NZ732171B2 - Human alpha-galactosidase variants - Google Patents

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)

The Claims defining the Invention are as follows:

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 ,. ____________________________________