OP/L293F/Q302K/V308G/S314F/M322I/A337P/K343E/E.-345 L441VK961/K206A/F2171VQ219P/N247D/M253K/S266F/D284
367N/R373K 429 41
_---—
961/K206A/F2 1 71VQ3O2K/M322I/A350G/K362Q/E367
L441UK961/K206A/M322I/A337P/E367N/T369S/R373K 40
_III
L44R/L1 OOF/K206A/F365L
L44R/L1OOF/K206A/I208V/Q219H/N247D/Q302K/M322V/K3
43D/R373K/I376V 416 37
L441UL1OOF/K206A/1208V/R221K/M322I/K343G/F365L/E367
K 442 40
L44R/L1OOF/K206A/I208V/R221K/N247D/Q302K/M322V/F3
65L/I376V 37
_IIIN/R373K 40
-_II350 3K/I376V 427 38
L44R/L1OOF/K206A/1208V/R221T/N247D/M322V/I376V
336 L44R/L100F/K206A/I208V/R221T/N247D/Q302K/M322I/K34
295 3D/F365L/R373K/I376V 424 37
392 L44R/L1OOF/K206A/I208V/R221T/Q302K/M322I/E367N/R37
3K/I376V 38
_%L44R/L1OOF/K206A/Q302K/M3221/E367N/R373K/I376V L441VL1OOF/K206A/R221K/M322I/F365L/E367N/R373K/1376
L441UL1OOF/K206A/R221T/M3221/F365L/E367N/R373K
36 L441UL1OOF/K206A/R221T/N247D/M3221/K343D/E367N/R37
II—II3K/I376V 38
356 3K 440 38
_II357 73K/I376V 427 38
400 L44R/L100F/Q181L/K206A/R221T/N247D/Q302K/M322V/E3
359 67N/R373K/I376V 429 38
441 L44R/L158C/K206A/F217R/N247D/Q302K/L316D/M322I/A3
400 37P/K362Q/E367N/R373K N. D. 34
_II37P/K362Q/E367N/R373K N D 34
_III37P/K362Q/E367N/R373K N D
L44R/L158H/K206A/F217R/N247D/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
2015/063329
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L44R/L158M/K206A/F217R/N247D/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L44R/L158Q/K206A/F217R/N247D/Q302K/L316D/M322I/A3
37P/K362Q/E367N/R373K
158R/K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K N D
3 84F 35
----37P/K362Q/E367N/R373K N D
L44R/N91M/Y92H/K206A/F217R/N247D/Q3O2K/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
565 L44R/N91V/Y92H/K206A/F2 1 7R/N247D/Q3O2K/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
566 L44R/Q76H/Y92H/K206A/F2 1 7R/N247D/Q3O2K/L3 1 6D/M32
2I/A337P/K362Q/E367N/W368A/R373K
464 L441VR162A/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
457 L441VR162G/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
451 162H/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
447 L441VR162K/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
462 L441VR162Q/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
45 8 L441VR162S/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
165H/K206A/F217R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367\/R373K
L441VR165K/K206A/F217R/N247D/Q302K/L316D/M322I/A3
37P/K362Q/E367/R373K
L44R/S 1 66A]K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L44R/S 1 66D/K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L44R/Sl66E/K206A/F217R/N247D/Q302K/L316D/M322I/A33
7P/K362Q/E367N/R373K
L441US]66F/K206A/F217R/N247D/Q302K/L316D/M322I/A33
7P/K362Q/E367N/R373K
L44R/S 1 66H/K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
42 L441US 1 66P/K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3 3
402 7P/K362Q/E367N/R373K \. D. 34
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
# ° Active Mutations
L441VS 1 66R/K206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3
37P/K362Q/E367N/R373K
L44R/Sl66T/K206A/F217R/N247D/Q302K/L316D/M322I/A33
7P/K362Q/E367N/R373K
L44R/S47D/K206A/F2 1 7D/Q3O2K/L316D/M322I/A3 3
7P/K362Q/E367N/R373K
L44R/S47I/K206A/F217R/N247D/Q302K/L316D/M322I/A337
Q/E367N/R373K
_-7P/K362Q/E367N/R373K N D
I.K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T
L441US47N/Sl66P/K206A/F217R/N247D/H271A/Q302K/L316
D/M322I/A337P/K362Q/E367N/R373K/M390Q
668 L44R/S47N/Y92H/S 1 66P/K206A/F2 1 7D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H 350 12
654 L44R/S47N/Y92H/S 1 66P/K206A/F2 1 7R/N247D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q 351 12
672 L44R/S47N/Y92H/S 1 66P/K206A/F2 1 7R/N247D/H271A/Q302
D/M322I/A337P/K362Q/E367N/R373K/M392T 352 12
667 L441US47N/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271
A]Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M390H
/M392T 31 1 5
663 L441US47N/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271
A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q
/M392T 305 5
656 L441US47N/Y92H/Sl66P/K206A/F217R/N247D/Q302K/L316
D/M322I/A337P/K362Q/E367N/R373K/M390H
402 L441US471UK206A/F2 1 7R/N247D/Q3O2K/L316D/M322I/A3 3
7P/K362Q/E367N/R373K
671 L44R/S47T/A53 S/Y92H/Sl66P/K206A/F217R/N247D/H271A/
Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M39OQ
420 L441US47T/K206A/F217R/N247D/Q302K/L316D/M322I/A337
P/K362Q/E367N/R373K . .
