Antibody specifically recognizing ROBO2 and use thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment specifically recognizing ROBO2, a preparation method therefor, and the use thereof.

Description

Antibodies specifically recognizing ROBO2 and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to patent application number PCT / CN2023 / 116161, application date August 31, 2023, entitled “Antibodies specifically recognizing ROBO2 and their applications,” and the entire contents of that application are incorporated herein by reference.

[0003] Submit sequence listing as XML text file

[0004] The contents of the following submitted xml text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (text name: ROBO2_WIPO seq.xml, recording date: 2023.08.28, size: 96KB). Technical Field

[0005] The present invention relates to antibodies that specifically recognize ROB02, as well as methods for preparing and using the antibodies, including using the antibodies to treat diseases associated with the ROB02 signaling pathway, such as kidney diseases, and diseases or conditions mediated by or associated with elevated protein levels in urine. Background Art

[0006] Circular junction proteins (ROBOs) are members of the immunoglobulin-like (Ig) superfamily of cell adhesion molecules (CAMs). To date, the ROBO proteins characterized in vertebrates include ROBO1 (also known as Dutt1), ROBO2, ROBO3 (also known as Rig1), and ROBO4 (also known as Magic Roundabout). ROBO1-4 are single-pass transmembrane receptors. With the exception of ROBO4, ROBO1-3 share high structural and functional similarities. ROBO1-3 share a similar extracellular domain (ECD) containing five immunoglobulin-like (Ig) domains (Ig1, Ig2, Ig3, Ig4, and Ig5) and three fibronectin type III (FNIII) repeats; in contrast, the ROBO4 extracellular region contains only two Ig domains and two FNIII repeats. Furthermore, the intracellular domains of ROBO1 and ROBO2 contain four conserved cytoplasmic (CC) sequences, designated CC0, CC1, CC2, and CC3, respectively. CC0 and CC1 contain multiple tyrosine residues, whereas CC2 and CC3 are rich in proline residues (Kidd T et al., 1998, Cell. 92(2):205–215; Zallen JA et al., 1998, Cell. 92(2):217–227).

[0007] The ligands of the ROBO receptor protein are Slit-directed ligands (SLITs). SLITs are secreted proteins associated with the extracellular matrix and include SLIT1, SLIT2, and SLIT3. Both Ig domains 1 and 2 of the ROBO2 protein interact with SLIT (Liu et al., 2004, Mol Cell Neurosci. 26(2):232–240), with Ig domain 1 being the primary binding site for SLIT (Morlot et al., 2007, Proc Natl Acad Sci USA; 104(38):14923–14928). After binding to SLIT, the ROBO1 / 2 receptor interacts with Rho GTPase-activating proteins (SRGAPs), downregulating the activity of the small GTPase Cdc42, thereby inhibiting F-actin polymerization (Wong et al., 2001, Cell, 107(2):209-221). Signaling components such as Ena / Abl, Dock, PAK, Sos, Cables and Ca2+ have been shown to be involved in the SLIT / ROBO repulsion signaling pathway (Fan et al., 2003, Neuron. 40(1):113-127; Guan et al., 2007, Cell. 129(2):385-395.).

[0008] Slit / Robo signaling was first identified as an "axon guidance" cue that plays a key role in preventing cerebellar axons from recrossing the midline and regulating the elongation and branching of sensory axons. This signaling was later shown to play an important role in the development of multiple tissues, including the lung, kidney, heart, and mammary gland (Wang et al., 1999, Cell. 96(6):771-84; Brose K et al., 1999, Cell. 96(6):795-806). In addition to kidney loss and heart defects, Slit3 mutant mice also show the presence of diaphragmatic hernia. Similar diaphragmatic hernia defects were also observed in Robo 1 / 2 mutant mice. Therefore, the Slit / Robo signaling pathway is an important regulator that controls tissue morphogenesis. In addition, Slit / Robo signaling has been shown to be involved in tumorigenesis, including cell proliferation, cell adhesion, and cell migration (Ballard et al., 2012, Adv Cancer Res. 114:187-235).

[0009] SLIT2 / ROBO2 signaling plays an important role in early kidney development and ureteric bud growth. Mouse mutants lacking Slit2 or Robo2 produce supernumerary ureteric buds, resulting in a wide range of urinary phenotypes, including double kidneys, abnormal ureterovesical junctions, and hydronephrosis (Grieshammer et al., Dev Cell. 2004; 6(5): 709-717; Lu et al., Am J Hum Genet. 2007; 80(4): 616-632). Disruption of human Robo2 leads to congenital anomalies of the kidney and urinary tract (CAKUT), and point mutations in Robo2, SLIT2, and SRGAP1 have also been found in patients with CAKUT and vesicoureteral reflux (Bertoli Avella et al., J Am Soc Nephrol, 2008; 19(4): 825-831; Hwang et al., Hum Genet. 2015; 134(8): 905-916).

[0010] Fan et al. reported that ROBO2 signaling affects the foot structure of glomerular podocytes by negatively regulating nephrin signaling. The study demonstrated that ROBO2 is expressed on the basal surface of mouse glomerular podocytes. After binding to SLIT2, ROBO2 forms a protein complex with nephrin in the glomerular filtration barrier through the adaptor protein NCK. In contrast to the role of nephrin in promoting actin polymerization, SLIT2-ROBO2 signaling inhibits actin polymerization induced by nephrin. In addition, the amount of nephrin-associated F-actin increases in robo2 gene knockout mice. Genetic interaction studies further showed that the loss of Robo2 partially repaired the podocyte foot process structural defects in nephrin-deficient mice (Fan X et al., 2012, Cell Rep. 2012; 2(1): 52-61). The mechanism by which SLIT2 / ROBO2 signaling regulates podocyte function was further revealed (Fan et al., 2016, JCI Insight, 17; 1(19): e86934). This study demonstrates a potential novel signaling pathway comprised of SLIT-ROBO Rho GTPase-activating protein 1 (SRGAP1) and non-muscle myosin IIA (NMIIA) that regulates podocyte adhesion downstream of Robo2. Myosin II regulatory light chain (MRLC), a subunit of NMIIA, directly interacts with SRGAP1 and forms a complex with Robo2 / SRGAP1 / NMIIA in the presence of SLIT2. Immunostaining revealed that SRGAP1, a podocyte protein, colocalizes with Robo2 on the basal surface of podocytes. Furthermore, SLIT2 stimulation inhibited NMIIA activity, reduced focal adhesion formation, and decreased podocyte attachment to collagen. In vivo studies further demonstrated that podocyte-specific knockout of Robo2 protected mice from hypertension-induced podocyte shedding and albuminuria and partially rescued the podocyte-depleted phenotype of Myh9 knockout mice. Therefore, the SLIT2 / ROBO2 / SRGAP1 / NMIIA pathway represents a potential novel signaling pathway in renal podocytes that may play a role in regulating podocyte adhesion and attachment.

[0011] Methods for inhibiting the activity of the Robo2 receptor have been described. Patent application WO2018222850A discloses the use of soluble fragments of the Robo2 receptor, including the Ig1 and Ig2 Slit binding domains of Robo2, to block or reduce the binding of the Robo2 polypeptide to SLIT2. Patent application WO2018227063A1 discloses the use of antibodies targeting the Robo2 Ig1 Slit binding domain to block the Slit-Robo interaction.

[0012] Therefore, there remains a need in the art for therapeutic antibodies that effectively inhibit or otherwise antagonize ROB02, as well as related methods for treating diseases or conditions mediated by ROB02, such as kidney disease, fibrotic disease, cancer, neurological disease, and orthopedic disease.

[0013] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.

[0014] Application Overview

[0015] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising DX1AMH (SEQ ID NO: 49), wherein X1 is F or Y; HC-CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 10); and HC-CDR3 comprising EGFYX1DGFDV (SEQ ID NO: 51), wherein X1 is A, D or S; and a light chain variable domain (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising RASQX1ISSWLA (SEQ ID NO:53), wherein X1 is G or R; a LC-CDR2 comprising X1ASTLQS (SEQ ID NO:54), wherein X1 is A, D or E; and a LC-CDR3 comprising QQANX1FPLT (SEQ ID NO:56), wherein X1 is S or T.

[0016] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40; (ii) V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40; (iii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40; (iv) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40; (v) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:41.

[0017] In some embodiments, an isolated anti-ROBO2 antibody is provided comprising: (i) V H , comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 69; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 59; and V L , comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 70; (iii) V H , comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60; and V L , comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 71; (iv) V H , comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 72; (v) V H , comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 61; and V L , comprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 73.

[0018] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising a heavy chain variable domain (V H ), the V HThe invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising SX1X2WWS (SEQ ID NO: 50), wherein X1 is E, M or S, and X2 is H or N; HC-CDR2 comprising EIYHSGSTNYNPSLKS (SEQ ID NO: 11); and HC-CDR3 comprising DGDDX1SGNDAFDI (SEQ ID NO: 52), wherein X1 is I, S or V; and a light chain variable domain (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising RASQSISSYLN (SEQ ID NO:26); a LC-CDR2 comprising X1ASX2RAT ​​(SEQ ID NO:55), wherein X1 is A or G, and X2 is E, S or T; and a LC-CDR3 comprising LQX1X2X3YPLT (SEQ ID NO:57), wherein X1 is H or L, X2 is M or N, and X3 is A or S.

[0019] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; (iii) V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44; (iv) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:36, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45.

[0020] In some embodiments, an isolated anti-ROBO2 antibody is provided comprising: (i) V H , comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 62; and V L , comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 74; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 63; and V L , comprising the amino acid sequence of SEQ ID NO: 75 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 75; (iii) V H , comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 64; and V L , comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 76; (iv) VH , comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 65; and V L , comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 77.

[0021] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:27, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46.

[0022] In some embodiments, any of the isolated anti-ROBO2 antibodies described above comprises: V H , comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 66; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 78 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 78.

[0023] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:47.

[0024] In some embodiments, any of the isolated anti-ROBO2 antibodies described above comprises: V H , comprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 67; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 79 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 79.

