Protein comprising ROBO domain and medical use thereof

Abstract

The invention relates to a protein containing a ROBO structural domain and a medical application thereof. Relates to a protein containing a ROBO structural domain and application of the protein to treatment of diseases with abnormal up-regulation of a ROBO-SLIT2 signal channel.

Description

Proteins containing ROBO domains and their medical uses

[0001] This application claims priority to patent application CN202311184604.1 filed on September 14, 2023. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and in particular to proteins containing a ROBO domain, pharmaceutical compositions thereof, preparation methods, medical uses, and methods for preventing or treating diseases associated with the ROBO-SLIT2 signaling pathway (e.g., kidney disease). Background Art

[0003] Vascular endothelial cells, basement membranes, and podocytes together form the glomerular filtration barrier. Podocyte foot process fusion and cell body shedding are the primary mechanisms of glomerular proteinuria (Kriz W, et al. 1998. Kidney Int. 54:687–97). Common glomerular diseases are associated with varying degrees of podocyte injury. Early intervention and improvement of podocyte injury are emerging therapeutic approaches for glomerular-related chronic kidney disease (JW Leeuwis et al. Advanced Drug Delivery Reviews 62 (2010) 1325-1336).

[0004] Renal podocytes express roundabout receptor 2 (ROBO2), which is autoinhibited when not bound to its ligand, SLIT (Barak et al., 2019, Cell 177, 272-285). Upon binding to SLIT2, ROBO2 forms a complex with nephrin via the downstream adaptor protein Nck, negatively regulating nephrin-mediated actin aggregation. In mouse models, ROBO2 deficiency alleviates the abnormal podocyte structure observed in nephrin-deficient mice (Xueping Fan et al., Cell Reports 2, 52–61, July 26, 2012). Furthermore, the ROBO2-SLIT2 signaling pathway inhibits the activity of non-muscle myosin IIA (NMIIA) through the downstream SLIT / ROBO Rho GTPase-activating protein 1 (SRGAP1), thereby impairing podocyte focal adhesion formation at the basement membrane and increasing podocyte detachment. In mouse models, ROBO2 deficiency can alleviate the glomerulosclerosis and proteinuria phenotype of NMIIA heavy chain-deficient mice (Xueping Fan et al. JCI Insight. 2016; 1(19): e86934). Upregulation of ROBO2 expression in podocytes has been observed in nephrotoxic serum-induced mouse renal damage models, rat passive Heymann membranous nephritis models, and patients with membranous nephropathy (Anna Pisarek-Horowitz et al. The American Journal of Pathology, Vol. 190, No. 4, April 2020). Therefore, in podocyte-related chronic kidney disease, blocking ROBO2-SLIT2 signaling can improve podocyte structure and function, thereby reducing proteinuria.

[0005] Currently, there are no drugs marketed worldwide that block ROBO2-SLIT2 signaling. Compared to recombinant native ROBO2 proteins, the present disclosure provides a class of recombinant ROBO proteins modified through mutations and other techniques. These proteins act as SLIT ligand traps, exhibiting enhanced SLIT2 neutralization activity and stability. This enhanced SLIT2 neutralization activity reduces dosing volume and frequency, and their excellent stability allows for development into more convenient formulations, improving compliance and ease of use for patients with chronic kidney disease. The recombinant ROBO proteins disclosed herein can be used to prevent or treat diseases associated with the ROBO-SLIT2 signaling pathway, such as podocyte-related nephropathy.

[0006] Summary of the Invention

[0007] The present disclosure provides proteins comprising a circular xenobiotic (ROBO) domain and polynucleotides encoding the proteins, pharmaceutical compositions, methods for treating or preventing diseases (eg, kidney disease), and related pharmaceutical uses.

[0008] ROBO domain-containing proteins

[0009] The present disclosure provides proteins comprising a ROBO domain comprising a circular Xceptor 2 immunoglobulin-like domain 1 (ROBO2 Ig1).

[0010] In some embodiments, the ROBO2 Ig1 has amino acid mutations at one or more positions 17, 30, 32, 66, and 68 compared to the wild-type ROBO2 Ig1 (SEQ ID NO: 20), and the position numbers are the natural sequence numbers relative to the amino acid sequence shown in SEQ ID NO: 1.

[0011] In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following compared to the wild-type ROBO2 Ig1:

[0012] A) 17F, 17T, 17V or 17K,

[0013] B) 30S, 30Y or 30H,

[0014] C) 32R, 32T or 32Q,

[0015] D) 66G, 66D, 66V or 66R, and / or

[0016] E)68H, 68K, 68D or 68E.

[0017] In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following compared to the wild-type ROBO2 Ig1:

[0018] F) S17F, S17T, S17V or S17K,

[0019] G) N30S, N30Y or N30H,

[0020] H) K32R, K32T or K32Q,

[0021] 1) S66G, S66D, S66V, or S66R, and / or

[0022] J) S68H, S68K, S68D or S68E.

[0023] In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following compared to the wild-type ROBO2 Ig1:

[0024] K)17F / 30S / 32R / 66G / 68H,

[0025] L)17T / 30Y / 32T / 66D / 68K,

[0026] M)17V / 30Y / 32R / 66V / 68D, or

[0027] N)17K / 30H / 32Q / 66R / 68E.

[0028] In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following compared to the wild-type ROBO2 Ig1:

[0029] O)S17F / N30S / K32R / S66G / S68H,

[0030] P)S17T / N30Y / K32T / S66D / S68K,

[0031] Q)S17V / N30Y / K32R / S66V / S68D, or

[0032] R)S17K / N30H / K32Q / S66R / S68E.

[0033] In some embodiments, the ROB02 Ig1 comprises an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%; the same below) identical to any one of SEQ ID NOs: 20, 11-14.

[0034] In other embodiments, ROB02 Ig1 is provided, wherein the amino acid mutation is present at one or more of positions 62, 64, 70, 72, and 73 compared to wild-type ROB02 Ig1, where the positions are numbered relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 1. For example, the amino acid mutation is present at one or more of positions M62, L64, F70, L72, and R73 compared to wild-type ROB02 Ig1.

[0035] In other embodiments, ROB02 Ig1 is provided, which has an amino acid mutation at one or more of positions 19, 26, 28, 30, and 70 compared to wild-type ROB02 Ig1, where the positions are numbered relative to the natural order of the amino acid sequence set forth in SEQ ID NO: 1. For example, compared to wild-type ROB02 Ig1, an amino acid mutation is present at one or more of positions V19, P26, T28, N30, and F70.

[0036] In some embodiments, the ROBO domain in the protein further comprises a circular Xceptor immunoglobulin-like domain 2 (ROBO Ig2).

[0037] In some embodiments, the ROBO Ig2 is circular Xceptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2) or circular Xceptor 2 immunoglobulin-like domain 2 (ROBO2 Ig2).

[0038] In some specific embodiments, the ROBO1 Ig2 comprises an amino acid sequence as set forth in SEQ ID NO: 25 or at least 90% (e.g., 95%) identical thereto, and the ROBO2 Ig2 comprises an amino acid sequence as set forth in SEQ ID NO: 24 or at least 90% (e.g., 95%) identical thereto.

[0039] In some embodiments, the ROBO domain in the protein further comprises one or more of the following i)-vii):

[0040] i) ROBO pre-immunoglobulin-like 1 (ROBO pre-Ig1),

[0041] ii) a linker between the ROBO immunoglobulin-like domain 1 and the immunoglobulin-like domain 2 (ROBO Ig1-2 linker),

[0042] iii) a linker between the ROBO immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 (ROBO Ig2-3 linker),

[0043] iv) ROBO immunoglobulin-like domain 3 (ROBO Ig3),

[0044] v) a linker between the ROBO immunoglobulin-like domain 3 and the immunoglobulin-like domain 4 (ROBO Ig3-4 linker),

[0045] vi) ROBO immunoglobulin-like domain 4 (ROBO Ig4),

[0046] vii) ROBO immunoglobulin-like domain 4 post-linker (ROBO Ig4 post-linker);

[0047] In some specific embodiments, ROBO in i)-vii) is ROBO1 or ROBO2.

[0048] In some specific embodiments, the ROBO pre-Ig1 comprises the amino acid sequence shown in SEQ ID NO:19.

[0049] In some specific embodiments, the ROBO Ig1-2 linker comprises the amino acid sequence shown in SEQ ID NO: 22 or 23.

[0050] In some specific embodiments, the ROBO Ig2-3 linker comprises the amino acid sequence shown in SEQ ID NO: 26 or 27.

[0051] In some embodiments, ROBO Ig3 comprises the amino acid sequence set forth in SEQ ID NO: 28 or 29, or an amino acid sequence at least 90% (eg, 95%) identical thereto.

