RU2791486C2 - Recombinant robo2 proteins, compositions, methods and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to recombinant production of fused proteins of Roundabout 2 receptor (hereinafter – ROBO2); it can be used in medicine for the treatment of a kidney disease. Fused ROBO2-Fc proteins contain Fc domain and part of an extracellular ROBO2 domain consisting of two immunoglobulin-like domains (Ig1 and Ig2) and two intradomain linkers (Ig1-Ig2 linker and Ig2-Ig3 linker), while the specified protein is deprived of three repeats of a fibronectin III type.

EFFECT: invention allows for obtainment of recombinant ROBO2-Fc proteins, which are capable of binding to slit guidance ligand SLIT2 and inhibiting ROBO2-dependent activity of SLIT2.

37 cl, 16 dwg, 24 tbl, 10 ex

Description

Cross-reference to related application

[0001] This application claims the benefit of U.S. Provisional Application No. 62/514242, filed June 2, 2017; and U.S. Provisional Application No. 62/663082, filed April 26, 2018, which are incorporated herein by reference in their entirety.

A group of specialists involved in joint research

[0002] The claimed invention was carried out by the following groups of specialists or with the participation of the following groups in accordance with a collaborative research agreement. This collaborative research agreement takes effect on or before the date on which the claimed invention is made, and the claimed invention was created as a result of the work performed under the collaborative research agreement. The parties to the Collaborative Research Agreement are BOSTON MEDICAL CENTER CORP. and PFIZER INC.

Sequence list

[0002.1] This application includes a Sequence Listing that has been filed in ASCII electronic format and is incorporated herein by reference in its entirety. The specified ASCII copy was created on May 3, 2018, named PCFC0043WO1_SL.txt, and is 54754 bytes in size.

Prerequisites for the creation of the invention

[0003] Chronic kidney disease (CKD) is a health problem worldwide and often leads to end-stage kidney failure. It is estimated that CKD affects 13% of the population or ~27 million people in the US and more than 500 million people worldwide. Experts predict that the incidence of CKD will increase due to the ongoing epidemic of diabetes and obesity in the general population. Approximately half a million patients with CKD in the US (~7 million worldwide) will progress to end-stage kidney disease (EKD) and these patients will need dialysis or kidney transplantation to save their lives. Morbidity and mortality from BPD is high and costs the US budget at least $40 billion a year. Proteinuria (i.e., the presence of excess serum proteins in the urine, usually defined as urinary albumin levels >30 mg/day) is an early biomarker, risk factor, and surrogate endpoint of CKD in patients with and without diabetes. Treatment aimed at reducing the level of proteinuria in the early stages of CKD can slow the progression of BPHD. However, there is currently no renal podocyte-specific treatment for proteinuria in patients with CKD and proteinuria.

[0004] Podocytes are specialized epithelial cells that undergo the primary and secondary stages of coating the outer surface of the glomerular basement membrane. Actin-rich tangles of secondary processes (eg, base processes) from adjacent podocytes create filtration slits bridged by a semi-permeable slit diaphragm that forms the final barrier to protein entry. Proteinuria is a clinical sign of podocyte damage in diabetic and non-diabetic kidney disease. There is now an expanding group of published studies showing that hereditary, congenital, or acquired abnormalities in the molecular component of podocytes lead to proteinuria. Since genetic mutations in slit diaphragm podocyte proteins such as nephrin and podocin are associated with inherited forms of proteinuric kidney disease, it is becoming increasingly clear that the proteins that make up and are associated with the slit diaphragm represent more than just a structural barrier. Thus, there is a wealth of data suggesting that these proteins form a balanced signal-transmitting network that can influence the structure of podocyte base processes and their function through interaction with the actin cytoskeleton.

Roundabout receptors (ROBOs)

[0005] Detour 2 receptor (ROBO2, also referred to herein as detour regulation 2 receptor or detour 2 homologue) is a ligand receptor for gap formation regulation ligand protein (SLIT). ROBO2 is expressed on the basal surface of glomerular podocytes in the kidney, and gap regulation ligand 2 (SLIT2) is present in the glomerulus of the kidney. After binding to SLIT2, ROBO2 forms a complex with nephrin in the glomerular filtration barrier and acts as a negative regulator of inhibition of nephrin-induced actin polymerization. Loss of ROBO2 increases the level of actin polymerization in the podocyte and attenuates the abnormal podocyte structural phenotype found in nephrin-deficient mice. Loss of ROBO2 also increases podocyte adhesion to the glomerular basement membrane in mice. These data, along with the observation that a patient with a ROBO2 chromosomal translocation does not have proteinuria, suggest that blocking the SLIT2-ROBO2 signaling pathway may increase the level of nephrin-induced actin polymerization and reduce the degree of proteinuria. Blocking ROBO2 signaling may also restore the glomerular filtration barrier in proteinuric disease by activating nephrin-induced actin polymerization.

[0006] SLIT1, SLIT2 and SLIT3. SLITs are secreted proteins associated with the extracellular matrix. The protein sequences of all SLITs show a high degree of conservatism and have the same structure: N-terminal signal peptide; four tandem leucine-rich repeat domains (LRRs), designated D1-D4; six epidermal growth factor (EGF) like domains; laminin G-like domain; an additional one EGF-like domain (invertebrates) or three EGF-like domains (vertebrates) and a C-terminal cysteine knot domain. SLIT ligands can be cleaved to form a short C-terminal fragment with unknown function (SLIT-C product) and a long N-terminal fragment (SLIT-N product) that is active and mediates binding to ROBO. SLIT ligands, as well as their cleavage products described here (eg, SLIT-N, SLIT2-D2), can be used to assess ROBO2 activity.

[0007] Four receptors have been characterized in vertebrates: ROBO ROBO1/Dutt1; ROBO2; ROBO3/Rig-1 and ROBO4/Magic Roundabout. ROBO1, ROBO2 and ROBO3 share an extracellular domain (ECD) structure that resembles that of cell adhesion molecules. This region contains five immunoglobulin-like (Ig-like) domains (Ig1, Ig2, Ig3, Ig4 and Ig5) followed by three fibronectin 3 (FN3) type repeats (FIG. 1A). In addition, ROBO2 has four cytoplasmic conserved (CC) sequences in its intracellular domain, as shown in FIG. 1A.

[0008] The sequence of the full length human ROBO2 precursor is shown as SEQ ID NO: 24. The 21 amino acid leader of ROBO2 (SEQ ID NO: 17; residues 1-21 according to the numbering given in SEQ ID NO: 24) is cleaved during production protein to form mature ROBO2 (Fig. 1A). Residues 22-859, as numbered in SEQ ID NO: 24, form the extracellular domain, residues 860-880, as shown in SEQ ID NO: 24, form the transmembrane domain, and residues 881-1378, as shown in SEQ ID NO: 24 form a cytoplasmic domain (FIG. 1A).

[0009] Representative sequences of the five Ig-like domains (Ig1, Ig2, Ig3, Ig4, and Ig5) of ROBO2 are shown in Table 23. Pre-Ig1 ROBO2 sequence (SEQ ID NO: 8), Ig1-Ig2 interdomain linker (SEQ ID NO: 10) and an Ig2-Ig3 interdomain linker (SEQ ID NO: 12) are also disclosed in Table 23.

[0010] The D2 SLIT LRR domain and the Ig1 and Ig2 ROBO domains are evolutionarily conserved and involved in binding. The Ig1 and Ig2 ROBO domains are also collectively referred to as the SLIT binding domain. Studies have shown that although the immunoglobulin-like (Ig-like) domains 1 and 2 (Ig1 and Ig2) of ROBO2 interact with SLIT, however, the first Ig-like domain (Ig1) is the main binding site for SLIT. In addition, previous studies have shown that deletion of three repeats of fibronectin type III (FNIII) has a more negative effect on ROBO binding to SLIT than deletion of the third and fourth immunoglobulin-like domains (Ig3 and Ig4) (see, for example, Liu et al., 2004, Molecular Cellular Neuroanatomy 39:256261).

[0011] After ROBO-SLIT binding, Rho GTPases and their regulators (GAP and GEF) are involved in the subsequent signaling pathway. In the presence of SLIT, SLITROBO, the Rho GTPase activation protein SLITROBO 1 (srGAP1) binds to the CC3 domain of ROBO and inactivates RhoA and Cdc42. These effector proteins are able to mediate, among others, repulsion, regulation of cytoskeletal dynamic impulses, and cell polarity. In the presence of SLIT, Vilse/CrossGAP can also bind to the CC2 domain of ROBO and inhibit Rac1 and Cdc42. Rac1 is also activated through the recruitment of the Son of sevenless (Sos) GEF protein by the Dreadlocks (Dock) adapter protein, which binds to the CC23 domain of ROBO. This leads to the activation of the downstream target of Rac1 and p21-activated kinase (Pak), which also binds to the CC23 domains of ROBO. These downstream ROBO signaling partners regulate charge repulsion and dynamic charge of the cytoskeleton. Abelson's tyrosine kinase (Abl) can also bind to the CC3 domain of ROBO and block ROBO signaling through phosphorylation of the CC1 domain, as well as mediate cell adhesion. ENABLED (Ena), an Abl substrate, also binds to the CC1 and CC2 domains of ROBO. All of these downstream ROBOSLIT molecules can be used to evaluate ROBO2 activity as well as to evaluate any neutralizing effect of the novel recombinant ROBO2 protein disclosed in this application.

[0012] In the kidney, ROBO2 is complexed with nephrin via the Nck adapter protein. In contrast to the role of nephrin, which stimulates actin polymerization, the SLIT-ROBO2 signal-transmitting complex inhibits nephrin-induced actin polymerization. Thus, the binding of the intracellular domain of ROBO2 and Nck can be used to assess the activity of ROBO2.

[0013] Currently, for patients suffering from many glomerular diseases (including focal segmental glomerular sclerosis), there is no available therapy to preserve kidney function or any other way to treat the disease. In addition, there is currently no available treatment for patients with CKD and proteinuria. Accordingly, there is a need to develop a therapeutic treatment that modulates ROBO2-SLIT signaling and thereby preserves or modulates podocyte function and reduces proteinuria or otherwise contributes to the treatment or prevention of kidney disease associated with binding and signal transmission ROBO2-SLIT or mediated by them.

The essence of the invention

[0014] The present invention relates to recombinant ROBO2 proteins that bind to the SLIT ligand, as well as their use and associated methods. Those skilled in the art will recognize, or be able to determine on their own, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described in this application. Such equivalents are within the scope of the following embodiments of the invention (E).

[0015] E1. Recombinant Roundabout Receptor 2 (ROBO2) protein containing amino acid residues 1-203 according to SEQ ID NO: 1 and also containing an immunoglobulin heavy chain constant domain.

[0016]E2. Recombinant Roundabout 2 receptor protein (ROBO2) consisting essentially of amino acid residues 1-203 according to SEQ ID NO: 1 and an immunoglobulin heavy chain constant domain.

[0017] E3. Recombinant receptor protein Roundabout 2 (ROBO2), containing (i) SLIT-binding fragment; and (ii) a fragment that increases the half-life, where the specified SLIT-binding fragment contains part of the extracellular domain of ROBO2.

[0018] E4. Recombinant ROBO2 E3 protein, where said part of said ROBO2 extracellular domain contains the first two immunoglobulin-like (Ig1 and Ig2) ROBO2 domains and a C-terminal sequence consisting of the sequence of SEQ ID NO: 12.

[0019] E5. Recombinant ROBO2 E3 protein, wherein said portion of said ROBO2 extracellular domain essentially consists of an immunoglobulin 1 (Ig1)-like ROBO2 pre-sequence (SRLRQEDFP (SEQ ID NO: 8), first immunoglobulin-like domain (Ig1), interdomain linker , located between the first and second immunoglobulin-like domains (interdomain linker Ig1-Ig2; VALLR (SEQ ID NO: 10)), the second immunoglobulin-like domain (Ig2) and the interdomain linker located between the second and third immunoglobulin-like domains (interdomain Ig2-Ig3 linker, VFER (SEQ ID NO: 12)).

[0020] E6. A recombinant ROBO2 protein of any one of E3-E5, wherein said portion of said ROBO2 extracellular domain essentially consists of amino acid residues 1-203 according to SEQ ID NO: 1.

[0021] E7. The recombinant ROBO2 protein of any of E3-E6, wherein said half-life increasing fragment contains an immunoglobulin domain.

[0022] E8. Recombinant ROBO2 protein of any of E1, E2 or E7, wherein said immunoglobulin domain is the Fc domain of IgA ₁ IgA ₂ , IgD, IgE, IgM, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ .

[0023] E9. Recombinant protein ROBO2 E8, where the specified domain Fc domain is the Fc domain of human IgG ₁ .

[0024] E10. Recombinant protein ROBO2 E9, where the specified Fc-domain has been modified to eliminate the effector function.

[0025] E11. Recombinant ROBO2 E10 protein, wherein said Fc domain is a human IgG ₁ Fc domain, and wherein said human IgG1 Fc domain contains at least one mutation selected from the group consisting of a leucine to alanine substitution at amino acid residue number 234 (L234A ), a leucine to alanine substitution at amino acid residue number 235 (L235A), and a glycine to alanine substitution at amino acid residue number 237 (G237A), where all residues are numbered according to Kabat's Eu numbering.

[0026] E12. Recombinant protein ROBO2 E9-E11, where the specified Fc-domain of human IgG1 does not contain lysine at residue number 447 in accordance with the Eu-numbering proposed by Kabat.

[0027]E13. Recombinant ROBO2 protein of any of E11-E12, where said Fc domain contains amino acid residues 210-440 according to SEQ ID NO: 1.

[0028]E14. Recombinant ROBO2 protein of any of E11-E12, where said Fc domain consists of amino acid residues 210-440 according to SEQ ID NO: 1.

[0029]E15. A recombinant ROBO2 protein of any of E1, E2 or E7, wherein said amino acid residues 1-203 according to SEQ ID NO: 1 are adjacent to said immunoglobulin domain.

[0030]E16. A recombinant ROBO2 protein of any of E1, E2 or E7, wherein said amino acid residues 1-203 according to SEQ ID NO: 1 are attached to said immunoglobulin domain via a linker.

[0031]E17. Recombinant protein ROBO2 E16, where the specified linker is a peptidyl linker containing approximately 1 to 30 amino acid residues.

[0032]E18. Recombinant ROBO2 E17 protein, wherein said peptidyl linker is selected from the group consisting of:

a) a glycine-rich peptide;

b) a peptide containing glycine and serine;

c) a peptide having the sequence (Gly-Gly-Ser) _n , where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 22); And

d) a peptide having the sequence (Gly-Gly-Gly-Gly-Ser)n, where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 23).

[0033]E19. Recombinant protein ROBO2 E18, where the specified peptidyl linker is (Gly-Gly-Ser) ₂ (SEQ ID NO: 15).

[0034]E20. Recombinant ROBO2-Fc protein containing the amino acid sequence of SEQ ID NO: 1.

[0035]E21. Recombinant ROBO2-Fc protein, consisting of the amino acid sequence of SEQ ID NO: 1.

[0036]E22. Recombinant ROBO2-Fc protein containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1.

[0037]E23. The recombinant ROBO2 protein of any of E1-E22, containing the amino acid sequence encoded by the plasmid insert deposited with ATCC and bearing the ATCC registration number No. PTA-124008.

[0038]E24. Recombinant ROBO2-Fc protein of any E4 or E5, wherein said ROBO2 Ig1 contains at least one of the following mutations; S17T and R73Y, each numbered according to SEQ ID NO: 1.

[0039] E25. Recombinant ROBO2-Fc containing the amino acid sequence of SEQ ID NO: 19.

[0040]E26. Recombinant ROBO2-Fc E25, wherein said protein does not contain C-terminal lysine at amino acid residue number 441 according to SEQ ID NO: 19.

[0041] E27. Recombinant ROBO2 protein of any of E1-E26, wherein said ROBO2 is human ROBO2.

[0042]E28. Recombinant ROBO2 protein of any of E1-E27, wherein said protein binds to SLIT2 with a binding affinity (K _D ) equal to or less than: about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, approximately 700 pM, approximately 600 pM, approximately 500 pM, approximately 400 pM, approximately 300 pM, approximately 250 pM, approximately 200 pM, approximately 150 pM, approximately 100 pM, approximately 50 pM, approximately 40 pM, approximately 30 pM , about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM.

[0043]E29. A recombinant ROBO2 protein of any of E1-E27, wherein said protein binds to SLIT2 with a K _D that is at least about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold, about 20 times, about 40 times, about 60 times, about 80 times, about 100 times, about 120 times, about 140 times, about 160 times lower than the K _D value for binding ROBO1 to SLIT2.

[0044]E30. A recombinant ROBO2 protein of any of E1-E29, wherein said protein binds to SLIT2 with a K _D value that is at least about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold, about 20-fold, approximately 40-fold, approximately 60-fold, approximately 80-fold, approximately 100-fold, approximately 120-fold, approximately 140-fold, approximately 160-fold lower than the K _D value for ROBO1-Fc protein binding with SLIT2.

[0045]E31. Recombinant protein ROBO2 of any of E1-E30, where the specified K _D is measured using surface plasmon resonance (SPR).

[0046]E32. Recombinant protein ROBO2 E31, where the specified K _D is measured on the device Biacore T200.

[0047]E33. Recombinant ROBO2 protein of any of E28-E30, where the specified K _D is measured using biolayer interferometry (BSI).

[0048]E34. Recombinant protein ROBO2 E33, where the specified K _D is measured on the device ForteBio Octet.

[0049] E35. Recombinant ROBO2 protein of any of E1-E34, wherein said protein inhibits SLIT ligand binding and ROBO2.

[0050]E36. Recombinant ROBO2 protein of any of E1-E34, wherein said protein inhibits ROBO2-dependent SLITx-N activity.

[0051]E37. A recombinant ROBO2 protein of any of E1-E36, wherein said protein inhibits SLIT and ROBO2 ligand binding and inhibits ROBO2-dependent SLIT-N activity.

[0052]E38. A recombinant ROBO2 protein of any one of E1-E37, wherein said ROBO2-dependent SLITx-N activity is selected from the group consisting of actin polymerization, podocyte adhesion, and inhibition of nerve cell migration.

[0053]E39. Recombinant ROBO2 protein of any of E1-E38, wherein said protein has a half-maximal inhibitory concentration (IC ₅₀ ) of no more than about 15 nM, about 13 nM, about 11 nM, about 9 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM.

[0054]E40. Recombinant ROBO2 E39 protein, wherein said IC ₅₀ is measured by time resolved homogeneous fluorescence (HTRF) analysis for inhibition of ROBO2 binding to SLIT2.

[0055] E41. Recombinant ROBO2 protein of any of E1-E40, wherein said protein has a half-maximal IC ₅₀ of not greater than about 75 nM, about 65 nM, about 55 nM, about 45 nM, about 35 nM, about 25 nM, about 15 nM, about 5 nM.

[0056]E42. Recombinant protein ROBO2 E41, where the specified IC ₅₀ is measured by assessing SLIT2-N-mediated inhibition of nerve cell migration.

[0057]E43. Recombinant ROBO2 protein of any of E28-E42, wherein said SLIT2 is human SLIT2.

[0058] E44. A recombinant ROBO2 protein of any one of E1-E43, wherein the two recombinant ROBO2 proteins are associated with each other to form a homodimer.

[0059] E45. An isolated nucleic acid molecule encoding a recombinant ROBO2 protein of any of E1-E44.

[0060] E46. An isolated E45 nucleic acid molecule containing the nucleic acid sequence of SEQ ID NO: 21.

[0061] E47. An isolated E45 nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO: 21.

[0062] E48. An isolated nucleic acid containing the nucleic acid sequence of the plasmid insert deposited with the ATCC and bearing ATCC Accession No. PTA-124008.

[0063] E49. Recombinant ROBO2 protein containing the amino acid sequence encoded by the sequence of SEQ ID NO: 21.

[0064]E50. Recombinant ROBO2 protein containing an amino acid sequence encoded by a sequence that is at least 85%, 90%, 95%, or 99% identical to the sequence of SEQ ID NO: 21.

[0065]E51. Recombinant ROBO2 protein containing an amino acid sequence encoded by a sequence capable of hybridizing under high stringency conditions with the sequence of SEQ ID NO: 21.

[0066]E52. A vector containing a nucleic acid molecule from any of E45-E48.

[0067] E53. A host cell containing a nucleic acid molecule of any of E45-E48.

[0068]E54. Host cell containing the E52 vector.

[0069]E55. An E53 or E54 host cell, wherein said cell is a mammalian cell.

[0070]E56. An E53 or E54 host cell, wherein said host cell is a CHO cell, a HEK-293 cell, or an Sp2.0 cell.

[0071]E57. A method for producing a recombinant ROBO2 protein, comprising cultivating a host cell of any of E53-E56 under conditions under which expression of said recombinant ROBO2 protein is observed.

[0072]E58. Method E57 also comprising isolating said recombinant ROBO2 protein.

[0073]E59. A pharmaceutical composition containing a recombinant ROBO2 protein of any of E1-E44A and a pharmaceutically acceptable carrier or excipient.

[0074]E60. A method for reducing the biological activity of ROBO2, comprising administering to an individual in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of any of E1-E44, or a pharmaceutical composition of E59.

[0075]E61. Method E60, wherein said ROBO2 biological activity is selected from the group consisting of binding to at least one SLIT ligand, actin polymerization, podocyte adhesion, inhibition of SLIT2-N-mediated inhibition of nerve cell migration, binding of ROBO2 to srGAP1, and binding of ROBO2 to Nck.

[0076]E62. A method for treating kidney disease, comprising administering to an individual in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59.

[0077]E63. A method for preserving the function of podocytes, which includes contacting the specified podocyte with the recombinant ROBO2 protein of any of E1-E44 or with the pharmaceutical composition E59.

[0078]E64. A method for modulating podocyte function, comprising contacting said podocyte with a recombinant ROBO2 protein of any of E1-E44 or with a pharmaceutical composition E59.

[0079]E65. A method for treating glomerulitis, comprising administering to an individual in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59.

[0080]E66. A method for treating focal segmental glomerular sclerosis (FSGS), comprising administering to an individual in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59.

[0081]E67. A method for treating nephropathy, comprising administering to an individual in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59.

[0082]E68. Method E67 wherein said nephropathy is IgA nephropathy.

[0083]E69. The method of any of E60-E68, wherein said subject is a human.

[0084]E70. The method of any of E60-E69, wherein said recombinant ROBO2 protein or pharmaceutical composition is administered intravenously.

[0085]E71. The method of any of E60-E69, wherein said recombinant ROBO2 protein or pharmaceutical composition is administered subcutaneously.

[0086]E72. The method of any of E60-E71, wherein said recombinant ROBO2 protein or pharmaceutical composition is administered approximately twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks , once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months or once every four months.

[0087]E73. Recombinant ROBO2 protein of any of E1-E44 or E59 pharmaceutical composition for use as a drug.

[0088]E74. A recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition for use in reducing ROBO2 activity in a cell.

[0089]E75. A recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition for use in reducing ROBO2 activity in an individual.

[0090] E76. A recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition for use in maintaining podocyte function in an individual.

[0091] E77. A recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for use in modulating podocyte function in an individual.

[0092] E78. A recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition for use in the treatment of glomerulitis in an individual.

[0093]E79. Recombinant protein ROBO2 E78, where the specified glomerulitis is FSGS.

[0094]E80. A recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition for use in the treatment of nephropathy in an individual.

[0095]E81. Recombinant protein ROBO2 E80, where the indicated nephropathy is IgA nephropathy.

[0096]E82. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 in order to prepare a drug for reducing the activity of ROBO2 in a cell.

[0097] E83. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for the preparation of a medicament for reducing ROBO2 activity in an individual.

[0098]E84. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for the preparation of a medicament for preserving podocyte function in an individual.

[0099] E85. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for the preparation of a medicament for modulating podocyte function in an individual.

[0100] E86. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for the preparation of a medicament for the treatment of glomerulitis in an individual.

[0101] E87. The use of a recombinant ROBO2 protein of any of E1-E44 or a pharmaceutical composition of E59 for the preparation of a medicament for the treatment of nephropathy in an individual.

[0102] E88. A kit comprising a container, a composition in this container containing a recombinant ROBO2 protein of any of E1-E44 or an E59 pharmaceutical composition and a package insert containing instructions for administering a therapeutically effective amount of a ROBO2 recombinant protein or a pharmaceutical composition for the treatment of a patient in need thereof.

Brief description of the drawings

[0103] In FIG. 1A is a graph illustrating human ROBO2 domains. The 21 amino acid leader sequence of ROBO2 (SEQ ID NO: 17; residues 1-21 according to SEQ ID NO: 24) is cleaved during protein production to form mature ROBO2. Residues 22-859, as numbered in SEQ ID NO: 24, form the extracellular domain, residues 860-880, as numbered in SEQ ID NO: 24, form the transmembrane domain, and residues 881-1378, as numbered in SEQ ID NO: 24 form a cytoplasmic domain. Also shown are the Ig1-ROBO2 pre-sequence (SEQ ID NO: 8), the Ig1-Ig2 interdomain linker (SEQ ID NO: 10)) and the Ig2-Ig3 interdomain linker (SEQ ID NO: 12).

[0104] In FIG. 1B is a graph illustrating the representative recombinant ROBO2-Fc fusion proteins described herein: ROBO2-Fc 1.0 (containing only the Ig1 domain, ie, amino acid residues 31-127 according to SEQ ID NO: 24); ROBO2-Fc 1.1 (comprising an Ig1 domain and an Ig1-Ig2 interdomain linker, ie amino acid residues 31-132 according to SEQ ID NO: 24); ROBO2-Fc 2.0 (comprising an Ig1 domain, an Ig1-Ig2 interdomain linker and an Ig2 domain, i.e., amino acid residues 31-220 according to SEQ ID NO: 24); ROBO2-Fc 2.1 (comprising an Ig1 domain, an Ig1-Ig2 interdomain linker, an Ig2 domain and an Ig2-Ig3 interdomain linker, i.e., amino acid residues 31-224 according to SEQ ID NO: 24); ROBO2-Fc 2.2 (containing an Ig1 pre-sequence, an Ig1 domain, an Ig1-Ig2 interdomain linker, an Ig2 domain, and an Ig2-Ig3 interdomain linker, i.e., amino acid residues 22-224 according to SEQ ID NO: 24); ROBO2-Fc 3.0 (comprising Ig1 domain, Ig1-Ig2 interdomain linker, Ig2 domain, Ig2-Ig3 interdomain linker, Ig3 domain, i.e., amino acid residues 31-309 according to SEQ ID NO: 24); and ROBO2-Fc 4.0 (comprising an Ig1 domain, an Ig1-Ig2 interdomain linker, an Ig2 domain, an Ig2-Ig3 interdomain linker, an Ig3 domain, an Ig3-Ig4 interdomain linker, and Ig4, that is, amino acid residues 31-409 according to SEQ numbering ID NO: 24).

[0105] Figure 2 shows the amino acid sequence of ROBO2-Fc 2.2 (SEQ ID NO: 1). The residues are numbered sequentially, starting from the N-terminus. The Ig1 and Ig2 domains are shown in uppercase letters, and the Fc domain is shown in lowercase letters. The Ig1 pre-sequence (SEQ ID NO: 8) and the Ig2-Ig3 interdomain linker (SEQ ID NO: 12) are shown in bold and italics, and the Ig1-Ig2 interdomain linker (SEQ ID NO: 10) is shown in italics. The predicted intra- and inter-chain disulfide bonds are shown as connecting lines. One polypeptide chain is present with disulfide bonds in the Fc hinge region, which can dimerize with a second Fc containing the polypeptide chain. Sites of the canonical N-linked glycosylation consensus sequence are indicated by circles (i.e., NXS/T, where the glycan is linked to an aspartic residue, and where X can be any amino acid other than proline, and the third amino acid is either serine or threonine), and dotted mutations lacking Fc effector function and located in A228, A229, and A231 are shown in bold. The 6 amino acid Gly-Ser linker sequence is shown in the box.

[0106] In FIG. 3 shows that ROBO2-Fc 2.1 (SEQ ID NO: 2) binds to SLIT2, while ROBO2-Fc 1.1 (SEQ ID NO: 4) and ROBO2-Fc 2.0 (SEQ ID NO: 3) do not bind to SLIT2. The ROBO2-Fc Octet Red proteins used were loaded onto sensors containing an anti-human crystallized fragment (AHFc) at a concentration of 10 μg/ml and incubated with 100 nM SLIT2 for 7 minutes, and then the sensors were transferred to a separate buffer for 640 seconds. ROBO2-Fc 4.0 (SEQ ID NO: 7) was included as a positive control for binding. Addition of the VFER sequence SEQ ID NO: 12) after the Ig2 domain of ROBO2 to produce ROBO2-Fc 2.1 (SEQ ID NO: 2) plays an important role in the production of a ROBO2-Fc fusion protein that binds to SLIT2.