;:—-/Q3O2K/L316D/M322I/A337P/K362Q/E367N/R373K/M392TL441US47T/P67T/Y92H/Sl66P/K182N/K206A/F217R/N247D/
H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T 338
L441US47T/Sl66P/K206A/F217R/N247D/H271A/Q302K/L316
D/M322I/A337P/K362Q/E367N/R373K/M390Q 378 21
_-nL44R/S47T/W64L/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392TA]Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
_-nA]Q3O2K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T
2015/063329
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L441VS47T/Y92H/Sl66P/K206A/F217R/L237P/N247D/H271A
/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q290
K/L3 1 2I/A337P/K362Q/E367N/W368A/R373K
/M392T
659 L441US47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302
K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K
660 L441US47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367\/R373K/M390H
661 47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367\/R373K/M39OQ
44 L441VS47T/Y92H/S 1 66P/K206A/F2 1 7R/\247D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367\/R373K/M392T
713 L441US47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302
K/L316D/M322I/A337P/K362Q/E367N/R373K/N377Y/M392T
733 L441US47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/Q302
K/L3 1 6D/M322I/A337P/K362Q/E367N/W368A/R373K/M392
L441US47T/Y92H/S 1 66P/K206A/F2 1 7R/\247D/H271A/Q302
K/L316Y/M322I/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/S 1 66P/K206A/F2 1 7R/\247D/H271A/Q302
K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T
729 L441US47T/Y92H/S 1 66P/K206A/F2 1 7D/H271A/Q302
K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T 345
724 L441VS47T/Y92H/S 1 66P/K206A/F2 1 7R/\247D/H271A/Q302
K/N305L/L3 1 6D/M322I/A337P/K362Q/E367N/W368A/R373K
/M392T 340 7
718 L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/M253W/A257
G/H271A/K2771VQ281L/Q302K/L316D/A319D/M322I/A337P
/E367N/R373K/M392T
717 L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/M253W/H271
D/P274S/K277R/Q302K/L316D/M322I/A337P/K362Q/
E367N/R373K/M392T
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/M259E/H271
A]Q302K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M39OQ
L441VS47T/Y92H/Sl66P/K206A/F217R/N247D/M259E/Q302 --
K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271
A]Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M390H
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/M259W/H271
A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M390Q 312 1
L441US47T/Y92H/Sl66P/K206A/F217R/N247D/P262S/H271A
/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T 8
L44R/S47T/Y92H/S 1 66P/K206A/F2 1 7R/N247D/Q3O2K/L316
D/M322I/A337P/K362Q/E367N/R373K
L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/W256L/H271
A]Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
—104—
WO 05889
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L441VS47T/Y92H/Sl66P/K206A/F217R/P228L/N247D/H271A
/Q3OZK/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/Sl66P/K206A/F217R/P228Q/N247D/H271
A]Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/Sl66P/K206A/F217R/P234H/N247D/H271
A]Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/Sl66P/K206A/F2171VV23 8I/N247D/H271A