[0052] In some specific embodiments, the ROBO Ig3-4 linker comprises the amino acid sequence shown in SEQ ID NO: 30 or 31.

[0053] In some embodiments, ROBO Ig4 comprises the amino acid sequence set forth in SEQ ID NO: 32 or 33, or an amino acid sequence at least 90% (eg, 95%) identical thereto.

[0054] In some specific embodiments, the ROBO Ig4 rear linker comprises the amino acid sequence shown in SEQ ID NO: 34 or 35.

[0055] In some embodiments, the ROBO domain in the protein comprises a structure represented by formula (II)-(IV):

[0056] [ROBO pre-Ig1]a2-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b2-[ROBO Ig2]c2-[ROBO Ig2-3 linker]d2-[ROBO Ig3]e2-[ROBO Ig3-4 linker]f2-[ROBO Ig4]g2-[ROBO Ig4 post-linker]h2 Formula (II)

[0057] [ROBO pre-Ig1]a2-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b2-[ROBO1 Ig2]c2-[ROBO Ig2-3 linker]d2-[ROBO Ig3]e2-[ROBO Ig3-4 linker]f2-[ROBO Ig4]g2-[ROBO Ig4 post-linker]h2 Formula (III)

[0058] [ROBO pre-Ig1]a2-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b2-[ROBO2 Ig2]c2-[ROBO Ig2-3 linker]d2-[ROBO Ig3]e2-[ROBO Ig3-4 linker]f2-[ROBO Ig4]g2-[ROBO Ig4 post-linker]h2 Formula (IV)

[0059] wherein - is a peptide bond, and a2, b2, c2, d2, e2, f2, g2, and h2 in formulas (II) to (IV) can be independently selected from 0 or 1. In some specific embodiments, a2, b2, c2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, a2 is 1, b2, c2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, b2 and c2 are 1, and a2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2 are 1, and d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2, d2 are 1, and e2, f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2, d2, e2 are 1, and f2, g2, and h2 are all 0. In some specific embodiments, a2, b2, c2, d2, e2, f2 are 1, and g2, h2 are 0. In some specific embodiments, a2, b2, c2, d2, e2, f2, g2 are 1, and h2 is 0. In some specific embodiments, b2, c2, d2 are 1, and a2, e2, f2, g2, and h2 are all 0. In some specific embodiments, b2, c2, d2, e2 are 1, and a2, f2, g2, and h2 are all 0. In some specific embodiments, b2, c2, d2, e2, f2 are 1, and a2, g2, and h2 are all 0. In some specific embodiments, b2, c2, d2, e2, f2 are 1, and a2, g2, and h2 are all 0. In some specific embodiments, b2, c2, d2, e2, f2, g2, and h2 are 1, and a2 is 0. In some specific embodiments, a2 and c2 are 1, and b2, d2, e2, f2, g2, and h2 are all 0. In some specific embodiments, c2 is 1, and a2, b2, d2, e2, f2, g2, and h2 are all 0.

[0060] In some embodiments, a ROBO domain is provided that comprises any one of SEQ ID NOs: 20, 11-14, or an amino acid sequence at least 90% (eg, 95%) identical thereto.

[0061] In some embodiments, a protein is provided that comprises any one of SEQ ID NOs: 20, 11-14, or an amino acid sequence that is or is at least 90% (eg, 95%) identical thereto.

[0062] The present disclosure provides proteins comprising a ROBO domain comprising the extracellular region of circular Xceptor 2 (ROBO2) or a portion thereof and the extracellular region of circular Xceptor 1 (ROBO1) or a portion thereof.

[0063] In some embodiments, the ROBO domain comprises an immunoglobulin-like domain of ROBO2 and an immunoglobulin-like domain of ROBO1.

[0064] In some embodiments, the ROBO domain comprises any one or any combination of the immunoglobulin-like domains Ig1, Ig2, Ig3, Ig4, and Ig5 of ROBO2, and any one or any combination of the immunoglobulin-like domains Ig1, Ig2, Ig3, Ig4, and Ig5 of ROBO1.

[0065] In some embodiments, the ROBO domain comprises a structure as shown in formula (I):

[0066] [ROBO pre-Ig1]a1-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b1-[ROBO1 Ig2]-[ROBO Ig2-3 linker]c1-[ROBO Ig3]d1-[ROBO Ig3-4 linker]e1-[ROBO Ig4]f1-[ROBO Ig4 post-linker]g1 Formula (I)

[0067] wherein - is a peptide bond, and a1, b1, c1, d1, e1, f1, and g1 in formula (I) can be independently selected from 0 or 1. In some specific embodiments, a1, b1, c1, d1, e1, f1, and g1 are all 0. In some specific embodiments, b1 is 1, and a1, c1, d1, e1, f1, and g1 are all 0. In some specific embodiments, a1, b1, c1 is 1, and d1, e1, f1, and g1 are all 0. In some specific embodiments, a1, b1, c1 is 1, and d1, e1, f1, and g1 are all 0. In some specific embodiments, a1, b1, c1, d1, e1, f1, and g1 are all 0. In some specific embodiments, a1, b1, c1, d1, e1, f1 are 1, and g1 is 0. In some specific embodiments, b1 is 1, and a1, c1, d1, e1, f1, and g1 are all 0. In some specific embodiments, b1 and c1 are 1, and a1, d1, e1, f1, and g1 are all 0. In some specific embodiments, b1, c1, d1 are 1, and a1, e1, f1, and g1 are all 0. In some specific embodiments, b1, c1, d1, e1 are 1, and a1, f1, and g1 are all 0. In some specific embodiments, b1, c1, d1, e1, f1 are 1, and a1, g1 are all 0. In some specific embodiments, b1, c1, d1, e1, f1, and g1 are 1, and a1 is 0. In some specific embodiments, a1 is 1, and b1, c1, d1, e1, f1, and g1 are all 0.

[0068] In some embodiments, the ROBO domain comprises any one of the following 1)-15):

[0069] 1) ROBO2 Ig1

[0070] 2) ROBO2 Ig1 and ROBO1 Ig2;

[0071] 3) ROBO2 Ig1 and ROBO2 Ig2;

[0072] 4) ROBO2 Ig1, ROBO1 Ig2, and ROBO1 Ig3;

[0073] 5) ROBO2 Ig1, ROBO1 Ig2, and ROBO2 Ig3;

[0074] 6) ROBO2 Ig1, ROBO2 Ig2, and ROBO1 Ig3;

[0075] 7) ROBO2 Ig1, ROBO2 Ig2, and ROBO2 Ig3;

[0076] 8) ROBO2 Ig1, ROBO1 Ig2, ROBO1 Ig3, and ROBO1 Ig4;

[0077] 9) ROBO2 Ig1, ROBO1 Ig2, ROBO2 Ig3, and ROBO1 Ig4;

[0078] 10) ROBO2 Ig1, ROBO2 Ig2, ROBO1 Ig3, and ROBO1 Ig4;

[0079] 11) ROBO2 Ig1, ROBO2 Ig2, ROBO2 Ig3, and ROBO1 Ig4;

[0080] 12) ROBO2 Ig1, ROBO1 Ig2, ROBO1 Ig3, and ROBO2 Ig4;

[0081] 13) ROBO2 Ig1, ROBO1 Ig2, ROBO2 Ig3, and ROBO2 Ig4;

[0082] 14) ROBO2 Ig1, ROBO2 Ig2, ROBO1 Ig3, and ROBO2 Ig4;

[0083] 15) ROBO2 Ig1, ROBO2 Ig2, ROBO2 Ig3, and ROBO2 Ig4.

[0084] In some embodiments, the ROBO domain may further comprise one or any combination of a ROBO1 pre-Ig1 sequence or a ROBO2 pre-Ig1 sequence, a ROBO1 Ig1-2 linker or a ROBO2 Ig1-2 linker, a ROBO1 Ig2-3 linker or a ROBO2 Ig2-3 linker, a ROBO1 Ig3-4 linker or a ROBO2 Ig3-4 linker, a ROBO1 Ig4 post-linker or a ROBO2 Ig4 post-linker.