[0107] In FIG. 4A-4C show that ROBO2-Fc 2.2 (SEQ ID NO: 1) binds to SLIT2 with high affinity. The K _D values were measured using surface plasmon resonance (SPR). The K _{D of} ROBO2-Fc for binding to human/canine simian SLIT2-D2 (100% identical ROBO2-binding domain) was 0.293 nM (FIG. 4A). The K _{D of} ROBO2-Fc 2.2 for binding to human SLIT2-N (with an N-terminal fragment) was 0.279 nM (Figure 4B) and the K _{D of} ROBO2-Fc 2.2 for binding to rat SLIT2-N was 0.543 nM (Figure 4C). ).

[0108] In FIG. 5 shows that ROBO2-Fc 2.2 (SEQ ID NO: 1) binds with a high affinity of 9 nM EC ₅₀ to human SLIT2-N, which is overexpressed on human embryonic kidney (HEK293) cells. 12-point 2-fold serial dilutions of ROBO2-Fc 2.2 labeled with Alexa Fluor 647 (AF647) were incubated with either SLIT2-N-expressing HEK293 cells or control HEK293 cells. Data are presented as geometric mean fluorescence intensity (Geo MFI) of ROBO2-Fc 2.2 AF647 on HEK293 SLIT2-N cells minus geometric mean fluorescence intensity of ROBO2-FC 2.2 AF647 on control HEK293 cells.

[0109] In FIG. 6A-6B demonstrate dose dependent inhibition of cell surface binding of SLIT2-N to ROBO2 by ROBO2-Fc 2.2 (SEQ ID NO: 1) as assessed by time resolved homogeneous fluorescence (HTRF). An 11-point 4-fold titratable dose of ROBO2-FC 2.2 (black squares) or isotype control antibody (black circles) was added to human SLIT2-N (Fig. 6A) or rat SLIT2-N (Fig. 6B) in the human HTRF assay. ROBO2. ROBO2-Fc is a potent binding neutralizer of human SLIT2-N:human ROBO2 (IC ₅₀ = 7 nM) and rat SLIT2-N: human ROBO2 (IC ₅₀ = 4 nM).

[0110] In FIG. 7 shows dose-dependent inhibition of SLIT2-mediated inhibition of nerve cell migration by ROBO2-Fc 2.2. Explants of nerve tissue cells of the subventricular zone (SVZ) were cultured overnight in the presence of 1 nM SLIT2-N and ROBO2-Fc 2.2 in the dose range. ROBO2-Fc 2.2 had the ability to restore nerve cell migration in a dose dependent manner with an IC ₅₀ =51 nM.

[0111] In FIG. 8 demonstrates the inhibition of proteinuria by administration of ROBO2-Fc 2.2 in a rat model of Hayman's passive nephritis using a representative prophylactic dose schedule. Twelve animals in each of the indicated groups were treated subcutaneously with the indicated dose of ROBO2-Fc 2.2 or an irrelevant isotype control monoclonal antibody (control) every three days, starting from the day of model induction on day 0. The y-axis plots the ratio of albumin to urinary creatinine (mg/ mg) as an indicator of protein penetration into the urine, as indicated by damage to podocytes. Lewis rats were injected with sheep antiserum against rat kidney brush border (against Fx1a, basement membrane and podocytes). Rats developed an immune response against sheep serum, which binds to rat podocytes. As podocytes are damaged and destroyed, the level of proteinuria increases. Treatment with ROBO2-Fc 2.2 administered at 25 mg/kg resulted in a reduction in proteinuria of up to 45% and a p- value of less than 0.001 as determined by performing repeated ANOVAs compared to control antibody treatment. This dose-dependent effect was also statistically significant with a p- value of less than 0.001.

[0112] In FIG. 9 demonstrates the inhibition of proteinuria by treatment with ROBO2-Fc 2.2 in a rat model of Hayman's passive nephritis using a representative prophylactic dose schedule. Twelve animals in each of the indicated groups were treated subcutaneously with the indicated dose of ROBO2-Fc 2.2 or irrelevant isotype control monoclonal antibody (control) every three days according to the respective dosing schedule: control antibody was administered on day 0 (circles) and ROBO2-Fc 2.2 was administered day 0 (squares), day 6 (triangles), or day 9 (inverted triangles). Plotted on the y-axis is the ratio of albumin to urinary creatinine (mg/mg) as an indicator of protein penetration into the urine, as indicated by podocyte damage. Lewis rats were injected with sheep antiserum against rat kidney brush border (against Fx1a, basement membrane and podocytes). Rats developed an immune response against sheep serum, which binds to rat podocytes. As podocytes are damaged and destroyed, the level of proteinuria increases. Treatment with ROBO2-Fc 2.2 administered on days 0, 6 and 9 resulted in a similar reduction in proteinuria of up to 40% and a p- value of less than 0.001 as determined by performing repeated ANOVAs for each ROBO2- Fc 2.2-treated group versus control antibody treatment.

[0113] In FIG. 10A-10B show that treatment with ROBO2-Fc 2.2 resulted in a reduction in the level of podocyte substructure damage in a model of Hayman's passive nephritis. Twelve animals in each of the indicated groups were treated subcutaneously with the indicated dose of ROBO2-Fc 2.2 or an irrelevant monoclonal control antibody every three days at a dose of 25 mg/kg until 100% coverage of the target was achieved, starting from the day of model induction on day 0. After killing the animals on day 16, collected kidney samples were digitally imaged on a transmission electron microscope. The three capillary loops of the first three glomeruli found at 200x were visualized at 5000x and 10000x and this procedure was not repeated. The ImageJ computer program (version 1.47v; National Institutes of Health, Bethesda, MD, USA) was used to manually monitor the width of adjacent ramified processes as well as the density of slit diaphragms per unit length of the glomerular basement membrane (GBM) on a high magnification transmission electron microscope. Samples were analyzed in 3 separate experiments. The base processes of the podocytes of the ROBO2-Fc 2.2-treated animal were significantly shorter than those of the control antibody-treated animal (Fig. 10A), and the density of the slit diaphragms was significantly higher in the ROBO2-Fc 2.2-treated animals (Fig. 10B; p -value was less than 0.01, as determined by a two-tailed T-test), indicating that these animals were less affected and were protected from glomerular injury.

[0114] FIG. 11 demonstrates that ROBO2-Fc S17T/R73Y binds to human SLIT2-N, which is overexpressed on human embryonic kidney (HEK293) cells with a high affinity of EC ₅₀ of 2.5 nM. 12-point 2-fold serial dilutions of ROBO2-Fc S17T/R73Y labeled with Alexa Fluor 647 (AF647) were incubated with either SLIT2-N-expressing HEK293 cells or control HEK293 cells. Data are presented as geometric mean fluorescence intensity (Geo MFI) of ROBO2-Fc S17T/R73Y AF647 on SLIT2-N HEK293 cells minus geometric mean fluorescence intensity of ROBO2-Fc S17T/R73Y AF647 on control HEK293 cells.

[0115] In FIG. 12 demonstrates dose dependent inhibition of SLIT2-N binding to ROBO2 on the cell surface by ROBO2-Fc S17T/R73Y as assessed by time resolved homogeneous fluorescence (HTRF). An 11-point 4-fold titratable dose of ROBO2-Fc S17T/R73Y (black squares) or isotype control antibodies (black circles) was added to human SLIT2-N or rat SLIT2-N in the human ROBO2 HTRF assay. ROBO2-Fc S17T/R73Y is a potent human SLIT2-N:human ROBO2 binding neutralizer with an IC ₅₀ =1.4 nM.

[0116] FIG. 13 shows dose-dependent inhibition of SLIT2-mediated inhibition of nerve cell migration by ROBO2-Fc S17T/R73Y. Subventricular zone (SVZ) neural tissue cell explants were cultured overnight in the presence of 1 nM SLIT2-N and titratable amounts of ROBO2-Fc S17T/R73Y. ROBO2-Fc S17T/R73Y had the ability to restore nerve cell migration in a dose dependent manner with an IC ₅₀ =11.5 nM.

[0117] In FIG. 14 is an image illustrating the crystal structure of the ROBO2-His construct, which consists of the ROBO2 Ig1 pre-sequence (SRLRQEDFP; SEQ ID NO: 8), the Ig1 domain, the Ig2 domain, and the ROBO2 Ig2-3 interdomain linker (VFER; SEQ ID NO: 12 ) with a histidine 6× tag (His6) (SEQ ID NO: 25) at the C-terminus. The crystal structure of ROBO2-His showed that Asp7, Phe8 and Pro9 play an important role in the interactions required to achieve the structural integrity of the ROBO2 Ig1 domain.

[0118] In FIG. 15 is an image illustrating the crystal structure of the ROBO2-His construct. The crystal structure of ROBO2-His6 ("His6", shown as SEQ ID NO: 25) showed that the Ig2-3 ROBO2 interdomain linker, valine200-phenylalanine201-glutamic acid202-arginine203 (SEQ ID NO:12) effectively stabilizes the C-fold. terminal region of the Ig2 ROBO2 domain.

[0119] FIG. 16 shows the crystal structure of the first immunoglobulin-like domain (Ig1) of ROBO2 S17T/R73Y in complex with SLIT2.

Detailed description of the invention

1. Overview

[0120] The present invention encompasses novel recombinant ROBO2 proteins capable of binding to SLIT ligands (e.g., the SLIT2 ligand) and thereby inhibiting the interaction of SLIT with ROBO2, and further inhibiting the SLIT2-ROBO2 signaling pathway. Previous studies have shown that although both immunoglobulin-like (Ig-like) domains 1 and 2 (Ig1 and Ig2) of ROBO2 interact with SLIT ligands, however, the first Ig-like domain (Ig1) is the primary binding site for SLIT. In addition, previous studies have shown that the removal of three repeats of type III fibronectin (FNIII) has a more negative effect on the binding of ROBO to SLIT ligands than the removal of the third and fourth immunoglobulin-like domains (Ig3 and Ig4). That is, the deletion of FNIII leads to a greater decrease in the level of binding of ROBO to SLIT ligands than the deletion of Ig3 and Ig4.

[0121] For the first time and unexpectedly, the ROBO2-Fc 2.1 construct (SEQ ID NO: 2; FIG. 1B) has been shown to contain only the first two immunoglobulin-like domains (Ig1 and Ig2) together with the Ig2-3 interdomain linker, VFER (SEQ ID NO: 12) and does not contain three repeats of type III fibronectin (FNIII) associated with SLIT2 (Fig. 3). In contrast, recombinant ROBO2 proteins do not contain the three repeats of fibronectin type III (FNIII), but are composed of:

(i) ROBO2 Ig1 domain (ROBO2-Fc 1.1; SEQ ID NO: 4; Fig. 1B),

(ii) ROBO2 Ig1 and Ig2 domains (ROBO2-Fc 2.0; SEQ ID NO: 3; FIG. 1B),

(iii) ROBO2 Ig1, Ig2 and Ig3 domains (ROBO2-Fc 3.0; SEQ ID NO: 6; FIG. 1B) that do not bind to SLIT2 (FIG. 3).

[0122] Thus, the attachment of VFER (SEQ ID NO: 12) to the C-terminus of the Ig1-Ig2 domains was necessary to generate a ROBO2-Fc construct (ROBO2-Fc 2.1; SEQ ID NO: 2) having a reliable binding profile to SLIT2 in the absence of FNIII repeats. This ROBO2-Fc 2.1 construct lacks not only the third, fourth, and fifth immunoglobulin-like domains (Ig3, Ig4, and Ig5), but also the three repeats of type III fibronectin (FNIII). Surprisingly, the differences in binding to SLIT2 and in the absence of binding to SLIT2 were found to be due to the presence of a four amino acid VFER sequence (SEQ ID NO: 12).

[0123] None of the above ROBO2 recombinant protein constructs contains the ROBO2 Ig1 pre-sequence (SEQ ID NO: 8). Surprisingly, it was found that the level of production of these recombinant ROBO2 proteins disclosed and described in this application can be dramatically increased due to the inclusion of the Ig1 ROBO2 pre-sequence (SEQ ID NO: 8). Attachment of this sequence results in an approximately 25-fold increase in protein production compared to constructs lacking this sequence but retaining high binding affinity for SLIT2 (FIGS. 3-4A-C). As shown in FIG. 14, this ROBO2 Ig1 pre-sequence bridges two β-folds of the ROBO2 Ig1 domain and likely stabilizes the N-terminal fold. Without wishing to be bound by any particular theory, the authors only note that the Ig1 pre-sequence appears to be involved in the upregulation of new proteins.

2. Definitions

[0124] In some of its aspects, the present invention relates to recombinant ROBO2 proteins capable of binding to SLIT and containing an immunoglobulin domain.

[0125] The term "recombinant protein" means a polypeptide that has been produced by recombinant DNA techniques, where essentially the DNA encoding the polypeptide is inserted into a suitable expression vector, which, in turn, will be inserted into a host cell to form a recombinant protein . As used herein, the term "protein" means any composition containing amino acids and known to those skilled in the art as a protein. As used herein, the terms "protein", "peptide" and "polypeptide" are synonymous. Amino acids may be referred to by their full names (eg, alanine), or they may be designated by conventional one-letter codes (eg, A) or three-letter codes (eg, Ala).

[0126] As used herein, the term "immunoglobulin domain" means a polypeptide derived from an immunoglobulin. In some embodiments, the immunoglobulin domain comprises an immunoglobulin heavy chain or a portion thereof. In some embodiments of the invention, part of the heavy chain is a crystallizable fragment (Fc) or part thereof. The term "Fc fragment" as used herein includes the heavy chain hinge region and the C _H 2 and C _H 3 domains of an immunoglobulin heavy chain. The heavy chain (or part thereof) can be derived from any known isotype of heavy chain: IgG (γ), IgM (μ), IgD (δ), IgE (ε) or IgA (α). In addition, the heavy chain (or part of it) can be derived from the heavy chain of any known isotype or subtype: IgG1 (γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2) . In some embodiments, the immunoglobulin domain comprises a contiguous native (ie, wild-type) immunoglobulin sequence. In some embodiments of the invention, the Fc domain of the immunoglobulin contains a modified Fc region.

[0127] For all amino acid positions of the heavy chain constant region discussed in this application, the numbering corresponds to the Eu index, first described by Edelman et al., 1969, Proc. Natl. Acad. sci. USA 63(1):78-85, which provides the amino acid sequence of the myeloma protein Eu, which was the first sequenced human IgG1. The Eu index described by Edelman et al. was also proposed by Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, "Cabat EU index" or "Cabat EU index" means the residue numbering system based on the human IgG1 Eu antibody described by Edelman et al., according to Kabat, 1991.

[0128] A "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of a native Fc region. A "modified Fc region" includes an amino acid sequence that differs from the native sequence of the Fc region by at least one amino acid modification. Preferably, the modified Fc region has at least one amino acid substitution compared to the native sequence Fc region, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions compared to Fc region with native sequence. The modified Fc region described herein will preferably have an amino acid sequence that is at least about 80%, more preferably at least about 90%, even more preferably at least about 95% at least about 96%, at least about 97%, at least about 98%, and most preferably at least about 99% amino acid sequence identical to the native sequence Fc region.

[0129] As used herein, the term "linker" refers to a molecule or group of molecules that links two separate molecules (e.g., the extracellular domain and the immunoglobulin domain of the recombinant ROBO2-Fc protein) to each other and can create a space between the two molecules and provide them with flexibility so so that their conformation allows them to specifically bind to, for example, their cognate ligand (eg, SLIT ligand). Particularly preferred are protein linkers, and these linkers can be expressed as a component of a recombinant protein by standard recombinant DNA techniques well known in the art.

[0130] The term "IC ₅₀ " or "half-maximal inhibitory concentration" means the concentration of recombinant ROBO2 protein that is required to inhibit 50% of the ROBO2-SLIT signaling pathway, for example, the ROBO2-SLIT2 signaling pathway. The IC ₅₀ is an indication of how much the recombinant ROBO2 protein is needed to inhibit the ROBO2-SLIT biological process by 50%, such as the binding of ROBO2 and the SLIT ligand; binding of intracellular signal-transmitting molecules (such as srGAP1 or Nck) to the intracellular domain of ROBO2; and/or downstream ROBO2-SLIT signaling activity (such as actin polymerization, podocyte adhesion, and/or SLITx-N-mediated inhibition of nerve cell migration). A lower IC ₅₀ indicates a stronger effect, since a decrease in the amount of recombinant ROBO2 protein mediates a stronger inhibitory effect.

[0131] As used herein, the term "SLITx" means essentially a SLIT ligand. Similarly, the terms "SLITx-N" and "SLITx-C" mean essentially the N-terminal and C-terminal fragments, respectively, of SLIT ligands. The SLIT ligand may be a mammalian SLIT ligand, and preferably a human SLIT ligand. In some embodiments, the SLIT ligand is selected from the group consisting of a SLIT1 ligand, a SLIT2 ligand, and a SLIT3 ligand. The SLIT ligand may be a SLIT2 ligand, and preferably a human SLIT2 ligand.

[0132] As used herein, the term "individual" refers to an animal, preferably a mammal, more preferably a non-human primate, and most preferably a human. As used herein, the terms "subject", "individual" and "patient" are synonymous. In all embodiments of the invention, human nucleic acids and human polypeptides are preferred. It is believed that the results obtained using the human, rat and cynomolgus molecules described herein may be predictive of those obtained using other homologous sequences.

[0133] As used herein, the term "treatment" means a method of achieving the desired or desired clinical results. In accordance with the objectives of the present invention, the desired or desired clinical results are, but are not limited to, one or more of the following factors, such as a decrease in the level of proteinuria (i.e., a decrease in the amount of protein in the urine compared to the amount of protein in the urine without drug administration). agents), reduction of edema and/or restoration of blood albumin levels. The term "treatment" includes prophylactic and/or therapeutic treatment. If the treatment is carried out before the appearance of clinical signs of the condition, then such treatment is called prophylactic. Therapeutic treatment includes, for example, attenuating or reducing the severity of the disease or shortening the duration of the disease.

[0134] The term "therapeutically effective amount" means an amount of a therapeutic agent of the invention effective to "treat" a disease or disorder in an individual. Thus, for example, a therapeutically effective amount may be an amount that relieves one or more symptoms of a disease or an amount necessary to maintain remission of the disease. In the case of focal segmental glomerulosclerosis (FSGS), a therapeutically effective amount means an amount that is appropriate to achieve at least one of the following effects: reduction in proteinuria (i.e., reduction in the amount of protein in the urine compared to the amount of protein in the urine without drug agents), reducing edema and/or restoring albumin levels in the blood.

[0135] As used herein, the terms "about" or "approximately" when used in conjunction with measurable numerical variables refer to the specified value of the variable and to all values of the variable that are within the experimental error of the specified value (e.g., within 95% confidence limit of the mean) or are ±10% of the specified value, which may exceed this value. Numerical limits include numbers that define the boundaries of the range of values.

Binding affinity

[0136] "Binding affinity" generally means the sum of the strengths of all non-covalent interactions between the contacting residue of one binding partner (eg, the recombinant ROBO2 protein described here) and the contacting residue of its binding partner (eg, the SLIT ligand). Unless otherwise noted, the term "binding affinity" as used herein means binding affinity that defines a 1:1 interaction between members of a binding pair or binding partners (eg, recombinant ROBO2 protein and SLIT2 ligand).

(например,

, используемый здесь в Примерах), от тяжелого атома аминокислотного остатка ROBO2 и тяжелого атома аминокислотного остатка SLIT. В другом аспекте, контактирующий остаток может быть охарактеризован как остаток, участвующий во взаимодействии водородной связи с когнатным партнером по связыванию или с молекулой воды, которая также представляет собой водород, связанный с партнером по связыванию (то есть, в образовании водородной связи, опосредуемой водой). В другом аспекте изобретения, контактирующий остаток может быть охарактеризован как остаток, образующий солевой мостик с остатком партнера по связыванию. В другом аспекте изобретения, контактирующий остаток может быть охарактеризован как остаток, имеющий ненулевое изменение площади скрытой поверхности (BSA) благодаря взаимодействию с контактирующим остатком партнера по связыванию. При менее детальном рассмотрении, аффинность связывания может быть охарактеризована по их функции, например, по конкурентному связыванию с другими белками.[0137] In more detail, the binding affinity between ROBO2 and the SLIT ligand can be determined from the spatial coordinates defining the atomic contacts involved in the ROBO2/SLIT interaction, as well as information about their respective participation in binding thermodynamics. At one level, the contact residue can be characterized by the spatial coordinates defining the atomic contacts between ROBO2 and SLIT. In one aspect of the invention, the contact residue may be defined by a specific criterion, such as the distance between atoms at an amino acid residue of a ROBO2 protein and atoms at an amino acid residue of a SLIT protein, such as a distance equal to or less than about

(For example,

, used here in the Examples), from the heavy atom of the ROBO2 amino acid residue and the heavy atom of the SLIT amino acid residue. In another aspect, the contact moiety can be characterized as a moiety involved in a hydrogen bond interaction with a cognate bonding partner or with a water molecule that is also a hydrogen bonded to the bonding partner (i.e., in a water-mediated hydrogen bonding) . In another aspect of the invention, the contact residue can be characterized as a residue that forms a salt bridge with the remainder of the binding partner. In another aspect of the invention, the contact moiety can be characterized as having a non-zero change in hidden surface area (BSA) due to interaction with the contact moiety of a binding partner. In less detail, binding affinity can be characterized by their function, for example by competitive binding to other proteins.

[0138] Low affinity recombinant proteins generally bind ligands slowly and tend to dissociate rapidly, while high affinity recombinant proteins generally bind ligands faster and tend to stay bound longer. A number of methods for measuring binding affinity are known to those skilled in the art, and any of these methods can be used to practice the invention. Specific illustrative and representative binding affinity measurements are described below.

[0139] Binding affinity can be expressed as a K _D value, which is the dissociation rate for a particular interaction of a recombinant ROBO2 SLIT ligand protein. K _D is the ratio of the rate of dissociation, also referred to as "dissociation (k _off )", to the rate of association or "association (k _on )". Thus, K _D is equal to k _off /k _on and is expressed as a molar concentration (M), and the smaller the K _D , the higher the binding affinity. The K _D values can be determined by methods well known to those skilled in the art. One representative method for measuring K _D is the surface plasmon resonance (SPR) method, that is, a method well known to those skilled in the art (e.g., Nguyen et al. Sensors (Basel), 2015 May 5; 15(5): 10481-510). The K _D value can be measured by SPR using a biosensor system such as BIACORE®. BIAcore kinetic analysis includes antigen binding and dissociation analysis from chips on the surface of which there are immobilized molecules (for example, molecules containing epitope-binding domains). Another well-known method for determining protein K _D is the method using biolayer interferometry (for example, Shah et al. J Vis Exp. 2014; (84): 51383). The K _D value can be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® assay (kinetic exclusion assay) developed by Sapidyne Instruments (Boise, Id.) can also be performed. Any known method for assessing binding affinity between two binding partners is within the scope of the present invention.

3. Recombinant ROBO2 proteins

[0140] In some of its aspects, the present invention relates to recombinant ROBO2 proteins. In some embodiments, the recombinant ROBO2 proteins described herein bind to SLIT ligands (and in particular the SLIT2 ligand) and thereby prevent SLIT from binding to cellular ROBO2 receptors and are therefore referred to herein as ligand uptake neutralizing SLIT proteins. As described in the Examples, the ROBO2-Fc 2.1 construct (SEQ ID NO: 2; FIG. 1B) was unexpectedly found to contain the first two immunoglobulin-like domains (Ig1 and Ig2), an Ig1-Ig2 interdomain linker, together with an Ig2 interdomain linker. -3, VFER (SEQ ID NO: 12), and lacks the three type III fibronectin (FNIII) repeats associated with SLIT2 (FIG. 3). Crystal structure studies have also shown that incorporation of the ROBO2, VFER (SEQ ID NO: 12) interdomain Ig2-Ig3 linker (V200-F201-E202-R203; SEQ ID NO: 12) effectively stabilizes the structural fold at the C-terminal region of the second Ig domain ROBO2 (Fig. 15) and markedly increases the level of expression of the recombinant ROBO2 protein.

[0141] In some of its aspects, the present invention relates to recombinant polypeptides containing a SLIT-binding molecule and a molecule that increases the half-life. The "SLIT binding molecule" imparts to the recombinant ROBO2 protein the ability to bind to SLIT. In some embodiments, the SLIT binding molecule contains a portion of the extracellular domain of ROBO2. In some embodiments, the extracellular domain portion of ROBO2 contains at least two immunoglobulin-like (Ig-like) domains and a C-terminal sequence consisting of VFER (SEQ ID NO: 12). In some embodiments of the invention, at least two Ig-like domains are selected from the group consisting of Ig1, Ig2, Ig3, Ig4 and Ig5. In some embodiments, at least two Ig-like domains are selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14. In some embodiments of the invention , part of the extracellular domain of ROBO2 contains the first two Ig-like domains (Ig1 and Ig2) of ROBO2. In some embodiments, the extracellular domain portion of ROBO2 comprises SEQ ID NO: 9 and/or SEQ ID NO: 11.

[0142] Protein production studies have also shown that the production of the recombinant ROBO2 proteins disclosed and described herein can be dramatically increased due to the inclusion of the ROBO2 Ig1 pre-sequence (SEQ ID NO: 8). Attachment of this sequence results in an approximately 25-fold increase in protein production in transiently and/or stably transfected mammalian cells and maintains high binding affinity to SLIT (FIGS. 3-5).

[0143] Accordingly, in some embodiments of the invention, part of the extracellular domain of ROBO2 also contains the Ig1 pre-sequence of ROBO2. In some embodiments, the Ig1 ROBO2 pre-sequence contains SEQ ID NO: 8.

[0144] In some embodiments, the ROBO2 extracellular domain portion comprises the ROBO2 Ig1 pre-sequence, Ig1, an Ig1-Ig2 interdomain linker, Ig2, and an Ig2-Ig3 interdomain linker. Representative sequences of the pre-sequence of Ig1 ROBO2 (SEQ ID NO: 8), Ig1 (SEQ ID NO: 9), Ig1-Ig2 interdomain linker (SEQ ID NO: 10), Ig2 (SEQ ID NO: 11), and Ig2-interdomain linker Ig3 (SEQ ID NO: 12) are shown in Table 23 and are also illustrated in FIG. 2. The present invention is not limited to the sequences disclosed here. Relevant residues of other homologues, isoforms, variants or fragments of ROBO2 can be identified by sequence alignments or structural alignments known to those skilled in the art. For example, alignment can be done manually or using well-known sequence alignment programs such as ClustalW2 or "BLAST 2 Sequences" using default parameters.

[0145] In some embodiments, the ROBO2 extracellular domain portion comprises amino acid residues 1-203 according to SEQ ID NO: 1. In some embodiments, the recombinant ROBO2 protein comprises a ROBO2 extracellular domain portion that is at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98% or at least 99% identical to amino acid residues 1-203 according to SEQ ID NO: 1. In some embodiments, the recombinant ROBO2 protein contains an extracellular domain consisting of amino acid residues 1-203 in accordance with the numbering of SEQ ID NO: 1.

[0146] In some aspects of the invention, ROBO2 is a human ROBO2. In some aspects of the invention, ROBO2 is rat ROBO2. In some aspects of the invention, ROBO2 is a murine ROBO2. In some aspects of the invention, ROBO2 is a primate ROBO2. In some aspects of the invention, ROBO2 is a simian ROBO2. In some aspects of the invention, ROBO2 is a simian ROBO2. In some aspects of the invention, ROBO2 is a cynomolgus monkey ROBO2.

[0147] The new recombinant ROBO2 proteins contain, in addition to the SLIT-binding molecule, a molecule that increases the half-life. A “half-life increasing molecule” increases the in vivo serum half-life of a recombinant ROBO2 protein compared to the same ROBO2 protein, but in the absence of a half-life increasing molecule. Examples of molecules that increase half-life include, but are not limited to, polyhistidine, Glu-Glu, glutathione-S-transferase (GST), thioredoxin, protein A, G protein, immunoglobulin domain, maltose binding protein (MBP), human serum albumin (HSA) or polyethylene glycol (PEG). In some embodiments, the half-life increasing molecule comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain contains an Fc domain. In some embodiments, the Fc domain is derived from a heavy chain of any known isotype: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In some embodiments, the Fc domain is derived from a heavy chain of any known isotype or subtype: IgG ₁ (γ1), IgG ₂ (γ2), IgG ₃ (γ3), IgG ₄ (γ4), IgA1 (α1), IgA2 ( α2). In some embodiments, the Fc domain is a human IgG ₁ Fc domain.

[0148] In some embodiments of the invention, the Fc domain contains a contiguous native sequence (ie, wild-type sequence) of the Fc domain. In some embodiments of the invention, the Fc domain of an immunoglobulin contains a modified Fc domain that results in a change in biological activity. For example, at least one point mutation or deletion can be included in the Fc domain to reduce or eliminate effector activity (eg WO 2005/063815) and/or to increase homogeneity during production of the recombinant protein. In some embodiments, the Fc domain is a human IgG ₁ Fc domain and contains one or more of the following effector deficient substitutions: L234A, L235A and G237A (Eu numbering) or L228A, L229A and G231A according to SEQ numbering ID NO: 1. In some embodiments, the Fc domain does not contain a lysine at the C-terminal position of human IgG1 (ie, K447 according to Eu numbering). The absence of lysine can increase homogeneity during the production of the recombinant protein. In some embodiments, the Fc domain contains a lysine at the C-terminal position (K447 according to Eu numbering).