[0025] In some embodiments, an isolated anti-ROBO2 antibody is provided, comprising: (i) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:29, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:39, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:48.

[0026] In some embodiments, any of the isolated anti-ROBO2 antibodies described above comprises: V H , comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 68; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 80 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 80.

[0027] In some embodiments, the isolated anti-ROB02 antibody binds to human ROB02 with a Kd value of 0.1 pM to about 1 nM.

[0028] In some embodiments, any of the isolated anti-ROB02 antibodies described above, the isolated anti-ROB02 antibody comprises an Fc fragment. In some embodiments, the isolated anti-ROB02 antibody is a full-length IgG antibody. In some embodiments, the isolated anti-ROB02 antibody is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated anti-ROB02 antibody is a chimeric, fully human, or humanized antibody. In some embodiments, the isolated anti-ROB02 antibody is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, and linear antibody.

[0029] In some embodiments, an isolated nucleic acid molecule encoding any of the anti-ROB02 antibodies described above is provided. In some embodiments, a vector comprising any of the nucleic acid molecules described above is provided. In some embodiments, a host cell comprising any of the anti-ROB02 antibodies described above, any of the nucleic acid molecules described above, or any of the vectors described above is provided. In some embodiments, a method for producing an anti-ROB02 antibody is provided, comprising: a) culturing any of the host cells described above under conditions effective to express the anti-ROB02 antibody; and b) obtaining the expressed anti-ROB02 antibody from the host cell.

[0030] In some embodiments, a method of treating a disease or condition in a desired individual is provided, comprising administering to the individual an effective amount of any of the anti-ROB02 antibodies described above. In some embodiments, use of any of the anti-ROB02 antibodies described above in the preparation of a pharmaceutical composition for treating a disease or condition in a desired individual is provided. In some embodiments, use of any of the anti-ROB02 antibodies described above, or a pharmaceutical composition comprising an anti-ROB02 antibody, in the preparation of a medicament for treating a disease or condition is provided. In some embodiments, the disease or condition is a disease and / or condition caused by dysregulation of the ROB02 signaling pathway, including kidney disease, or a disease or condition mediated by or associated with elevated protein levels in the urine. In some embodiments, the disease or disorder is selected from, for example, glomerular disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, reflux nephropathy.

[0031] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any of the anti-ROBO2 antibodies described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 shows the blocking activity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY63 on SLIT2-induced MPC shedding in immortalized mouse podocytes.

[0033] Figure 2A shows the binding affinity of anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, R2-HY60, and R2-HY63 to human ROBO2 protein as analyzed by biochemical ELISA. Figure 2B shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY60 to HEK293T-ROBO2 cells as analyzed by cell-based ELISA.

[0034] Figure 3A shows the binding affinity of exemplary affinity-matured anti-ROBO2 antibodies R2-HY27-2 and R2-HY27-3 to human ROBO2 protein as analyzed by biochemical ELISA. Figure 3B shows the binding affinity of exemplary affinity-matured anti-ROBO2 antibodies R2-HY27-2 and R2-HY27-3 to HEK293T-ROBO2 cells as analyzed by cell-based ELISA.

[0035] FIG4 shows the blocking activity of exemplary anti-ROBO2 antibodies R2-HY27-2 and R2-HY27-3 in binding between SLIT2 and ROB02 as analyzed by ELISA.

[0036] Figure 5A shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY63 to the human ROB02-Ig(1-5) domain as analyzed by biochemical ELISA. Figure 5B shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY63 to the human ROB02-Ig(1-4) domain as analyzed by biochemical ELISA. Figure 5C shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY63 to the human ROB02-Ig(1-2) domain as analyzed by biochemical ELISA. Figure 5D shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY27 and R2-HY13 to the human ROB02-Ig1 domain as analyzed by biochemical ELISA. FIG5E shows the binding affinity of exemplary anti-ROBO2 antibodies R2-HY24, R2-HY27, R2-HY13, and R2-HY63 to the human ROB02-Ig2 domain analyzed by biochemical ELISA.

[0037] Figure 6A shows the effect of the exemplary anti-ROBO2 antibody R2-HY27 on urine protein concentration in a passive Heimann nephritis rat model. Figure 6B shows the effect of the exemplary anti-ROBO2 antibody R2-HY27 on 24-hour urine protein in a passive Heimann nephritis rat model.

[0038] Figure 7A shows the effects of exemplary anti-ROBO2 antibodies R2-HY27, R2-HY27-2, and R2-HY27-3 on urine albumin concentration in a rat model of doxorubicin-induced focal granulomatous nephropathy (FSGS). Figure 7B shows the effects of exemplary anti-ROBO2 antibodies R2-HY27, R2-HY27-2, and R2-HY27-3 on 24-hour urine albumin levels in a rat model of doxorubicin-induced focal granulomatous nephropathy (FSGS). Figure 7C shows the effects of exemplary anti-ROBO2 antibodies R2-HY27, R2-HY27-2, and R2-HY27-3 on serum urea nitrogen levels in a rat model of doxorubicin-induced focal granulomatous nephropathy (FSGS). FIG7D shows the effects of exemplary anti-ROBO2 antibodies R2-HY27, R2-HY27-2, and R2-HY27-3 on serum total cholesterol levels in a rat model of focal granulomatous nephropathy (FSGS) induced by doxorubicin.

[0039] Detailed description of this application

[0040] In one aspect, the present application provides anti-ROBO2 antibody molecules. Through a combination of natural scFv phage library screening, yeast library screening, affinity maturation, and appropriately designed biochemical and biological experiments, highly potent antibody molecules capable of binding to human ROB02 and inhibiting the interaction between human ROB02 and its receptor have been identified. The results presented herein demonstrate that, compared to the known anti-ROBO2 antibody Abcs35J (Pfizer) or the known ROB02 ligand trap protein ROBO2-Fc2.2 (Pfizer), the antibodies described herein bind to different or identical regions or epitopes of ROB02. Surprisingly, in various biological experiments, the antibodies described herein have been shown to be even more potent than Abcs35J.

[0041] The anti-ROBO2 antibodies provided herein include, for example, full-length anti-ROBO2 antibodies, anti-ROBO2 single-chain antibodies (scFvs), anti-ROBO2 Fc fusion proteins, multispecific (eg, bispecific) anti-ROBO2 antibodies, anti-ROBO2 immunoconjugates, and the like.

[0042] On the other hand, the present application provides an anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising DX1AMH (SEQ ID NO: 49), wherein X1 is F or Y; HC-CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 10); and HC-CDR3 comprising EGFYX1DGFDV (SEQ ID NO: 51), wherein X1 is A, D or S; and a light chain variable domain (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising RASQX1ISSWLA (SEQ ID NO:53), wherein X1 is G or R; a LC-CDR2 comprising X1ASTLQS (SEQ ID NO:54), wherein X1 is A, D or E; and a LC-CDR3 comprising QQANX1FPLT (SEQ ID NO:56), wherein X1 is S or T.

[0043] On the other hand, the present application provides an anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V HThe invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising SX1X2WWS (SEQ ID NO: 50), wherein X1 is E, M or S, and X2 is H or N; HC-CDR2 comprising EIYHSGSTNYNPSLKS (SEQ ID NO: 11); and HC-CDR3 comprising DGDDX1SGNDAFDI (SEQ ID NO: 52), wherein X1 is I, S or V; and a light chain variable domain (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising RASQSISSYLN (SEQ ID NO:26); a LC-CDR2 comprising X1ASX2RAT ​​(SEQ ID NO:55), wherein X1 is A or G, and X2 is E, S or T; and a LC-CDR3 comprising LQX1X2X3YPLT (SEQ ID NO:57), wherein X1 is H or L, X2 is M or N, and X3 is A or S.

[0044] On the other hand, the present application provides an anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising DLSMD (SEQ ID NO: 7); HC-CDR2 comprising GFDPEDGDTDYAEKFRG (SEQ ID NO: 12); and HC-CDR3 comprising SGSVNDAFEF (SEQ ID NO: 21); and a light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising RASQSISTWLA (SEQ ID NO: 27); a LC-CDR2 comprising KTSNLES (SEQ ID NO: 37); and a LC-CDR3 comprising QQYNSYPYT (SEQ ID NO: 46).

[0045] On the other hand, the present application provides an anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises DYTMH (SEQ ID NO: 8); HC-CDR2 comprises GFDPEDGEAIFAQKFQG (SEQ ID NO: 13); and HC-CDR3 comprises VYDAGEFDF (SEQ ID NO: 22); and the light chain variable domain (V L ), the VL It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising RASQSIGSWLA (SEQ ID NO:28); a LC-CDR2 comprising KTSSLES (SEQ ID NO:38); and a LC-CDR3 comprising QQSYSTPFT (SEQ ID NO:47).

[0046] On the other hand, the present application provides an anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises DQYMD (SEQ ID NO: 9); HC-CDR2 comprises GISDNGGDAYYIDSVKG (SEQ ID NO: 14); and HC-CDR3 comprises DLVEKDAFDI (SEQ ID NO: 23); and a light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising RASQRINSYVN (SEQ ID NO: 29); a LC-CDR2 comprising GASTLQS (SEQ ID NO: 39); and a LC-CDR3 comprising QQSYQTPFT (SEQ ID NO: 48).

[0047] Also provided are nucleic acids encoding anti-ROBO2 antibodies, compositions comprising anti-ROBO2 antibodies, and methods of making and using anti-ROBO2 antibodies.

[0048] definition

[0049] As used herein, "treatment" or "treating" is a method of obtaining beneficial or desired results, including clinical results. For the purposes of this application, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partially or completely), reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or enhancing the quality of life, increasing body weight, and / or prolonging survival. At the same time, "treatment" also includes a reduction in the pathological consequences of the disease (e.g., urine albumin content for chronic kidney disease). The methods of the present application contemplate any one or more aspects of these treatments.

