WO2025056020A1 - Protein containing robo domain and medical use thereof - Google Patents

Abstract

The present disclosure relates to a protein comprising a ROBO domain and the medical use thereof, and particularly relates to a protein comprising a ROBO domain and the use thereof in the treatment of diseases related to abnormal upregulation of a ROBO-SLIT2 signalling pathway.

Description

Proteins containing ROBO domains and their medical uses

This application claims priority to patent application CN202311184604.1 filed on September 14, 2023.

Technical Field

The present disclosure relates to the field of biomedicine, and in particular to a protein comprising a ROBO domain and a pharmaceutical composition thereof, a preparation method, a medical use, and a method for preventing or treating diseases related to the ROBO-SLIT2 signaling pathway (eg, kidney disease).

Background Art

Endothelial cells, basement membrane and podocytes together constitute the filtration barrier of the glomerulus. Fusion of podocyte foot processes and shedding of cell bodies are the main mechanisms of glomerular proteinuria (Kriz W, et al. 1998. Kidney Int. 54: 687–97). Common glomerular diseases in clinical practice are accompanied by varying degrees of podocyte damage. Early intervention and improvement of podocyte damage are emerging treatments for glomerular-related chronic kidney disease (J. W. Leeuwis et al. Advanced Drug Delivery Reviews 62 (2010) 1325-1336).

Renal podocytes express Roundabout Receptor 2 (ROBO2), which is in an autoinhibitory state when not bound to the ligand SLIT (Barak et al., 2019, Cell 177, 272-285). After ROBO2 binds to the ligand SLIT2 as a receptor, it forms a complex with nephrin through the downstream adaptor protein Nck, negatively regulating actin aggregation mediated by nephrin. In a mouse model, ROBO2 deficiency can alleviate the abnormal podocyte structure of nephrin-deficient mice (Xueping Fan et al. Cell Reports 2, 52–61, July 26, 2012). In addition, 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 affecting the formation of podocyte adhesion spots on the basement membrane, making podocytes more likely to fall off. In mouse models, ROBO2 deficiency can alleviate the glomerular sclerosis and proteinuria phenotype of NMIIA heavy chain-deficient mice (Xueping Fan et al. JCI Insight. 2016; 1(19): e86934). In the mouse renal damage model induced by nephrotoxic serum, the passive Heimann membranous nephritis model in rats, and patients with membranous nephropathy, upregulation of podocyte ROBO2 expression was observed (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.

At present, there is no drug on the market that blocks ROBO2-SLIT2 signaling worldwide. Compared with recombinant natural ROBO2 protein, the present disclosure provides a class of recombinant ROBO proteins that have been modified by mutation, etc., which, as SLIT ligand traps, have stronger SLIT2 ligand neutralizing activity and stability, stronger SLIT2 neutralizing activity can reduce the volume and frequency of administration, and good stability, so that it can be developed into a more convenient formulation form to improve the compliance and ease of use of patients with chronic kidney disease. The recombinant ROBO protein disclosed in the present disclosure can be used to prevent or treat diseases related to the ROBO-SLIT2 signaling pathway, such as podocyte-related nephropathy.

Summary of the invention

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

ROBO domain-containing proteins

The present disclosure provides a protein comprising a ROBO domain, wherein the ROBO domain comprises a circular cross-receptor 2 immunoglobulin-like domain 1 (ROBO2 Ig1).

In some embodiments, the ROBO2 Ig1 has an amino acid mutation 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.

In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following 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.

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

F) S17F, S17T, S17V or S17K,

G) N30S, N30Y or N30H,

H) K32R, K32T or K32Q,

I) S66G, S66D, S66V or S66R, and/or

J) S68H, S68K, S68D or S68E.

In some specific embodiments, the ROBO2 Ig1 has an amino acid mutation selected from the following 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.

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

O)S17F/N30S/K32R/S66G/S68H,

P)S17T/N30Y/K32T/S66D/S68K,

Q)S17V/N30Y/K32R/S66V/S68D, or

R)S17K/N30H/K32Q/S66R/S68E.

In some embodiments, the ROBO2 Ig1 comprises any one of SEQ ID NOs: 20, 11-14 or An amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%; the same below) identity with the polypeptide.

In other embodiments, ROBO2 Ig1 is provided, which has an amino acid mutation at one or more positions 62, 64, 70, 72, 73 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. For example, compared to the wild-type ROBO2 Ig1, there is an amino acid mutation at one or more positions M62, L64, F70, L72, R73.

In other embodiments, ROBO2 Ig1 is provided, which has amino acid mutations at one or more positions 19, 26, 28, 30, 70 compared to wild-type ROBO2 Ig1, and the position numbers are natural sequence numbers relative to the amino acid sequence shown in SEQ ID NO: 1. For example, compared to wild-type ROBO2 Ig1, there are amino acid mutations at one or more positions V19, P26, T28, N30, F70.