[0149] In some embodiments, the recombinant ROBO2 polypeptide contains one, two, three, or four intrachain disulfide bonds that may be present in the ROBO2 extracellular domain or Fc domain. In some embodiments, the recombinant ROBO2 polypeptide contains four intrachain disulfide bonds, two of which are present in the extracellular domain of ROBO2 and two in the Fc domain. In some embodiments, intra-chain disulfide bonds present in the extracellular domain of ROBO2 are located between Cys31 and Cys89, and between Cys133 and Cys182, according to SEQ ID NO: 1. In some embodiments, intra-chain disulfide bonds present in Fc domain are located between Cys255 and Cys315 and between Cys362 and Cys419, according to SEQ ID NO: 1.

[0150] In some embodiments, two recombinant ROBO2 polypeptides are linked either covalently, such as via a disulfide bond, polypeptide bond, or cross-linking agent, or non-covalently, to form a homodimeric protein. In some embodiments, two recombinant ROBO2 polypeptides are covalently linked to form a homodimer via at least one, and more preferably two intra-chain disulfide bonds of cysteine residues, preferably located in the immunoglobulin Fc region of each polypeptide. In some embodiments of the invention, two intrachain disulfide bonds are located between Cys220 and Cys223. In some embodiments, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less less than 20%, less than 10%, less than 6%, less than 5%, less than 4%, less than 2%, less than 1% of recombinant ROBO2 polypeptides associate with each other to form a homodimer.

[0151] In some embodiments, two recombinant ROBO2 polypeptides are linked to another polypeptide either covalently, such as via a disulfide bond, polypeptide bond, or cross-linking agent, or non-covalently, to form a heterodimeric protein. In some embodiments, the heterodimeric protein is bispecific or multispecific. In some embodiments, the other polypeptide contains an immunoglobulin domain. In some embodiments of the invention, the polypeptides are covalently linked to form a heterodimer through at least one, and more preferably, two intra-chain disulfide bonds of cysteine residues, preferably located in the Fc region of the immunoglobulin of each polypeptide. In some embodiments, two intrachain disulfide bonds are located between Cys220 and Cys223 of the recombinant ROBO2 polypeptide. In some embodiments, the heterodimeric protein contains two different recombinant ROBO2 polypeptides.

[0152] In some embodiments, the Fc domain of the recombinant ROBO2 protein contains amino acid residues 210-440 according to SEQ ID NO: 1. In some embodiments, the recombinant ROBO2 protein contains an Fc domain that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% identical to amino acid residues 210-440 in accordance with the numbering of SEQ ID NO: 1. In some embodiments of the invention, the recombinant ROBO2 protein contains an Fc domain consisting of amino acid residues 210 -440 according to SEQ ID NO: 1.

[0153] In some embodiments, the extracellular domain of the recombinant ROBO2 protein is adjacent to an immunoglobulin domain. That is, the last C-terminal amino acid residue of the extracellular domain of the ROBO2 protein is covalently linked by a peptidyl bond to the first N-terminal amino acid residue of the immunoglobulin domain. In some embodiments, the extracellular domain of the recombinant ROBO2 protein is linked to the immunoglobulin domain via a linker. In some embodiments, the linker is a peptidyl linker. In some embodiments of the invention, the peptidyl linker contains from about 1 to 30 amino acid residues. In some embodiments, the peptidyl linker is selected from the group consisting of a glycine-rich peptide; a peptide containing glycine and serine; a peptide having the sequence [Gly-Gly-Ser] _n , where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 22); and a peptide having the sequence [Gly-Gly-GIy-Gly-Ser] _n , where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 23). In some embodiments, the peptidyl linker is Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 15). The glycine-rich peptide linker contains a peptide linker wherein at least 25% of the residues are glycine. Glycine-rich peptide linkers are well known in the art (eg, Chichili et al. Protein Sci. 2013 Feb; 22(2): 153-167).

[0154] In some embodiments, the recombinant ROBO2-Fc protein comprises the sequence of SEQ ID NO: 1. In some embodiments, the recombinant ROBO2-Fc protein consists of the sequence of SEQ ID NO: 1. In some embodiments, the recombinant protein ROBO2-Fc contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 1. In some embodiments of the invention, the recombinant ROBO2-Fc protein contains an amino acid sequence, which is at least 95% identical to the sequence of SEQ ID NO: 1. In some embodiments of the invention, the recombinant ROBO2-Fc protein contains an amino acid sequence that at least 96% identical to the sequence of SEQ ID NO: 1. In some embodiments, the recombinant ROBO2-Fc protein contains an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 1. In some embodiments, the recombinant the ROBO2-Fc protein contains an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 1. In some embodiments, the recombinant ROBO2-Fc protein contains an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: : 1.

[0155] In some embodiments, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than than 5, not more than 4, not more than 3, not more than 2 or not more than 1 substitution. In some embodiments of the invention, no more than 5 substitutions have been introduced into the sequence of SEQ ID NO: 1. In some embodiments of the invention, no more than 4 substitutions have been introduced into the sequence of SEQ ID NO: 1. In some embodiments of the invention, no more than 3 substitutions have been introduced into the sequence of SEQ ID NO: 1. In some embodiments of the invention, no more than 2 substitutions have been introduced in the sequence of SEQ ID NO: 1. In some embodiments of the invention, no more than 1 substitution has been introduced into the sequence of SEQ ID NO: 1. In some embodiments of the invention, the replacement(s) does not change(s) K _D more than 1000 times, more than 100 times or 10 times compared to the K _D of the protein containing the sequence of SEQ ID NO: 1. In In some embodiments, the substitutions are the conservative substitutions shown in Table 1.

Table 1: Amino acid substitutions

original balance Conservative substitutions Representative substitutions Ala (A) Val Val; Leu; ile Arg(R) Lys Lys; gln; Asn Asn(N) Gln gln; His; Asp, Lys; Arg Asp(D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln(Q) Asn asn; Glu Glu(E) asp asp; Gln Gly (G) Ala Ala His(H) Arg asn; gln; Lys; Arg Ile (I) Leu Leu; Val; met; Ala; Ph; norleucine Leu(L) ile Norleucine; Ile; Val; met; Ala; Phe Lys (K) Arg Arg; gln; Asn Met(M) Leu Leu; Ph; ile Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser TRP(W) Tyr Tyr; Phe Tyr (Y) Phe trp; Ph; Thr; Ser Val(V) Leu Ile; Leu; met; Ph; Ala; norleucine

[0156] In some embodiments, one or more of the ROBO2 amino acid residues listed in Tables 4-15 have not been substituted (e.g., E6, D7, F8, P9, V200, F201, E202, R203, each numbered in according to SEQ ID NO: 1). In some embodiments, none of the amino acid residues listed in Tables 4-15 (e.g., E6, D7, F8, P9, V200, F201, E202, R203, numbered according to SEQ ID NO: 1) was replaced. The ROBO2 amino acid residues listed in Tables 4-15 are amino acid residues that appear to play an important role in maintaining the structural integrity of the SLIT binding domain, as shown by crystal structure analysis (see Example 5). Amino acid substitutions at these positions can affect binding to SLIT. Accordingly, it may be desirable that no substitution be present in these provisions. In some embodiments, the recombinant ROBO2 protein contains a portion of the ROBO2 extracellular domain that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to amino acid residues 1-203 of the sequence shown in SEQ ID NO: 1 , and also contains one or more residues E6, D7, F8, P9, V200, F201, E202 and R203 (numbered according to SEQ ID NO: 1).

[0157] In some aspects of the invention, one or more point mutations can be introduced into the sequence of SEQ ID NO: 1 to increase the binding affinity of the recombinant ROBO2 protein to a SLIT ligand, such as SLIT2. As shown in the Examples, binding affinity for SLIT2 can be increased approximately 10-fold by introducing point mutations S17T and R73Y in the sequence of SEQ ID NO: 1 (Fig. 11-13). Accordingly, in some of its aspects, the present invention relates to a recombinant ROBO2 protein having one or more of the following mutations: S17T and R73Y compared to the sequence of SEQ ID NO: 1. In some embodiments of the invention, the recombinant ROBO2 protein contains the sequence shown in SEQ ID NO: 19. In some embodiments, the recombinant ROBO2 protein consists of the sequence shown in SEQ ID NO: 19. In some embodiments, the retroviral ROBO2 protein does not contain a C-terminal lysine at amino acid residue 441 in according to the numbering of SEQ ID NO: 19. In some embodiments, the recombinant ROBO2 proteins of the invention have a K _D of no more than about 1×10 ^-6 M, for example, no more than about 1×10 ^-7 M, not more than about 9×10 ^-8 M, not more than about 8×10 ^-8 M, not more than about 7×10 ^-8 M, not more than, than about 6×10 ^–8 M, not more than about 5× ^10–8 M, not more than about 4× ^10–8 M, not more than about 3× ^10–8 M, not more than about 2× ^10–8 M, not more than about 1× ^10–8 M, not more than about 9× ^10–9 M, not more than about 8× ^10–9 M, not more than about 7× ^10–9 M, not more than about 6× ^10–9 M, not more than about 5× ^10–9 M, not more than about 4× ^10–9 M, not more than about 3×10 ^{– 9} M, not more than about 2× ^10–9 M, not more than about 1× ^10–9 M, not more than about 9× ^10–10 M, not more than about 8× ^10–10 M , not more than about 7× ^10–10 M, not more than about 6× ^10–10 M, not more than about 5× ^10–10 M, not more than about 4× ^10–10 M, not more than about 3× ^10–10 M, not more than about 2× ^10–10 M, not more than about 1× ^10–10 M, not more than about 9× ^10–11 M, not more than about 8× ^10–11 M, not more than about 7× ^10–11 M, not more than about 6× ^10–11 M, not more than about 5× ^10–11 M, not more than than about 4× ^10–11 M, not more than about 3× ^10–11 M, not more than about 2× ^10–11 M, not more than about 1× ^10–11 M, not more than about 9×10 ^–12 M, not more than about 8×10 ^–12 M, not more than about 7×10 ^–12 M, not more than about 6×10 ^–12 M, not more than about 5 × ^10–12 M, not more than about 4× ^10–12 M, not more than about 3× ^10–12 M, not more than about 2× ^10–12 M, not more than about 1×10 ^–12 M, not more than about 9×10 ^–13 M, not more than about 8×10 ^–13 M, not more than about 7×10 ^–13 M, not more than about 6×10 ^–13 M, no more than about 5x10 ^-13 M, no more than about 4x10 ^-13 M, no more than about 3x10 ^-13 M, no more than approx. and approximately 2× ^10–13 M, not more than approximately 1× ^10–13 M.

[0158] In some embodiments, the recombinant ROBO2 proteins of the invention have a K _D ranging from about 1x10 ^-7 M to about 1x10 ^-14 M, from about 9x10 ^-8 M to about 1x10 ^{- 14} M, approximately 8× ^10–8 M to approximately 1× ^10–14 M, approximately 7× ^10–8 M to approximately 1× ^10–14 M, approximately 6× ^10–8 M to approximately 1×10 ^–14 M, approximately 5× ^10–8 M to approximately 1× ^10–14 M, approximately 4× ^10–8 M to approximately 1× ^10–14 M, approximately 3× ^10–8 M to approximately 1× ^10–14 M, approximately 2× ^10–8 M to approximately 1× ^10–14 M, approximately 1× ^10–8 M to approximately 1× ^10–14 M, approximately 9× ^10–9 M to approximately 1 ×10 ^–14 M, approximately 8 × 10 ^–9 M to approximately 1 × 10 ^–14 M, approximately 7 × 10 ^–9 M to approximately 1 × 10 ^–14 M, approximately 6 × 10 ^–9 M to approximately 1× ^10–14 M, approx. 5× ^10–9 M to approx. 1× ^10–14 M, approx. approximately 4× ^10–9 M to approximately 1× ^10–14 M, approximately 3× ^10–9 M to approximately 1× ^10–14 M, approximately 2× ^10–9 M to approximately 1× ^10–14 M , approximately 1× ^10–9 M to approximately 1× ^10–14 M, approximately 1× ^10–7 M to approximately 1× ^10–13 M, approximately 9× ^10–8 M to approximately 1× ^10–13 M, approximately 8× ^10–8 M to approximately 1× ^10–13 M, approximately 7× ^10–8 M to approximately 1× ^10–13 M, approximately 6× ^10–8 M to approximately 1×10 ^{– 13} M, approximately 5× ^10–8 M to approximately 1× ^10–13 M, approximately 4× ^10–8 M to approximately 1× ^10–13 M, approximately 3× ^10–8 M to approximately 1×10 ^–13 M, approximately 2× ^10–8 M to approximately 1× ^10–13 M, approximately 1× ^10–8 M to approximately 1× ^10–13 M, approximately 9× ^10–9 M to approximately 1× ^10–13 M, approximately 8× ^10–9 M to approximately 1× ^10–13 M, approximately 7× ^10–9 M to approximately 1× ^10–13 M, approximately 6 ×10 ^–9 M to approximately 1 × 10 ^–13 M, approximately 5 × 10 ^–9 M to approximately 1 × 10 ^–13 M, approximately 4 × 10 ^–9 M to approximately 1 × 10 ^–13 M, approximately 3× ^10–9 M to approximately 1× ^10–13 M, approximately 2× ^10–9 M to approximately 1× ^10–13 M, or approximately 1× ^10–9 M to approximately 1× ^10–13 M.

[0159] In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D equal to or less than: about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, approximately 700 pM, approximately 600 pM, approximately 500 pM, approximately 400 pM, approximately 300 pM, approximately 250 pM, approximately 200 pM, approximately 150 pM, approximately 100 pM, approximately 50 pM, approximately 40 pM, approximately 30 pM, approximately 25 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, or approximately 1 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 600 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 500 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 400 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 300 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 250 pM. In some embodiments, the recombinant ROBO2 protein binds to SLIT2 with a K _D of approximately 200 pM.

[0160] Generally speaking, a recombinant ROBO2-Fc protein must bind to a SLIT ligand (eg, SLIT2) with high affinity to effectively block ROBO2 activity. Desirably, the recombinant ROBO2-Fc protein has binding affinities (K _D ) in the low nanomolar and picomolar ranges, eg, about 1×10 ^-8 M or less.

[0161] In some embodiments, the recombinant ROBO2-Fc protein binds to SLIT2 with a K _D that is at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8 times, at least about 10 times, at least about 20 times, at least about 40 times, at least about 60 times, at least about 80 times, at least about 100 times times, at least about 120 times, at least about 140 times, at least about 160 times lower than the K _D value for binding ROBO1 to SLIT2. In some embodiments, the recombinant ROBO2-Fc protein binds to SLIT2 with a K _D that is at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold , at least about 10 times, at least about 20 times, at least about 40 times, at least about 60 times, at least about 80 times, at least about 100 times, at least about 120-fold, at least about 140-fold, at least about 160-fold lower than the K _D value for ROBO1-Fc protein binding to SLIT2.

Biological activity tests

[0162] In some embodiments, the recombinant ROBO2-Fc protein disclosed herein reduces at least one ROBO2-SLIT signaling biological activity. Such activities include, but are not limited to, binding of ROBO2 and the SLIT ligand; binding of intracellular signal-transmitting molecules (such as srGAP1 or Nck) to the intracellular domain of ROBO2; and/or subsequent ROBO2-SLIT signaling activity such as actin polymerization, podocyte adhesion and/or SLIT2-N-mediated inhibition of nerve cell migration, as well as other ROBO2-SLIT activities known to those skilled in the art. The reduction in ROBO2 activity by the recombinant ROBO2-Fc protein can be assessed using a number of assays well known to those skilled in the art. For example, assays known to those skilled in the art can be performed to determine if a recombinant ROBO2-Fc protein can: (a) inhibit SLIT binding to ROBO2; (b) reduce the level of srGAP1 binding to ROBO2; (c) reduce the level of Nck binding to ROBO2; (d) inhibit ROBO2-dependent SLIT2-N activity; (e) inhibit actin polymerization; (f) inhibit podocyte adhesion; and/or (g) inhibit SLIT2-N-mediated inhibition of nerve cell migration.

[0163] In some embodiments, the recombinant ROBO2-Fc protein inhibits the binding of the SLIT ligand to ROBO2 (for example, this can be assessed in an assay for competitive binding of the recombinant ROBO2-Fc protein and ROBO2 to SLIT2). For example, this assay can compare (i) ROBO2 and SLIT binding in the presence of recombinant ROBO2-Fc protein and (ii) ROBO2 and SLIT binding in the absence of recombinant ROBO2-Fc protein. The reduction in ROBO2 and SLIT binding may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in the presence of the recombinant ROBO2-Fc protein compared to binding of ROBO2 and SLIT in the absence of the tested recombinant ROBO2-Fc protein. The expected binding of SLIT to ROBO2 in the absence of the recombinant ROBO2-Fc protein can be taken as 100%.

[0164] In some embodiments, the recombinant ROBO2-Fc protein inhibits SLIT binding to ROBO2 with a half-maximal inhibitory concentration (IC ₅₀ ) of no more than about 1 x 10 ^-7 M, no more than about 1 x 10 ^{- 8} M, no more than about 1x10 ^-9 M, no more than about 1x10 ^-10 M, no more than about 1x10 ^-11 M, no more than about 1x10 ^-12 M , no more than about 1x10 ^-13 M, no more than about 1x10 ^-14 M, no more than about 1x10 ^-15 M, from about 1x10 ^-7 M to about 5x10 ^–14 M, approximately 1× ^10–7 M to approximately 1× ^10–14 M, approximately 1× ^10–7 M to approximately 5× ^10–13 M, approximately 1× ^10–7 M to approximately 1× 10 ^-13 M, about 1 x 10 ^-7 M to about 5 x 10 ^-12 M, or about 1 x 10 ^-7 M to about 1 x 10 ^-12 M. In some embodiments, the recombinant ROBO2-Fc protein has half max maximum inhibitory concentration (IC ₅₀ ) of not more than 15 nM, approximately 13 nM, approximately 11 nM, approximately 9 nM, approximately 7 nM, approximately 6 nM, approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, approximately 1 nM as measured by time resolved homogeneous fluorescence (HTRF) analysis for inhibition of ROBO2 binding to SLIT2. IC ₅₀ can be estimated using a fragment of SLIT or ROBO2, such as SLIT-N, and the Ig1 domain of ROBO2 or the Ig 1 and 2 domains of ROBO2.

[0165] The inhibitory activity of the recombinant ROBO2-Fc protein can also be assessed by measuring the level of ROBO2-dependent SLIT-N activity, such as actin polymerization, podocyte adhesion, and/or SLIT2-N-mediated inhibition of nerve cell migration. For example, this assay can compare (i) nerve cell migration in the presence of ROBO2, SLIT and recombinant ROBO2-Fc protein and (ii) nerve cell migration in the presence of ROBO2, SLIT but in the absence of recombinant ROBO2-Fc protein. Nerve cell migration may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97% by at least about 98%, or at least about 99%, in the presence of recombinant ROBO2-Fc protein compared to neural cell migration in the absence of recombinant ROBO2-Fc protein. The baseline migration of nerve cells in the absence of the recombinant ROBO2-Fc protein can be taken as 0%.

[0166] In some embodiments, the recombinant ROBO2-Fc protein inhibits ROBO2-dependent SLIT2-N activity, such as actin polymerization, podocyte adhesion, and/or SLIT2-N-mediated inhibition of nerve cell migration at a half-maximal inhibitory concentration (IC ₅₀ ) not more than about 1× ^10–7 M, not more than about 1× ^10–8 M, not more than about 1× ^10–9 M, not more than about 1× ^10–10 M, not more than than about 1× ^10–11 M, not more than about 1× ^10–12 M, not more than about 1× ^10–13 M, not more than about 1× ^10–14 M, not more than approximately 1× ^10–15 M, approximately 1× ^10–7 M to approximately 5× ^10–14 M, approximately 1× ^10–7 M to approximately 1× ^10–14 M, approximately 1× ^10–7 M to about 5× ^10–13 M, from about 1× ^10–7 M to about 1× ^10–13 M, from about 1× ^10–7 M to about 5× ^10–12 M, or from about 1× ^10–7 M up to approx. 1 x 10 ^-12 M. In some embodiments, the preferred IC ₅₀ is from about 1 x 10 ^-10 M to about 1 x 10 ^-13 M. In some embodiments, the preferred IC ₅₀ is from about 5 x 10 ^-11 M to about 5×10 ^-12 M. In some embodiments, the recombinant ROBO2-Fc protein has a half-maximal inhibitory concentration (IC ₅₀ ) of no more than about 75 nM, about 65 nM, about 55 nM, about 45 nM , approximately 35 nM, approximately 25 nM, approximately 15 nM, approximately 5 nM, as estimated by measuring the level of SLIT2-N-mediated inhibition of nerve cell migration.

[0167] In some embodiments, the properties of the recombinant ROBO2-Fc protein of the invention are also evaluated by other biological activity assays, such as assays for its activity, pharmacological activity, and potential efficacy as a therapeutic agent. Such assays are known to those skilled in the art and depend on the intended use of the recombinant protein. Examples are, for example, toxicity assays, immunogenicity assays, stability assays, anti-drug antibody assays and/or PK/PD profiling.

Nucleic acids and methods for producing recombinant ROBO2 proteins

[0168] The present invention also relates to polynucleotides encoding the recombinant ROBO2 proteins of the invention. The present invention also relates to a method for obtaining any of the polynucleotides described here. Polynucleotides can be obtained and expressed by methods known in the art.

[0169] In one of its aspects, the present invention relates to polynucleotides or compositions containing polynucleotides encoding a recombinant ROBO2 protein, including a part of the ROBO2 extracellular domain (ECD), as well as an immunoglobulin domain, where the extracellular domain contains at least two immunoglobulin-like (Ig-like) domain and a C-terminal sequence consisting of the sequence of SEQ ID NO: 12.

[0170] In one of its aspects, the present invention relates to polynucleotides or compositions containing polynucleotides encoding a recombinant ROBO2 protein comprising amino acid residues 1-203 in accordance with the numbering presented in SEQ ID NO: 1, as well as an immunoglobulin domain.

[0171] In some embodiments, the present invention relates to polynucleotides or compositions containing polynucleotides encoding any of the following recombinant ROBO2 proteins: ROBO2-Fc 2.2 (SEQ ID NO: 1), ROBO2-Fc 2.1 (SEQ ID NO: 2) , ROBO2-Fc 2.0 (SEQ ID NO: 3), ROBO2-Fc 1.1 (SEQ ID NO: 4), ROBO2-Fc 1.0 (SEQ ID NO: 5), ROBO2-Fc 3.0 (SEQ ID NO: 6), ROBO2 -Fc 4.0 (SEQ ID NO: 7) and ROBO2-Fc S17T R73Y (SEQ ID NO: 19). In some embodiments, the present invention relates to polynucleotides or compositions containing polynucleotides encoding ROBO2-Fc 2.2 (SEQ ID NO: 1). In some embodiments, the present invention relates to polynucleotides or compositions containing polynucleotides encoding ROBO2-Fc 2.1 (SEQ ID NO: 2). In some embodiments, the present invention relates to polynucleotides or compositions containing polynucleotides encoding ROBO2-Fc 2.0 (SEQ ID NO: 3).

[0172] The present invention also relates to polynucleotides or compositions containing these polynucleotides, where the polynucleotide contains the DNA sequence of the insert of a plasmid deposited with the ATCC and having the registration number ATCC No. PTA-124008.

[0173] In another aspect, the present invention provides polynucleotides and variants thereof encoding a recombinant ROBO2-Fc protein, wherein said polynucleotide variants have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of any nucleic acid disclosed in this application, such as, but not limited to, a nucleic acid containing the nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 19, and the nucleic acid sequence of a plasmid insert deposited with ATCC and having registration number ATCC no. PTA-124008. In some embodiments of the invention, such polynucleotide variants have a sequence that is at least 95% identical to the sequence of any nucleic acid disclosed herein, such as, but not limited to, a nucleic acid containing nucleic acid of SEQ ID NO: 1, 2, 3 , 4, 5, 6, 7, and 19. In some embodiments, such polynucleotide variants have a sequence that is at least 96% identical to the sequence of any nucleic acid disclosed herein, such as, but not limited to, a nucleic acid containing nucleic acid SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 19. In some embodiments, such polynucleotide variants have a sequence that is at least 97% identical to the sequence of any nucleic acid disclosed herein, such as, but not limited to, a nucleic acid containing the nucleic acid of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 19. In some embodiments, such polynucleotide variants have a sequence that is at least 98% identical to the sequence of any nucleic acid disclosed herein, such as, but not limited to, a nucleic acid comprising the nucleic acid of SEQ ID NO : 1, 2, 3, 4, 5, 6, 7, and 19. In some embodiments, such polynucleotide variants have a sequence that is at least 99% identical to the sequence of any nucleic acid disclosed herein, such as, but not limited to them, a nucleic acid containing the nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 19.

[0174] In another aspect, the present invention includes polynucleotides and variants thereof comprising the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, the present invention includes polynucleotides and variants thereof comprising the nucleic acid sequence set forth in SEQ ID NO: 21, as well as a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18 is N-terminal to the nucleic acid sequence shown in SEQ ID NO: 21.

[0175] In one embodiment of the invention, the extracellular domain of ROBO2 and the Fc domain of an immunoglobulin are encoded by separate polynucleotides. Alternatively, the extracellular domain of ROBO2 and the Fc domain of an immunoglobulin are encoded by the same polynucleotide.

[0176] Polynucleotides complementary to any such sequences are within the scope of the present invention. Polynucleotides can be single stranded (coding or antisense) or double stranded, and can be DNA molecules (recombinant DNA, cDNA or synthetic DNA) or RNA molecules. RNA molecules include hnRNA, which contains introns and corresponds one to one to the DNA molecule, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may optionally be present in the polynucleotide of the invention, and the polynucleotide may optionally be attached to other molecules and/or carrier materials.

[0177] Polynucleotides may contain a native sequence (ie, a wild-type sequence) or may contain a non-native sequence (ie, a variant). Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions that do not reduce the ability of SLIT to bind to the encoded polypeptide, as in the native molecule. The effect on the SLIT-binding activity of the encoded polypeptide can be essentially assessed as described in this application. In some embodiments, the variants are at least about 70%, in some embodiments, at least about 80%, in some embodiments, at least about 90%, and in some embodiments, at least about 95% identical to the polynucleotide sequence that encodes a recombinant ROBO2-Fc protein containing native (wild-type) ROBO2 and Fc domain sequences.

[0178] In some embodiments, the variants encode a recombinant ROBO2 protein containing amino acid residues having at least 20, at least 19, at least 18, at least 17, at least 16, at least 15, at least 14, at least 13, at least 12, at least 11, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2, or at least 1 amino acid substitution at amino acid residues 1-203 according to the numbering provided in SEQ ID NO: 1. In some embodiments, the variants encode a recombinant protein ROBO2 containing amino acid residues having at least 5 amino acid substitutions at amino acid residues 1-203 according to the numbering provided in SEQ ID NO: 1. In some embodiments of the invention, the variants encode a recombinant th ROBO2 protein containing amino acid residues having at least 4 amino acid substitutions at amino acid residues 1-203 according to the numbering provided in SEQ ID NO: 1. In some embodiments of the invention, the variants encode a recombinant ROBO2 protein containing amino acid residues, having at least 3 amino acid substitutions at amino acid residues 1-203 according to the numbering shown in SEQ ID NO: 1. In some embodiments, the variants encode a recombinant ROBO2 protein containing amino acid residues having at least 2 amino acid substitutions in amino acid residues 1-203 according to the numbering given in SEQ ID NO:1. the numbering shown in SEQ ID NO: 1. These amounts are not limiting, and increments may be observed between the indicated percentages, which are included in the scope of the present invention as part of its disclosure.

[0179] Two polynucleotide or polypeptide sequences are considered "identical" if the nucleotide or amino acid sequences in the two sequences are the same when aligned for maximum correspondence, as described below. Comparison of two sequences is usually carried out by comparing sequences across the comparison window to identify and compare the similarity of local regions of the sequences. The term "comparison window" as used herein means a segment of at least about 20 contiguous positions, and typically 50 to about 450 or 100 to about 300, in which the sequence can be compared to a reference sequence in the same row of contiguous positions after the optimal alignment of two sequences.

[0180] Optimal alignment of sequences for comparison can be performed using the MegAlign ^® program in the Lasergene ^® bioinformatics software package (DNASTAR ^® , Inc., Madison, Wl), using the default parameters. This program allows alignment according to several schemes described in the following works: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:1 1-17; Robinson, ED, 1971, Comb. Theor. 11 : 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. sci. USA 80:726-730.

[0181] In some embodiments of the invention, "percent sequence identity" is determined by comparing two optimally aligned sequences in a comparison window at least 20 positions, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps ) at 20 percent or less, and typically 5 to 15 percent or 10 to 12 percent, compared to reference sequences (which contain no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues are present in both sequences, obtaining the number of corresponding positions, then dividing the corresponding positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 s obtaining percent sequence identity.

[0182] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under conditions of moderate stringency to a natural DNA sequence encoding a recombinant ROBO2-Fc protein containing native (wild-type) ROBO2 sequences and an Fc domain (or complementary sequence).