[0050] The term "antibody" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include three hypervariable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having heavy chains of type α, δ, ε, γ, and μ, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0051] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain Fv (scFv), scFv dimers (divalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments can bind to the same antigen as a parent antibody or parent antibody fragment (such as a parent scFv). Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments. In some embodiments, an antigen-binding fragment may include one or more CDRs from a specific human antibody that are transplanted to a framework region from one or more different human antibodies.

[0052] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0053] As used herein, a first antibody "competes" for binding to a second antibody for a ROB02 target when the first antibody, at equimolar concentrations, inhibits binding of the second antibody to the ROB02 target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0054] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, e.g., binding of an antibody to a target that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes a target (which may be an epitope) means that the antibody binds to the target with greater affinity, avidity, more readily, and / or more persistently than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0055] As used herein, an "isolated" anti-ROBO2 antibody refers to an anti-ROBO2 antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not occur in nature.

[0056] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" is (1) unrelated to all or part of the polynucleotide sequence found in nature, (2) operably linked to a polynucleotide sequence with which it is not naturally associated, or (3) not found in nature as part of a longer sequence.

[0057] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions are described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody, transplanted antibody, or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by each of the above-cited references for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this article.

[0058] Table 1: CDR definitions

[0059] 1The amino acid residue numbers refer to the nomenclature of Kabat et al.

[0060] 2 The amino acid residue numbers refer to the nomenclature of Chothia et al.

[0061] 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.

[0062] 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.

[0063] 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0064] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0065] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and confer specificity for binding to the antibody. However, even a single variable domain (or half of an Fv fragment, which only contains 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0066] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0067] The term "diabody" is used in V H and V L A small antibody fragment is prepared by constructing scFv fragments (see above) using short linkers (e.g., 5-10 residues) between the domains. This allows the variable domains to pair between chains rather than within the chain, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the V domains of the two antibodies are H and V L The domains are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0068] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human mammal with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will comprise substantially all, at least one, and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0069] The "percent (%) amino acid sequence identity" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues in a candidate sequence and the polypeptide sequence being compared, when the sequences are aligned, considering conservative substitutions as part of the sequence identity. Percent amino acid sequence identity can be determined by various alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to maximize alignment over the full length of the sequences being compared. However, for the purposes of this article, percent amino acid sequence identity values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0070] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0071] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in their intact form within and between cells, protecting them from the default degradation pathway.

[0072] The "CH1 domain" of a human IgG heavy chain constant region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).

[0073] The "hinge region" is generally defined as stretching from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.

[0074] The "CH2 domain" of the human IgG Fc region typically extends from amino acids 231 to 340. The CH2 domain is unique in that it does not closely pair with another domain. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is hypothesized that carbohydrates may serve as a substitute for domain-to-domain pairing, helping to maintain CH2 domain stability. Burton, Molec. Immunol. 22:161-206 (1985).

[0075] The "CH3" domain includes the CH2 domain extending from the C-terminal residue within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 in IgG).

[0076] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0077] IgG Fc variant antibodies with "altered" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to FcRs have a higher binding affinity (e.g., lower apparent Kd or IC) to at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. 50In some embodiments, the binding capacity is enhanced by 3-fold, e.g., 5-, 10-, 25-, 50-, 60-, 100-, 150-, 200-, or even up to 500-fold, or the binding capacity is increased by 25% to 1000%, compared to the parent polypeptide. An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity for at least one FcR (e.g., a higher apparent Kd or IC) than the parent polypeptide. 50 Compared with the parent polypeptide, its binding capacity is reduced by 40% or more.

[0078] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0079] Polypeptides comprising Fc region variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. Such polypeptides comprising Fc region variants are capable of more effectively mediating ADCC, both in vitro and in vivo, when assayed with substantially the same number of wild-type IgG Fc polypeptides (or parent polypeptides). Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, for example, in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0080] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0081] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0082] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0083] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0084] An "effective amount" of an anti-ROBO2 antibody or composition disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods relevant to the stated purpose.

[0085] The term "therapeutically effective amount" refers to an amount of an anti-ROBO2 antibody or composition thereof disclosed herein that is effective in treating a disease or symptom in an individual. For example, in the case of cancer, a therapeutically effective amount of an anti-ROBO2 antibody or composition thereof refers to an amount that can reduce the urine albumin concentration of a patient with chronic kidney disease; reduce the total 24-hour urine albumin; reduce the serum urea nitrogen level; reduce the serum total cholesterol level; inhibit (i.e., slow down or preferably stop to some extent) the shedding of podocytes; and / or alleviate to some extent one or more symptoms associated with chronic kidney disease. The anti-ROBO2 antibodies or compositions thereof disclosed herein are capable of inhibiting and / or killing existing tumor cells to some extent, and can be cytostatic or cytotoxic. In some embodiments, a therapeutically effective amount refers to an amount that can prolong the patient's survival. In some embodiments, a therapeutically effective amount refers to an amount that can improve the patient's progression-free survival.

[0086] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0087]

[0066] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0088] Reference herein to "about" for a value or parameter includes (and describes) variations of the value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0089] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, "the method cannot be used to treat type X cancer" means that the method is generally used to treat other types of cancer besides type X cancer.

[0090] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0091] Anti-ROBO2 antibodies

[0092] In one aspect, the present application provides anti-human or / and cynomolgus monkey ROB02 antibodies that specifically bind to ROB02. The anti-ROB02 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs as described herein. In one aspect, the present application provides isolated antibodies that bind to ROB02. Contemplated anti-ROB02 antibodies include, for example, full-length anti-ROB02 antibodies (such as full-length IgG1 or IgG4), anti-ROB02 single-chain antibodies, anti-ROB02 Fc fusion proteins, multispecific (such as bispecific) anti-ROB02 antibodies, anti-ROB02 immunoconjugates, and the like. In some embodiments, the anti-ROB02 antibody is a full-length antibody (such as full-length IgG1 or IgG4) or an antigen-binding fragment thereof that specifically binds to ROB02. In some embodiments, the anti-ROBO2 antibody is a Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody or linear antibody. In some embodiments, an antibody that specifically binds to ROB02 means that the antibody binds to ROB02 with an affinity that is at least 10 times greater than the affinity for binding to a non-target (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 In some embodiments, a non-target antigen is an antigen that is not ROB02. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0093] Although anti-ROBO2 antibodies comprising human sequences (e.g., human heavy and light chain variable domains comprising human CDR sequences) are generally discussed herein, non-human anti-ROBO2 antibodies are also contemplated. In some embodiments, non-human anti-ROBO2 antibodies comprise human CDR sequences of the anti-ROBO2 antibodies described herein and non-human framework region sequences. In some embodiments, non-human framework region sequences include any sequence used to generate heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., small apes, macaques), etc. In some embodiments, non-human anti-ROBO2 antibodies include anti-ROBO2 antibodies generated by grafting one or more human CDR sequences described herein into non-human framework regions (e.g., mouse or chicken framework region sequences).

[0094] An exemplary amino acid sequence of the extracellular region of human ROB02 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85.

[0095] In some embodiments, the anti-ROB02 antibodies described herein specifically recognize an epitope in human ROB02. In some embodiments, the anti-ROB02 antibodies cross-react with ROB02 from species other than human. In some embodiments, the anti-ROB02 antibodies are completely specific for human ROB02 and do not cross-react with species other than human.

[0096] In some embodiments, the anti-ROB02 antibody cross-reacts with at least one allelic variant of a ROB02 protein (or a fragment thereof). In some embodiments, the allelic variant has up to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (e.g., conservative substitutions) compared to a naturally occurring ROB02 protein (or a fragment thereof). In some embodiments, the anti-ROB02 antibody does not cross-react with any allelic variant of a ROB02 protein (or a fragment thereof).

[0097] In some embodiments, the anti-ROB02 antibody cross-reacts with at least one interspecies variant of the ROB02 protein. In some embodiments, for example, the ROB02 protein (or fragment thereof) is human ROB02, and the interspecies variant of the ROB02 protein (or fragment thereof) is a variant in cynomolgus macaque. In some embodiments, the anti-ROB02 antibody does not cross-react with any interspecies variant of the ROB02 protein.

[0098] In some embodiments, as described herein, any anti-ROB02 antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-ROB02 antibody comprises an IgG1 heavy chain constant region. In some embodiments, the anti-ROB02 antibody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-ROB02 antibody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-ROB02 antibody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 82. In some embodiments, the anti-ROB02 antibody comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 83. In some embodiments, the anti-ROB02 antibody comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 84. In some embodiments, the anti-ROB02 antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.

[0099] In some embodiments, the anti-ROBO2 antibody comprises a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising DX1AMH (SEQ ID NO: 49), wherein X1 is F or Y; HC-CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 10); and HC-CDR3 comprising EGFYX1DGFDV (SEQ ID NO: 51), wherein X1 is A, D or S; and a light chain variable domain (V L ), the V L Comprising a light chain complementary determining region (LC-CDR) 1 comprising RASQX1ISSWLA (SEQ ID NO: 53), wherein X1 is G or R; a LC-CDR2 comprising X1ASTLQS (SEQ ID NO: 54), wherein X1 is A, D or E; and a LC-CDR3 comprising QQANX1FPLT (SEQ ID NO: 56), wherein X1 is S or T

[0100] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-2 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; an HC-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 10 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-17 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0101] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H It comprises: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-2, HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 10, and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-17.

[0102] In some embodiments, the anti-ROBO2 antibody comprises V L , the V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:24-25 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:30-32 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs:40-41 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0103] In some embodiments, the anti-ROBO2 antibody comprises V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:24-25, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:30-32, and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs:40-41.

[0104] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-2 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; an HC-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 10 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-17 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and V L , the V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:24-25 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:30-32 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs:40-41 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0105] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15-17; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:24-25, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:30-32, and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs:40-41.

[0106] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or the V HA variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0107] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40.

[0108] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0109] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40.

[0110] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0111] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:32, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40.

[0112] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0113] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40.

[0114] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0115] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:41.

