WO2023012314A1

WO2023012314A1 - Anti-plexin-b1 antibodies

Info

Publication number: WO2023012314A1
Application number: PCT/EP2022/072030
Authority: WO
Inventors: Thomas Worzfeld; Stefan Offermanns; David Matthews; Arkadiusz OLEKSY; Martina TROKTER; Seema Patel; Sharandip KAUR NIJJAR
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV; LifeArc
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV; LifeArc
Priority date: 2021-08-05
Filing date: 2022-08-04
Publication date: 2023-02-09
Anticipated expiration: 2024-02-05

Abstract

The invention relates to antibodies and antigen binding fragments thereof that specifically bind to Plexin-B1 and their therapeutic uses. The antibodies are useful in treating osteoporosis, multiple sclerosis, a neoplastic disease or a neurodegenerative disease in a subject.

Description

ANTI-PLEXIN-B1 ANTIBODIES

FIELD OF THE INVENTION

The invention relates to antibodies or antigen binding fragments thereof that specifically bind to Plexin- B1 and their therapeutic uses.

BACKGROUND

Plexins comprise a family of transmembrane receptors for semaphorins, which are membrane-bound or diffusible factors that regulate key cellular functions (Worzfeld & Offermanns, 2014, Nat Rev Drug Discov 13: 603-21). Plexin-B1 is one of the Plexin family members that binds semaphorin ligands. The receptorligand interactions result in alteration in the movement and differentiation of cells.

Semaphorins are the largest identified family of axonal guidance proteins and play an important role in the adhesion, migration, proliferation and differentiation of many other cells including tumor cells. They appear as soluble proteins, transmembrane proteins or as proteins bound via a glycosylphosphatidylinositol anchor (GPI-anchor) to cell membranes. The effects of the semaphorins are mediated via binding to plexins.

The Plexin-B1-Sema4D interaction has been implicated in a number of diseases, including multiple sclerosis, osteoporosis, arthritis, cancer, inflammation and neurodegenerative diseases. The semaphorin-plexin system is of critical importance in multiple tissues, and plays a pivotal role in cell-cell communication in the immune and bone system (Lee, Lee et al., 2019 Exp Neurobiol 28: 311-319, Nishide & Kumanogoh, 2018 Nat Rev Rheumatol 14: 19-31 , Verlinden, Vanderschueren et al., 2016, Mol Cell Endocrinol 432: 66-74). In mammals, based on homologies, plexins are divided into four subfamilies (A-D), and semaphorins are grouped into 5 classes (3-7). The interaction of plexins with semaphorins is mediated by a highly conserved seven-blade p-propeller domain, the “sema domain” (Janssen, Robinson et al., 2010 Nature 467:1118-1122, Liu, Juo et al. Cell 142:749-761 , 2010, Nogi, Yasui et al., 2010, Nature 467:1123-1 127)

The binding of semaphorins to plexins causes the activation of a plurality of signal pathways (6, 7). All plexins bear in their intracellular part a GTPase-activating protein (GTPase-activating protein, GAP) domain that mediates the guanine nucleotide exchange of R-Ras, M-Ras and Rap1 . The inhibition of R- Ras was thereby related to the inhibition of signal transduction by integrins. Plexins of the B-subfamily possess at their C-terminus a PDZ binding-motif interacting stably with the Rho-guanine nucleotide exchange factors (Rho guanine nucleotide exchange factors, RhoGEFs), LARG and PDZ-RhoGEF. Activation of the B-Plexins leads to activation of small GTPases RhoA and RhoC.

Plexin-B consists of three members, namely Plexin-B1 , Plexin-B2 and Plexin-B3. The plexin family member, Plexin-B1 , is expressed on microglia, a resident macrophage population in the brain, which is increasingly recognized as a pharmacological target in multiple sclerosis (Wang, Wang et al., 2019, Front Pharmacol 10: 286). In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, it has been demonstrated that Plexin-B1 -positive microglia are activated by T cells, which infiltrate the CNS and express the high-affinity ligand of Plexin-B1 , semaphorin 4D (Sema4D) (Okuno, Nakatsuji et al., 2010, J Immunol 184: 1499-1506).

Neuroinflammation and disease progression of EAE are strongly reduced in mice lacking Sema4D or Plexin-B1 (Okuno et al., 2010 J Immunol 184: 1499-1506). Moreover, human patients with virus-induced neuroinflammation and demyelination show Sema4D-positive T cells infiltrating the spinal cord and elevated concentrations of Sema4D in the cerebrospinal fluid (Giraudon, Vincent et al., 2004, Semaphorin CD 100).

Multiple sclerosis (MS) is a neurological disease. The disease is often progressive and leads to significant functional limitations. Hence, there is an urgent need for new therapies that are not only aimed at the delay of progression but are also aimed at the cure of the disease. Currently approved drugs in the treatment of multiple sclerosis are immunosuppressants and immunomodulators such as glucocorticoids, mitoxantrone, azathioprine, cyclophosphamide, p-interferons, glatiramer acetate, i.v. immunoglobulins, dimethyl fumarate, fingolimod, teriflunomide, natalizumab and fampridine. Some of the latest therapies are often related to undesirable effects and require a close patient monitoring.

The Plexin B1 -Serna 4D interaction has also been implicated in neurodegenerative diseases, with Sema4D targeted for the development of therapeutic agents. An anti-Sema4D antibody (Pepinemab) that blocks Sema4D binding to its Plexin-B1 and plexin-B2 receptors has been shown to reduce neurodegenerative processes in preclinical models, including Huntington’s disease and Alzheimer’s disease. (Evans EE et al, 2020, Drug Development, vol 16(S9) e043971).

In the bone system, Plexin-B1 is expressed on osteoblasts, while Sema4D localizes to osteoclasts (Negishi-Koga, Shinohara et al., 2011 , Nat Med 17: 1473-80). The binding of Sema4D to Plexin-B1 potently inhibits bone formation via activation of the small GTPase RhoA and suppression of insulin-like growth factor-1 (IGF-1) signaling in osteoblasts (Negishi-Koga et al., 2011 , Nat Med 17: 1473-80). Consistently, mice lacking Sema4D or Plexin-B1 , as well as mice expressing dominant-negative RhoA specifically in osteoblasts display significantly increased bone mass (Dacquin, Domenget et al., 2011 , PLoS One 6: e26627, Negishi-Koga et al., 2011 , Nat Med 17: 1473-80).

Osteoporosis has a prevalence of about 30 % in postmenopausal women and is a major risk factor for bone fractures, reduced quality of life and immobilization. Drugs approved in the treatment of osteoporosis include estrogens and selective estrogen-receptor-modulators, bisphosphonates, the anti- RANKL-antibody denosumab, the anti-sclerostin antibody romosozumab strontium ranelate and parathyroid hormones (such as teriparatide and abaloparatide). Despite advances in pharmacotherapy of osteoporosis, possibilities fortherapy are still insufficient and leave room for new drugs with significant additional benefits. Despite significant progress in the treatment of neoplastic diseases, existing therapies for many solid tumors continue to display decisive disadvantages. The majority of the new, targeted therapies delay the progression and metastasis only for a relatively short time, so that tumor diseases still represent the second leading cause of death worldwide.

Tumor-associated macrophages (TAMs) play a crucial role in tumor progression. Drugs specifically directed against the communication between TAM and tumor cells are not yet available. TAMs produce Sema4D promoting both the growth and invasion of tumor cells as well as acting on endothelial cells for the increased tumor angiogenesis (Basile, J.R. et al, 2006, Proc Natl Acad Sci U S A 103:9017-9022, Sierra, J.R et al, 2008, J Exp Med 205:1673-1685). In accordance herewith tumors, growing in Sema4D knockout mice, are smaller, metastasize less and are less vascularized than tumors growing in control animals (Sierra, J.R et al, 2008, J Exp Med 205:1673-1685).

Therefore, antibodies that target Plexin-B1 , and that block the Plexin-B1-Sema4D interaction may be useful in the treatment of a number of diseases, in particular immune-mediated diseases.

WO2016034733 discloses antibodies described therein as anti-Plexin-B1 antibodies.

There is a need for improved anti-Plexin-B1 antibodies, in particular those that inhibit the binding of Sema4D to Plexin-B1 and bind with higher affinity to Plexin-B1 , that can be used therapeutically, for example in the treatment of immune mediated diseases, including for diseases such as multiple sclerosis, and osteoporosis, and that are suitable for use as research and diagnostic tools.

SUMMMARY OF THE INVENTION

The inventors have identified anti-Plexin-B1 antibodies that specifically block the binding of Sema4D to Plexin-B1 . The antibodies specifically bind Plexin-B1 and are directed to the extracellular domain of Plexin-B1.

The antibodies have been shown to inhibit Sema4D-Plexin-B1 signalling. In particular the anti-Plexin- B1 antibodies and antigen binding fragments thereof are useful in the treatment of immune mediated diseases, osteoporosis, and cancer.

The present invention relates to antibodies that specifically bind Plexin-B1 .

In a first aspect the invention provides anti-Plexin-B1 antibodies and antigen-binding fragments thereof comprising a heavy chain variable region and a light chain variable region, wherein

- the heavy chain variable region amino acid sequence comprises:

- a HCDR1 comprising the sequence of SEQ ID NO: 2 (GFSFSGSYY) or SEQ ID NO: 1 (GIDLSSSA); - a HCDR2 comprising the sequence of SEQ ID NO: 4 (IYTGSTGST) or SEQ ID NO: 3 (IGSSDST); and

- a HCDR3 comprising the sequence of SEQ ID NO: 6 (ARDHAAYAGYGVPWYTRLDL) or SEQ ID NO: 5 (ARGLYSGMDP); and

- the light chain variable region amino acid sequence comprises:

- a LCDR1 comprising the sequence of SEQ ID NO: 8 (PSVLGNY) or SEQ ID NO: 7 (QSISNL);

- a LCDR2 comprising the sequence of SEQ ID NO: 10 (GAS) or SEQ ID NO: 9 (RAS); and

- a LCDR3 comprising the sequence of SEQ ID NO: 14 (LGGWSSASDNT); SEQ ID NO: 11 (QSNYGSINSDYGNA), SEQ ID NO: 12 (QSNYGSISSSYGNA) or SEQ ID NO: 13 (QSNYGSVSTNYGNA).

The anti-Plexin-B1 antibody or antigen-binding fragment thereof may comprise: i) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 2 (GFSFSGSYY), a HCDR2 comprising the sequence of SEQ ID NO: 4 (IYTGSTGST); and a HCDR3 comprising the sequence of SEQ ID NO: 6 (ARDHAAYAGYGVPWYTRLDL); and a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 8 (PSVLGNY), a LCDR2 comprising the sequence of SEQ ID NO: 10 (GAS); and a LCDR3 comprising the sequence of SEQ ID NO: 14 (LGGWSSASDNT); or ii) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a HCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 11 (QSNYGSINSDYGNA); or iii) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a HCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 12 (QSNYGSISSSYGNA) iv) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a VHCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 13 (QSNYGSVSTNYGNA); or The anti-Plexin-B1 antibody or antigen-binding fragment thereof may comprise a light chain variable region (VL region) comprising: a. a sequence having at least 90% sequence identity to SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26; b. a sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26; wherein five or fewer amino acids are substituted, deleted or inserted into the framework regions; or c. the sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26; and/or a heavy chain variable region (VH region) comprising: a. a sequence having at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25; b. a sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25; wherein five or fewer amino acids are substituted, deleted or inserted into the in the framework regions; or c. the sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25.

In particular the anti-Plexin-B1 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 24; a heavy chain variable region comprising SEQ ID NO: 25 and a light chain variable region comprising SEQ ID NO: 26; a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 16; a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18; a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20; or a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 22.

In a second aspect the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof that specifically binds to Plexin-B1 and inhibits the binding of Sema4D to Plexin-B1 .

In a third aspect the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof that specifically binds to an epitope of human Plexin-B1 wherein the epitope is comprised in the region of amino acids 20 to 535 of SEQ ID NO: 28 or an epitope of mouse Plexin-B1 wherein the epitope is comprised in the region of amino acids 20 to 535 of SEQ ID NO: 30. In one embodiment the anti-Plexin-B1 antibody or antigen-binding fragment thereof specifically binds a conformational epitope that comprises at least one amino acid selected from amino acid residues 106, 131 , 132, 135- 137 141 , 188, 190-196, 199-203, 256 of SEQ ID NO: 30. Preferably the anti- Plexin-B1 antibody or antigen-binding fragment specifically binds a conformational epitope comprising the amino acids 106, 131 , 132, 135-137, 141 , 188, 190-196, 199-203, and 256 of SEQ ID NO: 30.

The anti-Plexin-B1 antibody or antigen-binding fragment thereof may be a monoclonal antibody.