/Q3OZK/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L441US47T/Y92H/Sl66P/K206A/F217R/W246P/N247D/A261
G/H271A/Q3OZK/N3OSL/L3 1 6D/M3221/A337P/K362Q/E367N
/R373K/M392T
G/H271A/L441US47T/Y92H/Sl66P/K206A/F217R/W246P/N247D/A261Q3OZK/N3OSL/L3 1 6D/M3221/A337P/K362Q/E367N/W368A/R373K/M392T
L44R/S47T/Y92H/Sl66P/P174S/K206A/F217R/N247D/H271A
/Q3OZK/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L44R/S47T/Y92H/Sl66P/W195C/K206A/F217R/N247D/H271
A]Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
L44R/S47V/K206A/F217R/N247D/Q302K/L316D/M3221/A33
7P/K362Q/E367\/R373K
163 S/K206A/F217R/N247D/Q302K/L316D/M3221/A33
7P/K362Q/E367\/R373K
L44R/V93L/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
2Q/E367\/R373K
L44R/V93 S/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
L44R/V93T/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
L44R/Y92A/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
92C/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
L44R/Y92E/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
L44R/Y92G/K206A/F217R/\247D/Q302K/L316D/VI3221/A33
7P/K362Q/E367\/R373K
569 L441UY92H/D130Q/K206A/F217R/\1247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K
570 L441UY92H/K182A/K206A/F217R/\1247D/Q302K/L316D/M3
37P/K362Q/E367\/R373K
57] L44R/Y92H/K1 82E/K206A/F2 1 47D/Q302K/L3 1 6D/M3
221/A337P/K362Q/E367\/R373K
572 L441UY92H/K182H/K206A/F217R/\1247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K
573 L44R/Y92H/K182M/K206A/F217R/N247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K
574 L441UY92H/K182Q/K206A/F217R/N247D/Q302K/L316D/M3
221/A337P/K362Q/E367\/R373K
Table 7.1 Total genicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L44R/Y92H/K182R/K206A/F217R/N247D/Q302K/L316D/VI3
22I/A337P/K362Q/E367\/R373K
L44R/Y92H/K182T/K206A/F217R/N247D/Q302K/L316D/VI3
22I/A337P/K362Q/E367\/R373K
L441UY92H/K182V/K206A/F217R/N247D/Q302K/L316D/VI3
37P/K362Q/E367\/R373K
L441UY92H/K182Y/K206A/F217R/N247D/Q302K/L316D/VI3
22I/A337P/K362Q/E367\/R373K
579 L44R/Y92H/K206A/F2 1 7R/N247D/A287C/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367\/R373K
580 L441VY92H/K206A/F2 1 7R/N247D/A287H/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367\/R373K
58] 92H/K206A/F2 1 7R/N247D/A287M/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\/R373K
582 L441UY92H/K206A/F2 1 7R/N247D/K283A/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\/R373K
583 L441VY92H/K206A/F2 1 7R/N247D/K283G/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\/R373K
584 L44R/Y92H/K206A/F2 1 7R/N247D/K283M/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\/R373K
585 L441VY92H/K206A/F2 1 7R/\247D/K283V/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367\/R373K
586 L441VY92H/K206A/F2 1 7R/\247D/K295A/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367\/R373K
587 L44R/Y92H/K206A/F217R/\247D/K295E/Q302K/L316D/VI3
22I/A337P/K362Q/E367\/R373K
588 L44R/Y92H/K206A/F217R/\247D/K295L/Q302K/L316D/VI3
22I/A337P/K362Q/E367\/R373K
L441UY92H/K206A/F2 1 7D/K295N/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367\/R373K
L441UY92H/K206A/F2 1 7R/\247D/K295Q/Q3O2K/L3 1 6D/V13
22I/A337P/K362Q/E367/R373K
L44R/Y92H/K206A/F217R/\247D/K295T/Q302K/L3 1 6D/M3