In some embodiments, the ROBO domain in the protein also comprises a circular cross-receptor immunoglobulin-like domain 2 (ROBO Ig2).

In some specific embodiments, ROBO Ig2 is circular cross-receptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2) or circular cross-receptor 2 immunoglobulin-like domain 2 (ROBO2 Ig2).

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

In some embodiments, the ROBO domain in the protein 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);

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

In some specific embodiments, ROBO pre-Ig1 comprises an amino acid sequence as shown in SEQ ID NO: 19.

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

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

In some specific embodiments, ROBO Ig3 comprises an amino acid sequence as shown in SEQ ID NO: 28 or 29 or an amino acid sequence that has at least 90% (e.g., 95%) identity thereto.

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

In some specific embodiments, ROBO Ig4 comprises an amino acid sequence as shown in SEQ ID NO: 32 or 33 or an amino acid sequence that has at least 90% (e.g., 95%) identity thereto.

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

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

[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)

[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)

[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)

Wherein, - is a peptide bond, and a2, b2, c2, d2, e2, f2, g2, and h2 in formula (II)-(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 and 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.

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

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

The present disclosure provides a protein comprising a ROBO domain comprising an extracellular region of circular X receptor 2 (ROBO2) or a portion thereof and an extracellular region of circular X receptor 1 (ROBO1) or a portion thereof.

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

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.

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

[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)

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, is 1, and 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, d1 is 1, and e1, f1, and g1 are all 0. In some specific embodiments, a1, b1, c1, d1, e1 is 1, and 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.

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

1) ROBO2 Ig1

2) ROBO2 Ig1 and ROBO1 Ig2;

3) ROBO2 Ig1 and ROBO2 Ig2;

4) ROBO2 Ig1, ROBO1 Ig2, and ROBO1 Ig3;

5) ROBO2 Ig1, ROBO1 Ig2, and ROBO2 Ig3;

6) ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3;

7) ROBO2 Ig1, ROBO2 Ig2 and ROBO2 Ig3;

8) ROBO2 Ig1 and ROBO1 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

9) ROBO2 Ig1 and ROBO1 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

10) ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

11) ROBO2 Ig1 and ROBO2 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

12) ROBO2 Ig1 and ROBO1 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

13) ROBO2 Ig1 and ROBO1 Ig2 and ROBO2 Ig3 and ROBO2 Ig4;

14) ROBO2 Ig1 and ROBO2 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

15)ROBO2 Ig1 and ROBO2 Ig2 and ROBO2 Ig3 and ROBO2 Ig4.

In some embodiments, the ROBO domain may also include 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.

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

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

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

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

19) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO1 Ig3;

20) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO2 Ig3;

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

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

23) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO1 Ig3 and ROBO1 Ig4;

24) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO2 Ig3 and ROBO1 Ig4;

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

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

27) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO1 Ig3 and ROBO2 Ig4;

28) ROBO2 pre-Ig1 sequence (or ROBO1 pre-Ig1 sequence) and ROBO2 Ig1 and ROBO1 Ig2 and ROBO2 Ig3 and ROBO2 Ig4;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

47) 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;

48) 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;

49) 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 Ig3;

50) 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 ROBO2 Ig3;

51) 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 Ig3;

52) 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 ROBO2 Ig3;

53) 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 Ig3 and ROBO1 Ig4;

54) 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 ROBO2 Ig3 and ROBO1 Ig4;

55) 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 Ig3 and ROBO1 Ig4;

56) 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 ROBO2 Ig3 and ROBO1 Ig4;

57) 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 Ig3 and ROBO2 Ig4;

58) 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 ROBO2 Ig3 and ROBO2 Ig4;

59) 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 Ig3 and ROBO2 Ig4;

60) 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 ROBO2 Ig3 and ROBO2 Ig4;

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);

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);

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;

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;

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;

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;

67) 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);

68) 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);

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;

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;

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;

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;

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);

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);

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);

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);

77) ROBO2 Ig1 and ROBO1 Ig1-2 connexon (or ROBO2 Ig1-2 connexon) and ROBO1 Ig2 and ROBO1 Ig2-3 connexon (or ROBO2 Ig2-3 connexon) and ROBO1 Ig3 (or ROBO2 Ig3) and ROBO1 Ig3-4 connexon (or ROBO2 Ig3-4 connexon) and ROBO1 Ig4 (or ROBO2 Ig4);

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);

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);

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);

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);

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);

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);

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).

In some embodiments, the ROBO domain comprises structures selected from 1)-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.