The anti-Plexin-B1 antibody or antigen-binding fragment thereof may be a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, an Fv fragment, an scFv fragment, dAb, Fd, a diabody, or a single chain antibody.

The anti-Plexin-B1 antibody or antigen-binding fragment thereof may be a human antibody, murine antibody, a rabbit antibody, a humanised antibody or a chimeric antibody. Preferably the antibody is a chimeric or a humanised antibody.

The anti-Plexin-B1 antibody or antigen-binding fragment thereof may be an IgG antibody, preferably an IgG 1 , lgG2, lgG3 or lgG4-type antibody, more preferably the antibody is a IgG 1 antibody.

In a further aspect of the invention the anti-Plexin-B1 antibody or antigen-binding fragment thereof is comprised in a bispecific antibody, a multispecific antibody, or conjugated to a further therapeutic or diagnostic agent.

In a further aspect the invention provides a pharmaceutical composition comprising an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention. The pharmaceutical composition may further comprise an additional therapeutically active agent, or is for use in combination with another therapy or additional therapeutically active agent.

In a further aspect the invention provides a kit comprising an anti-Plexin-B1 antibody or antigenbinding fragment thereof of the invention or a pharmaceutical composition of the invention, optionally further comprising an additional therapeutically active agent.

In a further aspect the invention provides a nucleic acid encoding an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

In a further aspect of the invention, there is provided a vector or plasmid comprising a nucleic acid encoding an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention. The vector comprising the nucleic acid may be operably linked to a promoter. In a further aspect of the invention, there is provided a host cell comprising the nucleic acid encoding an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention or a vector or plasmid comprising a nucleic acid encoding an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

In a further aspect of the invention, there is provided a method of making an anti-Plexin-B1 antibody or antigen-binding fragment thereof, the method comprising culturing a host cell of the invention. The host cell may be cultured in a cell culture medium under conditions to express the conditions to express the encoding nucleic acid sequence of the plasmid or vector inside the cell. The method may further comprise recovering the antibody from the cell culture.

In a further aspect the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof or a pharmaceutical composition for use in medicine.

In a further aspect the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof for use in the treatment of osteoporosis, multiple sclerosis, a neoplastic disease, or a neurodegenerative disease.

In a further aspect the invention provides the use of an anti-Plexin-B1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of osteoporosis, multiple sclerosis, a neoplastic disease, or a neurodegenerative disease.

In a further aspect the invention provides a method of inhibiting the binding of Plexin-B1 to Sema4D for the treatment of a disease associated with Sema4D signalling via Plexin B1 , the method comprising administering an effective amount of an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

Diseases associated with Sema4D signalling via Plexin-B1 include but are not limited to immune- mediated disease, for example multiple sclerosis, arthritis and amyotrophic lateral sclerosis (ALS), osteoporosis, cancer, inflammation and neurodegenerative diseases, for example Huntington’s disease and Alzheimer’s disease.

In a further aspect the invention provides a method of treating osteoporosis, multiple sclerosis, a neoplastic disease or a neurodegenerative disease in a subject, the method comprising administering an effective amount of an anti-Plexin-B1 antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention to the subject thereof.

The method may further comprise administering, simultaneously or sequentially, in any order, at least one further therapeutically active agent. In one embodiment the neoplastic disease may be cancer, and the further therapeutically active agent is one or more an immune modulating agent, preferably wherein the immune modulating agent is immune checkpoint inhibitor. The method may further comprise administering one or more immune checkpoint inhibitor selected from an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof.

In a further aspect the invention provides a method of inhibiting the binding of Plexin-B1 to Sema4D, comprising contacting the Plexin-B1 with an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

Brief description of Figures

FIGURE 1. Amino acid sequences of variable domains of anti-Plexin-B1 antibodies (A) RbPLX11 ; (B) RbPLX18; (C) RbPLX19; (D) RbPLX20; (E) RbPLX7; and (F) HuPLX7. Each CDR is underlined within the framework sequences of the heavy (top) and light chain (bottom) of the antibody as initially identified.

FIGURE 2: Binding kinetics of anti-Plexin-B1 antibodies (A) RbPLX11 and HuPLX11 , (B) RbPLX18, RbPLX19 and RbPLX20 generated by SPR method (Biacore). Representative figures show the kinetics sensograms using ranges of concentration of human or mouse Plexin-B1.

FIGURE 3: Cross-reactivity and Sema4D competition characteristics of anti-Plexin-B1 antibodies analysed by biolayer interferometry (OctetRED384). Representative figures show the Octet binding sensograms for (A) HuPLX7 antibody binding only to human Plexin-B1 and (B) RbPLX20 cross-reactive antibody binding both human and mouse Plexin-B1. Both, HuPLX7 and RbPLX20 also block the interaction of Plexin-B1 with Sema4D (A, B). The buffer control (C) confirms Plexin-B1-Sema4D interaction without the presence of the antibody.

FIGURE 4: Collapse assay in Cos-7 cells over-expressing human and mouse Plexin-B1-FLAG. (A) Representative images of cells demonstrating collapsed morphology upon Sema4D treatment. Antibodies blocking Plexin-B1-Sema4D interaction rescues this phenotype, while isotype control (HuG1 K) does not. Cellular staining is indicated in coloured font (Plexin-B1 - Green, Hoechst - blue, Phalloidin - red). (B) Sema4D dose dependency. Cells were incubated with indicated concentrations of Sema4D, followed by image analysis to assess the collapse morphology of individual wells. Dosedependent inhibition of cellular collapse activity by anti-Plexin-B1 antibodies tested using human Plexin-B1 (C) and mouse Plexin-B1 (D). The number of collapsed cells per well was quantified manually and collapse response values obtained from two technical replicates are shown as means ±SD. EC50 were calculated using non-linear regression analysis (five parameters). Bottom graphs in (C) and (D) show normalised data expressed as fraction of the collapsed cells.

FIGURE 5: Binding of the anti-Plexin-B1 antibodies to Plexin-B1 expressed on Expi293 cell measured by flow cytometry. (A) RbPLX7, RbPLX11 and isotype control binding to human Plexin- B1 in comparison to human Plexin-B2 and Plexin-B3 and (B) RbPLX11 , RbPLX18, RbPLX19, RbPLX20 and isotype control binding to mouse Plexin-B1 in comparison to mouse Plexin-B2 and Plexin-B3. The corresponding IgG antibody (hlgG1) was used as a control.

FIGURE 6. The anti-Plexin-B1 antibody RbPLX7 blocks Sema4D-induced inhibition of osteoblast differentiation and mineralization. (A) Human osteoblasts were exposed to 150 nM of Sema4D in osteogenic medium without or in combination with 150 nM anti-Plexin-B1 antibody RbPLX7 or the corresponding IgG control antibody (IgG). Osteoblast differentiation was determined after 7 days via measurement of Alkaline Phosphatase (ALP) activity, and osteoblast mineralization was analyzed after 21 days via Alizarin Red S staining. (B, C) Quantification of the results in (A). Mean values shown with error bars representing standard error of the mean (SEM), and statistical significances evaluated by One-Way ANOVA Tukey’s Post Tests. *p < 0.05, **p < 0.01 , ***p < 0.001 .

FIGURE 7. Therapeutic effect of the anti-Plexin-B1 antibody - RbPLX7 in an in vivo mouse model of postmenopausal osteoporosis. Humanized Plexin-B1 mice were ovariectomized and treated with the anti-Plexin-B1 antibody RbPLX7 or the corresponding IgG control antibody at day 4, 7, 14, 21 , 28, 35, 42, 49 and 56. Analysis was performed on day 63. (A) Microcomputed tomography (pCT) and morphometric analysis of the distal femur of humanized Plexin-B1 mice treated with RbPLX7 or with the corresponding IgG control antibody. Plexin-B1 knockout mice (plxnbl¹') were used as a control and were treated with the IgG control antibody. Top: longitudinal view; bottom: transaxial view of the metaphyseal region. (B) Quantification of the results, plxnbl human, treated with IgG: n=13; plxnbl human, treated with RbPLX7: n=15; plxnbl ¹-, treated with IgG: n=2. Scale bar, 1 mm. Mean values shown with error bars representing standard error of the mean (SEM), and statistical significances evaluated by One-Way ANOVA Tukey’s Post Tests. *p < 0.05, **p < 0.01 .

FIGURE 8. Therapeutic effect of the anti-Plexin-B1 antibody - RbPLX7 in a mouse model of Multiple Sclerosis. (A) Penetration of RbPLX7 into the central nervous system (CNS). Healthy mice or mice with EAE were injected without or with RbPLX7 according to the schemes depicted in panel (B) and (C). At day 26 (24 hours after the last antibody injection), mice were perfused with PBS, spinal cords were harvested and lysed. Each lane represents the spinal cord lysate of an individual mouse. Western Blot analysis was performed by using an anti-human Fc-antibody. (B) Clinical score and (C) weight change of mice with EAE treated with IgG isotype control (IgG) or the anti-Plexin-B1 antibody RbPLX7 over the course of 35 days, plxnbl human: n=8; plxnbl ¹ n=8. Mean values shown with error bars representing standard error of the mean (SEM), and statistical significances evaluated by One-Way ANOVA Tukey’s Post Tests. *p < 0.05, **p < 0.01 .

FIGURE 9. Characterization of primary osteoblasts derived from the humanized Plexin-B1 mouse line. (A) RNA was isolated from the femora of mice carrying one functional plxnbl allele (plxnb1^+/- ; mice carry one wildtype allele and one knockout allele), from the femora of Plexin-B1 knockout mice (plxnb ^A), from the femora of “humanized Plexin-B1 mice” expressing the human plxnbl gene instead of the endogenous murine plxnbl gene (^‘plxnbl human”), or from the human breast cancer cell line BT-474. Shown is an RT-PCR, which specifically detects the human plxnbl mRNA (but not the murine plxnbl mRNA). (B) RNA was isolated from the femora of mice with the indicated genotypes. Shown is an RT- PCR, which specifically detects the murine plxnbl mRNA (but not the human plxnbl mRNA). (C) Osteoblasts isolated from humanized Plexin-B1 mice were exposed to 150 nM of Sema4D in osteogenic medium, and osteoblast mineralization was analyzed after 21 days via Alizarin Red S staining. (D) Quantification of the results in (C). (E, F) Osteoblasts isolated from Plexin-B1 knockout mice or from humanized Plexin-B1 mice were exposed to 150 nM of Sema4D in osteogenic medium, and after 14 days, mRNA expression levels of the osteoblast differentiation markers (E) Bglap2 (Osteocalcin-2) and of (F) Alpl (alkaline phosphatase) were determined by qPCR. plxnbl human: n=4; plxnbl ¹ n=3. Mean values shown with error bars representing standard error of the mean (SEM).

FIGURE 10. Validation of the humanized Plexin-B1 mouse line in experimental autoimmune encephalomyelitis (EAE). (A) Microglia were isolated from Plexin-B1 knockout mice (plxnbl ¹) and from humanized Plexin-B1 mice (plxnbl human). Shown is an RT-PCR, which specifically detects the human plxnbl mRNA (but not the murine plxnbl mRNA). Each lane represents microglia from an individual mouse. (B) Microglia isolated from a Plexin-B1 knockout mouse and from a humanized Plexin- B1 mouse were immunostained for the microglia marker iba1 (red) and for Plexin-B1 (green). (C, D) Humanized Plexin-B1 mice and Plexin-B1 knockout mice were subjected to EAE, and clinical score and weight change were analyzed, plxnbl human: n=7; plxnbl ¹ n=7. Mean values shown with error bars representing standard error of the mean (SEM), and statistical significances evaluated by One-Way ANOVA Tukey’s Post Tests. *p < 0.05.

FIGURE 11. Therapeutic effect of the anti-Plexin-B1 antibody - RbPLX20 in an in vivo mouse model of postmenopausal osteoporosis. (A) Human osteoblasts were exposed to 150 nM of Sema4D in osteogenic medium without or in combination with 150 nM anti-Plexin-B1 antibody RbPLX20 or the corresponding IgG control antibody (IgG). Alkaline Phosphatase (ALP) activity was determined after 7 days activity. (B) Quantification of the results in (A). (C) Humanized Plexin-B1 mice were ovariectomized mice and treated with the anti-Plexin-B1 antibody RbPLX20 or the corresponding IgG control antibody. Sham-operated humanized Plexin-B1 mice were treated with the IgG control antibody and were used as controls. Scale bar: 1 mm. Shown are microcomputed tomography (pCT) images of the distal femur. Top: longitudinal view; bottom: transaxial view of the metaphyseal region. Shown are mean values ± SEM. **p < 0.01.