[0183] Suitable "moderate stringency conditions" include a pre-wash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50-65°C, 5x SSC overnight; followed by double washing at 65°C for 20 minutes with each of 2×, 0.5× and 0.2× SSC containing 0.1% SDS.

[0184] As used herein, "highly stringent conditions" or "high stringency conditions" means conditions under which: (1) low ionic strength and high wash temperature, such as 0.015M sodium chloride/0.0015M sodium citrate /0.1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent during hybridization such as formamide, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM phosphate- sodium buffer at pH 6.5 together with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) 50% formamide, 5× SSC (0.75 M NaCl, 0.75 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, treated sonicated salmon sperm DNA (50 µg/mL), 0.1% SDS and 10% dextran sulfate at 42°C followed by washing at 42°C in 0.2× SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C and a high stringency wash consisting of a 0.1x SSC wash containing EDTA at 55°C. A person skilled in the art can independently adjust the temperature, ionic strength, and the like. depending on factors such as probe length, etc.

[0185] For a person skilled in the art it is obvious that as a result of the degeneracy of the genetic code, many nucleotide sequences will encode a ROBO2-Fc polypeptide containing the amino acid sequence described here. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon occurrence are specifically contemplated in the present invention. In addition, alleles of genes containing the polynucleotide sequences described herein are within the scope of the present invention. Alleles are endogenous genes that have been modified by one or more mutations such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may optionally have an altered structure or function. Alleles can be identified by standard methods (such as hybridization, amplification, and/or database sequence comparison).

[0186] The present invention also includes codon-optimized polynucleotides, where the nucleic acid sequence has been optimized to maximize its expression in a particular cell. Generally speaking, codon optimization means a method of modifying a nucleic acid sequence to increase expression levels in host cells of interest by changing at least one codon (e.g., about or about more than 1, 2, 3, 4, 5, 10, 15 , 20, 25, 50 or more codons) of the native sequence by codons that occur more frequently or very frequently in the genes of that host cell and retain the native amino acid sequence. In different species, a specific shift of some codons of a particular amino acid is observed. Codon bias (differences in the occurrence of codons in different organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which, in turn, probably depends on the properties, translated codons and the availability of specific transfer RNA (tRNA) molecules, as well as other factors. The predominance of selected tRNAs in the cell is probably due to the most common codons used in peptide synthesis. Accordingly, genes can be specifically designed for their optimal expression in a given organism based on codon optimization. Codon usage tables are readily available and these tables can be adapted in a number of ways (e.g., Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28 :292 (2000)). Computer algorithms for codon optimization of a particular sequence for its expression in a particular host cell are also available, for example, the Gene Forge program (Aptagen; Jacobus, Pa.) also exists. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in the sequence encoding the recombinant ROBO2-Fc protein correspond to the most common codons for a particular amino acid.

[0187] Thus, in one aspect of the invention, the modified nucleic acid sequence provides a detectably higher level of expression of the recombinant ROBO2 protein in the cell compared to the expression of the recombinant ROBO2 protein from a wild-type nucleic acid sequence, for example, a nucleic acid encoding ROBO2-Fc 2.2 (SEQ ID NO: 21) in another identical cage. This nucleic acid may be referred to herein as "optimized expression" or "overexpressed" or simply "modified nucleic acid".

[0188] The terms "optimized" or "codon-optimized" are synonymous and refer to a coding sequence that has been optimized compared to a wild-type coding sequence (e.g., a sequence encoding a human ROBO2 and/or a human Fc domain) to increase the level of expression of the coding sequence, for example, by minimizing the presence of rare codons, reducing the number of CpG dinucleotides, removing hidden splicing donor or acceptor sites, removing Kozak sequences, removing ribosome binding sites, and the like.

[0189] Examples of modifications are the elimination of one or more cis-acting motifs and the introduction of one or more Kozak sequences. In one embodiment of the invention, one or more cis-acting motifs are removed and one or more Kozak sequences are introduced.

[0190] Examples of cis-acting motifs that can be removed are internal TATA boxes; chi sites; ribosome binding sites; sequence elements ARE, INS, and/or CRS; repeating sequences and/or secondary structures of RNA; (hidden) donor and/or acceptor splicing sites; branch points; and Sall.

[0191] In one embodiment, the GC content (e.g., the number of G and C nucleotides present in a nucleic acid sequence) has been increased compared to the sequence of the wild-type ROBO2 gene and/or human IgG1 Fc domain in the novel ROBO2 proteins of the invention . The GC content is preferably at least 5%, more preferably at least 6%, even more preferably at least 7%, even more preferably at least 8%, even more preferably at least at least 9%, even more preferably at least 10%, even more preferably at least 12%, even more preferably at least 14%, even more preferably at least 15%, still more preferably at least 17%, even more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, and most preferably at least 70% higher than the GC content of the wild-type gene (eg, SEQ ID NO: 21).

[0192] In another embodiment, the GC content is expressed as the percentage of G (guanine) and C (cytosine) nucleotides in the sequence. That is, the GC content of the wild-type nucleic acid encoding ROBO2-Fc 2.2 (SEQ ID NO: 21) is lower than the GC content of the codon-optimized nucleic acid sequence encoding ROBO2-Fc 2.2.

[0193] In one embodiment of the invention, the content of GC in the modified nucleic acid according to the invention exceeds the content of GC in the wild-type nucleic acid encoding ROBO2-Fc 2.2 and containing the nucleic acid sequence of SEQ ID NO: 21. Those skilled in the art will know that due to degeneracy of the nucleic acid code, regardless of the nucleic acid sequence encoding the protein, the amino acid sequence of ROBO2-Fc 2.2 is expressed, preferably, as the amino acid sequence of SEQ ID NO: 1.

[0194] CpG dinucleotide methylation is known to play an important role in the regulation of gene expression in eukaryotes. In particular, methylation of CpG dinucleotides in eukaryotes mainly serves to silence gene expression by influencing the transcriptional mechanism. Thus, since gene silencing is caused by methylation of CpG motifs, nucleic acids and vectors according to the invention having a reduced number of CpG dinucleotides will provide a high and long-term level of transgene expression.

[0195] Potential local CpG regions can be identified using a publicly available computer program available, for example, at http://www.bioinformatics.org/sms2/cpg_islands.html. The local CpG region program allows potential local CpG regions to be identified by the method described by Gardiner-Garden and Frommer, 1987, J. Mol. Biol. 196(2):261-282, as well as many other methods well known to those skilled in the art in identifying potential local CpG regions. The calculation can be performed using a 200 base pair (bp) window moving through the entire sequence at 1 bp intervals. Local CpG regions are defined as intervals of sequences where the Obs/Exp value is greater than 0.6 and the GC content is greater than 50%. The expected number of CpG dimers in the window can be calculated as the number of "C" in the window, multiplied by the number "G" in the window and divided by the length of the window. Thus, potential local CpG regions present in a nucleic acid sequence can be easily identified by entering the desired sequence into a given window created by a computer program (followed by instructions for "embedding the original sequence or one or more FASTA sequences into a text area, below. The input limit is 100,000 characters"). Local CpG regions are often found in the 5' regions of vertebrate genes, and therefore such a program can be used to identify potential genes in genomic sequences.

[0196] Polynucleotides according to the invention can be obtained by chemical synthesis, recombinant methods or using PCR. Methods for the chemical synthesis of polynucleotides are well known to those skilled in the art and are therefore not described in detail in this application. One skilled in the art can use the sequences described herein and a commercially available DNA synthesizer to obtain the desired DNA sequence.

[0197] To obtain polynucleotides by recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and this vector can, in turn, be introduced into a suitable host cell for replication and amplification, as discussed in detail in this application. The polynucleotides can be inserted into host cells by any methods known to those skilled in the art. Cells are transformed by introducing an endogenous polynucleotide via direct uptake, endocytosis, transfection, F-crossing, or electroporation. After introduction, the exogenous polynucleotide may be maintained in the cell as a non-integrated vector (such as a plasmid) or as a vector integrated into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known to those skilled in the art. See, for example, Sambrook et al., 1989.

[0198] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known to those skilled in the art and is described in US Pat. Nos. 4,683,195; 4,800,159;

[0199] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. If the cell replicates and the DNA is transcribed into RNA, then the RNA can then be isolated by methods well known in the art, see e.g. Sambrook et al., 1989.

[0200] Suitable cloning vectors can be constructed by standard methods, or they can be selected from a large number of cloning vectors available to those skilled in the art. Although the selected cloning vector may vary depending on the host cell used, however, suitable cloning vectors are those that are substantially capable of self-replication, may have a single target for a particular restriction endonuclease, and/or may have marker genes that can be used to select clones containing the vector. Suitable examples are plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0201] The present invention also relates to expression vectors. Expression vectors are typically replicable polynucleotide constructs containing a polynucleotide of the invention. This provides that the expression vector should be replicated in host cells either as episomes or as an integrated part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vector(s) disclosed in PCT Publication WO 87/04462. The components of the vectors may be, in essence, but are not limited to, one or more of the following components: a signal sequence; origin of replication; one or more marker genes; suitable transcriptional regulatory elements (such as promoters, enhancers and terminators). Expression (ie, translation) also typically requires one or more translational regulatory elements, such as ribosome binding sites, translation initiation sites, and stop codons.

[0202] In some embodiments, the vector comprises a nucleic acid molecule as set forth in SEQ ID NO: 21. In some embodiments, the vector comprises a nucleic acid molecule as set forth in SEQ ID NO: 21 and a nucleic acid sequence encoding an amino acid the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18 is N-terminal relative to the nucleic acid sequence shown in SEQ ID NO: 21.

[0203] The vectors containing the polynucleotides of interest and/or the polynucleotides themselves can be introduced into the host cell by any of various suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection and infection (eg, where the vector is an infectious agent such as vaccinia virus). The choice of method for introducing vectors or polynucleotides often depends on the properties of the host cell.

[0204] Examples of host cells are E. coli cells, yeast cells, insect cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. Preferred host cells are CHO cells, human embryonic kidney (HEK) 293 cells or Sp2.0 cells, as well as a variety of other cells well known to those skilled in the art.

[0205] Host cells can be cultured under conditions conducive to expression of the encoded recombinant ROBO2 protein. In some embodiments, the encoded recombinant ROBO2-Fc protein comprises the ROBO2 leader sequence set forth in SEQ ID NO: 17 or the Ig leader sequence set forth in SEQ ID NO: 18. In some embodiments, the ROBO2 leader sequence (SEQ ID NO: 17) is cleaved during protein production to form mature ROBO2-Fc. In some embodiments, the Ig leader sequence (SEQ ID NO: 18) is cleaved during protein production to form mature ROBO2-Fc, eg ROBO2-Fc 2.2.

4. Compositions and their use

[0206] The recombinant ROBO2 proteins of the invention may be formulated into a pharmaceutical composition. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier, excipient and/or stabilizer (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover) in the form of a lyophilized preparation or an aqueous solution. The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein include any substance which, in combination with an active ingredient, allows that ingredient to retain biological activity and inability to react with the immune system of an individual. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil-in-water emulsion, and wetting agents of various types. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are prepared by well-known standard methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

[0207] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used, and may contain buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described in this application.

Application in diagnostics

[0208] The recombinant ROBO2 proteins of the invention can be used in a variety of therapeutic or diagnostic applications. For example, the recombinant ROBO2 proteins of the invention can be used as an affinity purification agent (e.g., in vitro purification of SLIT ligands such as SLIT2) and as a diagnostic agent (e.g., detection of SLIT ligand expression (e.g., SLIT2 in specific cells, tissues or serum)). Representative diagnostic assays for a SLIT ligand, such as SLIT2, may include, for example, contacting a patient sample with a recombinant ROBO2 protein of the invention, where the recombinant ROBO2 protein is labeled with a detectable label or reporter molecule.

[0209] The present invention encompasses the use of the recombinant ROBO2 proteins disclosed herein in diagnostic imaging techniques for a SLIT ligand, such as SLIT2, in a sample, in a cell, in a tissue, or in a patient. For example, a recombinant ROBO2 protein can be conjugated to an imaging agent such that the presence of the recombinant ROBO2 protein can be detected and then the presence of a SLIT ligand, such as SLIT2, can be detected.

Therapeutic use

[0210] A representative therapeutic use of the recombinant ROBO2 proteins of the invention is in the treatment of kidney disease such as glomerulitis, focal segmental glomerulitis (FSH). The recombinant ROBO2 proteins of the invention can also be used for prophylaxis (eg, administration to an individual who is asymptomatic but susceptible to developing kidney disease such as glomerulitis, FSH).

[0211] In another aspect, the present invention includes the treatment of any disorder, disease, or condition mediated by, or associated with, elevated levels of urinary protein relative to urinary protein in the absence of such disease, disorder, or condition. Such diseases, disorders, or conditions include, but are not limited to, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), minimal replacement disease (MBD), fibrosis (eg, liver fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria , glomerulonephritis, diabetic nephropathy, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, kidney transplant rejection, and reflux nephropathy.

[0212] For therapeutic use, the recombinant ROBO2 proteins of the invention may be administered to a mammal, and in particular to a human, by standard methods, e.g., intravenously (as a bolus or continuous infusion over a period of time), intramuscularly, intraperitoneally, into liquid, subcutaneously, intraarticularly, into the synovial fluid, intrathecally, orally, topically or by inhalation. The recombinant ROBO2 proteins according to the invention can also be appropriately introduced into the interior of the tumor, in the peritumor sites, in or around the affected area.

[0213] Accordingly, in one of its aspects, the present invention provides a method for reducing ROBO2 activity, comprising administering to an individual (e.g., human) in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of the invention.

[0214] In another aspect, the present invention provides a method for maintaining or modulating podocyte function, comprising administering to an individual (eg, human) in need thereof a therapeutically effective amount of a recombinant ROBO2 protein of the invention.

[0215] In some embodiments of the invention, the individual suffers from kidney disease or is susceptible to kidney disease. In some embodiments, the kidney disease is glomerulitis. In some embodiments, the kidney disease is FSGS.

[0216] In some embodiments of the invention, the individual suffers from nephropathy or is susceptible to nephropathy.

[0217] In another aspect, the present invention provides a recombinant ROBO2 protein of the invention for use in the treatment method disclosed herein. For example, the present invention relates to a recombinant ROBO2 protein of the invention for its use in reducing ROBO2 activity in a cell, reducing ROBO2 activity in an individual, maintaining podocyte function in an individual, modulating podocyte function in an individual, treating glomerulitis in an individual, and treating nephropathy in an individual. individual.

[0218] In another aspect, the present invention relates to the use of the recombinant ROBO2 protein of the invention for the preparation of a medicament for reducing ROBO2 activity in a cell, reducing ROBO2 activity in an individual, maintaining podocyte function in an individual, modulating podocyte function in an individual, treating glomerulitis in an individual; and treating nephropathy in an individual.

Doses and administration

[0219] In some embodiments, the recombinant ROBO2 protein of the invention is administered subcutaneously. In some embodiments of the invention, the recombinant ROBO2 protein of the invention is administered intravenously.

[0220] Pharmaceutical compositions may be administered to an individual in need thereof at a frequency that may vary depending on the severity of the kidney disease. In the case of prophylactic therapy, such frequency may vary depending on the susceptibility or predisposition of the individual to kidney disease. In some embodiments of the invention, the pharmaceutical composition is administered in a single dose, subcutaneously or intravenously. In some embodiments of the invention, the pharmaceutical composition is administered in subcutaneous or intravenous divided doses.

[0221] The compositions may be administered to patients in need thereof as a bolus dose or as a continuous infusion. So, for example, the introduction of the recombinant ROBO2 protein in a loading dose can be carried out in an amount of from 0.0025 to 200 mg/kg of body weight, from 0.025 to 0.25 mg/kg, from 0.010 to 0.10 mg/kg, or 0, 10-0.50 mg/kg. For continuous infusion, recombinant ROBO2 protein can be administered at a dose of 0.001 to 200 mg/kg bw/minute, 0.0125 to 1.25 mg/kg/minute, 0.010 to 0.75 mg/kg/minute, 0.010 to 1.0 mg/kg/minute or 0.10 to 0.50 mg/kg/minute for 1-24 hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours or 1-2 hours.

[0222] For administration of recombinant ROBO2 proteins, doses may be administered in amounts from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 3 mg/kg to about 10 mg/kg, about 4 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 20 mg/kg, about 2 mg/kg to about 20 mg/kg, about 3 mg/kg to about 20 mg/kg, about 4 mg/kg to about 20 mg/kg, about 5 mg/kg to about 20 mg/kg, about 1 mg/kg or more, about 2 mg/kg or more, about 3 mg/kg or more, about 4 mg/kg or more, about 5 mg/kg or more, about 6 mg/kg or more, about 7 mg/kg or more , approximately 8 mg/kg or more, approximately 9 mg/kg or more, approximately 10 mg/kg or more, approximately 11 mg/kg or more , about 12 mg/kg or more, about 13 mg/kg or more, about 14 mg/kg or more, about 15 mg/kg or more, about 16 mg/kg or more, about 17 mg/kg or more, approximately 19 mg/kg or more, or approximately 20 mg/kg or more. The frequency of administration depends on the severity of the condition. The introduction can be carried out with a frequency of 3 times a week to 1 time in two or three weeks.

[0223] In addition, the compositions can be administered to patients by subcutaneous injection. For example, a dose of 1 to 200 mg of the recombinant ROBO2 protein can be administered to patients by subcutaneous or intravenous injection twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months or once every three months.

[0224] In some embodiments, the half-life of the recombinant ROBO2 protein in humans is about 24 hours, about 2 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days , approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, 28 days, approximately 29 days, approximately 30 days, approximately 5 days to approximately 40 days, approximately 5 days to approximately 35 days, about 5 days to about 30 days, about 5 days to about 25 days, about 10 days to about 40 days, about 10 days to about 35 days, about 10 days to about 30 days, about 10 days to about 25 days, about 15 days to about 40 days, about 15 days to about 35 days, about 15 days to about 30 days, or about 15 days to about 25 days.

[0225] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2-6 weeks at a dose of about 0.1 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 10 mg /kg, from about 1 mg/kg to about 10 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 0.1 mg /kg to about 8 mg/kg, about 0.5 mg/kg to about 8 mg/kg, about 1 mg/kg to about 8 mg/kg, about 1.5 mg/kg to about 8 mg/ kg, approximately 2 mg/kg to approximately 8 mg/kg, approximately 0.1 mg/kg to approximately 5 mg/kg, approximately 0.5 mg/kg to approximately 5 mg/kg, approximately 1 mg/kg kg to about 5 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 2 mg/kg to about 5 mg/kg, about 0.5 mg/kg, approx. 1.0 mg/kg approximately 1.5 mg/kg approximately 2.0 mg/kg approximately 2.5 mg/kg approximately 3.0 mg/kg approximately 3.5 mg/kg approximately 4 .0 mg/kg, approximately 4.5 mg/kg, approximately 5.0 mg/kg, approximately 5.5 mg/kg, approximately 6.0 mg/kg, approximately 6.5 mg/kg, approximately 7.0 mg/kg, 7.5 mg/kg, approximately 8.0 mg/kg, approximately 8.5 mg/kg, approximately 9.0 mg/kg, approximately 9.5 mg/kg, or approximately 10.0 mg/kg kg.

[0226] In some embodiments of the invention, the pharmaceutical composition is administered subcutaneously or intravenously every 2-6 weeks at a dose of approximately 3.0 mg/kg. In some embodiments of the invention, the pharmaceutical composition is administered subcutaneously or intravenously every 2-6 weeks at a dose of from about 2.0 mg/kg to about 10.0 mg/kg.

[0227] In some embodiments, the pharmaceutical composition is administered subcutaneously or intravenously weekly or every 2 weeks at a dose of approximately 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg , 250 mg, 275 mg or 300 mg.

[0228] In one representative embodiment of the invention, the pharmaceutical composition is administered subcutaneously weekly or every 2 weeks. In some embodiments of the invention, the pharmaceutical composition is administered subcutaneously once a week at a dose of approximately 2 mg/kg. In some embodiments of the invention, the pharmaceutical composition is administered subcutaneously once a week at a dose of approximately 150 mg.

[0229] In some embodiments of the invention, the pharmaceutical composition is administered intravenously or subcutaneously every 2-6 weeks at a dose of approximately 10.0 mg/kg. In some embodiments of the invention, the pharmaceutical composition is administered intravenously or subcutaneously every 2-6 weeks at a dose of from about 1.0 mg/kg to about 10.0 mg/kg.

[0230] In one representative embodiment of the invention, the pharmaceutical composition is administered intravenously every month.

[0231] The recombinant ROBO2 protein of the invention can be used as monotherapy or in combination with other therapies to treat, for example, kidney disease. Other therapies for the treatment of kidney disease are well known to those skilled in the art and are not listed here.

5. Sets

[0232] The present invention also relates to kits or to an industrial product containing the recombinant ROBO2 protein according to the invention and instructions for their use. Accordingly, in some embodiments, the present invention provides a kit or article of manufacture comprising a container, a composition present in the container containing the recombinant ROBO2 protein, and a package insert containing instructions for administering a therapeutically effective amount of the recombinant ROBO2 protein to treat a patient in need thereof.

[0233] In some embodiments, the kit may comprise a first container having a desiccated protein and a second container having an aqueous formulation. In some embodiments, kits containing single-chamber and multi-chamber pre-filled syringes (eg, syringes for liquids and syringes for lyophilized preparation) are included.

[0234] Instructions for use of the recombinant ROBO2 proteins of the invention typically include information on dosages, dosing schedules, and how to administer the appropriate treatment. The containers may be unit doses, bulk packs (eg, packs for fractional doses), or subunit doses. Instructions supplied with kits according to the invention are usually text instructions on a label or package insert (for example, in the form of a paper information sheet included in the kit), but electronic instructions (for example, magnetic or optical instructions) may also be used. storage disk).

[0235] Kits according to the invention are in appropriate packaging. Suitable packages include, but are not limited to, vials, bottles, jugs, flexible packaging (eg, Mylar sealed bags or plastic bags), and the like. Also contemplated are packages for use in combination with a particular device such as an inhaler, a nasal device (eg an aerosol dispenser) or an infusion device such as a minipump. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a cap pierced by a hypodermic needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a cap pierced by a hypodermic needle). The container may also contain a second pharmaceutically active agent.

[0236] Kits may optionally contain additional components such as buffers and explanatory information. Typically, the kit contains a container and a label affixed to the container, or insert(s) nested(s) in the container.

6. Biological deposit

[0237] Representative materials of the invention have been deposited with the American Type Culture Collection, 10801, University Boulevard, Manassas, Va. 201 10-2209, USA, February 23, 2017. Vector ROBO2-Fc 2.2 under registration ATCC no. PTA-124008 contains a DNA insert encoding SEQ ID NO: 1. The deposits were created in accordance with the Budapest Treaty on the International Deposit of Microorganisms in the Patent Procedure and Related Regulations (Budapest Treaty). This guarantees the preservation of a viable depository culture for 30 years from the date of creation of the deposit. The deposit may be made available under the ATCC number under the Budapest Agreement and the Agreement between Pfizer Inc. and ATCC, which guarantee the permanent and unrestricted release of depository culture progeny to a specific individual upon request by a U.S. patent holder or upon public access to the holder of any U.S. or foreign patent application, regardless of the order in which the request is made, and guarantee the availability of progeny to a person as determined by the Patent and Trademark Commission. U.S. marks pursuant to section 35 of title 122 of the United States Code and in accordance with rules approved by the Commission (including section 37 of title 1.14 of the United States Code with specific reference to 886 OG 638).

[0238] Pfizer Inc., that is, the applicant of the present invention, has agreed that if the culture of materials on deposit dies or is lost or destroyed when cultivated under certain conditions, then such materials will be immediately replaced with other similar materials, which will be appropriate notice has been given. The availability of deposited material should not be construed as a license to practice the invention, otherwise it would be an infringement of the rights of any governmental body that made a grant under patent law.

Examples

[0239] This application describes representative methods and materials, although methods and materials similar or equivalent to the methods and materials described herein may also be used to practice the present invention or to conduct tests according to the invention. Such materials, methods, and examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Preparation of ROBO2-Fc 2.2

Selection of ROBO2-Fc 2.2 by constructing multiple ROBO2-Fc fusion proteins

[0240] In order to select the optimal ROBO2-Fc decoy ligand, a variety of proteins were generated, consisting of a ROBO2 extracellular domain of various lengths, attached to the Fc domain of IgG1 via a glycine-serine (GS) linker. Studies have shown that for the ROBO1 and ROBO2 receptors, their Ig1 domain is sufficient to bind to the leucine-rich D2 repeat domain (LRR) of SLIT ligands. ROBO2 contains 5 Ig domains (Ig 1-5). Selection criteria were first applied to generate a ROBO2-Fc fusion protein with a minimal ROBO2 sequence that can bind to SLIT2, and then to optimize this molecule for expression of the recombinant protein in HEK 293 cells. First, 4 DNA constructs were generated for the expression of polypeptide sequences consisting from Ig1 domain (ROBO2-Fc 1.0; SEQ ID NO: 5), Ig1 and Ig2 domains (ROBO2-Fc 2.0; SEQ ID NO: 3), Ig1, Ig2 and Ig3 domains (ROBO2-Fc 3.0; SEQ ID NO: 6 ) and Ig1, Ig2, Ig3 and Ig4 domains (ROBO2-Fc 4.0; SEQ ID NO: 7) linked to the Fc portion of human IgG1 via a GS linker (Table 23). Expression of the constructs was induced by the Ig leader sequence (SEQ ID NO: 18). The ROBO2-Fc 1.0, 2.0, and 3.0 proteins did not bind to SLIT2. ROBO2-Fc 4.0 contacted SLIT2. To minimize the Fc fusion portion of ROBO2, more proteins were generated, including a variant with an Ig1 domain containing an Ig1-Ig2 interdomain linker at the C-terminus (SEQ ID NO: 10) (ROBO2-Fc 1.1 (SEQ ID NO: 4). Other the resulting construct included the Ig1 and Ig2 domains and the Ig2-Ig3 interdomain linker (VFER (SEQ ID NO: 12)) (ROBO2-Fc 2.1; SEQ ID NO: 2). Ig1-Ig2 to produce the ROBO2-Fc 2.1 protein provided reliable binding to SLIT2 (Figure 3) ROBO2-Fc 1.1 did not bind to SLIT2 Using Octet Red, ROBO2-Fc proteins were loaded onto sensors coated with anti-human Fc (AHC) at 10 μg/ml and incubated with 100 nM SLIT2 for 7 minutes and then the sensors were moved into buffer for 640 seconds ROBO2-Fc 4.0 was included as a positive control for binding.

[0241] Expression of the recombinant ROBO2-Fc 2.1 protein in HEK 293 cells was very low (3 μg/ml). The ROBO2 Ig1 pre-sequence (SEQ ID NO: 8) was added to increase the expression level, and the ROBO2 leader sequence (SEQ ID NO: 17) was also included in the ROBO2-Fc 2.2 expression construct to give the ROBO2-Fc 2.2 expression construct. The ROBO2 leader sequence (SEQ ID NO: 17) was cleaved during protein production to form mature ROBO2-Fc 2.2. Attachment of the Ig1 ROBO2 pre-sequence (SEQ ID NO: 8) resulted in a more than 25-fold increase in the expression level of the protein compared to another identical construct that does not encode the Ig1 SRLRQEDFP pre-sequence (SEQ ID NO: 8) in another identical host cell. The increase in expression level did not affect the biological activity of ROBO2-Fc 2.2, which still bound to SLIT2 with high affinity (Fig. 3-4A-C).

Example 2 Characterization of binding and neutralization activity of ROBO2-Fc 2.2

[0242] ROBO2-Fc 2.2 has been screened in a variety of assays for binding to SLIT2-N, to neutralize binding to SLIT2-N, and to inhibit SLITx-N functional activity. Binding of ROBO2-Fc 2.2 to SLIT2 was assessed in two ways, first by surface plasmon resonance (SPR) and then by cellular flow cytometry analysis to detect binding of ROBO2-Fc 2.2 to human SLIT2-N expressed by the cell.