In some specific embodiments, the ROBO2 Ig1 is wild-type ROBO2 Ig1 (e.g., shown in SEQ ID NO: 20) or an amino acid sequence that has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO2 Ig1 is any of the amino acids provided herein. A mutated ROB02 Ig1 (eg, any one of SEQ ID NOs: 11-14, or an amino acid sequence having at least 90% (eg, at least 95%) identity thereto, or any one of A)-R) above).

In some specific embodiments, the ROBO2 Ig 2 is the amino acid sequence shown in SEQ ID NO: 24 or has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO1 Ig 2 is the amino acid sequence shown in SEQ ID NO: 25 or has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO2 Ig 3 is the amino acid sequence shown in SEQ ID NO: 28 or has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO1 Ig 3 is the amino acid sequence shown in SEQ ID NO: 29 or has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO2 Ig 4 is the amino acid sequence shown in SEQ ID NO: 32 or has at least 90% (e.g., at least 95%) identity thereto.

In some specific embodiments, the ROBO1 Ig 4 is the amino acid sequence shown in SEQ ID NO: 33 or has at least 90% (e.g., at least 95%) identity thereto.

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

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

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

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

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

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

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

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

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

In some specific embodiments, the ROBO1 Ig4 post-linker is the amino acid sequence shown in SEQ ID NO: 35.

In some specific embodiments, a variant of ROB0 pre-Ig1 (ROB01 pre-Ig1 or ROB02 pre-Ig1) is provided, which has one or more (e.g., 2, 3, 4, 5, 6) compared to SEQ ID NO: 19. Amino acid substitutions, deletions and/or additions, while still maintaining the function of ROBO pre-Ig1. For example, a variant of ROBO pre-Ig1 is provided, which has one or more (e.g., 2, 3, 4, 5, 6) conservative amino acid substitutions compared to SEQ ID NO: 19, and the variant still maintains the function of ROBO pre-Ig1.

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 invention, and can still achieve the function of the linker.

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) extension domain.

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

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, and also an anti-HSA single domain antibody), and an immunoglobulin domain.

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 include mutations, exemplary mutations include: any or any combination of L234A, L235A, G237A on IgG1; for example, L234A/L235A, L234A/L235A/G237A; any or any combination of F234A, L235A, S228P on IgG4, for example, F234A/L235A, S228P/F234A/L235A. In some specific embodiments, the Fc region includes C220S. In some specific embodiments, the IgG Fc region contains SEQ ID NO: 37-39 or an amino acid sequence that is at least 80% or at least 90% identical thereto.

In some embodiments, (i) the ROBO domain and (ii) the half-life (e.g., in vivo half-life) extension domain may be directly connected or connected via a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary structures. For example, the linker is a flexible linker; for example, G ₄ S (SEQ ID NO: 54), GS, GAP, (G ₄ S) ₂ (SEQ ID NO: 55), (G ₄ S) ₃ (SEQ ID NO: 56), (G ₄ S) ₄ (SEQ ID NO: 57), (G ₄ S) ₅ (SEQ ID NO: 58), ASGS (SEQ ID NO: 59), etc.; for example, the linker is a (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), (G ₄ S) ₂ ; in some embodiments, the linker is absent, that is, y is 0.

In some embodiments, a protein is provided, which comprises an amino acid sequence shown in any of SEQ ID NOs: 15-18, 40-53 or having at least 80%, at least 90% (e.g., at least 95%) identity thereto.

The protein containing the ROBO domain provided by the present disclosure has at least one property selected from the following:

(a) binds to human SLIT2 with a K _D value of ≤1×10 ^-7 M, such as ≤1×10 ^-8 M, ≤1×10 ^-9 M, ≤1×10 ^-10 M, ≤1×10 ^-11 M, ≤1×10 ^-12 _M , preferably ≤1× ₁₀ ^-11 M. The detection method is conventional in the art, such as that provided in Example 4 of the present disclosure.

(b) When the protein comprising the ROBO domain comprises an immunoglobulin Fc region (e.g., an IgG1 Fc region comprising 234A/L235A/237A), it has a prolonged 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 more than 20 times compared to when the immunoglobulin Fc region is not included; 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 when the immunoglobulin Fc region is not included.

(c) blocking the binding of ROB02 expressed on the cell surface to SLIT2 with an _IC50 of ≤500nM, 400nM, 300nM, 200nM, 100nM, 50nM, preferably _≤100nM , more preferably ≤50nM. The experimental method for blocking the binding of ROB02 expressed on the cell surface to SLIT2 and the method for detecting the _IC50 value are conventional in the art, such as those provided in Example 5 of the present disclosure.

(d) Restoring neuronal migration with an EC ₅₀ of ≤200nM, 100nM, 50nM, 20nM, 10nM, 5nM, 3nM, and relieving the repulsive guidance effect dependent on ROBO2-SLIT2, wherein the EC ₅₀ is preferably ≤10nM, more preferably ≤5nM, 2nM. The experimental method for neuronal axon migration and the method for detecting the EC ₅₀ value are conventional in the art, such as provided in Example 6 of the present disclosure.