[0085] Exemplarily, the ROBO domain comprises any one of the following 16)-84):

[0086] 16) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1;

[0087] 17) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2;

[0088] 18) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2;

[0089] 19) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, and ROBO1 Ig3;

[0090] 20) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, and ROBO2 Ig3;

[0091] 21) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3;

[0092] 22) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO2 Ig3;

[0093] 23) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, ROBO1 Ig3, and ROBO1 Ig4;

[0094] 24) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, ROBO2 Ig3, and ROBO1 Ig4;

[0095] 25) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

[0096] 26) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

[0097] 27) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, ROBO1 Ig3, and ROBO2 Ig4;

[0098] 28) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig2, ROBO2 Ig3, and ROBO2 Ig4;

[0099] 29) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

[0100] 30) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO2 Ig2 and ROBO2 Ig3 and ROBO2 Ig4;

[0101] 31) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker);

[0102] 32) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2;

[0103] 33) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2;

[0104] 34) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig3;

[0105] 35) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO2 Ig3;

[0106] 36) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig3;

[0107] 37) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO2 Ig3;

[0108] 38) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

[0109] 39) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

[0110] 40) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

[0111] 41) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

[0112] 42) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

[0113] 43) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO2 Ig3 and ROBO2 Ig4;

[0114] 44) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

[0115] 45) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO2 Ig3 and ROBO2 Ig4;

[0116] 46) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker);

[0117] 47) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), and ROBO1 Ig2;

[0118] 48) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), and ROBO2 Ig2;

[0119] 49) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, and ROBO1 Ig3;

[0120] 50) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, and ROBO2 Ig3;

[0121] 51) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, and ROBO1 Ig3;

[0122] 52) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, and ROBO2 Ig3;

[0123] 53) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, ROBO1 Ig3, and ROBO1 Ig4;

[0124] 54) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, ROBO2 Ig3, and ROBO1 Ig4;

[0125] 55) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, ROBO1 Ig3, and ROBO1 Ig4;

[0126] 56) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, ROBO2 Ig3, and ROBO1 Ig4;

[0127] 57) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, ROBO1 Ig3, and ROBO2 Ig4;

[0128] 58) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO1 Ig2, ROBO2 Ig3, and ROBO2 Ig4;

[0129] 59) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, ROBO1 Ig3, and ROBO2 Ig4;

[0130] 60) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), ROBO2 Ig2, ROBO2 Ig3, and ROBO2 Ig4;

[0131] 61) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker);

[0132] 62) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker);

[0133] 63) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3;

[0134] 64) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3;

[0135] 65) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3;

[0136] 66) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3;

[0137] 67) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1, and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker);

[0138] 68) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence), ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker), and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker);

[0139] 69) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3;

[0140] 70) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3;

[0141] 71) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3;

[0142] 72) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3;

[0143] 73) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker);

[0144] 74) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker);

[0145] 75) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3 and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker);

[0146] 76) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker);

[0147] 77) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4);

[0148] 78) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4);

[0149] 79) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3 and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4);

[0150] 80) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4);

[0151] 81) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4) and ROBO1 Ig4 post-linker (or ROBO2 Ig4 post-linker);

[0152] 82) ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4) and ROBO1 Ig4 post-linker (or ROBO2 Ig4 post-linker);

[0153] 83) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO1 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO2 Ig3 and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4) and ROBO1 Ig4 post-linker (or ROBO2 Ig4 post-linker);

[0154] 84) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig1-2 linker (or ROBO2 Ig1-2 linker) and ROBO2 Ig2 and ROBO1 Ig2-3 linker (or ROBO2 Ig2-3 linker) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 linker (or ROBO2 Ig3-4 linker) and ROBO1 Ig4 (or ROBO2 Ig4) and ROBO1 Ig4 post-linker (or ROBO2 Ig4 post-linker).

[0155] In some embodiments, the ROBO domain comprises structures selected from 1) to 84) from N-terminus to C-terminus, for example, "2) ROBO2 Ig1 and ROBO1 Ig2" means that the protein comprises ROBO2 Ig1 and ROBO1 Ig2 from N-terminus to C-terminus.

[0156] In some embodiments, the ROB02 Ig1 is wild-type ROB02 Ig1 (eg, as shown in SEQ ID NO: 20) or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0157] In some specific embodiments, the ROB02 Ig1 is any of the ROB02 Ig1 with amino acid mutations provided herein (e.g., any of SEQ ID NOs: 11-14 or an amino acid sequence having at least 90% (e.g., at least 95%) identity thereto, or any of the ROB02 Ig1 described in A)-R) above).

[0158] In some specific embodiments, the ROB02 Ig 2 is represented by SEQ ID NO: 24, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0159] In some specific embodiments, the ROBO1 Ig 2 is represented by SEQ ID NO: 25, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0160] In some specific embodiments, the ROBO2 Ig 3 is as shown in SEQ ID NO: 28, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0161] In some specific embodiments, the ROBO1 Ig 3 is represented by SEQ ID NO: 29, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0162] In some specific embodiments, the ROBO2 Ig 4 is represented by SEQ ID NO: 32, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0163] In some specific embodiments, the ROBO1 Ig 4 is represented by SEQ ID NO: 33, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto.

[0164] In some specific embodiments, the ROBO2 pre-Ig1 sequence is the amino acid sequence shown in SEQ ID NO: 19.

[0165] In some specific embodiments, the ROBO1 pre-Ig1 sequence is the amino acid sequence shown in SEQ ID NO: 19.

[0166] In some specific embodiments, the ROBO2 Ig1-2 linker is the amino acid sequence shown in SEQ ID NO: 22.

[0167] In some specific embodiments, the ROBO1 Ig1-2 linker is the amino acid sequence shown in SEQ ID NO: 23.

[0168] In some specific embodiments, the ROBO2 Ig 2-3 linker is the amino acid sequence shown in SEQ ID NO: 26.

[0169] In some specific embodiments, the ROBO1 Ig 2-3 linker is the amino acid sequence shown in SEQ ID NO: 27.

[0170] In some specific embodiments, the ROBO2 Ig 3-4 linker is the amino acid sequence shown in SEQ ID NO: 30.

[0171] In some specific embodiments, the ROBO1 Ig 3-4 linker is the amino acid sequence shown in SEQ ID NO: 31.

[0172] In some specific embodiments, the ROBO2 Ig4 rear linker is the amino acid sequence shown in SEQ ID NO: 34.

[0173] In some specific embodiments, the ROBO1 Ig4 rear linker is the amino acid sequence shown in SEQ ID NO: 35.

[0174] In some specific embodiments, variants of ROBO pre-Ig1 (ROBO1 pre-Ig1 or ROBO2 pre-Ig1) are provided, which have one or more (e.g., 2, 3, 4, 5, 6) amino acid substitutions, deletions, and / or additions compared to SEQ ID NO: 19, while still maintaining the function of ROBO pre-Ig1. For example, variants of ROBO pre-Ig1 are provided, which have one or more (e.g., 2, 3, 4, 5, 6) conservative amino acid substitutions compared to SEQ ID NO: 19, while still maintaining the function of ROBO pre-Ig1.

[0175] In some specific embodiments, variants of ROBO2 Ig1-2 linker, ROBO1 Ig1-2 linker, ROBO2 Ig2-3 linker, ROBO1 Ig2-3 linker, ROBO2 Ig3-4 linker, ROBO1 Ig3-4 linker, ROBO2 Ig4 post-linker, and ROBO1 Ig4 post-linker are provided, which have one or more (e.g., 2, 3, 4) amino acid substitutions, deletions, and / or additions (e.g., conservative amino acid substitutions) compared with the aforementioned amino acid sequences provided by the present disclosure, and can still achieve the function of the linker.

[0176] The present disclosure provides a protein comprising (i) any one of the aforementioned ROBO domains of the present disclosure and (ii) a half-life (eg, in vivo half-life) extending domain.

[0177] In some embodiments, the (ii) half-life (eg, in vivo half-life) extending domain may be located at the N-terminus or C-terminus of the (i) ROBO domain.

[0178] In some embodiments, the (ii) half-life (e.g., in vivo half-life) extending domain includes but is not limited to human serum albumin (HSA), an HSA binding domain (e.g., an anti-HSA antibody, such as an anti-HSA single domain antibody), and an immunoglobulin domain.

[0179] In some embodiments, the immunoglobulin domain is an immunoglobulin Fc region. In some specific embodiments, the immunoglobulin Fc region is derived from the Fc region of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM. In some specific embodiments, the Fc region may comprise a mutation, exemplary mutations including: any one or any combination of L234A, L235A, G237A on IgG1; for example, L234A / L235A, L234A / L235A / G237A; any one or any combination of F234A, L235A, S228P on IgG4, for example, F234A / L235A, S228P / F234A / L235A. In some specific embodiments, the Fc region comprises C220S. In some specific embodiments, the IgG Fc region comprises SEQ ID NO: 37-39, or an amino acid sequence at least 80% or at least 90% identical thereto.