[0116] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 58-61 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 69-73 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0117] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 58 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 69 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0118] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 59 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 70 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0119] In some embodiments, the anti-ROBO2 antibody comprises V H, which comprises V as shown in the amino acid sequence SEQ ID NO: 60 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 71 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0120] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 58 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 72 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0121] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO:61 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 73 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0122] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 58-61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 58-61; and V L , the V L comprising the amino acid sequence of any one of SEQ ID NOs: 69-73 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-73. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 58-61, and VL , the V L Comprising the amino acid sequence shown in any one of SEQ ID NOs: 69-73.

[0123] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 58, and V L , the V L Comprising the amino acid sequence SEQ ID NO:69.

[0124] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 59; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 59, and V L , the V L Comprising the amino acid sequence SEQ ID NO:70.

[0125] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 60; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 60, and V L , the V L Comprising the amino acid sequence SEQ ID NO:71.

[0126] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 58, and V L , the V L Comprising the amino acid sequence SEQ ID NO:72.

[0127] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 61; and V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 61, and V L , the V L Comprising the amino acid sequence SEQ ID NO:73.

[0128] In some embodiments, the anti-ROBO2 antibody comprises a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising SX1X2WWS (SEQ ID NO: 50), wherein X1 is E, M or S, and X2 is H or N; HC-CDR2 comprising EIYHSGSTNYNPSLKS (SEQ ID NO: 11); and HC-CDR3 comprising DGDDX1SGNDAFDI (SEQ ID NO: 52), wherein X1 is I, S or V; and a light chain variable domain (V L ), the V L Comprising: a light chain complementary determining region (LC-CDR) 1 comprising RASQSISSYLN (SEQ ID NO: 26); a LC-CDR2 comprising X1ASX2RAT ​​(SEQ ID NO: 55), wherein X1 is A or G, and X2 is E, S, or T; and a LC-CDR3 comprising LQX1X2X3YPLT (SEQ ID NO: 57), wherein X1 is H or L, X2 is M or N, and X3 is A or S

[0129] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-6 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; an HC-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 11 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 18-20 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0130] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H It comprises: HC-CDR1, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 3-6, HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 11, and HC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 18-20.

[0131] In some embodiments, the anti-ROBO2 antibody comprises V L , the V L Comprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 33-36 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-45 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0132] In some embodiments, the anti-ROBO2 antibody comprises V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 26, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 33-36, and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-45.

[0133] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 3-6 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; an HC-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 11 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 18-20 or a variant thereof, wherein the variant comprises a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and V L , the V LComprising: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 33-36 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids; and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-45 or a variant thereof, wherein the variant comprises substitutions of up to about 3 (e.g., 1, 2 or 3) amino acids.

[0134] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 3-6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 18-20; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 26, LC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 33-36, and LC-CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-45.

[0135] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 33, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0136] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:33, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:42.

[0137] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0138] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:43.

[0139] In some embodiments, the anti-ROBO2 antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0140] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:44.

[0141] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0142] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:36, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45.

[0143] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 62-65 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , the V L Comprising V as shown in any one of the amino acid sequences of SEQ ID NOs: 74-77 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0144] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 62 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 74 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0145] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 63 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 75 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0146] In some embodiments, the anti-ROBO2 antibody comprises V H, which comprises V as shown in the amino acid sequence SEQ ID NO: 64 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 76 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0147] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 65 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 77 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0148] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 62-65 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 62-65; and V L , the V L comprising the amino acid sequence of any one of SEQ ID NOs: 74-77 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 74-77. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 62-65, and V L , the V L Comprising the amino acid sequence shown in any one of SEQ ID NOs:74-77.

[0149] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the anti-ROBO2 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 62, and V L , the V L Comprising the amino acid sequence SEQ ID NO:74.

[0150] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 75 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 63, and V L , the V L Comprising the amino acid sequence SEQ ID NO:75.

[0151] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64; and V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 64, and V L , the V L Comprising the amino acid sequence SEQ ID NO:76.

[0152] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 65, and V L , the V L Comprising the amino acid sequence SEQ ID NO:77.

[0153] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 27, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46, or the V LA variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0154] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:27, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46.

[0155] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 66 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 78 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0156] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 78 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 66, and V L , the V L Comprising the amino acid sequence SEQ ID NO:78.

[0157] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0158] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:47.

[0159] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 67 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 79 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0160] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V Hcomprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 67; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 79 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 67, and V L , the V L Comprising the amino acid sequence SEQ ID NO:79.

[0161] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 39, LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0162] In some embodiments, the anti-ROBO2 antibody comprises V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and V L , the V LIt comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:29, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:39, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:48.

[0163] In some embodiments, the anti-ROBO2 antibody comprises V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 68 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:80 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0164] In some embodiments, the anti-ROBO2 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 68; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 80 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the anti-ROB02 antibody comprises V H , the V H Comprising the amino acid sequence SEQ ID NO: 68, and V L , the V L Comprising the amino acid sequence SEQ ID NO:80.

[0165] In some embodiments, the above-mentioned amino acid substitutions are limited to the "exemplary substitutions" shown herein in Table 4. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown herein in Table 4.

[0166] In some embodiments, functional epitopes can be resolved using combinatorial alanine scanning. In this process, combinatorial alanine scanning can be used to identify amino acids within the Robo2 protein that are essential for interaction with anti-RobO2 antibodies. In some embodiments, the epitope is conformational, and the epitope can be identified using a crystal structure of an anti-RobO2 antibody bound to the Robo2 protein.

[0167] In some embodiments, the present application provides antibodies that compete with any of the anti-ROB02 antibodies described herein for binding to ROB02. In some embodiments, antibodies are provided that can compete with any of the anti-ROB02 antibodies described herein for binding to an epitope on ROB02. In some embodiments, anti-ROB02 antibodies are provided that are combined with a V H and V L The anti-ROBO2 antibody molecules bind to the same epitope, wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 58-68, and said V L In some embodiments, an anti-ROBO2 antibody is provided, which is combined with a V H and V L The anti-ROBO2 antibody competitively binds to ROBO2, wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 58-68, and said V L Comprising the amino acid sequence shown in any one of SEQ ID NOs: 69-80.

[0168] In some embodiments, competition assays can be used to identify monoclonal antibodies that compete with the anti-ROBO2 antibodies described herein for binding to ROB02. Competition assays can determine whether two antibodies bind to the same epitope by recognizing identical or spatially overlapping epitopes or by competitively inhibiting antigen binding of one antibody to the other. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein. Some exemplary competition assays include, but are not limited to, conventional assays described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody. In some embodiments, the antibodies that compete with the anti-ROBO2 antibodies described herein are chimeric, humanized, or fully human antibodies.

[0169] Exemplary anti-ROBO2 antibody sequences are shown in Tables 2 and 3, where CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining the variable regions of antibody light and heavy chains. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, are also within the scope of this application.

[0170] Table 2 Exemplary anti-ROBO2 antibody CDR sequences

[0171] Table 3 Exemplary sequences

[0172] ROBO2

[0173] The roundabout gene, robo, was originally discovered in Drosophila during a screen for mutants controlling midline crossing of axons in the central nervous system (CNS). Caenorhabditis elegans (C. elegans) expresses a ROBO homolog. Three ROBOs are found in Drosophila, chicken, and Xenopus laevis. Four ROBO proteins are found in zebrafish and mammals. Robos, consisting of 1000–1600 amino acids, are highly conserved transmembrane receptors belonging to the Ig superfamily of cell adhesion molecules. They lack autocatalytic and enzymatic activity. With the exception of Robo4, Robo1–3 share a high degree of structural and functional similarity. They share a similar extracellular domain consisting of five immunoglobulin-like (Ig) structures followed by three fibronectin type III (FNIII) repeats. The Robo4 extracellular domain contains only two Ig domains and two FNIII repeats. The intracellular domain of Robos is quite variable, but it contains several conserved motifs for recruiting various adaptor proteins, such as the CC0-3 motif in Robo1-2 molecules, the CC0, CC2, and CC3 motifs in Robo3, and the CC0 and CC2 motifs in Robo4 (Wu et al., 2017, Tissue Barriers; 5(2):e1331155).

[0174] SLIT2 ligands

[0175] Slit-guide ligand proteins (SLITs) are evolutionarily conserved secreted polypeptides that bind to transmembrane circular interdigitating protein (ROBO) receptors. SLITs are secreted proteins associated with the extracellular matrix and include SLIT1, SLIT2, and SLIT3. All SLIT protein sequences display a highly conserved and identical structure, consisting of an N-terminal signal peptide, four tandem leucine-rich repeat domains (LRRs), designated D1-D4, six epidermal growth factor (EGF)-like domains, a laminin G-like domain, one additional (invertebrate) or three (vertebrate) EGF-like domains, and a C-terminal cysteine ​​knot domain (Itoh et al., 1998, Brain Res. 62, 175-186; Rothberg et al., 1990, Genes Dev, 4, 2169-2187). SLIT ligands bind to the Ig1 domain of ROBO1 and ROBO2 via the second leucine-rich repeat region (D2). However, mammalian ROBO3 and ROBO4 lack SLIT-binding residues and therefore do not bind to SLITs. SLIT ligands can be cleaved to produce a short C-terminal fragment (SLIT-C product) of unknown function and a long N-terminal fragment (SLIT-N product), which is active and mediates binding to ROBOs. In addition to axon repulsion, Slits have other properties including regulation of axonal branching from the dorsal root ganglion (Wang et al., 1999, Cell. 96, 771-784). Slit2 promotes neuronal migration from the subventricular zone in the developing vertebrate central nervous system (Hu et al., 1999, Neuron. 23, 703-711; Nguyen Ba-Charvet et al., 2002, J Physiol. 96, 91-98).