(e) capable of inhibiting or reducing proteinuria in a subject, the subject being, for example, a rat, more for example, a rat passive Heimann nephritis model. The construction method, administration regimen, and proteinuria detection method of the rat passive Heimann nephritis model are conventional in the art, such as those provided in Example 7 of the present disclosure.

The present disclosure provides variants of a protein 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 the function.

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

Polynucleotides and vectors

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

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.

The polynucleotides of the present disclosure may also be in the form of a vector, may be present in a vector and/or may be part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may be, in particular, an expression vector, i.e., a vector that can provide 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 the elements and their sequences for expression in a particular host. Regulatory elements and other elements useful or necessary for the expression of the protein comprising the ROBO domain of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

The polynucleotides of the present invention can be prepared by known methods based on the amino acid sequence information of the polypeptides of the present invention. The polypeptides may be prepared or obtained (eg by automated DNA synthesis and/or recombinant DNA techniques) and/or may be isolated from suitable natural sources.

Host cells

In some embodiments, a host cell is provided that expresses or is capable of expressing one or more ROBO domain-containing proteins of the present disclosure, and/or a recombinant host cell containing a polynucleotide or vector of the present disclosure.

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

Bacterial cells include, for example, 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).

Fungal cells include, for example, cells of species of Trichoderma, Neurospora and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica) and Hansenula.

Illustratively, mammalian cells are such as HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

The present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

The cells of the present disclosure are not capable of developing into complete plants or animals.

Production or preparation method

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

- culturing a host cell of the present disclosure under conditions that allow expression of a protein of the present disclosure comprising a ROBO domain; and

- recovering the expressed protein from the host cell from the culture; and

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

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.

Methods and reagents for recombinant production of polypeptides or proteins, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, separation and purification techniques suitable for making target proteins such as binding molecules or antibodies disclosed herein are well known to those skilled in the art. Methods for producing and purifying antibodies are well known and can be found in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Technology (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect cells. Mammalian expression systems lead to glycosylation of antibodies, especially 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 and cultured in serum-free medium in bioreactors to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or freeze-dried.

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

Composition

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

In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of a protein containing a ROBO domain in a unit dose. In other specific embodiments, the amount of a protein containing a ROBO domain in a unit dose of the pharmaceutical composition is 0.1-2000 mg. In some specific embodiments, it is 1-1000 mg.

Kits and Tests

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

The present disclosure provides a composition for detecting SLIT2, the composition 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, which comprises using the protein comprising the ROBO domain of the present disclosure.

In some embodiments, the in vitro detection method, system or device may, for example, include: (1) contacting a sample with a protein containing a ROBO domain of the present disclosure; (2) detecting a complex formed between the protein containing a ROBO domain of the present disclosure and the sample; and/or (3) contacting a reference sample (e.g., a control sample) with a protein containing a ROBO domain; and (4) determining the extent of complex formation between the protein containing a ROBO domain 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.

In other embodiments, the in vivo detection method, system or device may include: (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 include 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 can be detected by measuring or visualizing antibodies that bind or do not bind to the ROBO domain-containing protein. Conventional detection assays may be used, such as 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. Detection or determination The living sample can be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture medium.

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

Methods for treating and preventing diseases and pharmaceutical uses

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.

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.

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

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

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

In some embodiments, the disease is the glomerular disease, focal segmental glomerulosclerosis (FSGS).

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG. 2 shows that ROBO2-Fc2.2 and the chimeric ligand trap of the present disclosure are able to neutralize the ligand SLIT2-N, restoring the migration of neuronal cells in a dose-dependent manner.

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

definition

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.

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

Roundabout Receptor (ROBO) is a receptor for Slit-guided ligand (SLIT) protein ligands. Four ROBO receptors in vertebrates have been characterized: ROBO1/Dutt1; ROBO2; ROBO3/Rig-1 and ROBO4/Magic Roundabout. Among them, the sequence of human ROBO2 can be found in Uniprot Q9HCK4, and the sequence of human ROBO1 can be found in Uniprot Q9Y6N7. Both ROBO1 and ROBO2 have an extracellular domain (ECD) structure, and the ECD contains five immunoglobulin-like (Ig-like) domains (Ig1, Ig2, Ig3, Ig4 and Ig5), followed by three fibronectin type 3 (FN3) repeat sequences. Exemplary, roundabout The fork receptor immunoglobulin-like domain is abbreviated as ROBO Ig, the circular cross receptor 1 immunoglobulin-like domain 2 is abbreviated as ROBO1 Ig2, the circular cross 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 of expression only, illustratively, the linker between ROBO Ig2 and ROBO Ig3 can be abbreviated as ROBO Ig2-3 linker, and the ROBO Ig1-2 linker includes ROBO1 Ig1-2 linker and 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.