[0180] In some embodiments, (i) the ROBO domain and (ii) the half-life (e.g., in vivo half-life) extension domain can be directly connected or connected through a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. For example, the linker is a flexible linker; for example, G4S (SEQ ID NO: 54), GS, GAP, (G4S)2 (SEQ ID NO: 55), (G4S)3 (SEQ ID NO: 56), (G4S)4 (SEQ ID NO: 57), (G4S)5 (SEQ ID NO: 58), ASGS (SEQ ID NO: 59), etc.; for example, the linker is (G x S) y Linker, wherein x is selected from an integer of 1-5 (e.g., 2), and y is selected from an integer of 1-6; for example, the linker is GGSGGS (SEQ ID NO: 36), (G4S)2; in some embodiments, the linker is absent, i.e., y is 0.

[0181] In some embodiments, a protein is provided that comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 15-18, 40-53, or at least 80%, at least 90% (e.g., at least 95%) identical thereto.

[0182] The protein comprising the ROBO domain provided by the present disclosure has at least one property selected from the following:

[0183] (a) ≤1×10 -7 M, for example, ≤1×10 -8 M, ≤1×10 -9M, ≤1×10 -10 M, ≤1×10 -11 M, ≤1×10 -12 M's K D value for binding to human SLIT2, the K D The value is preferably ≤1×10 -11 M. K D The method for detecting the value is conventional in the art, such as that provided in Example 4 of the present disclosure.

[0184] (b) When the protein comprising the ROBO domain comprises an immunoglobulin Fc region (e.g., an IgG1 Fc region comprising 234A / L235A / 237A), the protein has an extended in vivo (or plasma) half-life. The in vivo half-life is increased by at least 1.5 times, preferably at least 2 times, such as at least 5 times, such as at least 10 times or greater than 20 times, compared to the protein without the immunoglobulin Fc region; for example, the increased in vivo half-life can be greater than 1, 2, 6, 12 hours, or greater than 24, 48, or 72 hours compared to the protein without the immunoglobulin Fc region.

[0185] (c) IC≤500nM, 400nM, 300nM, 200nM, 100nM, 50nM 50 Blocking the binding of cell surface expressed Robo2 to SLIT2, the IC 50 Preferably ≤100 nM, more preferably ≤50 nM. The experimental method and IC 50 The method for detecting the value is conventional in the art, such as that provided in Example 5 of the present disclosure.

[0186] (d) EC values ​​≤ 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 3 nM 50 Restoring neuronal migration and relieving the repulsive guidance effect of Robo2-SLIT2 50 Preferably ≤10nM, more preferably ≤5nM, 2nM. The experimental method of neuronal axon migration and EC 50 The method for detecting the value is conventional in the art, such as that provided in Example 6 of the present disclosure.

[0187] (e) capable of inhibiting or reducing proteinuria in a subject, for example, a rat, more preferably a rat passive Heimann nephritis model. The method for constructing the rat passive Heimann nephritis model, the dosing regimen, and the method for detecting proteinuria are conventional in the art, such as those provided in Example 7 of the present disclosure.

[0188] The present disclosure provides variants of proteins comprising a ROBO domain, wherein the variant has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acid mutations compared to any one of SEQ ID NOs: 7-18; the amino acid mutations may be conservative amino acid substitutions and / or deletions or additions that do not affect function.

[0189] Also, the proteins disclosed herein encompass pharmaceutically acceptable salts thereof.

[0190] Polynucleotides and vectors

[0191] The present disclosure provides polynucleotides encoding the ROBO domain-containing proteins of the present disclosure.

[0192] In some embodiments, the polynucleotide may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the polynucleotide of the present disclosure is an isolated polynucleotide.

[0193] The polynucleotides of the present disclosure may also be in the form of, may be present in and / or may be part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may in particular be an expression vector, i.e., a vector that provides for expression of a protein comprising a ROBO domain in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector typically comprises at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select such elements and their sequences for expression in a particular host. Regulatory elements and other elements that are useful or necessary for the expression of the protein comprising a ROBO domain of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0194] The polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0195] host cells

[0196] In some embodiments, provided are host cells that express or are capable of expressing one or more ROBO domain-containing proteins of the present disclosure, and / or recombinant host cells containing a polynucleotide or vector of the present disclosure.

[0197] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0198] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0199] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0200] For example, mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0201] The present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0202] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0203] Production or preparation method

[0204] The present disclosure provides a method for preparing a protein comprising a ROBO domain of the present disclosure, comprising:

[0205] - culturing the host cell of the present disclosure under conditions that allow expression of the ROBO domain-containing protein of the present disclosure; and

[0206] - recovering the protein expressed by the host cell from the culture; and

[0207] - Optionally, further purification and / or modification of the ROBO domain-containing protein of the present disclosure is included.

[0208] The ROBO domain-containing proteins of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0209] Methods and reagents for recombinantly producing polypeptides or proteins, such as specifically suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for producing target proteins, such as binding molecules or antibodies, are well known to those skilled in the art. Methods for producing and purifying antibodies are well known in the art and can be found in the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture fluid secreting the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0210] However, the ROBO domain-containing proteins of the present disclosure can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0211] Composition

[0212] In some embodiments, the composition is a pharmaceutical composition, which contains a preventively or therapeutically effective amount of the protein containing a ROBO domain as disclosed above, and / or a polynucleotide encoding the protein containing a ROBO domain, and one or more pharmaceutically acceptable carriers or excipients.

[0213] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the protein containing a ROBO domain per unit dose. In other embodiments, the pharmaceutical composition may contain 0.1 to 2000 mg of the protein containing a ROBO domain per unit dose. In some embodiments, the pharmaceutical composition may contain 1 to 1000 mg of the protein containing a ROBO domain per unit dose.

[0214] Kits and Tests

[0215] The present disclosure provides a kit or product comprising any of the aforementioned proteins comprising a ROBO domain and / or a nucleic acid molecule encoding a protein comprising a ROBO domain.

[0216] The present disclosure provides a composition for detecting SLIT2, comprising the protein comprising the ROBO domain of the present disclosure. The present disclosure also provides a method, system or device for detecting SLIT2 in vivo or in vitro, comprising using the protein comprising the ROBO domain of the present disclosure.

[0217] In some embodiments, an in vitro detection method, system, or device may, for example, comprise: (1) contacting a sample with a ROBO domain-containing protein of the present disclosure; (2) detecting complex formation between the ROBO domain-containing protein of the present disclosure and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with the ROBO domain-containing protein; and (4) determining the extent of complex formation between the ROBO domain-containing protein and the sample by comparison with the reference sample. A change (e.g., a statistically significant change) in complex formation in the sample, as compared to the control sample or subject, indicates the presence of SLIT2 in the sample.

[0218] In other embodiments, the in vivo detection method, system or device may comprise: (1) administering a ROBO domain-containing protein of the present disclosure to a subject; and (2) detecting the formation of a complex between the ROBO domain-containing protein of the present disclosure and the subject. Detection may comprise determining the location or time of complex formation. Antibodies that bind to the ROBO domain-containing protein may be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include a variety of enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Complex formation between the ROBO domain-containing protein of the present disclosure and serum albumin may be detected by measuring or visualizing antibodies that are bound or unbound to the ROBO domain-containing protein. Conventional detection assays may be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. In some embodiments, the presence of serum albumin in a sample is analyzed by a competitive immunoassay using a standard labeled with a detectable substance and an unlabeled ROBO domain-containing protein of the present disclosure. The living sample to be detected or measured can be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture medium.

[0219] In some embodiments, the ROBO domain-containing proteins of the present disclosure can be labeled with a fluorophore or chromophore for detection purposes.

[0220] Methods for treating and preventing diseases and pharmaceutical uses

[0221] The present disclosure provides a method for treating and / or preventing a disease, comprising administering a therapeutically and / or preventively effective amount of a protein comprising a ROBO domain of the present disclosure to a subject in need thereof.

[0222] The present disclosure also provides a protein comprising a ROBO domain for use in a method for treating and / or preventing a disease, the method comprising administering a therapeutically and / or preventively effective amount of the protein comprising a ROBO domain of the present disclosure to a subject in need thereof.

[0223] The present disclosure also provides use of a protein comprising a ROBO domain in preparing a medicament for treating and / or preventing a disease.

[0224] In some embodiments, the disease is caused by abnormal upregulation of the ROBO-SLIT2 signaling pathway.

[0225] In some embodiments, the disease is a kidney disease, including podocyte-related kidney disease.

[0226] In some embodiments, the disease is the glomerular disease, focal segmental glomerulosclerosis (FSGS). BRIEF DESCRIPTION OF THE DRAWINGS

[0227] FIG1 shows that ROB02-Fc2.2 and the chimeric ligand trap of the present disclosure inhibit the binding of the ligand SLIT2-D2 to the cell surface human ROB02 receptor in a dose-dependent manner.

[0228] FIG2 shows that ROBO2-Fc2.2 and the chimeric ligand trap of the present disclosure are able to neutralize the ligand SLIT2-N and restore neuronal cell migration in a dose-dependent manner.

