FUSION PROTEIN FOR USE IN THERAPY

The present invention relates to soluble SIRPa binding proteins, for use as a medicament, in particular for the prevention or treatment of autoimmune and inflammatory disorders, for example allergic asthma and inflammatory bowel diseases. The invention more specifically relates to a soluble SIRPa binding protein comprising a complex of at least two bivalent heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a first SIRPa binding domain fused to the N-terminal part of a heavy chain constant region of an antibody; and

(ii) a second monovalent single chain polypeptide comprising a second SIRPa binding domain fused to the N-terminal part of a light chain constant region of an antibody. One specific embodiment of the invention is further illustrated by Figure 1.

SIRPa (CD172a) is an immunoreceptor expressed by myeloid lineage cells including macrophages, granulocytes and conventional dendritic cells (DCs), as well as on neuronal cells (van den Berg, et al. 2008, Trends in Immunol., 29(5):203-6). SIRPa is a low affinity ligand for CD47 (Rebres, et al. 2001 , J.Biol.Chem.; 276(37):34607-16; Hatherley, et al. 2007; J.Biol.Chem.; 282(19): 14567-75; Hatherley, et al. 2008; Mol. Cell; 31 (2) 266-77) and the interaction of SIRPa with CD47 composes a cellular communication system based on adhesion and bidirectional signaling controlling, which regulates multiple cellular functions in the immune- and neuronal system. These functions include migration, cellular maturation, macrophage phagocytosis and cytokine production of myeloid dendritic cells (van den Berg, et al. 2008 Trends in Immunol. 29(5):203-6; Sarfati 2009, Curr. Drug. Targets, 9(10):852- 50).

Data from animal models suggest that the SIRPa/CD47 interaction may contribute to or even control the pathogenesis of several disorders including autoimmune, inflammatory (Okuzawa, et al. 2008, BBRC; 371(3):561-6; Tomizawa, et al. 2007, J Immunol; 179(2):869- 877); ischemic (Isenberg, et al. 2008, Arter.Thromb Vase. Biol., 28(4):615-21 ; Isenberg 2008, Am. J. Pathol., 173(4): 1100-12) or oncology-related (Chan, et al. 2009, PNAS, 106(33): 14016-14021 ; Majeti, et al. 2009, Cell, 138(2): 286-99) diseases. Modulating the SIRPa/CD47 pathway may therefore be a promising therapeutic option for multiple diseases.

The use of antibodies against CD47, SIRPa or CD47-derived SIRPa-binding polypeptides has been suggested as therapeutic approaches (WO 1998/40940, WO 2004/108923, WO 2007/133811 , WO 2009/046541 ). Besides, SIRPa binding CD47-derived fusion proteins were efficacious in animal models of disease such as TNBS-colitis (Fortin, et al. 2009, J Exp Med., 206(9): 1995-201 1 ), Langerhans cell migration (J Immunol. 2004, 172: 4091-4099), and arthritis (VLST Inc, 2008, Exp. Opin.Therap. Pat., 18(5): 555-561 ).

In addition, SIRPa/CD47 is suggested to be involved in controlling phagocytosis (van den Berg, et al. 2008, Trends in Immunol., 29(5):203-6) and intervention by SIRPa binding polypeptides was claimed to augment human stem cell engraftment in a NOD mouse strain (WO 2009/046541 ) suggesting the potential benefits of CD47 extracellular domain (ECD) containing therapeutics for use in human stem cell transplantation.

The present invention provides soluble binding proteins comprising heterodimers of first and second polypeptide chains, each chain comprising a binding moieity fused to an antibody constant region sequence. The soluble proteins are for use as therapeutics.

The present invention further provides improved soluble SIRPa binding proteins for use as therapeutics. SIRPa-binding antibody-like proteins as defined in the present invention may provide means to increase avidity to targeted SIRPa expressing cells compared to prior art CD47 protein fusions while maintaining excellent developability properties. Additionally, without being bound by any theory, a higher avidity is expected to result in longer pharmacodynamic half-life thus providing enhanced therapeutic efficacy. These new findings offer new therapeutic tools to target SIRPa expressing cells and represent therapeutic perspectives, in particular for multiple autoimmune and inflammatory disorders, cancer disorders or stem cell transplantation.

Therefore, in one aspect, the invention provides a soluble protein, comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the heavy chain constant region of an antibody; and

(ii) a second monovalent single chain polypeptide comprising a region of the same binding molecule fused to the light chain constant region of an antibody. In another aspect the invention provides a soluble protein, comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the CH1 constant heavy chain region of an antibody; and

(ii) a second monovalent single chain polypeptide comprising a region of the same binding molecule fused to the CL constant light chain region of an antibody.

In preferred embodiments, each single chain polypeptide is monovalent, each heterodimer is divalent, and each complex is at least tetravalent. The heterodimers and soluble proteins of the invention have a valency of one per polypeptide chain. Compared to prior art molecules, the soluble proteins of the invention have increased valency. By incorporation of the same binding molecule in each first and second single chain polypeptide, the valency of each heterodimer is two, i.e. each chain within the heterodimer can bind a separate binding partner, or two times on the same binding partner. This is to be contrasted with prior art molecules (for example those disclosed in WO 01/46261) where the valency of a heterodimer of first and second polypeptide chains is one (i.e. both chains are required to bind the binding partner), to the extent that a complex of two heterodimers has a valency of two. Thus, a complex of two divalent heterodimers of the invention has a valency of four (tetravalent), i.e. the complex can bind up to four binding partners, or up to four times on the same binding partner. The heterodimers of the invention are bivalent and a complex of heterodimers has a valency of n x 2, where n is the number of heterodimers comprised within the complex. In preferred embodiments, the complex comprises two heterodimers, and has a valency of 4. Complexes comprising more than two heterodimers have a valency greater than 4, for example 6, 8, or 10. The increased valency of the soluble proteins of the invention results in a higher avidity, with advantageous effects on half-life and efficacy.

Therefore, in one aspect, the invention provides a soluble protein having at least tetravalency (or being at least tetravalent), comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the constant region heavy chain of an antibody; and

(ii) a second monovalent single chain polypeptide comprising a region of the same mammalian binding molecule fused to the constant region light chain of an antibody. ln another aspect, the invention provides a soluble protein having at least tetravalency, comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the CH1 constant heavy chain region of an antibody; and

(ii) a second monovalent single chain polypeptide comprising a region of the same binding molecule fused to the CL constant light chain region of an antibody.

In a preferred aspect the region of the binding molecule is the same. Therefore, the invention provides a soluble protein having at least tetravalency, comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the constant region heavy chain of an antibody; and

(ii) a second monovalent single chain polypeptide comprising the same region of the same mammalian binding molecule fused to the constant region light chain of an antibody.

In another aspect, the invention provides a soluble protein having at least tetravalency, comprising a complex of at least two heterodimers, wherein each heterodimer essentially consists of:

(i) a first monovalent single chain polypeptide comprising a region of a mammalian binding molecule fused to the CH1 constant heavy chain region of an antibody; and

(ii) a second monovalent single chain polypeptide comprising the same region of the same binding molecule fused to the CL constant light chain region of an antibody. In a preferred embodiment, the region of a mammalian binding molecule is fused to the N- terminal part of the antibody sequence (i.e. to the CH1 and CL contstant regions).

In one embodiment the binding molecule is a cytokine, growth factor, hormone, signaling protein, low molecular weight compound (drug), ligand, or cell surface receptor. Preferably, the binding molecule is a mammalian monomeric or homo-polymeric cell surface receptor. The region of the binding molecule may be the whole molecule, or a portion or fragment thereof, which may retain its biological activity. The region of the binding molecule may be an extracellular region or domain. In one embodiment, said mammalian monomeric or homo-polymeric cell surface receptor comprises an immunoglobulin superfamily (IgSF) domain, for example it comprises the extracellular domain of CD47.

In one preferred embodiment, the soluble protein is an antibody-like protein (also called and defined hereafter as a Fusobody) wherein the variable regions of both arms of the antibody are replaced by SIRPa binding domains, thereby providing a multivalent soluble protein.

One example of such a SIRPa binding Fusobody is shown in Figure 1.

In one embodiment, the invention relates to isolated soluble SIRPa-binding proteins or SIRPa-binding Fusobodies, comprising a tetravalent complex of two divalent heterodimers, wherein each heterodimer essentially consists of: (i) a first single chain polypeptide comprising a first SIRPa-binding domain fused at the N- terminal part of a constant C _H 1 heavy chain region of an antibody; and,

(ii) a second single chain polypeptide comprising a second SIRPa-binding domain fused at the N-terminal part of constant C _L light chain region of an antibody.

In a preferred embodiment, said first single chain polypeptide of each heterodimer of the soluble protein or SIRPa binding Fusobody further comprises the C _H 2 and C _H 3 regions of an immunoglobulin fused to said C _H 1 region, thereby reconstituting a full length constant heavy chain of an antibody. Said C _H 1 , C _H 2 and C _H 3 regions can be derived from wild type or mutant variants of human lgG1 , lgG2, lgG3 or lgG4 corresponding regions with silent effector functions and/or reduced cell killing, ADCC or CDC effector functions, for example reduced ADCC effector functions.

In one embodiment, said soluble protein or SIRPa-binding Fusobody binds to human SIRPa with a K _D of 10μΜ or less, for example of 4μΜ or less, for example 1 μΜ or less, 0.1 μΜ or less, as measured by surface plasmon resonance, such as a BiaCORE assay. In one embodiment, the soluble protein or SIRPa-binding Fusobody binds to human SIRPa with a K _D in a range of 0.1 to 10 μΜ.

In another embodiment, said soluble protein or SIRPa-binding Fusobody promotes the adhesion of SIRPa+ leukocytes, such as SIRPa+ U937 cells with an EC ₅₀ of 20nM or less, for example 2nM or less, for example between 200pM and 20nM, as measured in a plate- based cellular adhesion assay. In another embodiment, said soluble protein or SIRPa binding Fusobody inhibits the Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells.

For example, said soluble protein or SIRPa binding Fusobody inhibits the Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells, with an IC ₅₀ of 2nM or less, 0.2nM or less, for example between 20pM and 2nM, as measured in a dendritic cell cytokine release assay.

In another related embodiment, said first and second single chain polypeptides of each heterodimer are covalently bound by a disulfide bridge, for example using a natural disulfide bridge between cysteine residues of the corresponding C _H 1 and C _L regions.

In one embodiment, the first and second SIRPa binding domains may be fused to the C _H 1 and C _L regions respectively via a peptide linker. In another embodiment, the first and/or second SIRPa binding domain is directly fused to the respective C _H 1 and C _L regions in the absence of a peptide linker. In one preferred embodiment, said soluble protein or SIRPa binding Fusobody essentially consists of two heterodimers, wherein said first single chain polypeptide of each heterodimer comprises the hinge region of an immunoglobulin constant part, and the two heterodimers are stably associated with each other by a disulfide bridge between the cysteines at their hinge regions. In one embodiment, the soluble protein of the invention comprises at least one SIRPa binding domain selected from the group consisting of:

(i) an extracellular domain of human CD47;

(ii) a polypeptide of SEQ ID NO:4 or a fragment of SEQ ID NO:4 retaining SIRPa binding properties; and,

(iii) a variant polypeptide of SEQ ID NO:4 having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NO:4 and retaining SIRPa binding properties.

In one specific embodiment, all SIRPa binding domains have identical amino acid sequences. For example, all SIRPa binding domains consist of SEQ ID NO:4 or SEQ ID NO:3 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:27. ln one specific embodiment, said soluble protein of the invention or SIRPa binding Fusobody comprises two heterodimers, wherein each heterodimer essentially consists of: a first single chain polypeptide of SEQ ID NO: 5 and a second single chain polypeptide of SEQ ID NO:6. Said first and second single chain polypeptides are stably associated at least via one disulfide bond, similar to the heavy and light chains of an antibody. In a related embodiment, the soluble protein or SIRPa binding Fusobody comprises two heterodimers, wherein the first and second single chain polypeptides of each heterodimer have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to corresponding first and second single chain polypeptide of SEQ ID NO:5 and SEQ ID NO:6 respectively, while retaining the advantageous functional properties of a SIRPa binding Fusobody as described above.

In particular, in one specific embodiment, such soluble protein or SIRPa binding Fusobody binds to human SIRPa with a K _D of 10μΜ, or less, 4μΜ or less, or 2μΜ or less, for example between 0.1 μΜ and 10μΜ.

In one specific embodiment, the four SIRPa binding domains of a SIRPa binding Fusobody according to the invention are identical in sequence. For example, said SIRPa binding Fusobody is made of a first and second single chain polypeptide of SEQ ID NO.5 and SEQ ID NO:6 respectively.

The invention further relates to such soluble proteins or Fusobodies, in particular SIRPa- binding proteins or Fusobodies for use as a drug or diagnostic tool, for example in the treatment or diagnosis of autoimmune and acute and chronic inflammatory disorders. In particular SIRPa-binding proteins or Fusobodies are for use in a treatment selected from the group consisting of Th2-mediated airway inflammation, allergic disorders, asthma, inflammatory bowel diseases and arthritis.

The soluble proteins or Fusobodies of the invention may also be used in the treatment or diagnosis of ischemic disorders, leukemia or other cancer disorders, or in increasing hematopoietic stem engraftment in a subject in need thereof.

Definitions

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. The term SIRPa refers to the human Signal Regulatory Protein Alpha (also designated CD172a or SHPS-1) which shows adhesion to CD47 integrin associated protein. Human SIRPa includes SEQ ID NO:1 but further includes, without limitation, any natural polymorphic variant, for example, comprising single nucleotide polymorphisms (SNPs), or splice variants of human SIRPa. Examples of splice variants or SNPs in SIRPa nucleotide sequence found in human are described in Table 1.

Table 1 : Variants of SIRPa Protein

Variant Type Variant ID Description

Splice Variant NP_542970.1 reference; short variant; sequence NO:2

ENSP00000382941 long variant, insertion of four amino acids close to C-terminus

Single Nucleotide rs17855609 DNA: A or T; protein: T or S (pos. 50 of Polymorphism NP_542970.1)

rs 17855610 DNA: C or T; protein: T or I (pos. 52 of

NP_542970.1)

rs 17855611 DNA: G or A; protein: R or H (pos. 54 of

NP_542970.1)

rs17855612 DNA: C or T; protein: A or V (pos. 57 of

NP_542970.1)

rs1057114 DNA: G or C; protein: G or A (pos. 75 of

NP_542970.1)

rs 1135200 DNA: C or G; protein: D or E (pos. 95 of

NP_542970.1)

rs17855613 DNA: A or G; protein: N or D (pos. 100 of

NP_542970.1)

rs17855614 DNA: C or A; protein: N or K (pos. 100 of

NP_542970.1)

rs17855615 DNA: C or A; protein: R or S (pos. 107 of

NP_542970.1)

rs1135202 DNA: G or A; protein: G or S (pos. 109 of

NP_542970.1)

rs17855616 DNA: G or A; protein: G or S (pos. 109 of

NP_542970.1)

rs2422666 DNA: G or C; protein: V or L (pos. 302 of

NP_542970.1)

rs 12624995 DNA: T or G; protein: V or G (pos. 379 of

NP_542970.1)

rs41278990 DNA: C or T; protein: P or S (pos. 482 of

NP_542970.1) The term CD47 refers to the cell surface mammalian integrin associated protein. Human CD47 includes SEQ ID NO:2 but also any natural polymorphic variant, for example, comprising single nucleotide polymorphisms (SNPs), or splice variants of human CD47. Examples of splice variants or SNPs in CD47 nucleotide sequence found in human are described in Table 2.

Table 2: Variants of CD47 Protein

As used herein, the term "protein" refers to any organic compounds made of amino acids arranged in one or more linear chains and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" further includes, without limitation, peptides, single chain polypeptide or any complex molecules consisting primarily of two or more chains of amino acids. It further includes, without limitation, glycoproteins or other known post-translational modifications. It further includes known natural or artificial chemical modifications of natural proteins, such as without limitation, glycoengineering, pegylation, hesylation and the like, incorporation of non-natural amino acids, and amino acid modification for chemical conjugation with another molecule.

As used herein, a "complex protein" refers to a protein which is made of at least two single chain polypeptides, wherein said at least two single chain polypeptides are associated together under appropriate conditions via either non-covalent binding or covalent binding, for example, by disulfide bridge. A "heterodimeric protein" refers to a protein that is made of two single chain polypeptides forming a complex protein, wherein said two single chain polypeptides have different amino acid sequences, in particular, their amino acid sequences share not more than 90, 80, 70, 60 or 50% identity between each other. To the contrary, a "homodimeric protein" refers to a protein that is made of two identical or substantially identical polypeptides forming a complex protein, wherein said two single chain polypeptides share 100% identity, or at least 95% or at least 99% identity, the amino acid differences consisting of amino acid substitution, addition or deletion which does not affect the functional and physical properties of the polypeptide compared to the other one of the homodimer, for example conservative amino acid substitutions.

As used herein, a protein is "soluble" when it lacks any transmembrane domain or protein domain that anchors or integrates the polypeptide into the membrane of a cell expressing such polypeptide. In particular, the soluble proteins of the invention may likewise exclude transmembrane and intracellular domains of CD47. As used herein the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V _H ) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C _H 1 , C _H 2 and C _H 3. Each light chain is comprised of a light chain variable region (abbreviated herein as V _L ) and a light chain constant region. The light chain constant region is comprised of one domain, C _L . The V _H and V _L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V _H and V _L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system. The term "Fusobody" is used in the present text by analogy with the term "antibody", for ease of reading. As used in the present text, the term "Fusobody" refers to an antibody-like soluble protein comprising two heterodimers, each heterodimer consisting of one heavy and one light chain of amino acids, stably associated together, for example via one or more disulfide bond(s). Each heavy or light chain comprises constant regions of an antibody, referred hereafter respectively as the heavy and light chain constant regions of the Fusobody. The heavy chain constant region comprises at least the C _H 1 region of an antibody and may further comprise C _H 2 and C _H 3 regions, including the hinge region. The light chain constant region comprises the C _L region of an antibody. In a Fusobody, the variable regions of an antibody are replaced by heterologous soluble binding domains. The term "heterologous" means that these domains are not naturally found associated with constant regions of an antibody. In particular, such heterologous binding domains do not have the typical structure of an antibody variable domain consisting of 4 framework regions, FR1 , FR2, FR3 and FR4 and the 3 complementarity determining regions (CDRs) in- between. Each arm of the Fusobody therefore comprises a first single chain polypeptide comprising a first binding domain covalently linked at the N-terminal part of a constant C _H 1 heavy chain region of an antibody, and a second single chain polypeptide comprising a second binding domain covalently linked at the N-terminal part of a constant C _L light chain region of an antibody. The covalent linkage may be direct, for example via peptidic bound or indirect, via a linker, for example a peptidic linker. The two heterodimers of the Fusobody are covalently linked, for example, by at least one disulfide bridge at their hinge region, like an antibody structure. Figure 1 is a schematic representation of an example of a Fusobody molecule. Examples of molecules with a Fusobody structure have been described in the Art, in particular, Fusobodies comprising ligand binding region of heterodimeric receptor (see for example WO 01/46261).