37P/K362Q/E367/R373K
L441UY92H/K206A/F217R/\247D/Q302K/L316D/A317D/M3
22I/A337P/K362Q/E367\/R373K
594 L441UY92H/K206A/F217R/\247D/Q302K/L316D/A317Q/M3
22I/A337P/K362Q/E367\/R373K
595 92H/K206A/F217R/\247D/Q302K/L316D/M322I/A33
7P/A346G/K362Q/E367\/R373K
596 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/M322I/A33
7P/G344A/K362Q/E367\/R373K
597 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/M322I/A33
7P/G344D/K362Q/E367\/R373K
598 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/M322I/A33
557 7P/G344S/K362Q/E367N/R373K 388 24
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
3L/K362Q/E367N/R373K
L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/L372W/R373K
L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/R373K
L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/W368A/R373K
602 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/W368L/R373K
603 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
2Q/E367N/W368N/R373K
604 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/W368R/R373K
605 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/K362Q/E367N/W368V/R373K
606 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/N348E/K362Q/E367N/R373K
607 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/N348M/K362Q/E367N/R373K
608 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/N348Q/K362Q/E367N/R373K
L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/N3481VK362Q/E367N/R373K
610 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/N348W/K362Q/E367N/R373K
611 L44R/Y92H/K206A/F217R/\247D/Q302K/L316D/VI322I/A33
7P/T354S/K362Q/E367N/R373K
612 L441UY92H/K206A/F2 1 7R/\247D/Q302K/N305K/L3 1 6D/M3
22I/A337P/K362Q/E367N/R373K
613 L44R/Y92H/K206A/F217R/\247D/Q302K/N305L/L316D/M3
22I/A337P/K362Q/E367N/R373K
L44R/Y92H/K206A/F217R/N247D/Q302K/S314A/L316D/M32
2I/A337P/K362Q/E367N/R373K
L44R/Y92H/K206A/F2 1 7R/N247D/Q302K/S3 14H/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
L44R/Y92H/K206A/F2 1 7D/Q302K/S3 14N/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
L44R/Y92H/K206A/F2 1 7R/N247D/Q302K/S3 14Y/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
L441UY92H/K206A/F2 1 6A/N247D/Q302K/L3 1 6D/M3
577 22I/A337P/K362Q/E367N/R373K
-578
-579 221/A337P/K362Q/E367N/R373K 3
621 L44R/Y92H/K206A/F2 1 71VW246R/N247D/Q3OZK/L3 1 6D/M3
580 221/A337P/K362Q/E367N/R373K 396 24
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L441UY92H/K206A/F2171VW246S/N247D/Q302K/L316D/M3
22I/A337P/K362Q/E367N/R373K
92H/K206A/S21OA/F217R/\247D/Q3O2K/L3 1 6D/V132
2I/A337P/A350T/K362Q/E367N/R373K
L44R/Y92H/K206A/S21OA/F217R/\247D/Q3O2K/L3 1 6D/V132
2I/A337P/K362Q/E367\/R373K
L44R/Y92H/K206A/S21OE/F217R/\247D/Q3O2K/L3 1 6D/V132
2I/A337P/K362Q/E367/R373K
-III21/A337P/K362Q/E367\/R373K 24
—-L44R/Y92H/K206A/S210N/F217R/\247D/Q302K/L316D/VI3221/A337P/K362Q/E367\/R373K
L441VY92H/K206A/82101VF217R/\247D/Q302K/L316D/VI32
2I/A337P/K362Q/E367\/R373K
92H/K96A/K206A/F2 1 7D/Q3O2K/L3 1 6D/M32
2I/A337P/K362Q/E367\/R373K
L44R/Y92H/K96W/K206A/F2 1 7R/N247D/Q3O2K/L3 1 6D/M32
2I/A337P/K362Q/E367\/R373K
L44R/Y92H/L1 36V/S 1 66P/K206A/F2 1 7R/N247D/M259A/Q30
2K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q
-III22I/A337P/K362Q/E367N/R373K 29