[0176] Full-length anti-ROBO2 antibody

[0177] In some embodiments, the anti-ROB02 antibody is a full-length anti-ROB02 antibody. In some embodiments, the full-length anti-ROB02 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-ROB02 antibody comprises an IgG constant region, such as a constant region of IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the full-length anti-ROB02 antibody comprises a lambda light chain constant region. In some embodiments, the full-length anti-ROB02 antibody comprises a kappa light chain constant region. In some embodiments, the full-length anti-ROB02 antibody is a full-length human anti-ROB02 antibody. In some embodiments, the full-length anti-ROB02 antibody comprises a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-ROB02 antibody comprises an altered or otherwise modified Fc sequence such that it has enhanced effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0178] Thus, for example, in some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody specifically binds to ROB02. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0179] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG2 constant region is provided, wherein the anti-ROB02 antibody specifically binds to ROB02. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0180] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG3 constant region is provided, wherein the anti-ROB02 antibody specifically binds to ROB02. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0181] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody specifically binds to ROB02. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:84.

[0182] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 10-14 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-23 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 24-29 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 30-39 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40-48 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:84.

[0183] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG2 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 10-14 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-23 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 24-29 of any one of the amino acid sequences or variants thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 30-39 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40-48 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG2 is a human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0184] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG3 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 10-14 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-23 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 24-29 of any one of the amino acid sequences or variants thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 30-39 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40-48 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG3 is a human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0185] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 10-14 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 15-23 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 24-29 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 30-39 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40-48 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:84.

[0186] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-9, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 10-14, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15-23, or a variant of the heavy chain variable domain comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequence; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 24-29, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 30-39, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NOs: The amino acid sequence of any one of NOs:40-48, or a variant of the light chain variable domain, comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in its LC-CDR sequence. In some embodiments, the IgG1 is human

[0187] IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0188] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-9, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 10-14, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15-23, or a variant of the heavy chain variable domain comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequence; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 24-29, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 30-39, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NOs: NOs: 40-48, or a variant of the light chain variable domain, comprising a substitution of up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acids in its LC-CDR sequence. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0189] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-9, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 10-14, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15-23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 24-29, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 30-39, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 40-48. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0190] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-9, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 10-14, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15-23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 24-29, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 30-39, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 40-48. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0191] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0192] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0193] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0194] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0195] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0196] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0197] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0198] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0199] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0200] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 27, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0201] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0202] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG1 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0203] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0204] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0205] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0206] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0207] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0208] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0209] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0210] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0211] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 26, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0212] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 27, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0213] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0214] In some embodiments, a full-length anti-ROB02 antibody comprising an IgG4 constant region is provided, wherein the anti-ROB02 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0215] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 58-68 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 58-68; and a light chain variable domain (V L ), the V Lcomprising the amino acid sequence of any one of SEQ ID NOs: 69-80 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-80. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0216] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG2 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 58-68 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 58-68; and a light chain variable domain (V L ), the V L The invention also provides an IgG2 antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 69-80, or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 69-80. In some embodiments, the IgG2 antibody is a human IgG2 antibody. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0217] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG3 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V Hcomprising the amino acid sequence of any one of SEQ ID NOs: 58-68 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 58-68; and a light chain variable domain (V L ), the V L The invention also provides an IgG3 antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 69-80, or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 69-80. In some embodiments, the IgG3 antibody is a human IgG3 antibody. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0218] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 58-68 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 58-68; and a light chain variable domain (V L ), the V Lcomprising the amino acid sequence of any one of SEQ ID NOs: 69-80 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69-80. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0219] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 58-68, and a light chain variable domain (V L ), the V L comprising the amino acid sequence shown in any one of SEQ ID NOs: 69-80. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence

[0220] Consists of SEQ ID NO:84.

[0221] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: a heavy chain variable domain (V H ), the V Hcomprising the amino acid sequence shown in any one of SEQ ID NOs: 58-68, and a light chain variable domain (V L ), the V L comprising the amino acid sequence of any one of SEQ ID NOs: 69-80. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0222] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , which comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0223] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 59; and V L , which comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0224] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60; and V L , which comprises the amino acid sequence of SEQ ID NO: 71 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0225] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , which comprises the amino acid sequence of SEQ ID NO: 72 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0226] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 61; and V L , which comprises the amino acid sequence of SEQ ID NO: 73 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0227] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 62; and V L , which comprises the amino acid sequence of SEQ ID NO: 74 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0228] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 63; and V L , which comprises the amino acid sequence of SEQ ID NO: 75 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0229] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 64; and V L , which comprises the amino acid sequence of SEQ ID NO: 76 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 76. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0230] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 65; and V L , which comprises the amino acid sequence of SEQ ID NO: 77 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0231] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 66; and V L , which comprises the amino acid sequence of SEQ ID NO: 78 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0232] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 67; and V L , which comprises the amino acid sequence of SEQ ID NO: 79 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 79. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0233] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG1 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 68; and V L , which comprises the amino acid sequence of SEQ ID NO: 80 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0234] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , which comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 69. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0235] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 59; and V L , which comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0236] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60; and V L , which comprises the amino acid sequence of SEQ ID NO: 71 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 71. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0237] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , which comprises the amino acid sequence of SEQ ID NO: 72 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 72. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0238] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 61; and V L , which comprises the amino acid sequence of SEQ ID NO: 73 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0239] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 62; and V L , which comprises the amino acid sequence of SEQ ID NO: 74 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0240] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 63; and V L , which comprises the amino acid sequence of SEQ ID NO: 75 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0241] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 64; and V L , which comprises the amino acid sequence of SEQ ID NO: 76 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 76. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0242] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 65; and V L , which comprises the amino acid sequence of SEQ ID NO: 77 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0243] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 66; and V L , which comprises the amino acid sequence of SEQ ID NO: 78 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0244] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 67; and V L , which comprises the amino acid sequence of SEQ ID NO: 79 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 79. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0245] In some embodiments, a full-length anti-ROBO2 antibody comprising an IgG4 constant region is provided, wherein the anti-ROBO2 antibody comprises: V H, comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 68; and V L , which comprises the amino acid sequence of SEQ ID NO: 80 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0246] Binding affinity

[0247] Binding affinity is expressed as Kd, Koff, Kon or Ka. As used herein, the term Koff refers to the rate constant for the dissociation of an antibody from an antigen / antibody complex, as measured by a kinetic selection apparatus. The term Kon refers to the association rate constant for the binding of an antibody to an antigen to form an antigen / antibody complex. The equilibrium dissociation constant, Kd, ​​used herein refers to the dissociation constant for a specific antibody-antigen interaction, and is the concentration of antigen required for the antigen to occupy half of all antibody binding sites in a solution of antibody molecules and to reach equilibrium, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antibody is attached to the cell wall, such as in a yeast expression system, the corresponding equilibrium dissociation rate constant is expressed as EC 50 It is expressed as , which is a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0248] The dissociation constant (Kd) can be used as an indicator of the affinity of the reactive antibody portion for the antigen. For example, a simple analysis can be performed using antibodies labeled with various markers using the Scatchard method and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, a Kd of ≤10 -7 M, ≤10 -8 M, ≤10-9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0249] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0250] In some embodiments, the anti-ROBO2 antibody specifically binds to the ROB02 target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Therefore, in some embodiments, the Kd value of the binding between the anti-ROBO2 antibody and ROB02 is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 - 13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 - 13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10-9 M to 1×10 -11 M, 5×10 -9 M to 1×10 - 10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 - 13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 - 13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the anti-ROBO2 antibody and ROB02 is 10 -7 M to 10 -13 M.

[0251] In some embodiments, the Kd value of the anti-ROBO2 antibody binding to the non-target is higher than the Kd value of the anti-ROBO2 antibody binding to the target, and in some embodiments cited herein, the binding affinity of the anti-ROBO2 antibody to the target (e.g., ROB02) is higher than the binding affinity of the anti-ROBO2 antibody to the non-target. In some embodiments, the non-target is an antigen other than ROB02. In some embodiments, the Kd value of the anti-ROBO2 antibody (directed to ROB02) binding to the non-ROBO2 target differs by at least 10 times, e.g., 10-100 times, 100-1000 times, 10 3 -104 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0252] In some embodiments, the anti-ROBO2 antibody has a Kd value of 10 for binding to a non-target -1 M to 10 -6 M (e.g. 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target is an antigen other than ROB02. Thus, in some embodiments, the Kd value of the binding between the anti-ROBO2 antibody and the non-ROBO2 target is 10 -1 M to 10 - 6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10 -4 M, 10 -1 M to 10 -3 M, 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 -5 M, 1×10 -2 M to 5×10-5 M, 10 -2 M to 10 -4 M, 1×10 -2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0253] In some embodiments, when it is stated that an anti-ROBO2 antibody specifically recognizes a ROB02 target with high binding affinity and binds to a non-target with low binding affinity, the Kd value of the anti-ROBO2 antibody for binding to the ROB02 target is 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -12 M), and the Kd value for binding to non-target is 10 -1 M to 10 -6 M (e.g. 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0254] In some embodiments, when an anti-ROB02 antibody is referred to as specifically recognizing ROB02, the binding affinity of the anti-ROB02 antibody is compared to the binding affinity of a control anti-ROB02 antibody (e.g., Abcs35J). In some embodiments, the Kd value for binding between the control anti-ROB02 antibody and ROB02 can be at least 2 times, e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10-100 times, 100-1000 times, 1 ... 3 -10 4 times.

[0255] Nucleic Acids

[0256] Nucleic acid molecules encoding anti-ROBO2 antibodies are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-ROBO2 antibody is provided, including any of the full-length anti-ROBO2 antibodies described herein. In some embodiments, the nucleic acid (or set of nucleic acids) encoding the anti-ROBO2 antibody described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His tag, an HA tag).

[0257] Also contemplated herein are isolated host cells comprising an anti-ROBO2 antibody, isolated nucleic acids encoding an anti-ROBO2 antibody polypeptide component, or vectors comprising nucleic acids encoding an anti-ROBO2 antibody polypeptide component as described herein.

[0258] The present application also includes variants of these nucleic acid sequences. For example, the variant includes a nucleotide sequence that hybridizes to the nucleic acid sequence encoding the anti-ROBO2 antibody of the present application under at least moderately stringent hybridization conditions.

[0259] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0260] Briefly, a natural or synthetic nucleic acid encoding an anti-ROBO2 antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody). The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0261] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0262] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0263] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0264] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0265] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are interchanged or moved. In the thymidine kinase (Tk) promoter, activity only begins to decline when the spacing between promoter elements increases to 50 bp.