In a preferred embodiment, the extracellular domain of a mammalian monomeric or homopolymeric cell surface receptor or a variant or region of such extracellular domain retaining ligand binding activities, is fused to the constant regions of the heavy and light chains of an antibody. The resulting molecule is a multivalent protein retaining the advantageous properties of an antibody molecule for use as a therapeutic molecule.

The term "mammalian binding molecule" as used herein is any molecule, or portion or fragment thereof, that can bind to a target molecule, cell, complex and/or tissue, and which includes proteins, nucleic acids, carbohydrates, lipids, low molecular weight compounds, and fragments thereof, each having the ability to bind to one or more of members selected from the group consisting of: soluble protein, cell surface protein, cell surface receptor protein, intracellular protein, carbohydrate, nucleic acid, a hormone, or a low molecular weight compound (small molecule drug), or a fragment thereof. The mammalian binding molecule may be a protein, cytokine, growth factor, hormone, signaling protein, inflammatory mediator, ligand, receptor, or fragment thereof. In preferred embodiments, the mammalian binding molecule is a native or mutated protein belonging to the immunoglobulin superfamily; a native hormone or a variant thereof being able to bind to its natural receptor; a nucleic acid or polynucleotide sequence being able to bind to complementary sequence and/or soluble cell surface or intracellular nucleic acid/polynucleotide binding proteins; a carbohydrate binding moiety being able to bind to other carbohydrate binding moieties and/or soluble, cell surface or intracellular proteins; a low molecular weight compound (drug) that binds to a soluble or cell surface or intracellular target protein. In particular the definition includes the following molecules:

- a cytokine selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1. IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IL-34, IL-35, granulocyte macrophage colony stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia-inhibitory factor (LIF), CNTF, Cardiotropin-1 , NNT-1/BSF-3, growth hormone, Prolactin, Erythropoietin, Thrombopoietin, Leptin, G-CSF, or receptor or ligand thereof;

- a member of the interferon family of cytokines selected from the group consisting of: IFN- gamma, IFN-alpha, and IFN-beta; - a member of the immunoglobulin superfamily of cytokines selected from the group consisting of B7.1 (CD80) and B7.2 (B70);

- a member of the TNF family of cytokines selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1 BBL;

- a member of the TGF-β/ΒΜΡ family selected from the group consisting of TGF-βΙ , TGF- β2, TGF- 3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b,

BMP-9, BMP-10, BMP-11 , BMP-15, BMP-16, endometrial bleeding associated factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, mullerian inhibiting substance (MIS), activin-1 , activin-2, activin-3, activin-4, and activin-5; - a cluster of differentiation (CD) molecule selected from the group consisting of: CD1 (a-c, 1A, 1 D, 1 E), CD2, CD3 (γ, δ, ε), CD4, CD5, CD6, CD7, CD8 (a), CD9, CD10, CD1 1 (a, b, c), CD13, CD14, CD15, CD16 (A, B), CD18, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31 , CD32 (A, B), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41 , CD42 (a, b, c, d), CD43, CD44, CD45, CD46, CD47, CD48, CD49 (a, b, c, d, e, f), CD50, CD51 , CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD61 , CD62 (E, L, P), CD63, CD64 (A, B, C), CD66 (a, b, c, d, e, f), CD68, CD69, CD70, CD71 , CD72, CD73, CD74, CD78, CD79 (a, b), CD80, CD81 , CD82, CD83, CD84, CD85 (a, d, e, h, j, k), CD86, CD87, CD88, CD89, CD90, CD91 , CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101 , CD102, CD103, CD104, CD105, CD106, CD107 (a, b), CD108, CD109, CD110, CD111 , CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120 (a, b), CD121 (a, b), CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131 , CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD140b, CD141, CD142, CD143, CD144, CD146, CD147, CD148, CD150, CD151 , CD152, CD153, CD154, CD155, CD156 (a, b, c), CD157, CD158 (a, d, e, i, k), CD159 (a, c), CD160, CD161 , CD162, CD163, CD164, CD166, CD167 (a, b), CD168, CD169, CD170, CD171 , CD172 (a, b, g), CD174, CD177, CD178, CD179 (a, b), CD181 , CD182, CD183, CD184, CD185, CD186, CD191 , CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CDw199, CD200, CD201 , CD202b, CD204, CD205, CD206, CD207, CD208, CD209, CDw210 (a, b), CD212, CD213a (1 , 2), CD217, CD218 (a, b), CD220, CD221 , CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD233, CD234, CD235 (a, b), CD236, CD238, CD239, CD240CE, CD241 , CD243, CD244, CD246, CD247- CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261 , CD262, CD264, CD265, CD266, CD267, CD268, CD269, CD271 , CD272, CD273, CD274, CD275, CD276, CD278, CD279, CD280, CD281 , CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD297, CD298, CD299, CD300A, CD301 , CD302, CD303, CD304, CD305, CD306, CD307, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD320, CD321 , CD322, CD324, CD325, CD326, CD328, CD329, CD331 , CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350;

- a molecule selected from the group consisting of ADAM10, ADAM17, ADAM8, ALCAM, ART4, ATP1B3, ABCG2, Alvircept sudotox, Anaplastic lymphoma kinase, B3GAT1 , BCAM, BMPR1A, BMPR1B, BST1 , BTLA, Band 3, Basigin, C-C chemokine receptor type 6, C-C chemokine receptor type 7, CCR1 , CCR2, CCR4, CCR5, CCR8 (gene), CCR9, CD1 , CD109, CD11c, Tissue factor, CD15, CD151 , CD155, CD16, CD160, CD163, CD177, CD19, CD1A, CD1E, CD2, CD20, CD200, CD226, CD23, CD244, CD247, CD248, CD25, CD276, CD278, CD28, CD300A, CD31 , CD32, CD320, CD37, CD38, CD3D, CD3G, CD4, CD40 (protein), CD43, CD44, CD46, CD48, CD5, CD5 (protein), CD53, Neural cell adhesion molecule, CD59, CD6, CD63, CD64 (biology), CD68, CD69, CD7, CD70, CD72, CD78, CD79, CD79A, CD79B, CD8, CD80, CD82 (gene), CD83, CD84, CD86, CD8A, CD90, CD93, CD96, CD98, CD99, CDCP1 , CDH1 (gene), CDH2, CEACAM1 , CEACAM3, CEACAM5, CEACAM6, CEACAM8, CLEC4M, CTLA-4, CXCR3, CXCR5, CXCR6, CCR3 (gene), CD11 , CD134, CD14, CD154, CD3 (immunology), CD34, CD36, CD47, CD74, CD81 , Colony stimulating factor 1 receptor, Complement receptor 1 , DC-SIGN, DDR1 , Discoidin domain-containing receptor 2, Duffy antigen system, E-selectin, EMR2, ENTPD1 , Endoglin, Endothelial protein C receptor, Epithelial cell adhesion molecule, F11 receptor, FCAR, FCGR2B, FCGR3A, FCGR3B, FCRL5, FZD10, FZD4, FZD9, Fas ligand, FCGR2A, Fibroblast growth factor receptor 1 , Fibroblast growth factor receptor 2, Fibroblast growth factor receptor 3, Fibroblast growth factor receptor 4, User:Frog21/Cd36 using MGI Gene box, Fucosyltransferase 3, GGT1 , GP1 BA, GP1 BB, GP5, GPR44, GYPA, GYPB, Glutamyl aminopeptidase, Glycophorin C, Glycoprotein IX, Granulocyte colony-stimulating factor receptor, Granulocyte macrophage colony-stimulating factor receptor, Group 1 CD1 , HER2/neu, Hyaluronan-mediated motility receptor, ICAM2, ICAM3, ICOSLG, IFITM1 , IGLL1 , IGSF2, IGSF8, IL13RA2, IL17RA, IL18R1 , IL18RAP, IL3RA, ITGA2B, ITGA5, ITGAV, ITGB4, Insulin receptor, Insulin-like growth factor 1 receptor, Insulin-like growth factor 2 receptor, Interferon gamma receptor 1 , Interleukin 1 receptor, type I, Interleukin 1 receptor, type II, Interleukin 10 receptor, alpha subunit, Interleukin 10 receptor, beta subunit, Interleukin 12 receptor, beta 1 subunit, Interleukin 13 receptor, alpha 1 , Interleukin 5 receptor alpha subunit, Interleukin 8 receptor, alpha, Interleukin 8 receptor, beta, Interleukin- 18 receptor, lnterleukin-4 receptor, lnterleukin-6 receptor, lnterleukin-7 receptor, Interleukin- 9 receptor, ITGA6, JAG1 , JAM2, KIR2DL1 , KIR2DL4, KIR2DS4, KIR3DL1 , KIR3DL2, KLRB1 , KLRC2, KLRD1 , KLRK1 , Kell antigen system, Kinase insert domain receptor, L1 (protein), LAG3, LAIR1 , LAMP1 , LAMP2, LAMP3, LILRA2, LILRA3, LILRB1 , LILRB2, LILRB3, LILRB4, LRP1 , LY75, LY9, Leptin receptor, Leukemia inhibitory factor receptor, Low-affinity nerve growth factor receptor, MFI2, MSR1 , Magnetic-activated cell sorting, MUC1 , Myeloproliferative leukemia virus oncogene, NCR1 , NCR2, NCR3, NKG2, NT5E, OX40L, P-glycoprotein, P-selectin glycoprotein ligand-1 , PD-L1 , PDCD1 LG2, PDGFRB, PSG1 (gene), PTGFRN, PVRL1 , PVRL2, PVRL3, PRNP, Programmed cell death 1 , RANK, RANKL, RHAG, RHCE (gene), SEMA4D, SEMA7A, SIGLEC5, SIGLEC7, SIGLEC8, SIRPB1 , SIRPG, SLAMF1 , SLC44A1 , Sialoadhesin, Signal-regulatory protein alpha, SuPAR, T-cell surface glycoprotein CD3 epsilon chain, TLR 1 , TLR 2, TLR 4, TLR10, TLR6, TLR8, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF17, TNFRSF1A, TNFSF13, TNFSF14, TRAIL, TEK tyrosine kinase, Tetherin, TFRC, Thrombomodulin, TLR 3, TLR9, Urokinase receptor, VE-cadherin, VPREB1 ; - a hormone selected from the group consisting of: Growth hormone (GH), Adrenocorticotropic hormone (ACTH), Leutinizing hormone (LH), Follicle stimulating hormone (FSH), Thyroid stimulating hormone (TSH), Prolactin hormone, Oxytosin, Antidiuretic hormone (ADH), Thyroxin, Calcitonin, Parathyroid hormone (PTH), Epinephrine, Nor-epinephrine, mineralocorticoids, glucocorticoids, androgens, Testosterone, Melatonin, Thymosin, thymopoetin, Glucagon, Insulin, Estrogen, and Progesterone; or fragment or receptor thereof.

The term "IgSF-domains" refers to the Immunoglobulin super-family domain containing proteins comprising a vast group of cell surface and soluble proteins that are involved in the immune system by mediating binding, recognition or adhesion processes of cells. The immunoglobulin domain of the IgSF-domain molecules share structural similarity to immunoglobulins. IgSF-domains contain about 70-110 amino acids and are categorized according to their size and function. Ig-domains possess a characteristic Ig-fold, which has a sandwich-like structure formed by two sheets of antiparallel beta strands. The Ig-fold is stabilized by a highly conserved disulfide bonds formed between cysteine residues as well as interactions between hydrophobic amino acids on the inner side of the sandwich. One end of the Ig domain has a section called the complementarity determining region that is important for the specificity of the IgSF domain. Most Ig domains are either variable (IgV) or constant (IgC). Examples of proteins displaying one or more IgSF domains are cell surface co-stimulatory molecules (CD28, CD80, CD86), antigen receptors (TCR/BCR) co-receptors (CD3/CD4/CD8). Other examples are molecules involved in cell adhesion (ICAM-1 , VCAM- 1) or with IgSF domains forming a cytokine binding receptor (IL1 R, IL6R) as well as intracellular muscle proteins. In many examples, the presence of multiple IgSF domains in close proximity to the cellular environment is a requirement for efficacy of the signaling triggered by said cell surface receptor containing such IgSF domain. A prominent example is the clustering of IgSF domain containing molecules (CD28, ICAM-1 , CD80 and CD86) in the immunologic synapse that enables a microenvironment allowing optimal antigen- presentation by antigen-presenting cells as well as resulting in controlled activation of naive T cells (Dustin, 2009, Immunity). Other examples for other IgSF containing molecules that need clustering for proper function are CD2 (Li, et al. 1996, J. Mol. Biol., 263(2):209-26) and ICAM-1 (Jun, et al. 2001 , J. Biol. Chem.; 276(31 ):29019-27). Therefore, by mimicking an oligovalent structure containing IgSF domain, the Fusobodies of the invention comprising several IgSF domains may advantageously be used for modulating the activity of their corresponding binding partner.

As used herein, the term SIRPy refers to CD172g. Human SIRPy includes SEQ ID NO:26 but also any natural polymorphic variant, for example, comprising single nucleotide polymorphisms (SNPs), or splice variants of human SIRPy. Examples of splice variants or SNPs in SIRPy nucleotide sequence found in human are described in Table 3.

Table 3: Variants of SIRPy Protein

The term "K _assoc " or "K _a ", as used herein, is intended to refer to the association rate of a particular protein-protein interaction, whereas the term "K _dis " or "K _d ," as used herein, is intended to refer to the dissociation rate of a particular protein-protein interaction. The term "K _D ", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K _d to K _a (i.e. K _d /K _a ) and is expressed as a molar concentration (M). K _D values for protein-protein interaction can be determined using methods well established in the art. A method for determining the K _D of a protein/protein interaction is by using surface plasmon resonance, or using a biosensor system such as a BiaCORE ^® system. At least one assay for determining the K _D of the proteins of the invention interacting with SIRPa is described in the Examples below. As used herein, the term "affinity" refers to the strength of interaction between the polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts through weak non-covalent forces with its target at numerous sites; the more interactions, the stronger the affinity. As used herein, the term "high affinity" for a binding polypeptide or protein refers to a polypeptide or protein having a K _D of 1 μΜ or less for its target.

As used herein, a protein that "promotes adhesion of SIRPa expressing leukocytes" refers to a protein that antagonizes the interaction of cellular SIRPa with cellular CD47 by binding to functional cellular SIRPa. Enhanced cellular adhesion of human leukocytes expressing SIRPa (SIRPa+ cells) to recombinant SIRPa binding proteins can serve as surrogate assessment for the antagonizing activity. Representative for SIRPa+ leukocytes are inflammatory myeloid leukocytes or malignant SIRPa+ leukocyte cell lines for example U937, Monomac 6, MUTZ-3, KG-1 , THP-1. Such improved promotion of adhesion can be measured by plate-based cellular adhesion assays. An example of such plate-based cellular adhesion assay using SIRPa+ U937 cells is described in the Examples. In a specific embodiment, a protein that "promotes adhesion of SIRPa expressing leukocytes" is a protein that promotes adhesion of SIRPa U937 cells with an EC ₅₀ of 20nM or less, for example 2nM or less, for example 20pM and 200pM and 2nM, as measured in a plate- based cellular binding assay, for example, as described in the Examples.

As used herein, a protein that "inhibits immune complex-stimulated cell cytokine release" is a protein that inhibits cytokine (e.g. IL-6, IL-10, IL-12p70, IL-23, IL-8 and/or TNF-a) release from peripheral blood monocytes, conventional dendritic cells (DCs) and/or monocyte- derived DCs stimulated with Staphylococcus aureus Cowan 1 (Pansorbin) or soluble CD40L and IFN-γ. One example of an immune complex-stimulated dendritic cell cytokine release assay is the Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells described in more details in the Examples below. In a preferred embodiment, a protein that "inhibits immune complex-stimulated cell cytokine release" is a protein that inhibits the Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in of in vitro generated monocyte-derived dendritic cells with an IC ₅₀ of 2nM or less, 0.2nM or less, for example between 2nM and 20pM, as measured in a dendritic cell cytokine release assay. As used herein, unless otherwise defined more specifically, the term "inhibition", when related to a functional assay, refers to any statistically significant inhibition of a measured function when compared to a negative control.

Assays to evaluate the effects of the soluble proteins or Fusobodies of the invention on functional properties of SIRPa are described in further detail in the Examples.

As used herein, the term "subject" includes any human or non-human animal.

The term "non-human animal" includes all vertebrates, e.g. mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, either a eukaryotic cell, for example, a cell of Pichia or Saccharomyces, a cell of Trichoderma, a Chinese Hamster Ovary cell (CHO) or a human cell, or a prokaryotic cell, for example, a strain of Escherichia coli. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence. The optimized sequences herein have been engineered to have codons that are preferred in the corresponding production cell or organism, for example a mammalian cell, however optimized expression of these sequences in other prokaryotic or eukaryotic cells is also envisioned herein. The amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

Various aspects of the invention are described in further detail in the following subsections.

Preferred embodiments of the invention are soluble SIRPa binding proteins selected among the group consisting of (Fab)-like Proteins, (Fab) ₂ -like Proteins, Fusobodies and their derivatives, and that comprise SIRPa-binding domain as described hereafter. For ease of reading, (Fab)-like Proteins, (Fab) ₂ -like Proteins, Fusobodies and their derivatives, comprising SIRPa binding domains are referred as the SIRPa binding Proteins of the Invention.

SIRPa-binding domain As used herein, a "SIRPa binding domain" refers to any single chain polypeptide domain that is necessary for binding to SIRPa under appropriate conditions. A SIRPa binding domain comprises all amino acid residues directly involved in the physical interaction with SIRPa. It may further comprise other amino acids that do not directly interact with SIRPa but are required for the proper conformation of the SIRPa binding domain to interact with SIRPa. SIRPa binding domains may be fused to heterologous domains without significant alteration of their binding properties to SIRPa. SIRPa binding domain may be selected among the binding domains of proteins known to bind to SIRPa such as CD47 protein. SIRPa binding domain may further consist of artificial binders to SIRPa. In particular, binders derived from single chain immunoglobulin scaffolds, such as single domain antibody, single chain antibody (scFv) or camelid antibody. In one embodiment, the term "SIRPa binding domain" does not contain SIRPa antigen-binding regions derived from variable regions, such as V _H and V _L regions of an antibody that binds to SIRPa.