FIGURE 12. Quantification of the results from Figure 11. plxnbl human, treated with IgG: n=6; plxnbl human, treated with RbPLX20: n=7; plxnbl human, sham-operated and treated with IgG: n=5. Mean values shown with error bars representing standard error of the mean (SEM), and statistical significances evaluated by One-Way ANOVA Tukey’s Post Tests. *p < 0.05, **p < 0.01 . Detailed description

The present inventors have identified antigen binding molecules that specifically bind Plexin-B1 . The anti-Pexin-B1 antibodies block the binding of Sema4D to Plexin-B1 . In particular in a first aspect, the present invention provides antibodies and antigen-binding fragment thereof that specifically bind to Plexin-B1 . Preferably an antibody or antigen-binding fragment thereof specifically binds to the extracellular domain of Plexin-B1 , in particular to an epitope in the region of amino acids 20 to 535 of human Plexin-B1 .

The antibodies and antigen-binding fragments thereof as described herein may bind human Plexin-B1 , mouse Plexin-B1 , and /or cynomolgus Plexin-B1 , preferably human Plexin-B1 . For example, the antibody and antigen-binding fragments may bind human Plexin-B1 and show no binding or substantially no binding to mouse Plexin-B1 and/or cynomolgus Plexin-B1. The antibodies and antigen-binding fragments may be cross reactive with human, mouse and cynomolgus Plexin-B1. For example, in one embodiment a cross reactive anti-Plexin-B1 antibody or antigen-binding fragment thereof may bind human Plexin-B1 , mouse Plexin-B1 and cynomolgus Plexin-B1. In one embodiment a cross reactive anti-Plexin-B1 antibody or antigen-binding fragment thereof may bind human Plexin- B1 and cynomolgus Plexin-B1 and not bind mouse Plexin-B1. Typically, specificity may be determined by means of a binding assay such as ELISA.

The antibodies and antigen-binding fragments described herein will generally be specific for Plexin-B1 . In other words, an antibody or antigen-binding fragment thereof may bind Plexin-B1 but show no binding or substantially no binding to other members of the Plexin family. Preferably, an antibody or antigen-binding fragment thereof specific for Plexin-B1 binds Plexin-B1 but shows no binding or substantially no binding to plexins in the subfamilies A, C and D. The antibody or antigen-binding fragment thereof specific for Plexin-B1 , preferably shows no binding or substantially no binding to Plexin-B2 or Plexin-B3.

The anti-Plexin-B1 antibodies and antigen-binding fragments of the invention have been shown to inhibit Sema4D-Plexin-B1 signalling. The antibodies have been shown not to block signalling with other Plexin-B family members, i.e. Sema4D-Plexin-B2. Signalling inhibition can be measured by using a standard Cos-7 collapse assay. For example, a Cos-7 collapse assay to measure Sema4D- Plexin-B1 signalling may involve culturing Cos-7 cells expressing Plexin-B1 in the presence of Sema4D and anti-Plexin-B1 antibody for 1 hour. Cells can then be immunostained by incubating the cells with anti-Plexin-B1 antibody and then stained to visualize cell collapse and analysed via fluorescence microscopy.

The amino acid sequences of Plexin-B1 to which the antibody and binding fragments thereof of the invention bind are as provided below:

Pre-processed human Plexin-B1 (huPlexin-B1) can have the amino acid sequence as identified in UniProt reference - 043157 (PLXB1 _HUMAN) (SEQ ID NO: 28). The extracellular domain of huPlexin-B1 comprises amino acids residues 20-535 of huPlexin-B1 and is set out below:

LQPLPPTAFTPNGTYLQHLARDPTSGTLYLGATNFLFQLSP

GLQLEATVSTGPVLDSRDCLPPVMPDECPQAQPTNNPNQLLLVSPGALVVCGSVHQGVCE QRRLGQLEQLLLRPERPGDTQYVAANDPAVSTVGLVAQGLAGEPLLFVGRGYTSRGVGGG IPPITTRALWPPDPQAAFSYEETAKLAVGRLSEYSHHFVSAFARGASAYFLFLRRDLQAQ SRAFRAYVSRVCLRDQHYYSYVELPLACEGGRYGLIQAAAVATSREVAHGEVLFAAFSSA APPTVGRPPSAAAGASGASALCAFPLDEVDRLANRTRDACYTREGRAEDGTEVAYIEYDV NSDCAQLPVDTLDAYPCGSDHTPSPMASRVPLEATPILEWPGIQLTAVAVTMEDGHTIAF LGDSQGQLHRVYLGPGSDGHPYSTQSIQQGSAVSRDLTFDGTFEHLYVMTQSTLLKVPVA SCAQHLDCASCLAHRDPYCGWCVLLGRCSRRSECSRGQGPEQWLWSFQPELGCLQ (SEQ ID NO: 27)

Pre-processed mouse Plexin-B1 (moPlexin-B1) can have the amino acid sequence as identified in UniProt reference - Q8CJH3 (PLXB1_MOUSE) (SEQ ID NO: 30).

The extracellular domain of moPlexin-B1 comprises amino acid residues 20-535 of moPlexin-B1 and is set out below: LRSPLPAAFTANGTHLQHI-ARDPTTGTLYVGATNFLFQLSPGLQLEAVVSTGPVNDSRDC LPPVIPDECPQAQPTNNPNQLLLVSPEALVVCGSVHQGICELRSLGQIRQLLLRPERPGD TQYVAANDPAVSTVGLVAQGLVGEPLLFVGRGYTSRGVGGGIPPITTRALRPPDPQAAFS YEETAKLAVGRLSEYSHHFVSAFVRGASAYFLFLRRDLKAPSRAFRAYVSRVCLQDQHYY SYVELPLACQGGRYGLIQAAAVATSKEVARGDVLFAAFSSVAPPTVDWPLSASTGASGTS VLCAFPLDEVDQLANYTRDACYTREGRAENGTKVADIAYDVLSDCAQLPVDTPDAFPCGS DHTPSPMVSCVPLEATPILELPGVQLTAVAVTMEDGHTIAFLGDSQGQLHRVYLGPGRSA APYSKQSIQPGSPVNRDLTFDGTFEHLYVATQTTLVKVPVAPCAQHLDCDSCLAHRDPYC GWCVLLGRCSRRSECSRDQGPEQWLWSFQPELGCLR (SEQ ID NO: 29)

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The terms "immunoglobulin" and "antibody" may be used interchangeably to refer to any protein comprising an antibody antigen-binding site which has the ability to specifically bind one or more antigens. The term “antibody” also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain.

Antibodies may be polyclonal or monoclonal. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Antibodies are polypeptides that typically contain two identical heavy chains and two identical light chains, which are smaller than the heavy chains. In mammals there are two types of light chain, which are called lambda (A) and kappa (K) based on the amino acid sequence of the light chain constant region. Each of the heavy chains and each of the light chains are composed of a variable region and a constant region. The heavy chain variable region is referred to as the VH region and the light chain variable region is referred to as the VL region. For kappa light chains, the VL region can also be referred to as the VK region. Each of the variable regions of the light and heavy chains comprise three complementary determining regions (CDRs), CDR1 , CDR2 and CDR3. These are named LCDR1 , LCDR2, LCDR3, HCDR1 , HCDR2 and HCDR3 respectively. The CDRs are interspersed among relatively conserved framework regions (FRs). The sequences of the CDRs may be readily identified using standard techniques.

Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses. An antibody described herein is preferably an immunoglobulin G (IgG) antibody, such as lgG1 , lgG2, lgG3 or lgG4, most preferably an lgG1 antibody.

The antibodies of the invention are typically monoclonal antibodies. In some embodiments, the anti- Plexin-B1 antibody is a fully human or humanised monoclonal antibody

The antibody or antigen-binding fragment thereof of the invention may be from any animal species including murine, rat, human, or any other origin. In some embodiments the antibody or antigenbinding fragment thereof may be a chimeric or humanised antibody. In some embodiments, the anti- Plexin-B1 antibody or antigen-binding antibody fragment thereof is monoclonal, e,g. a monoclonal anti-Plexin-B1 antibody. In some embodiments, the antibody or antigen- binding fragment thereof is a human or humanised antibody or antigen-binding fragment thereof. A non-human antibody or antigenbinding fragment thereof may be humanised by recombinant methods to reduce its immunogenicity in humans. In some embodiments, the anti-Plexin-B1 antibody of the present invention is a chimeric antibody.

As used herein, a "humanised antibody" refers to an antibody in which some, most or all of the amino acids outside the CDRs of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In some embodiments, humanised antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The humanised antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance. In one embodiment of a humanised form of an antibody, some, most or all the amino acids outside the CDRs have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible provided they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanised" antibody retains an antigenic specificity similar to that of the original antibody. A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a rabbit antibody and the constant regions are derived from a human antibody or vice versa.

The invention also provides a fragment of the anti-Plexin-B1 antibody, specifically an antigen-binding fragment of an anti-Plexin-B1 antibody. The antigen binding fragments comprise one or more antigen binding regions. Examples of binding fragments are Fab fragment consisting of VL, VH, CL and CH1 domains; Fab’; F(ab’)2; Fd fragment consisting of the VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single antibody; the dAb fragment which consists of a VH domain; F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments; single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; di-scFv (divalent scFv), bispecific single chain Fv dimers; diabodies, bispecific diabody, trispecific triabody, scFv-Fc, sdAb (single domain antibody) and multivalent or multi-specific fragments constructed by gene fusion. Typically, the fragment is a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, an Fv fragment, an scFv fragment, dAb, Fd, a diabody, or a single chain antibody. Fragments of the antibodies retain the ability to bind Plexin-B1 and can be produced using standard methods known in the art.

In some embodiments the antibody or antigen binding fragment thereof is provided as part of a multispecific binding agent such as for example as part of a bispecific or multispecific. A bispecific antibody is one which can bind to two target molecules simultaneously, such as two antigens or two epitopes. In some embodiments, the antibody or antigen -binding fragment thereof of the invention may be bispecific for Plexin-B1 and another antigen or epitope. Bispecific and multispecific antibodies may be produced by a variety of methods known in the art.

In some embodiments, the antigen binding molecules of the invention are monovalent for Plexin-B1 , for example a monovalent antibody or antigen-binding fragment thereof. A monovalent antigen binding molecule (also known as a monomeric antibody) is an antigen binding molecule with only one binding site for an epitope or antigen. The present invention therefore also provides monovalent antibodies or antibody fragments that specifically bind to Plexin-B1 .

"Specific binding", "bind specifically", and "specifically bind" are terms well understood and methods to determine such specific binding are also well known in the art. An antibody is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell, protein or substance than it does with alternative cells, proteins or substances. As used herein it is understood to mean that the antibody or antigen-binding fragment has a dissociation constant (Kd) for the antigen of interest of less than about 10⁶M, 10'⁷M, 10⁸M, 10⁹M, 10'¹⁰M, 10'¹¹M or 10'¹²M. In a preferred embodiment, the dissociation constant is less than 10⁸M, for instance in the range 10^-9M, 1 O^-1CIM, 10^-11M or 10^-12M. In accordance with some embodiments of the invention. In some embodiments, the affinity of the anti-Plexin-B1 antibody is from 10¹¹ to 10⁹ (for example about 10¹⁰).

In some embodiments the anti-Plexin-B1 antibody or antigen-binding fragment thereof may be afucosylated. It is well known that antibody glycosylation may have impact on the activity, pharmacokinetics and pharmacodynamics of antibodies (e.g., monoclonal antibodies, recombinant antibodies, and/or antibodies that are otherwise engineered or isolated) and Fc-fusion proteins and specific technology may be exploited to obtain an antibody with the desired glycosylation profile.

Antibodies or antigen binding fragments thereof of the invention may also be conjugated to other molecules such as therapeutic agents, prodrugs or toxic moieties. This may serve to target the antibodies to the target cell. The other molecule may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art.

In some embodiments the antibodies may be conjugated to detectable labels. Any detectable label that may be readily measured may be conjugated to an antibody described herein. Detectable labels include but are not limited to enzymes for use assays, fluorescent materials, bioluminescent materials, and radioactive materials. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. The detectable label may assist the antibodies use as a diagnostic antibody or as a research tool.

A summary of the antibodies provided by the present invention is provided below, with identification of the assigned SEQ ID NO. in the accompanying sequence listing. Antigen binding variants, derivatives and fragments thereof are also provided as part of the present invention:

Table 1

In some embodiments, an anti-Plexin-B1 antibody, variant or fragment thereof is provided comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of: a) a VH comprising the amino acid sequence of SEQ ID NO: 23 and a VL comprising the amino acid sequence of SEQ ID NO: 24; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 23 and SEQ ID NO: 24, respectively; b) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 26; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 25 and SEQ ID NO: 26, respectively; c) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 15 and SEQ ID NO: 16, respectively; d) a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 17 and SEQ ID NO: 18, respectively; e) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 20; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and f) a VH comprising the amino acid sequence of SEQ ID NO: 21 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or comprising VH and VL sequences that are at least 90% identical to SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

In some embodiments, the % sequence identity for the above antibody and antigen-binding fragments thereof is calculated without the sequences of all 6 CDRs of the anti-Plexin-B1 antibody. For example, the anti-Plexin-B1 antibody, or antigen-binding fragment thereof, may comprise a variable light chain region sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26 and/or a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23 and SEQ ID NO: 25 wherein any amino acid variations occur only in the framework regions of the variable heavy and light chain region sequences.