[0266] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to the use of only constitutive promoters, as inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when it is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0267] In some embodiments, expression of the anti-ROBO2 antibody is inducible. In some embodiments, the nucleic acid sequence encoding the anti-ROBO2 antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0268] Inducible promoter

[0269] The use of an inducible promoter provides a molecular switch that turns on the expression of an operably linked polynucleotide sequence when expression is desired, and turns off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system used to express anti-ROBO2 antibodies is the Tet system. In some embodiments, the inducible promoter system used to express anti-ROBO2 antibodies is the E. coli lac repression system.

[0270] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0271] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0272] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0273] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transcriptional activation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0274] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0275] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0276] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and that encodes a polypeptide whose expression is expressed as some easily detectable properties, such as enzymatic activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is detected at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see, Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Generally, the construct of the minimum 5' flanking region that can show the highest expression level of the reporter gene is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0277] In some embodiments, nucleic acids encoding any of the full-length anti-ROB02 antibodies described herein are provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-ROB02 antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, a mammalian expression vector and a viral vector (e.g., a vector derived from a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, and a lentivirus).

[0278] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0279] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0280] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0281] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0282] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with lipids. The nucleic acid combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, be dispersed in a solution containing lipids, mix with lipids, combine with lipids, be suspended in lipids, be contained in micelles or mix with micelles, or otherwise combine with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist with a bilayer structure, with micelles or with a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates of uneven size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0283] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or identification by the assays described herein, all fall within the scope of the present application.

[0284] Preparation of anti-ROBO2 antibodies

[0285] In some embodiments, the anti-ROBO2 antibody is a monoclonal antibody or is derived from a monoclonal antibody. In some embodiments, the anti-ROBO2 antibody comprises a V from a monoclonal antibody. H and V L , or variants thereof. In some embodiments, the anti-ROBO2 antibody further comprises the CH1 and CL regions from a monoclonal antibody, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, including hybridoma cell methods, phage display methods, or recombinant DNA methods. Exemplary phage display methods are described herein and in the examples below.

[0286] In the hybridoma method, an immunizing agent is usually used to immunize hamsters, mice or other suitable host animals to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0287] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0288] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0289] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0290] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0291] In some embodiments, according to any of the anti-ROB02 antibodies described herein, the anti-ROB02 antibody comprises the sequence of a clone selected from an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening combinatorial libraries of antibody fragments for desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0292] In some phage display methods, V H and V LAll components of the gene are randomly recombined in a phage library, and then phage that can bind to the antigen is screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phage of immune origin provides high affinity antibodies to immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0293] The anti-ROBO2 antibody is prepared by screening the anti-ROBO2 antibody portion of the library by phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of unique human antibody fragments with different diversity. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all heavy and light chain subfamilies. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0294] Phage clones with high affinity for the target ROB02 can be screened by iterative binding of phage to the target ROB02, wherein the target ROB02 is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. Subsequently, the bound phage clones are eluted and used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Phage clones that specifically bind to ROB02 can be enriched by multiple rounds of panning (e.g., 2, 3, 4, 5, 6, or more rounds), such as solution panning, cell panning, or a combination of both. Specific binding of the enriched phage clones to the target ROB02 can be detected by any method known in the art, including, for example, ELISA and FACS.

[0295] Another method for screening antibody libraries is to display proteins on the surface of yeast cells. Wittrup et al. (U.S. Patents 6,699,658 and 6,696,251) developed a method for displaying libraries on yeast cells. In this yeast display system, one component includes a yeast lectin protein (Aga1) anchored to the yeast cell wall, and the other component includes the second subunit of the lectin protein Aga2, which can bind to the Aga1 protein through a disulfide bond and then be displayed on the yeast cell surface. The Aga1 protein is expressed by integrating the Aga1 gene into the yeast chromosome. A single-chain variable fragment (scFv) library is fused to the Aga2 gene in a yeast display plasmid, and after transformation, the library can be retained in yeast due to the presence of an additional nutritional marker. Both Aga1 and Aga2 proteins are expressed under the control of a galactose-inducible promoter.

[0296] Human antibody V gene library (V H and V K The fragments were obtained by PCR using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998). The PCR templates were obtained from commercially available RNA or cDNA, including PBMC, spleen, lymph node, bone marrow and tonsil. H and V K After the PCR libraries were combined, they were assembled into scFv formats by overlap extension PCR (Sheets, MD et al, Proc. Natl. Acad. Sci. USA 95, 6157–6162 1998). To construct a yeast scFv display library, the resulting scFv PCR products were cloned into a yeast display plasmid in yeast by homologous recombination. (Chao, G, et al, Nat Protoc. 2006; 1(2): 755-68. Miller KD, et al. Current Protocols in Cytometry 4.7.1-4.7.30, 2008).

[0297] Mammalian cell display systems can be used to screen for anti-ROBO2 antibodies, in which the antibody portion is displayed on the cell surface and antibodies that specifically target ROBO2 are isolated by antigen-directed screening methods (as described in US Patent No. 7,732,195B2). A Chinese hamster ovary (CHO) cell library displaying a large number of human IgG antibody genes can be established and used to discover clones expressing high-affinity antibody genes. Another display system has been developed that allows the same protein to be simultaneously displayed and secreted on the cell surface through alternative splicing, in which the displayed protein phenotype remains associated with the genotype, allowing the secreted soluble antibody to be characterized in both biophysical and cell-based functional assays. This approach overcomes many of the limitations of previous mammalian cell display and enables direct screening and maturation of full-length, glycosylated IgGs (Peter M. Bowers, et al, Methods 2014, 65: 44-56). Transient expression systems are suitable for a single round of antigen selection before antibody gene recovery and are therefore most useful for selecting antibodies from smaller libraries. Stable exosome vectors provide an attractive alternative. Exosome vectors can be efficiently transfected and stably maintained at low copy numbers, allowing multiple rounds of panning and the resolution of more complex antibody repertoires.

[0298] The IgG library was constructed based on the ligation of germline sequence V gene segments isolated from a group of human donors and rearranged (D)J regions. RNA collected from 2000 human blood samples was reverse transcribed into cDNA. H and V K Specific primers amplify V H and V K The fragment was purified by gel extraction. H and V K The fragments were subcloned into display vectors containing IgG1 or K constant regions, and then electroporated or transduced into 293T cells to prepare IgG libraries. H and V K To generate scFv, the fragments are then subcloned into a display vector and electroporated or transduced into 293T cells. As is well known, IgG libraries are constructed based on germline sequence V gene segments and rearranged (D)J regions isolated from a group of donors, which can be mice, rats, rabbits or monkeys.

[0299] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that specifically bind to the genes encoding the light and heavy chains of murine antibodies). Hybridoma cells as described above or the ROBO2-specific phage clones of this application can be used as a source of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in the recombinant host cell. The DNA can also be modified, for example, by replacing the human heavy and light chain constant regions with coding sequences and / or replacing homologous non-human sequences with framework regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequence for an immunoglobulin with the coding sequence for a non-immunoglobulin polypeptide. This non-immunoglobulin polypeptide can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable domain of the antibody in the present application to form a chimeric bivalent antibody.

[0300] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0301] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0302] The antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant region. Preferably, the fusion is performed with an immunoglobulin heavy chain constant region, which includes at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1) containing the necessary sites for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion, and if desired, the DNA encoding the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism.

[0303] Fully human and humanized antibodies

[0304] The anti-ROBO2 antibody (such as a full-length anti-ROBO2 antibody) can be a fully human antibody or a humanized antibody. The humanized form of a non-human (such as a mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequences of an antibody), which generally includes minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibodies), in which the residues of the receptor CDRs are replaced by CDR residues of a non-human source (donor antibody) having the desired specificity, affinity, and performance, such as mouse, rat, or rabbit CDRs. In some embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain amino acid residues that are neither in the receptor antibody nor in the introduced CDR or framework region sequences. Generally, a humanized antibody comprises at least one, and typically two, variable domains, in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all framework regions are human immunoglobulin consensus sequences.

[0305] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable domain. According to some embodiments, humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences. Therefore, this "humanized" antibody portion (US Patent No. 4,816,567) is substantially less than a complete human antibody, and its variable domains have been replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0306] Fully human antibodies are an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies after immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that produced in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0307] Fully human antibodies can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1):86-95 (1991).

[0308] Anti-ROBO2 antibody variants

[0309] In some embodiments, the amino acid sequences of anti-ROB02 antibody variants (e.g., full-length anti-ROB02 antibodies) provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequence of the antibody variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. The final construction can be completed by any combination of deletions, insertions, and substitutions of amino acid residues to provide the desired characteristics, such as antigen binding.

[0310] In some embodiments, anti-ROBIO2 antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity, e.g., improved biological activity, maintained / improved antigen binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0311] Conservative substitutions are shown in Table 4 below.

[0312] Table 4 Conservative substitutions

[0313] Amino acids are divided into different categories based on the properties of their side chains:

[0314] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leucine

[0315] Leu, isoleucine Ile;

[0316] b. Neutral hydrophilic amino acids: cysteine ​​Cys, serine Ser, threonine Thr, asparagine Asn, glutamine Gln;

[0317] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0318] d. Basic amino acids: histidine His, lysine Lys, arginine Arg;

[0319] e. Contains amino acids that affect chain direction: glycine Gly, proline Pro;

[0320] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0321] Non-conservative amino acid substitutions include substituting one class for another.

[0322] An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. Briefly, one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants having a specific biological activity (e.g., biological activity based on SLIT2-induced podocyte shedding assay or binding affinity) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved biological activity based on SLIT2-induced podocyte shedding assay or binding affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variants are tested. H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0323] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0324] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0325] A useful method for identifying amino acid residues or regions in antibodies that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or additionally, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0326] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides comprising 100 or more residues, and also include insertions of one or more amino acid residues within a sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include fusions of an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody molecule to the N- or C-terminus of the antibody molecule.