In one preferred embodiment, the SIRPa binding domain is selected from the group consisting of:

(i) an extracellular domain of human CD47;

(ii) a polypeptide of SEQ ID NO:4 or a fragment of SEQ ID NO:4 retaining SIRPa binding properties; and,

(iii) a variant polypeptide of SEQ ID NO:4 having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NO:4 and retaining SIRPa binding properties.

The SIRPa binding proteins of the invention should retain the capacity to bind to SIRPa. The binding domain of CD47 has been well characterized and one extracellular domain of human CD47 is a polypeptide of SEQ ID NO:4. Fragments of the polypeptide of SEQ ID NO:4 can therefore be selected among those fragments comprising the SIRPa binding domain of CD47. Those fragments generally do not comprise the transmembrane and intracellular domains of CD47. In non-limiting illustrative embodiments, SIRPa-binding domains essentially consist of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21 , SEQ ID NO:23 or SEQ ID NO:27. Fragments include without limitation shorter polypeptide wherein between 1 and 10 amino acids have been truncated from C-terminal or N-terminal of SEQ ID NO:4, SEQ ID NO:21 or SEQ ID NO:3, for example SEQ ID NO:23 or SEQ ID NO:27. SIRPa- binding domains further include, without limitation, a variant polypeptide of SEQ ID NO:4, where amino acids residues have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent identity to SEQ ID NO:4; so long as changes to the native sequence do not substantially affect the biological activity of the SIRPa binding proteins, in particular its binding properties to SIRPa. In some embodiments, it includes mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion or substitution in the SIRPa-binding domain when compared with SEQ ID NO:4. Examples of mutant amino acid sequences are those sequences derived from single nucleotide polymorphisms (see Table 2).

As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the algorithm of E. Myers and W. Miller (Comput. Appl. Biosci. 4:11-17, 1988) which has been incorporated into the ALIGN program. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:443- 453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package. Yet another program to determine percent identity is CLUSTAL (M. Larkin et a/., Bioinformatics 23:2947-2948, 2007; first described by D. Higgins and P. Sharp, Gene 73:237-244, 1988) which is available as stand-alone program or via web servers (see http:/ www.clustal.org/).

In a specific embodiment, the SIRPa binding domain includes changes to SEQ ID NO:4 or SEQ ID NO:3 wherein said changes to SEQ ID NO:4 or SEQ ID NO:3 essentially consist of conservative amino acid substitutions.

Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the SIRPa binding domain of SEQ ID NO:4 or SEQ ID NO:3 can be replaced with other amino acid residues from the same side chain family, and the new polypeptide variant can be tested for retained function using the binding or functional assays described herein.

In another embodiment, the SIRPa binding domains are selected among those that cross- react with non-human primate SIRPa such as cynomolgus or rhesus monkeys.

In another embodiment, the SIRPa binding domains are selected among those that do not cross-react with human proteins closely related to SIRPa, such as SIRPy.

In some embodiments, the SIRPa binding domains are selected among those that retain the capacity for a SIRPa-binding Protein that comprises such SIRPa binding domain, to inhibit the binding of CD47-Fc fusion to SIRPa+ U937 cells, at least to the same extent as a SIRPa binding Protein comprising the extracellular domain of human SIRPa of SEQ ID NO:4, as measured in a plate-based cellular adhesion assay.

In other embodiments, the SIRPa binding domains are selected among those that retain the capacity for a SIRPa-binding Protein, that comprises such SIRPa binding domain, to inhibit Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro differentiated myeloid dendritic cells, at least to the same extent as a SIRPa binding Protein comprising the extracellular domain of human SIRPa of SEQ ID NO:4, as measured in a dendritic cell cytokine release assay.

(Fab)-like or (Fab like SIRPa binding Proteins of the invention

In one embodiment, the SIRPa binding Proteins of the invention are (Fab)-like or (FabV'ike Proteins, which binds to SIRPa. Fab fragments of antibodies are known as the fragments containing the binding region of an antibody, consisting of C _L and V _L regions of the light chain and C _H 1 and V _H regions of the heavy chain. (Fab)-like proteins are proteins similar to (Fab) fragments wherein V _H and V _L regions are replaced by heterologous binding domains, e.g. SIRPa binding domain. In an embodiment where the SIRPa binding domains are identical, the resulting (Fab)-like Protein of the invention comprises two identical binding domains and may therefore be bivalent with respect to SIRPa binding.

(Fab')2-Nke Proteins further comprise the hinge region of an antibody, enabling the covalent association of two (Fab)-like Proteins via disulfide bridge at the hinge region. The resulting protein comprises four binding domains. In one embodiment, such heterologous binding domains are binding domains derived from IgSF domains.

In one embodiment, a SIRPa-binding Protein of the invention is a (Fab)-like Protein consisting of (i) a first single chain polypeptide comprising a first SIRPa binding domain covalently linked to a constant C _H 1 heavy chain region of an antibody, and (ii) a second single chain polypeptide comprising a second SIRPa binding domain covalently linked to the constant C _L light chain region of an antibody.

The SIRPa binding domain can be fused directly in frame with the constant regions or via a polypeptide linker (spacer). Such spacer may be a single amino acid (such as, for example, a glycine residue) or between 5-100 amino acids, for example between 5-20 amino acids. The linker should permit the SIRPa binding domain to assume the proper spatial orientation to form a binding site with SIRPa. Suitable polypeptide linkers may be selected among those that adopt a flexible conformation. Examples of such linkers are (without limitation) those linkers comprising Glycine and Serine residues, for example, (Gly ₄ Ser) _n wherein n is an integer between 1-12, for example between 1 and 4, for example 2. (Fab)-like or (Fab) ₂ -like SIRPa binding Proteins of the Invention can be conjugated or fused together to form multivalent proteins.

The skilled person can further advantageously use the background technologies developed for engineering antibody molecules, either to increase the valencies of the molecule, or improve or adapt the properties of the engineered molecules for their specific use. In another embodiment, the (Fab)-like or (Fab) ₂ -like SIRPa binding Proteins of the invention, can be fused to another heterologous protein, which is capable of increasing half life of the resulting fusion protein in blood.

Such heterologous protein can be, for example, an immunoglobulin, serum albumin and fragments thereof. Such heterologous protein can also be a polypeptide capable of binding to serum albumin proteins to increase half life of the resulting molecule when administered in a subject. Such approach is for example described in EP0486525.

Alternatively or in addition, the (Fab)-like or (Fab) ₂ -like Proteins further comprises a domain for multimerization. SIRPg binding Fusobody

In a further aspect, the invention relates to a Fusobody comprising at least one SIRPa binding domain or (Fab)-like Proteins as described in the above paragraphs.

The two heterodimers of the Fusobody may contain different binding domains with different binding specificities, thereby resulting in a bispecific Fusobody. For example, the Fusobody may comprise one heterodimer containing SIRPa binding domain and another heterodimer containing another heterologous binding domain. Alternatively, both heterodimers of the Fusobody comprise SIRPa binding domains. In the latter, the structure or amino acid sequence of such SIRPa binding domains may be identical or different. In one preferred embodiment, both heterodimers of the Fusobody comprise identical SIRPa binding domains.

In one specific embodiment the heavy chain of each heterodimer comprises the C _H 2 and C _H 3 regions of an antibody, referred as the Fc part or Fc moiety of the Fusobody, by analogy to antibody structure. Detailed description of the Fc part of a Fusobody is described in a paragraph further below. Specific Examples of SIRPa binding Fusobodies of the Invention

Fusobodies of the invention include without limitation the Fusobodies structurally characterized as described in Table 4 in the Examples. The SIRPa binding domain used in these examples are shown in SEQ ID NO:4, SEQ ID NO:21 , SEQ ID NO:23 or SEQ ID NO:27. Specific examples of heavy chain amino acid sequences of SIRPa binding Fusobodies of the invention are polypeptide sequences selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. Specific examples of light chain amino acid sequences of SIRPa binding Fusobodies of the invention are polypeptide sequences selected from the group consisting of: SEQ ID NO:6, SEQ ID NO: 13, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57.

Other SIRPa binding Fusobodies of the invention comprise SIRPa binding domains that have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity in any one of the corresponding SIRPa binding domains of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21 , SEQ ID NO:23 or SEQ ID NO:27. In some embodiments, Fusobodies of the invention comprise SIRPa binding domains which include mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been changed by amino acid deletion or substitution in the SIRPa binding domains when compared with the SIRPa binding domains as depicted in any one of the sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:27.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#1 , comprises a heavy chain of SEQ ID NO:5 and a light chain of SEQ ID NO:6. In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#2, comprises a heavy chain of SEQ ID NO: 18 and a light chain of SEQ ID NO:6.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#3, comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO:20.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#4, comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#5, comprises a heavy chain of SEQ ID NO:24 and a light chain of SEQ ID NO:25.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#6, comprises a heavy chain of SEQ ID NO:36 and a light chain of SEQ ID NO:37. In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#7, comprises a heavy chain of SEQ ID NO:38 and a light chain of SEQ ID NO:39.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#8, comprises a heavy chain of SEQ ID NO:40 and a light chain of SEQ ID NO:41. ln one embodiment, a SIRPa binding Fusobody of the invention, described as Example#9, comprises a heavy chain of SEQ ID NO:42 and a light chain of SEQ ID NO:43.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#10, comprises a heavy chain of SEQ ID NO:44 and a light chain of SEQ ID NO:45. In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#11 , comprises a heavy chain of SEQ ID NO:46 and a light chain of SEQ ID NO:47.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#12, comprises a heavy chain of SEQ ID NO:48 and a light chain of SEQ ID NO:49.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#13, comprises a heavy chain of SEQ ID NO:50 and a light chain of SEQ ID NO:51.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#14, comprises a heavy chain of SEQ ID NO:52 and a light chain of SEQ ID NO:53.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#15, comprises a heavy chain of SEQ ID NO:54 and a light chain of SEQ ID NO:55. In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#16, comprises a heavy chain of SEQ ID NO:56 and a light chain of SEQ ID NO:57.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#17, comprises a heavy chain of SEQ ID NO:58 and a light chain of SEQ ID NO:20.

In one embodiment, a SIRPa binding Fusobody of the invention, described as Example#18, comprises a heavy chain of SEQ ID NO:29 and a light chain of SEQ ID NO:20.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#1 , having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO: 10; and a light chain encoded by a nucleotide sequence of SEQ ID NO:11.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#3, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:59; and a light chain encoded by a nucleotide sequence of SEQ ID NO:60. In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#4, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:61 ; and a light chain encoded by a nucleotide sequence of SEQ ID NO:62.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#5, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:63; and a light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:64.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#6, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:65; and a light chain encoded by a nucleotide sequence of SEQ ID NO:66.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#7, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:67; and a light chain encoded by a nucleotide sequence of SEQ ID NO:68.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#8, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:69; and a light chain encoded by a nucleotide sequence of SEQ ID NO:70.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#9, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:71 ; and a light chain encoded by a nucleotide sequence of SEQ ID NO:72. In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#10, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:73; and a light chain encoded by a nucleotide sequence of SEQ ID NO:74.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#11 , having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:75; and a light chain encoded by a nucleotide sequence of SEQ ID NO:76.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#12, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:77; and a light chain encoded by a nucleotide sequence of SEQ ID NO:78. In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#13, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:79; and a light chain encoded by a nucleotide sequence of SEQ ID NO:80.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#14, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:81 ; and a light chain encoded by a nucleotide sequence of SEQ ID NO:82.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#15, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:83; and a light chain encoded by a nucleotide sequence of SEQ ID NO:84. In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#16, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:85; and a light chain encoded by a nucleotide sequence of SEQ ID NO:86.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#17, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:87; and a light chain encoded by a nucleotide sequence of SEQ ID NO:60.

In another aspect, the invention provides an isolated Fusobody of the invention, described as Example#18, having: a heavy chain encoded by a nucleotide sequence of SEQ ID NO:88; and a light chain encoded by a nucleotide sequence of SEQ ID NO:60.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p3HC_5460_ID59 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24361 , and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p3LC_5461_ID60 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24362.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p4HC_5444_ID61 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24363, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p4LC_5445_ID62 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24364.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid pHC_5466_ID63 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 10, 2010 with accession number DSM 24330, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p5LC_5467_ID64 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24365. In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p6HC_5440_ID65 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24366, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p6LC_5441_ID66 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24367.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p7HC_5450_ID67 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24368, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p7LC_5451_ID68 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24369.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p8HC_5442_ID69 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24370, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p8LC_5443_ID70 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24371.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p9HC_5452_ID71 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 10, 2010 with accession number DSM 24331 , and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p9LC_5453_ID72 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24372.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p10HC_5454_ID73 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24373, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p10LC_5455_ID74 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24374.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p11 HC_5446_ID75 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24375, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p11 LC_5447_ID76 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24376. In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p12HC_5456_ID77 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 10, 2010 with accession number DSM 24332, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p12LC_5457_ID78 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24377.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p13HC_5448_ID79 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24378, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p13LC_5449_ID80 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24379.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p14HC_5468_ID81 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24380, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p14LC_5469_ID82 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24381. In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p15HC_5458_ID83 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 10, 2010 with accession number DSM 24333, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p15LC_ 5459JD84 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24382.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p16HC_5464_ID85 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 10, 2010 with accession number DSM 24334, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p16LC_5465_ID86 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24383.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p31 HC_5471_ID89 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24384, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p32LC_5471_ID90 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24385.

In another aspect the invention provides an isolated Fusobody of the invention, having: a heavy chain encoded by a corresponding nucleotide sequence contained within plasmid p34HC_5472_ID91 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24386, and a light chain encoded by a corresponding nucleotide sequence contained within plasmid p35LC_5473_ID92 as deposited by Novartis Pharma AG, Novartis Campus, CH-4002 Basel, Switzerland, at DSMZ on December 13, 2010 with accession number DSM 24387.

Other SIRPa binding Fusobodies of the invention comprise a heavy chain encoded by nucleotide sequences which have been mutated by nucleotide deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NO:10 or SEQ ID NO:14 or SEQ ID NO:59 or SEQ ID NO:63 or SEQ ID NO:67 and a light chain encoded by nucleotide sequences which have been mutated by nucleotide deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NO:11 or SEQ ID NO:15 or SEQ ID NO:60 or SEQ ID NO:64 or SEQ ID NO:68. In some embodiments, Fusobodies of the invention comprise a heavy chain encoded by a nucleotide sequence which includes mutant nucleotide sequence wherein no more than 1 , 2, 3, 4 or 5 nucleotide have been changed by nucleotide deletion, insertion or substitution when compared with SEQ ID NO:10 or SEQ ID NO:14 or SEQ ID NO:59 or SEQ ID NO:63 or SEQ ID NO:67 and a light chain encoded by a nucleotide sequence which includes mutant nucleotide sequence wherein no more than 1 , 2, 3, 4 or 5 nucleotide have been changed by nucleotide deletion, insertion or substitution when compared with SEQ ID NO:11 or SEQ ID NO:15 or SEQ ID NO:60 or SEQ ID NO:64 or SEQ ID NO:68.

Functional Fusobodies

In yet another embodiment, a SIRPa binding Fusobody of the invention has heavy and light chain amino acid sequences; heavy and light chain nucleotide sequences or SIRPa binding domains fused to heavy and light chain constant regions, that are homologous to the corresponding amino acid and nucleotide sequences of the specific SIRPa binding Fusobodies described in the above paragraph, in particular, Examples#1-18 as described in Table 4, and wherein said Fusobodies retain substantially the same functional properties of at least one of the specific SIRPa binding Fusobodies described in the above paragraph, in particular, Examples#1 -18 as described in Table 4.

For example, the invention provides an isolated Fusobody comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein: the heavy chain has an amino acid sequence that is at least 80%, at least 90%, at least 95% or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58; the light chain has an amino acid sequence that is at least 80%, at least 90%, at least 95% or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57; the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro generated monocyte derived dendritic cells. As used herein, a Fusobody that "specifically binds to SIRPa" is intended to refer to a Fusobody that binds to human SIRPa polypeptide of SEQ ID NO:1 with a K _D of 4μΜ or less, 2μΜ or less, 400 nM or less, within at least one of the binding affinity assays described in the Examples, for example by surface plasmon resonance in a BiaCORE assay. A Fusobody that "cross-reacts with a polypeptide other than SIRPa" is intended to refer to a Fusobody that binds that other polypeptide with a K _D of 4μΜ or less, 2μΜ or less, 400nM or less. A Fusobody that "does not cross-react with a particular polypeptide" is intended to refer to a Fusobody that binds to that polypeptide, with a K _D of at least ten fold higher, preferably at least hundred fold higher than the K _D measuring binding affinity of said Fusobody to human SIRPa under similar conditions. In certain embodiments, such Fusobodies that do not cross- react with the other polypeptide exhibit essentially undetectable binding against these proteins in standard binding assays.

In various embodiments, the Fusobody may exhibit one or more or all of the functional properties discussed above.

In other embodiments, the SIRPa-binding domains may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to at least one of the specific sequences of SIRPa binding domains set forth in the above paragraph related to "SIRPa binding domains", including without limitation SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21 , SEQ ID NO:23 or SEQ ID N0.27. In other embodiments, the SIRPa-binding domains may be identical to at least one of the specific sequences of SIRPa binding domains set forth in the above paragraph related to "SIRPa binding domains", including without limitation SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21 , SEQ ID NO:23 or SEQ ID NO:27 except for an amino acid substitution in no more than 1 , 2, 3, 4 or 5 amino acid positions of said specific sequence.

A Fusobody having SIRPa-binding domains with high (i.e., at least 80%, 90%, 95%, 99% or greater) identity to specifically described SIRPa-binding domains, can be obtained by mutagenesis (e.g. site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding said specific SIRPa-binding domains respectively, followed by testing of the encoded altered Fusobody for retained function (i.e., the functions set forth above) using the functional assays described herein.