In one embodiment there is provided an anti-Plexin-B1 , or antigen-binding fragment thereof, of the invention comprising from 1 to 10, preferably from 1 to 5, more preferably from 1 to 2 amino acid substitutions in the antibody-binding domain or antigen-binding domains. For example, in one embodiment of the invention, there is provided an anti-Plexin-B1 antibody or antigen-binding fragment thereof, wherein the anti-Plexin-B1 antibody or antigen-binding fragment thereof comprises the 6 CDR regions of an antibody selected from the group consisting of RbPLX11 , RbPLXI 8, RbPLXI 9, RbPLX20, RbPLX7, an HuPLX7, optionally wherein the antigen-binding molecule has from 1 to 10 amino acid substitutions across all of its CDR regions, preferably from 1 to 5 amino acid substitutions across all of its CDR regions. In a further embodiment of the invention, there is provided an anti- Plexin-B1 antibody or antigen-binding fragment thereof, wherein the anti-PLexin-B1 antibody or antigen-binding fragment thereof comprises the VH and VL sequences of an antibody selected from the group consisting of RbPLX11 , RBPLX18, RbPLX19, RbPLX20, RbPLX7, an HuPLX7, optionally wherein the antibody or antigen-binding fragment thereof has from 1 to 10 amino acid substitutions across its VH and VL sequences, preferably from 1 to 5 amino acid substitutions across its VH and VL sequences. In a still further embodiment of the invention, there is provided an anti-Plexin-B1 antibody, wherein the anti-Plexin-B1 antibody is an antibody selected from the group consisting of RbPLX11 , RbPLX18, RbPLX19, RbPLX20, RbPLX7, an HuPLX7, wherein the antibody has from 1 to 10 amino acid substitutions, preferably from 1 to 5 amino acid substitutions. Substitutions are of course substitutions with reference to the original CDR or variable chain sequences of the starting antibody.

For example, in some embodiments there is provided an anti-Plexin-B1 antibody, wherein the anti- Plexin-B1 antibody is an antibody selected from the group consisting of RbPLX11 , RbPLXI 8, RbPLX19, RbPLX20, RbPLX7, an HuPLX7, wherein the antibody has from 1 to 10 amino acid substitutions across its all of its framework regions, preferably from 1 to 5 amino acid substitutions across its all of its framework regions (i.e. the substitutions appear in the framework regions relative to reference antibody, and the CDR sequences are unchanged).

In one embodiment, an anti-Plexin-B1 antibody, variant or fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: a) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 23; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 24; b) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 25; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 26; c) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 15; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 16; d) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 17; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 18; e) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 19; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 20; and f) a VH sequence having 1 to 5 amino acid substitutions compared to the VH sequence of SEQ ID NO: 21 ; and/or a VL sequence having 1 to 5 amino acid substitutions compared to the VL of SEQ ID NO: 22.

In some embodiments, the amino acid substitutions all occur in the CDRs of the variable regions. In some embodiments, all the amino acid substitutions occur in the framework regions of the variable regions. Preferably there are four or fewer, three or fewer, two or fewer or one substitution in the sequence. Variant antibodies having the one or more amino acid substitutions may retain the functional activity of the antibody from which the variant antibody is derived, i.e. are functionally active variants.

RbPLX7

In one embodiment an antibody or antigen-binding fragment thereof that bind Plexin-B1 is provided comprising:

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23, and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24.

In one embodiment, an antigen-binding molecule, for example an antibody, fragment or variant thereof is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody is or is derived from the antibody designated herein as RbPLX7, a rabbit/human chimeric antibody.

HuPLX7

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26.

In one embodiment, an antigen-binding molecule, for example an antibody, fragment or variant thereof is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody is or is derived from the antibody designated herein as HuPLX7, a humanised rabbit/human chimeric antibody.

RbPLXI 1

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 16.

In one embodiment, an antigen-binding molecule for example an antibody or antigen-binding fragment is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is or is derived from the antibody designated herein as RbPLXI 1 , a rabbit/human chimeric antibody.

RbPLXI 8

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17, and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

In one embodiment, an antigen-binding molecule, for example an antibody, fragment or variant thereof is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody is or is derived from the antibody designated herein as RbPLX18, a rabbit/human chimeric antibody.

RbPLXI 9

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19, and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

In one embodiment, an antigen-binding molecule, for example an antibody, fragment or variant thereof is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is or is derived from the antibody designated herein as RbPLXI 9, a rabbit/human chimeric antibody.

RbPLX20

(a) a heavy chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 , and/or

(b) a light chain variable region comprising: an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22.

In one embodiment, an antigen-binding molecule, for example an antibody, fragment or variant thereof is provided comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody is or is derived from the antibody designated herein as RbPLX20, a rabbit/human chimeric antibody.

There is also provided an anti-Plexin-B1 antibody or antigen-binding fragment thereof that specifically binds to Plexin-B1 and inhibits the binding of the antibodies designated herein as RbPLX11 , RbPLXI 8, RbPLXI 9, RbPLX20, RbPLX7, an HuPLX7 to Plexin-B1.

For example, in one embodiment the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof that specifically binds to an epitope of Plexin-B1 that is bound by an antibody selected from the group consisting of RbPLX11 , RbPLXI 8, RbPLXI 9, RbPLX20, RbPLX7, and HuPLX7. Preferably the epitope is within amino acids 20 to 535 of human or mouse Plexin-B1 .

In one embodiment the invention provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof that specifically binds to a conformational epitope comprising at least one amino acid selected from the amino acid residues 106, 131 , 132, 135-141 , 188, 190-196, 199- 203, 256 of SEQ ID NO: 30. Preferably the epitope comprises at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, at least fifteen amino acids, at least twenty amino acids or more comprised in amino acids residues 106, 131 , 132, 135-141 , 188, 190-196, 199- 203, 256 of SEQ ID NO: 30. Preferably the epitope comprises amino acids residues 106, 131 , 132, 135-141 , 188, 190-196, 199- 203, 256 of SEQ ID NO: 30. More preferably the conformational epitope consists essentially of amino acids residues 106, 131 , 132, 135- 141 , 188, 190-196, 199- 203, 256 of SEQ ID NO: 30. As used herein, the term "epitope" refers to a portion of an antigen that is bound by an antibody or antigen-binding fragment. In some embodiments, where the antigen is a polypeptide, an epitope is conformational in that it is comprised of portions of an antigen that are not covalently contiguous in the antigen but that are near to one another in three-dimensional space when the antigen is in a relevant conformation. Means for determining the exact sequence and/or particular amino acid residues of the epitope for the Plexin-B1 antibodies, and whether two antibodies bind to identical or overlapping epitopes are known in the literature, including competition with peptides, from antigen sequences, binding to Plexin-B1 sequence from different species, truncated, and/or mutagenized (e.g. by alanine scanning or other site-directed mutagenesis), phage display-based screening, yeast presentation technologies, or (co-) crystallography techniques. An antibody binds “the same epitope” as another antibody when they both recognize identical epitopes (i.e. all contact points between the antigen and the antibody are the same). For example, an antibody may bind the same epitope as another antibody when all contact points across a specified region of an antigen are identified as the same with aid of a characterization method such as antibody/antigen cross-linking-coupled MS, HDX, X-ray crystallography, cryo-EM, or mutagenesis.

In some embodiments there is provided an anti-Plexin-B1 antibody that competes for binding to the extracellular domain of Plexin-B1 with an anti-Plexin-B1 antibody or antigen-binding fragment thereof as described herein, for examples competes for binding to the extracellular domain of Plexin-B1 with HuPLX7, RbPLX7, RbPLX11 , RbPLX18, RbPLXI 9 and/or RbPLX20. Such antibodies that compete for binding can be used in the methods, compositions, and uses as described herein.

The present invention also extends to variants of amino acid sequences referred to herein. As used herein the term “variant” relates to proteins that have a similar amino acid sequence and/or that retain the same Plexin-B1 binding function as the antibodies described above. For instance, the term “variant” encompasses an antibody or antigen binding fragment which include one or more amino acid additions, deletions, substitutions, or the like. An example of a variant of the present invention is an antibody or antigen binding fragment thereof comprising an amino acid sequence as defined above, apart from the substitution of one or more amino acids with one or more other amino acids. Amino acid substitutions may be made to, for example, reduce or eliminate liabilities in the amino acid sequences. Alternatively, amino acid substitutions may be made to improve antigen affinity or to humanise or deimmunise the antibodies, if required. Affinity matured variants, humanised variants and deimmunised variants of the specified antibodies are provided herein, as well as variants comprising amino acid substitutions to reduce or eliminate any liabilities in the sequences of the antibodies.

In some embodiments, the one or more amino acid substitutions are in the CDR region or regions. In other embodiments, the one or more amino acid substitutions are in the framework regions, i.e., in the variable heavy and light chains but not in the CDR region or regions. In other embodiments, the one or more amino acid substitutions may be at any position in the variable heavy and/or variable light regions. In some embodiments, the amino acid substitutions do not adversely affect the binding specificity and/or affinity of the antibody. Accordingly, the variant antibody may have the same or superior functional profile as the antibody from which is it derived.

The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. In some embodiments, the amino acid substitutions may be conservative amino acid substitutions. References to “conservative” amino acid substitutions refer to amino acid substitutions in which one or more of the amino acids in the sequence of the antibody (e.g. in the CDRs or in the VH or VL sequences) is substituted with another amino acid in the same class. Conservative amino acid substitutions may also occur in the framework regions or in the CDR regions.

Amino acid substitutions or insertions can be made using naturally occurring or non-naturally occurring amino acids, although naturally occurring amino acids may be preferred. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

Amino acid changes relative to the sequence given above can be made using any suitable technique e.g. by using site-directed mutagenesis or solid-state synthesis.

“Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptides or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e. , % identity = number of identical positions/total number of positions x 100). Generally, references to % identity herein refer to % identity along the entire length of the molecule, unless the context specifies or implies otherwise. The variant amino acid sequences provided herein having a particular identity, are typically calculated for example using the default parameters for the above computer programs. Antibody residues positions described herein are numbered according to the scheme set out in Kabat, E.A., Wu, T.T., Perry, H.M., Gottesmann, K.S & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242. U.S. Department of Health and Human Services. Where appropriate, the position of a substitution may be described relative to a Kabat numbered residue which is invariant in immunoglobulin sequences.

In one aspect of the invention, there is provided nucleic acid sequences encoding polypeptides capable of forming the anti-Plexin-B1 antibodies, antigen-binding fragments thereof and variants thereof. In one embodiment the nucleic acid molecules may encode just the polypeptide sequence that comprises the VL domain of the anti-Plexin-B1 antibody or fragment thereof. In one embodiment the nucleic acid molecules may encode just the polypeptide sequence that comprises the VH domain of the anti-Plexin-B1 antibody or fragment thereof.

In one embodiment, nucleotides encoding an anti-Plexin-B1 antibody comprising a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26, and/or a heavy chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23 and SEQ ID NO: 25 are provided.

In one embodiment, nucleotides encoding an antibody that binds to Plexin-B1 comprising a light chain variable region having the amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26, and/or a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23 and SEQ ID NO: 25, are provided.

In one embodiment, the invention provides a nucleotide encoding an antibody that binds to Plexin-B1 comprising: a) a heavy chain variable region having the amino acid sequence SEQ ID NO: 15 and a light chain variable region having the amino acid sequence SEQ ID NO: 16; b) a heavy chain variable region having the amino acid sequence SEQ ID NO: 17 and a light chain variable region having the amino acid sequence SEQ ID NO: 18; c) a heavy chain variable region having the amino acid sequence SEQ ID NO: 19 and a light chain variable region having the amino acid sequence SEQ ID NO: 20; d) a heavy chain variable region having the amino acid sequence SEQ ID NO: 21 and a light chain variable region having the amino acid sequence SEQ ID NO: 22; e) a heavy chain variable region having the amino acid sequence SEQ ID NO: 23 and a light chain variable region having the amino acid sequence SEQ ID NO: 24; or f) a heavy chain variable region having the amino acid sequence SEQ ID NO: 25 and a light chain variable region having the amino acid sequence SEQ ID NO: 26.

The present invention also provides nucleic acid molecules encoding all of the antigen binding fragments thereof and variant antibody sequences disclosed herein comprising one or more amino acid substitutions.

Also provided are nucleic acid molecules that encode an amino acid sequence according to any one of SEQ ID NOs 1-26.