"Recombinant protein" refers to a polypeptide produced by recombinant DNA technology. Generally speaking, the DNA encoding the polypeptide is inserted into a suitable expression vector and then introduced into a host cell to produce the recombinant protein. In the present 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, the protein comprising a ROBO domain of the present disclosure is a recombinant protein. The recombinant ROBO protein of the present disclosure binds to the SLIT ligand, thereby hindering the binding of SLIT to the cellular ROBO receptor, and is therefore called a SLIT neutralizing ligand trap.

Typically, the ROBO domain-containing proteins of the present disclosure will bind to SLIT2 with an equilibrium dissociation constant (KD) of preferably ^10-7 to ^10-10 moles/liter (M), more preferably ^10-8 to ^10-10 moles/liter, even more preferably ^10-9 to ^10-10 or less as measured in a Biacore or _KinExA or Fortibio assay. Any _KD value greater than ^10-4 M is generally considered to indicate non-specific binding. Specific binding of a receptor to a ligand can be determined in any suitable manner known, 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 in the present disclosure.

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

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 a spacer between the two entities to enable them to achieve a conformation in which they, for example, 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.

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

In the present disclosure, "homology" and "identity" refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of 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 are matched or homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences are matched or homologous, then the two sequences are 95% homologous. In general, the comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

"Conservative amino acid substitution" refers to the replacement of one or more amino acid residues in a protein or polypeptide with conservative amino acids, where the chemical structure of the amino acid residue before the replacement is similar to that of the amino acid residue after the replacement, and the replacement The function, activity or other biological properties of the protein or polypeptide are less affected or substantially unaffected. The conservative amino acid substitution is well known in the art, for example, a conservative amino acid substitution is preferably a substitution of an amino acid in the following groups (i)-(v) by another amino acid residue in the same group:

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

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

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

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

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

"Nucleic acid," "nucleic acid molecule," or "polynucleotide" are used interchangeably in the present disclosure and refer to any DNA molecule or RNA molecule that is single-stranded or double-stranded and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in 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.

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

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 should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different designations are intended, this is clear from the context.

"Host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporating a nucleic acid insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected and/or transformed in vivo with a nucleic acid of the present disclosure. "Cell", "Cell line" and "cell culture" are used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

"Pharmaceutical composition" refers to a mixture containing one or more fusion proteins, proteins containing ROBO domains, polynucleotides and other components described in the present disclosure, such as physiologically/pharmaceutically acceptable carriers, diluents, buffers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient and thus exert biological activity.

"Administering," "applying," and "treating" when applied to an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering," "applying," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, 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. "Administering," "applying," and "treating" also mean treating a cell in vitro and ex vivo by an agent, a diagnostic, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent (such as a fusion protein disclosed herein) to a subject who has 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 in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may 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 disease symptom has been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating every target disease symptom, 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.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. "And/or" should be regarded as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and/or" used in phrases such as "A and/or B" in the present 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".

The “subject” and “patient” of the present disclosure refer to mammals, especially primates, and especially humans.

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 foregoing 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 foregoing 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 foregoing 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.

Example

The following embodiments are used to further describe the present disclosure, but these embodiments are not intended to limit the scope of the present disclosure.

Experimental methods without specific conditions in the embodiments or test examples disclosed herein are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. See Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents without specific sources are conventional reagents purchased from the market.

Example 1. Preparation of ligand trap and detection protein

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

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

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, which removes the Fc-mediated effect of hIgG1-Fc with C220S/L234A/L235A/G237A.

>ROBO2 Ig1-hIgG1-FcS

The italic double underlined part is the ROBO2 pre-Ig1 sequence; the underlined part is ROBO2 Ig1; the italic part is the GS linker; the rest is the antibody constant region, which is the hIgG1-Fc with C220S/L234A/L235A/G237A that removes the Fc-mediated effect.

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

>ROBO1 Ig1-hIgG1-FcS

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

The amino acid sequences of SLIT2 and detection proteins were designed using the extracellular region of human SLIT2 (Uniprot Entry: O94813) as a template for SLIT2 (hereinafter, SLIT2 refers to human SLIT2 unless otherwise specified).

>human SLIT2-D2-avi-his

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.

> Human SLIT2-D2-his

> Rat SLIT2-D2-his

In the above SEQ ID NO: 5-6, the dotted part is his-tag, and the rest are human SLIT2-D2 and rat SLIT2-D2, respectively.

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

Based on the crystal structure of the complex of SLIT2-D2 and ROBO1 Ig1 (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, and three random mutation phage libraries were designed (Table 1).