In other embodiments, the heavy chain and light chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the heavy and light chains of the specific Fusobody Examples#1-18 set forth above, while retaining at least one of the functional properties of SIRPa binding Fusobody described above. A SIRPa binding Fusobody having a heavy chain and light chain having high (i.e., at least 80%, 90%, 95% or greater) identity to the corresponding heavy chains of any of SEQ ID NO: 5, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58; and light chains of any of SEQ ID NO:6, SEQ ID NO: 13, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57, respectively, can be obtained by mutagenesis (e.g. site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding heavy chains SEQ ID NO: 5, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58; and light chains SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57; respectively, followed by testing of the encoded altered SIRPa binding Fusobody for retained function (i.e., the functions set forth above) using the functional assays described herein.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#1 , having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:5 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:6, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#2, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 18 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:6, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#3, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 19 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:20, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#4, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 12 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 13, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#5, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:24 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:25, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#6, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:36 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:37, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#7, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:38 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:39, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#8, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:40 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:41 , the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#9, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:42 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:43, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#10, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:44 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:45, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others. In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#11 , having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:46 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:47, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#12, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:48 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:49, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPoc+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others. In one embodiment, a SIRPoc binding Fusobody of the invention is a variant of Example#13, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:50 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:51 , the Fusobody specifically binds to SIRPoc, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPoc+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPoc binding Fusobody of the invention is a variant of Example#14, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 52 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:53, the Fusobody specifically binds to SIRPoc, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPoc+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPoc binding Fusobody of the invention is a variant of Example#15, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 54 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:55, the Fusobody specifically binds to SIRPoc, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPoc+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPoc binding Fusobody of the invention is a variant of Example#16, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:56 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:57, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#17, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:58 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:20, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

In one embodiment, a SIRPa binding Fusobody of the invention is a variant of Example#18, having a heavy chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:29 and a light chain at least 80%, 90%, 95% or 99% identical to SEQ ID NO:20, the Fusobody specifically binds to SIRPa, and the Fusobody exhibits at least one of the following functional properties: it promotes the adhesion of SIRPa+ leukocytes, or it inhibits release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells elicited by various bacterial derivatives such as Staphylococcus aureus Cowan strain particles or others.

Fc Domain of Fusobody

An Fc domain comprises at least the C _H 2 and C _H 3 domain. As used herein, the term Fc domain further includes, without limitation, Fc variants into which an amino acid substitution, deletion or insertion at one, two, three, four of five amino acid positions has been introduced compared to natural Fc fragment of antibodies, for example, human Fc fragments.

The use of Fc domain for making soluble constructs with increased in vivo half life in human is well known in the art and for example described in Capon et al. (US 5,428, 130). In one embodiment, it is proposed to use a similar Fc moiety within a Fusobody construct. However, it is appreciated that the invention does not relate to known proteins of the Art sometimes referred as "Fc fusion proteins" or "immunoadhesin". Indeed, the term "Fc fusion proteins" or "immunoadhesins" generally refer in the Art to a heterologous binding region directly fused to C _H 2 and C _H 3 domain, but which does not comprise at least either of C _L or C _H 1 region. The resulting protein comprises two heterologous binding regions. The Fusobody may comprise an Fc moiety fused to the N-terminal of the C _H 1 region, thereby reconstituting a full length constant heavy chain which can interact with a light chain, usually via C _H 1 and C _L disulfide bonding.

In one embodiment, the hinge region of C _H 1 of the Fusobody or SIRPa binding Proteins is modified such that the number of cysteine residues in the hinge region is altered, e.g. increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 (Bodmer et a/.). The number of cysteine residues in the hinge region of C _H 1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the fusion polypeptide.

In another embodiment, the Fc region of the Fusobody or SIRPa binding Proteins is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following positions can be mutated: 252, 254, 256, as described in U.S. Patent No. 6,277,375, for example: M252Y, S254T, T256E.

In yet other embodiments, the Fc region of the Fusobody or SIRPa binding Proteins is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the Fc portion. For example, one or more amino acids can be replaced with a different amino acid residue such that the Fc portion has an altered affinity for an effector ligand. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter ef a/. In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the resulting Fc portion has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 (Idusogie et al.)

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the Fc region to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al. ln yet another embodiment, the Fc region of the Fusobody or SIRPa binding Proteins is modified to increase the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase or decrease the affinity of the Fc region for an Fey receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072. Moreover, the binding sites on human lgG1 for FcyRI, FcyRI I, FcyRI 11 and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chem. 276:6591-6604).

In one embodiment, the Fc domain of the Fusobody or SIRPa binding Proteins is of human origin and may be from any of the immunoglobulin classes, such as IgG or IgA and from any subtype such as human lgG1 , lgG2, lgG3 and lgG4 or chimera of lgG1 , lgG2, lgG3 and lgG4. In other embodiments the Fc domain is from a non-human animal, for example, but not limited to, a mouse, rat, rabbit, camelid, shark, non-human primate or hamster.

In certain embodiments, the Fc domain of lgG1 isotype is used in the Fusobody or SIRPa binding Proteins. In some specific embodiments, a mutant variant of lgG1 Fc fragment is used, e.g. a silent lgG1 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor. An example of an lgG1 isotype silent mutant, is a so-called LALA mutant, wherein leucine residues are replaced by alanine residues at amino acid positions 234 and 235, as described by Hezareh et al. (J. Virol 2001 Dec;75(24):12161-8). Another example of an lgG1 isotype silent mutant comprises the D265A mutation. In certain embodiments, the Fc domain is a mutant preventing glycosylation at residue at position 297 of Fc domain, for example, an amino acid substitution of asparagine residue at position 297 of the Fc domain. Example of such amino acid substitution is the replacement of N297 by a glycine or an alanine. In another embodiment, the Fc domain is derived from lgG2, lgG3 or lgG4.

In one embodiment, the Fc domain of the Fusobody or SIRPa binding Proteins comprises a dimerization domain, preferably via cysteine capable of making covalent disulfide bridge between two fusion polypeptides comprising such Fc domain.

Glycosylation modifications In still another embodiment, the glycosylation pattern of the Soluble Proteins of the invention, including in particular the SIRPa-binding Proteins or Fusobodies, can be altered compared to typical mammalian glycosylation pattern such as those obtained in CHO or human cell lines. For example, an aglycoslated protein can be made by using prokaryotic cell lines as host cells or mammalian cells that has been engineered to lack glycosylation. Carbohydrate modifications can also be accomplished by; for example, altering one or more sites of glycosylation within the SIRPa binding Fusobody.

Additionally or alternatively, a glycosylated protein can be made that has an altered type of glycosylation. Such carbohydrate modifications can be accomplished by, for example, expressing the soluble proteins of the invention in a host cell with altered glycosylation machinery, i.e the glycosylation pattern of the soluble protein is altered compared to the glycosylation pattern observed in corresponding wild type cells. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant soluble proteins to thereby produce such soluble proteins with altered glycosylation. For example, EP 1 ,176,195 (Hang et a/.) describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that glycoproteins expressed in such a cell line exhibit hypofucosylation. WO 03/035835 describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of glycoproteins expressed in that host cell (see also Shields, R.L. et a/., 2002 J. Biol. Chem. 277:26733- 26740). Alternatively, the soluble proteins can be produced in yeast, e.g. Pichia pastoris, or filamentous fungi, e.g. Trichoderma reesei, engineered for mammalian-like glycosylation pattern (see for example EP1297172B1). Advantages of those glycoengineered host cells are, inter alia, the provision of polypeptide compositions with homogeneous glycosylation pattern and/or higher yield.

Pegylated Soluble Proteins and other conjugates Another embodiment of the Soluble Proteins herein that is contemplated by the invention is pegylation. The Soluble Proteins of the invention, for example, SIRPa-binding Proteins or Fusobodies can be pegylated. Pegylation is a well-known technology to increase the biological (e.g. serum) half-life of the resulting biologies as compared to the same biologies without pegylation. To pegylate a polypeptide, the polypeptide is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptides. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy- polyethylene glycol or polyethylene glycol-maleimide. Methods for pegylating proteins are known in the art and can be applied to the soluble proteins of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et a/.

Alternative conjugates or polymeric carrier can be used, in particular to improve the pharmacokinetic properties of the resulting conjugates. The polymeric carrier may comprise at least one natural or synthetic branched, linear or dendritic polymer. The polymeric carrier is preferably soluble in water and body fluids and is preferably a pharmaceutically acceptable polymer. Water soluble polymer moieties include, but are not limited to, e.g. polyalkylene glycol and derivatives thereof, including PEG, PEG homopolymers, mPEG, polypropyleneglycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copoloymers are unsubstituted or substituted at one end e.g. with an acylgroup; polyglycerines or polysialic acid; carbohydrates, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethylcellulose; starches (e.g. hydroxyalkyl starch (HAS), especially hydroxyethyl starch (HES) and dextrines, and derivatives thereof; dextran and dextran derivatives, including dextransulfat, crosslinked dextrin, and carboxymethyl dextrin; chitosan (a linear polysaccharide), heparin and fragments of heparin; polyvinyl alcohol and polyvinyl ethyl ethers; polyvinylpyrrollidon; alpha, beta-poly[(2-hydroxyethyl)-DL-aspartamide; and polyoxy- ethylated polyols.

Use of the SIRPa binding Proteins as a medicament

The SIRPa binding Proteins and in particular the SIRPa binding Fusobodies may be used as a medicament, in particular to decrease or suppress (in a statistically or biologically significant manner) the inflammatory and/or autoimmune response, in particular, a response mediated by SIRPa+ cells in a subject. When conjugated to cytotoxic agents or with cell- killing effector functions provided by Fc moiety, the SIRPa binding Proteins and in particular the SIRPa binding Fusobodies can also be advantageously used in treating, decrease or suppress cancer disorders or tumors, such as, in particular myeloid lymphoproliferative diseases such as acute myeloid lymphoproliferative (AML) disorders or bladder cancer. Nucleic acid molecules encoding the Soluble Proteins of the Invention Another aspect of the invention pertains to nucleic acid molecules that encode the soluble Proteins of the invention, including without limitation, the embodiments related to Fusobody, for example as described in Table 4 of the Examples. Non-limiting examples of nucleotide sequences encoding the SIRPa binding Fusobodies comprise SEQ ID NOs: 10 and 11 , encoding respectively the heavy and light chains of a SIRPa binding Fusobody.

The nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In an embodiment, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector.

Once DNA fragments encoding the soluble SIRPa-binding Proteins are obtained, for example, SIRPa binding Fusobodies, as described above and in the Examples, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to include any signal sequence for appropriate secretion in expression system, any purification tag and cleavable tag for further purification steps. In these manipulations, a DNA fragment is operatively linked to another DNA molecule, or to a fragment encoding another protein, such as a purification/secretion tag or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under control of a desired promoter.

Generation of transfectomas producing the SIRPa-binding Proteins

The Soluble Proteins of the Invention, for example SIRPa-binding Proteins of Fusobodies can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art. For expressing and producing recombinant Fusobodies in host cell transfectoma, the skilled person can advantageously use its own general knowledge related to the expression and recombinant production of antibody molecules or antibody-like molecules.

For example, to express the Soluble Proteins of the Invention or intermediates thereof, DNAs encoding the corresponding polypeptides, can be obtained by standard molecular biology techniques (e.g. PCR amplification or cDNA cloning using a hybridoma that expresses the polypeptides of interest) and the DNAs can be inserted into expression vectors such that the corresponding gene is operatively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The gene encoding the Soluble Proteins of the invention, e.g. the heavy and light chains of the SIRPa binding Fusobodies or intermediates are inserted into the expression vector by standard methods (e.g. ligation of complementary restriction sites on the gene fragment and vector, or blunt end ligation if no restriction sites are present). Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the polypeptide chain(s) from a host cell. The gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the polypeptide chain. In specific embodiments with CD47 derived sequences as SIRPa binding region, the signal peptide can be a CD47 signal peptide or a heterologous signal peptide (i.e., a signal peptide not naturally associated with CD47 sequence). In addition to the polypeptide encoding sequence, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the gene in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g. polyadenylation signals) that control the transcription or translation of the polypeptide chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g. the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al., 1988 Mol. Cell. Biol. 8:466-472). In addition to this, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g. U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et a/.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the protein, the expression vector(s) encoding the Soluble Proteins or intermediates such as heavy and light chain sequences of the SIRPa binding Fusobody is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g. electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is theoretically possible to express the Soluble Proteins of the invention in either prokaryotic or eukaryotic host cells. Expression of glycoprotein in eukaryotic cells, in particular mammalian host cells, is discussed because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and biologically active glycoprotein such as the SIRPa binding Fusobodies. The Fusobodies can be advantageously produced using well known expression systems developed for antibodies molecules.

Mammalian host cells for expressing the Soluble Proteins and intermediates such as heavy and light chains of SIRPa binding Fusobody of the invention include Chinese Hamster Ovary cells (CHO cells), including dhfr- CHO cells, (described by Urlaub and Chasin, 1980, Proc. Natl. Acad. Sc ^' i. USA 77:4216-4220) used with a DH FR selectable marker, e.g. as described in R.J. Kaufman and P.A. Sharp, 1982 Mol. Biol. 159:601-621 ), NSO myeloma cells, COS cells and SP2 cells or human cell lines (including PER-C6 cell lines, Crucell or HEK293 cells, Yves Durocher et a/., 2002, Nucleic acids research vol 30, No 2 e9). When recombinant expression vectors encoding polypeptides are introduced into mammalian host cells, the Soluble Proteins and intermediates such as heavy and light chains of SIRPa- binding Fusobody of the invention are produced by culturing the host cells for a period of time sufficient to allow for expression of the recombinant polypeptides in the host cells or secretion of the recombinant polypeptides into the culture medium in which the host cells are grown. The polypeptides can then be recovered from the culture medium using standard protein purification methods.

Multivalent SIRPa binding Proteins In another aspect, the present invention provides multivalent proteins comprising at least two identical or different soluble SIRPa binding Proteins of the invention. In one embodiment, the multivalent protein comprises at least two, three or four Soluble SIRPa binding Proteins of the invention. The Soluble SIRPa binding Proteins can be linked together via protein fusion or covalent or non covalent linkages. The multivalent proteins of the present invention can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each binding specificity of the multivalent protein can be generated separately and then conjugated to one another.

A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g. Karpovsky et a/., 1984 J. Exp. Med. 160:1686; Liu, MA et a/., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et a/., 1985 Science 229:81-83), and Glennie et a/., 1987 J. Immunol. 139: 2367- 2375). Covalent linkage can be obtained by disulfide bridge between two cysteines, for example disulfide bridge from cysteine of an Fc domain.

Conjugated SIRPa binding Proteins

In another aspect, the present invention features a SIRPa binding Proteins, in particular, SIRPa binding Fusobody, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g. an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as "Conjugated SIRPa binding Proteins". A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g. kills) cells. Such agents have been used to prepare conjugates of antibodies or immunoconjugates. Such technologies can be applied advantageously with SIRPa binding Proteins, in particular, SIRPa binding Fusobody. Examples of cytotoxin or cytotoxic agent include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), ablating agents (e.g. mechlorethamine, thioepa chloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to SIRPa binding Proteins or Fusobodies of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.

Cytoxins can be conjugated to SIRPa binding Proteins or Fusobodies of the invention using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to SIRPa binding Proteins or Fusobodies of the invention include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g. cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. ef a/., 2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P.A. ef a/., 2003 Cancer Immunol. Immunother. 52:328-337; Payne, G., 2003 Cancer Cell 3:207-212; Allen, T.M., 2002 Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J., 2002 Curr. Opin. Investig. Drugs 3:1089-1091 ; Senter, P.D. and Springer, C.J., 2001 Adv. Drug Deliv. Rev. 53:247-264. SIRPa binding Proteins or Fusobodies of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals. Examples of radioactive isotopes that can be conjugated to the SIRPa binding Proteins or Fusobodies of the present invention for use diagnostically or therapeutically include, but are not limited to, iodinel31 , indium111 , yttrium90, and Iutetium177. Method for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (DEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radiopharmaceuticals using SIRPa binding Proteins or Fusobodies of the present invention of the invention. Furthermore, techniques for conjugating toxin or radioisotopes to antibodies are well known, see, e.g. Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Inmunol. Rev., 62:1 19-58 (1982).

Pharmaceutical compositions

In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing one or a combination of the Soluble SIRPa binding Proteins or Fusobodies of the present invention, formulated together with a pharmaceutically acceptable carrier.

Pharmaceutical formulations comprising a Soluble SIRPa binding Protein or Fusobody of the invention may be prepared for storage by mixing the proteins having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions, lyophilized or other dried formulations. The invention further relates to a lyophilized composition comprising at least the Soluble Protein of the invention, e.g. the SIRPa binding Fusobodies of the invention and appropriate pharmaceutically acceptable carrier. The invention also relates to syringes pre-filled with a liquid formulation comprising at least the Soluble Protein of the invention, e.g. the SIRPa binding Fusobodies, and appropriate pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a Soluble SIRPa binding Protein or Fusobody of the present invention combined with at least one other anti-inflammatory or another chemotherapeutic agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the soluble SIRPa binding Proteins of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). Depending on the route of administration, the active principle may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate the active principle.

The pharmaceutical composition of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g. Berge, S.M., et al., 1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, one can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the Soluble Proteins, e.g. the SIRPa binding Proteins or Fusobodies in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microflltration. Generally, dispersions are prepared by incorporating the active principle into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, from about 0.1 per cent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g. a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. For administration of the Soluble SIRPa binding Proteins or Fusobodies of the invention, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-30 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Dosage regimens for a Soluble SIRPa binding Proteins or Fusobodies of the invention include 1 mg/kg body weight or 3 mg/kg body weight by intravenous administration, with the protein being given using one of the following dosing schedules: every four weeks for six dosages, then every three months; every three weeks; 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

The Soluble SIRPa binding Proteins or Fusobodies is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of Soluble Polypeptide in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of about 0.1-1000 pg/ml and in some methods about 5- 300 Mg/ml.

Alternatively, the Soluble SIRPa binding Proteins or Fusobodies can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the Soluble Proteins in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of Soluble SIRPa binding Proteins or Fusobodies can result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

A composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for Soluble Proteins of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intraocular, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

Alternatively, a Soluble SIRPa binding Proteins or Fusobodies can be administered by a nonparenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active principles can be prepared with carriers that will protect the proteins against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are published or generally known to those skilled in the art. See, e.g. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Patent Nos. 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which shows an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which shows a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which shows a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which shows a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which shows an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which shows an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the Soluble SIRPa binding Proteins or Fusobodies can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g. U.S. Patents 4,522,81 1 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g. V.V. Ranade, 1989 J. Cline Pharmacol. 29:685). Uses and methods of the invention

The Soluble SIRPa binding Proteins or Fusobodies have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or in vivo, or in a subject, e.g. in vivo, to treat, prevent or diagnose a variety of disorders. In one embodiment, the Soluble SIRPa binding Fusobodies can be used in in vitro expansion of stem cells or other cell types like pancreatic beta cells in the presence of other cell types which otherwise would interfere with expansion. In addition, in particular the Soluble SIRPa binding proteins or Fusobodies are used to in vitro qualify and quantify the expression of functional SIRPa at the cell surface of cells from a biological sample of an organism such as human. This application may be useful as commercially available SIRPa antibodies cross-react with various isoforms of SIRPp making difficult to unambigously quantify SIRPa protein expression on the cell surface. Quantification of Soluble SIRPa binding Proteins or Fusobodies can therefore be used for diagnostic purpose for example to assess the correlation of the quantity of SIRPa protein expression with immune or cancer disorders and therefore allow selection of patients (patient stratification) for treatment with, for example, Conjugated SIRPa binding Proteins or antibody-based therapies targeted against SIRPa. The methods are particularly suitable for treating, preventing or diagnosing autoimmune and inflammatory disorders mediated by SIRPa+ cells e.g. allergic asthma or ulcerative colitis. These include acute and chronic inflammatory conditions, allergies and allergic conditions, autoimmune diseases, ischemic disorders, severe infections, and cell or tissue or organ transplant rejection including transplants of non-human tissue (xenotransplants). The methods are particularly suitable for treating, preventing or diagnosing autoimmune and inflammatory or malignant disorders mediated by cells expressing aberrant or mutated variants of the activating SIRPp receptor which are reactive to CD47 and dysfunction via binding to CD47 or other SIRPa ligands.