[0229] FIG3 shows the effect of ROBO2-Fc2.2 and the chimeric ligand trap of the present disclosure in suppressing proteinuria in a rat passive Heimann nephritis model using a prophylactic dosing regimen. DETAILED DESCRIPTION

[0230] definition

[0231] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0232] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0233] Roundabout receptors (ROBOs) are receptors for Slit-guided ligand (SLIT) protein ligands. Four ROBO receptors have been characterized in vertebrates: ROBO1 / Dutt1; ROBO2; ROBO3 / Rig-1; and ROBO4 / Magic Roundabout. The sequence of human ROBO2 is Uniprot Q9HCK4, and the sequence of human ROBO1 is Uniprot Q9Y6N7. Both ROBO1 and ROBO2 have an extracellular domain (ECD) structure containing five immunoglobulin-like (Ig-like) domains (Ig1, Ig2, Ig3, Ig4, and Ig5), followed by three fibronectin type 3 (FN3) repeats. Illustratively, the circular X receptor immunoglobulin-like domain is abbreviated as ROBO Ig, the circular X receptor 1 immunoglobulin-like domain 2 is abbreviated as ROBO1 Ig2, the circular X receptor 2 immunoglobulin-like domain 1 is abbreviated as ROBO2 Ig1, and the ROBO pre-immunoglobulin-like 1 is abbreviated as ROBO pre-Ig1; and for the sake of simplicity, illustratively, the linker between ROBO Ig2 and ROBO Ig3 can be abbreviated as ROBO Ig2-3 linker, and the ROBO Ig1-2 linker includes the ROBO1 Ig1-2 linker and the ROBO2 Ig1-2 linker; illustratively, the ROBO2 Ig1-2 linker is shown in SEQ ID NO: 22, and the ROBO1 Ig1-2 linker is shown in SEQ ID NO: 23.

[0234] A "recombinant protein" refers to a polypeptide produced by recombinant DNA technology. Generally, DNA encoding the polypeptide is inserted into a suitable expression vector and then introduced into a host cell to produce the recombinant protein. In this disclosure, "protein" or "protein" refers to any composition comprising amino acids and recognized as a protein by those skilled in the art. The terms "protein," "protein," "peptide," and "polypeptide" are used interchangeably herein. As an example, a protein comprising a ROBO domain of the present disclosure is a recombinant protein. The recombinant ROBO protein of the present disclosure binds to a SLIT ligand, thereby preventing SLIT from binding to a cellular ROBO receptor and is therefore referred to as a SLIT neutralizing ligand trap.

[0235] Typically, the ROBO domain-containing proteins of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10mol / L, even more preferably 10 -9 to 10 -10 or lower equilibrium dissociation constant (K D ) combined with SLIT2. Any greater than 10 -4 M's K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of a receptor to a ligand can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (ELISA), and sandwich competitive assays as described herein.

[0236] The constant region amino acid position numbering in this disclosure is based on the Eu index.

[0237] A "linker" is a molecule or group of molecules that binds two separate entities (e.g., a ROBO domain and an immunoglobulin Fc region) to each other, and the linker can provide spacing between the two entities to enable them to achieve a conformation in which they specifically bind to their cognate ligand (e.g., a SLIT ligand). The linker is, for example, a polypeptide linker and can be expressed as a component of a recombinant protein using standard recombinant DNA techniques well known in the art.

[0238] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking.

[0239] As used herein, "homology" and "identity" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Generally speaking, a comparison is made when the two sequences are aligned to obtain the maximum percent homology.

[0240] "Conservative amino acid substitution" refers to the substitution of one or more amino acid residues of a protein or polypeptide with a conservative amino acid residue, wherein the chemical structure of the amino acid residue before the substitution is similar to that of the amino acid residue after the substitution, and the substitution has little or substantially no effect on the function, activity, or other biological properties of the protein or polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions preferably involve the substitution of an amino acid residue within the following groups (i)-(v) with another amino acid residue within the same group:

[0241] (i) Small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly;

[0242] (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln;

[0243] (iii) polar positively charged residues: His, Arg, and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and

[0244] (v) Aromatic residues: Phe, Tyr and Trp.

[0245] Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0246] "Nucleic acid," "nucleic acid molecule," or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded sequences, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0247] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, a vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, a vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector in the present disclosure can autonomously replicate in the host cell having been introduced therein (e.g., a bacterial vector and an additional mammalian vector having a bacterial origin of replication) or can be integrated into the genome of the host cell after introducing the host cell, thereby replicating (e.g., a non-additional mammalian vector) with the host genome.

[0248] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It will also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0249] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating nucleic acid inserts. Host cells include the progeny of a single host cell, and due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the nucleic acids of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0250] A "pharmaceutical composition" refers to a mixture containing one or more fusion proteins, proteins containing ROBO domains, polynucleotides, and other components described herein, such as physiologically / pharmaceutically acceptable carriers, diluents, buffers, or excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0251] "Administer," "apply," and "treat" as applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of a cell by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0252] "Treatment" means administering an internal or external therapeutic agent (such as a fusion protein of the present disclosure) to a subject having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered to a treated patient or population in an amount effective to alleviate one or more symptoms of a disease to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0253] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0254] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0255] In the present disclosure, "at least 95% (sequence) identity" encompasses at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity, and ranges between any two of the aforementioned values, including integers and decimals; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and ranges between any two of the aforementioned values, including integers and decimals; "at least 80% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity, and ranges between any two of the aforementioned values, including integers and decimals; "% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, 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%, at least 99% or 100% (sequence) identity, and ranges between any two of the foregoing values, including integers and decimals.

[0256] Example

[0257] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0258] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0259] Example 1. Preparation of ligand traps and detection proteins

[0260] The extracellular region of human ROBO2 (Uniprot Entry: Q9HCK4) was used as a template for ROBO2 to design the amino acid sequences of the ROBO2 ligand trap and detection protein (hereinafter, ROBO2 ligand trap refers to human ROBO2 unless otherwise specified).

[0261] >ROBO2-Fc2.2(Pfizer&Boston medical center,WO2018222850Al)

[0262] The italic double underline is ROBO2 pre-Ig1; the underlined part is ROBO2 Ig1; the double underline is the linker between ROBO2Ig1 and ROBO2 Ig2, and the curved line is ROBO2 Ig2; the dotted line is the ROBO2 Ig2-Ig3 linker; the italic part is the GS linker; the rest is the antibody constant region, minus the hIgG1-Fc with C220S / L234A / L235A / G237A that removes the Fc-mediated effect.

[0263] >ROBO2 Ig1-hIgG1-FcS

[0264] The double-underlined italicized portion is the ROBO2 pre-Ig1 sequence; the underlined portion is the ROBO2 Ig1; the italicized portion is the GS linker; the rest is the antibody constant region, with the hIgG1-Fc with C220S / L234A / L235A / G237A removed from the Fc-mediated effect.

[0265] The amino acid sequence of the ROBO1 ligand trap and detection protein was designed using the extracellular region of human ROBO1 (Uniprot Entry: Q9Y6N7) as a template for ROBO1 (hereinafter, ROBO1 ligand trap refers to human ROBO1 unless otherwise specified).

[0266] >ROBO1 Ig1-hIgG1-FcS

[0267] The italic double-underlined portion is the ROBO1 pre-Ig1 sequence; the underlined portion is ROBO1 Ig1; the italic portion is the GS linker; the rest is the antibody constant region, which is hIgG1-Fc containing C220S / L234A / L235A / G237A.

[0268] The amino acid sequences of SLIT2 and detection proteins were designed using the extracellular region of human SLIT2 (Uniprot Entry: 094813) as a template for SLIT2 (hereinafter, SLIT2 refers to human SLIT2 unless otherwise specified).

[0269] >human SLIT2-D2-avi-his

[0270] The underlined part is the extracellular region of human SLIT2, the second LRR domain of human Slit2 (SLIT2 D2); the italic part is the GS linker; the curved part is the avi-tag; and the dotted part is the his-tag.

[0271] >human SLIT2-D2-his

[0272] >Rat SLIT2-D2-his

[0273] In the above SEQ ID NOs: 5-6, the dotted part is the his-tag, and the remaining parts are human SLIT2-D2 and rat SLIT2-D2, respectively.

[0274] Example 2. Design, construction and screening of saturation mutation phage library

[0275] Based on the crystal structure of the SLIT2-D2 and ROBO1 Ig1 complex (PDB ID: 2V9T) and the sequence alignment of ROBO2Ig1 and ROBO1 Ig1, some key amino acid residues in the interaction interface between ROBO2 Ig1 and SLIT2-D2 were selected to design three random mutation phage libraries (Table 1).