Analysis with PPR

[0243] Human SLIT2 has a high degree of homology with cynomolgus SLIT2, where the overall homology is 99%, and the homology in the leucine-rich repeat domain 2 (D2) of SLIT2, which contains the ROBO2 binding site, is 100%. Similar to cynomolgus SLIT2, human and rat SLIT2 have a high degree of homology with a common sequence identity and in the D2 binding domain, such homology is 97%. Binding of ROBO2-Fc 2.2 to the N-terminal fragment of human and rat SLIT2 containing the D2 region (SLIT2-N) and to the specific D2 domain of human and cynomolgus SLIT2 (100% identity) was assessed by SPR. For each cycle of the kinetic titration experiment (FIGS. 4A-4C), the ROBO2-Fc 2.2 molecule was not covalently linked to the anti-human Fc (AHFc) attached to the C1 chip, which bound to the Fc region of the human IgG1 ROBO2-Fc 2.2. The captured ROBO2-Fc 2.2 was then treated with various concentrations of SLIT2 ligands to monitor association and dissociation kinetics. ROBO2-Fc 2.2 was diluted to 2 nM. HBS-EP was used as a diluent. Human/cynomolgus SLIT2-D2, human SLIT2-N and rat SLIT2-N were diluted to 2 nM in HBS-EPB based on their starting concentrations. An 8-point 2-fold serial dilution was performed on SLIT2 ligands at concentrations ranging from 2 nM to 15.6 pM using HBS-EPB as diluent. ROBO2-Fc 2.2 was captured until reads of 10-15 FU equivalents were made and then treated with titratable amounts of a specific SLIT2 analyte (i.e., human/canine simian SLIT2-D2, human SLIT2-N, or rat SLIT2 -N). The association of said SLIT2 ligand was monitored to be 120 seconds and the dissociation was 180 seconds. Apparent binding affinity was determined using a simple 1:1 interaction model for kinetic binding rate constants. The binding K _D of ROBO2-Fc 2.2 to human/canine simian SLIT2-D2 (ROBO2-binding domain 100% identical) was determined to be 0.293 nM (FIG. 4A). The K _D of ROBO2-Fc 2.2 binding to human SLIT2-N (N-terminal fragment) was determined to be 0.279 nM (FIG. 4B) and finally the K _D of ROBO2-Fc 2.2 binding to rat SLIT2-N was as was determined to be 0.543 nM (Fig. 4C).

Analysis by flow cytometry

[0244] ROBO2-Fc 2.2 was conjugated to Alexa Fluor (AF) 647 according to the manufacturer's instructions. Human embryonic kidney cells (HEK293) overexpressing human SLIT2-N were resuspended in buffer containing 5% fetal bovine serum in a stain preparation. To stain cells with ROBO2-Fc 2.2-AF647, 2x stock solutions were prepared and 11-12-point 2-fold serial dilutions were made in Fluorescence Activated Cell Sorting (FACS) buffer. Cells and appropriate dilutions of ROBO2-Fc 2.2-AF647 were pooled in a 96-well U-bottomed plate and incubated at 4° C. for 45 minutes. After incubation, 150 μl of FACS buffer was added per cell wash well. After washing, cells were resuspended in buffer to obtain information on a Fortessa flow cytometer. Data were analyzed using FlowJo software and presented as the geometric mean of fluorescence intensity (Geo MFI) of SLIT2-N-expressing HEK293 cells minus Geo MFI of control HEK293 cells. ROBO2-Fc 2.2 bound human SLIT2-N overexpressed on HEK293 cells in a dose dependent manner with high affinity for EC ₅₀ 9 nM (FIG. 5).

Time Resolved Homogeneous Fluorescence (HTRF)

[0245] ROBO2-Fc inhibits the binding of a SLIT ligand, such as SLIT2, to the cellular ROBO2 receptor, likely through its action as a decoy ligand. An HTRF labeling assay of ROBO2-SLITx-N was performed to evaluate the ability of ROBO2-Fc 2.2 to neutralize SLIT ligand binding to human ROBO2. In this assay, HEK293 cells expressing human ROBO2 tagged with terbium (Tb) (donor) and SNAP tagged (SNAP-tag® New England Biolabs; see also Keppler et al., 2003, Nat. Biotechnol. 21:86 ), were incubated with 5 nM multispecies-derived d2-labeled (acceptor) SLITx-N (produced using a Cisbio d2 labeling kit according to the manufacturer's instructions) in the presence of titratable amounts of ROBO2-Fc 2.2 for 1 hour. After incubation, fluorescence was measured at 665 nm and 620 nm on an Envision multilabeled plate reader. The HTRF ratio was calculated as: fluorescence at 665 nm/fluorescence at 620 nm × 10,000. The maximum signal was defined as the HTRF ratio for Tb-labeled ROBO2 cells with d2-labeled SLIT2-N in the absence of ROBO2-Fc 2.2, and the minimum signal was defined as HTRF ratio only for HEK293 cells expressing Tb-labeled ROBO2. This assay evaluated the neutralization of three different SLIT ligands (SLIT1-N, SLIT2-N and SLIT3-N) from several species including humans, cynomolgus monkeys, rabbits, rats and mice. The IC ₅₀ for each SLITx-N was determined using an 11-point 4-fold serial dilution with the highest concentration of 4000 nM. The geometric mean IC ₅₀ for 3 independent experiments was calculated and summarized for all evaluated ligands (Table 2).

Table 2. Table of systematic results of the HTRF assay for inhibition of SLITx-N: human ROBO2 by ROBO2-Fc 2.2

Ligand SLIT View IC ₅₀ (geom. average, nM) 95% CI (nM) n SLIT1N Human 5.9 4.48.0 3 SLIT1N dog-like monkey 6.5 4.88.9 3 SLIT1N Rabbit 9.8 6,415.0 3 SLIT1N Rat 8.3 5.512.5 3 SLIT2N Human 3.9 1,510.6 4 SLIT2N Rabbit 5.7 2,910.9 3 SLIT2N Mouse 5.4 4.66.4 3 SLIT3N Human 5.1 3.57.4 3 SLIT3N dog-like monkey 3.6 2.06.4 3 SLIT3N Rabbit 10.2 7,513.9 3 SLIT3N Rat 3.0 1.75.2 3 SLIT3N Mouse 4.3 2.28.5 3

CI = confidence interval; Geom. mean = geometric mean; HTRF = Homogeneous Time Resolved Fluorescence; IC ₅₀ = Inhibitory concentration at 50% activity; n = number of definitions; ROBO2 = roundabout regulation receptor 2; SLIT = gap regulation ligand; x=1, 2 or 3.

Dose-dependent inhibition of multispecies-derived SLIT ligand binding to human ROBO2 by ROBO2-Fc 2.2 was assessed by HTRF. IC ₅₀ was determined using an 11-point 4-fold dose titration of ROBO2-Fc 2.2 in an HTRF assay for a human, cynomolgus monkey, rabbit, rat, or mouse SLIT ligand panel. ROBO2-Fc 2.2 is a potent neutralizer of human, cynomolgus, rabbit, and rat SLIT1-N binding with human ROBO2 with an IC ₅₀ ranging from a low value of 5.9 nM (human) to a high value of 9.8 nM ( rabbit); ROBO2-Fc 2.2 also demonstrated dose-dependent inhibition of SLIT2-N binding to human ROBO2 with an IC ₅₀ ranging from 3.9 nM (human) to 5.7 nM (rabbit). ROBO2-Fc 2.2 is a strong neutralizer of SLIT3-N binding to human ROBO2 with an IC ₅₀ ranging from 3.6 nM (canine monkey) to 10.2 nM (rabbit). Finally, ROBO2-Fc 2.2 is a strong binding neutralizer of human SLIT2-N:human ROBO2 (FIG. 6A) and rat SLIT2-N:human ROBO2 (FIG. 6B) with IC _50s of 7 nM or 4 nM, respectively.

Nerve cell migration assay

[0246] The final screening selection was carried out to assess the functional neutralization of ROBO2-dependent SLIT2-N activity. SLIT2-ROBO2 interactions are key regulators of axon migration during development. It is known that SLIT2 is a repulsive chemical for subventricular neurons, and that such activity is dependent on ROBO2. Then, explants of nervous tissue were isolated from the subventricular zone (SVZ) of rats and placed in a collagen matrix. In the presence of SLIT2-N, inhibition of nerve cell migration was observed (SVZ assay), and therefore tissue explants were incubated in the presence of 1 nM SLIT2-N and in the presence or absence of titratable amounts of ROBO2-Fc 2.2. After incubation, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342. Wide field fluorescent images were obtained on an Operetta High Content Imager (Perkin Elmer) with a high numerical aperture (NA) objective and 10x magnification. Nine pitches per hole with 5% overlap were taken to cover the entire center area of the hole. Information was obtained on the Z-stacking interaction for each field, consisting of 6 planes with a distance of 1 μm between each plane to capture the entire depth of the tissue explant. The analysis was performed using the Volocity software (Perkin Elmer). All fields in each well were pooled. The tissue explant area at the center and in each nucleus outside the tissue explant was detected by staining with Hoechst 33342 dye. Individual nuclei were counted and the distance from the center of each nucleus to the nearest edge of the tissue explant was measured in µm. The average migration distance of the nuclei in the well was multiplied by the number of nuclei to obtain the total migration distance in each well. ROBO2-Fc 2.2 had the ability to restore nerve cell migration in a dose dependent manner with an IC ₅₀ of 51 nM (FIG. 7). These results showed that ROBO2-Fc 2.2 can not only inhibit SLIT2 binding to ROBO2, but also has a strong dose-dependent neutralizing effect on SLIT2's ROBO2-dependent chemical repulsion ability of SVZ neurons.

Example 3 in vivo effects of new ROBO2 proteins

[0247] Passive Hayman's nephritis is a rat model of nephrotoxic injury affecting glomerular podocytes and leading to increased levels of proteinuria. This model was used to evaluate the effectiveness of ROBO2-Fc 2.2 in reducing proteinuria. Treatment of rats with ROBO2-Fc 2.2 resulted not only in a reduction in proteinuria, but also protected the architecture of podocyte base processes. As shown in FIG. 8 and FIG. 9, administration of prophylactic or therapeutic doses of ROBO2-Fc 2.2 to rats resulted in a reduction in proteinuria. Lewis rats were injected with sheep antiserum against Fxla of rat kidney brush border (Probetex Inc), basement membrane and podocytes. Rats developed an immune response to sheep serum. Complement activation resulted in podocyte deletion and an increase in proteinuria on days 3-12, and then a plateau was observed. This mechanism is very similar to that of membranous nephropathy, in which autoantibodies against the PLA2R podocyte protein (in 70% of cases) cause podocyte elimination and nephrotic proteinuria after complement uptake. Rats were pretreated 24 hours prior to ovine antiserum administration at dose intervals covering approximately 50%, 90%, and 99% (1, 5, and 25 mg/kg) of ROBO2 glomeruli, and then these doses were administered every 72 hours (prophylactic administration schedule). doses). For therapeutic dosing, ROBO2-Fc 2.2 was administered on day 6 or day 9 (5 or 8 days after sheep antiserum administration), and prior to sheep antiserum administration, the 24 hour dose was included in this study. The maximum reduction in proteinuria was 45% when treatment was initiated with prophylactic doses (FIG. 8), and reanalysis by ANOVA confirmed a dose-response with a p- value of less than 0.001. Treatment with ROBO2-Fc 2.2 on days 0, 6 and 9 resulted in a similar reduction in proteinuria (FIG. 9) of up to 40% and a p- value of less than 0.001 as indicated by reanalysis by statistical analysis. ANOVA, for each ROBO2-Fc 2.2 treated group compared to control antibody treated group. No reduction in renal immune complex deposition was observed, as determined by IHC complement assessment, indicating that this response was the result of a protective effect of podocytes.

[0248] To further validate the modulation of podocyte function and structure, quantitative analysis of electron micrographs of podocyte substructures was performed as described below.

Collection, sampling and sectioning

[0249] Completely purified kidney samples (one kidney per animal) were obtained, fixed by immersion (in 4% formaldehyde/1% glutaraldehyde), and then these samples were cut to include only the cortical substance, and five samples of each kidney were embedded into epoxy. A section of the first embedded sample of each kidney was then taken. If this sample contained three glomeruli, then thin sections were obtained and visualized. If this first sample did not contain glomeruli, then successive sections of other embedded kidney samples were taken and these samples were similarly evaluated until a sample with three glomeruli was found.

Overview and visualization

[0250] Collected kidney samples were digitally visualized with a transmission electron microscope (Hitachi H-7100) and a charge-coupled digital camera system (Advanced Microscopy Techniques, Danvers, MA). The three capillary loops of the first three glomeruli found at 200x were visualized at 5000x and 10000x and these procedures were not repeated. This resulted in 18 digital images per kidney (ie, three glomeruli per kidney sample x three areas per glomerulus at x2 magnification). For blind evaluation, each image was identified by number of studies, number of animals, number of samples, and magnification only.

Estimation of the width of the processes of the endings of podocytes and the density of the slit diaphragm

[0251] ImageJ software (version 1.47v; National Institutes of Health, Bethesda, MD, USA) was used to manually monitor and measure the width of contiguous end processes per unit length of the glomerular basement membrane (GBM) by imaging with a transmission electron microscope with big increase. Briefly, measurements were made on six rats per group and nine 5000x TEM images were obtained for each rat. The width of the podocyte base processes was measured from one end of the slit diaphragm to the other end by drawing a line parallel to the GBM. This procedure was repeated for all ending processes in the image. Then the obtained results were scaled and the harmonic mean was calculated for each image.

[0252] Slit Diaphragm Density: Slit Diaphragm Density was calculated by counting the number of slits and dividing the result by the total GBM length spanning the area of those slits. Briefly, measurements were made on six rats per group and nine 5000x TEM images were obtained for each rat. Density was calculated by dividing the number of slotted diaphragms by the length of the GBM. If more than one area of the GBM was visible, or if the GBM was separated due to lack of visibility of the base processes, then the above procedure was repeated and the average of these measurements was used to calculate the slit density. The distance between the slit diaphragms of the intertwined processes of the bases was calculated for the entire set of capillary loops, which made it possible to determine the average width of the processes of the bases. The width of the processes of the bases of the removed podocytes exceeded the width of normal non-removed podocytes.

[0253] As shown in FIG. 10A, the width of the base processes in animals treated with ROBO2-Fc 2.2 at 25 mg/kg was significantly lower than in animals treated with control antibody. These data indicated that the reduction in proteinuria was due to a modification of the podocyte substructure. In addition, as shown in FIG. 10B, ROBO2-Fc 2.2 treatment increased the density of slit diaphragms ( p- value less than 0.01 as determined by two-tailed T-test), indicating that they are less wrinkled and protected from glomerular damage. .

[0254] These results demonstrate that ROBO2-Fc 2.2 effectively inhibits two hallmarks of glomerulitis: proteinuria and diffuse podocyte shrinkage. Thus, these results support the use of ROBO2-Fc 2.2 as a potential therapeutic agent for the treatment of glomerulitis such as, but not limited to, focal segmental glomerulosclerosis (FSGS).

[0255] Key pharmacological properties of ROBO2-Fc 2.2 are summarized in Table 3.

Table 3. Summary of key pharmacological properties of ROBO2-Fc 2.2

Analysis Pharmacodynamic activity Analyzes in vitro Surface plasmon resonance Man and dog-like monkey KD=293±70 pM Monkey SLIT2D2 Human SLIT2N KD=279±37 pM Rat SLIT2N KD=543±72 pM Cellular Binding Assays (Flow Cytometry) Human SLIT2N EC50=8.6±5.0 nM Homogeneous fluorescence with time resolution Human SLIT2N IC ₅₀ =7.0±3.9 nM Rat SLIT2N IC ₅₀ =4.0±.7 nM Analysis in vivo Rat Passive Hayman Jade Dose given before nephrotoxic injury or on day 6 or 9 (5 or 8 after injury) resulted in a significant reduction in proteinuria

[0256] In vitro pharmacological studies have shown that ROBO2-Fc 2.2 binds to human SLIT2-N with the same high affinity as the identical domain of human and cynomolgus SLIT2-D2. ROBO2-Fc 2.2 binds to rat SLIT2-N with the same high affinity. ROBO2-Fc 2.2 binds to cells expressing human SLIT2-N in a dose dependent manner. In addition, ROBO2-Fc 2.2 can inhibit the binding of rat and human SLIT2-N to human ROBO2 in a cellular HTRF binding assay. Mechanistic, pharmacological and in vivo efficacy studies were also performed in a rat model of proteinuria-associated glomerulitis. Administration of ROBO2-Fc 2.2 on a prophylactic or therapeutic dosing schedule resulted in a statistically significant reduction in proteinuria in a PNC model. These results support the use of ROBO2-Fc 2.2 in the treatment of glomerulitis such as focal segmental glomerulosclerosis (FSGS).

Example 4 Characterization of ROBO2-Fc S17T/R73Y Binding and Neutralizing Activity

Analysis by flow cytometry

[0257] ROBO2-Fc S17T/R73Y was conjugated with Alexa Fluor (AF) 647 according to the manufacturer's instructions. Cells overexpressing human SLIT2-N were resuspended in buffer containing 5% fetal bovine serum in a stain preparation. To stain ROBO2-Fc cells with S17T/R73Y-AF647, 2x stock solutions were prepared and 11-12-point 2-fold serial dilutions were made in FACS buffer. Cells and appropriate dilutions of ROBO2-Fc S17T/R73Y-AF647 were pooled in a 96-well U-bottom plate and left for 45 minutes at 4°C. After incubation, 150 μl of FACS buffer was added per cell wash well. After washing, cells were resuspended in buffer to obtain information on a Fortessa flow cytometer. Data were analyzed using FlowJo software and presented as the geometric mean of fluorescence intensity (Geo MFI) of SLIT2-N-expressing HEK293 cells minus Geo MFI of control HEK293 cells. ROBO2-Fc S17T/R73Y bound human SLIT2-N overexpressed on human embryonic kidney cells (HEK293) with high affinity with an EC ₅₀ of 2.5 nM (FIG. 11).

Time Resolved Homogeneous Fluorescence (HTRF)

[0258] A time resolved homogeneous fluorescence assay for ROBO2-SLIT2-N (HTRF) was performed to evaluate the ability of ROBO2-Fc S17T/R73Y to block the interaction of SLIT2-N and ROBO2 expressed by cells. In this assay, HEK293 cells expressing ROBO2 labeled with terbium (Tb) (donor) and labeled with SNAP were incubated with 5 nM d2-labeled (acceptor) human SLITx-N in the presence of titratable amounts of ROBO2-Fc S17T/R73Y for 1 hour according to the manufacturer's instructions (d2 HTRF labeling kit Cisbio 62D2DPEA and terbium cryptate labeling kit 62TBSPEA). After incubation, fluorescence was measured at 665 nm and 620 nm on an Envision multilabeled plate reader. The HTRF ratio was calculated by the formula: fluorescence at 665 nm/fluorescence at 620 nm × 10000. The maximum signal was determined as the ratio of HTRF for Tb-labeled ROBO2 cells with d2-labeled SLIT2-N in the absence of ROBO2-Fc S17T/R73Y, and the minimum signal was defined as the ratio of HTRF only for HEK293 cells expressing Tb-labeled ROBO2. ROBO2-Fc S17T/R73Y is a potent human SLIT2-N binding neutralizer: human ROBO2 with an IC _{50 of} 1.4 nM (FIG. 12).

Nerve cell migration assay

[0259] As with ROBO2-Fc 2.2, ROBO2-Fc S17T/R73Y was evaluated for its ability to reverse SLIT2-N-mediated inhibition of nerve cell migration in a dose-dependent manner. Explants of nervous tissue were isolated from the subventricular zone (SVZ) of rats and embedded in a collagen matrix. In the presence of SLIT2-N, inhibition of nerve cell migration was observed (SVZ assay), and therefore tissue explants were incubated in the presence of 1 nM SLIT2-N and in the presence or absence of titratable amounts of ROBO2-Fc S17T/R73Y. After incubation, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342. Wide field fluorescent images were obtained on an Operetta High Content Imager (Perkin Elmer) with a high numerical aperture (NA) objective and 10x magnification. Nine pitches per hole with 5% overlap were taken to cover the entire center area of the hole. Information was obtained on the Z-stacking interaction for each field, consisting of 6 planes with a distance of 1 μm between each plane to capture the entire depth of the tissue explant. The analysis was performed using the Volocity software (Perkin Elmer). All fields in each well were pooled. The tissue explant area at the center and in each nucleus outside the tissue explant was detected by staining with Hoechst 33342 dye. Individual nuclei were counted and the distance from the center of each nucleus to the nearest edge of the tissue explant was measured in µm. The average migration distance of the nuclei in the well was multiplied by the number of nuclei to obtain the total migration distance in each well. ROBO2-Fc S17T/R73Y had the ability to restore nerve cell migration in a dose dependent manner with an IC ₅₀ of 11.5 nM (FIG. 13).

[0260] These results showed that ROBO2-Fc S17T/R73Y, like ROBO2-Fc 2.2, can not only inhibit SLIT2 binding to ROBO2, but also has a strong dose-dependent neutralizing effect on SLIT2's ROBO2-dependent chemical repulsion ability of SVZ neurons. Thus, these data suggest that ROBO2-Fc S17T/R73Y, like ROBO2-Fc 2.2, may represent a potential therapeutic agent that can be used to treat glomerulitis associated with ROBO2-SLIT interaction, such as focal segmental glomerulosclerosis ( FSGS).

Example 5 Structural Analysis of ROBO2-Fc 2.2

Material preparation, crystallization, data collection and structure determination

Expression and purification of the ROBO2-His construct

[0261] A ROBO2 construct consisting of an Ig1 ROBO2 pre-sequence (SEQ ID NO: 8), an Ig1 domain, an Ig2 domain, and an Ig2-3 ROBO2 interdomain linker (SEQ ID NO: 12) with a 6x histidine tag (SEQ ID NO: 25) at the C-terminus (ROBO2-His6 ("His6" disclosed as SEQ ID NO: 25)), was expressed in HEK293 cells. The construct was purified on an Excel Ni-column by gradient elution with imidazole. The construct was then purified to homogeneity by size exclusion chromatography on a HiLoad 26/200 Superdex 200 (GE Healthcare).

Crystallization

.[0262] ROBO2-His6 crystals were prepared under the following conditions: 100 mM sodium citrate, pH 4.5, 12% PEG 8000 to form crystals having a diffraction grating up to

.

Data collection

, α=γ=90°, β=114,92, с двумя копиями человеческого ROBO2 (Ig1 +Ig2) на асимметрическую ячейку.[0263] The crystals were subjected to temporary cryoprotection, and data collection was carried out remotely at the synchrotron in the presence of an activated photon source (APS). The cropped images were processed using the AutoPROC software (Global Phasing Ltd). These data belong to the space group P2 ₁ , where elementary cells have the following parameters:

, α=γ=90°, β=114.92, with two copies of human ROBO2 (Ig1 +Ig2) per asymmetric cell.

Structure definition and refinement

равны 0,2119 и 0,2425, соответственно, и где RMSD связи составляет 0,010

, а RMSD углов составляет 1,19°.[0264] Searching for molecular substitutions using the ROBO2 (Ig1+Ig2, PDB code: 2V9Q) homologous model allows each component of the solution to be identified. Refinement was performed using the autoBUSTER computer program (Global Phasing Ltd), where the end factors R/Rfree at 2.19

are 0.2119 and 0.2425, respectively, and where the bond RMSD is 0.010

, and the RMSD of angles is 1.19°.

Results and structure analysis

Increased expression level via the Ig1 ROBO2 pre-sequence

или менее). Кроме того, Asp7 (D7), Phe8 (F8) и Pro9 (P9) также активно участвуют в образовании ван-дер-ваальсовых связей с соседними остатками, что дает более, чем 50% площади скрытой поверхности (Таблицы 6-9). Не ограничиваясь какой-либо конкретной теорией, авторы лишь отмечают, что полученные данные показали, что пре-последовательность Ig1 ROBO2 образует мостиковую связь с двумя β-складками первого домена Ig ROBO2 и, тем самым, значительно повышает стабильность структурной укладки N-концевой области. Эти данные, как было неожиданно обнаружено, позволяют предположить, что стабилизация структурной укладки опосредует повышение уровня экспрессии ROBO2-Fc 2.2 с правильной укладкой по сравнению с уровнем экспрессии конструкций ROBO2-Fc, не содержащих пре-последовательность Ig1.[0265] Example 1 demonstrated that incorporation of the wild-type Ig1 ROBO2 pre-sequence SRLRQEDFP (SEQ ID NO: 8) resulted in a dramatic increase in the expression level of ROBO2-Fc 2.2. From the crystal structure of ROBO2-His6 ("His6" disclosed as SEQ ID NO: 25), Asp7 (D7), Phe8 (F8), and Pro9 (P9) are mainly involved in interactions that play an important role in structural integrity. the first Ig domain of ROBO2 (Fig. 14). Asp7 forms a hydrogen bond with Tyr94 and Phe8 forms a double bond with Arg36 and Asn93 (Tables 4-5; distance between adjacent residues is 3.8

or less). In addition, Asp7 (D7), Phe8 (F8) and Pro9 (P9) are also actively involved in the formation of van der Waals bonds with neighboring residues, which gives more than 50% of the hidden surface area (Tables 6-9). Without wishing to be bound by any particular theory, the authors only note that the data obtained showed that the Ig1 ROBO2 pre-sequence bridges two β-sheets of the first domain of Ig ROBO2 and thereby significantly increases the stability of the N-terminal fold. These data were surprisingly found to suggest that fold stabilization mediates an increase in the expression level of correctly folded ROBO2-Fc 2.2 relative to the level of expression of ROBO2-Fc constructs lacking the Ig1 pre-sequence.

Table 4. Hydrogen bonds included in the N-terminal residues of ROBO2 (copy 1)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) A.S.P. 7 TYR 94 2.73 PHE 8 ARG 36 3.3 PHE 8 ASN 93 2.84 PHE 8 ARG 36 3.01

Table 5. Hydrogen bonds included in the N-terminal residues of ROBO2 (copy 2)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) A.S.P. 7 TYR 94 2.58 PHE 8 ARG 36 3.14 PHE 8 ASN 93 2.97 PHE 8 ARG 36 2.79

Table 6. Table of interactions at the minimum distance from the N-terminal residues of ROBO2 (copy 1)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) GLU 6 ARG 36 3.44 A.S.P. 7 ARG 36 3.18 A.S.P. 7 TYR 94 2.73 A.S.P. 7 LEU 95 3.54 PHE 8 GLY 35 3.55 PHE 8 ARG 36 3.01 PHE 8 GLU 34 3.89 PHE 8 LEU 95 3.78 PHE 8 ASN 93 2.84 PRO 9 ASN 93 3.71 PRO 9 LEU 95 3.59 PRO 9 ARG eleven 3.82

Table 7. Table of interactions at the minimum distance from the N-terminal residues of ROBO2 (copy 2)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) GLU 6 ARG 36 3.74 A.S.P. 7 ARG 36 3.19 A.S.P. 7 TYR 94 2.58

A.S.P. 7 LEU 95 3.56 PHE 8 GLY 35 3.4 PHE 8 ARG 36 2.79 PHE 8 GLU 34 3.91 PHE 8 LEU 95 3.6 PHE 8 ASN 93 2.97 PRO 9 ASN 93 3.85 PRO 9 LEU 95 3.58 PRO 9 ARG eleven 3.75

Table 8. N-terminal residue hidden in surface area (BSA) assay (copy 1)

Remainder Remainder# A.S.A. complex Free ASAs BSA BSA% GLU 6 197.12 218.69 21.57 9.86 A.S.P. 7 64.6 148.81 84.21 56.59 PHE 8 82.91 179.66 96.75 53.85 PRO 9 80.69 173.68 92.99 53.54

Table 9. N-terminal residue hidden in surface area (BSA) assay (copy 2)

Remainder Remainder# A.S.A. complex Free ASAs BSA BSA% GLU 6 191.1 216.4 25.3 11.69 A.S.P. 7 59.52 147.69 88.17 59.7 PHE 8 82.87 181.29 98.42 54.29 PRO 9 78.78 172.08 93.3 54.22

Increasing the expression level through the interdomain linker Ig2-3 ROBO2

[0266] Inclusion of the interdomain Ig2-3 linker ROBO2 V200-F201-E202-R203 (VFER; SEQ ID NO: 12) results in a marked increase in the expression level of ROBO2-Fc 2.2. Val200 is part of the last β chain in the Ig2 domain of ROBO2. V200 is deeply hidden by neighboring remnants due to intense van der Waals bonds (Tables 10-11); and therefore 85% of the surface area is hidden (Tables 12-13). Phe201-Glu202-Arg203, in addition to Val200, is also largely involved in the formation of hydrogen bonds and van der Waals interactions in this area (Tables 10-11, 14-15). Overall, these data suggest that these four amino acid residues effectively stabilize the fold in the C-terminal region of the Ig2 domain of ROBO2 (FIG. 15). Without wishing to be bound by any particular theory, the authors only hypothesize based on their findings that fold stabilization increases the expression level of correctly folded ROBO2-Fc 2.2 compared to a decrease in the expression level of ROBO2-Fc constructs lacking the Ig2-3 interdomain linker.