Examples of autoimmune diseases include, without limitation, arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarhropathies including ankolsing spondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis, and enterophathis arthritis, hypersensitivity (including both airways hypersensitivity and dermal hypersensitivity) and allergies. Autoimmune diseases include autoimmune haematological disorders (including e.g. hemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), systemic lupus erythematosus, inflammatory muscle disorders, polychondritis, sclerodoma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue, endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis and glomerulonephritis (with and without nephrotic syndrome, e.g. including gout, langerhans cell histiocytosis, idiopathic nephrotic syndrome or minimal change nephropathy), tumors, multiple sclerosis, inflammatory disease of skin and cornea, myositis, loosening of bone implants, metabolic disorders, such as atherosclerosis, diabetes, and dislipidemia.

The Soluble SIRPa binding Proteins or Fusobodies are also useful for the treatment, prevention, or amelioration of asthma, bronchitis, pneumoconiosis, pulmonary emphysema, and other obstructive or inflammatory diseases of the airways.

The Soluble SIRPa binding Proteins or Fusobodies are also useful for the treatment, prevention, or amelioration of immunesystem-mediated or inflammatory myopathies including coronar myopathies. The Soluble SIRPa binding Proteins or Fusobodies are also useful for the treatment, prevention, or amelioration of disease involving the endothelial or smooth muscle system which express SIRPa.

The Soluble SIRPa binding Proteins or Fusobodies are also useful for the treatment of IgE- mediated disorders. IgE mediated disorders include atopic disorders, which are characterized by an inherited propensity to respond immunologically to many common naturally occurring inhaled and ingested antigens and the continual production of IgE antibodies. Specific atopic disorders include allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastroenteropathy.

However, disorders associated with elevated IgE levels are not limited to those with an inherited (atopic) etiology. Other disorders associated with elevated IgE levels, that appear to be IgE-mediated and are treatable with the formulations of this present invention include hypersensitivity (e.g. anaphylactic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-lgE syndrome, ataxia- telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and graft- versus-host reaction.

The Soluble SIRPa binding Proteins or Fusobodies are useful as first line treatment of acute diseases involving the major nervous system in which inflammatory pathways are mediated by SIRPa+ cells such as activated microglia cells. A particular application for instance can be the silencing of activated microglia cells after spinal cord injury to accelerate healing and prevent the formation of lymphoid structures and antibodies autoreactive to parts of the nervous system. The Soluble SIRPa binding Proteins or Fusobodies may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant to or in combination to, other drugs e.g. immunosuppressive or immunomodulating agents or other anti-inflammatory agents, e.g. for the treatment or prevention of diseases mentioned above. For example, the Soluble SIRPa binding Proteins or Fusobodies may be used in combination with DMARD, e.g. Gold salts, sulphasalazine, antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, glococorticoids; a calcineurin inhibitor, e.g. cyclosporin A or FK 506; a modulator of lymphocyte recirculation, e.g. FTY720 and FTY720 analogs; a mTOR inhibitor, e.g. rapamycin, 40-O-(2- hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; an ascomycin having immuno-suppressive properties, e.g. ABT-281 , ASM981 , etc.; corticosteroids; cyclo-phos- phamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid; myco- pheno-late mofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogue or derivative thereof; immunosuppressive monoclonal antibodies, e.g. monoclonal antibodies to leukocyte receptors, e.g. MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, e.g. LEA29Y; adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent, e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or 5-fluorouracil; anti TNF agents, e.g. monoclonal antibodies to TNF, e.g. infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII, e.g. Etanercept, PEG-TNF-RI; blockers of proinflammatory cytokines, IL-1 blockers, e.g. Anakinra or IL-1 trap, AAL160, ACZ 885, IL-6 blockers; chemokines blockers, e.g inhibitors or activators of proteases, e.g. metalloproteases, anti-IL-15 antibodies, anti-IL- 6 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-IL17 antibodies, anti-IL12 antibodies, anti-IL12R antibodies, anti-IL23 antibodies, anti-IL23R antibodies, anti-IL21 antibodies, NSAIDs, such as aspirin, ibuprophen, paracetamol, naproxen, selective Cox2 inhibitors, combined Cox1 and 2 inhibitors like diclofenac, or an anti-infectious agent (list not limited to the agent mentioned).

The Soluble SIRPa binding Proteins or Fusobodies are also useful as co-therapeutic agents for use in conjunction with anti-inflammatory or bronchodilatory drug substances, particularly in the treatment of obstructive or inflammatory airways diseases such as those mentioned hereinbefore, for example as potentiators of therapeutic activity of such drugs or as a means of reducing required dosaging or potential side effects of such drugs. An agent of the invention may be mixed with the anti-inflammatory or bronchodilatory drug in a fixed pharmaceutical composition or it may be administered separately, before, simultaneously with or after the anti-inflammatory or bronchodilatory drug. Such anti-inflammatory drugs include steroids, in particular glucocorticosteroids such as budesonide, beclamethasone, fluticasone or mometasone, and dopamine receptor agonists such as cabergoline, bromocriptine or ropinirole. Such bronchodilatory drugs include anticholinergic or antimuscarinic agents, in particular ipratropium bromide, oxitropium bromide and tiotropium bromide.

Combinations of agents of the invention and steroids may be used, for example, in the treatment of COPD or, particularly, asthma. Combinations of agents of the invention and anticholinergic or antimuscarinic agents or dopamine receptor agonists may be used, for example, in the treatment of asthma or, particularly, COPD.

In accordance with the foregoing, the present invention also provides a method for the treatment of an obstructive or inflammatory airways disease which comprises administering to a subject, particularly a human subject, in need thereof a Soluble SIRPa binding Proteins or Fusobodies, as hereinbefore described. In another aspect, the invention provides a Soluble SIRPa binding Proteins or Fusobodies, as hereinbefore described for use in the preparation of a medicament for the treatment of an obstructive or inflammatory airways disease.

The Soluble SIRPa binding Proteins or Fusobodies are also particularly useful for the treatment, prevention, or amelioration of chronic gastrointestinal inflammation, such as inflammatory bowel diseases, including Crohn's disease and ulcerative colitis.

"Chronic gastrointestinal inflammation" refers to inflammation of the mucosal of the gastrointestinal tract that is characterized by a relatively longer period of onset, is long- lasting (e.g. from several days, weeks, months, or years and up to the life of the subject), and is associated with infiltration or influx of mononuclear cells and can be further associated with periods of spontaneous remission and spontaneous occurrence. Thus, subjects with chronic gastrointestinal inflammation may be expected to require a long period of supervision, observation, or care. "Chronic gastrointestinal inflammatory conditions" (also referred to as "chronic gastrointestinal inflammatory diseases") having such chronic inflammation include, but are not necessarily limited to, inflammatory bowel disease (IBD), colitis induced by environmental insults (e.g. gastrointestinal inflammation (e.g. colitis) caused by or associated with (e.g. as a side effect) a therapeutic regimen, such as administration of chemotherapy, radiation therapy, and the like), colitis in conditions such as chronic granulomatous disease (Schappi et al. Arch Dis Child. 2001 February; 1984(2): 147- 151), celiac disease, celiac sprue (a heritable disease in which the intestinal lining is inflamed in response to the ingestion of a protein known as gluten), food allergies, gastritis, infectious gastritis or enterocolitis (e.g. Helicobacter pylori-infected chronic active gastritis) and other forms of gastrointestinal inflammation caused by an infectious agent, and other like conditions.

As used herein, "inflammatory bowel disease" or "IBD" refers to any of a variety of diseases characterized by inflammation of all or part of the intestines. Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease and ulcerative colitis. Reference to IBD throughout the specification is often referred to in the specification as exemplary of gastrointestinal inflammatory conditions, and is not meant to be limiting.

In accordance with the foregoing, the present invention also provides a method for the treatment of chronic gastrointestinal inflammation or inflammatory bowel diseases, such as ulcerative colitis, which comprises administering to a subject, particularly a human subject, in need thereof, a Soluble SIRPa binding Proteins or Fusobodies, as hereinbefore described. In another aspect, the invention provides a Soluble SIRPa binding Proteins or Fusobodies, as hereinbefore described for use in the preparation of a medicament for the treatment of chronic gastrointestinal inflammation or inflammatory bowel diseases. The present invention is also useful in the treatment, prevention or amelioration of leukemias or other cancer disorders. For example, a Soluble SIRPa binding Proteins or Fusobodies can be used in treating, preventing or ameliorating cancer disorders selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin lymphoma, hodgkin disease, bladder cancer, malignant forms of langerhans cell histiocytosis.

Modulating SIRPa-CD47 interaction can be used to increase hematopoietic stem cell engraftment (see e.g. WO2009/046541 related to the use of CD47-Fc fusion proteins). The present invention, and for example, Soluble SIRPa binding Proteins or Fusobodies are therefore useful for increasing human hematopoietic stem cell engraftment. Hematopoietic stem cell engraftment can be used to treat or reduce symptoms of a patient that is suffering from impaired hematopoiesis or from an inherited immunodeficient disease, an autoimmune disorder or hematopoietic disorder, or having received any myelo-ablative treatment. For example, such hematopoietic disorder is selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin lymphoma, hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, fanconi anemi, thalassemia major, Sickle cell anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis and inborn errors of metabolism. Therefore, in one embodiment, the invention relates to Soluble SIRPa binding Proteins or Fusobodies for use in treating hematopoietic disorder is selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin lymphoma, hodgkin disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, fanconi anemi, thalassemia major, Sickle cell anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis and inborn errors of metabolism in particular, after treatment with an expanded cell population containing hematopoietic stem cell, in order to improve hematopoietic stem cell engraftment.

Also encompassed within the scope of the present invention is a method as defined above comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of a Soluble SIRPa binding Proteins or Fusobodies, and at least one second drug substance, said second drug substance being a immuno-suppressive / immunomodulatory, anti-inflammatory chemotherapeutic or anti-infectious drug, e.g. as indicated above.

Or, a therapeutic combination, e.g. a kit, comprising of a therapeutically effective amount of a) a Soluble SIRPa binding Proteins or Fusobodies and b) at least one second substance selected from an immuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeutic or anti-infectious drug, e.g. as indicated above. The kit may comprise instructions for its administration.

Where the Soluble SIRPa binding Proteins or Fusobodies are administered in conjunction with other immuno-suppressive / immunomodulatory, anti-inflammatory chemotherapeutic or anti-infectious therapy, dosages of the co-administered combination compound will of course vary depending on the type of co-drug employed, on the condition being treated and so forth. The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not meant to be further limiting.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of an example of a SIRPa binding Fusobody

Figure 2. SIRPa Binding activity of recombinant SIRPa binding Fusobody compared to prior art divalent SIRPa binding protein (CD47-Fc). SIRPa binding Fusobody Example#4 is compared to a divalent SIRPa binding protein in the capacity to compete with the binding of divalent biotinylated SIRPa binding protein (CD47- Fc) to immobilized SIRPa-Fc as described in under 2.2. SIRPa binding Fusobody Example#4 (triangles) competes >5 fold more potently with the binding of biotinylated CD47- Fc (used at 5nM) compared to the divalent SIRPa binding protein (black circles). Since the affinity of the single CD47 moieties of both competitors is identical these data demonstrate improvement of avidity of SIRPa binding Fusobody over prior art CD47-Fc fusion proteins.

Figure 3 Binding activity of recombinant SIRPa binding Fusobody to cellular SIRPa.

SIRPa binding Fusobody Example#4 is compared in its ability to support SIRPa-dependent cellular adhesion. Fluorescently labelled U937 cells are allowed to adhere for 30 min under static conditions to various concentrations of immobilized SIRPa binding Fusobody Example#4 or a divalent SIRPa binding protein (CD47-Fc). Loosely adhering or non bound cells are removed by fluidic shear force e.g. repeated washing steps as described in 2.3. Data show that SIRPa binding Fusobody Example#4 (triangles) supports >5 fold more potently (Table 5) the firm adherence of SIRPa ⁺ U937 cells compared to the divalent SIRPa binding protein (CD47-Fc) (black circles). Since the affinity of both competitors is identical these data demonstrate improvement of avidity of SIRPa binding Fusobody to its cell bound target over prior art CD47-Fc fusion proteins.

Figure 4. Specific binding of a SIRPa binding Fusobody (Example#4), to human SIRPa+ monocytes in whole blood and competition with unlabeled SIRPa binding proteins.

SIRPa binding Fusobody Example#4 efficiently binds to to CD14 ⁺ monocytes in whole blood, e.g. in the presence of CD47 high expressing erythrocytes. Binding was quantified by flow cytometry in whole human blood using an Ax647-fluorochrome-labeled SIRPa binding Fusobody Example#4 (Method as in 2.4). Binding is concentration-dependently blocked by unlabelled SIRPa binding Fusobody (triangles) or a prior art SIRPa binding protein (CD47- Fc) (black circles)). Ax647-fluorochrome-labeled SIRPa binding Fusobody Example#4 was unable to interact with CD14+ monocytes when blood samples were treated with of 20 ig/m\ anti-SIRPa antibody (clone 148) before addtion of Ax647-fluorochrome-labeled SIRPa binding Fusobody Example#4. No binding to lymphocytic T or B cells was observed (not shown). The superior binding of the SIRPa binding Fusobody to human SIRPa+ monocytes in whole blood is reflected by the clearly less potent competition (ca 20-50 fold higher IC50 values obtained, Table 5) with non-labeled prior art divalent SIRPa binding protein (CD47- Fc). Control human lgG1 (boxes) was not affecting binding of Ax647-fluorochrome-labeled SIRPa binding Fusobody to CD14+ monocytes.

Figure5. SIRPa binding Fusobody Example#4 silences the cytokine release from in 'froonocyte-derived human dendritic cells with pM potency.

GMSCF/IL4-differentiated monocyte-derived dendritic cells are stimulated with SAC particles (Staphylococcus aureus Cowan strain, 0.01 %) over night in the presence of SIRPa binding Fusobody Example#4 or human lgG1 as control. SIRPa binding Fusobody Example#4 blocked the cytokine release of TNFa, IL6 and IL12 into supernatants with pM potency.

Figure 6. Murine surrogates of the SIRPa binding fusobodies protect animals from development of antigen-triggered lung inflammation, a model mimicking disease parameters of human allergic asthma. Treatment of mice with two administrations of 100 g/animal i.p. of murine SIRPa binding fusobodies (mCD47 C15G Fusobody (heavy chain SEQ ID: 31 , light chain SEQ ID: 32, left graph) or mCD47 Fusobody, (heavy chain SEQ ID: 33, light chain SEQ ID: 34, right graph) reduced the total cell counts as well as the numbers of eosinophils (eos), neutrophils (neu) and lymphocytes (lymp) in the BALF after airosol antigen challenge compared to controls. Both murine SIRPa binding fusobodies thus potently protected mice from development of allergic asthma. n= number of animals used per group.

Figure 7. Murine surrogate of the SIRPa binding fusobodies decrease severity of TNBS-colitis a model mimicking pathology aspects of human colitis.

Treatment of mice with 3-4 administrations of 100 g/animal i.p. of murine SIRPa binding Fusobody (mCD47 C15G Fusobody (heavy chain SEQ ID: 31 , light chain SEQ ID: 32) statistically significantly reduced the severity of the inflammatory colitis elicited by TNBS as indicated by body weight loss. After disease reinduction at day 7 with TNBS, mCD47 C15G Fusobody treated animals maintained bodyweights above PBS or Control IgG controls. Injection of murine SIRPa-binding protein (mCD47-C15G Fusobody) thus actively blocks the severity of disease development. Data are a summary of 2 different experiments with either 3 or 4 consecutive administrations of test compounds. n= number of animals used per group.

EXAMPLES

1. Examples of SIRPa binding Fusobodies of the invention

The following table 4 provides examples of SIRPa binding Fusobodies of the invention that may be produced by recombinant methods using DNA encoding the disclosed heavy and light chain amino acid sequences.