Table 1. ROBO2-Ig1 saturation mutation phage library

The above three saturation mutation libraries were constructed based on the wild-type ROBO2 Ig1 domain, and clones were randomly selected for sequencing to verify the quality of the library. Subsequently, the phage library and the corresponding SLIT2-D2 were screened.

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 combined with 100 uL Dynabeads ^TM M-280 Streptavidin, and then placed at 37°C for one hour and blocked with 2% skim milk at room temperature for 1 hour. The ROBO2 Ig1 domain saturated mutant phage display library was added and allowed to act at room temperature for 1 hour. The phages that did not bind were removed by washing 9 times with PBST (0.05% Tween-20) solution. The phages that specifically bound to human SLIT2-D2 were eluted with 1 mg/mL trypsin and infected with Escherichia coli TG1 in logarithmic phase growth to produce and purify phages for the next round of screening. The above steps were repeated 5 times for screening, and 96 monoclonal colonies were selected from the positive clones enriched by screening and packaged into phage single-chain antibodies for phage ELISA test. 2 μg/mL of SLIT2-D2 protein was coated on the ELISA plate, and the phage supernatant diluted with blocking solution was added, and detected with anti-M13 HRP (Sino Biological, Cat.#11973-MM05T-H). The clones with OD450 value/background value>5 in the ELISA binding experiment were sequenced, the sequences were cloned into eukaryotic expression vectors, the purified proteins were expressed, and further screened by cell blocking experiments. A total of 4 mutants with improved affinity and function were screened (Table 2).

Table 2. Ligand trap in vitro cell blocking binding _IC50 determination

In the above SEQ ID NO: 7-10, the italicized one is ROBO2-Ig1, the single underline is the amino acid mutation site, the double underline is the ROBO2 Ig1-2 linker, the dotted line is ROBO2 Ig2, the curved line is the ROBO2 Ig2-3 linker, the hyperbola is the GS linker, and the rest is the antibody constant region, which is hIgG1-Fc of C220S/L234A/L235A/G237A; the italic double underline is the ROBO2 pre-Ig1 sequence.

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

Example 3. Construction and screening of chimeric ligand traps

In the stability experiment, it was found that ROBO2 Ig2 had a site that was prone to breakage. Replacing it with the ROBO1 Ig2 sequence could avoid this breakage site. Based on the 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.

In the above SEQ ID NO: 15-18, the italicized one is ROBO2-Ig1 (SEQ ID NO: 11-14), the double underlined straight line is ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double underlined curved line is 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 one is the ROBO2 pre-Ig1 sequence.

Example 4. Affinity determination of chimeric ligand trap and ligand

In this example, the affinity of the example 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.

Experimental method: The ligand trap protein to be detected was captured on the chip surface using the chip Series S sensor chip Protein A (GE Healthcare, Cat.#29127556), and then different concentrations of protein human/rat SLIT2 D2-his (SEQ ID NO: 5-6) were passed over the chip surface. The reaction signal was detected in real time to obtain the binding Dissociation curve, the binding force constant is obtained by fitting. The solution used in the experiment is HBS-EP solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chip was washed and regenerated with pH1.5 Glycine (GE Healthcare, Cat.#BR-1003-54) solution. The affinity results are shown in Table 3. The results show that the exemplary mutant 0044-0520 disclosed in the present invention has a better affinity for human SLIT2D2-his and rat SLIT2 D2-his than the positive control ROBO2-Fc2.2.

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

Example 5. Experiment on chimeric ligand trap blocking the binding of ROBO2 receptor and SLIT2 in vitro

This example detects whether the chimeric ligand trap protein acts as a ligand trap by competitively binding to the ligand SLIT2 with the ROBO2 receptor expressed on the cell surface.

Experimental method: HEK293E cells were suspended and transfected with human ROBO2 receptor plasmid (SinoBiological, Cat.#HG10310-CF) by Lipofectamine ^TM 2000 (Invitrogen, Cat.#11668027). 60 hours after transfection, cells were collected by centrifugation, washed once with PBS buffer, counted the live cells and resuspended to a concentration of 500,000 cells/50uL PBS, incubated with 5nM human SLIT2-D2 (SEQ ID NO: 5), different concentrations of ligand trap protein, and 10ug/mL heparin sodium in PBS, incubated at 4°C for 1 hour, and washed twice with PBS to wash away unbound proteins. Add anti-His-APC fluorescent labeled secondary antibody (biolegend, Cat.#362605), continue incubation at 4°C for 1 hour, add working concentration 7AAD (abcam, Cat.#ab142391), incubate at room temperature for 10 minutes, wash twice with PBS to wash away excess antibody. Detection was performed on a flow cytometer (BD Accuri C6 Plus), and the results were analyzed by FlowJo software. 7AAD-negative live cells were selected to analyze the proportion of APC-positive cell populations. The concentration of ligand trap protein and the percentage of APC-positive cells/live cells were analyzed by GraphPad Prism9 software for three-parameter fitting to obtain IC ₅₀ . See Table 4 for example measurement results.