Also provided are vectors comprising a nucleic acid sequence encoding an anti-Plexin-B1 antibody or antigen-binding fragment thereof. Vectors include but are not limited to plasmid vectors and viral vectors. Nucleic acids as described above, encoding light and/or heavy chain variable regions optionally linked to constant regions, may be inserted into expression vectors. Vectors which comprise nucleic acids encoding antibodies described herein are themselves an aspect of the invention. The light and heavy chains may be cloned in the same or different expression vectors. The nucleic acids encoding the antibody chains described herein may be operably linked to one or more control sequences in the expression vectors) that ensure the expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS, CHO, or Expi293 cells). The vectors may be incorporated into an appropriate host, whereby the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the antibodies.

The expression vectors for use as described herein are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.

Vectors described herein containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) may be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, or viral-based transfection may be used for other cellular hosts. (See generally Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 4th ed., 2012). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). Host cells may be transformed with the expression vectors and cultured in conventional nutrient media as appropriate for inducing promoters, selecting transformants, and/or amplifying the genes encoding the required sequences. Accordingly, the invention also provides host cells comprising a nucleic acid, a plasmid or vector as described above. Preferably a eukaryotic or mammalian cell host comprising a nucleic acid or vector described herein is provided. A number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact antibodies) have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, Expi293 cells, ExpiCHO cells, myeloma cell lines, or transformed B-cells or hybridomas. The cells may be human or non-human e.g. non-human mammalian cells.

Also provided are methods for the production of an anti-Plexin-B1 antibody comprising culturing a host cell of the invention in a cell culture medium under conditions to express the encoding nuclide acid sequence of the plasmid or vector inside the cell. The method may further comprise obtaining the expressed anti-Plexin-B1 antibody. The method of producing a cell that expresses an anti-Plexin-B1 antibody, can further comprise transfecting the host cell with a plasmid or vector of the invention. The cells can then be cultured for the production of the anti-Plexin-B1 antibody

Methods for the production of monoclonal antibodies are well known to the skilled person. Therefore, anti-Plexin-B1 antibodies described herein may be produced for examples as described by any suitable technique including techniques described herein, as well as other techniques known in the art.

In one aspect of the invention, a pharmaceutical composition comprising an anti-Plexin-B1 antibody and antigen-binding fragments thereof of the invention is provided. Pharmaceutical composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.

The composition can be formulated for use by any convenient route. The pharmaceutical composition will normally include a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer or stabiliser in addition to an anti-Plexin-B1 antibody or antigen-binding fragment thereof. Such carriers include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol and combinations thereof.

The pharmaceutical composition may be in any suitable form depending upon the desired method of administering it to a patient.

The pharmaceutical compositions may be adapted for administration by any appropriate route, for example by the oral, rectal, intranasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Parental administration may be preferred. Such formulations may be prepared by any method known in the art of pharmacy. See Remington: The Science and Practice of Pharmacy (22nd ed., Pharmaceutical

Press, London, Pa. (2013)

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, preservatives and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Additional excipients which may be included in the compositions include but are not limited to surfactants (e.g. polysorbate 80 or polysorbate 20), tonicity agents, chelating agents, sugars, amino acids and salts.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules, tablets, powders, granules, solutions or suspensions in aqueous or non-aqueous liquids.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question.

The pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of each active ingredient per dose. Such a unit may be adapted to provide for example 0.5- 100 mg/kg of the compound to the subject, preferably either 0.5-50mg/kg, 1-10mg/kg, 1-5mg/kg, 5- 10mg/kg or 10-50mg/kg. Such doses can be provided in a single dose or as a number of discrete doses. The ultimate dose will of course depend on the condition being treated, the route of administration and the age, weight and condition of the subject.

The pharmaceutical compositions can also contain one or more other therapeutically active agents in addition to anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

The antibodies or antigen-binding fragments thereof may be provided as part of a kit. Such kits may include instructions for use and/or additional pharmaceutically active components. The antibodies and binding fragments thereof and the additional pharmaceutically active components may be disposed separately within the kit, or in some embodiments the antibodies and binding fragments thereof and the additional pharmaceutically active components may be formulated together. The anti-Plexin-B1 antibodies and binding fragments thereof of the invention are useful in preventing and/or treating diseases or disorders associated with Plexin-B1 . Therefore, the invention also includes a method for the treatment of a Plexin-B1 mediated disorder or disease in a subject, comprising administering to the subject an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention. The invention also provides the use of the anti-Plexin-B1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for use in the treatment of a Plexin-B1 mediated disorder or disease and use of the anti-Plexin-B1 antibody or antigen-binding fragment thereof in prevention and/or treatment of such conditions. The invention also provides an anti-Plexin-B1 antibody or antigen-binding fragment thereof for use in the treatment of a Plexin-B1 mediated disorder or disease.

Also provided are methods of inhibiting the binding of Plexin-B1 to Sema4D for the treatment of a disease associated with Sema4D signalling via Plexin-B1 , the method comprising administering an effective amount of an anti-Plexin-B1 antibody or antigen-binding fragment thereof of the invention.

Diseases associated with Sema4D signalling via Plexin-B1 include but are not limited to immune mediated diseases, for example multiple sclerosis, arthritis and Amyotrophic lateral sclerosis (ALS), osteoporosis, neoplastic disease, for example cancer, inflammation, and neurodegenerative diseases, for example Huntington’s disease and Alzheimer’s Disease.

In one embodiment, the anti-Plexin-B1 antibodies and binding fragments thereof of the invention are for use in the treatment of osteoporosis, multiple sclerosis or a neoplastic disease. Preferably in the treatment of osteoporosis or multiple sclerosis.

Neoplastic diseases include the treatment of cancer, in particular the treatment of tumors in a subject with cancer. Cancers that can be treated with the anti-Plexin-B1 antibody or antigen-binding fragment thereof include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, melanoma and stomach cancer.

The terms "patient", “individual” or “subject” include human and other animal subjects. Non-human animals include for example mammals such as mice, rats, rabbits, non-human primates. The treatment can be of a human or another animal subject and the invention extends equally to uses in both human and/or veterinary medicine. As used herein, “treatment” includes any regime that can benefit a human or non-human animal, preferably mammal. The treatment may be in respect of an existing condition (therapeutic treatment) or may be prophylactic (preventative treatment). The method may be an in vitro method. The method may be an in vivo method.

The anti-Plexin-B1 antibody or antigen-binding fragment thereof is preferably administered to an individual in a “therapeutically effective amount”. As used herein, a “therapeutically effective amount” or an “effective dosage” or a “sufficient amount” (or equivalent terms thereof) of an antibody described herein refers to an amount of antibody or composition described herein that is effective to produce a desired effect, which is optionally a therapeutic or prophylactic effect, i.e. an amount to ameliorate a symptom of a disease or disorder. A disease or disorder is “ameliorated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. Other factors that will determine the frequency and dosage regime of the antibody, includes form of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered. Subjects may be administered doses of the antibody multiple times per day, daily, on alternative days, weekly or according to any other determined schedule determined. A treatment may involve administration in multiple dosages over a prolonged period, for example, of at least six months. Antibodies and compositions described herein may be administered by parenteral, topical, intravenous, oral, gastric, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal or intramuscular methods, as described herein. Intramuscular injection or intravenous infusion are preferred for administration of antibodies.

The anti-Plexin-B1 antibodies and antigen-binding fragments thereof may be used in combination with other pharmaceutically active components for simultaneous, separate or sequential use. The anti- Plexin-B1 antibodies may be used in combination therapy. As used herein the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously. Alternatively, such agents may be administered sequentially; otherwise, such agents are administered in overlapping dosing regimens.

The other therapeutic agents that the anti-Plexin-B1 antibodies may be used in combination may depend on the intended therapeutic use.

For example, when treating osteoporosis, the anti-Plexin-B1 antibodies and binding fragments thereof may be used in combination with another therapy or additional therapeutically active agent, for example in combination with estrogens, selective estrogen-receptor-modulators, bisphosphonates, an anti-RANKL-antibody, an anti-sclerostin antibody, strontium, and/or parathyroid hormone. When treating or preventing multiple sclerosis the anti-Plexin-B1 antibodies and binding fragments thereof may be used in combination with another therapy or additional therapeutically active agent, for example in combination with immunosuppressants, and/or immunomodulators .

The anti-Plexin-B1 antibodies and binding fragments thereof may also be used in the treatment of a neoplastic disease, for example in the treatment of tumors, preferably solid tumors, in a subject with cancer. In one embodiment when treating a subject with cancer the anti-Plexin-B1 antibodies and binding fragments thereof may be used in combination with another therapy or additional therapeutically active agent, for example in combination with other immunotherapies. The immune modulating therapy or agent can be selected from the group consisting of administration of a cancer vaccine, administration of an immunostimulatory agent, adoptive T cell or antibody therapy, administration of an immune checkpoint inhibitor, administration of a regulatory T cell (Treg) modulator, and a combination thereof. In some embodiments the immune checkpoint inhibitor may be selected from an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof, including but not limited to ipilimumab, tremelimumab, nivolumab, pembrolizumab, spartalizumab, avelumab, durvalumab, atezolizumab or combinations thereof. In some embodiments the Treg modulator may be a cyclophosphamide therapy including but not limited to ipilimumab, nivolumab, and/or avelumab.

The anti-Plexin-B1 antibodies of the invention can also be used in applicable diagnostic methods and as research tools, for example, in western blots and in methods based on immunofluorescence. The antibodies of the invention are particular useful in immunostaining. Therefore, the invention also provides kits and methods for analytical and/or diagnosis. The kits can include an anti-Plexin-B1 antibody or antigen-binding fragment thereof. Depending on the detection method the antibody can be conjugated to another molecule, such as a detectable label, and/or can be immobilized on a solid support (substrate). The kit may also comprise a second antibody for the detection of Plexin- B1/antibody-complex. The anti-Plexin-B1 antibody or antigen-binding fragment thereof may be free or immobilized on a solid support. The kit can also comprise instructions that describe the use of anti- Plexin-B1 antibody or functional fragment thereof in a diagnostic assay.

Therefore, also provided are methods for using the anti-Plexin-B1 antibody in assays. The assays include but are not limited to an immunohistochemistry assay, a competitive-binding assay, a Western Blot analysis, an ELISA assay, a radioimmunoassays, an enzyme immunoassay, a sandwich immunoassays, an immunodiffusion assay, a fluorescent immunoassay, and an immunoelectrophoresis assay. Also provided are methods of detecting anti-Plexin-B1 . The method comprising administering the anti-Plexin-B1 antibodies or antigen-binding fragments thereof of the invention to a sample and detecting the binding of the anti-Plexin-B1 antibody to Plexin-B1. In a further embodiment, the antibody is immobilized on a substrate and the method comprises contact the sample with the substrate. The detection of binding indicates the presence of Plexin-B1 in the sample. The kit can further comprise suitable reagents for the detection of markers or for marking positive and negative controls, washing solutions, and dilution buffers.

Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of’ or “consisting of’.

The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of and vice versa, wherever they occur herein.

It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope described herein.

The contents of all publications cited herein are incorporated herein by reference in their entirety into this application to more fully describe the state of the art to which this invention pertains.

Examples

The following examples describe the generation and properties of anti-Plexin B1 antibodies.

Example 1

The anti-Plexin-B1 antibodies, RbPLX11 , RbPLXI 8, RbPLXI 9, RbPLX20, RbPLX7, HuPLX7 having the sequences as shown in Figure 1 originate from phage display libraries generated from the rabbits immunized by extracellular region comprising Serna domain and PSI domain (residues 20-535) of human Plexin-B1 .

Generation of recombinant extracellular region of Plexin-B1 for immunization

Human Plexin-B1 (1-535) fragment was cloned to pcDNA5 vector using Hindlll (R0104S, NEB) and Xhol (R0146S, NEB) restriction sites with addition of the C-terminal 6xHis tag (KHHHHHH) and expressed in Expi293F cells (Invitrogen) according to manufacturer’s instructions. Supernatant containing secreted Plexin-B1 (20-535)-6xHis (1-19 signal peptide cleaved off) was collected after 7 days and processed by two step purification protocol developed at LifeArc. Briefly, an overexpressed protein was captured on Excel HisTrap (GE) column in 20mM HEPES, pH 8.0, 0.3M NaCI and 10 mM imidazole buffer. After washing the column with 10 column volumes of washing buffer (20mM HEPES, pH 8.0, 0.3M NaCI and 20 mM imidazole) the protein was eluted with elution buffer (20mM HEPES, pH 8.0, 0.3M NaCI and 250 mM imidazole), following with gel filtration chromatography using Superdex 200 16/60 column equilibrated with PBS. Collected fractions were analysed on SDS-PAGE, pooled, quantified by NanoDrop, aliquoted and flash-freezed for storage at- 80°C. The same method was used to clone, express, and purify corresponding fragments of mouse and cynomolgus Plexin-B1 - moPlexin- B1 (20-535)-6xHis and cynoPlexin-B1 (20-535)-6xHis, respectively.