The DNA encoding the heavy and/or light chain may further comprise coding sequence of the CD47 signal sequence (see for example SEQ ID NO: 10). The CD47 signal sequence is for example expressed at the N-terminal part of the heavy and light chain to direct the secretion of the Fusobody outside of the producing cells. Table 4:

Example Fc Part CH1 region Linker SIRPa SEQ ID or CL region binding

region

#1 heavy SEQ ID NO:9 SEQ ID NO:7 No linker SEQ ID NO:4 SEQ ID NO:5 chain

#1 light Not applicable SEQ ID NO:8 No linker SEQ ID NO:4 SEQ ID NO:6 chain

#2 heavy SEQ ID NO:22 SEQ ID NO:7 No linker SEQ ID NO:4 SEQ ID chain NO: 18

#2 light Not applicable SEQ ID NO:8 No linker SEQ ID NO:4 SEQ ID NO:6 chain

#3 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO.19

#3 light Not applicable SEQ ID NO:8 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO:20

#4 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)2 SEQ ID NO: SEQ ID chain 21 NO: 12

#4 light Not applicable SEQ ID NO:8 (Gly4Ser)2 SEQ ID NO: SEQ ID chain 21 NO:13

#5 heavy SEQ ID N0:9 SEQ ID NO:7 No linker SEQ ID SEQ ID chain NO:23 NO:24

#5 light Not applicable SEQ ID NO:8 No linker SEQ ID SEQ ID chain N0.23 N0.25

#6 heavy SEQ ID NO:9 SEQ ID NO:7 No linker SEQ ID NO: SEQ ID chain 21 NO:36

#6 light Not applicable SEQ ID NO:8 No linker SEQ ID NO: SEQ ID chain 21 NO:37

#7 heavy SEQ ID NO:9 SEQ ID NO:7 No linker SEQ ID NO: SEQ ID chain 27 NO:38

#7 light Not applicable SEQ ID NO.8 No linker SEQ ID NO. SEQ ID chain 27 NO:39

#8 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)1 SEQ ID NO: SEQ ID chain 21 NO:40

#8 light Not applicable SEQ ID NO:8 (Gly4Ser)1 SEQ ID NO: SEQ ID chain 21 NO:41

#9 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)1 SEQ ID NO: SEQ ID chain 27 NO:42

#9 light Not applicable SEQ ID NO:8 (Gly4Ser)1 SEQ ID NO: SEQ ID chain 27 NO:43

#10 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)2 SEQ ID NO: SEQ ID chain 27 NO:44

#10 light Not applicable SEQ ID NO:8 (Gly4Ser)2 SEQ ID NO: SEQ ID chain 27 NO:45

#11 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)3 SEQ ID NO: SEQ ID chain 21 NO:46

#11 light Not applicable SEQ ID NO:8 (Gly4Ser)3 SEQ ID NO: SEQ ID chain 21 NO:47 #12 heavy SEQ ID N0:9 SEQ ID NO:7 (Gly4Ser)3 SEQ ID NO: SEQ ID chain 27 NO:48

#12 light Not applicable SEQ ID NO:8 (Gly4Ser)3 SEQ ID NO: SEQ ID chain 27 NO:49

#13 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)5 SEQ ID NO: SEQ ID chain 21 NO:50

#13 light Not applicable SEQ ID NO:8 (Gly4Ser)5 SEQ ID NO: SEQ ID chain 21 NO:51

#14 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)1 SEQ ID SEQ ID chain NO:23 NO:52

#14 light Not applicable SEQ ID NO:8 (Gly4Ser)1 SEQ ID SEQ ID chain NO:23 NO:53

#15 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)1 SEQ ID NO:4 SEQ ID chain NO:54

#15 light Not applicable SEQ ID NO:8 (Gly4Ser)1 SEQ ID NO:4 SEQ ID chain NO: 55

#16 heavy SEQ ID NO:9 SEQ ID NO:7 (Gly4Ser)5 SEQ ID NO:4 SEQ ID chain NO:56

#16 light Not applicable SEQ ID NO:8 (Gly4Ser)5 SEQ ID NO:4 SEQ ID chain NO:57

#17 heavy SEQ ID NO:22 SEQ ID NO:7 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO:58

#17 light Not applicable SEQ ID NO:8 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO:20

#18 heavy SEQ ID NO:28 SEQ ID NO:7 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO:29

#18 light Not applicable SEQ ID NO:8 (Gly4Ser)2 SEQ ID NO:4 SEQ ID chain NO:20 2. Affinity determination

2.1. Binding assay to monovalent SIRPa (BiaCORE assay)

The monovalent affinity of human monomeric SIRPa-APP CD47 can be assessed by BiaCORE using for example a BiaCORE T100 instrument. A CM5 chip is immobilized with Protein A applying the standard amine coupling procedure. Flow cell 1 is blank immobilized to serve as a reference. SIRPa binding proteins are immobilized via Fc binding properties of Protein A. Monovalent - for example an APP-tagged SIRPa V domain protein is expressed in HEK293 cells. APP-SIRPa is serially diluted twelve times by a factor of 1 :2. Starting concentrations are 25 μΜ- 0.5μΜ. Affinity data are acquired by subsequent injections of the APP-SIRPa concentration series on the reference and measuring flow cells. The chip surface is regenerated after each analyte injection by 50mM Citrate solution.

The monovalent interaction with SIRPa-APP is measured as K _D of 3 μΜ which shows similar affinity as the monovalent interaction of CD47 V-domain with SIRPa reported (1-2 μΜ, Heatherley et al. 2008 Mol Cell.) or measured (3 μΜ) using a bivalent SIRPa binding protein (CD47-FC).

Alternatively, binding of SIRPa binding Proteins to divalent recombinant SIRPa can be characterized by BiaCORE. For this human SIRPa-Fc (10 μg/mL, R&D systems, UK) in can be immobilizing in acetate buffer pH4.5, on a BiaCORE chip alike CM5 (carboxymethylated dextran matrix) after surface activation/deactivation by standard procedures like EDC/NHS or ethanolamine respectively. Assessment can be done by contact time for 120s, dissociation times for 240s and flow rates for 50μΙ/ηιϊη. After each injection of analyte, the chip can be regenerated with Gentle elution buffer (ThermoScientific).

2.2. Competition assay with recombinant CD47 -fusion protein binding to SIRPa

Experiments are performed in 384-well microtiter plates (Nunc). Immobilized human SIRPa- Fc fusion protein (0.5 μg/mL, R&D systems, UK) is incubated with a mixture of biotinylated SIRPa binding protein consisting of either a CD47-ECD lgG1 Fc fusion protein (CD47-Fc, 5nM) or a biotinylated CD47 Fusobody (Example #4, 1 nM) and varying concentrations (30nM-0.003nM) of unlabelled SIRPa binding proteins or unlabelled SIRPa binding Fusobodies. After complex formation for 18 h at RT unbound proteins are removed by extensive washing. Bound biotinylated CD47-fusion protein is detected via Streptavidin- Europium (PerkinElmer reagents). The label, Eu ³⁺ , is measured using dissociation- enhanced time-resolved fluorometry (TRF) using a VICTOR2 reader (PerkinElmer)

2.3 Plate-based cellular adhesion assay using U937 cells

U937 cells, a histiocytic cell line expressing SIRPa (ATCC) is grown under standard cell culture conditions in RPMI1640 supplemented with 10% fetal bovine serum and antibiotics (all from Invitrogen). Cells are split 1 :1 on day before an experiment. Cells are harvested and resuspended in phosphate buffered saline (PBS, SIGMA) containing bovine serum albumin (BSA, SIGMA) (PBS/BSA). Cells can be labeled with 5 g/mL BCECF-AM (Invitrogen) or equivalent dyes like Calcein AM (Invitrogen) for 20 min at 37°C. Unbound BCECF-AM is removed by a washing step. Cells are counted and number adjusted to 1x10 ⁶ cells/mL in RPMI 1640 supplemented with 0.5% BSA. 96 well plates are coated with 60μΙ per well of 3μg/ml anti-human Fc goat IgG (Jackson ImmunoResearch Laboratories) in 0.1 M NaHC9 ₃ /Na ₂ C9 ₃ buffer overnight. Plates are washed twice with PBS, blocked with 1.5% BSA in PBS for 30min (250 L/well) and then incubated with varying concentrations of SIRPa binding proteins like soluble SIRPa binding Fusobodies or CD47-ECD lgG1 Fc fusion protein (CD47-Fc, Seq1 of CD47 ECD) (CD47-Fc) (0.01 and 30nM). After 2h at RT, plates are washed 2 times with PBS/BSA before adding BCECF-labeled U937 cells (100000 cells per well). After 30min incubation at 37 °C, U937 cells are subjected to fluidic shear stress by repeated manual or automatic washing steps using RPMI 1640 supplemented with 0.5% BSA. Generally 4-5 washing steps are required to remove loosely adhering or unbound cells. The fluorescence of the remaining U937 adherent cells is quantified by using a VICTOR2 plate reader (PerkinElmer).

2.4 Whole blood human cell binding assay

Human Blood from healthy volunteers is collected into Na-Heparin coated vacutainers (BectonDickinison, BD) applying ethical guidelines. Blood is aliquoted into 96- well deep well polypropylene plates (Costar) and incubated with various concentrations of SIRPa binding proteins like soluble SIRPa binding Fusobodies or CD47-ECD lgG1 Fc fusion protein (CD47-FC, Seq1 of CD47 ECD) (CD47-Fc) in the presence of final 0.1 % w/v sodium azide on ice. The fluorochrome Alexa Fluor 647 (AX647) can be conjugated to SIRPa binding Proteins using an labelling kit (Invitrogen). AX647-conjugated SIRPa binding Proteins like the Fusobody listed in the EXAMPLE #4 can be added to the whole blood samples at a concentration of 1-10 nM for 30 min on ice. During the last 15 minutes concentration- optimized antibodies against phenotypic cell surface markers are added: CD14-PE (clone MEM18, Immunotools, Germany), CD3 Percp-Cy5.5 (clone SK7, BD), CD16 FITC (clone 3G8, BD). Whole blood is lysed by addition of 10x volume of FACSLYSING solution (BD) and incubation for 10 min at RT. Samples are washed 2x with phosphate-buffered solution containing 0.5% bovine serum albumin (SIGMA-ALDRICH). Samples are acquired on a Facs Canto II (BD) within 24 hrs after lysing. Cell subsets are gated according to the monocyte light scatter profile and by CD14+ and CD3- expression. Of these cell subset, fluorescence histograms can be drawn and statistically evaluated taking the median fluroescence intensity as readout.

3. Dendritic Cell cytokine release assay for measuring inhibition of Staphylococcus aureus Cowan 1 strain particles stimulated release of proinflammatory cytokines

Peripheral blood monocytes (CD14+) as well as monocyte-derived dendritic cells (DCs) are prepared as described (Latour et al., J of Immunol, 2001 : 167:2547). Conventional (DCs) are isolated as CD11c+, lineage-, by a FACS Aria (BD Biosciences) by using allophycocyanin (APC)-labeled anti-CD11c (B-ly6), a mixture of FITC-labeled mAbs against lineage markers, CD3, CD14, CD15, CD16, CD19 and CD56 and APC-Cy7-labeled CD4 (RPA-T4) to reach >99% purity. APCs are stimulated with Staphylococcus aureus Cowan 1 particles at 1/40.000 (Pansorbin) in the presence of various concentrations of human SIRPa binding Fusobodies (1 to 10000 pM) in HB101 or X-VIV015 serum-free medium. Cytokine (IL-1 , IL-6, IL-10, IL-12p70, IL-23, IL-8 and TNF-a) release is assessed by ELISA in the 24h or 48h culture supernatants.

4. A mouse model of inflammatory lung disease (OVA-asthma) for use of SIRPa- binding proteins to prevent lung inflammation

Female BALB/c (6 to 8 weeks old) were purchased from Charles River maintained under specific pathogen free conditions. BALB/c mice were sensitized on days 0 and 5 by intraperitoneal (IP) injection of 10 pg OVA adsorbed to 1 mg Imject Alum (Pierce) in the absence (PBS control) or presence of 100 g of murine SIRPa binding Fusobodies containing murine CD47 extracellular IgSF domains with (mCD47 C15G Fusobody) or without C15G mutation (mCD47 Fusobody) fused to a human lgG1 backbone (mCD47 Fusobody: heavy chain SEQ ID: 34, light chain SEQ ID: 35,or mCD47 C15G Fusobody: heavy chain SEQ ID: 31 , light chain SEQ ID: 32) or control human IgGl On days 12, 16 and 20, mice are challenged for 30 minutes with a 0.5% OVA aerosol (Sigma, Grade V). Mice are sacrificed 24 hours after the last challenge. Bronchoalveolar lavage fluid (BALF) is collected 4 times with 0.5 mL physiologic saline. A schematic representation of the model is depicted in Figure 6.

Total cells in the BALF were stained with anti-CCR3, anti-B220 (R&D systems) and anti- CD3 (clone 145-2C11) and analyzed by flow cytometry. All the data were acquired on a FACSAria II (BD Biosciences). Statistical analyses were performed using unpaired student's T test and the non-parametric Mann-Whitney U test. ***P<0.001 , **P<0.01 , *P<0.05.

5. A murine animal model of colitis for the use of SIRPa-binding proteins

Trinitrobenzene sulfonic acid (TNBS) (2 or 3 mg) is dissolved in 50% ethanol and instilled into the colons of male Balb/c mice (WT and CD47 KO) via a 3.5F catheter. Control mice are given ethanol alone. TNBS colitis is reinduced on day 7 in several animals (as indicated in Figure 7) by instilation of 1.5 mg of TNBS mice. Mice are weighed every 24 hours. Mice are sacrificed on day 14. Serum, mesenteric lymph nodes and colons are harvested for further analysis. Colons can be scored macroscopically using the Wallace criteria which takes into account the presence of diarrhea, adhesions, thickening of the bowel wall and ulceration. They can also evaluated for microscopic markers of inflammation using the Ameho criteria, a scoring system based upon thickening of the submucosa, infiltration of the submucosa and lamina propria with mononuclear cells, mucous depletion, loss of crypt architecture, and edema (data not shown). A recombinant mouse SIRPa-binding protein (mCD47 C15G Fusobody) is administered intraperitoneally (100 g/mouse) just prior to TNBS colitis induction and 24, and 48 and in some animals 72 hours thereafter. Control mice receive phosphate buffered saline alone (PBS) or a Control lgG1.

Results

Binding and other functional properties of a SIRPa binding Fusobody (Example #4) as described in Table 4 are presented in the following Table 5 and compared with the properties of divalent CD47-Fc fusion.

Table 5:

Assays Example #4 CD47-FC Binding assay to monovalent

SIRPa [μΜ] 3 μΜ 3 μΜ

(Method 2.1)

Competition assay with divalent

CD47-FC binding to SIRPa 0.4-0.6 nM 3-6 nM

(Method 2.2) IC ₅₀ [nM]

Plate-based cellular adhesion

assay using U937 cells 0.3-0.6nM 3-5nM

(Method 2.3) EC ₅₀ [nM]

Whole blood human cell binding

1-2nM > 90nM

assay (Method 2.4) IC ₅₀ [nM]

Impairment of cytokine release

from SAC triggered monocyte- derived dendritic cells <0.25nM <0.25nM

TNFo/IL6/IL12 [nM]

(Method 3)

Functional properties of the heavy chains of the Examples of the invention are detailed in Table 6.

Table 6:

Competition assay Whole blood

Affinity to

with divalent human cell

SIRPa-Fc

Example CD47-Fc binding binding assay

KD [nM]

to SIRPa (Method (Method 2.4)

(BiaCORE)

2.2) ICso [nM] ICso [nM]

#3 heavy chain 0.06 4.8 18-20

#4 heavy chain 0.03-0.07* 30-52

#5 heavy chain 0.03-0.04 1.7 22

#6 heavy chain 0.03-0.05 ^* #7 heavy chain 0.03

#8 heavy chain 0.12

#9 heavy chain 0.08

#10 heavy chain 0.04-0.05*

#11 heavy chain 0.04-0.05*

#12 heavy chain 0.08*

#13 heavy chain 0.09

#14 heavy chain 0.05-0.06 1.5 28

#15 heavy chain 0.04-0.05 5.3 30

#16 heavy chain 0.06 11.8 35

#17 heavy chain 33

#18 heavy chain 7.3 33

* competition with huCD47-Fusobody

In vivo efficacy of SIRPa binding Fusobodies in a model of inflammatory lung disease (OVA-asthma)

Since interspecies cross-reacitvity between human and rodent CD47/SIRPa proteins is not given (not shown) murine SIRPa binding fusobodies were generated in analogy to human SIRPa binding proteins. SIRPa binding fusobodies containing either a wild-type (SEQ ID: 33) or a C15G-mutated (SEQ ID: 30) CD47 moiety (mCD47 Fusobody: heavy chain SEQ ID: 34, light chain SEQ ID: 35, or mCD47 C15G Fusobody: heavy chain SEQ ID: 31 , light chain SEQ ID: 32) were generated as human IgG fusion proteins in mammalian transient expression systems and purified to generate aggregate-free and endotoxin-free material by standard procedures.

Treatment of mice with murine SIRPa binding fusobodies (mCD47 C15G Fusobody or mCD47 Fusobody) potently protected mice from development of allergic asthma. As shown in Figure 6 treatment of mice with 2 x 100 pg/animal i.p. of either of the SIRPa binding fusobodies potently reduced the total cell counts as well as the numbers of eosinophils, neutrophils and lymphocytes in the bronchoalveolar lavage fluid (BALF) after aerosol antigen challenge compared to controls. In contrast, in control groups treated with either a human lgG1 with irrelevant specificity or PBS, a fulminant infiltration of leukocytes into BALF was observed. The influx of these various leukocyte subsets into BALF is generally regarded a marker correlating strongly with the severity of inflammatory lung disease. This model also is regarded useful to mimick aspects of pathology seen in human allergic asthma. These data demonstrate that a) the Fusobody protein formats are active in vivo and b) that SIRPa binding fusobodies mediate potent in vivo efficacy and c) that C15 of CD47 e.g. the amino acid that normally forms a disulfide bridge to C235 of a transmembrane loop of cellular CD47 (Rebres et a/. Biol Chem 2001) is not required for potent efficacy in vivo.

In vivo efficacy of SIRPa binding Fusobodies in a model of inflammatory colonic disease (TNBS colitis)

Treatment of mice with 3-4 administrations of 100 pg/animal i.p. of murine SIRPa binding Fusobody (mCD47 C15G Fusobody, heavy chain SEQ ID: 31 , light chain SEQ ID: 32) reduced the severity of the inflammatory colitis elicited by TNBS as indicated by the statistically significantly reduced body weight loss. After disease reinduction at day 7 with TNBS, mCD47 C15G Fusobody treated animals maintained bodyweights above PBS or Control IgG controls. Injection of murine SIRPa-binding protein (mCD47-C15G Fusobody) thus actively blocks the severity of disease development in TNBS colitis. Data are a summary of 2 different experiments with either 3 or 4 consecutive administrations of test compounds. n= number of animals used per group.