[0327] Fc region variants

[0328] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., a full-length anti-ROBO2 antibody or an anti-ROBO2 antibody fusion protein), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC potency, typically associated with receptors that bind to Fc (FcRs). In some embodiments, the Fc region variant has reduced ADCC potency. There are many examples of how changes or mutations in Fc sequences affect their potency, for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0329] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) when antigens on the target cell membrane are bound by specific antibodies (e.g., anti-ROBO2 antibodies). The ADCC effect typically involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the target cell surface. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of Fc receptors to the Fc region of antibodies leads to NK cell activation, release of cytolytic granules, and subsequent apoptosis of the target cell. The tumor cell-killing effect of ADCC can be measured using specificity assays using NK-92 cells transfected with high-affinity FcRs. The results are compared with wild-type NK-92 cells that do not express FcRs.

[0330] In some embodiments, the present application also provides anti-ROBO2 antibody variants (e.g., full-length anti-ROBO2 antibody variants) that comprise an Fc region with some, but not all, effector functions, resulting in an extended half-life in vivo. However, specific effector functions (e.g., CDC or ADCC) are unnecessary or deleterious, making such anti-ROBO2 antibodies ideal candidates for the present application. Reduction / elimination of CDC and / or ADCC activity can be confirmed by in vitro and / or in vivo cytotoxicity assays. For example, an Fc receptor (FcR) binding assay can be used to confirm that the antibody lacks FcγR binding ability (and therefore may lack ADCC activity) but still retains FcRn binding ability. Among the primary cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assessment of ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be employed (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0331] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0332] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0333] In some embodiments, an anti-ROB02 antibody (e.g., a full-length anti-ROB02 antibody) variant is provided, comprising an Fc region variant having one or more amino acid substitutions that enhance ADCC effects. In some embodiments, the Fc region variant comprises one or more amino acid substitutions that enhance ADCC effects, and the positions of these substitutions are at positions 298, 333, and / or 334 of the Fc region (EU residue numbering). In some embodiments, the anti-ROB02 antibody (e.g., a full-length anti-ROB02 antibody) variant comprises amino acid substitutions at positions S298A, E333A, and K334A of the Fc region.

[0334] In some embodiments, alterations in the Fc region result in altered (ie, enhanced or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described in US Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0335] In some embodiments, an anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody) variant is provided, comprising an Fc region variant having one or more amino acid substitutions that can extend half-life or enhance binding to an Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( U.S. Pat. No. 7,371,826 ).

[0336] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc region variants.

[0337] The present application contemplates anti-ROB02 antibodies (eg, full-length anti-ROB02 antibodies) comprising any one of the Fc variants described herein, or a combination thereof.

[0338] Glycosylation variants

[0339] In some embodiments, the anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) provided herein are altered to increase or decrease the extent of glycosylation of the anti-NGF antibody. Adding or deleting glycosylation sites on the anti-ROBO2 antibody can be conveniently achieved by altering the amino acid sequence of the anti-NGF antibody or its polypeptide portion to add or remove one or more glycosylation sites.

[0340] Wherein the anti-ROBO2 antibody comprises an Fc region, the sugars attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically N-linked to the CH2 domain of the Fc region at Asn297 (see, for example, Wright et al., TIBTECH 15:26-32 (1997)). Such oligosaccharides can include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the anti-ROBO2 antibodies of the present application can be subjected to oligosaccharide modification to generate anti-ROBO2 antibody variants with certain improved properties.

[0341] The N-glycans attached to the CH2 domain of the Fc region are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for binding to FcγRs; however, non-fucosylated N-glycans enhance the binding of Fc to FcγRIIIa. Enhanced binding to FcRIIIa results in enhanced ADCC, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0342] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or CH2 domain is non-glycosylated. In some embodiments, glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.

[0343] In some embodiments, anti-ROBO2 antibody variants (e.g., full-length anti-ROBO2 antibodies) are provided that comprise an Fc region in which the carbohydrate structure attached to the Fc region has reduced or no fucose, which may enhance ADCC function. Specifically, anti-ROBO2 antibodies are provided herein that have reduced fucose relative to the same anti-ROBO2 antibody produced in wild-type CHO cells. That is, they are characterized by having a lower amount of fucose compared to antibodies produced in native CHO cells (e.g., CHO cells that produce native glycosylated forms, CHO cells that contain the native FUT8 gene). In some embodiments, the N-linked glycans of the anti-ROBO2 antibody have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the anti-ROBO2 antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycans of the anti-ROBO2 antibody do not contain fucose, i.e., the anti-ROBO2 antibody is completely fucose-free, or is devoid of fucose or defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all glycostructures (e.g., complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0344] Anti-ROBO2 antibody (e.g., full-length anti-ROBO2 antibody) variants further provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the anti-ROBO2 antibody is bisected by GlcNAc. Such anti-ROBO2 antibody (e.g., full-length anti-ROBO2 antibody) variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Anti-ROBO2 antibody (e.g., full-length anti-ROBO2 antibody) variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such anti-ROBO2 antibody variants may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0345] In some embodiments, the anti-ROB02 antibody (e.g., full-length anti-ROB02 antibody) variant comprises an Fc region that binds to FcγRIII. In some embodiments, the anti-ROB02 antibody (e.g., full-length anti-ROB02 antibody) variant comprising an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has enhanced ADCC activity in the presence of human effector cells compared to an otherwise identical anti-ROB02 antibody (e.g., full-length anti-ROB02 antibody) having a human wild-type IgG1 Fc region.

[0346] Cysteine ​​engineered variants

[0347] In some embodiments, it is desirable to prepare cysteine-engineered anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) in which one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the anti-ROBO2 antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the anti-ROBO2 antibody, which can be used to conjugate the anti-ROBO2 antibody to other moieties, such as drug moieties or linker-drug moieties, to prepare anti-ROBO2 immunoconjugates as further described herein. Cysteine-engineered anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) can be prepared, for example, as described in U.S. Pat. No. 7,521,541.

[0348] derivative

[0349] In some embodiments, the anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) provided herein can be further modified to include other non-protein moieties known in the art and readily available. Suitable moieties for derivatizing anti-ROBO2 antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the anti-ROB02 antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, whether the desired property or function of the anti-ROB02 antibody is to be improved, whether the anti-ROB02 antibody derivative is to be used for the treatment of a particular condition, etc.

[0350] Pharmaceutical composition

[0351] Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any anti-ROBO2 antibody (e.g., full-length anti-ROBO2 antibody), nucleic acids encoding the antibodies, vectors comprising nucleic acids encoding the antibodies, or host cells comprising nucleic acids or vectors described herein. In some embodiments, a pharmaceutical composition is provided comprising any anti-ROBO2 antibody described herein and a pharmaceutically acceptable carrier.

[0352] Suitable anti-ROBO2 antibody formulations can be obtained by mixing an anti-ROBO2 antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and prepared in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418, expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the subject to be treated herein. Cationic liposomes or liposomes can be used to deliver the anti-ROBO2 antibodies of the present application to cells.

[0353] In addition to anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies), the formulations described herein may also contain one or more other active substances necessary for treating a specific condition, preferably substances with complementary activities and no adverse reactions to each other. For example, in addition to the anti-ROBO2 antibody, it may be necessary to further include an angiotensin-converting enzyme (ACE) inhibitor, angiotensin II receptor antagonist (ARB), a mineralocorticoid receptor (MR) antagonist, and a diuretic. These molecules are present in combination in amounts effective for the intended purpose. The effective amount of the other substances depends on the amount of anti-ROBO2 antibody in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are generally used in the same dosages and routes of administration as described herein, or at 1% to 99% of the currently used dosage.

[0354] The anti-ROBO2 antibody (e.g., full-length anti-ROBO2 antibody) can also be entrapped in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Sustained-release formulations can be prepared.

[0355] Sustained-release formulations of anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing the antibody (or fragment thereof), which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels can release proteins over shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of biological activity or altered immunogenicity. Rational strategies can be designed to stabilize anti-ROBO2 antibodies based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0356] In some embodiments, the anti-ROB02 antibody (eg, full-length anti-ROB02 antibody) is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.

[0357] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0358] Treatment using anti-ROBO2 antibodies

[0359] Anti-ROBO2 antibodies (e.g., full-length anti-ROBO2 antibodies) and / or compositions described herein can be administered to an individual (e.g., a mammal, such as a human) to treat diseases and / or conditions resulting from dysregulation of the ROB02 signaling pathway (e.g., kidney diseases, diseases mediated by or associated with elevated levels of protein in the urine), including but not limited to glomerular diseases, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, and reflux nephropathy. Therefore, in some embodiments, the present application provides a method for treating diseases and / or conditions caused by dysregulation of the ROB02 signaling pathway (e.g., kidney disease, diseases mediated by or associated with elevated protein levels in urine), comprising administering to an individual an effective amount of a composition (e.g., a pharmaceutical composition) comprising an anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody), such as any anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody) described herein, wherein in some embodiments, the individual is human.

[0360] For example, in some embodiments, a method for treating an individual suffering from a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody (e.g., a full-length anti-ROB02 antibody) that specifically binds to an epitope on human ROB02, wherein the epitope comprises an amino acid residue at position 20 of human ROB02. In some embodiments, the anti-ROB02 antibody is a full-length antibody. In some embodiments, the full-length anti-ROB02 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, glomerular disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, reflux nephropathy. In some embodiments, the individual is a human.

[0361] For example, in some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody), wherein the antibody comprises: a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising DX1AMH (SEQ ID NO: 49), wherein X1 is F or Y; HC-CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 10); and HC-CDR3 comprising EGFYX1DGFDV (SEQ ID NO: 51), wherein X1 is A, D or S; and a light chain variable domain (V L ), the V L The invention also provides an antibody comprising a light chain complementary determining region (LC-CDR) 1 comprising RASQX1ISSWLA (SEQ ID NO: 53), wherein X1 is G or R; a LC-CDR2 comprising X1ASTLQS (SEQ ID NO: 54), wherein X1 is A, D, or E; and a LC-CDR3 comprising QQANX1FPLT (SEQ ID NO: 56), wherein X1 is S or T. In some embodiments, the anti-ROB02 antibody is a full-length antibody. In some embodiments, the full-length anti-ROB02 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, glomerular disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, reflux nephropathy. In some embodiments, the individual is a human.