Table 10. Table of interactions at the minimum distance from the C-terminal residues of ROBO2 (copy 1)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) VAL 200 ARG 173 3.66 VAL 200 VAL 123 3.17 VAL 200 THR 172 3.33 VAL 200 LYS 174 3.28 VAL 200 ALA 125 3.84 PHE 201 LYS 174 3.87 PHE 201 VAL 122 3.62 PHE 201 ALA 124 3.54 PHE 201 ALA 125 2.87 PHE 201 VAL 123 2.93 GLU 202 LYS 174 3.54 ARG 203 ALA 125 3.62 ARG 203 GLU 127 3.53

Table 11. Table of interactions at the minimum distance from the C-terminal residues of ROBO2 (copy 2)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) VAL 200 ARG 173 3.55 VAL 200 VAL 123 3.27 VAL 200 THR 172 3.27 VAL 200 LYS 174 3.88 VAL 200 ALA 125 3.75 PHE 201 VAL 122 3.6 PHE 201 ALA 124 3.56 PHE 201 ALA 125 2.84 PHE 201 VAL 123 2.94 GLU 202 LYS 174 3.43 ARG 203 ALA 125 3.03 ARG 203 GLU 127 3.12

Table 12 C-terminal residue hidden in surface area (BSA) assay (copy 1)

Remainder Remainder # A.S.A. complex Free ASA BSA BSA% VAL 200 21.95 207.67 185.72 89.43 PHE 201 93.47 167.02 73.55 44.04 GLU 202 138.33 166.75 28.42 17.04 ARG 203 96.04 153.35 57.31 37.37

Table 13 C-terminal residue hidden in surface area (BSA) assay (copy 2)

Remainder Remainder # A.S.A. complex Free ASA BSA BSA% VAL 200 27.85 206.09 178.24 86.49 PHE 201 99.37 171.31 71.94 41.99 GLU 202 113.98 153.45 39.47 25.72 ARG 203 77.8 159.3 81.5 51.16

Table 14. Hydrogen bonds in the C-terminal residues of ROBO2 (copy 1)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) VAL 200 LYS 174 3.28 PHE 201 ALA 125 2.87 PHE 201 VAL 123 2.93

Table 15. Hydrogen bonds in the C-terminal residues of ROBO2 (copy 2)

Remainder_1 Remainder_1 # Remainder_2 Remainder_2 #

) Distance (

) PHE 201 ALA 125 2.84 PHE 201 VAL 123 2.94 GLU 202 LYS 174 3.43 ARG 203 ALA 125 3.03 ARG 203 GLU 127 3.23 ARG 203 GLU 127 3.12

Example 6 Structural Analysis of ROBO2-Fc S17T/R73Y

Obtaining materials, crystallization, data collection and structure determination:

Expression

[0267] The S17T/R73Y-His6 human ROBO2 construct ("His6" disclosed as SEQ ID NO: 25) consists of a human Ig1 ROBO2 domain with 2 point mutations (S17T and R73Y) followed by a His x6 C-terminal tag (SEQ ID NO: 25). The human SLIT2 construct consists of the D2 domain of SLIT2 (271-479) followed by the His x6 C-terminal tag (SEQ ID NO: 25). These constructs were expressed separately in HEK293 cells and conditioned media were collected 120 hours after transfection.

Purification of human ROBO2 S17T/R73Y-His6 ("His6", disclosed as SEQ ID NO : 25) and SLIT2 D2 domain

[0268] Human ROBO2 S17T/R73Y-His6 ("His6" disclosed as SEQ ID NO: 25) was purified from conditioned media using HP Nickel Sepharose (GE Healthcare) and an imidazole gradient. Peak fractions were pooled and diluted to 20 mM NaCl, adjusted to pH 6.0, and loaded onto a HiTrap Q-Sepharose Fast Flow (Q FF) column in tandem with a HiTrap column containing strong sulfopropyl (SP), i.e., with high performance column (HP). After extensive washing, the Q FF column was removed and a NaCl gradient was applied to the SP HP column. Fractions were collected depending on the degree of glycosylation and dialyzed against TBS.

[0269] The human D2 domain of SLIT2 was captured from conditioned media using HP Nickel Sepharose (GE Healthcare) and purified in an imidazole gradient. Peak fractions were pooled and purified again by size exclusion chromatography on Superdex 200 in buffer containing 50 mM Tris-HCl pH 7.5, 1000 mM NaCl. Fractions containing D2 SLIT2 were pooled and adjusted to 500 mM NaCl.

Formation of the human ROBO2 S17T/R73Y-His complex and the D2 domain of SLIT2

[0270] Due to an unexpected interaction between SLIT2 and resin on the gel filtration column, the ROBO2 S17T/R73Y-His6 complex ("His6", disclosed as SEQ ID NO: 25) and SLIT2 could not be obtained using size exclusion chromatography. Instead, purified ROBO2 S17T/R73Y-His6 ("His6", disclosed as SEQ ID NO: 25) was mixed with the D2 domain of SLIT2 in a 1:1 molar ratio and concentrated for crystallization purposes.

Crystallization

.[0271] Crystals of ROBO2 S17T/R73Y-His6 ("His6" disclosed as SEQ ID NO: 25) complexed with the D2 domain of SLIT2 were prepared under the following conditions: 100 mM sodium citrate, pH 5.5, 15% PEG 6000. B as a result, crystals were obtained with a diffraction grating up to

.

Data collection

, α=β=γ=90°, с одной копией комплекса на асимметрическую ячейку.[0272] The crystals were subjected to temporary cryoprotection, and data collection was carried out remotely at the synchrotron in the presence of an activated photon source (APS). The cropped images were processed using the AutoPROC software (Global Phasing Ltd). These data belong to the space group P2 ₁ , where elementary cells have the following parameters:

, α=β=γ=90°, with one copy of the complex per asymmetric cell.

Structure definition and refinement

равны 0,1848 и 0,2354, соответственно, и где RMSD связи составляет 0,010

, а RMSD углов составляет 1,10°.[0273] A search for molecular substitutions using a homologous model of the Ig1 domain of ROBO1 and the D2 domain of SLIT2 (PDB code: 2V9T) identified each component of the solution. Refinement was performed using the autoBUSTER computer program (Global Phasing Ltd), where the end factors R/Rfree at 1.78

are 0.1848 and 0.2354, respectively, and where the bond RMSD is 0.010

, and the RMSD of angles is 1.10°.

Results and structure analysis

[0274] The crystal structure of ROBO2 S17T/R73Y-His6-SLIT2 ("His6" disclosed as SEQ ID NO: 25) is largely consistent with ROBO1-SLIT2, and the ROBO-SLIT border region is highly conserved between ROBO2 and ROBO1. Therefore, the publicly available ROBO1-SLIT2 structure can be used as a substitute for ROBO2-SLIT2 to compare structures in order to probe the effect on S17T and R73Y.

[0275] Ser17 (S17) ROBO2 interacts with Arg287 SLIT2 via hydrogen bonding (FIG. 16). The hydrogen bond to arginine is conserved regardless of the mutation, however, Thr17 communicates an additional van der Waals interaction with Arg287, conferring some energetic advantage over wild-type Ser17.

[0276] R73Y ROBO2 interacts with Tyr404 SLIT2 (FIG. 16). Van der Waals interactions caused by pi-pi-stacknig interactions of two tyrosine residues are much more efficient than interactions between tyrosine and the flexible side chain of arginine. This is indicated by the percentage of hidden surface area (42% for Tyr73 and 22% for Arg73; Tables 16-19). The Arg to Tyr substitution in ROBO2 S17T/R73Y is likely to be the main factor involved in the increase in affinity over wild-type ROBO2 containing S17 and R73.

[0277] In addition, S17 and R73 are present at opposite ends of the ROBO2-SLIT2 border region (FIG. 16). Without wishing to be bound by any particular theory, the authors only note that stabilization of the interactions at these positions probably gives ROBO2 a greater ability to bind to SLIT2, and thereby increases the overall binding affinity of the two proteins.

Table 16 Interaction table for key mutations in ROBO2

Remainder_1 Remainder_1 # Chain Remainder _2 Remainder _2 # Chain

) Distance (

) THR 17 ROBO2 ARG 287 SLIT2 3.28 TYR 73 ROBO2 TYR 404 SLIT2 3.43

Table 17. Table of interactions of the corresponding wild-type residues in ROBO1

Remainder _1 Remainder _1 # Chain Remainder _2 Remainder _2 # Chain

) Distance (

) SER 17 ROBO1 ARG 287 SLIT2 3.25 ARG 73 ROBO1 TYR 404 SLIT2 3.01

Table 18. Latent surface area analysis for key mutations in ROBO2

Remainder Remainder # Chain A.S.A. complex Free ASAs BSA BSA% THR 17 ROBO2 64.76 99.97 35.21 35,.22 TYR 73 ROBO2 44.17 75.85 31.68 41.77

Table 19 Latent surface area analysis for corresponding wild-type residues in ROBO10

Remainder Remainder # Chain A.S.A. complex Free ASAs BSA BSA% SER 17 ROBO1 53.39 79.32 25.93 32.69 ARG 73 ROBO1 98.23 125.94 27.71 22

Example 7 Posttranslational Modifications of ROBO2-Fc 2.2

LC/MS - Peptide Mapping

[0278] Analysis of post-translational modifications of ROBO2-Fc 2.2 was performed by peptide mapping. Briefly, ROBO2-Fc 2.2 was reduced, alkylated and cleaved with a lysine-specific protease, lysyl endopeptidase (Lys-C). Lys-C proteolytic peptides were analyzed by reverse phase high performance liquid chromatography (RP-HPLC) with UV detection at 214 nm.

[0279] All major and minor peaks in the peptide map for ROBO2-Fc 2.2 were identified by online electrospray ionization mass spectrometry (RP-HPLC/IEP-MS or LC/MS). The observed masses for each peak corresponded to the expected Lys-C proteolytic peptides from ROBO2-Fc 2.2. These LC/MS peptide mapping results indicated that ROBO2-Fc 2.2 contained the correct amino acid sequence as predicted from the cDNA sequence. LC/MS analysis indicated the presence of N-linked oligosaccharides in the K6 peptide (which was detected as K4K5K6) containing the AS consensus sequence N ¹⁰² for N-linked glycosylation. The main oligosaccharide structures identified in the K4K5K6 glycopeptide include two-antennary complex-type structures containing two galactose residues with one terminal N-acetylneuraminic acid residue (G2F+1NeuAc) or with two terminal N-acetylneuraminic acid residues (G2F+2NeuAc). A small amount of K4K5K6 glycopeptides containing two terminal galactose residues (G2F) was also observed. In addition, a small content of other molecules was also observed, which are three-antennary and four-antennary structures of a complex type with fucose, which has a substitution in the core and contains up to four terminal residues of N-acetylneuraminic acid.

[0280] LC/MS analysis also indicated the presence of N-linked oligosaccharides in the K19 peptide (which was detected as K18K19) containing the ST consensus sequence N ²⁹¹ for N-linked glycosylation. The major oligosaccharide structures identified in K18K19 include complex-type asialo-biantennary structures that do not contain galactose residues (G0F) or contain a 1 (G1F) terminal galactose residue. A small amount of K18K19 was also observed with an asialo-two-antenna structure of a complex type containing two galactose residues (G2F). A small amount and trace amounts of the K18K19 glycopeptide containing truncated N-glycans and complex N-glycans including up to two terminal residues of N-acetylneuraminic acid were detected.

Table 20. LC/MS-N-linked glycosylation

Properties results N-linked glycosylation Occupation of the N-linked glycosylation site confirmed in ST N ²⁹¹ in the K18K19 peptide
(major >40%, minor 540%, trace <5%):
Detected N-glycans (identical and corresponding excess amounts):
Major: G0F, G1F
Auxiliary: G0, G2F
Trace amounts: non-glycosylated, G0 minus GlcNAc (truncated), G0F minus GlcNAc (truncated), Man5 (high mannose), G1, G1F+1NeuAc, G2F+1NeuAc, and G2F+2NeuAc
Occupation of the N-linked glycosylation site, confirmed in N ¹⁰² AT in peptides K4K5K6 and K5K6 on the peptide map
(Major >40%, Minor 540%, Trace <5%):
Detected N-glycans (identical and corresponding excess amounts):
Major: G2F+1NeuAc, G2F+2NeuAc
Auxiliary: G2F, G3TriF+2NeuAc, G3TriF+3NeuAc, G4TetraF+3NeuAc and G2F+2NeuAc (peptide K5K6)
Trace amounts: G0 minus GlcNAc (truncated), G1F+1NeuAc, G4TetraF+2NeuAc, G4 TetraF+4NeuAc, G2F+1NeuAc (K5K6 peptide)

Example 8 Studies for Safe Pharmacological Effects

[0281] Male monkeys were telemetrically administered ROBO2-Fc 2.2 once a week by intravenous (IV) injection for 6 weeks at 50 mg/kg/dose (6 doses total) to assess cardiovascular disease (CV) endpoints. There were statistically significant differences in heart rate (HR) and activity. However, these differences were small in magnitude, sporadic in nature, and were not long lasting, and therefore, they were not considered to be associated with ROBO2-Fc 2.2. IV administration of ROBO2-Fc 2.2 at 50 mg/kg/dose once a week for 6 weeks did not produce any ROBO2-Fc 2.2-associated changes in blood pressure (BP), heart rate, or activity at any time during the study. C _max values were 1060, 1030 and 1070 μg/ml on days 1, 22 and 36, respectively.

[0282] A Repeat Dose Toxicity Telemetry Experimental Study (ETS) in male and female cynomolgus monkeys was performed to evaluate the effect of once weekly administration of ROBO2-Fc 2.2 by intravenous injection at 50 mg/kg/dose for 29 days (total 5 doses) on electrocardiogram (ECG) and blood pressure (KP) parameters, as well as activity. No ROBO2-Fc 2.2-associated effect on CV parameters taken on day 9 was observed. On day 29, C _max for animals of both sexes was 497 µg/ml.

[0283] Measurements of ECG and heart rate parameters were included in a 3-month repeat dose toxicity study according to standard laboratory practice (GLP) for male and female cynomolgus monkeys administered doses of 20, 100, or 300 mg/kg/dose intravenously or 300 mg/kg/dose subcutaneously (SC). In this study, no ROBO2-Fc 2.2-associated effect on ECG or HR was observed with an average C _max for animals of both sexes up to 9210 µg/ml. In addition, there were no ROBO2-Fc 2.2-associated clinical observations that could indicate any systemic effects in the respiratory tract or the central nervous system.

[0284] In addition, neurofunctional endpoints were assessed in a 3-month rat toxicity study. However, no ROBO2-Fc 2.2-associated effects were observed on locomotor activity or the combination of functional effects at doses administered intravenously at 425 mg/kg/dose or subcutaneously at 425 mg/kg/dose (mean C _max for animals of both sexes was 7610 µg/ml).

Example 9 Pharmacokinetics and Metabolism of Products in Animals

Analysis Methods

I. Quantification of ROBO2-Fc 2.2 in rat and monkey serum

[0285] Electrochemiluminescent (ECL) analysis was performed to determine the amount of ROBO2-Fc 2.2 in the serum of Wistar Han rats and cynomolgus monkeys on Meso Scale Discovery (MSD®) analysis equipment (15-1611, 15-1609). In these assays, samples containing ROBO2-Fc 2.2 were incubated on streptavidin-coated MSD plates sensitized with a biotinylated anti-ROBO2-Fc antibody capture reagent. Bound ROBO2-Fc 2.2 was detected using a rutenylated mouse anti-human IgG Fc fragment. Final detection was achieved by adding an MSD read buffer to generate an ECL signal that was read on an MSD plate reader. The obtained ECL signal was directly proportional to the concentration of ROBO2-Fc 2.2. Sample concentrations were determined by interpolation from a standard curve that was generated using a logarithmic 4PL model (automatically estimated) with a weighting factor of 1/y. The quantitation interval in 100% serum ranged from 50 to 1280 ng/ml with a minimum required dilution factor (MRD) of 20x.

II. Detection of anti-ROBO2-Fc 2.2 antibodies in the serum of rats and monkeys

[0286] ECL analysis was performed to detect the presence of anti-drug antibodies (ADA) in the sera of Wistar Han rats and cynomolgus monkeys on MSD® assay equipment. In these assays, ROBO2-Fc 2.2 labeled with biotin and ruthenium was co-incubated with experimental samples, positive control (with anti-ROBO2-Fc 2.2 antibodies in Wistar Han rat or cynomolgus rat sera) or negative control (with pooled normal rat sera). Wistar Han or dog-like monkeys). Anti-ROBO2-Fc 2.2 antibodies present in the samples should bind to biotin and ruthenium-labeled ROBO2-Fc 2.2 variants detectable in these assays. Bound ADA was captured on streptavidin-coated MSD plates. Final detection was achieved using tripropylamine producing an ECL signal which was measured on an MSD plate reader and expressed in relative light units (RLU).

[0287] Experimental samples were tested for ADA using a number of strategies. Samples were first tested in a screening assay at the minimum required dilution of 1:50. Samples that gave RLUs below the assay cutoff were reported as negative (<1.70, log10 of the minimum required dilution factor, 50). Samples that gave an RLU equal to or greater than the cut-off value of the assay were re-analyzed in a full dilution series to confirm a positive result and determine the antibody titer. The antibody titer was determined as the reciprocal of the dilution factor of the sample that gave an RLU equivalent to the RLU of the assay cutoff and reported as the log (base 10) of that titer.

[0288] Conclusions related to the induction of ADA were made based on a comparison of the results for the samples taken before the dose on day 1, and for the sample taken after the dose. If a pre-dose test sample was ADA negative and the corresponding post-dose test sample was ADA positive, then that animal was considered positive for inducing an ADA response to ROBO2-Fc 2.2. If pre-dose and post-dose test samples were positive for ADA, then that animal was considered positive for induction of an ADA response only if the post-dose sample titer was also at least 0.48 (log 3, serial dilution factor) or above the titer of the sample prior to dosing.

results

I. Pharmacokinetics

A. Single dose pharmacokinetic assays

приблизительно 5 и 8 дней у крыс и обезьян, соответственно. После введения крысам ROBO2-Fc 2.2 подкожно в дозе 10 мг/кг, оцененная биодоступность составляла 56%.[0289] Serum pharmacokinetic properties of ROBO2-Fc 2.2 were determined in Lewis rats (n=3) and cynomolgus monkeys (n=2) following a single intravenous dose of 10 mg/kg. ROBO2-Fc 2.2 showed a clearance (CL) in rats at 1.5 ml/h/kg and in monkeys at 0.89 ml/h/kg and a stationary volume of distribution (Vss) in rats at 0.19 l/kg and in monkeys at 0.09 L/kg, and gave a terminal half-life

approximately 5 and 8 days in rats and monkeys, respectively. Following subcutaneous administration of ROBO2-Fc 2.2 to rats at a dose of 10 mg/kg, the estimated bioavailability was 56%.

B. Repeated Dose Pharmacokinetics (Toxicokinetics; TK)

Toxicokinetics in rats

[0290] TK and ADA were assessed after intravenous administration of 25, 125 or 425 mg/kg/dose, and after subcutaneous administration of 425 mg/kg/dose to male and female Wistar Han rats once every 3 days (n=6/sex /dose group) during a 3-month experimental GLP toxicity study with a 6-week recovery phase.

[0291] The concentrations of ROBO2-Fc 2.2 in the samples taken and analyzed before the dose on day 1 or in the samples taken and analyzed from the group, which was administered a vehicle control, was not determined. Quantitative concentrations of ROBO2-Fc 2.2 were observed on day 88 (last sample collection) and until the last day of the recovery phase (day 135) in the ROBO2-Fc 2.2 dosed group.

[0292] At the same time, there were no obvious sex-associated differences in the systemic response (as measured by the maximum concentration [C _max ] and the area under the concentration curve (AUC) for a period of 0 to 72 hours [AUC ₇₂ ]) in the group with any dose. The mean systemic response increased with dose approximately in proportion to the administered dose from day 1 to day 88. The systemic bioavailability of ROBO2-Fc 2.2 was 24.4% and 25.4% after subcutaneous dosing on days 1 and 88, respectively. Accumulation ratios (AUC, day 88/day 1) were < 2.0 for groups dosed intravenously and subcutaneously (Table 21).

[0293] The frequency of induction of ADA in response to ROBO2-Fc 2.2 in groups that received intravenous doses of 25, 125 and 425 mg/kg was 0.0% (0/12 animals), 0.0% (0/12 animals ), and 0.0% (0/12 animals), respectively, and 41.7% in the 425 mg/kg subcutaneous dose group (5/12 animals). Serum concentrations of ROBO2-Fc 2.2 in ADA positive animals were similar to those in ADA negative animals. However, it should be noted that circulating levels of ROBO2-Fc 2.2 present in samples may interfere with ADA detection.

Toxicokinetics in cynomolgus monkeys

[0294] Assessment of TK and ADA was performed after intravenous administration of 20, 100 or 300 mg/kg/dose, and after subcutaneous administration of 300 mg/kg/dose to male and female cynomolgus monkeys once a week (n=3 or 5/sex/ dose group) during a 3-month experimental GLP toxicity study with a 6-week recovery phase.

[0295] The concentrations of ROBO2-Fc 2.2 in the samples taken and analyzed before the dose on day 1, or in the samples taken and analyzed from the group that was injected with the vehicle control, was not determined. Quantitative ROBO2-Fc 2.2 concentrations were observed on day 78 (last sample collection) and until the last day of the recovery phase (day 127 or 128) in the ROBO2-Fc 2.2 dosed group.

[0296] At the same time, there were no obvious sex-associated differences in the systemic response (as measured by C _max and AUC ₁₆₈ ) in the group with any dose. The mean systemic response increased with dose approximately proportional to the administered dose from day 1 to day 78. The systemic bioavailability of ROBO2-Fc 2.2 (at 300 mg/kg IV and SC) was 53.7% and 77.0% after SC administration. doses on day 1 and day 78, respectively. The accumulation ratios (AUC, day 78/day 1) were <2.0 for the intravenously dosed groups and 2.1 for the subcutaneously dosed group (Table 21).

[0297] The frequency of induction of ADA in response to ROBO2-Fc 2.2 in groups that received intravenous doses of 20, 100 or 300 mg/kg was 0% (0/6 animals), 0% (0/6 animals) and 20% (2/10 animals), respectively, and 33% in the subcutaneously administered dose of 300 mg/kg (2/6 animals). Serum concentrations of ROBO2-Fc 2.2 in ADA positive animals were similar to those in ADA negative animals. However, it should be noted that circulating levels of ROBO2-Fc 2.2 present in samples may interfere with ADA detection.

II. Distribution

[0298] Stationary volume of distribution (Vss) of ROBO2-Fc 2.2 in rats (0.19 L/kg) and monkeys (0.09 L/kg) was low after a single intravenous dose, consistent with limited distribution of Fc-containing protein during extracellular fluids.

III. Pharmacokinetics-pharmacodynamics and PK prediction in humans

[0299] Based on PK/PD modeling (incorporating predicted human PK data, measured serum ROBO2 and SLIT2 data, and measured renal concentrations), it was predicted that the estimated subcutaneous dose of 2 mg/kg (150 mg) administered to a human weekly, maintained coverage of the target C _min >90% in the kidneys. A predicted effective human serum concentration (C _eff ) of ~11 μg/mL gave >90% target coverage in the kidneys. The predicted steady-state _Cmax and AUCtau in humans following a weekly human subcutaneous dose of 2 mg/kg (150 mg) were 18.2 μg/mL and 2610 μg h/mL, respectively.

[0300] PK prediction for ROBO2-Fc 2.2 in humans was made by scaling PK data after intravenous dosing in cynomolgus monkeys based on allometric exponents. It was predicted that in humans, the clearance of ROBO2-Fc 2.2 was 0.5 ml/h/kg, and the stationary volume of distribution at a dose of 90 ml/kg gave a terminal half-life of approximately 14 days. The predicted bioavailability when administered subcutaneously to humans was 60%.

[0301] In initial clinical studies, the exposure limit was set to C _max 2930 µg/ml, and AUC 74500 µg h/ml was determined to be associated with exposure limits of 161× and 29×, that is, the predicted effective exposure in humans, respectively, at the experimental effective human dose (150 mg SC once a week). This exposure limit is based on the No Observed Adverse Effect Level (NOAEL) following a 3-month GLP rat toxicity study.

[0302] Although ROBO2-Fc 2.2-associated effects on serum cytokine levels were not observed during the 3-month GLP toxicity studies in rats or cynomolgus monkeys in ETS following administration of ROBO2-Fc 2.2 once a week subcutaneously at a dose of 10 mg/kg/dose or intravenously at 50 or 200 mg/kg/dose, respectively, however, an increase in TNF-α and/or IL-6 was seen in 1 in 8 human donors and 1 in 12 monkeys in vitro . Although the in vitro data have not yet been accurately interpreted, procedures such as slow administration of ROBO2-Fc 2.2 via intravenous infusion (in a small fraction of the total dose administered in the first hour, followed by the remaining dose in the second hour) have been carried out. in a single phase dose escalation study to assess dosing interruption/end, close monitoring of vital signs, clinical symptoms, and assessment of cytokine levels.

Table 21 Mean total (female + male) toxicokinetic parameters ± standard deviation ^A in rats and monkeys

Types/number of studies Dose (mg/kg/dose) ^b Way Day _Cmax (µg/ml) T _max (hours) AUC ₇₂ or AUC ₁₆₈ (mcg h/mL) ^s AUC ₇₂ or AUC ₁₆₈ //dose ([mcg h/mL]/[mg/kg]) Rat ^d 16GR156 (3 months old) 25 IV 1 431 0.50 10700 428 IV 88 674 0.50 20900 836 125 IV 1 2370 0.50 47100 377 IV 88 2930 0.50 74500 596 425 IV 1 9270 0.50 180000 424 IV 88 7610 0.50 199000 468 425 ^t SC 1 760 48 43900 103 SC 88 842 24 50500 119 Canine monkey ^t 16GR143 (3 months old) 20 IV 1 403±48.7 0.5±0.0 17200±2480 859±124 IV 78 470±56.5 0.5±0.0 24300±3640 1210±182 100 IV 1 2470±246 0.5±0.0 95100±15100 951±151 IV 78 2350±503 0.5±0.0 113000±29300 1130±293 300 IV 1 7160±565 0.5±0.0 287000±15500 958±51.5 IV 78 9210±1080 0.5±0.0 427000±49800 1420±166 ^300g SC 1 1260±250 32±20 155000±29000 515±96.7 SC 78 2340±480 36±29 328000±73200 1090±244 AUC ₁₆₈ = Area under the concentration curve from time 0 to 168 hours; AUC ₇₂ = Area under the concentration curve from time 0 to 72 hours;
C _max = maximum observed concentration; IV=intravenously; SC=subcutaneous; T _max = time to first observation C _max .
A. Standard deviation was not recorded for 16GR156 because the sample used is non-serial.
b. In the 16GR156 study, animals were dosed every 3 days. In the 16GR143 study, animals were dosed once a week.
c. AUC ₇₂ was used in the 16GR156 study and AUC ₁₆₈ was used in the 16GR143 study.
d. N=6/gender/dosing group (3/gender/time point).
e. Total bioavailability (F%) after SC dose, based on AUC ₇₂ = 24.4 on day 1 and 25.4 on day 88.
f. N=3 or 5/gender/dosed group.
g. Total bioavailability (F%) after SC-dose, based on AUC ₁₆₈ = 53.7 on day 1 and 77.0 on day 78

Example 10 Toxicology

[0303] ROBO2-Fc 2.2 was administered to rats and cynomolgus monkeys by intravenous and subcutaneous injection in experimental toxicity studies (which were conducted in a non-standard laboratory practice [GLP] regimen) and in pilot toxicity studies (conducted in accordance with GLP) over a period of time up to 3 months (13 weeks). However, no side effect of NOAEL was observed after 3 months of dosing:

(i) 125 mg/kg/dose IV (C _max 2930 µg/mL and AUC ₇₂ 74500 µg h/mL) and 425 mg/kg/dose SC (C _max 842 µg/mL and AUC ₇₂ 50500 µg h/mL) h/ml) in rats, and

(ii) 300 mg/kg/dose, IV (C _max 9210 µg/mL and AUC ₁₆₈ 427000 µg h/mL) and SC (C _max 2340 µg/mL and AUC ₁₆₈ 328000 µg h/mL) in monkeys.

I. Repeated dose toxicity

[0304] Experimental and pilot studies on repeated dose toxicity were performed using ROBO2-Fc 2.2 in rats and cynomolgus monkeys.

A. Rat studies

(i) Experimental Toxicity Study (ETS)

[0305] In an experimental toxicity study (ETS) in male rats, ROBO2-Fc 2.2 was administered once every 3 days for 14 days (5 doses in total) intravenously at a dose of 200 mg/kg/dose or subcutaneously at a dose of 10.50 or 200 mg/kg/dose, all of which were acceptable doses. However, no ROBO2-Fc 2.2-associated clinical signs and changes in body weight parameters or food digestibility were observed.

[0306] ROBO2-Fc 2.2-associated effects included minimal to moderate perivascular inflammation at subcutaneous injection sites at ≥10 mg/kg/SC dose; an increase in the absolute mass of the thymus (1.21×-1.44× compared to the control) and the relative mass of the thymus (1.17×-1.39× compared to the control body weight and 1.22×-1.46× compared with control brain mass) at a dose of ≥10 mg/kg/dose of SC and IV that are not associated with a correlate obtained under a microscope; increased urinary creatinine (1.70× and 1.83× compared to control) and lower urine volume (0.52× and 0.55× compared to control) at 200 mg/kg/dose SC and IV; high mean urine specific gravity (1.011×-1.012× compared to control) at 200 mg/kg/dose SC and IV; and higher mean serum cholesterol levels (1.38× compared to control) at 200 mg/kg/dose IV. Clinical chemistry and organ weight data were not associated with any microscopic data.