Generation of recombinant extracellular region of Plexin-B1 for biopanninq and screening

To enable an indirect coating of antigen via the streptavidin, an Avi-tag versions of huPlexin-B1 (20- 535) and moPlexin-B1 (20-535) were generated. The sequence encoding the Avi-tag (GLNDIFEAQKIEWHE) was inserted between the C-terminus of the Plexin-B1 fragments and the 6xHis by site-directed mutagenesis (SDM) using QuikChange Lightning Site-Directed Mutagenesis Kit (# 210518, Agilent Technologies) and confirmed by Sanger sequencing analysis. Both versions - huPlexin- B1 (20-535)-Avi-6xHis and moPlexin-B1 (20-535)-Avi-6xHis - were expressed and purified using the same method as described in paragraph above for non-Avi tag versions. The in vitro biotinylation of the Avi-tag proteins was carried out using the BirA enzyme (BirA500 standard reaction kit, Avidity) according to manufacturer protocol. A free biotin was removed by desalting method using Zeba™ Spin 2ml 7K desalting columns (89889, ThermoFisher) and the biotinylation was verified by Western Blotting analysis using Streptavidin-HRP conjugate (N100, ThermoFisher).

Rabbit Immunisation and sera testing

Protein immunisation was outsourced to BioGenes Antibodies (Germany). The purified antigen, human Plexin-B1 (20-535)-6xHis, was injected four times to each rabbit at day 0, 7, 14 and 21 , using fast 35 days immunisation schedule. Pre-immune, intermediate and final serum was collected at day 0, 21 and 35, respectively. Spleens were extracted and submerged in RNAIater reagent (AM7020, ThermoFisher), frozen at -20°C and shipped to LifeArc on dry ice.

Sera of individual rabbits were analysed for antigen-specific response by standard ELISA assay using the biotinylated huPlexin-B1 (20-535)-Avi-6xHis. Briefly, HBC Streptavidin plate (Pierce) was coated with 50 pl of 5 pg/ml of biotinylated antigen diluted in PBS. After washing, 50 pl of sera diluted in PBS were added and incubated for 1 h following with goat anti-rabbit -HRP conjugate (65-6120, Invitrogen) incubation for 1 h. 50 pl of TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added and the reaction was stopped with 50 pl of 0.5 M H2SO4. The absorbance was recorded at 450 nm and the data were fitted to dose response four-parameter equation using Prism software. To assess cross-reactivity against cynomolgus and mouse orthologs, a similar ELISA assay was carried out. In this case, diluted sera were pre-incubated with 20 pg/ml of mouse or cynomolgus Plexin-B1 (20-535)-6xHis before adding to the plate coated with the biotinylated human Plexin-B1 (20-535)-Avi-6xHis.

Generation of rabbit immune phage display library

The total RNA was extracted from rabbits’ spleen and bone marrow using RNeasy Maxi kit (75162, Qiagen) according to the manufacturer protocol and used to synthesise the cDNA using SuperScript® III First-Strand Synthesis System (18080-051 , Invitrogen) with oligo (dT)2o primers according to manufacturer protocol. The gene fragments encoding antibody variable domains (VH and VL) were amplified from cDNA using germline specific oligos, similar to previously described (Rader C et al, 2000, J Biol Chem vol 275(18) p13668-76). The amplified antibody VH and VL repertoires were assembled into single-chain Fv (scFv) library format by two-stage overlap extension PCR and cloned into the pHEN1 H6 phagemid vector. The ligation product was electroporated to TG1 cells using standard method, and the library collected from the 2xYTAG agar bioassay plates and stored at -80°C.

Isolation of Plexin-B1 specific antibodies by phage library biopanninq

Before antigen biopanning, the phage library was rescued by M13KO7 helper phage using 1 :20 cells/phage ratio and phage particles precipitated with 20%PEG 8,000/2.5 M NaCI solution were purified using the standard protocol (Lennard S, Methods in Molecular Biology (Eds O’Brien PM and Aitken R) vol. 178 (2002), p. 59-71). Three rounds of “in solution” panning were performed using streptavidin- coated magnetic beads (Dynabeads M-280 Streptavidin). Briefly, 10¹¹-10¹² purified phage were incubated (blocked) in 2%milk/PBS for 1 h at room temperature to block non-specific interactions. The same incubation was performed for the M-280 Streptavidin magnetic beads. For round one of biopanning, the blocked phage library was mixed with the biotinylated human or mouse Plexin-B1 (20- 535)-Avi-6xHis (to final antigen concentration of 10 nM). After 1 hour incubation at room temperature, the magnetic beads were added to capture the biotinylated target. Beads were pelleted after 15 minutes incubation and washed six times with PBS/0.1 % Tween solution before bound phage were eluted with 100 mM triethylamine (TEA) and used for infection of TG1 cells (in logarithmic growth phase). The infected TG1 cells were plated on 2xYTAG agar bioassay plates and collected next day, providing enriched library for the next rounds of the biopanning. The same steps were performed for subsequent rounds, however with lower target concentration, 5 nM and 0.5 nM in round two and round three, respectively.

Screening for positive binders by phage ELISA

Random clones from enriched library after round two and three were picked and phage-containing supernatants were produced using a standard method (Coomber, David W. J., Methods in Molecular Biology (Eds O’Brien PM and Aitken R) vol. 178 (2002), p. 133-145) and subjected to antigen binding standard ELISA assay. Briefly, 96-well Nunc-lmmuno Maxisorp plates were coated 3 pg/ml streptavidin (ThermoFisher, 21122) and following overnight at 4°C, a 50 pl of 1 pg/ml biotinylated antigen (or non- relevant target) was added. After 30 minutes incubation, plates were washed with PBS/0.1 % Tween and blocked with 2% milk/PBS for 1 hour at room temperature. 50 pl of prepared phage supernatants (blocked for 1 h with 2%milk/PBS), were added to each well and incubated for 1 hour at room temperature. After washing with PBS/0.1 % Tween, the M13-HRP antibody (GE, 27-9421-01) diluted in 2% milk/PBS (1/5,000) was added to each well and incubated for 30 minutes. Finally, bound phage were detected with TMB substrate (Sigma, T4444) and colorimetric reactions quenched by 0.5 M sulfuric acid. Absorbance signal was measured at 450 nm. All single clone plates analysed by ELISA assay were subjected to Sanger sequencing.

Reformatting to full length chimeric (rabbit/human) antibodies

Based on the ELISA assay and sequencing results, the pool of antibodies was reformatted from scFv to full length chimeric (rabbit/human) IgG 1 isotype for further characterization. Construction of chimeric expression vectors was carried out by cloning of the amplified rabbit heavy and light chain variable regions into separate vectors containing human constant domains, pHuG1 (lgG1 isotype) and pHuK (kappa), respectively, using ligase-independent cloning (LIC) and transformed into chemically competent TOP10 cells. Several clones were isolated, prepped using the QIAprep Spin Miniprep Kit (27103, QIAGEN) and sequences verified by Sanger sequencing.

Generation of the chimeric antibodies

To produce chimeric IgG candidates, a pair of plasmids, encoding heavy and light chains, was cotransfected into Expi293 suspension cells using ExpiFectamine293 reagent (The Expi293™ Expression System Kit - Invitrogen - manufacture’s standard instructions). After 5 days of cultivation, culture supernatants were collected and IgG concentration quantified by OctetRED384 (ForteBio). Supernatants from large-scale cultures were purified by standard antibody purification methods using Protein A or G affinity chromatography followed by polishing step by size exclusion chromatography. Concentrations of the purified antibodies were measured by absorbance at 280 nm.

Humanisation of PLX7 antibody

Humanisation of RbPLX7 antibody was carried out by the CDR grafting method. Homology models (10) of the variable regions of RbPLX7 were produced using the Bioluminate 1.9 software (Schrodinger) to determine residues which were within 4A of the CDR loops. Optimal human frameworks were selected by interrogation of human VH and VL in-house curated databases using LifeArc’s proprietary antibody sequence analysis software. The selected human VH and VL frameworks were used to design the humanised variants; the first variant is comprised of the IMGT CDRs from the antibody grafted straight into the selected VH and VL frameworks, the second variant comprises the first variant with additional rabbit back-mutations at key proximity, VCI or interface positions and the remaining variants are composed of the first variant with single rabbit back-mutations in key positions. The first and second humanisation variants were synthesised (Genscript) and SDM performed on the first variant to produce the remaining variants. Gene sequences for heavy and light variable domains were cloned into pHuG1 and pHuK vectors, respectively. Assembled expression vectors were sequenced and used to transfect Expi293 cells. Cells were cultured for 5-7 days in serum free media, whereupon the conditioned medium containing secreted antibody was harvested. The concentrations of IgGiK antibodies in Expi293 cell conditioned media were measured by Octet RED384 (ForteBio). Most generated PLX7 humanization variants were produced at good expression levels and tested for binding, specificity, and range of biophysical properties (results not shown). The preferred humanised variant comprised the sequence of SEQ ID NO: 25 and 26.

Engineering of PLX11 antibody

Cross-reactive RbPLX11 antibody was engineered for higher expression using standard methods of light chain shuffling and site-directed mutagenesis known to those skilled in the art. The improved versions - RbPLXI 8, RbPLXI 9 and RbPLX20 were expressed and purified using the same methods as described above.

Binding characterisation

Binding kinetics analysis

Binding kinetics and affinity analysis of anti-Plexin-B1 antibodies were performed by SPR method using BIAcoreT200 instrument. 0.5 pg/ml of antibody was captured on Protein A chip in the 1xHBS-P+ buffer and series of Plexin-B1 (20-535) concentrations were injected over the flow cell to carry out kinetics. After each dissociation stage the chip was regenerated with 10 mM glycine, pH 2.0. Experimental data were fitted by 1 :1 Langmuir model using Biacore software to calculate KD values for each antibody.

Cross-reactivity and Sema4D competition characteristics

Cross-reactivity and capability of anti-Plexin-B1 antibodies to block interaction between Plexin-B1 and Sema4D were analysed by biolayer interferometry using OctetRED384 (ForetBio). Briefly, a streptavidin biosensor (ForteBio) was used to capture 5 pg/ml of biotinylated human or mouse Plexin-B1 (20-535) in the IxKinetics buffer (ForteBio). In the next step, the binding response was measured towards 50 nM of anti-Plexin-B1 or buffer, followed by the 50 nM of human Sema4D (7470-S4, R&D). Qualitative analysis of individual binding association curves was performed. Binding to full-length Plexin-B 1 expressed on Expi293F cells

Expi293F cells were plated into 24-well plates (2.5x10⁶ cells per well in a total volume 845 pl per well) and then transiently transfected with human or mouse Plexin-B1/Plexin-B2/Plexin-B3-FLAG pcDNA3.1 plasmids, using the ExpiFectamine (Gibco, A14524) transfection reagent according to the manufacturer instructions. Briefly, 1 pg DNA was mixed with 2.7 pl ExpiFectamine in serum-free medium (OptiMem, Gibco, 31985 in total volume 100 pl per well. Following 20 min incubation DNA mixture was added to cells and cultured in a shaker incubator at 225 rpm overnight. The following day, 5 pl of Enhancer 1 and 50 pl of Enhancer 2 were added. 48 hours after transfection cells were harvested, resuspended in PBS buffer (Gibco, 10010015) substituted with 0.1 % BSA (Sigma) and transferred into 96-well U-bottom plates (Corning, 3799) (2.5x10⁵ cells per well in a total volume 50 pl per well). Then anti-Plexin-B1 antibodies or the isotype control in a final concentration 10 pg/ml were added to cells. After 30-60 min incubation on ice, cells were washed in PBS-0.1 % BSA buffer and incubated with secondary AlexaFluor 488 anti-human antibody (Life Technology, A11013) for 30 min at room temperature. 3 pM of fluorescent dye Draq7 (Biostatus, DR7 1000) were added to cells in the last 10 minutes of secondary antibody incubation to label dead cells. Following PBS-0.1 % BSA washing cells were analysed on IntelliCyt iQue Screener instrument (Sartorius). Median Fluorescence Intensity (MFI) was calculated with ForeCyt software.