-_II591 22I/A337P/K362Q/E367N/R373K 390 24
_II592 22I/A337P/K362Q/E367N/R373K 398 24
I.666 L44R/Y92H/Sl66P/K206A/F217R/N247D/H271A/Q302K/L31 21/A337P/K362Q/E367N/R373K/M390Q
652 L44R/Y92H/Sl66P/K206A/F217R/N247D/Q302K/L316D/M32
21/A337P/K362Q/E367N/R373K/M390Q 14
-_II21/A337P/K362Q/E367N/R373K/M392T 14
_-L44R/Y92H/S95A/K206A/F217R/N247D/Q302K/L316D/M322--I/A337P/K362Q/E367NR373K
L441UY92H/S95E/K206A/F217R/N247D/Q302K/L316D/M322
I/A337P/K362Q/E367NR373K
L44R/Y92H/T186A/K206A/F217R/N247D/Q302K/L316D/M3
22I/A337P/K362Q/E367\I/R373K 393 24
L44R/Y92H/T186G/K206A/F217R/N247D/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\I/R373K 393 24
L44R/Y92H/T186V/K206A/F217R/N247D/Q3O2K/L3 1 6D/M3
22I/A337P/K362Q/E367\I/R373K
L441VY92H/Y120H/K206A/F217R/N247D/Q302K/L316D/M3 —2-
221/A337P/K362Q/E367\I/R373K 24
-III21/A337P/K362Q/E367N/R373K
L44R/Y92H/Y12OS/K206A/F217R/N247D/Q302K/L316D/M32
21/A337P/L341F/K362Q/E367N/R373K
Table 7.1 Total genicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
L44R/Y92K/K206A/F217R/\247D/Q302K/L316D/V13221/A33
7P/K362Q/E367\/R373K
L44R/Y92Q/K206A/F217R/\247D/Q302K/L316D/V13221/A33
2Q/E367\/R373K
L441UY921UK206A/F217R/\247D/Q302K/L316D/V13221/A33
7P/K362Q/E367\/R373K
L44R/Y9ZS/K206A/F2 1 7R/\247D/Q302K/L316D/V13221/A3 3
2Q/E367\/R373K
433 92T/K206A/F217R/\247D/Q302K/L316D/V13221/A33
7P/K362Q/E367\/R373K
425 L44R/Y92V/K206A/F217R/\247D/Q302K/L316D/V13221/A33
7P/K362Q/E367\/R373K
410 L44S/K206A/F217R/N247D/Q302K/L316D/M3221/A337P/K36
2Q/E367N/R373K
166 L44T
L44T/K206A/F217R/N247D/Q302K/L316D/M3221/A337P/K3
62Q/E367N/R373K
-III62Q/E367N/R373K N D 36
-_II371 62Q/E367N/R373K N. D. 32
—_nM39OR 31
M39OT
I. I
-j——--21/A337P/K362Q/E367N/R373KM39E/E43D/L44R/S47T/Y92H/S166P/K206A/F2171UW246P/
N247D/M253W/H271A/SZ73D/Q3OZK/L3 1 6D/V13221/A337P/
K362Q/E367N/W368A/R373K/M392T
700 V139E/L441US47T/Y92H/Sl 66P/K206A/F21 7R/\1247D/A261 G
/Q302K/N305L/L316D/M3221/A337P/K362Q/E367N/R
373K/M392T
708 V139E/L441US47T/Y92H/Sl 66P/K206A/F21 7R/\1247D/H271A
/Q3OZK/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
716 V139E/L441VS47T/Y92H/Sl 66P/K206A/F21 7R/\1247D/H271A
/Q302K/L316D/M3221/A337P/K362Q/E367N/W368A/R373K/
M392T
V139E/L441US47T/Y92H/Sl 66P/K206A/F2 1 7R/\1247Y/H271A
/Q3OZK/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/M392T
V139E/L441VS47T/Y92H/Sl66P/K206A/F217R/W246P/N247D
/H271A/Q302K/L3 1 6D/M3221/A337P/K362Q/E367N/R373K/
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
Active Mutations
M392T
M39E/L44R/Y92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
2I/A337P/K362Q/E367N/R373K
405 M39H/L44R/K206A/F2 1 7D/Q302K/L3 1 6D/M322I/A33
364 7P/K362Q/E367N/R373K
408 M391VL441UK206A/F2 1 7R/N247D/Q302K/L3 1 2I/A33
367 7P/K362Q/E367N/R373K N. D. 32
_-I603 2I/A337P/K362Q/E367N/R373K 368 19
_-I377 7P/K362Q/E367N/R373K N. D. 32
645 M39V/L44R/Y92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M32
604 2I/A337P/K362Q/E367N/R373K 393 24
132 176 M39Y 451 3 7
417 M41P/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A33
376 7P/K362Q/E367N/R373K N. D. 35
414 M411VL441VK206A/F217R/N247D/Q302K/L316D/M3221/A33 _-
373 7P/K362Q/E367N/R373K N. D. 36
133 177 N388R 454 38
134 178 N91Q 438 32
26 72 P179S/R373K 430 37