[0276] Table 1. ROBO2-Ig1 saturation mutation phage library

[0277] The three saturation mutation libraries described above were constructed based on the wild-type ROBO2 Ig1 domain. Randomly selected clones were sequenced to verify library quality. Subsequently, phage libraries were screened against the corresponding SLIT2-D2.

[0278] The ROBO2 Ig1 domain with high affinity to human SLIT2-D2-avi-his (SEQ ID NO: 4) protein was obtained by screening the phage library. 20 μg of human SLIT2-D2-avi-his biotin protein was bound to 100 μL Dynabeads. TMAfter incubation at 37°C for one hour, the plate was blocked with 2% skim milk at room temperature for one hour. A saturated mutant phage display library containing the ROBO2 Ig1 domain was added and allowed to react at room temperature for one hour. Unbound phage was removed by washing nine times with PBST (0.05% Tween-20). Phage specifically binding to human SLIT2-D2 was eluted with 1 mg / mL trypsin and infected with Escherichia coli TG1 cells growing in logarithmic phase to generate and purify phage for the next round of screening. This screening process was repeated five times. From the enriched positive clones, 96 monoclonal colonies were selected and packaged into phage-based single-chain antibodies for phage ELISA testing. ELISA plates were coated with 2 μg / mL of SLIT2-D2 protein, and phage supernatants diluted in blocking buffer were added. The plates were then detected using anti-M13 HRP (Sino Biological, Cat. #11973-MM05T-H). Clones with an OD450 / background value > 5 in the ELISA binding assay were sequenced, cloned into a eukaryotic expression vector, expressed, purified, and further screened using cell blocking assays. Four mutants with improved affinity and function were identified (Table 2).

[0279] Table 2. Ligand traps blocking in vitro cell binding IC 50 Determination

[0280] In the above SEQ ID NOs: 7-10, the italicized text represents ROBO2-Ig1, the single underlined text represents the amino acid mutation site, the double underlined text represents the ROBO2 Ig1-2 linker, the dotted line represents ROBO2 Ig2, the curved line represents the ROBO2 Ig2-3 linker, the hyperbola represents the GS linker, and the remainder represents the antibody constant region, which is hIgG1-Fc with C220S / L234A / L235A / G237A; the italicized double underlined text represents the ROBO2 pre-Ig1 sequence.

[0281] The ROBO2 Ig1 mutant sequences obtained by screening are as follows:

[0282] Example 3. Construction and screening of chimeric ligand traps

[0283] Stability experiments revealed a breakage-prone site in ROBO2 Ig2, which could be circumvented by replacing it with the ROBO1 Ig2 sequence. Based on mutants 0044-0504, 0044-0505, 0044-0506, and 0044-0507, ROBO2 Ig1 / ROBO1 Ig2 chimeras were constructed, and the ROBO1Ig1-2 linker, ROBO1 Ig2-3 linker, GS linker, and hIgG1 Fc hinge region sequences were replaced or optimized to further screen for target proteins with better stability.

[0284] In the above SEQ ID NOs: 15-18, the italicized portion is ROBO2-Ig1 (SEQ ID NOs: 11-14), the double-underlined straight line is the ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double-underlined curved line is the ROBO1 Ig2-3 linker, and the rest are hIgG1-Fc with L234A / L235A / G237A and EPKSC removed in the hinge region; the italicized double-underlined portion is the ROBO2 pre-Ig1 sequence.

[0285] Example 4. Affinity determination of chimeric ligand trap and ligand

[0286] In this example, the affinity of the exemplary chimeric ligand trap 0044-0520 and the positive control ROBO2-Fc2.2 to human or rat SLIT2 D2 was determined using a Biacore T200 (GE Healthcare) instrument.

[0287] Experimental Methods: The ligand-trap protein to be detected was captured onto the chip surface using a Series S sensor chip Protein A (GE Healthcare, Cat. #29127556). Human / rat SLIT2 D2-his (SEQ ID NOs: 5-6) was then passed over the chip at varying concentrations. The reaction signal was monitored in real time, and binding and dissociation curves were obtained. Binding constants were then calculated by fitting. The experimental solution used was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chip was washed and regenerated with pH 1.5 Glycine (GE Healthcare, Cat. #BR-1003-54). Affinity results are shown in Table 3. The results showed that the exemplary mutant 0044-0520 of the present disclosure had better affinity for both human SLIT2D2-his and rat SLIT2 D2-his than the positive control ROBO2-Fc2.2.

[0288] Table 3. Affinity determination of ligand traps to human / rat SLIT2 D2

[0289] Example 5. Experiment on the chimeric ligand trap blocking the binding of ROBO2 receptor to SLIT2 in vitro

[0290] This example tests whether the chimeric ligand trap protein acts as a ligand trap by competing with the ROBO2 receptor expressed on the cell surface for the ligand SLIT2.

[0291] Experimental method: HEK293E cells were suspended and transfected with Lipofectamine TM 2000 (Invitrogen, Cat.#11668027) was transfected with a human ROBO2 receptor plasmid (SinoBiological, Cat.#HG10310-CF). 60 hours after transfection, cells were harvested by centrifugation, washed once with PBS, and viable cells were counted and resuspended at a concentration of 500,000 cells / 50 μL PBS. The cells were incubated with 5 nM human SLIT2-D2 (SEQ ID NO: 5), various concentrations of ligand trap protein, and 10 μg / mL heparin sodium in PBS for 1 hour at 4°C. The cells were then washed twice with PBS to remove unbound proteins. Anti-His-APC fluorescently labeled secondary antibody (biolegend, Cat.#362605) was added and incubated at 4°C for another 1 hour. A working concentration of 7AAD (abcam, Cat.#ab142391) was added and incubated at room temperature for 10 minutes. The cells were then washed twice with PBS to remove excess antibody. Flow cytometry (BD Accuri C6 Plus) was used for detection and FlowJo software was used to analyze the results. 7AAD-negative live cells were selected and the proportion of APC-positive cell populations was analyzed. GraphPad Prism9 software was used to analyze the concentration of ligand trap protein and the percentage of APC-positive cells / live cells, and a three-parameter fitting was performed to obtain the IC 50 See Table 4 for example measurement results.

[0292] Table 4. Ligand traps blocking IC of cell binding in vitro 50 Determination

[0293] GraphPad Prism 9 was used to plot the inhibitor-response model using nonlinear fitting and a three-parameter non-logarithmic inhibitor-response model. Example results are shown in Figure 1 . 0044-0520 inhibited the binding of the ligand SLIT2-D2 to the human ROBO2 receptor in a dose-dependent manner, and the blocking effect of 0044-0520 was significantly better than that of the positive control ROBO2-Fc2.2.

[0294] Example 6. Neuronal cell migration experiment

[0295] This example tested the ability of chimeric ligand traps to neutralize SLIT2-N, thereby disabling the repulsive guidance effect of Robo2-SLIT2 on migrating subependymal neurons in the rat forebrain. Within two weeks of birth, rat forebrain subependymal neurons migrate along the rostral migratory canal to the olfactory bulb and develop into olfactory bulb interneurons. SLIT2 exerts a repulsive guidance effect on these neuronal migrations, which is Robo2-dependent.

[0296] Experimental Methods: Forebrain subependymal neuronal tissue blocks were isolated from Sprague-Dawley rat pups (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., gender unrestricted, 3-4 days old) and cut into 0.5 mm x 0.5 mm x 0.5 mm pieces. Matrigel and DMEM (DMEM) supplemented with 10% serum were mixed at a volume ratio of 4:1 (Matrigel, BD, Cat.#354234; DMEM (thermo), Cat.#11995073; fetal bovine serum, Gibco, Cat.#10099-141c). Approximately 50 μL of this mixture was pipetted onto the center of the glass bottom of a 35 mm glass-bottom confocal culture dish (Biyuntian, Cat.#FCFC020) and embedded within the dish. The dish was incubated at 37°C for half an hour to allow the Matrigel to solidify. Different concentrations of ligand-trap proteins were added to DMEM containing a working concentration of 5 nM human SLIT2-N (PeproTech, Cat. #150-11-50). 2 mL was pipetted into confocal microplates and covered with solidified Matrigel. The cells were incubated at 37°C for 24 hours. The neuronal tissue blocks were imaged using bright-field imaging using an Olympus inverted microscope. The perimeter (C1) of the tissue block and the perimeter (C2) of the distal end of the migrating neurons were circled using ImageJ. The radii (R1 = C1 / (π*2) and R2 = C2 / (π*2)) corresponding to each perimeter were calculated. The migration distance (L = R2-R1) of the neurons in each tissue block was calculated. At least five tissue blocks were included in each experimental group for statistical analysis.

[0297] Ligand traps can restore neuronal cell migration in a dose-dependent manner. The EC values ​​were obtained by three-parameter fitting based on the ligand trap protein concentration and neuronal migration distance analyzed by GraphPad Prism9 software. 50 See Table 5 for example measurement results.