(ii) Repeat dose toxicity studies

[0307] In a pilot repeated dose toxicity study in rats, ROBO2-Fc 2.2 was administered once every 3 days for 3 months (total 31 doses) intravenously at a dose of 25, 125, or 425 mg/kg/dose or subcutaneously at a dose of 425 mg/kg/dose, followed by a 6-week recovery phase (control and 425 mg/kg/dose IV). The neurofunctional effects of ROBO2-Fc 2.2 were also evaluated. ROBO2-Fc 2.2-associated clinical signs included skin lesions (dorsal, thoracic, cranial, or injection site) seen in 4 out of 10 male rats at a dose of 425 mg/kg/dose SC during the dosing phase . These changes were not considered as side effects since their frequency was only slightly higher than the frequency observed in the control group (2/15 males), and such changes were only sporadic in the dosing phase and were not observed in animals administered ROBO2-Fc 2.2 at doses up to 425 mg/kg/dose IV. No data were observed regarding the association of ROBO2-Fc 2.2 with body weight, food digestibility, ophthalmic parameters, neurofunctional properties, or macroscopic parameters.

[0308] In males, renal symptoms consisted of minimal glomerulopathy at a dose of ≥125 mg/kg/dose IV. In females, minimal glomerulopathy was also observed only at 425 mg/kg/dose IV and was accompanied by minimal to mild tubular basophilia and hyaline deposits in the tubules, as well as increased urinary protein (100 mg/dL). Morphological signs of glomerulopathy, tubular basophilia, and hyaline deposits in the tubules in females were considered side effects because their combination with a non-side effect, such as an increase in urinary protein, led to a deterioration in kidney function. In males, glomerulopathy was not an adverse effect, since such a sign was much less intense in its distribution, observed in the absence of basophilia and hyaline deposits in the tubules, and was not associated with an increase in urinary protein levels. Glomerulopathy was characterized by the presence of aggregates of granular eosinophilic matter in the glomeruli, present in the renal cortex and observed with an optical microscope, as well as fusion of podocyte base processes surrounding glomerular capillary loops, observed with a transmission electron microscope. At the end of the recovery phase, in males, glomerulopathy completely disappeared and partially disappeared in females and was considered as a non-side effect. Females also showed complete disappearance of tubular basophilia, hyaline deposits in the tubules, and protein in the urine. Higher total serum protein ((1.09×-1.18×) and serum albumin (1.10×-1.20×) was observed in the group of males administered 425 mg/kg/dose IV, and in a group of females treated with a dose of ≥25 mg/kg/dose IV and 425 mg/kg/dose SC This effect was observed in a group of females administered a dose of 425 mg/kg/dose IV, despite an increase in urinary protein levels .

[0309] Additional ROBO2-Fc 2.2-associated signs observed under the microscope were observed in animals that were injected with 425 mg/kg/dose of SC, and consisted of an increase in the frequency and/or severity of (moderate) hemorrhage and inflammation at the injection site compared to with control (from minimal to weak) and in an increase in the frequency of minimal lymphoid hyperplasia in the draining lymph node. An increase in the incidence of hemorrhage and severity of inflammation at the SC injection site was not considered as a side effect, since such effects only slightly exceeded those observed in parallel controls and were not associated with tissue damage, which should be significantly higher, as expected during the injection procedure. An increase in the frequency of minimal lymphoid hyperplasia in the draining lymph node was not considered as a side effect, since, in its morphology, such hyperplasia is similar to control hyperplasia and probably represents a small immune response to ROBO2-Fc 2.2 and/or to the dosing procedure. It is expected that these signs will disappear after the injection is stopped.

[0310] ROBO2-Fc 2.2-associated increase in liver weight observed at the end of the dosing phase was not considered as a side effect and consisted of an increase in the average absolute and relative weight of the body and brain (1.11 × -1.17 × compared with controls) in groups of females treated with ≥25 mg/kg/dose IV and 425 mg/kg/dose SC. This increase in liver mass was not considered as a side effect as there were no correlated macroscopic and microscopic signs and there were no changes in liver enzymes indicative of tissue damage. There was a complete recovery of the mass of the liver.

[0311] Additional changes in clinical pathological parameters of blood coagulation and clinical chemical parameters were not considered as side effects, and such changes consisted of an increase in fibrinogen levels (1.21x-1.48x) in groups of females and males who were dosed ≥125 mg/kg/dose IV, in an increase in cholesterol (1.37×-2.52×) and triglycerides (1.58×-3.11×) in a group of males dosed ≥125 mg/kg/dose IV , and in the female group treated with ≥25 mg/kg/dose IV and 425 mg/kg/dose SC, and in the increase in globulin (1.16×) and calcium (1.09×) in the female group, who were dosed at 425 mg/kg/dose IV. The male and female groups showed complete recovery of fibrinogen, cholesterol, triglycerides, total protein, serum albumin, and globulin levels, but only partial recovery of calcium (1.04× compared to the control group at the end of the recovery phase) in the female group that was dosed 425 mg/kg/dose IV. These clinical pathological changes were not side effects, considering the small differences between the parameters for the group administered ROBO2-Fc 2.2 and the control group, as well as the absence of correlated macroscopic and microscopic signs.

[0312] A systematic description of toxicokinetic data in rats after 3-label administration of ROBO2-Fc 2.2 can be found in Table 22. After IV administration of ROBO2-Fc 2.2 to rats at 25, 125, or 425 mg/kg/dose or after SC administration doses of 425 mg/kg/dose once every 3 days for 3 months (total 31 doses), doses of 125 mg/kg/dose IV and 425 mg/kg/dose SC were identified as NOAELs.

B. Monkey studies

(i) Experimental Toxicity Study (ETS)

[0313] In ETS, male and female cynomolgus monkeys were administered ROBO2-Fc 2.2 once a week for 29 days (5 doses total) at 50 or 200 mg/kg/dose IV or 10 mg/kg/dose SC. Cardiovascular effects were evaluated in telemetrically implanted animals in the 50 mg/kg/dose IV group. On day 30 after the last dose, selected animals were subjected to autopsy. The two monkeys in the 50 mg/kg/dose IV group were kept until day 71 to assess tolerance and toxicokinetic properties. All administered doses of ROBO2-Fc 2.2 were tolerated by the animals. However, no ROBO2-Fc 2.2-associated effects were observed with respect to survival, clinical signs, body weight, food digestibility, cardiovascular parameters, in vivo cytokine assessment, hematological and blood coagulation parameters, and macroscopic and microscopic parameters. ROBO2-Fc 2.2-associated effects included a slight increase in aspartate aminotransferase levels in both females treated with 50 mg/kg/dose IV (1.26x-1.51x vs control) and in male and female animals treated with 200 mg/kg/dose IV (1.59× and 1.55× compared to control, respectively), and a slight increase in alanine aminotransferase levels in the same females administered 50 mg/kg/dose IV (1 .29x-2.05x versus control), and 200 mg/kg/dose IV (1.29x versus control). Elevated levels of these enzymes were not associated with microscopic changes associated with ROBO2-Fc 2.2 in the liver of these animals.

(ii) Repeat dose toxicity studies

[0314] In a pilot study on repeated dose toxicity in cynomolgus monkeys, ROBO2-Fc 2.2 was administered once a week for 3 months (13 doses in total) intravenously at a dose of 20, 100 or 300 mg/kg/dose or subcutaneously at a dose of 300 mg/kg/dose, followed by a 6-week recovery phase (control and 300 mg/kg/dose IV). In this study, no side effects caused by ROBO2-Fc 2.2 were observed at any of the endpoints. However, there were no clinical signs associated with the administration of ROBO2-Fc 2.2, namely, changes in body weight, food digestibility, electrocardiogram/heart rate, hematological parameters, blood coagulation, urinalysis, ophthalmic parameters, organ weight or macroscopic parameters. ROBO2-Fc 2.2-associated changes in clinical chemistry parameters included an increase in cholesterol levels (1.25×-1.61× compared with control) at a dose of 300 mg/kg/dose IV or SC, an increase in triglycerides (1.58× -4.59x vs. control) when administered at a dose of ≥20 mg/kg/dose IV, or 300 mg/kg/dose SC, and decreased globulin levels (0.83x -0.87x vs. control) when administered at a dose of ≥100 mg/kg/dose IV or 300 mg/kg/dose SC. All clinical chemical changes were non-side effects, as indicated by the small magnitude of the changes and the absence of associated tissue changes. Clinical chemical changes completely disappeared, except for the increase in triglycerides, which persisted in males who were dosed at 300 mg/kg/dose IV after the recovery phase (2.78×-6.79× compared to control); however, the quantified concentrations of ROBO2-Fc 2.2 were maintained until day 128/127 of the recovery phase.

[0315] An increased concentration of cells in the lymphoid follicles was observed in the draining lymph nodes (in the left axillary) and in the axillary lymph nodes (right axillary) in animals that were administered a dose of 300 mg/kg/dose of SC. These parameters were characterized by minimal-slight increase in the size and number of lymphoid follicles with predominant formation of the germinal center and corresponded to the response to the antigenic stimulator. These parameters were not considered as side effects due to minimal to moderate severity. It is expected that these signs will disappear after the injection is stopped. In contrast, microscopic signs associated with ROBO2-Fc 2.2 were not observed in animals treated with ROBO2-Fc 2.2 at doses up to 300 mg/kg/dose IV.

[0316] A systematic description of toxicokinetic data for cynomolgus monkeys after 3 months of ROBO2-Fc 2.2 administration can be found in Table 22. After IV administration of ROBO2-Fc 2.2 to monkeys at 20, 100 or 300 mg/kg/dose or after SC- administration of a dose of 300 mg/kg/dose once a week for 3 months (total 13 doses), doses of 300 mg/kg/dose IV and 300 mg/kg/dose SC were identified as NOAEL.

II. Local tolerance

[0317] IV and SC injection sites were assessed microscopically in experimental and pilot studies for repeated dose toxicity in rats and cynomolgus monkeys. No ROBO2-Fc 2.2-associated changes were observed at IV injection sites in rats. ROBO2-Fc 2.2-associated changes were observed only at sites and SC injections in rats. In ETS in rats, minimal-to-moderate subcutaneous perivascular inflammation at ≥10 mg/kg/dose of SC was associated with physical injury at the injection site and was not considered as a side effect. In the pilot study in rats, an increase in the severity of (moderate) hemorrhage was not considered as a side effect, since this effect was only slightly higher than that observed in the parallel control and was not associated with tissue damage, which should be significantly higher, as expected during the injection procedure. . In addition, in a pilot study in rats, ROBO2-Fc 2.2-associated skin lesions (dorsal, thoracic, cranial, or injection site) were observed in males at a dose of 425 mg/kg/dose SC during the administration phase. doses. These changes were not considered as side effects, since their frequency of occurrence was only slightly higher than the frequency in the control group and was observed sporadically during the dosing phase.

[0318] In cynomolgus monkeys, no ROBO2-Fc 2.2-associated changes were observed at IV injection sites. Hemorrhage and/or neutrophilic inflammation of varying severity observed at IV injection sites in most animals, including controls, with ETS at doses up to 200 mg/kg/dose IV was not considered to be associated with ROBO2-Fc 2.2, nor was it no ROBO2-Fc 2.2-associated changes were observed at IV injection sites in a pilot study in cynomolgus monkeys at doses up to 300 mg/kg/IV dose. No ROBO2-Fc 2.2-associated changes were observed at the sites of SC injections in monkeys.

III. Cytokine release assays

[0319] In an in vitro cytokine release assay (CRA) performed using human whole blood samples, ROBO2-Fc 2.2 promoted the production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in whole blood samples taken from 1 in 8 human donors participating in the trial. In human whole blood samples incubated with ROBO2-Fc 2.2, no ROBO2-Fc 2.2-associated release of interferon-gamma (IFN-γ) was observed.

[0320] In an in vitro CRA assay using pre-dose whole blood samples from cynomolgus monkeys, ROBO2-Fc 2.2 stimulated IL-6 production in whole blood samples from 1 of 12 cynomolgus monkeys tested and however, no ROBO2-Fc 2.2-associated release of TNF-α or IFN-γ was observed.

[0321] In addition, blood samples were taken from cynomolgus monkeys in ETS after administration of ROBO2-Fc 2.2 once a week at 10 mg/kg/dose SC or 50 or 200 mg/kg/dose IV to characterize changes in cytokine concentrations. No ROBO2-Fc 2.2-associated changes in serum TNF-α, IL-6 or IFN-γ concentrations were observed.

IV. C1q and FcγR binding assays

[0322] ROBO2-Fc 2.2 was evaluated in vitro in a Complement 1q (C1q) binding ELISA to test its ability to produce complement-dependent cytotoxicity (CDC) and in a crystallizable gamma fragment receptor (FcγR) binding assay to test its ability to produce antibody-dependent cell-mediated cytotoxicity (ADCC). ROBO2-Fc 2.2 did not bind to C1q at the concentrations tested and was therefore considered to have a low ability to induce CDC. The binding of ROBO2-Fc 2.2 to all FcγRs tested was similar to or lower than that observed for the test control antibody, and lower than the binding to the positive control antibody. These data suggest that ROBO2-Fc 2.2 has a low ability to generate ADCC activity.

V. Interrelation of changes and pharmacokinetics

[0323] Exposure to ROBO2-Fc 2.2 (measured by C _max and AUC) increased with increasing dose in approximately direct proportion after repeated administration of IV and SC doses to rats and cynomolgus monkeys over the dose range tested. No clear sex-associated differences in observed effects were found. Cut-off concentrations of ROBO2-Fc 2.2 associated with key responses and calculated exposure limits compared to these key responses can be found in Table 22.

Table 22. ROBO2-Fc 2.2 Concentrations Associated with Key Responses

Key answer(s) Dose (mg/kg/day) C _max ^a (µg/ml) AUC _last ^a (µg*h/ml) C _max Border exposure ^b AUC _last Borderline exposure ^b 14-day IV/SC ETS repeated dose toxicity studies in male rats (13MA059; 5/group) Target organs: SC injection site: perivascular inflammation, lymphoid tissues: ↑ thymus mass 10SC 33.7 2040 1.9 <1 Same as described above 50SC 73.7 3820 4.1 1.5 Same as above plus Kidney: ↑ urine creatinine, SG, ↑ urine volume 200SC 245 12500 13 4.8 Same as above plus Liver: ↑CHOL 200IV 6550 68600 360 26 3-month IV/SC toxicity study in rats with 6-week recovery (16GR156; 10 or 15/sex/group) Target Organs: Liver: ↑ CHOL (F), TRIG (F) Other results: ↑ ALB (F) 25IV 674 20900 37 8.0 Kidney: glomerulopathy (M) Liver: ↑ CHOL (M), TRIG (M), mass (F) Other results: ↑ FIB ↑ TP (F) 125IV (NOAEL) 2930 74500 161 29 Same as above plus Kidney: glomerulopathy (F), tubular basophilia (F), hyaline sheath (F), ↑ urine protein (F) Liver: ↑ CHOL (M), TRIG (M) Other results: ↑ GLOB (F), CA (F), FIB, TP (M), ALB (M) Recovery: partial resolution of glomerulopathy and CA(F), otherwise complete 425IV 7610 199000 418 76 SC injection site: inflammation and hemorrhage Liver: ↑ CHOL (F), TRIG (F), mass (F) Lymphoid tissues: ↑ lymphoid hyperplasia in draining lymph nodes Other findings: ↑ ALB (F), skin lesions (M) 425SC (NOAEL) 842 50500 46 19

Table 22. ROBO2-Fc 2.2 Concentrations Associated with Key Responses (continued)

Key answer(s) Dose (mg/kg/day) C _max ^a (µg/ml) AUC _last ^a (µg*h/ml) C _max Border exposure ^b AUC _last Borderline exposure ^b 29-day IV ETS studies with telemetric evaluation of monkeys in the SC group (14MA014; 1 or 2/gender/group) Target Organs: Liver: ↑ AST (F), ALT (F) 50IV 1050 19100 58 7.3 Same as above Liver: ↑ AST (M) 200IV 6420 96200 353 37 3-month monkey IV/SC toxicity study with 6-week recovery (16GR143; 3 or 5/sex/group) Target Organs: Liver: ↑TRIG 20IV 470 24300 26 9.3 Same as above plus Liver: ↑ GLOB 100IV 2350 113000 129 43 Same as above plus Liver: ↑ CHOL Recovery: ↑ TRIG; full analysis on GLOB and CHOL 300IV (NOAEL) 9210 427000 506 164 Lymphoid tissues: ↑ cell colonization of lymphoid follicles Liver: ↑TRIG, CHOL, ↑ GLOB 300SC (NOAEL) 2340 328000 129 126 ALB=albumin; ALT = alanine aminotransferase; AST = aspartate aminotransferase; AUC = area under the concentration-time curve; CHOL = cholesterol; C _max = maximum (mean) plasma concentration; ETS = experimental toxicity study; F = females; FIB = fibrinogen; GLOB = globulin; IV = intravenously;
M = males; NOAEL = no observed side effects; SC=subcutaneous; SG = specific gravity; TP = total protein; TRIG=triglyceride.
A. AUC and C _max values mean mean serum concentrations. The registered values were obtained almost at the end of the experiment.
b. Breakpoint exposures were calculated in animal toxicity studies by dividing C _max and AUC _last by the experimental human C _max of 18.2 µg/mL and AUC _last , = 1610 µg*h/mL at the experimental human effective dose. 2 mg/kg [150 mg] administered by SC weekly.

[0324] In general, these data suggest that ROBO2-Fc 2.2 is a therapeutic agent that can be used to treat various diseases, disorders and conditions in humans.

[0325] Thus, the present invention has been widely disclosed and illustrated with reference to the representative embodiments of the invention described above. A person skilled in the art will appreciate that various modifications can be made to the present invention without departing from the spirit and scope of the invention. All publications, patent applications and granted patents are incorporated herein by reference, as if each individual publication, patent application or patent granted were specifically and separately incorporated herein by reference in their entirety. Definitions in the text incorporated by reference should be excluded if they conflict with the definitions given in this application.

[0326] It should be noted that certain features of the invention, which are described for clarity in the context of separate embodiments of the invention, may also be presented in combination in one embodiment of the invention. Conversely, various features of the invention, which for brevity are described in the context of one embodiment of the invention, may also be presented separately or in any suitable subcombination.

[0327] In particular, it is contemplated that any limitation discussed for one embodiment of the invention may be applicable to any other embodiment of the invention. In addition, any composition according to the invention can be used in any method according to the invention, and any method according to the invention can be used to obtain or use any composition according to the invention. In particular, any aspect of the invention described in the claims and taken alone or in combination with one or more additional claims and/or aspects of the description should be considered as aspects that can be combined with other aspects of the invention presented in the claims, and/or in the description of the invention, and/or in the sequence listings, and/or in the description of the drawings.

[0328] If the specific examples given here are not included in the scope of the present invention, then the specified specific example can be completely excluded from the claims.

[0329] As used herein, the conjunction "or" means "and/or", and unless otherwise noted, has only a separative sense or mutually exclusive sense, although in the present description there are definitions that have only a separative sense and "and/or". As used herein, the articles "a" or "an" may mean one or more unless otherwise noted. The articles "a" or "an", when used in a claim in conjunction with the word "comprising", may mean one or more than one. As used herein, the word "other" may mean at least a second or more. Unless specifically defined in the present invention, the scientific and technical terms used in the present invention have their generally accepted meaning, understandable to a person skilled in the art. In addition, if it does not contradict the context of the invention, then terms used in the singular may refer to plural nouns, and terms used in the plural may refer to nouns in the singular. The words "comprises/comprising" and the words "having/comprising", when used in the present invention, indicate the specific presence of the claimed features, integers, steps or components, but do not exclude the presence or addition of one or more other features, integers, stages, components or groups.

[0330] Although the description disclosed herein is given with reference to various uses, methods and compositions, however, it is obvious that various changes and modifications can be made to the present invention without going beyond the scope of the invention described here and the following claims. The examples are given to better illustrate the disclosed description and are not construed as limiting the scope of the invention presented here. Although the present description is based on these representative embodiments of the invention, however, various changes and modifications can be made to it, which can be carried out without undue experimentation. All such changes and modifications are within the scope of the present invention.

[0331] If aspects or embodiments of the invention are described in terms of the "Markush group" or other alternative groups, then the present invention covers not only the whole group, presented as a whole, but also as each individual member of this group and all possible subgroups of the main group, and as a main group that does not contain one or more members of this group. The present invention also considers the complete exclusion of one or more of any members of the group present in the claimed invention.

[0332] All documents cited herein, including patents, patent applications, articles, manuals, and the like. and the references cited therein, if they have not already been introduced, are incorporated herein in their entirety. In the event that one or more of the included documents and similar materials differ from or contradict the documents and materials of this application, including, but not limited to, certain terms, the use of these terms, the methods described, or the like, then preference should be given to the terms used in this application.

[0333] In the description and in the examples, some specific embodiments of the invention are presented in detail and the best method considered by the inventors is described. However, it should be noted that the description of the invention may not give details of these options, and therefore the present invention can be carried out by any means and should be considered in accordance with the attached claims and any equivalents thereof.

Table 23. Sequences

SEQ Description Subsequence 1 ROBO2FC 2.2 SRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 2 ROBO2FC 2.1 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 ROBO2FC 2.0 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 ROBO2FC 1.1 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5 ROBO2FC 1.0 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 6 ROBO2FC 3.0 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 7 ROBO2FC 4.0 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 8 Pre-Ig1 ROBO2 sequence SRLRQEDFP 9 ROBO2 Ig1 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLE 10 ROBO2 Ig12 linker VALLR eleven ROBO2 Ig2 DDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELT 12 Ig23 linker ROBO2 VFER 13 ROBO2 Ig3 PTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLT 14 ROBO2 Ig4 VRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVT 15 Linker GS GGSGGS 16 Fc3mut IgG1 EPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTTPPGVLDSDGSFFLYSKLTVQHLS 17 ROBO2 leader sequence MSLLMFTQLLLCGFLYVRVDG 18 Ig leader sequence MGWSCIIFLVATAGAHS 19 ROBO2Fc S17T/R73Y SRLRQEDFPPRIVEHPTDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLYIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20 ROBO2 Ig1 S17T R73Y PRIVEHPTDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLYIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLE 21 ROBO2FC 2.2 TCGCGTCTTCGCCAGGAGGACTTTCCCCCGCGGATTGTGGAGCATCCTTCCGATGTCATCGTCTCTAAGGGCGAGCCCACGACTCTGAACTGCAAGGCGGAGGGCCGGCCAACGCCCACCATTGAGTGGTACAAAGATGGGGAGCGAGTGGAGACTGACAAGGACGATCCCCGGTCCCACAGGATGCTTCTGCCCAGCGGATCCTTATTCTTCTTGCGCATCGTGCACGGGCGCAGGAGTAAACCTGATGAAGGAAGCTACGTTTGTGTTGCGAGGAACTATCTTGGTGAAGCAGTGAGTCGAAATGCGTCTCTGGAAGTGGCATTGTTACGAGATGACTTCCGACAAAACCCCACAGATGTTGTAGTGGCAGCTGGAGAGCCTGCAATCCTGGAGTGCCAGCCTCCCCGGGGACACCCAGAACCCACCATCTACTGGAAAAAAGACAAAGTTCGAATTGATGACAAGGAAGAAAGAATAAGTATCCGTGGTGGAAAACTGATGATCTCCAATACCAGGAAAAGTGATGCAGGGATGTATACTTGTGTTGGTACCAATATGGTGGGAGAAAGGGACAGTGACCCAGCAGAGCTGACTGTCTTTGAACGAGGCGGCAGCGGCGGCAGCGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA 22 Peptidyl linker [GlyGlySer] _n, n=1, 2, 3, 4, 5, or 6 23 Peptidyl linker [GlyGlyGlyGlySer] _n , n=1, 2, 3, 4, 5 or 6 24 Human ROBO2
(ROBO2 leader sequence underlined) MSLLMFTQLLLCGFLYVRVDG

Table 24. ID Sequence Explanations

Construction of ROBO2Fc Complete construction ROBO2Fct component Leader Sequence SRLRQEDFP (SEQ ID NO: 8) Ig1 Linker Ig12 Ig2 Linker Ig23 Ig3 Ig4 Linker GS IgG1 Fc 3mut ROBO2FC 1.0 5 18 X 9 X X X X X 15 16 ROBO2FC 1.1 4 18 X 9 10 X X X X 15 16 ROBO2FC 2.0 3 18 X 9 10 eleven X X X 15 16 ROBO2FC 2.1 2 18 X 9 10 eleven 12 X X 15 16 ROBO2FC 2.2 1 17 8 9 10 eleven 12 X X 15 16 ROBO2FC 3.0 6 18 X 9 10 eleven 12 13 X 15 16 ROBO2FC 4.0 7 18 X 9 10 eleven 12 13 14 15 16 ROBO2Fc S17T R73Y 19 17 8 20 10 eleven 12 X X 15 16

X = Component that is not part of the structure.

--->

SEQUENCE LIST

<110> PFIZER INC.

BOSTON MEDICAL CENTER CORPORATION

<120> RECOMBINANT ROBO2 PROTEINS, COMPOSITIONS, METHODS AND THEIR

APPLICATION

<130> PCFC-0043-WO1

<140>

<141>

<150> 62/663.082

<151> 2018-04-26

<150> 62/514.242

<151> 2017-06-02

<160> 25

<170>PatentIn version 3.5

<210> 1

<211> 440

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 1

Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val Glu His Pro

1 5 10 15

Ser Asp Val Ile Val Ser Lys Gly Glu Pro Thr Thr Leu Asn Cys Lys

20 25 30

Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys Asp Gly Glu

35 40 45

Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg Met Leu Leu

50 55 60

Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val His Gly Arg Arg Ser

65 70 75 80

Lys Pro Asp Glu Gly Ser Tyr Val Cys Val Ala Arg Asn Tyr Leu Gly

85 90 95

Glu Ala Val Ser Arg Asn Ala Ser Leu Glu Val Ala Leu Leu Arg Asp

100 105 110

Asp Phe Arg Gln Asn Pro Thr Asp Val Val Val Ala Ala Gly Glu Pro

115 120 125

Ala Ile Leu Glu Cys Gln Pro Pro Arg Gly His Pro Glu Pro Thr Ile

130 135 140

Tyr Trp Lys Lys Asp Lys Val Arg Ile Asp Asp Lys Glu Glu Arg Ile

145 150 155 160

Ser Ile Arg Gly Gly Lys Leu Met Ile Ser Asn Thr Arg Lys Ser Asp

165 170 175

Ala Gly Met Tyr Thr Cys Val Gly Thr Asn Met Val Gly Glu Arg Asp

180 185 190

Ser Asp Pro Ala Glu Leu Thr Val Phe Glu Arg Gly Gly Ser Gly Gly

195 200 205

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

210 215 220

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

340 345 350

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 2

<211> 432

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 2

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Val Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val

100 105 110

Val Val Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg

115 120 125

Gly His Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile

130 135 140

Asp Asp Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile

145 150 155 160

Ser Asn Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr

165 170 175

Asn Met Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Val Phe

180 185 190

Glu Arg Gly Gly Ser Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser

210 215 220

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

225 230 235 240

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

245 250 255

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

260 265 270

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

275 280 285

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

290 295 300

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

305 310 315 320

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

325 330 335

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

385 390 395 400

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

420 425 430

<210> 3

<211> 428

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 3

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Val Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val

100 105 110

Val Val Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg

115 120 125

Gly His Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile

130 135 140

Asp Asp Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile

145 150 155 160

Ser Asn Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr

165 170 175

Asn Met Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Gly Gly

180 185 190

Ser Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro

195 200 205

Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe

210 215 220

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

225 230 235 240

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

245 250 255

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

260 265 270

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

275 280 285

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

290 295 300

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

305 310 315 320

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

325 330 335

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

340 345 350

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

355 360 365

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

370 375 380

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

385 390 395 400

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

405 410 415

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

420 425

<210> 4

<211> 340

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 4

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Val Ala Leu Leu Arg Gly Gly Ser Gly Gly Ser Glu Pro Lys Ser

100 105 110

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

115 120 125

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

130 135 140

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

145 150 155 160

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

165 170 175

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

180 185 190

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

195 200 205

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

210 215 220

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

225 230 235 240

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

245 250 255

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

260 265 270

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

275 280 285

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

290 295 300

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

305 310 315 320

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

325 330 335

Ser Pro Gly Lys

340

<210> 5

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 5

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Gly Gly Ser Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His

100 105 110

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

115 120 125

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

130 135 140

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

145 150 155 160

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

165 170 175

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

180 185 190

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

195 200 205

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

210 215 220

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

225 230 235 240

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

245 250 255

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

260 265 270

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

275 280 285

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

290 295 300

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

305 310 315 320

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330 335

<210> 6

<211> 517

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 6

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Val Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val