Sema4D signaling inhibition by Cos-7 collapse assay

Cos-7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with GlutaMAX, pyruvates (Gibco, 31966) and 10% Fetal Bovine Serum (Gibco, 16250078). 24 hours prior to transfection, Cos-7 cells were seeded in 96-well plates (Greiner, 655090) (6500 cells per well in a total volume 100 pl per well) and then transiently transfected 24 hours later with human or mouse Plexin- B1/Plexin-B2Z -FLAG pcDNA3.1 plasmids, using the X-tremeGene 9 transfection reagent according to the manufacture instructions (Sigma, 06365779001). Briefly, 0.05 pg DNA was mixed with 0.2 pl X- treme Gene 9 in serum-free medium (OptiMem, Gibco, 31985) to reach total volume 10 pl per well. Following 30 min incubation the DNA mixture was added to cells. After transfection for 48 hours, anti- Plexin-B1 antibodies or the isotype control in concentration 0.01-10 nM (for Plexin-B1) or 150 nM (for Plexin-B2) were added (10 pl/well), incubated for 1 hour, following by an additional 1 hour treatment with 50 nM Sema4D (for Plexin-B1), 150 nM Sema4D (for Plexin-B2). Cells mock-treated with 10 pl media instead of antibodies were incubated with 0.08-50 nM Sema4D for a Sema4D dose response (10 pl/well). The cells were fixed with 4% formaldehyde, permeabilised and incubated with anti-FLAG antibody (Sigma, F1804) following by staining with anti-mouse-Alexa Fluor 488 antibody (LifeTechnology, A1 1001), Hoechst (Invitrogen, H3570) and Phalloidin Texas Red (Life Technology, T7471). After staining, the plates were imaged using a high-content screening system (IN Cell Analyser 2000, GE Healthcare). Images were analysed and number of collapsed cells per well was quantified manually. Non-linear regression in GraphPad Prism was used to calculate EC50 values. Structural characterization of epitope

The epitope for RbPLX19 was determined from the crystal structure of the complex between Fab fragment of RbPLX19 and moPlexin-B1 (20-535).

The complex of moPlexin-B1 (20-535) with RbPLX19 was formed, deglycosylated with EndoH and purified using size exclusion chromatography, on a S200 16/600 column, in a buffer of 25 mM Tris-HCI pH 7.5, 50 mM NaCI. Optimal crystals of the complex were grown by hanging-drop vapour diffusion in 22% (w/v) PEG 1000, 0.4 M LiSO4, 0.1 M Phosphate/Citrate pH 4.2 at a protein concentration of 5 mg/ml. A cryo-protectant of 22% ethylene glycol in mother liquor was used. X-ray diffraction data was collected at the Diamond Light Source (Oxford, UK) using beamline I04.

Diffraction data for the moPlexin-B1 :RbPLX19 complex was indexed, integrated and scaled in DIALS (Winter G, 2018, Acta Crystallogr D Struct Biol. 2018 Feb 1 ; 74(Pt 2): 85-97), and merged with STARANISO (Tickle, 2018, STARANISO (Global Phasing Ltd)). The structure was determined using molecular replacement, using the program PHASER (McCoy, 2007, J Appl Crystallogr. vol. 40(Pt 4):658-674.).

The structural epitope has been defined based on the Plexin-B1 residues showing the buried surface area in the complex. The analysis was performed using PISA software (https://www.ebi.ac.uk/pdbe/pisa/).

Example 2

RT-PCR

To isolate RNA from mouse femora, bones were pestled in liquid nitrogen, vortexed in ribozol (VWR) for 45 minutes at room temperature, and centrifuged. The supernatant was extracted with chloroform. After adding an equal volume of ethanol, RNA was purified using Zymo-Spin IIICG columns (Zymo Research). RNA purification from primary microglia was performed using a Direct-zol RNA Microprep Kit (Zymo Research). cDNA was synthesized by reverse transcription. Quantitative PCR was performed using the Light-Cycler 480 Probes Master system (Roche). The following primers were used: for human plxnbl forward 5 -GACCGAGGTGGCCTACATCGAG-3’ (SEQ ID NO: 31) and reverse 5'- ACCTTCAGAAGTGTGCTCTGGGTCATG-3' (SEQ ID NO: 32) (“primer pair #1 ”), and forward 5'- CGGGACCGCTGCAAGAAGGAATTC-3' (SEQ ID NO: 33) and reverse 5'- TCCACAGTGGGCCGTCTGCTC-3' (SEQ ID NO: 34) (“primer pair #2”); for mouse plxnbl forward 5'- GTGTGCTGGAGCTAGGGAGTCGG-3' (SEQ ID NO: 35) and reverse 5'- CATGCAGCCCATCGGCACTG-3' (SEQ ID NO: 36) (“primer pair #1 ”), and forward 5'- GCCCGAGGAGCAGCGAGTG-3' (SEQ ID NO: 37) and reverse 5'-TCCTCCCCGCTGGCTCC-3' (SEQ ID NO: 38) (“primer pair #2”); for mouse bglap2: forward 5'-AGACTCCGGCGCTACCTT-3' (SEQ ID NO: 39) and reverse 5'-CTCGTCACAAGCAGGGTTAAG-3' (SEQ ID NO: 40); for mouse alpl: forward 5'- CGGATCCTGACCAAAAACC-3' (SEQ ID NO: 41) and reverse 5 -TCATGATGTCCGTGGTCAAT-3' (SEQ ID NO: 42). Western Blotting

Spinal cord tissue was sonicated in ice-cold radioimmunoprecipitation buffer (150 mM NaCI, 50 mM Tris pH 7.4, 5 mM EDTA, 1 % Triton X-100, 0.1 % SDS, 0.5% sodium deoxycholate, protease inhibitors), tissue lysates were centrifuged, and supernatants were used for Western blotting according to standard laboratory protocols.

Osteoblast assays

Human osteoblasts (HOB) were purchased from PromoCell (cat. no. C-12720), and cultured in osteoblast growth medium (PromoCell, cat. no. C-27001). To induce differentiation, cells were seeded into a 96-well plate (1 x 10⁴ cells/well), and, after cells attained confluency, the osteoblast growth medium was replaced by osteogenic medium (PromoCell, cat. no. C-27020). Sema4D (150 nM), anti- Plexin-B1 or IgG control antibodies (150nM) were added to the osteogenic medium; medium was changed every third day. ALP activity was analyzed after 7 days, using BCIP/NBT as a substrate (SigmaFast BCIP/NBT, Sigma Aldrich, cat. no. B5655-25TAB). To do so, cells were carefully washed once with PBS, and then fixed in cold 4% paraformaldehyde for 60 seconds at room temperature. Fixed cells were washed in 0.05% Tween 20/PBS, and stained with BCIP/NBT substrate solution in the dark for 5-10 min at room temperature. After one washing step with 0.05% Tween 20/PBS, PBS was added to the wells, and the optical density at 450 nm was analyzed in a microplate reader (Thermo Scientific Multiskan Spectrum). Alizarin Red staining (Sigma Aldrich, cat. no. A5533-25G) was done 21 days after the addition of osteogenic medium to human osteoblasts. Cells were washed in PBS and fixed in cold 4% paraformaldehyde for 20 min at room temperature. Alizarin Red S solution (2%) was added for 45 minutes, then cells were rinsed with water, and absorption at 590 nm was measured in a microplate reader (Thermo Scientific Multiskan Spectrum).

Primary mouse osteoblast progenitor cells were isolated from the calvarial bone of 3-day-old newborn mice. To do so, connective tissue was mechanically removed from bone tissue using forceps, and bone pieces were digested in a-MEM (Gibco, cat. no. 41061-029) containing 0.2% collagenase A and dispase II at 37°C for 10 minutes. Cells extracted in this step (fibroblasts) were discarded. The digestion of bone pieces was repeated 4 times and extracted cells were pooled. Cells were pelleted, resuspended in a- MEM containing 10% FBS and penicillin/streptomycin, and seeded into 6-well plates. After 48 hours, cells were trypsinized and seeded into 96-well plates (1 x 10⁴ cells/well) in osteogenic medium containing a-MEM, 10% FBS, penicillin/streptomycin, 5 mM p-glycerophosphate (Sigma Aldrich, cat. no. G9422), and 100 pg/ml ascorbic acid (Sigma Aldrich, cat. no. A4544). Medium was changed every third day. After 21 days, alizarin red S staining was performed as described above.

Generation of “humanized Plexin-B1 ” mice

The generation of Plexin-B1 knockout mice (plxnbl ¹') was described previously (Worzfeld et al., 2012, J Clin Invest 122: 1296-305). The BAC clone RP11 -47K17, carrying the human plxnbl gene, was obtained from the BACPAC Resource Center (Children’s Hospital Oakland Research Institute, Oakland, CA). BAC DNA was linearized by restriction digest, purified using a Sepharose column (Sepharose CL- 4B, Sigma) and injected into pronuclei of zygotes (C57BL/6 background). The resulting BAC transgenic mouse line, B6.Cg-Tg(human plxnb1)334383Soff, was crossed twice with the Plexin-B1 knockout mouse line (as described above) to obtain mice which lack a functional endogenous murine plxnbl gene, and instead express a transgenic human plxnbl gene. These mice were termed “humanized Plexin-B1 ” mice. Mice were kept heterozygous for the human plxnbl transgene. For genotyping, the following primers were used: forward 5’-CTGATACCGGTCCATGTGGAACGC-3’ (SEQ ID NO: 43) and reverse 5 -GGAAGCTGGGTCCTGAAGGCTG-3’ (SEQ ID NO: 44) (size of PCR products: 231 bp for the endogenous plxnbl wildtype allele, no product for the plxnbl knockout allele, no product for the transgenic human plxnbl allele), forward 5’-GTGGCTTTTCCAGGAGTGTTTGCC-3’ (SEQ ID NO: 45) and reverse 5’- GTGGCTCTTCAACAGTCCTTCCG-3’ (SEQ ID NO: 46) (size of PCR products: 1649 bp for the endogenous plxnbl wildtype allele, 430 bp for the plxnbl knockout allele, no product for the transgenic human plxnbl allele), forward 5 -GTCGTGTGGTTTGGGGCTGGG-3’ (SEQ ID NO: 47) and reverse 5’- GTGTCCTACTTGCTGGCTACTGCAG -3’ (SEQ ID NO: 48) (size of PCR products: no product for the endogenous plxnbl wildtype allele, no product for the plxnbl knockout allele, 291 bp for the transgenic human plxnbl allele)

Ovariectomy-induced bone loss and analysis of bone density

The mouse model of postmenopausal osteoporosis was performed as described (Negishi-Koga et al., 2011 , Nat Med 17: 1473-80). Briefly, 8-week-old female mice were ovariectomized or sham-operated. Mice were intravenously injected with anti-Plexin-B1 or IgG control antibodies (10 pg/g body weight) via the tail vein at days 4, 7, 14, 21 , 28, 35, 42, 49 and 56 after surgery. At day 63, femora were collected, fixed in 80% ethanol for at least 24 hours before scanning, and subjected to microcomputed tomography (Bruker Skyscan 1276) with the following settings: X-ray source at a voltage of 70 kV, with a current 200 pA, pixel size 4 microns, rotation range 180°, rotation step 0.200 degrees and averaging frame 7. After reconstruction (Bruker, Skyscan NRecon software), the images were morphometrically analyzed to obtain quantitative information on trabecular bone structures (Bruker, CTAn software).

Microglia isolation

Primary microglia were isolated from newborn mice. Brain tissue was harvested, placed into HBSS and meninges were removed. After washing with HBSS, tissue was digested with trypsin and DNAse, cells were pelleted by centrifugation, and seeded into poly-L-lysine coated flasks in medium containing DMEM, heat-inactivated FBS, penicillin/streptomycin, sodium pyruvate and glutamine. Cells were incubated for 8 days at 37°C and the medium was changed daily for 4 days after isolation. At day 9 post isolation, cells were stimulated with 30% L-929 conditioned DMEM medium, which had been harvested after 14 days continuous cultivation of L-929 fibroblasts. At day 14, microglia were harvested from the astrocyte layer by shaking the culture for at least 30 min at 37°C at a frequency of 90/min. Cells were pelleted and seeded on poly-L-lysine coated cover slips (1 x 10⁵ cells/ml). For immunostainings, primary microglia were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton/PBS, blocked in 1.5% horse serum/1 %BSA/PBS, incubated with primary antibodies, washed, incubated with fluorescently- labelled secondary antibodies, and analyzed using a Zeiss Ob-server Z1 AX10 fluorescence microscope.