[0362] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-2, HC-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 15-17, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 24-25, an LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 30-32, and an LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40-41, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0363] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 58-61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 58-61; and V L , the V L The invention also provides a method for preparing a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 69-73 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 69-73.

[0364] In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0365] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0366] In some embodiments, the anti-ROBO2 antibodies described herein comprise: V H , the V H comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the anti-ROB02 antibody described herein is a full-length anti-ROB02 antibody comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0367] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0368] In some embodiments, the anti-ROBO2 antibodies described herein comprise: V H , the V H comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 59; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0369] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0370] In some embodiments, the anti-ROBO2 antibodies described herein comprise: V H , the V H comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 60; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0371] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 40, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0372] In some embodiments, the anti-ROBO2 antibodies described herein comprise: V H , the V H comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 58; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0373] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 41, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0374] In some embodiments, the anti-ROBO2 antibodies described herein comprise: V H , the V H comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 61; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0375] For example, in some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-ROBO2 antibody (e.g., a full-length anti-ROBO2 antibody), wherein the antibody comprises: a heavy chain variable domain (V H ), the V H The invention comprises: a heavy chain complementary determining region (HC-CDR) 1 comprising SX1X2WWS (SEQ ID NO: 50), wherein X1 is E, M or S, and X2 is H or N; HC-CDR2 comprising EIYHSGSTNYNPSLKS (SEQ ID NO: 11); and HC-CDR3 comprising DGDDX1SGNDAFDI (SEQ ID NO: 52), wherein X1 is I, S or V; and a light chain variable domain (V L ), the V L The invention also provides an antibody comprising a light chain complementary determining region (LC-CDR) 1 comprising RASQSISSYLN (SEQ ID NO: 26); a LC-CDR2 comprising X1ASX2RAT ​​(SEQ ID NO: 55), wherein X1 is A or G, and X2 is E, S, or T; and a LC-CDR3 comprising LQX1X2X3YPLT (SEQ ID NO: 57), wherein X1 is H or L, X2 is M or N, and X3 is A or S. In some embodiments, the anti-ROB02 antibody is a full-length antibody. In some embodiments, the full-length anti-ROB02 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or disorder is selected from, for example, glomerular disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, reflux nephropathy. In some embodiments, the individual is a human.

[0376] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3-6, HC-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 18-20, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, LC-CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 33-36, and LC-CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 42-45, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0377] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising an amino acid sequence as shown in any one of SEQ ID NOs: 62-65 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 62-65; and V L , the V L The invention also provides a method for preparing a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 74-77 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 74-77.

[0378] In some embodiments, the anti-ROB02 antibodies described herein are full-length anti-ROB02 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 81. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 82. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0379] In some embodiments, a method for treating an individual with a disease associated with the ROB02 signaling pathway (e.g., kidney disease, a disease mediated by or associated with elevated protein levels in urine) is provided, comprising administering to the individual an effective amount of a composition comprising an anti-ROBO2 antibody, wherein the antibody comprises: H , the V H comprising: HC-CDR1 c...

Claims

1. An isolated anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: Heavy chain variable domain (V H ), the V H Include: a heavy chain complementarity determining region (HC-CDR) 1 comprising DX1AMH (SEQ ID NO: 49), wherein X1 is F or Y; HC-CDR2 comprising GISWNSGSIGYADSVKG (SEQ ID NO: 10); and HC-CDR3 comprising EGFYX1DGFDV (SEQ ID NO: 51), wherein X1 is A, D or S; and Light chain variable domain (V L ), the V L Include: a light chain complementarity determining region (LC-CDR) 1 comprising RASQX1ISSWLA (SEQ ID NO: 53), wherein X1 is G or R; LC-CDR2 comprising X1ASTLQS (SEQ ID NO: 54), wherein X1 is A, D or E; and LC-CDR3 comprising QQANX1FPLT (SEQ ID NO: 56), wherein X1 is S or T.

2. The isolated anti-ROBO2 antibody according to claim 1, comprising: (i)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; (iii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:32, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; (iv)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L , the V L comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:25, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; or (v)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:24, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:31, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

41.

3. The isolated anti-ROBO2 antibody according to any one of claims 1-2, comprising: (i)V H , comprising the amino acid sequence of SEQ ID NO:58 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:58; and V L , comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 69; (ii) V H , comprising the amino acid sequence of SEQ ID NO:59 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:59; and V L , comprising the amino acid sequence of SEQ ID NO:70 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:70; (iii)V H , comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 60; and V L , comprising the amino acid sequence of SEQ ID NO:71 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:71; (iv)V H , comprising the amino acid sequence of SEQ ID NO:58 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:58; and V L , which comprises the amino acid sequence of SEQ ID NO:72 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:72; or (v)V H , comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 61; and V L , which comprises the amino acid sequence of SEQ ID NO:73 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

73.

4. An isolated anti-ROBO2 antibody, wherein the anti-ROBO2 antibody comprises: Heavy chain variable domain (V H ), the V H Include: a heavy chain complementarity determining region (HC-CDR) 1 comprising SX1X2WWS (SEQ ID NO: 50), wherein X1 is E, M or S, and X2 is H or N; HC-CDR2 comprising EIYHSGSTNYNPSLKS (SEQ ID NO: 11); and HC-CDR3, which comprises DGDDX1SGNDAFDI (SEQ ID NO: 52), wherein X1 is I, S or V; and a light chain variable domain (V L ), the V L Include: Light chain complementarity determining region (LC-CDR) 1, comprising RASQSISSYLN (SEQ ID NO: 26); LC-CDR2 comprising X1ASX2RAT ​​(SEQ ID NO: 55), wherein X1 is A or G, and X2 is E, S or T; and LC-CDR3, which comprises LQX1X2X3YPLT (SEQ ID NO: 57), wherein X1 is H or L, X2 is M or N, and X3 is A or S.

5. The isolated anti-ROBO2 antibody according to claim 4, comprising: (i)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:33, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:42; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:43; (iii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:26, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:44; or (iv)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:26, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:36, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

45.

6. The isolated anti-ROBO2 antibody according to any one of claims 4-5, comprising: (i)V H , comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 62; and V L , comprising the amino acid sequence of SEQ ID NO:74 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:74; (ii) V H , comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 63; and V L , comprising the amino acid sequence of SEQ ID NO:75 or a variant thereof, wherein the variant has at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:75; (iii)V H , comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 64; and V L , which comprises the amino acid sequence of SEQ ID NO:76 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:76; or (iv)V H , comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 65; and V L , which comprises the amino acid sequence of SEQ ID NO:77 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO:

77.

7. An isolated anti-ROBO2 antibody comprising: (i)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:27, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46; (ii) V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and V L , the V L comprising: a LC-CDR1 comprising the amino acid sequence of SEQ ID NO:28, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:47; or (iii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and V L , the V L It comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:29, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:39, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:

48.

8. The isolated anti-ROBO2 antibody according to claim 7, comprising: (i)V H , comprising the amino acid sequence shown in SEQ ID NO:66 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:66; and V L , comprising the amino acid sequence shown in SEQ ID NO:78 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:78; (ii) V H , comprising the amino acid sequence shown in SEQ ID NO: 67 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 67; and V L , which comprises the amino acid sequence shown in SEQ ID NO:79 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:79; or (iii)V H , which comprises the amino acid sequence shown in SEQ ID NO:68 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:68; and V L , which comprises the amino acid sequence shown in SEQ ID NO:80 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

80.

9. The isolated anti-ROBO2 antibody of any one of claims 1-8, wherein the anti-ROBO2 antibody binds to human ROB02 with a Kd value of about 0.1 pM to about 1 nM.

10. The isolated anti-ROBO2 antibody according to any one of claims 1-8, wherein the anti-ROBO2 antibody comprises an Fc fragment.

11. The isolated anti-ROBO2 antibody according to claim 10, wherein the anti-ROBO2 antibody is a full-length IgA, IgD, IgE, IgG or IgM antibody.

12. The isolated anti-ROBO2 antibody according to claim 11, wherein the anti-ROBO2 antibody is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.

13. The isolated anti-ROBO2 antibody according to any one of claims 1 to 12, wherein the anti-ROBO2 antibody is a chimeric, human or humanized antibody.

14. The isolated anti-ROBO2 antibody according to any one of claims 1 to 9, wherein the anti-ROBO2 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody and linear antibody.

15. An isolated nucleic acid molecule encoding the anti-ROBO2 antibody of any one of claims 1-14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. An isolated host cell comprising the anti-ROBO2 antibody of any one of claims 1-14, the nucleic acid molecule of claim 15, or the vector of claim 16.

18. A method for preparing an anti-ROBO2 antibody, comprising: a) culturing the host cell of claim 17 under conditions effective to express the anti-ROBO2 antibody; and b) Obtaining the expressed anti-ROBO2 antibody from the host cells.

19. A pharmaceutical composition comprising the anti-ROBO2 antibody of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16, the isolated host cell of claim 17 or the antibody prepared by the method of claim 18, and a pharmaceutically acceptable carrier.

20. Use of the antibody of any one of claims 1-14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, the antibody prepared by the method of claim 18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating a disease or condition in an individual in need thereof.

21. The use according to claim 20, wherein the disease or condition is a disease and / or condition caused by dysregulation of the ROB02 signaling pathway, such as kidney disease, a disease or condition mediated by or associated with elevated protein levels in urine.

22. The method of claim 21, wherein the disease or disorder is selected from the group consisting of glomerular disease, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, lupus nephritis, IgA nephropathy, membranous nephropathy (MN), fibrosis (e.g., liver fibrosis, kidney fibrosis), non-alcoholic steatohepatitis (NASH), proteinuria, albuminuria, glomerulonephritis, nephrotic syndrome, focal glomerulosclerosis, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, renal transplant rejection, and reflux nephropathy.

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