138 182 435
139 183 447
140 184 445
141 185 449
142 186 450
143 187 Q 450
146--—
_--—
148--—
149 193 38
151 195 R1628 450 37
225 226 R16SS/K206A 427 39
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Variant
N0: Active Mutations TIS
1:: U) 0
3W1 707 U) \l
32 78 R373K/1376V
705 R7CVL44R/S47TFY92EUS166Pni206AuF217an247Dn12712u
Q302K/L316D/M3221/A337P/K362Q/E367N/W368A/R373K/
665 hA392T
690 R7H/T10P/L44ms47T/Y92H/s166P/K206A/F217R/N247D/H2
71A]Q302K/L3 1 6D/M3221/A3237P/K362Q/E367N/R373K/M39
66 I 302K/L316D/M3221/A337P/K362Q/E367N/R373K/M392T
316D/M3221/A337P/K362Q/E367N/R373K/M392T
S34D/M392P
O\ S34G
O\ S34H/M390R
174 450
175 219 459
176 220 433
177 221 422
178 222 414
179 223 446
728 T10Pn317CVL44R/S47TFY92EUS166Pnz206AuF217an247Dn1
271A/Q302K/L316D/M3221/A337P/K362Q/E367N/R373K/M3
686 92T 352
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
T1OP/E43D/L44R/S47T/Y92H/Sl66P/K206A/F217R/N247D/A
261 G/H271A/Q302K/N305L/L3 1 6D/M322I/A337P/K362Q/E3
67N/W368A/R373K/M392T
T10P/L44R/S47T/Y92H/M156V/Sl66P/K206A/F217R/N247D/
H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T
T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/A261G/
H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T
T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/
Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M392T
T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/
L3 1 2I/A337P/K362Q/E367N/R373K/M392T
711 T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/
Q3O2K/L3 1 2I/A337P/K362Q/E367N/W368A/R373K/
M392T
683 T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/H271A/
Q3O2K/L3 1 6D/M322I/R325S/A337P/K362Q/E367N/R373K/M
392T
704 T1OP/L44R/S47T/Y92H/Sl66P/K206A/F217R/\247D/Q252H/
M253R/A254E/A261G/H271A/Q302K/L316D/M322I/A337P/K
362Q/E367N/R373K/M392T
T1OP/L44R/S47T/Y92H/Sl66P/K206A/F2171VW246P/N247D/
A261 G/H271A/Q302K/L3 1 6D/M322I/A337P/K362Q/E367N/R
373K/M392T
T1OP/L44R/S47T/Y92H/Sl66P/K206A/F2171VW246P/N247D/
H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R373K/M
392T
T1OP/L44R/Y92H/K206A/F217R/N247D/Q3O2K/L3 1 6D/M322
I/A337P/K362Q/E367N/R373K
T10P/L44R/Y92H/R189L/K206A/F217R/N247D/Q302K/L316
D/M322I/A337P/K362Q/E367N/R373K
I39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N2
47D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R37
3K/M392T
T1OP/VI39E/L441VS47T/Y92H/Sl66P/K206A/F217R/N247D/A
271A/Q302K/L316D/M322I/A337P/K362Q/E367N/W3
68A/R373K/M392T
720 T1OP/VI39E/L441US47T/Y92H/Sl66P/K206A/F217R/N247D/H
271A]Q3O2K/L3 1 2I/A337P/K362Q/E367N/R373K/M3
719 T1OP/VI39E/L441US47T/Y92H/Sl66P/K206A/F217R/N247D/H
271A]Q3O2K/N305L/L3 1 6D/M322I/A337P/K362Q/E367N/R37
3K/M392T
681 T1 OP/M39E/L441US47T/Y92H/S 1 66P/K206A/F21 7R/N247D/S
266P/H271A/Q3O2K/L3 1 6D/M322I/A337P/K362Q/E367N/R37
3K/M392T
T1OP/M39E/L44R/S47T/Y92H/Sl66P/K206A/F2171VW246P/
N247D/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R
Table 7.1 Total Immunogenicity Score (T18), and Immunogenic Hit Count (IHC) for GLA
Variants
Active Mutations
373K/M392T
T369D
T3698
T3 898 WWW t—‘OOOO
T8L/L441UY92H/K206A/F2 1 7R/N247D/Q3OZK/L3 1 6D/M3221
/K362Q/E367N/R373K
T8Q/L441VY92H/K206A/F2 1 7R/N247D/Q302K/L3 1 6D/M3221
/A337P/K362Q/E367N/R373K
V1331
N.D. — Not determined.