[0298] Table 5. Ligand traps restore neuronal cell migration in EC 50 Determination

[0299] GraphPad Prism 9 was used to plot the responses using a nonlinear fitting, three-parameter non-logarithmic agonist-response model, and two-way ANOVA ( Test, *<0.05), as shown in Figure 2 , the exemplary results show that the ligand trap restored the migration of neuronal cells in a dose-dependent manner, and the SLIT2-N neutralizing activity of the 0044-0520 ligand was significantly better than that of the positive control ROBO2-Fc2.2.

[0300] Example 7. In vivo efficacy verification of chimeric ligand trap in rat nephritis model

[0301] This example tests the effect of chimeric ligand traps in inhibiting proteinuria in a rat passive Heimann nephritis model.

[0302] Experimental Methods: Lewis rats (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., male, 8 weeks old) were acclimated for 1 week. Rats were randomly grouped according to body weight and injected intravenously with sheep anti-rat Fx1A serum (Probetex, Cat.# PTX-002S) to induce an immune response to the serum, leading to complement activation and podocyte foot process effacement, resulting in a proteinuric renal phenotype. A prophylactic dosing regimen was used, with ligand trap protein administered subcutaneously starting the day before anti-rat serum injection at a dose of 25 mg / kg and continuing every two days for a total of 6 doses. Urine samples were collected from rats before and after serum modeling in metabolic cages for analysis of urinary albumin and creatinine (Invitrogen, Cat.# EIACUN). The urine albumin / creatinine ratio (UACR, mg / mg) was calculated, and statistical differences in UACR between treatment groups were analyzed using GraphPad Prism 9.

[0303] Table 6. Proteinuria reduction rate in the ligand trap-treated group

[0304] As shown in Table 6 and Figure 3, the rats began to experience proteinuria around day 2-3 of modeling, reaching a peak around day 11. On day 11, compared to the PBS control group, the positive control ROBO2-Fc2.2-treated group showed a 30.4% reduction in proteinuria, while the 0044-0520-treated group showed a 45.8% reduction. Proteinuria levels were significantly different between the two treatment groups, as determined by one-way ANOVA (Tukey test, *<0.05).

[0305] The sequences disclosed herein also include:

[0306] >ROBO1 / 2 pre-Ig1 sequence

[0307] >ROBO2 Ig1

[0308] >ROBO1 Ig1

[0309] >ROBO2 Ig1-2 linker

[0310] >ROBO1 Ig1-2 linker

[0311] >ROBO2 Ig2

[0312] >ROBO1 Ig2

[0313] >ROBO2 Ig2-3 linker

[0314] >ROBO1 Ig2-3 linker

[0315] >ROBO2 Ig3

[0316] >ROBO1 Ig3

[0317] >ROBO2 Ig3-4 linker

[0318] >ROBO1 Ig3-4 linker

[0319] >ROBO2 Ig4

[0320] >ROBO1 Ig4

[0321] >ROBO2 Ig4 post-linker

[0322] >ROBO1 Ig4 post-linker

[0323] >GS linker

[0324] >IgG1 Fc(C220S)

[0325] >IgG1 Fc(C220S / L234A / L235A / G237A)

[0326] >IgG1 Fc (L234A / L235A / G237A; missing EPKSC)

[0327] In addition, the present disclosure also constructs and prepares the following chimeric ligand trap:

[0328] >0044-0545

[0329] In the above sequence, the italic double-underlined element is ROBO2 pre-Ig1, the italic element is ROBO2-Ig1 (SEQ ID NO: 13), the double-underlined straight line is the ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double-underlined curved line is the ROBO1 Ig2-3 linker, the single-underlined element is the ROBO1 Ig3 and ROBO1 Ig3-4 linkers, and the rest are hIgG1-Fc.

[0330] >0044-0546

[0331] In the above sequence, the italic double-underlined line represents ROBO2 pre-Ig1, the italicized line represents ROBO2-Ig1 (SEQ ID NO: 13), the double-underlined straight line represents ROBO1 Ig1-2 linker, the dotted line represents ROBO1-Ig2, the double-underlined curved line represents ROBO1 Ig2-3 linker, the single underline represents ROBO1 Ig3-linker and ROBO1 Ig3-4 linker, the single curved line represents ROBO1 Ig4 and ROBO1 Ig4 post-linker, and the rest represents hIgG1-Fc.

[0332] >0044-0547

[0333] In the above sequence, the italic double underlined line represents ROBO2 pre-Ig1, the italic line represents ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line represents ROBO1 Ig1-2 linker, the dotted line represents ROBO1-Ig2, the double underlined curved line represents ROBO1 Ig2-3 linker, the single underline represents ROBO1 Ig3-linker and ROBO1 Ig3-4 linker, the single curved line represents ROBO2 Ig4 and ROBO2 Ig4 post-linker, and the rest represents hIgG1-Fc.

[0334] >0044-0548

[0335] In the above sequence, the italic double-underlined element is ROBO2 pre-Ig1, the italic element is ROBO2-Ig1 (SEQ ID NO: 13), the double-underlined straight line is the ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double-underlined curved line is the ROBO1 Ig2-3 linker, the single-underlined element is ROBO2 Ig3 and ROBO2 Ig3-4 linkers, and the rest are hIgG1-Fc.

[0336] >0044-0549

[0337] In the above sequence, the italic double underlined line represents ROBO2 pre-Ig1, the italic line represents ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line represents the ROBO1 Ig1-2 linker, the dotted line represents ROBO1-Ig2, the double underlined curved line represents the ROBO1 Ig2-3 linker, the single underline represents ROBO2 Ig3 and the ROBO2 Ig3-4 linker, the single curved line represents ROBO2 Ig4 and the ROBO2 Ig4 post-linker, and the rest represents hIgG1-Fc.

[0338] >0044-0550

[0339] In the above sequence, the italic double underlined line represents ROBO2 pre-Ig1, the italic line represents ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line represents the ROBO1 Ig1-2 linker, the dotted line represents ROBO1-Ig2, the double underlined curved line represents the ROBO1 Ig2-3 linker, the single underline represents ROBO2 Ig3 and the ROBO2 Ig3-4 linker, the single curved line represents ROBO1 Ig4 and the ROBO1 Ig4 post-linker, and the rest represents hIgG1-Fc.

[0340] >0044-0518-1

[0341] >0044-0519-1

[0342] >0044-0520-1

[0343] >0044-0521-1

[0344] In the above SEQ ID NOs: 46-49, the italic double-underlined sequence is the ROBO2 pre-Ig1 sequence, the italicized sequence is ROBO2-Ig1 (SEQ ID NOs: 11-14), the double-underlined straight line is the ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, and the double-underlined curved line is the ROBO1 Ig2-3 linker.

[0345] >0044-0518-2

[0346] >0044-0519-2

[0347] >0044-0520-2

[0348] >0044-0521-2

[0349] In the above SEQ ID NOs: 50-53, the italicized ones are ROBO2-Ig1 (SEQ ID NOs: 11-14), the double-underlined straight line is the ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, and the double-underlined curved line is the ROBO1 Ig2-3 linker.

Claims

1. A protein comprising a circular X receptor (ROBO) domain, wherein the ROBO domain comprises: Circular Xceptor 2 Immunoglobulin-like domain 1 (ROBO2 Ig1), and Circular X-receptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2).

2. The protein according to claim 1, wherein the ROBO domain further comprises one or more of the following i)-vii): i) Robo pre-immunoglobulin-like 1 (Robo pre-Ig1), ii) a linker between the ROBO immunoglobulin-like domain 1 and the immunoglobulin-like domain 2 (ROBO Ig1-2 linker), iii) a linker between the ROBO immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 (ROBO Ig2-3 linker), iv) ROBO immunoglobulin-like domain 3 (ROBO Ig3), v) a linker between the ROBO immunoglobulin-like domain 3 and the immunoglobulin-like domain 4 (ROBO Ig3-4 linker), vi) ROBO immunoglobulin-like domain 4 (ROBO Ig4), vii) ROBO immunoglobulin-like domain 4 post-linker (ROBO Ig4 post-linker); Preferably, the ROBO in i)-vii) is ROBO1 or ROBO2.