Experimental Autoimmune Encephalomyelitis (EAE)

EAE was induced by injection of a total of 250 pg of myelin oligodendrocyte glycoprotein peptide MOG35- 55 (Gene Script) emulsified in Complete Freund’s Adjuvant (CFA) containing Incomplete Freund’s Adjuvant (BD, cat. no. 263910) and Mycobacterium tuberculosis H37 Ra (BD, cat. no. 231141). Injections were subcutaneous and bilateral into the flanks under isoflurane anesthesia. Pertussis toxin (Sigma Aldrich, cat. no. P7208) was injected intraperitoneally at a dose of 300 ng on the day and after 2 days of immunization with MOG35-55/CFA. Under isoflurane anesthesia, mice were intravenously injected with anti-Plexin-B1 or IgG control antibodies (10 pg/g body weight) via the tail vein at days 4, 8, 11 , 18, and 25 post immunization. Mice were evaluated daily for changes in weight and clinical symptoms for 35 days using the following scoring system with observers blinded to the genotype and treatment of mice: 0 = No obvious changes in motor function; when picked up by base of tail, the tail has tension and is erect. 0.5 = Tip of tail is limp; when picked up by base of tail, the tail has tension except for the tip; muscle straining is felt in the tail, while the tail continues to move; intact righting reflex. 1 = Limp tail; when picked up by base of tail, instead of being erect, the whole tail drapes over finger, no signs of tail movement are observed; intact righting reflex. 1.5 = Limp tail and hind leg inhibition; when the mouse is dropped on a wire rack, at least one hind leg falls through consistently; walking is very slightly wobbly; righting reflex delayed. 2 = Limp tail and weakness of hind legs; when the mouse is dropped on a wire rack, at least both hind legs fall through consistently; no righting reflex. 2.5 = Limp tail and dragging of hind legs; slight paralysis of the hind legs. 3 = complete paralysis of one hind leg or moderate paralysis of both hind legs. 3.5 = Limp tail and complete paralysis of hind legs. 4 = additional mild paralysis of the front legs. 4.5 = moribund. 5 = Death.

RESULTS

Antibodies binding to Plexin-B1 were produced having the sequences as shown in Figure 1 , with the identified CDRs underlined. Their format and specificity are shown below.

Table 2 - Format and specificity of anti-Plexin-B1 antibodies

The binding properties of the antibodies were evaluated as set out in the methods above. The results are summarised in the below tables.

Table 3 - Binding affinity and cross-reactivity of the anti-Plexin-B1 antibodies

Table 4 - Binding to full length human and mouse Plexin-B1/Plexin-B2/Plexin-B3 overexpressed on Expi293 cells

Antibodies that specifically bind Plexin-B1 were identified, which had cross reactivity to human and cynomolgus Plexin-B1. The antibody RbPLX7 and HuPLX7 did not bind mouse Plexin-B1. RbPLX11 , RbPLX18, RbPLX19 and RbPLX20 showed cross-reactivity to human, mouse and cynomolgus Plexin- B1. All antibodies (RbPLX7, HuPLX7, RbPLX11 , RbPLX18, RbPLX19 and RbPLX20) were shown to compete with Sema4D binding. The anti-Plexin-B1 antibodies bind human Plexin-B1 with a KD value in the 10^-9 M to 1O^-1CI M range (Table 3). The anti-Plexin-B1 antibodies did not bind Plexin-B2 or Plexin-B3. The results in Figures 2, 3 and 5 show further binding characteristics of the antibodies to human and mouse Plexin-B1.

Evidence of Sema4D-Plexin-B1 signalling inhibition by Cos-7 collapse assay has been demonstrated (Figure 4). Inhibition of Sema4D signalling was assessed via Cos7 collapse assay the results are summarised in the Table below. Table 5 - Cos-7 collapse assay (Sema4D blocking), ECsonM

RbPLXI 8, RbPLXI 9 and RbPLX20 are engineered versions of RbPLX11 parental antibody isolated from the rabbit phage display library. The primary aim of engineering was improvement of the expression properties of RbPLX11 antibody. RbPLX18, RbPLX19 and RbPLX20 demonstrated significantly higher expression (8-10-fold) without losing their potency, specificity, or cross-reactivity. RbPLX20 demonstrated activity in vitro and efficacy in vivo, in animal models (humanized transgenic mice lines that carry the human Plexin-B1 gene). Increased bone volume in ovariectomy-induced osteoporosis model was seen with RbPLX20 (Figures 11 and 12).

The chimeric (rabbit/human) anti-Plexin-B1 antibodies RbPLX11 , RbPLX18, RbPLX19 and RbPLX20 showed cross-reactivity to human, mouse and cynomolgus Plexin-B1. RbPLX11 , RbPLX18, RbPLX19 and RbPLX20 have the same heavy chain variable region CDR sequences, with similar light chain variable region CDR sequences (LCDR1 and LCDR2 were identified as being the same).

PLX19 was shown to bind to the second and third blades of the moPlexin-B1 sema domain, specifically to the D2 and D3 strands, as well as the A2-B2, D2-A3, C3-D3 and D3-A4 loops. Based on the crystal structure of the complex between RbPLX19 Fab and moPlexin-B1 (20-535) the conformational epitope for RbPLX19 antibody was determined to comprise the amino acids residue as followed:

106, 131 , 132, 135, 136, 137, 138, 139, 140, 141 , 188, 190, 191 , 192, 193, 194, 195, 196, 199, 200, 201 , 202, 203, 256 (numbers corresponding to amino acid residues of moPlexin-B1 (Uniprot: Q8CJH3)).

Although only the RbPLX19 epitope structure was determined, due to high similarity of the CDRs of RbPLX11 , RbPLX18, and RbPLX20 with RbPLX19, the epitope is highly likely to be the same for at least these antibodies.

RbPLX7 demonstrated activity in vitro and efficacy in vivo, in animal models (humanized transgenic mice lines that carry the human Plexin-B1 gene). In vitro, inhibition of osteoblast differentiation and mineralization was seen with RbPLX7 (Figure 6). Increased bone volume in ovariectomy-induced osteoporosis model was seen with RbPLX7 (Figures 7). Decreased clinical score in EAE model was seen with RbPLX7 (Figure 8). Penetration to the CNS in EAE model was seen (Figure 8).

Summary

Monoclonal antibodies directed against the extracellular region of human Plexin-B1 (comprising the Serna and PSI domains), which specifically block the binding of Sema4D to Plexin-B1 and inhibit Sema4D-Plexin-B1 signaling were produced. Some antibodies were further engineered for higher expression levels compared to their parental clone. In vitro, these anti-Plexin-B1 antibodies have been shown to block the inhibitory effects of Sema4D on human osteoblast differentiation and mineralization.

The anti-Plexin-B1 antibodies have also been shown to exhibit beneficial effects in mouse models of MS and postmenopausal osteoporosis. In summary, the data shows the anti-Plexin-B1 antibodies as potential therapeutic agents in conditions such as multiple sclerosis and osteoporosis, which can be produced at sufficient expression levels for subsequent use.

Exemplary sequences of the antibodies and antigens to which they may bind referred to herein, are provided in Table 6.

Table 6:

Claims

1 . An anti-Plexin-B1 antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein a) the heavy chain variable region amino acid sequence comprises:

- a HCDR1 comprising the sequence of SEQ ID NO: 2 (GFSFSGSYY) or SEQ ID NO: 1 (GIDLSSSA)

- a HCDR2 comprising the sequence of SEQ ID NO: 4 (IYTGSTGST) or SEQ ID NO: 3 (IGSSDST); and

- a HCDR3 comprising the sequence of SEQ ID NO: 6 (ARDHAAYAGYGVPWYTRLDL) or SEQ ID NO: 5 (ARGLYSGMDP); and b) the light chain variable region amino acid sequence comprises

- a LCDR3 comprising the sequence of SEQ ID NO: 14 (LGGWSSASDNT), SEQ ID NO: 11 (QSNYGSINSDYGNA) or SEQ ID NO: 12 (QSNYGSISSSYGNA); or SEQ ID NO: 13 (QSNYGSVSTNYGNA).

2. An antibody or antigen binding fragment thereof according to claim 1 , wherein i) the heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 2 (GFSFSGSYY), a HCDR2 comprising the sequence of SEQ ID NO: 4 (IYTGSTGST); and a HCDR3 comprising the sequence of SEQ ID NO: 6 (ARDHAAYAGYGVPWYTRLDL); and the light chain variable region comprises a LCDR1 of SEQ ID NO: 8 (PSVLGNY), a LCDR2 of SEQ ID NO: 10 (GAS); and a LCDR3 of SEQ ID NO: 14 (LGGWSSASDNT); or ii) the heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO:

1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a HCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and the light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 11 (QSNYGSINSDYGNA); or iii) the heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO:

1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a HCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and the light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 12 (QSNYGSISSSYGNA); or

48 iv) the heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO:

1 (GIDLSSSA), a HCDR2 comprising the sequence of SEQ ID NO: 3 (IGSSDST) and a HCDR3 comprising the sequence of SEQ ID NO: 5 (ARGLYSGMDP); and the light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 7 (QSISNL), a LCDR2 comprising the sequence of SEQ ID NO: 9 (RAS), and a LCDR3 comprising the sequence of SEQ ID NO: 13 (QSNYGSVSTNYGNA); or An antibody or antigen binding fragment thereof according to any one of the preceding claims wherein the light chain variable region (VL region) comprises: a. a sequence having at least 90% sequence identity to SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26; b. a sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26; wherein five or fewer amino acids are substituted, deleted or inserted into the sequence; or c. the sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO: 24 or SEQ ID NO: 26. An antibody or antigen binding fragment thereof according to any one of the preceding claims wherein the heavy chain variable region (VH region) comprises; a. a sequence having at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25; b. a sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25; wherein one amino acid is substituted, deleted or inserted into the sequence; or c. the sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 19, SEQ ID NO: 21 , SEQ ID NO: 23 or SEQ ID NO: 25. An antibody or antigen binding fragment thereof according to any one of the preceding claims wherein: the heavy chain variable region comprises SEQ ID NO: 23 and the light chain variable region comprises SEQ ID NO: 24; the heavy chain variable region comprises SEQ ID NO: 25 and the light chain variable region comprises SEQ ID NO: 26; the heavy chain variable region comprises SEQ ID NO: 15 and the light chain variable region comprises SEQ ID NO: 16; the heavy chain variable region comprises SEQ ID NO: 17 and the light chain variable region comprises SEQ ID NO: 18; the heavy chain variable region comprises SEQ ID NO: 19 and the light chain variable region comprises SEQ ID NO: 20; or

49 the heavy chain variable region comprises SEQ ID NO: 21 and the light chain variable region comprises SEQ ID NO: 22. An anti-Plexin-B1 antibody or antigen binding fragment that specifically binds to an epitope of human Plexin-B1 wherein the epitope is comprised in the region of amino acids 20 to 535 of SEQ ID NO: 28 or 30. An anti-Plexin-B1 antibody or antigen binding fragment that specifically binds to an epitope of Plexin-B1 wherein the epitope is comprised of at least one amino acid selected amino acid residues 106, 131 , 132, 135-141 , 188, 190-196, 199- 203, 256 of SEQ ID NO: 30. The anti-Plexin-B1 antibody or antigen binding fragment according to claim 7 wherein the epitope comprises the amino acid residues 106, 131 , 132, 135-137, 141 , 188, 190-196, 199-203, and 256 of SEQ ID NO: 30. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody is a monoclonal antibody. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody is a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, an Fv fragment, an scFv fragment, dAb, Fd, a diabody, and a single chain antibody. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody is human antibody, murine antibody, a rabbit antibody, a humanised antibody or a chimeric antibody. The antibody or antigen-binding fragment thereof according to anyone of the preceding claims, wherein the antibody is an IgG antibody, preferably and lgG1 , lgG2, lgG3 or lgG4-type antibody, more preferably wherein the antibody is an IgG 1 antibody. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the wherein the antibody or antigen-binding fragment is comprised in a bispecific antibody, a multispecific antibody, or is conjugated to a further therapeutic or diagnostic agent. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier. A pharmaceutical composition of claim 14 further comprising an additional therapeutically active agent, or wherein the pharmaceutical compositions is for use in combination with another therapy or additional therapeutically active agent.

50 A nucleic acid encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to

13. A vector comprising the nucleic acid of claim 16 operably linked to a promoter. A host cell comprising the nucleic acid of claim 16 or vector of claim 17. A method of making an antibody or antigen-binding fragment thereof according to anyone of claims 1 to 13, the method comprising culturing a host cell of claim 18. An antibody according to any one of claims 1 to 13 or a pharmaceutical composition of any one of claims 14 or 15 for use in medicine An antibody according to any one of claims 1 to 13 for use in the treatment of osteoporosis, multiple sclerosis, a neoplastic disease or a neurodegenerative disease. A method of inhibiting the binding of Plexin-B1 to Sema4D for the treatment of a disease associated with Sema4D signalling via Plexin-B1 , the method comprising administering an effective amount of antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 or 15 to a subject thereof. A method of treating osteoporosis, multiple sclerosis, a neoplastic disease or a neurodegenerative disease in a subject, the method comprising administering an effective amount of antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 or 15 to the subject. The method according to claim 23 further comprising administering simultaneously or sequentially, in any order, a further therapeutically active agent. The method according to claim 24 wherein the neoplastic disease is cancer, and the further therapeutically active agent is an immune modulating agent, preferably wherein the immune modulating agent is immune checkpoint inhibitor.

51