While the invention has been described With reference to the specific embodiments, various
changes can be made and equivalents can be substituted to adapt to a particular situation, material,
composition of matter, process, process step or steps, thereby achieving benefits of the ion
Without departing from the scope of What is claimed.
For all purposes in the United States of America, each and every ation and patent
document cited in this application is incorporated herein by reference as if each such ation or
document was specifically and dually indicated to be incorporated herein by reference. Citation
of publications and patent documents is not intended as an tion that any such document is
pertinent prior art, nor does it constitute an admission as to its contents or date.
-ll3-
Claims (22)
1. A recombinant alpha galactosidase A and/or biologically active recombinant alpha galactosidase A fragment sing an amino acid sequence comprising at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5, wherein said alpha galactosidase A comprises a mutation at on 206 wherein the position is numbered with reference to SEQ ID NO:5.
2. The recombinant alpha galactosidase A of Claim 1, wherein said alpha galactosidase A further comprises at least one mutation in at least one position as provided in Table 2.3, wherein the positions are numbered with reference to SEQ ID NO:10.
3. The recombinant alpha galactosidase A of Claim 1, wherein said alpha galactosidase A further comprises at least one mutation in at least one position as provided in Tables 2.1, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and/or 7.1, wherein the ons are ed with reference to SEQ ID NO:5.
4. The recombinant alpha galactosidase A of Claim 1, wherein said alpha galactosidase A further ses at least one mutation at a position selected from position 2, 7, 8, 10, 14, 15, 17, 20, 21, 23, 24, 30, 31, 34, 36, 37, 39, 40, 41, 43, 44, 47, 48, 52, 53, 56, 59, 64, 65, 66, 67, 74, 76, 77, 80, 84, 87, 88, 91, 92, 93, 94, 95, 96, 100, 102, 105, 113, 120, 123, 124, 125, 130, 133, 136, 143, 144, 147, 155, 156, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 169, 170, 174, 177, 178, 179, 180, 181, 182, 186, 187, 189, 190, 195, 198, 199, 208, 210, 217, 219, 221, 228, 230, 234, 237, 238, 246, 247, 249, 252, 253, 254, 255, 256, 257, 258, 259, 261, 262, 263, 266, 269, 270, 271, 273, 274, 276, 277, 281, 283, 284, 287, 290, 293, 295, 299, 301, 302, 303, 305, 308, 314, 316, 317, 319, 322, 325, 326, 337, 339, 343, 344, 345, 346, 348, 349, 350, 352, 353, 354, 359, 362, 365, 367, 368, 369, 371, 373, 374, 375, 376, 377, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, and 398, wherein the ons are ed relative to SEQ ID NO:5.
5. The recombinant alpha galactosidase A of any one of Claims 1 to 4, wherein said recombinant alpha galactosidase A is a recombinant human alpha galactosidase A.
6. The inant alpha galactosidase A of Claim 1, wherein said mutation at position 206 is a 206A, 206M, 206Q, 206R, 206T, 206E, 206G, or 206S mutation.
7. The recombinant alpha galactosidase A of Claim 6, wherein said mutation at position 206 is a 206A mutation.
8. The recombinant alpha galactosidase A of Claim 1, wherein the recombinant alpha galactosidase A ses the polypeptide sequence of SEQ ID NO:15, 13, 10, 18, 40, 42, 44, or 46.