Table 4. Ligand trap in vitro cell blocking binding _IC50 determination

GraphPad Prism 9 was used to plot the nonlinear fitting and 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.

Example 6. Neuronal cell migration experiment

This example detects the neutralizing ability of chimeric ligand traps on SLIT2-N, and relieves the repulsive guiding effect of ROBO2-SLIT2 on the migration of subependymal neurons in the forebrain of rat pups. Within two weeks after birth, the subependymal neurons of the rat forebrain migrate along the rostral migration channel to the olfactory bulb and develop into olfactory bulb interneurons. SLIT2 has an repulsive guiding effect on the migration of the above-mentioned nerve cells, and is ROBO2-dependent.

Experimental method: Forebrain subependymal neuron tissue blocks were isolated from SD rat pups (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., gender not limited, 3-4 days old) and cut into 0.5mm*0.5mm*0.5mm size. Matrigel and DMEM complete culture medium containing 10% serum were mixed in a volume ratio of 4:1 (Matrigel BD, Cat.#354234; DMEM culture medium thermo, Cat.#11995073; fetal bovine serum Gibco, Cat.#10099-141c), and about 50μL of the above mixture was pipetted to the center of the glass bottom of a 35mm glass-bottomed confocal culture dish (Biyuntian, Cat.#FCFC020), and the tissue blocks were embedded therein and placed at 37℃ for half an hour to allow the matrigel to solidify. Different concentrations of ligand trap proteins were added to DMEM complete medium containing 5 nM human SLIT2-N (PeproTech, Cat.#150-11-50), and 2 mL was pipetted into the confocal culture dish and covered with the solidified matrix gel. Incubate in a cell culture incubator at 37 °C for 24 hours, and photograph each neuronal tissue block with an Olympus inverted microscope in bright field. ImageJ was used to circle the tissue block perimeter C1 and the farthest perimeter C2 of neuronal migration, and the radii R1 = C1/(π*2) and R2 = C2/(π*2) corresponding to each perimeter were calculated, and the migration distance L = R2-R1 of neurons in each tissue block was calculated. Each experimental group required no less than 5 tissue blocks to be included in the statistical analysis.

The ligand trap can restore the migration of neuronal cells in a dose-dependent manner, and the ligand trap protein concentration and neuronal migration distance were analyzed by GraphPad Prism9 software to perform three-parameter fitting to obtain EC ₅₀ . See Table 5 for example measurement results.

Table 5. Ligand trap _EC50 determination for restoration of neuronal cell migration

GraphPad Prism 9 was used to plot the responses using a nonlinear, three-parameter non-log agonist-response model and a two-way ANOVA ( Test, *<0.05), the example results are shown in Figure 2, 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.

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

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

Experimental methods: Lewis rats (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., male, 8 weeks old) were adaptively raised for 1 week. Rats were randomly grouped and numbered according to their weight, and sheep anti-rat Fx1A (Probetex, Cat. #PTX-002S) serum was injected through the tail vein to induce the rats to produce an immune response to the injected serum, complement activation, causing podocyte foot process elimination and then proteinuria renal damage phenotype. A preventive dosing regimen was adopted, and the ligand trap protein was initially administered subcutaneously one day before the injection of anti-rat serum, with a dose of 25 mg/kg, once every two days, for a total of 6 times. Rat urine samples were collected before and after serum modeling through metabolic cages, and urine albumin and urine creatinine (Invitrogen, Cat. #EIACUN) were detected respectively, and urine albumin/creatine was calculated. The difference of UACR among treatment groups was analyzed by GraphPad Prism9.

Table 6. Proteinuria reduction rate in the ligand trap treatment group

The sample results are shown in Table 6 and Figure 3. Rats began to have proteinuria on the 2nd to 3rd day of modeling and reached a peak around the 11th day. On the 11th day of modeling, compared with the PBS control group, the proteinuria reduction rate in the positive control ROBO2-Fc2.2 treatment group was 30.4%, and the proteinuria reduction rate in the 0044-0520 treatment group was increased to 45.8%. And the proteinuria levels of the two treatment groups were significantly different, one-way analysis of variance (Tukey test, *<0.05).