3. The protein according to claim 1 or 2, wherein the ROBO2 Ig1 has an amino acid mutation at one or more positions 17, 30, 32, 66, 68 compared to the wild-type ROBO2 Ig1, and the position number is the natural sequence number relative to the amino acid sequence shown in SEQ ID NO: 1; Preferably, the ROBO2 Ig1 has an amino acid mutation selected from A) to E) compared to the wild-type ROBO2 Ig1: A) 17F, 17T, 17V or 17K, B) 30S, 30Y or 30H, C) 32R, 32T or 32Q, D) 66G, 66D, 66V or 66R, and / or E) 68H, 68K, 68D or 68E; More preferably, the ROBO2 Ig1 has an amino acid mutation selected from K)-N) compared to the wild-type ROBO2 Ig1: K)17F / 30S / 32R / 66G / 68H, L)17T / 30Y / 32T / 66D / 68K, M)17V / 30Y / 32R / 66V / 68D, or N)17K / 30H / 32Q / 66R / 68E.

4. The protein according to claim 2 or 3, wherein the ROBO domain comprises the structure shown in formula (I) from the amino terminus to the carboxyl terminus, or the structure shown in formula (I): [ROBO pre-Ig1]a-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b-[ROBO1 Ig2]-[ROBO Ig2-3 linker]c-[ROBO Ig3]d-[ROBO Ig3-Ig4 linker]e-[ROBO Ig4]f-[ROBO Ig4 post-linker]g Formula (I) in, - is a peptide bond, and a, b, c, d, e, f, and g can be independently selected from 0 or 1.

5. The protein according to any one of claims 1 to 4, wherein ROBO2 Ig1 comprises any one of SEQ ID NOs: 20, 11-14, or an amino acid sequence having at least 90% identity thereto.

6. The protein according to any one of claims 2 to 5, wherein ROBO1 Ig2 comprises the amino acid sequence shown in SEQ ID NO: 25; ROBO pre-Ig1 comprises the amino acid sequence shown in SEQ ID NO: 19; The ROBO Ig1-2 linker comprises the amino acid sequence shown in SEQ ID NO: 22 or 23; The ROBO Ig2-3 linker comprises the amino acid sequence shown in SEQ ID NO: 26 or 27; ROBO Ig3 comprises the amino acid sequence shown in SEQ ID NO: 28 or 29; The ROBO Ig3-4 linker comprises the amino acid sequence shown in SEQ ID NO: 30 or 31; ROBO Ig4 comprises the amino acid sequence shown in SEQ ID NO: 32 or 33; and / or, The ROBO Ig4 rear linker comprises the amino acid sequence shown in SEQ ID NO: 34 or 35.

7. The protein according to any one of claims 1 to 6, further comprising an immunoglobulin Fc region, Preferably, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; More preferably, the Fc is a human IgG1 Fc region comprising one or more mutations of 220S, 234A, 235A, 237A; Most preferably, the Fc region comprises any one of SEQ ID NOs: 37-39 or an amino acid sequence at least 90% identical thereto.

8. The protein of claim 7, wherein the ROBO domain is connected to the Fc region directly or through a linker; Preferably, the linker is (G x S) y As shown, x is selected from an integer of 1-5, y is selected from an integer of 1-6; More preferably, the linker is represented by (G2S)2.

9. The protein of any one of claims 1 to 8, comprising any one of SEQ ID NOs: 15-18, 40-53 or an amino acid sequence having at least 90% identity thereto.

10. A protein comprising a cyclic cross-receptor (ROBO) domain, wherein the ROBO domain comprises a cyclic cross-receptor 2 immunoglobulin-like domain 1 (ROBO2 Ig1), wherein the ROBO2 Ig1 has an amino acid mutation at one or more positions 17, 30, 32, 66, and 68 compared to wild-type ROBO2 Ig1, wherein the position numbering is the natural sequence numbering relative to the amino acid sequence of SEQ ID NO: 1; Preferably, the ROBO2 Ig1 has an amino acid mutation selected from A) to E) compared to the wild-type ROBO2 Ig1: A) 17F, 17T, 17V or 17K, B) 30S, 30Y or 30H, C) 32R, 32T or 32Q, D) 66G, 66D, 66V or 66R, and / or E) 68H, 68K, 68D or 68E; More preferably, the ROBO2 Ig1 has an amino acid mutation selected from K)-N) compared to the wild-type ROBO2 Ig1: K)17F / 30S / 32R / 66G / 68H, L)17T / 30Y / 32T / 66D / 68K, M)17V / 30Y / 32R / 66V / 68D, or N)17K / 30H / 32Q / 66R / 68E; Most preferably, the ROBO2 Ig1 comprises any one of SEQ ID NOs: 11-14 or an amino acid sequence having at least 90% identity thereto.

11. The protein according to claim 10, wherein the ROBO domain further comprises a circular X receptor immunoglobulin-like domain 2 (ROBO Ig2), preferably a circular X receptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2) or a circular X receptor 2 immunoglobulin-like domain 2 (ROBO2 Ig2); More preferably, the ROBO1 Ig2 comprises an amino acid sequence as shown in SEQ ID NO: 25, or a sequence at least 90% identical thereto, and the ROBO2 Ig2 comprises an amino acid sequence as shown in SEQ ID NO: 24, or a sequence at least 90% identical thereto.

12. The protein of claim 10 or 11, wherein the ROBO domain further comprises one or more of the following i)-vii): i) Robo pre-immunoglobulin-like 1 (Robo pre-Ig1), ii) a linker between the ROBO immunoglobulin-like domain 1 and the immunoglobulin-like domain 2 (ROBO Ig1-2 linker), iii) a linker between the ROBO immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 (ROBO Ig2-3 linker), iv) ROBO immunoglobulin-like domain 3 (ROBO Ig3), v) a linker between the ROBO immunoglobulin-like domain 3 and the immunoglobulin-like domain 4 (ROBO Ig3-4 linker), vi) ROBO immunoglobulin-like domain 4 (ROBO Ig4), vii) ROBO immunoglobulin-like domain 4 post-linker (ROBO Ig4 post-linker); Preferably, ROBO in i)-vii) is ROBO1 or ROBO2; more preferably, ROBO pre-Ig1 comprises the amino acid sequence shown in SEQ ID NO: 19, The ROBO Ig1-2 linker comprises the amino acid sequence shown in SEQ ID NO: 22 or 23, The ROBO Ig2-3 linker comprises the amino acid sequence shown in SEQ ID NO: 26 or 27, ROBO Ig3 comprises the amino acid sequence shown in SEQ ID NO: 28 or 29, The ROBO Ig3-4 linker comprises the amino acid sequence shown in SEQ ID NO: 30 or 31, ROBO Ig4 comprises the amino acid sequence shown in SEQ ID NO: 32 or 33, and / or, The ROBO Ig4 rear linker comprises the amino acid sequence shown in SEQ ID NO: 34 or 35.

13. The protein according to any one of claims 10 to 12, wherein the ROBO domain comprises a structure as shown in formula (II): [ROBO pre-Ig1]a-[ROBO2 Ig1]-[ROBO Ig1-2 linker]b-[ROBO Ig2]c-[ROBO Ig2-3 linker]d-[ROBO Ig3]e-[ROBO Ig3-Ig4 linker]f-[ROBO Ig4]g-[ROBO Ig4 post-linker]h Formula (II) in, - is a peptide bond, and a, b, c, d, e, f, g, and h can be independently selected from 0 or 1.

14. The protein according to any one of claims 10 to 13, further comprising an immunoglobulin Fc region, Preferably, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4; More preferably, when the immunoglobulin Fc region is the Fc region of human IgG1, it comprises one or more mutations of 220S, 234A, 235A, 237A; Most preferably, the immunoglobulin Fc region comprises any one of SEQ ID NOs: 37-39 or an amino acid sequence at least 90% identical thereto.

15. The protein of claim 14, wherein the ROBO domain is connected to the Fc region directly or through a linker; Preferably, the linker is (G x S) y As shown, x is selected from an integer of 1-5, y is selected from an integer of 1-6; More preferably, the linker is represented by (G2S)2.

16. The protein of any one of claims 10 to 15, comprising any one of SEQ ID NOs: 7-18 or an amino acid sequence having at least 90% identity thereto.

17. A polynucleotide encoding the protein according to any one of claims 1 to 16.

18. A vector comprising or expressing the polynucleotide of claim 17.

19. A host cell comprising or expressing the vector of claim 18.

20. A method for producing or preparing the protein according to any one of claims 1 to 16, comprising: Cultivating the host cell of claim 19; recovering the protein, and Optionally, the protein is isolated and / or purified.

21. A pharmaceutical composition comprising: The protein according to any one of claims 1 to 16, the polynucleotide according to claim 17, or the vector according to claim 18; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

22. Use of the protein according to any one of claims 1 to 16, the polynucleotide according to claim 17 or the vector according to claim 18 in preparing a drug for treating or preventing a disease; Preferably, the disease is a disease in which the ROBO-SLIT2 signaling pathway is abnormally upregulated; Preferably, the disease is kidney disease; More preferably, the renal disease is the glomerular disease, focal segmental glomerulosclerosis (FSGS).

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