100 105 110

Val Val Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg

115 120 125

Gly His Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile

130 135 140

Asp Asp Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile

145 150 155 160

Ser Asn Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr

165 170 175

Asn Met Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Val Phe

180 185 190

Glu Arg Pro Thr Phe Leu Arg Arg Pro Ile Asn Gln Val Val Leu Glu

195 200 205

Glu Glu Ala Val Glu Phe Arg Cys Gln Val Gln Gly Asp Pro Gln Pro

210 215 220

Thr Val Arg Trp Lys Lys Asp Asp Ala Asp Leu Pro Arg Gly Arg Tyr

225 230 235 240

Asp Ile Lys Asp Asp Tyr Thr Leu Arg Ile Lys Lys Thr Met Ser Thr

245 250 255

Asp Glu Gly Thr Tyr Met Cys Ile Ala Glu Asn Arg Val Gly Lys Met

260 265 270

Glu Ala Ser Ala Thr Leu Thr Gly Gly Ser Gly Gly Ser Glu Pro Lys

275 280 285

Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

290 295 300

Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

305 310 315 320

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

325 330 335

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

340 345 350

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

355 360 365

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

370 375 380

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

385 390 395 400

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

405 410 415

Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

420 425 430

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

435 440 445

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

450 455 460

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

465 470 475 480

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

485 490 495

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

500 505 510

Leu Ser Pro Gly Lys

515

<210> 7

<211> 617

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 7

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu Val Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val

100 105 110

Val Val Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg

115 120 125

Gly His Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile

130 135 140

Asp Asp Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile

145 150 155 160

Ser Asn Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr

165 170 175

Asn Met Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Val Phe

180 185 190

Glu Arg Pro Thr Phe Leu Arg Arg Pro Ile Asn Gln Val Val Leu Glu

195 200 205

Glu Glu Ala Val Glu Phe Arg Cys Gln Val Gln Gly Asp Pro Gln Pro

210 215 220

Thr Val Arg Trp Lys Lys Asp Asp Ala Asp Leu Pro Arg Gly Arg Tyr

225 230 235 240

Asp Ile Lys Asp Asp Tyr Thr Leu Arg Ile Lys Lys Thr Met Ser Thr

245 250 255

Asp Glu Gly Thr Tyr Met Cys Ile Ala Glu Asn Arg Val Gly Lys Met

260 265 270

Glu Ala Ser Ala Thr Leu Thr Val Arg Ala Pro Pro Gln Phe Val Val

275 280 285

Arg Pro Arg Asp Gln Ile Val Ala Gln Gly Arg Thr Val Thr Phe Pro

290 295 300

Cys Glu Thr Lys Gly Asn Pro Gln Pro Ala Val Phe Trp Gln Lys Glu

305 310 315 320

Gly Ser Gln Asn Leu Leu Phe Pro Asn Gln Pro Gln Gln Pro Asn Ser

325 330 335

Arg Cys Ser Val Ser Pro Thr Gly Asp Leu Thr Ile Thr Asn Ile Gln

340 345 350

Arg Ser Asp Ala Gly Tyr Tyr Ile Cys Gln Ala Leu Thr Val Ala Gly

355 360 365

Ser Ile Leu Ala Lys Ala Gln Leu Glu Val Thr Gly Gly Ser Gly Gly

370 375 380

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

385 390 395 400

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

405 410 415

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

420 425 430

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

435 440 445

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

450 455 460

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

465 470 475 480

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

485 490 495

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

500 505 510

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

515 520 525

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

530 535 540

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

545 550 555 560

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

565 570 575

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

580 585 590

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

595 600 605

Lys Ser Leu Ser Leu Ser Pro Gly Lys

610 615

<210> 8

<211> 9

<212> PRT

<213> Homo sapiens

<400> 8

Ser Arg Leu Arg Gln Glu Asp Phe Pro

15

<210> 9

<211> 97

<212> PRT

<213> Homo sapiens

<400> 9

Pro Arg Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu

<210> 10

<211> 5

<212> PRT

<213> Homo sapiens

<400> 10

Val Ala Leu Leu Arg

15

<210> 11

<211> 88

<212> PRT

<213> Homo sapiens

<400> 11

Asp Asp Phe Arg Gln Asn Pro Thr Asp Val Val Val Ala Ala Gly Glu

1 5 10 15

Pro Ala Ile Leu Glu Cys Gln Pro Arg Gly His Pro Glu Pro Thr

20 25 30

Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile Asp Asp Lys Glu Glu Arg

35 40 45

Ile Ser Ile Arg Gly Gly Lys Leu Met Ile Ser Asn Thr Arg Lys Ser

50 55 60

Asp Ala Gly Met Tyr Thr Cys Val Gly Thr Asn Met Val Gly Glu Arg

65 70 75 80

Asp Ser Asp Pro Ala Glu Leu Thr

85

<210> 12

<211> 4

<212> PRT

<213> Homo sapiens

<400> 12

Val Phe Glu Arg

1

<210> 13

<211> 85

<212> PRT

<213> Homo sapiens

<400> 13

Pro Thr Phe Leu Arg Arg Pro Ile Asn Gln Val Val Leu Glu Glu Glu

1 5 10 15

Ala Val Glu Phe Arg Cys Gln Val Gln Gly Asp Pro Gln Pro Thr Val

20 25 30

Arg Trp Lys Lys Asp Asp Ala Asp Leu Pro Arg Gly Arg Tyr Asp Ile

35 40 45

Lys Asp Asp Tyr Thr Leu Arg Ile Lys Lys Thr Met Ser Thr Asp Glu

50 55 60

Gly Thr Tyr Met Cys Ile Ala Glu Asn Arg Val Gly Lys Met Glu Ala

65 70 75 80

Ser Ala Thr Leu Thr

85

<210> 14

<211> 100

<212> PRT

<213> Homo sapiens

<400> 14

Val Arg Ala Pro Pro Gln Phe Val Val Arg Pro Arg Asp Gln Ile Val

1 5 10 15

Ala Gln Gly Arg Thr Val Thr Phe Pro Cys Glu Thr Lys Gly Asn Pro

20 25 30

Gln Pro Ala Val Phe Trp Gln Lys Glu Gly Ser Gln Asn Leu Leu Phe

35 40 45

Pro Asn Gln Pro Gln Gln Pro Asn Ser Arg Cys Ser Val Ser Pro Thr

50 55 60

Gly Asp Leu Thr Ile Thr Asn Ile Gln Arg Ser Asp Ala Gly Tyr Tyr

65 70 75 80

Ile Cys Gln Ala Leu Thr Val Ala Gly Ser Ile Leu Ala Lys Ala Gln

85 90 95

Leu Glu Val Thr

100

<210> 15

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

peptide"

<400> 15

Gly Gly Ser Gly Gly Ser

15

<210> 16

<211> 232

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 16

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 17

<211> 21

<212> PRT

<213> Homo sapiens

<400> 17

Met Ser Leu Leu Met Phe Thr Gln Leu Leu Leu Cys Gly Phe Leu Tyr

1 5 10 15

Val Arg Val Asp Gly

20

<210> 18

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

peptide"

<400> 18

Met Gly Trp Ser Cys Ile Ile Phe Leu Val Ala Thr Ala Gly Ala His

1 5 10 15

Ser

<210> 19

<211> 441

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 19

Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg Ile Val Glu His Pro

1 5 10 15

Thr Asp Val Ile Val Ser Lys Gly Glu Pro Thr Thr Leu Asn Cys Lys

20 25 30

Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp Tyr Lys Asp Gly Glu

35 40 45

Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser His Arg Met Leu Leu

50 55 60

Pro Ser Gly Ser Leu Phe Phe Leu Tyr Ile Val His Gly Arg Arg Ser

65 70 75 80

Lys Pro Asp Glu Gly Ser Tyr Val Cys Val Ala Arg Asn Tyr Leu Gly

85 90 95

Glu Ala Val Ser Arg Asn Ala Ser Leu Glu Val Ala Leu Leu Arg Asp

100 105 110

Asp Phe Arg Gln Asn Pro Thr Asp Val Val Val Ala Ala Gly Glu Pro

115 120 125

Ala Ile Leu Glu Cys Gln Pro Pro Arg Gly His Pro Glu Pro Thr Ile

130 135 140

Tyr Trp Lys Lys Asp Lys Val Arg Ile Asp Asp Lys Glu Glu Arg Ile

145 150 155 160

Ser Ile Arg Gly Gly Lys Leu Met Ile Ser Asn Thr Arg Lys Ser Asp

165 170 175

Ala Gly Met Tyr Thr Cys Val Gly Thr Asn Met Val Gly Glu Arg Asp

180 185 190

Ser Asp Pro Ala Glu Leu Thr Val Phe Glu Arg Gly Gly Ser Gly Gly

195 200 205

Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

210 215 220

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

340 345 350

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 20

<211> 97

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<400> 20

Pro Arg Ile Val Glu His Pro Thr Asp Val Ile Val Ser Lys Gly Glu

1 5 10 15

Pro Thr Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile

20 25 30

Glu Trp Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro

35 40 45

Arg Ser His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Tyr

50 55 60

Ile Val His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys

65 70 75 80

Val Ala Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu

85 90 95

Glu

<210> 21

<211> 1320

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polynucleotide"

<400> 21

tcgcgtcttc gccaggagga ctttcccccg cggattgtgg agcatccttc cgatgtcatc 60

gtctctaagg gcgagcccac gactctgaac tgcaaggcgg agggccggcc aacgcccacc 120

attgagtggt acaaagatgg ggagcgagtg gagactgaca aggacgatcc ccggtcccac 180

aggatgcttc tgcccagcgg atccttattc ttcttgcgca tcgtgcacgg gcgcaggagt 240

aaacctgatg aaggaagcta cgtttgtgtt gcgaggaact atcttggtga agcagtgagt 300

cgaaatgcgt ctctggaagt ggcattgtta cgagatgact tccgacaaaa ccccacagat 360

gttgtagtgg cagctggaga gcctgcaatc ctggagtgcc agcctccccg gggacaccca 420

gaacccacca tctactggaa aaaagacaaa gttcgaattg atgacaagga agaaagaata 480

agtatccgtg gtggaaaact gatgatctcc aataccagga aaagtgatgc agggatgtat 540

acttgtgttg gtaccaatat ggtgggagaa agggacagtg acccagcaga gctgactgtc 600

tttgaacgag gcggcagcgg cggcagcgag cccaaatctt ctgacaaaac tcacacatgc 660

ccaccgtgcc cagcacctga agctgcaggg gcaccgtcag tcttcctctt ccccccaaaa 720

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 780

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 840

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 900

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 960

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1020

caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt cagcctgacc 1080

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1140

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200

tatagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1260

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtccccggga 1320

<210> 22

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

peptide"

<220>

<221> MISC_FEATURE

<222> (1)..(18)

<223> /note="This sequence can span 1-6

repeating links 'Gly Gly Ser'"

<400> 22

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 23

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial Sequence Description: Synthetic

polypeptide"

<220>

<221> MISC_FEATURE

<222> (1)..(30)

<223> /note="This sequence can span 1-6

repeating links 'Gly Gly Gly Gly Ser'

<400> 23

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 24

<211> 1378

<212> PRT

<213> Homo sapiens

<400> 24

Met Ser Leu Leu Met Phe Thr Gln Leu Leu Leu Cys Gly Phe Leu Tyr

1 5 10 15

Val Arg Val Asp Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg

20 25 30

Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu Pro Thr

35 40 45

Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp

50 55 60

Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser

65 70 75 80

His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val

85 90 95

His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys Val Ala

100 105 110

Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu Glu Val

115 120 125

Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val Val Val

130 135 140

Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg Gly His

145 150 155 160

Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile Asp Asp

165 170 175

Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile Ser Asn

180 185 190

Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr Asn Met

195 200 205

Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Val Phe Glu Arg

210 215 220

Pro Thr Phe Leu Arg Arg Pro Ile Asn Gln Val Val Leu Glu Glu Glu

225 230 235 240

Ala Val Glu Phe Arg Cys Gln Val Gln Gly Asp Pro Gln Pro Thr Val

245 250 255

Arg Trp Lys Lys Asp Asp Ala Asp Leu Pro Arg Gly Arg Tyr Asp Ile

260 265 270

Lys Asp Asp Tyr Thr Leu Arg Ile Lys Lys Thr Met Ser Thr Asp Glu

275 280 285

Gly Thr Tyr Met Cys Ile Ala Glu Asn Arg Val Gly Lys Met Glu Ala

290 295 300

Ser Ala Thr Leu Thr Val Arg Ala Pro Pro Gln Phe Val Val Arg Pro

305 310 315 320

Arg Asp Gln Ile Val Ala Gln Gly Arg Thr Val Thr Phe Pro Cys Glu

325 330 335

Thr Lys Gly Asn Pro Gln Pro Ala Val Phe Trp Gln Lys Glu Gly Ser

340 345 350

Gln Asn Leu Leu Phe Pro Asn Gln Pro Gln Gln Pro Asn Ser Arg Cys

355 360 365

Ser Val Ser Pro Thr Gly Asp Leu Thr Ile Thr Asn Ile Gln Arg Ser

370 375 380

Asp Ala Gly Tyr Tyr Ile Cys Gln Ala Leu Thr Val Ala Gly Ser Ile

385 390 395 400

Leu Ala Lys Ala Gln Leu Glu Val Thr Asp Val Leu Thr Asp Arg Pro

405 410 415

Pro Pro Ile Ile Leu Gln Gly Pro Ala Asn Gln Thr Leu Ala Val Asp

420 425 430

Gly Thr Ala Leu Leu Lys Cys Lys Ala Thr Gly Asp Pro Leu Pro Val

435 440 445

Ile Ser Trp Leu Lys Glu Gly Phe Thr Phe Pro Gly Arg Asp Pro Arg

450 455 460

Ala Thr Ile Gln Glu Gln Gly Thr Leu Gln Ile Lys Asn Leu Arg Ile

465 470 475 480

Ser Asp Thr Gly Thr Tyr Thr Cys Val Ala Thr Ser Ser Ser Ser Gly Glu

485 490 495

Thr Ser Trp Ser Ala Val Leu Asp Val Thr Glu Ser Gly Ala Thr Ile

500 505 510

Ser Lys Asn Tyr Asp Leu Ser Asp Leu Pro Gly Pro Pro Ser Lys Pro

515 520 525

Gln Val Thr Asp Val Thr Lys Asn Ser Val Thr Leu Ser Trp Gln Pro

530 535 540

Gly Thr Pro Gly Thr Leu Pro Ala Ser Ala Tyr Ile Ile Glu Ala Phe

545 550 555 560

Ser Gln Ser Val Ser Asn Ser Trp Gln Thr Val Ala Asn His Val Lys

565 570 575

Thr Thr Leu Tyr Thr Val Arg Gly Leu Arg Pro Asn Thr Ile Tyr Leu

580 585 590

Phe Met Val Arg Ala Ile Asn Pro Gln Gly Leu Ser Asp Pro Ser Pro

595 600 605

Met Ser Asp Pro Val Arg Thr Gln Asp Ile Ser Pro Pro Ala Gln Gly

610 615 620

Val Asp His Arg Gln Val Gln Lys Glu Leu Gly Asp Val Leu Val Arg

625 630 635 640

Leu His Asn Pro Val Val Leu Thr Pro Thr Thr Val Gln Val Thr Trp

645 650 655

Thr Val Asp Arg Gln Pro Gln Phe Ile Gln Gly Tyr Arg Val Met Tyr

660 665 670

Arg Gln Thr Ser Gly Leu Gln Ala Thr Ser Ser Trp Gln Asn Leu Asp

675 680 685

Ala Lys Val Pro Thr Glu Arg Ser Ala Val Leu Val Asn Leu Lys Lys

690 695 700

Gly Val Thr Tyr Glu Ile Lys Val Arg Pro Tyr Phe Asn Glu Phe Gln

705 710 715 720

Gly Met Asp Ser Glu Ser Lys Thr Val Arg Thr Thr Glu Glu Ala Pro

725 730 735

Ser Ala Pro Pro Gln Ser Val Thr Val Leu Thr Val Gly Ser Tyr Asn

740 745 750

Ser Thr Ser Ile Ser Val Ser Trp Asp Pro Pro Pro Pro Asp His Gln

755 760 765

Asn Gly Ile Ile Gln Glu Tyr Lys Ile Trp Cys Leu Gly Asn Glu Thr

770 775 780

Arg Phe His Ile Asn Lys Thr Val Asp Ala Ala Ile Arg Ser Val Ile

785 790 795 800

Ile Gly Gly Leu Phe Pro Gly Ile Gln Tyr Arg Val Glu Val Ala Ala

805 810 815

Ser Thr Ser Ala Gly Val Gly Val Lys Ser Glu Pro Gln Pro Ile Ile

820 825 830

Ile Gly Arg Arg Asn Glu Val Val Ile Thr Glu Asn Asn Asn Ser Ile

835 840 845

Thr Glu Gln Ile Thr Asp Val Val Lys Gln Pro Ala Phe Ile Ala Gly

850 855 860

Ile Gly Gly Ala Cys Trp Val Ile Leu Met Gly Phe Ser Ile Trp Leu

865 870 875 880

Tyr Trp Arg Arg Lys Lys Arg Lys Gly Leu Ser Asn Tyr Ala Val Thr

885 890 895

Phe Gln Arg Gly Asp Gly Gly Leu Met Ser Asn Gly Ser Arg Pro Gly

900 905 910

Leu Leu Asn Ala Gly Asp Pro Ser Tyr Pro Trp Leu Ala Asp Ser Trp

915 920 925

Pro Ala Thr Ser Leu Pro Val Asn Asn Ser Asn Ser Gly Pro Asn Glu

930 935 940

Ile Gly Asn Phe Gly Arg Gly Asp Val Leu Pro Pro Val Pro Gly Gln

945 950 955 960

Gly Asp Lys Thr Ala Thr Met Leu Ser Asp Gly Ala Ile Tyr Ser Ser

965 970 975

Ile Asp Phe Thr Thr Lys Thr Ser Tyr Asn Ser Ser Ser Gln Ile Thr

980 985 990

Gln Ala Thr Pro Tyr Ala Thr Thr Gln Ile Leu His Ser Asn Ser Ile

995 1000 1005

His Glu Leu Ala Val Asp Leu Pro Asp Pro Gln Trp Lys Ser Ser

1010 1015 1020

Ile Gln Gln Lys Thr Asp Leu Met Gly Phe Gly Tyr Ser Leu Pro

1025 1030 1035

Asp Gln Asn Lys Gly Asn Asn Gly Gly Lys Gly Gly Lys Lys Lys

1040 1045 1050

Lys Asn Lys Asn Ser Ser Lys Pro Gln Lys Asn Asn Gly Ser Thr

1055 1060 1065

Trp Ala Asn Val Pro Leu Pro Pro Pro Pro Val Gln Pro Leu Pro

1070 1075 1080

Gly Thr Glu Leu Glu His Tyr Ala Val Glu Gln Gln Glu Asn Gly

1085 1090 1095

Tyr Asp Ser Asp Ser Trp Cys Pro Pro Leu Pro Val Gln Thr Tyr

1100 1105 1110

Leu His Gln Gly Leu Glu Asp Glu Leu Glu Glu Asp Asp Asp Arg

1115 1120 1125

Val Pro Thr Pro Pro Val Arg Gly Val Ala Ser Ser Pro Ala Ile

1130 1135 1140

Ser Phe Gly Gln Gln Ser Thr Ala Thr Leu Thr Pro Ser Pro Arg

1145 1150 1155

Glu Glu Met Gln Pro Met Leu Gln Ala His Leu Asp Glu Leu Thr

1160 1165 1170

Arg Ala Tyr Gln Phe Asp Ile Ala Lys Gln Thr Trp His Ile Gln

1175 1180 1185

Ser Asn Asn Gln Pro Pro Gln Pro Pro Val Pro Pro Leu Gly Tyr

1190 1195 1200

Val Ser Gly Ala Leu Ile Ser Asp Leu Glu Thr Asp Val Ala Asp

1205 1210 1215

Asp Asp Ala Asp Asp Glu Glu Glu Ala Leu Glu Ile Pro Arg Pro

1220 1225 1230

Leu Arg Ala Leu Asp Gln Thr Pro Gly Ser Ser Met Asp Asn Leu

1235 1240 1245

Asp Ser Ser Val Thr Gly Lys Ala Phe Thr Ser Ser Gln Arg Pro

1250 1255 1260

Arg Pro Thr Ser Pro Phe Ser Thr Asp Ser Asn Thr Ser Ala Ala

1265 1270 1275

Leu Ser Gln Ser Gln Arg Pro Arg Pro Thr Lys Lys His Lys Gly

1280 1285 1290

Gly Arg Met Asp Gln Gln Pro Ala Leu Pro His Arg Arg Glu Gly

1295 1300 1305

Met Thr Asp Glu Glu Ala Leu Val Pro Tyr Ser Lys Pro Ser Phe

1310 1315 1320

Pro Ser Pro Gly Gly His Ser Ser Ser Gly Thr Ala Ser Ser Lys

1325 1330 1335

Gly Ser Thr Gly Pro Arg Lys Thr Glu Val Leu Arg Ala Gly His

1340 1345 1350

Gln Arg Asn Ala Ser Asp Leu Leu Asp Ile Gly Tyr Met Gly Ser

1355 1360 1365

Asn Ser Gln Gly Gln Phe Thr Gly Glu Leu

1370 1375

<210> 25

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: Synthetic label

6xHis"

<400> 25

His His His His His His His

15

<---

Claims (41)

1. Recombinant Roundabout 2 receptor (ROBO2) protein binding to the SLIT2 ligand, containing (i) part of the extracellular domain of ROBO2, and (ii) an immunoglobulin domain, which is an Fc domain of IgA ₁ , IgA ₂ , IgD, IgE, IgM, IgG ₁ , IgG ₂ , IgG ₃ or IgG _4, where the specified part of the extracellular domain ROBO2 contains sequentially the first immunoglobulin-like domain (Ig1) with SEQ ID NO: 9 or SEQ ID NO: 20, an interdomain linker located between the first and the second immunoglobulin-like domains (interdomain linker Ig1-Ig2) with SEQ ID NO: 10; a second immunoglobulin-like domain (Ig2) with SEQ ID NO: 11, and an interdomain linker located between the second and third immunoglobulin-like domains (Ig2-Ig3 interdomain linker) with SEQ ID NO: 12, and lacking three repeats of the fibronectin III type ( FNIII) of the extracellular domain of ROBO2. 2. The recombinant ROBO2 protein according to claim 1, wherein said ROBO2 Ig1 contains at least one of the following mutations: S17T and R73Y, each of which is numbered according to SEQ ID NO: 1. 3. The recombinant ROBO2 protein according to claim 1, wherein said part of the ROBO2 extracellular domain contains (i) amino acid residues 31-224 according to SEQ ID NO: 24 or (ii) amino acid residues 31-409 according to SEQ ID NO. : 24. 4. The recombinant ROBO2 protein according to claim 1, wherein said portion of the ROBO2 extracellular domain further comprises an immunoglobulin 1 (Ig1)-like ROBO2 pre-sequence, SEQ ID NO: 8. 5. The recombinant ROBO2 protein according to claim 1, where the specified part of the extracellular domain of ROBO2 contains or consists of amino acid residues 1-203 in accordance with the numbering of SEQ ID NO: 1. 6. The recombinant ROBO2 protein according to claim 1, wherein said Fc domain is the Fc domain of human IgG ₁ . 7. Recombinant ROBO2 protein according to claim 6, where the specified Fc-domain of human IgG ₁ contains the substitutions L234A, L235A and G237A (Eu-numbering) and does not contain K447 (Eu-numbering). 8. Recombinant ROBO2 protein according to claim 7, where the specified Fc-domain contains amino acid residues 210-440 in accordance with the numbering of SEQ ID NO: 1. 9. Recombinant protein ROBO2 according to any one of paragraphs. 1-8, where the specified part of the extracellular domain of ROBO2 is adjacent to the specified immunoglobulin domain or linked to the specified immunoglobulin domain through a linker. 10. The recombinant ROBO2 protein according to claim 9, wherein said linker is a peptidyl linker containing approximately 1 to 30 amino acid residues. 11. The recombinant ROBO2 protein according to claim 10, wherein said peptidyl linker is selected from the group consisting of: a) a glycine-rich peptide; b) a peptide containing glycine and serine; c) a peptide having the sequence (Gly-Gly-Ser) _n , where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 22); And d) a peptide having the sequence (Gly-Gly-Gly-Gly-Ser)n, where n is 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 23). 12. Recombinant ROBO2 protein according to claim 11, wherein said peptidyl linker is (Gly-Gly-Ser) ₂ (SEQ ID NO: 15). 13. Recombinant Roundabout 2 receptor (ROBO2)–Fc protein binding to the SLIT2 ligand, essentially consisting of the amino acid sequence of SEQ ID NO: 1. 14. Recombinant Roundabout 2 receptor (ROBO2)-Fc protein binding to the SLIT2 ligand, essentially consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and retains the activity of the protein (ROBO2)- fc. 15. Recombinant ROBO2-Fc protein according to claim 13, containing the amino acid sequence encoded by the plasmid insert deposited in ATCC and having the registration number ATCC No. PTA-124008. 16. The recombinant ROBO2 protein of any one of the preceding claims, wherein said protein binds to SLIT2 with a K _D equal to or less than: about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, approximately 700 pM, approximately 600 pM, approximately 500 pM, approximately 400 pM, approximately 300 pM, approximately 250 pM, approximately 200 pM, approximately 150 pM, approximately 100 pM, approximately 50 pM, approximately 40 pM, approximately 30 pM, approximately 25 pM, about 20 pM, about 15 pM, about 10 pM, at least about 5 pM, or about 1 pM. 17. The recombinant ROBO2 protein of any one of the preceding claims, wherein said protein binds to SLIT2 with a K _D that is at least about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold, approximately 20-fold, approximately 40-fold, approximately 60-fold, approximately 80-fold, approximately 100-fold, approximately 120-fold, approximately 140-fold, approximately 160-fold lower than the K _D value for binding ROBO1 to SLIT2 . 18. The recombinant ROBO2 protein of any one of the preceding claims, wherein said protein binds to SLIT2 with a K _D that is at least about 2-fold, about 4-fold, about 6-fold, about 8-fold, about 10-fold, approximately 20-fold, approximately 40-fold, approximately 60-fold, approximately 80-fold, approximately 100-fold, approximately 120-fold, approximately 140-fold, approximately 160-fold lower than the K _D value for ROBO1– protein binding Fc with SLIT2. 19. The recombinant ROBO2 protein according to any one of claims 16-18, wherein said K _D is measured by surface plasmon resonance (SPR). 20. The recombinant ROBO2 protein according to any one of claims 16-18, wherein said K _D is measured by biolayer interferometry (BSI). 21. A recombinant ROBO2 protein according to any one of the preceding claims, wherein said protein inhibits SLIT and ROBO2 binding and/or inhibits ROBO2-dependent SLIT-N activity. 22. The recombinant ROBO2 protein of any one of the preceding claims, wherein said protein has a half-maximal inhibitory concentration (IC ₅₀ ) of no more than about 15 nM, about 13 nM, about 11 nM, about 9 nM, about 7 nM, about 6 nM , approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, approximately 1 nM, as determined by time resolved homogeneous fluorescence (HTRF) analysis for inhibition of ROBO2 binding to SLIT2. 23. Recombinant ROBO2 protein according to any one of paragraphs. 1-21, wherein said protein has a half-maximal inhibitory concentration (IC ₅₀ ) of no more than about 75 nM, about 65 nM, about 55 nM, about 45 nM, about 35 nM, about 25 nM, about 15 nM, about 5 nM, as assessed by determining SLIT2-N-mediated inhibition of nerve cell migration. 24. An isolated nucleic acid molecule encoding a recombinant ROBO2 protein according to any one of paragraphs. 1–23. 25. An isolated nucleic acid molecule according to claim 24, wherein said molecule contains the nucleic acid sequence of SEQ ID NO: 21. 26. An expression vector containing the isolated nucleic acid molecule according to claim 24 or 25. 27. A host cell expressing a recombinant ROBO2 protein according to any one of paragraphs. 1-23, containing the vector according to item 26. 28. Pharmaceutical composition for the treatment of kidney disease associated with binding and signaling ROBO2-SLIT or mediated by them, containing the recombinant ROBO2 protein according to any one of paragraphs. 1-23 and a pharmaceutically acceptable carrier or excipient. 29. The use of recombinant protein ROBO2 according to any one of paragraphs. 1-23 for the treatment of kidney disease associated with or mediated by ROBO2-SLIT signaling and signaling. 30. Use of the recombinant ROBO2 protein according to claim 29, wherein said kidney disease is glomerulitis, focal segmental glomerular sclerosis (FSGS), or nephropathy. 31. The use of the recombinant ROBO2 protein according to claim 30, wherein said kidney disease is nephropathy, wherein said nephropathy is IgA nephropathy. 32. A method for producing a recombinant ROBO2 protein, comprising culturing a host cell according to claim 27 under conditions under which the recombinant ROBO2 protein is expressed. 33. The method of claim 32, further comprising isolating the recombinant ROBO2 protein. 34. Recombinant ROBO2-Fc protein binding to the SLIT2 ligand, essentially consisting of the amino acid sequence of SEQ ID NO: 2, 7 or 19, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, 7 or 19 and retains the activity of the (ROBO2)–Fc protein. 35. The recombinant ROBO2-Fc protein according to claim 34, wherein the recombinant ROBO2-Fc protein contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, 7, or 19. 36. The recombinant ROBO2-Fc protein according to claim 34, wherein the recombinant ROBO2-Fc protein contains an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2, 7, or 19. 37. The recombinant ROBO2-Fc protein according to claim 34, wherein the recombinant ROBO2-Fc protein contains the amino acid sequence of SEQ ID NO: 2, 7, or 19.

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