Useful amino acid and nucleotide sequences for practicing the invention

Table 7A: Brief description of useful amino acid and nucleotide sequences for practicing the invention

Light chain of Fusobody example #13 (w/o signal sequence)

Heavy chain of Fusobody example #14 (w/o signal sequence)

Light chain of Fusobody example #14 (w/o signal sequence)

Heavy chain of Fusobody example #15 (w/o signal sequence)

Light chain of Fusobody example #15 (w/o signal sequence)

Heavy chain of Fusobody example #16 (w/o signal sequence)

Light chain of Fusobody example #16 (w/o signal sequence)

Heavy chain of Fusobody example #17 (w/o signal sequence)

Nucleotide sequence of SEQ ID 19 heavy chain of Fusobody example #3 (w/o signal sequence)

Nucleotide sequence of SEQ ID 20 light chain of Fusobody example #3, #17 and #18 (w/o signal sequence)

Nucleotide sequence of SEQ ID 12 heavy chain of Fusobody example #4 (w/o signal sequence)

Nucleotide sequence of SEQ ID 13 light chain of Fusobody example #4 (w/o signal sequence)

Nucleotide sequence of SEQ ID 24 heavy chain of Fusobody example #5 (w/o signal sequence)

Nucleotide sequence of SEQ ID 25 light chain of Fusobody example #5 (w/o signal sequence)

Nucleotide sequence of SEQ ID 36 heavy chain of Fusobody example #6 (w/o signal sequence)

Nucleotide sequence of SEQ ID 37 light chain of Fusobody example #6 (w/o signal sequence)

Nucleotide sequence of SEQ ID 38 heavy chain of Fusobody example #7 (w/o signal sequence)

Nucleotide sequence of SEQ ID 39 light chain of Fusobody example #7 (w/o signal sequence)

Nucleotide sequence of SEQ ID 40 heavy chain of Fusobody example #8 (w/o signal sequence)

Nucleotide sequence of SEQ ID 41 light chain of Fusobody example #8 (w/o signal sequence)

Nucleotide sequence of SEQ ID 42 heavy chain of Fusobody example #9 (w/o signal sequence)

Nucleotide sequence of SEQ ID 43 light chain of Fusobody example #9 (w/o signal sequence)

Nucleotide sequence of SEQ ID 44 heavy chain of Fusobody example #10 (w/o signal sequence)

Nucleotide sequence of SEQ ID 45 light chain of Fusobody example #10 (w/o signal sequence)

Nucleotide sequence of SEQ ID 46 heavy chain of Fusobody example #11 (w/o signal sequence)

Nucleotide sequence of SEQ ID 47 light chain of Fusobody example #11 (w/o signal sequence)

Nucleotide sequence of SEQ ID 48 heavy chain of Fusobody example #12 (w/o signal sequence)

Nucleotide sequence of SEQ ID 49 light chain of Fusobody example #12 (w/o signal sequence)

Nucleotide sequence of SEQ ID 50 heavy chain of Fusobody example #13 (w/o signal sequence) 80 Nucleotide sequence of SEQ ID 51 light chain of Fusobody example #13 (w/o signal sequence)

81 Nucleotide sequence of SEQ ID 52 heavy chain of Fusobody example #14

(w/o signal sequence)

82 Nucleotide sequence of SEQ ID 53 light chain of Fusobody example #14

(w/o signal sequence)

83 Nucleotide sequence of SEQ ID 54 heavy chain of Fusobody example #15

(w/o signal sequence)

84 Nucleotide sequence of SEQ ID 55 light chain of Fusobody example #15

(w/o signal sequence)

85 Nucleotide sequence of SEQ ID 56 heavy chain of Fusobody example #16

(w/o signal sequence)

86 Nucleotide sequence of SEQ ID 57 light chain of Fusobody example #16

(w/o signal sequence)

87 Nucleotide sequence of SEQ ID 58 heavy chain of Fusobody example #17

(w/o signal sequence)

88 Nucleotide sequence of SEQ ID 29 heavy chain of Fusobody example #18

(w/o signal sequence)

89 Nucleotide sequence of SEQ ID 31

90 Nucleotide sequence of SEQ ID 32

91 Nucleotide sequence of SEQ ID 34

92 Nucleotide sequence of SEQ ID 35

Table 7B: Sequence listing

SEQ Amino acid or Nucleotide Sequence

ID

NO:

1 MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTA

TSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPA D

AGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPWSGPAARATPQHTVSFTCE

SHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKWLTREDVHSQVIC

EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQL

TWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQP

AVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIWGWCTLLVALLMAALYLVRIR

QKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYA

SIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

2 MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVK

WKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTC

EVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGM DEK

TIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAI GLTSFVI

AILVIQVIAYILAWGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQK TIQP

PRKAVEEPLNAFKESKGMMNDE

3 QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNE QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNEN

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

GK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNENRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRV

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK

ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGC

TCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTG

TCGTCATTCCATGC I I I GTTACTAATATGGAGGCACAAAACACTACTGAAGTATAC

GTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCTTTGATGGAGCTCTAAACA

AGTCCACTGTCCCCACTGAC I I I AGTAGTGCAAAAATTGAAGTCTCACAATTACTA

AAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGA

AACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGC

TAAAATATCGTGTTGTTTCATGG I I I I CTCCAAATGAAAATTCAGCTAGCACCAAG

GGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCA

CAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCT

GCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCA

GCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCA

AGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCC

CCCTGCCCAGCCCCAGAGGCAGCGGGCGGACCCTCCGTGTTCCTGTTCCCCCC

CAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGG

TGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGAC

GGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAG

CACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACG

GCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAA

AGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTG

CCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGT

GAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGC CCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCT TCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTG TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGC CTGAGCCTGTCCCCCGGCAAG

ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGC

TCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTTGTAATGACACTG

TCGTCATTCCATGCTTTGTTACTAATATGGAGGCACAAAACACTACTGAAGTATAC

GTAAAGTGGAAATTTAAAGGAAGAGATATTTACACC I I I GATGGAGCTCTAAACA

AGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTGAAGTCTCACAATTACTA

AAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGA

AACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGC

TAAAATATCGTGTTGTTTCATGG I I I I CTCCAAATGAAAATCGTACGGTGGCCGC

TCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCG

CCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG

TGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGA

GCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCA

AGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGC

CTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA V

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC

ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGC

TCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTGGTAATGACACTG

TCGTCATTCCATGC I I I GTTACTAATATGGAGGCACAAAACACTACTGAAGTATAC

GTAAAGTGGAAATTTAAAGGAAGAGATATTTACACCTTTGATGGAGCTCTAAACA

AGTCCACTGTCCCCACTGACTTTAGTAGTGCAAAAATTGAAGTCTCACAATTACTA

AAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGA

AACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGC

TAAAATATCGTGTTGTTTCATGGTTTTCTCCAAATGAAAATGGAGGTGGTGGATC

TGGAGGTGGAGGTAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGG

CCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGT

GAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA

CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGC

CTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGC

CCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGGCA

GCGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGAT

GATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAG

GACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGC

CAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAG

GTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAG

GGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGAT

GACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGA

CATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCA

CCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACC

GTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCA

CGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCA

AG

ATGTGGCCCCTGGTAGCGGCGCTGTTGCTGGGCTCGGCGTGCTGCGGATCAGC

TCAGCTACTATTTAATAAAACAAAATCTGTAGAATTCACGTTTGGTAATGACACTG

TCGTCATTCCATGC I I I GTTACTAATATG GAGG CACAAAACACTACTG AAGTATAC

GTAAAGTG G AAATTTAAAG G AAGAG ATATTTACACCTTTG ATG GAG CTCTAAACA

AGTCCACTGTCCCCACTGAC I I I AGTAGTGCAAAAATTGAAGTCTCACAATTACTA

AAAGGAGATGCCTCTTTGAAGATGGATAAGAGTGATGCTGTCTCACACACAGGA

AACTACACTTGTGAAGTAACAGAATTAACCAGAGAAGGTGAAACGATCATCGAGC

TAAAATATCGTGTTG I I I CATG GTTTTCTCCAAATGAAAATGG AG GTG GTG GATC

TGGAGGTGGAGGTAGCCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCC

CCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAAC

AACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCA

GAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCT

ACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAG

GTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAG

CTTCAACAGGGGCGAGTGC

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

G

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA V

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPG

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

GK QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA V

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNEN

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VS

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

MPVPASWPHPPGPFLLLTLLLGLTEVAGEEELQMIQPEKLLLVTVGKTATLHCTVTS L

LPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVG T

YYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPWLGPAARTTPEHTVSFTCESHG

FSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARWLDPWDVRSQVICEVA

HVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKFYPQSLQLTW

SENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDWLTCQVKHDGQLAVS

KRLALEVTVHQKDQSSDATPGPASSLTALLLIAVLLGPIYVPWKQKT

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VS

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC\A VDV

SH EDPEVKFN WYVDGVEVH NAKTKPRE EQ YASTYRWSVLTVLHQ DWLNG KEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA V

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPGK

QLLFSNVNSIEFTSGNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSWF SPNEKI

QLLFSNVNSIEFTSGNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T

DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSW F

SPNEKIGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK

RVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCNA VDVSHED

PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN

KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE S

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

QLLFSNVNSIEFTSGNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T

DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSW F

SPNEKIGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

QLLFSNVNSIEFTSCNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSWF SPNEKI

QLLFSNVNSIEFTSCNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T

DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSW F

SPNEKIGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK

RVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCNA VDVSHED

PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN

KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE S

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

QLLFSNVNSIEFTSCNETWIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTT T

DQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSW F

SPNEKIGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCNA VDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNENRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDP

EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES N

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK

THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT

C\A/VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN

GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNN

FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKNA'AC

EVTHQGLSSPVTKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSGGGGSGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV

DKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV

SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE W

ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY

TQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSGGGGSGGGGSGGGGSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS

SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN

QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFGNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNENGGGGSGGGGSGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK

THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV

DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKR

VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP

EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES N

GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSGGGGSGGGGSGGGGSSASTKGPSVFPLAPSSKST

SGGTAALGC LVKDYFP EPVTVS WN SGALTSGVHTF PAVLQSSG LYS LSSWTVPSS

SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN

QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV VSWFSPNENGGGGSGGGGSGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKS

TVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYR V

VSWFSPNENGGGGSGGGGSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA V

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPGK cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtgg tggatctggaggtggaggtag ctcagctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcaccag cggcggcacagccg ccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcg gagccctgacctccggc gtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtg acagtgcccagcagcag cctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtgga caagagagtggagc ccaagagctgcgacaagacccacacctgccccccctgcccagccccagaggcagcgggcg gaccctccgtgttcc tgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcg tggtggtggacgtgag ccacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgc caagaccaagccca gagaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccagg actggctgaacggca aggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccatca gcaaggccaagggc cagccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaac caggtgtccctgacc tgtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccag cccgagaacaactac aagaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgacc gtggacaagtccaggt ggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactaca cccagaagagcctga gcctgtcccccggcaag

Cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtg gtggatctggaggtggaggta gccgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaaga gcggcaccgccagcgt ggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaa cgccctgcagagcgg caacagccaggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagcag caccctgaccctga gcaaggccgactacgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgt ccagccccgtgacca agagcttcaacaggggcgagtgc

cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtc gtcattccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtg gtggatctggaggtggaggta gctcagctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcacca gcggcggcacagcc gccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagc ggagccctgacctccgg cgtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggt gacagtgcccagcagca gcctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtgg acaagagagtggag cccaagagctgcgacaagacccacacctgccccccctgcccagccccagaggcagcgggc ggaccctccgtgttc ctgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgc gtggtggtggacgtga gccacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacg ccaagaccaagccc agagaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccag gactggctgaacggc aaggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccatc agcaaggccaaggg ccagccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaa ccaggtgtccctgac ctgtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggcca gcccgagaacaactac aagaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgacc gtggacaagtccaggt ggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactaca cccagaagagcctga gcctgtcccccggcaag

Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtg gtggatctggaggtggaggta gccgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaaga gcggcaccgccagcgt ggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaa cgccctgcagagcgg caacagccaggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagcag caccctgaccctga gcaaggccgactacgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgt ccagccccgtgacca agagcttcaacaggggcgagtgc

cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtc gtcattccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcaagcgctagcaccaagggccccagcgtgtt ccccctggcccccagcagc aagagcaccagcggcggcacagccgccctgggctgcctggtgaaggactacttccccgag cccgtgaccgtgtcct ggaacagcggagccctgacctccggcgtgcacaccttccccgccgtgctgcagagcagcg gcctgtacagcctgtc cagcgtggtgacagtgcccagcagcagcctgggcacccagacctacatctgcaacgtgaa ccacaagcccagcaa caccaaggtggacaagagagtggagcccaagagctgcgacaagacccacacctgcccccc ctgcccagcccca gaggcagcgggcggaccctccgtgttcctgttcccccccaagcccaaggacaccctgatg atcagcaggacccccg aggtgacctgcgtggtggtggacgtgagccacgaggacccagaggtgaagttcaactggt acgtggacggcgtgga ggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcacctacagggtggt gtccgtgctgaccgt gctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccct gccagcccccatcga aaagaccatcagcaaggccaagggccagccacgggagccccaggtgtacaccctgccccc ctcccgggaggag atgaccaagaaccaggtgtccctgacctgtctggtgaagggcttctaccccagcgacatc gccgtggagtgggagag caacggccagcccgagaacaactacaagaccacccccccagtgctggacagcgacggcag cttcttcctgtacag caagctgaccgtggacaagtccaggtggcagcagggcaacgtgttcagctgcagcgtgat gcacgaggccctgca caaccactacacccagaagagcctgagcctgtcccccggcaag

Cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcacgtacggtggccgctcccagcgtgttca tcttcccccccagcgacgag cagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaacttctacccccgggag gccaaggtgcagtgga aggtggacaacgccctgcagagcggcaacagccaggagagcgtcaccgagcaggacagca aggactccaccta cagcctgagcagcaccctgaccctgagcaaggccgactacgagaagcataaggtgtacgc ctgcgaggtgaccca ccagggcctgtccagccccgtgaccaagagcttcaacaggggcgagtgc

cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtc gtcattccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatagcgcta gcaccaagggccccagcgtgt tccccctggcccccagcagcaagagcaccagcggcggcacagccgccctgggctgcctgg tgaaggactacttccc cgagcccgtgaccgtgtcctggaacagcggagccctgacctccggcgtgcacaccttccc cgccgtgctgcagagc agcggcctgtacagcctgtccagcgtggtgacagtgcccagcagcagcctgggcacccag acctacatctgcaacg tgaaccacaagcccagcaacaccaaggtggacaagagagtggagcccaagagctgcgaca agacccacacctg ccccccctgcccagccccagaggcagcgggcggaccctccgtgttcctgttcccccccaa gcccaaggacaccctg atgatcagcaggacccccgaggtgacctgcgtggtggtggacgtgagccacgaggaccca gaggtgaagttcaact ggtacgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggagcagtaca acagcacctacag ggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtg caaggtctccaacaag gccctgccagcccccatcgaaaagaccatcagcaaggccaagggccagccacgggagccc caggtgtacaccct gcccccctcccgggaggagatgaccaagaaccaggtgtccctgacctgtctggtgaaggg cttctaccccagcgac atcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccca gtgctggacagcg acggcagcttcttcctgtacagcaagctgaccgtggacaagtccaggtggcagcagggca acgtgttcagctgcagc gtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtcccccggc aag

Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatcgtacgg tggccgctcccagcgtgttcatc ttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctgctgaac aacttctacccccggg aggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggagagcg tcaccgagcagg acagcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgactacg agaagcataaggtgt acgcctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaacaggg gcgagtgc cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaagcgctagcaccaagggccccagcgtgt tccccctggcccccagcag caagagcaccagcggcggcacagccgccctgggctgcctggtgaaggactacttccccga gcccgtgaccgtgtcc tggaacagcggagccctgacctccggcgtgcacaccttccccgccgtgctgcagagcagc ggcctgtacagcctgtc cagcgtggtgacagtgcccagcagcagcctgggcacccagacctacatctgcaacgtgaa ccacaagcccagcaa caccaaggtggacaagagagtggagcccaagagctgcgacaagacccacacctgcccccc ctgcccagcccca gaggcagcgggcggaccctccgtgttcctgttcccccccaagcccaaggacaccctgatg atcagcaggacccccg aggtgacctgcgtggtggtggacgtgagccacgaggacccagaggtgaagttcaactggt acgtggacggcgtgga ggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcacctacagggtggt gtccgtgctgaccgt gctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccct gccagcccccatcga aaagaccatcagcaaggccaagggccagccacgggagccccaggtgtacaccctgccccc ctcccgggaggag atgaccaagaaccaggtgtccctgacctgtctggtgaagggcttctaccccagcgacatc gccgtggagtgggagag caacggccagcccgagaacaactacaagaccacccccccagtgctggacagcgacggcag cttcttcctgtacag caagctgaccgtggacaagtccaggtggcagcagggcaacgtgttcagctgcagcgtgat gcacgaggccctgca caaccactacacccagaagagcctgagcctgtcccccggcaag

Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcacgtacggtggccgctcccagcgtgttca tcttcccccccagcgacgag cagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaacttctacccccgggag gccaaggtgcagtgga aggtggacaacgccctgcagagcggcaacagccaggagagcgtcaccgagcaggacagca aggactccaccta cagcctgagcagcaccctgaccctgagcaaggccgactacgagaagcataaggtgtacgc ctgcgaggtgaccca ccaggg cctgtccag ccccgtgaccaagagcttcaacagggg cgagtg c

cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtc gtcattccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggcggcg gcggatccagcgctagcacc aagggccccagcgtgttccccctggcccccagcagcaagagcaccagcggcggcacagcc gccctgggctgcct ggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcggagccctgacctc cggcgtgcacaccttcc ccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtgacagtgcccagca gcagcctgggcaccca gacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagagagtgga gcccaagagctgcg acaagacccacacctgccccccctgcccagccccagaggcagcgggcggaccctccgtgt tcctgttcccccccaa gcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcgtggtggtggacgt gagccacgaggaccc agaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagcc cagagaggagcagt acaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacg gcaaggaatacaagtg caaggtctccaacaaggccctgccagcccccatcgaaaagaccatcagcaaggccaaggg ccagccacgggag ccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaaccaggtgtccctg acctgtctggtgaagg gcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaact acaagaccaccccc ccagtgctggacagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagtcc aggtggcagcagggca acgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcc tgagcctgtcccccgg caag

cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtc gtcattccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggcggcg gcggatcccgtacggtggcc gctcccagcgtgttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagc gtggtgtgcctgctgaa caacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcgg caacagccaggag agcgtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctg agcaaggccgacta cgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgac caagagcttcaacag gggcgagtgc

cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtc gtcattccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggcggcggcggatccagcgctagcacca agggccccagcgtgttcc ccctggcccccagcagcaagagcaccagcggcggcacagccgccctgggctgcctggtga aggactacttccccg agcccgtgaccgtgtcctggaacagcggagccctgacctccggcgtgcacaccttccccg ccgtgctgcagagcag cggcctgtacagcctgtccagcgtggtgacagtgcccagcagcagcctgggcacccagac ctacatctgcaacgtg aaccacaagcccagcaacaccaaggtggacaagagagtggagcccaagagctgcgacaag acccacacctgc cccccctgcccagccccagaggcagcgggcggaccctccgtgttcctgttcccccccaag cccaaggacaccctgat gatcagcaggacccccgaggtgacctgcgtggtggtggacgtgagccacgaggacccaga ggtgaagttcaactg gtacgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggagcagtacaa cagcacctacagg gtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgc aaggtctccaacaagg ccctgccagcccccatcgaaaagaccatcagcaaggccaagggccagccacgggagcccc aggtgtacaccctg cccccctcccgggaggagatgaccaagaaccaggtgtccctgacctgtctggtgaagggc ttctaccccagcgacat cgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccacccccccagt gctggacagcgac ggcagcttcttcctgtacagcaagctgaccgtggacaagtccaggtggcagcagggcaac gtgttcagctgcagcgt gatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtcccccggcaa g