9. The recombinant alpha galactosidase A of any one of Claims 1 and 3 to 8, wherein said recombinant alpha galactosidase A is: a) more thermostable than the alpha osidase A of SEQ ID NO:5; b) is more stable at pH 7.4 than the alpha galactosidase A of SEQ ID NO:5; optionally wherein said recombinant alpha osidase A is: i) more stable at pH 4.3 than the alpha galactosidase A of SEQ ID NO:5; or ii) more stable to exposure to serum than the alpha galactosidase A of SEQ ID NO:5; c) is a deimmunized alpha galactosidase A; d) is a deimmunized alpha galactosidase A provided in Table 7.1; e) is purified; and/or f) exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) sed tolerance to pH 7.4; iii) increased tolerance to pH 4.3; iv) increased tolerance to serum; or v) reduced genicity; or vi) a combination of any one of i), ii), iii), iv), or v), as ed to a reference sequence wherein said reference sequence is SEQ ID NO:5 or SEQ ID NO:10.
10. A recombinant polynucleotide sequence ng at least one recombinant alpha galactosidase A as set forth in any one of Claims 1 to 9, optionally wherein said cleotide sequence is codon-optimized.
11. An expression vector comprising the recombinant polynucleotide sequence of Claim 10, optionally wherein said recombinant polynucleotide sequence is ly linked to a control sequence, optionally wherein said control sequence is a promoter, optionally wherein said promoter is a heterologous promoter.
12. An in vitro host cell comprising the expression vector of Claim 11, optionally wherein said host cell is an in vitro eukaryotic cell.
13. A method of producing an alpha osidase A variant, comprising culturing said in vitro host cell of Claim 12, under conditions such that said alpha galactosidase A encoded by said recombinant polynucleotide is produced, optionally further comprising the step of recovering said alpha galactosidase A, ally further comprising the step of purifying said alpha galactosidase A.
14. A composition comprising the recombinant alpha galactosidase A of any one of Claims 1 to 9.
15. A pharmaceutical composition formulated to be used in the treatment of Fabry disease, comprising the composition of Claim 14.
16. The pharmaceutical composition of Claim 15, wherein the ition: a) further ses a pharmaceutically acceptable carrier or excipient; b) is formulated to be used in parenteral injection or infusion to a human; and/or c) is formulated to be used in the treatment of Fabry disease.
17. The pharmaceutical composition of Claim 15 or Claim 16, wherein the pharmaceutical composition is ated to be used in treating and/or preventing the symptoms of Fabry disease in a subject.
18. The pharmaceutical composition of Claim 17, wherein a) said ms of Fabry disease are ameliorated; and/or b) said composition is formulated to be co-administered to the subject together with a diet that is less restricted in its fat content than diets required by subjects exhibiting the symptoms of Fabry disease; and/or c) said subject is i) an infant or child; or ii) said t is an adult or young adult.
19. The composition of any one of Claims 14 to 18, formulated to be used as a medicament.
20. The composition of any one of Claims 14 to 18, ated to be used as a nutritional supplement.
21. Use of a recombinant alpha galactosidase A of any one of Claims 1 to 9 in the manufacture of a medicament for treating and/or preventing the symptoms of Fabry disease in a subject having Fabry disease.
22. The use of Claim 21, wherein a) said symptoms of Fabry disease are ameliorated; and/or b) said the medicament is formulated to be co-administered to the subject er with a diet that is less restricted in its fat content than diets required by subjects exhibiting the symptoms of Fabry disease; and/or c) said subject is an infant or child; or d) said subject is an adult or young adult. 1 i 8 ”500900 00000-------------------------------------------------------------------- 3m? --------------------------------------------------------------------- 3 ' 448) 00000~ ?0000~ g .000000000: 0 00~ «000000N0: *0 :0? “0—000 :0 N0: Q 0 ,. ____________________________________
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462095313P | 2014-12-22 | 2014-12-22 | |
US62/095,313 | 2014-12-22 | ||
US201562216452P | 2015-09-10 | 2015-09-10 | |
US62/216,452 | 2015-09-10 | ||
PCT/US2015/063329 WO2016105889A1 (en) | 2014-12-22 | 2015-12-02 | Human alpha-galactosidase variants |
Publications (2)
Publication Number | Publication Date |
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NZ732171A NZ732171A (en) | 2021-01-29 |
NZ732171B2 true NZ732171B2 (en) | 2021-04-30 |
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ID=
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