The sequences disclosed herein also include:

>ROBO1/2 pre-Ig1 sequence

>ROBO2 Ig1

>ROBO1 Ig1

>ROBO2 Ig1-2 linker

>ROBO1 Ig1-2 linker

>ROBO2 Ig2

>ROBO1 Ig2

>ROBO2 Ig2-3 linker

>ROBO1 Ig2-3 linker

>ROBO2 Ig3

>ROBO1 Ig3

>ROBO2 Ig3-4 linker

>ROBO1 Ig3-4 linker

>ROBO2 Ig4

>ROBO1 Ig4

>ROBO2 Ig4 post-linker

>ROBO1 Ig4 post-linker

>GS linker

>IgG1 Fc(C220S)

>IgG1 Fc(C220S/L234A/L235A/G237A)

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

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

>0044-0545

In the above sequence, the italic double underline is ROBO2 pre-Ig1, the italic is ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line is ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double underlined curved line is ROBO1Ig2-3 linker, the single underline is ROBO1 Ig3 and ROBO1 Ig3-4 linker, and the rest are hIgG1-Fc.

>0044-0546

In the above sequence, the italic double underline is ROBO2 pre-Ig1, the italic is ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line is ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double underlined curved line is ROBO1Ig2-3 linker, the single underline is ROBO1 Ig3-linker and ROBO1 Ig3-4 linker, the single curved line is ROBO1 Ig4 and ROBO1 Ig4 post-linker, and the rest are hIgG1-Fc.

>0044-0547

In the above sequence, the italic double underline is ROBO2 pre-Ig1, the italic is ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line is ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double underlined curved line is ROBO1Ig2-3 linker, the single underline is ROBO1 Ig3-linker and ROBO1 Ig3-4 linker, the single curved line is ROBO2 Ig4 and ROBO2 Ig4 post-linker, and the rest are hIgG1-Fc.

>0044-0548

In the above sequence, the italic double underline is ROBO2 pre-Ig1, the italic is ROBO2-Ig1 (SEQ ID NO: 13), the double underlined straight line is ROBO1 Ig1-2 linker, the dotted line is ROBO1-Ig2, the double underlined curved line is ROBO1Ig2-3 linker, the single underline is ROBO2 Ig3 and ROBO2 Ig3-4 linker, and the rest are hIgG1-Fc.

>0044-0549

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

>0044-0550

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

>0044-0518-1

>0044-0519-1

>0044-0520-1

>0044-0521-1

In the above SEQ ID NOs: 46-49, the italic double underline is the ROBO2 pre-Ig1 sequence, the italic 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.

>0044-0518-2

>0044-0519-2

>0044-0520-2

>0044-0521-2

In the above SEQ ID NOs: 50-53, the italic 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.

Claims (22)

A protein comprising a circular X receptor (ROBO) domain, wherein the ROBO domain comprises: Circular cross-receptor 2 immunoglobulin-like domain 1 (ROBO2 Ig1), and Circular cross-receptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2). The protein of 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. 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)-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. The protein according to claim 2 or 3, wherein the ROBO domain comprises a structure as shown in formula (I) from the amino terminus to the carboxyl terminus, or a structure as 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) Wherein, - is a peptide bond, and a, b, c, d, e, f, and g can be independently selected from 0 or 1. A protein as described in 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 that is at least 90% identical thereto. 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. 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 that is at least 90% identical thereto. The protein of claim 7, wherein the ROBO domain is connected to the Fc region directly or through a linker; Preferably, the linker is represented by (G _x S) _y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; More preferably, the linker is represented by (G ₂ S) ₂ . The protein according to 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. A protein comprising a circular crossover receptor (ROBO) domain, wherein the ROBO domain comprises a circular crossover 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, and the position numbering is the natural sequence numbering relative to the amino acid sequence shown in SEQ ID NO: 1; Preferably, the ROBO2 Ig1 has an amino acid mutation selected from A)-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 that is at least 90% identical thereto. The protein of claim 10, wherein the ROBO domain further comprises a circular cross receptor immunoglobulin-like domain 2 (ROBO Ig2), preferably a circular cross receptor 1 immunoglobulin-like domain 2 (ROBO1 Ig2) or a circular cross 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 having at least 90% identity thereto, and the ROBO2 Ig2 comprises an amino acid sequence as shown in SEQ ID NO: 24 or a sequence having at least 90% identity thereto. 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. 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) Wherein, - is a peptide bond, and a, b, c, d, e, f, g, and h can be independently selected from 0 or 1. 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 NO: 37-39 or an amino acid sequence that is at least 90% identical thereto. The protein of claim 14, wherein the ROBO domain is connected to the Fc region directly or through a linker; Preferably, the linker is represented by (G _x S) _y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; More preferably, the linker is represented by (G ₂ S) ₂ . A protein as described in any one of claims 10 to 15, which comprises any one of SEQ ID NOs: 7-18 or an amino acid sequence that is at least 90% identical thereto. A polynucleotide encoding the protein according to any one of claims 1 to 16. A vector comprising or expressing the polynucleotide according to claim 17. A host cell comprising or expressing the vector according to claim 18. 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. 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. 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 for 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).