72 Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggcggcggcggatcccgtacggtggccg ctcccagcgtgttcatcttcc cccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaact tctacccccgggagg ccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggagagcgtca ccgagcaggaca gcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgactacgaga agcataaggtgtacg cctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaacaggggcg agtgc

73 cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggcggcggcggcagcggcggcggcggat ccagcgctagcaccaa gggccccagcgtgttccccctggcccccagcagcaagagcaccagcggcggcacagccgc cctgggctgcctggt gaaggactacttccccgagcccgtgaccgtgtcctggaacagcggagccctgacctccgg cgtgcacaccttccccg ccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtgacagtgcccagcagca gcctgggcacccaga cctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagagagtggagc ccaagagctgcgac aagacccacacctgccccccctgcccagccccagaggcagcgggcggaccctccgtgttc ctgttcccccccaagc ccaaggacaccctgatgatcagcaggacccccgaggtgacctgcgtggtggtggacgtga gccacgaggacccag aggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagccca gagaggagcagtac aacagcacctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggc aaggaatacaagtgca aggtctccaacaaggccctgccagcccccatcgaaaagaccatcagcaaggccaagggcc agccacgggagcc ccaggtgtacaccctgcccccctcccgggaggagatgaccaagaaccaggtgtccctgac ctgtctggtgaagggct tctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactaca agaccaccccccca gtgctggacagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagtccagg tggcagcagggcaacg tgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctga gcctgtcccccggcaa g

74 Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggcggcggcggcagcggcggcggcggat cccgtacggtggccgct cccagcgtgttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtg gtgtgcctgctgaacaa cttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaa cagccaggagagc gtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctgagc aaggccgactacga gaagcataaggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgaccaa gagcttcaacagggg cgagtgc

75 cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcg gaggatctggcggcggagg aagtggcggaggaggatccagcgctagcaccaagggccccagcgtgttccccctggcccc cagcagcaagagca ccagcggcggcacagccgccctgggctgcctggtgaaggactacttccccgagcccgtga ccgtgtcctggaacag cggagccctgacctccggcgtgcacaccttccccgccgtgctgcagagcagcggcctgta cagcctgtccagcgtgg tgacagtgcccagcagcagcctgggcacccagacctacatctgcaacgtgaaccacaagc ccagcaacaccaag gtggacaagagagtggagcccaagagctgcgacaagacccacacctgccccccctgccca gccccagaggcag cgggcggaccctccgtgttcctgttcccccccaagcccaaggacaccctgatgatcagca ggacccccgaggtgacc tgcgtggtggtggacgtgagccacgaggacccagaggtgaagttcaactggtacgtggac ggcgtggaggtgcaca acgccaagaccaagcccagagaggagcagtacaacagcacctacagggtggtgtccgtgc tgaccgtgctgcacc aggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgccagccc ccatcgaaaagacc atcagcaaggccaagggccagccacgggagccccaggtgtacaccctgcccccctcccgg gaggagatgaccaa gaaccaggtgtccctgacctgtctggtgaagggcttctaccccagcgacatcgccgtgga gtgggagagcaacggcc agcccgagaacaactacaagaccacccccccagtgctggacagcgacggcagcttcttcc tgtacagcaagctgac cgtggacaagtccaggtggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggc cctgcacaaccactac acccagaagagcctgagcctgtcccccggcaag

76 Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcg gaggatctggcggcggagg aagtggcggaggaggatcccgtacggtggccgctcccagcgtgttcatcttcccccccag cgacgagcagctgaag agcggcaccgccagcgtggtgtgcctgctgaacaacttctacccccgggaggccaaggtg cagtggaaggtggac aacgccctgcagagcggcaacagccaggagagcgtcaccgagcaggacagcaaggactcc acctacagcctga gcagcaccctgaccctgagcaaggccgactacgagaagcataaggtgtacgcctgcgagg tgacccaccagggc ctgtccagccccgtgaccaagagcttcaacaggggcgagtgc

77 cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggaggcggaggatctggcggcggaggaa gtggcggaggaggatc cagcgctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcaccag cggcggcacagccg ccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcg gagccctgacctccggc gtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtg acagtgcccagcagcag cctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtgga caagagagtggagc ccaagagctgcgacaagacccacacctgccccccctgcccagccccagaggcagcgggcg gaccctccgtgttcc tgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcg tggtggtggacgtgag ccacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgc caagaccaagccca gagaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccagg actggctgaacggca aggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccatca gcaaggccaagggc cagccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaac caggtgtccctgacc tgtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccag cccgagaacaactac aagaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgacc gtggacaagtccaggt ggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactaca cccagaagagcctga gcctgtcccccggcaag 78 Cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggaggcggaggatctggcggcggaggaa gtggcggaggaggatc ccgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaagag cggcaccgccagcgtg gtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaac gccctgcagagcggc aacagccaggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagcagc accctgaccctga gcaaggccgactacgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgt ccagccccgtgacca agagcttcaacaggggcgagtgc

79 cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcg gaggatctggcggcggagg aagcggaggcggcggaagtggagggggaggatcagggggaggaggatccagcgctagcac caagggcccca gcgtgttccccctggcccccagcagcaagagcaccagcggcggcacagccgccctgggct gcctggtgaaggact acttccccgagcccgtgaccgtgtcctggaacagcggagccctgacctccggcgtgcaca ccttccccgccgtgctgc agagcagcggcctgtacagcctgtccagcgtggtgacagtgcccagcagcagcctgggca cccagacctacatctg caacgtgaaccacaagcccagcaacaccaaggtggacaagagagtggagcccaagagctg cgacaagaccca cacctgccccccctgcccagccccagaggcagcgggcggaccctccgtgttcctgttccc ccccaagcccaaggac accctgatgatcagcaggacccccgaggtgacctgcgtggtggtggacgtgagccacgag gacccagaggtgaag ttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggag cagtacaacagcac ctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaata caagtgcaaggtctcca acaaggccctgccagcccccatcgaaaagaccatcagcaaggccaagggccagccacggg agccccaggtgta caccctgcccccctcccgggaggagatgaccaagaaccaggtgtccctgacctgtctggt gaagggcttctacccca gcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccc ccccagtgctgga cagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagtccaggtggcagca gggcaacgtgttcagct gcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtccc ccggcaag

80 cagctactatttaataaaacaaaatctgtagaattcacgtttggtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcg gaggatctggcggcggagg aagcggaggcggcggaagtggagggggaggatcagggggaggaggatcccgtacggtggc cgctcccagcgtg ttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctg ctgaacaacttctacccc cgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggag agcgtcaccgag caggacagcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgac tacgagaagcataa ggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaa caggggcgagtgc

81 cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcaggcggcggcggatccagcgctagcaccaa gggccccagcgtgttccc cctggcccccagcagcaagagcaccagcggcggcacagccgccctgggctgcctggtgaa ggactacttccccga gcccgtgaccgtgtcctggaacagcggagccctgacctccggcgtgcacaccttccccgc cgtgctgcagagcagc ggcctgtacagcctgtccagcgtggtgacagtgcccagcagcagcctgggcacccagacc tacatctgcaacgtga accacaagcccagcaacaccaaggtggacaagagagtggagcccaagagctgcgacaaga cccacacctgccc cccctgcccagccccagaggcagcgggcggaccctccgtgttcctgttcccccccaagcc caaggacaccctgatg atcagcaggacccccgaggtgacctgcgtggtggtggacgtgagccacgaggacccagag gtgaagttcaactggt acgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggagcagtacaaca gcacctacagggt ggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgcaa ggtctccaacaaggcc ctgccagcccccatcgaaaagaccatcagcaaggccaagggccagccacgggagccccag gtgtacaccctgcc cccctcccgggaggagatgaccaagaaccaggtgtccctgacctgtctggtgaagggctt ctaccccagcgacatcg ccgtggagtgggagagcaacggccagcccgagaacaactacaagaccacccccccagtgc tggacagcgacgg cagcttcttcctgtacagcaagctgaccgtggacaagtccaggtggcagcagggcaacgt gttcagctgcagcgtgat gcacgaggccctgcacaaccactacacccagaagagcctgagcctgtcccccggcaag

82 Cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcaggcggcggcggatcccgtacggtggccg ctcccagcgtgttcatcttcc cccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctgctgaacaact tctacccccgggagg ccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggagagcgtca ccgagcaggaca gcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgactacgaga agcataaggtgtacg cctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaacaggggcg agtgc

83 cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggcggcgg cggatccagcgctagcacca agggccccagcgtgttccccctggcccccagcagcaagagcaccagcggcggcacagccg ccctgggctgcctg gtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcggagccctgacctcc ggcgtgcacaccttccc cgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtgacagtgcccagcag cagcctgggcaccca gacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagagagtgga gcccaagagctgcg acaagacccacacctgccccccctgcccagccccagaggcagcgggcggaccctccgtgt tcctgttcccccccaa gcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcgtggtggtggacgt gagccacgaggaccc agaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagcc cagagaggagcagt acaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacg gcaaggaatacaagtg caaggtctccaacaaggccctgccagcccccatcgaaaagaccatcagcaaggccaaggg ccagccacgggag ccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaaccaggtgtccctg acctgtctggtgaagg gcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaact acaagaccaccccc ccagtgctggacagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagtcc aggtggcagcagggca acgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcc tgagcctgtcccccgg caag

84 Cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggcggcg gcggatcccgtacggtggcc gctcccagcgtgttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagc gtggtgtgcctgctgaa caacttctacccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcgg caacagccaggag agcgtcaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgaccctg agcaaggccgacta cgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgac caagagcttcaacag gggcgagtgc

85 cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcgg aggatctggcggcggagga agcggaggcggcggaagtggagggggaggatcagggggaggaggatccagcgctagcacc aagggccccag cgtgttccccctggcccccagcagcaagagcaccagcggcggcacagccgccctgggctg cctggtgaaggacta cttccccgagcccgtgaccgtgtcctggaacagcggagccctgacctccggcgtgcacac cttccccgccgtgctgca gagcagcggcctgtacagcctgtccagcgtggtgacagtgcccagcagcagcctgggcac ccagacctacatctgc aacgtgaaccacaagcccagcaacaccaaggtggacaagagagtggagcccaagagctgc gacaagacccac acctgccccccctgcccagccccagaggcagcgggcggaccctccgtgttcctgttcccc cccaagcccaaggaca ccctgatgatcagcaggacccccgaggtgacctgcgtggtggtggacgtgagccacgagg acccagaggtgaagtt caactggtacgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggagca gtacaacagcacc tacagggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatac aagtgcaaggtctccaa caaggccctgccagcccccatcgaaaagaccatcagcaaggccaagggccagccacggga gccccaggtgtac accctgcccccctcccgggaggagatgaccaagaaccaggtgtccctgacctgtctggtg aagggcttctaccccag cgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccacccc cccagtgctggac agcgacggcagcttcttcctgtacagcaagctgaccgtggacaagtccaggtggcagcag ggcaacgtgttcagctg cagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgtcccc cggcaag

86 Cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatg gaggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttac acctttgatggagctcta aacaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattacta aaaggagatgcctctttgaa gatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatt aaccagagaaggtgaaa cgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggcg gaggatctggcggcggagg aagcggaggcggcggaagtggagggggaggatcagggggaggaggatcccgtacggtggc cgctcccagcgtg ttcatcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtgtgcctg ctgaacaacttctacccc cgggaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccaggag agcgtcaccgag caggacagcaaggactccacctacagcctgagcagcaccctgaccctgagcaaggccgac tacgagaagcataa ggtgtacgcctgcgaggtgacccaccagggcctgtccagccccgtgaccaagagcttcaa caggggcgagtgc

87 cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtgg tggatctggaggtggaggtag ctcagctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcaccag cggcggcacagccg ccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcg gagccctgacctccggc gtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtg acagtgcccagcagcag cctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtgga caagagagtggagc ccaagagctgcgacaagacccacacctgccccccctgcccagccccagagctgctgggcg gaccctccgtgttcct gttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcgt ggtggtggacgtgagc cacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgcc aagaccaagcccag agaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccagga ctggctgaacggcaa ggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccatcag caaggccaagggcc agccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaacc aggtgtccctgacct gtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagc ccgagaacaactaca agaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgaccg tggacaagtccaggtg gcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacac ccagaagagcctgag cctgtcccccggcaag

88 I cagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtc attccatgctttgttactaatatgg aggcacaaaacactactgaagtatacgtaaagtggaaatttaaaggaagagatatttaca cctttgatggagctctaa acaagtccactgtccccactgactttagtagtgcaaaaattgaagtctcacaattactaa aaggagatgcctctttgaag atggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaatta accagagaaggtgaaac gatcatcgagctaaaatatcgtgttgtttcatggttttctccaaatgaaaatggaggtgg tggatctggaggtggaggtag ctcagcctccaccaagggtccatcggtcttccccctggcaccctcctccaagagcacctc tgggggcacagcggccct gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgc cctgaccagcggcgtgc acaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccg tgccctccagcagcttggg cacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagag agttgagcccaaatc ttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtc agtcttcctcttcccccca aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagacc ctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagc cgcgggaggagcagt acgccagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatg gcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagg gcagccccgagaac cacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaag gcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaact acaagaccacgcctc ccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagca ggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggtaaa

89 I caactactgtttagtaacgtcaactccatagagttcacttcaggcaatgaaactgtggtc atcccttgcatcgtccgtaatg tggaggcgcaaagcaccgaagaaatgtttgtgaagtggaagttgaacaaatcgtatattt tcatctatgatggaaataa aaatagcactactacagatcaaaactttaccagtgcaaaaatctcagtctcagacttaat caatggcattgcctctttgaa aatggataagcgcgatgccatggtgggaaactacacttgcgaagtgacagagttatccag agaaggcaaaacagtt atagagctgaaaaaccgcacggtttcgtggttttctccaaatgaaaagatcggaggtggt ggatctggaggtggaggt agctcagctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcacc agcggcggcacagc cgccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacag cggagccctgacctccg gcgtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtgg tgacagtgcccagcagc agcctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtg gacaagagagtgga gcccaagagctgcgacaagacccacacctgccccccctgcccagccccagaggcagcggg cggaccctccgtgtt cctgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctg cgtggtggtggacgtg agccacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaac gccaagaccaagcc cagagaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcacca ggactggctgaacgg caaggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccat cagcaaggccaagg gccagccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaaga accaggtgtccctga cctgtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggcc agcccgagaacaacta caagaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgac cgtggacaagtccaggt ggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactaca cccagaagagcctga gcctgtcccccggcaag

90 caactactgtttagtaacgtcaactccatagagttcacttcaggcaatgaaactgtggtc atcccttgcatcgtccgtaatg tggaggcgcaaagcaccgaagaaatgtttgtgaagtggaagttgaacaaatcgtatattt tcatctatgatggaaataa aaatagcactactacagatcaaaactttaccagtgcaaaaatctcagtctcagacttaat caatggcattgcctctttgaa aatggataagcgcgatgccatggtgggaaactacacttgcgaagtgacagagttatccag agaaggcaaaacagtt atagagctgaaaaaccgcacggtttcgtggttttctccaaatgaaaagatcggaggtggt ggatctggaggtggaggt agccgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaag agcggcaccgccagcg tggtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggaca acgccctgcagagcgg caacagccaggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagcag caccctgaccctga gcaaggccgactacgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgt ccagccccgtgacca agagcttcaacaggggcgagtgc

91 caactactgtttagtaacgtcaactccatagagttcacttcatgcaatgaaactgtggtc atcccttgcatcgtccgtaatgt ggaggcgcaaagcaccgaagaaatgt tgtgaagtggaagttgaacaaatcgtatattttcatctatgatggaaataaa aatagcactactacagatcaaaactttaccagtgcaaaaatctcagtctcagacttaatc aatggcattgcctctttgaaa atggataagcgcgatgccatggtgggaaactacacttgcgaagtgacagagttatccaga gaaggcaaaacagttat agagctgaaaaaccgcacggtttcgtggttttctccaaatgaaaagatcggaggtggtgg atctggaggtggaggtag ctcagctagcaccaagggccccagcgtgttccccctggcccccagcagcaagagcaccag cggcggcacagccg ccctgggctgcctggtgaaggactacttccccgagcccgtgaccgtgtcctggaacagcg gagccctgacctccggc gtgcacaccttccccgccgtgctgcagagcagcggcctgtacagcctgtccagcgtggtg acagtgcccagcagcag cctgggcacccagacctacatctgcaacgtgaaccacaagcccagcaacaccaaggtgga caagagagtggagc ccaagagctgcgacaagacccacacctgccccccctgcccagccccagaggcagcgggcg gaccctccgtgttcc tgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgcg tggtggtggacgtgag ccacgaggacccagaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgc caagaccaagccca gagaggagcagtacaacagcacctacagggtggtgtccgtgctgaccgtgctgcaccagg actggctgaacggca aggaatacaagtgcaaggtctccaacaaggccctgccagcccccatcgaaaagaccatca gcaaggccaagggc cagccacgggagccccaggtgtacaccctgcccccctcccgggaggagatgaccaagaac caggtgtccctgacc tgtctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccag cccgagaacaactac aagaccacccccccagtgctggacagcgacggcagcttcttcctgtacagcaagctgacc gtggacaagtccaggt ggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactaca cccagaagagcctga gcctgtcccccggcaag

caactactgtttagtaacgtcaactccatagagttcacttcatgcaatgaaactgtg gtcatcccttgcatcgtccgtaatgt ggaggcgcaaagcaccgaagaaatgtttgtgaagtggaagttgaacaaatcgtatatttt catctatgatggaaataaa aatagcactactacagatcaaaactttaccagtgcaaaaatctcagtctcagacttaatc aatggcattgcctctttgaaa atggataagcgcgatgccatggtgggaaactacacttgcgaagtgacagagttatccaga gaaggcaaaacagttat agagctgaaaaaccgcacggtttcgtggttttctccaaatgaaaagatcggaggtggtgg atctggaggtggaggtag ccgtacggtggccgctcccagcgtgttcatcttcccccccagcgacgagcagctgaagag cggcaccgccagcgtg gtgtgcctgctgaacaacttctacccccgggaggccaaggtgcagtggaaggtggacaac gccctgcagagcggc aacagccaggagagcgtcaccgagcaggacagcaaggactccacctacagcctgagcagc accctgaccctga gcaaggccgactacgagaagcataaggtgtacgcctgcgaggtgacccaccagggcctgt ccagccccgtgacca agagcttcaacaggggcgagtgc

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